Zona-Free Hamster Eggs: Their Use in Assessing Fertilizing Capacity and Examining Chromosomes of Human Spermatozoa

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1 Gamete Research 10: (1984) Review Article ZonaFree Hamster Eggs: Their Use in Assessing Fertilizing Capacity and Examining Chromosomes of Human Spermatozoa R. Yanagimachi Department of Anatomy and Reproductive Biology, University of Hawaii School of Medicine, Honolulu Key words: spermatozoa, zonafree egg, egg, fertilizing capacity, sperm chromosome INTRODUCTION As a rule, spermatozoa of one species cannot fertilize eggs of other spscies. One of the major sites of the specificity of fertilization in mammals is the zona pellucida, a glycoprotein coat surrounding the egg proper. When the zona is removed, the specificity of fertilization may be reduced, but not always. In fact zonafree eggs of most mammalian species retain very strong or fairly strong species specificity, rejecting entry of spermatozoa of most other species. The golden hamster (Mesocricetus auratus) is exceptional. Its zonafree eggs permit entry of spermatozoa of not only the same species, but also those of a wide variety of other species provided the spermatozoa have completed capacitation and acrosome reaction. As capacitation and the acrosome reaction are essential preliminaries to normal fertilization of all mammalian species [Yanagimachi, 19811, zonafree hamster eggs can be used in assessing the fertilizing capacity of spermatozoa of various other species. This would be particularly useful for species in which spermatozoa are relatively easy to collect but the eggs are not. The human is a good example. In this review, I will discuss how zonafree hamster eggs have been used in assessing the fertilizing capacity of human spermatozoa. In addition, I will review some studies that have utilized zonafree hamster eggs to visualize human sperm chromosomes. Before going further, I will describe how mammalian spermatozoa effect fertilization under normal conditions. This information is of absolute necessity in understanding correctly which sperm functions we can (or cannot) assess using zonafree eggs. Received January 16, 1984; accepted February 22, Address reprint requests to Dr. R. Yanagimachi, Department of Anatomy and Reproductive Biology, University of Hawaii School of Medicine, 1951 EastWest Road, Honolulu, HI Alan R. Liss, Inc.

2 188 Yanagimachi OUTLINE OF THE PROCESSES AND MECHANISMS OF NORMAL FERTILIZATION Freshly ejaculated mammalian spermatozoa are unable to fertilize eggs immediately even if they are brought directly into contact with them. The spermatozoa acquire their fertilizing capacity during their sojourn in the female genital tract. This phenomenon is referred to as capacitation (Fig. 1A). Although capacitation normally occurs within the female genital tract, it can be induced under a variety of experimental (in vitro) conditions. The time required for capacitation varies from species to species. It is generally believed that human spermatozoa need several hours to be capacitated. However, one must be aware that capacitation time is by no means fixed. It varies depending on the physiological (eg, hormonal) state of the female as well as the condition of the medium to which spermatozoa are exposed. We should not expect that all the spermatozoa are capacitated at the same rate. Even among spermatozoa in the same ejaculate, some are capacitated much faster than others. Furthermore, in the human, for example, there seems to be considerable variation in capacitation time from man to man (cf Fig. 10 of this review). The biochemical basis of sperm capacitation is not fully understood, but a major component is believed to be biochemical changes in the sperm plasma membrane including the removal or alteration of substances incorporated onto or into the membrane during maturation of the spermatozoa in the epididymis and upon exposure to the secretions of the male accessory glands at ejaculation. Without capacitation, spermatozoa are unable to undergo the acrosome reaction (Fig. lb), which is essential for spermatozoa to pass through the egg investments. When capacitated spermatozoa come into close approximation with Fig. 1. Possible relationships among the major events that occur before and during normal fertilization in mammals.

3 Zonafree Hamster Egg 189 the eggs in the ampulla or contact the egg investments, the outer acrosomal membrane and the overlying plasma membrane undergo multiple fusions (called the acrosome reaction), releasing or exposing acrosomal enzymes. Some enzymes (eg, hyaluronidase) thus released are generally believed to depolymerize the cumulus matrix and assist the passage of spermatozoa through the cumulus oophorus. Some other enzymes (eg, acrosin) tightly bound to the surface of the acrosomereacted spermatozoa are believed to dissolve zona material and so assist sperm passage through the zona (Fig. 1C). Shortly before or about the time of the acrosome reaction, capacitated spermatozoa of many species begin very active movement called hyperactivation (Fig. 1D). This movement seems to provide strong thrusting power to the spermatozoa passing througn the egg investments, particularly the zona. Thus, the spermatozoa seem to use both mechanical and biochemical (enzymatic) means to pass through the egg investments. If spermatozoa become weak or their zonadissolving enzymes (zona lysins) become inactivated or exhausted before or during their passage through the zona, they will fail to pass through it. A spermatozoon that has passed through the zona fuses with the egg plasma membrane (Fig. 1E). Under normal conditions, a spermatozoon that is about to fuse with the egg plasma membrane has always completed its acrosome raction. Must the spermatozoon be acrosome reacted to fuse with the egg? The answer is yes. If spermatozoa with intact acrosomes (either uncapacitated or capacitated) are placed with zonafree eggs, for instance, the spermatozoa may attach to the egg surfaces, but never fuse with them. In other words, the acrosome reaction renders the sperm membrane (more specifically, the plasma membrane over the equatorial segment of the acrosome and the anterior region of postacrosomal area, which is not directly involved in the acrosome reaction) capable of fusing with the egg plasma membrane (Fig. 1F). Soon after the head of the spermatozoon is incorporated into the egg cytoplasm (Fig. lg), the nuclear envelope of the sperm nucleus disappears (Fig. 1H) and decondensation of the nuclear chromatin begins (Fig. 11). Upon spermegg fusion, the properties of the egg plasma membrane change (electrically?) (Fig. 1J) such that it rejects fusion with further spermatozoa. This is called the plasma membrane block (Fig. 1K). Spermegg fusion also triggers activation of the egg cytoplasm (Fig. 1L) causing exocytosis of cortical granules (Fig. 1M). The cortical granule material interacts with and modifies the zona so that it becomes impenetrable by further spermatozoa. This is called the zona reaction (Fig. 1N). The precise mechanism used for the block to polyspermy depends on the species. Eggs of some species (eg, the rabbit) depend almost solely on the egg plasma membrane block, and those of some other species (eg, the mouse) seem to use both the egg plasma membrane block and the zona reaction in preventing polyspermy. Eggs of some other species totally depend on the zona reaction. The eggs of the golden hamster are an example of the last. They have no distinct (at least, no rapid) egg plasma membrane block to polyspermy. This is one of the unique and useful features of the hamster egg. Activation of the egg cytoplasm allows the decondensed sperm nucleus to continue to develop into a sperm pronucleus (Fig. 10). Meantime, the egg chromosomes arrested at metaphase of the second meiotic division before sperm entry complete meiosis (Fig. 1P) and form an egg pronucleus (Fig. 1Q). DNA synthesis and chromosome duplication in both sperm and egg pronuclei occur more or less synchronously with the development of the pronuclei. Activation and rearrangement

4 190 Yanagimachi of cytoskeletal systems (Fig. 1R) bring developing sperm and egg pronuclei to the center of the egg. Fully developed pronuclei come into close approximation, their nuclear envelopes disintegrate, and their chromosomes mingle (Fig. 1s) for the first cleavage (Fig. 1T). The time interval between sperm entry and the first cleavage is more than 12 hr in most mammals. It is about 16 hr in the golden hamster and about 28 hr in the human. WHAT IS REQUIRED FOR SPERM PENETRATION INTO ZONAFREE EGGS? When the eggs are freed from their zonae pellucidae so spermatozoa can make direct contact with the egg plasma membrane, the spermatozoa to not need to have the vigorous motility and zona lysins that are essential for successful zona penetration. As long as they are alive and acrosomereacted, they will be able to fuse with (penetrate) zonafree eggs. According to Aitken et a1 [1983c], the spermatozoa of a man with Kartagener s syndrome, which are completely immotile due to the lack of dyenin arms, can undergo capacitation and the acrosome reaction in vitro and be incorporated by zonafree eggs if they meet the egg surface. Under ordinary conditions, the acrosome reaction occurs as a result of capacitation. Therefore, if spermatozoa in question become capable of penetrating zonafree eggs, they must have both completed capacitation and undergone the acrosome reaction. If not, either capacitation or the acrosome reaction or both must have failed to occur in these spermatozoa. Under very special in vitro conditions, spermatozoa may undergo an acrosome reaction while bypassing the changes of capacitation. These spermatozoa will be able to penetrate zonafree eggs if they are alive. PENETRATION OF HUMAN SPERMATOZOA INTO ZONAFREE HAMSTER EGGS Uniqueness of Zonafree Hamster Egg Table 1 summarizes the results of experiments in which zonafree eggs of six species were inseminated in vitro with spermatozoa of the same (homologous) and different (heterologous) species. Although data are incomplete, it is obvious that zonafree eggs of the golden hamster are rather unique in that they permit entry of spermatozoa of a wide variety of foreign species. So far, it is the zonafree eggs of only the golden hamster that allow the entry of human spermatozoa. It is somewhat unexpected that zonafree eggs of the Chinese hamster fail to incorporate human spermatozoa. Human spermatozoa are unable to attach to zonafree eggs of this species. No one knows at the moment why zonafree eggs of the golden hamster are so special. Morphology and Criteria for Sperm Penetration Into the Egg Freshly ejaculated human spermatozoa with intact acrosomes (Fig. 2) cannot attach to and penetrate zonafree hamster eggs. Those that collide with egg surfaces may stay there for a few seconds but then swim away. Preincubated (capacitated) spermatozoa, on the other hand, can attach to and penetrate into the eggs. Electron microscopic examination of spermatozoa attached to egg surfaces have revealed that all or almost all of them are acrosomereacted (Fig. 3) [Yanagimachi et al, 1976; Barros et al, 1979; Koehler et al, 1982; Talbot and Chacon, 1982; Gould et al,

5 Zonafree Hamster Egg 191 TABLE 1. Penetration of Zonafree Eggs by Spermatozoa of Homologous and Heterologous Species* Zonafree eggs Golden Chinese Guinea Spermatozoa hamster hamster Mouse Rat pig Rabbit Golden hamster Yes NO'^ yes, NO^ Yes, NO^ Yes NO^ Chinese hamster yes Mouse yes2,5,6 yes2sj5 yes2s,26 yes21 ye$ Deer mouse yes3 NO^ ~ ~ 3 ~ 2 6 Rat yes2s,6 Yes, NO^ ~es~, ~ Yes6 Guinea pig ~ e s ~. ~ NO^ NO^ Yes NO^ Bat yes7 Rabbit yes3 Dog No8 Dolphin yes yes1013,27 Pig Bull yes131s Goat Yes 6 Horse Yes Marmoset monkey YesI7 Human Yes s ~ ~ 1 ~~20,21 9 No20,21 No *References: (1)Yanagimachi and Noda [1970a,b]; Barros et a1 [ 19731; Barros and Herrera [1977]; (2) Hanada and Chang [1972]; (3) Hanada and Chang [1978]; (4) Yanagimachi [1972a]; (5) Pavlok [1979]; (6) Hanada and Chang [1976]; (7) Lambert [1981]; (8) Yanagimachi and MahiBrown (unpublished data); (9) Fleming et a1 [1981]; (10) Imai et al [1977, 1979, 19801; (11) Pavlok [1981]; (12) Pavlok et a1 [1982]; (13) Hanada and Nagase [1981]; (14) Bousquet and Brackett [1982]; (15) Brackett et a1 [1982]; (16) Kim et a1 [1980]; (17) Moore [1981]; (18) Yanagimachi et a1 [I9761 and see this review; (19) Kamiguchi, Mikamo, and Yanagimachi (unpublished data); (20) Quinn [ 19791; (21) Yanagimachi (unpublished data); (22) Yanagimachi [1972b]; (23) Brackett et a1 [1971]; Gordon and Dandekar [ 19761; (24) Toyoda and Chang [1968]; (25) Wolf et a1 [1976]; Wolf and Armstrong [1978]; (26) Thadani [1980]; (27) Smith et a1 [1983]. Although some acrosomeintact spermatozoa may attach to egg surfaces [Gould et al, 1983; Koehler et al, 19831, it is obvious that it is acrosomereacted ones that are able to fuse with (penetrate) the eggs (Figs. 4 and 5). It has been reported that acrosomeintact human spermatozoa can penetrate zonafree human eggs [Soupart and Strong, However, the micrographic evidence presented by these authors is not convincing enough to substantiate their statement. No one else has ever seen eggs of any species penetrated by acrosomeintact spermatozoa. Therefore, we can say with confidence that all human spermatozoa that penetrate zonafree hamster eggs have been acrosomereacted. In this context, it is interesting to note that acrosomeless human spermatozoa, which are obviously unable to undergo the acrosome reaction, cannot penetrate even a single zonafree hamster egg [Weissenberg et al, 1982; Jeyendran et al, The most commonly used criterion for egg penetration by spermatozoa is the presence of swollen sperm heads or sperm pronuclei within the egg cytoplasm. Swollen sperm heads or pronuclei can be readily observed with either a phasecontrast or interferencecontrast microscope after compressing living eggs between a slide and coverslip (Fig. 6) [Yanagimachi et al, However, the presence of a pronucleus or pronuclei alone is not a dependable criterion for sperm penetration because hamster eggs may activate spontaneously without sperm penetration. The presence of swollen

6 Figs. 24. Semisagittal sections of human spermatozoa. Figure 2 shows a freshly ejaculated spermatozoon with an intact acrosome. Figure 3 shows an acrosomereacted spermatozoon trapped by microvilli of a zonafree hamster egg. Figure 4 shows an acrosomereacted spermatozoon fusing with a zonafree hamster egg. acp, acrosomal cap; c, cortical granule; eq, equatorial segment of the acrosome; iam, inner acrosomal membrane; m, microvilli of the egg; n, nucleus; oam, outer acrosomal membrane; p, plasma membrane. Bar = 1 pm.

7 Zonafree Hamster Egg 193 Fig. 5. Human sperm nuclei decondensing in the cytoplasm of zonafree hamster eggs. (A,B) Micrographs showing chromatin decondensation. (C) Micrograph showing an advanced stage of chromatin decondensation. Bar = 0.5 pm.

8 194 Yanagimachi Fig. 6. Phasecontrast micrographs of zonafree hamster eggs penetrated by human spermatozoa. (A) Egg with four swollen sperm heads (arrows). (B,C) Swollen sperm heads under a higher magnification. (D) Comparison of swollen (s) and unswollen (u) sperm heads. AC, living specimens; D, fixed and stained specimen. pb, Second polar body; s, swollen sperm head in the egg cytoplasm; t, tail of spermatozoon that has penetrated; u, unswollen sperm head probably lying on the egg surface. Bar = 10 pm.

9 Zonafree Hamster Egg 195 sperm heads or pronuclei with accompanying sperm tails is the only dependable criterion of sperm penetration. Warning must be given here that timing of egg examination is important because the human sperm tail disintegrates and becomes undetectable by light microscopy several hours after its entry into the cytoplasm. Then, only electron microscopy can detect sperm tail components in the cytoplasm. The disappearance (or a sharp reduction in number) of cortical granules from the egg cortex is another good indication of sperm penetration. Cortical granules of the hamster egg are large enough to visualize with a 1OOX objective (Fig. 7) [Austin, Although this criterion has been rarely used by investigators, it is useful particularly when the earliest time of sperm penetration must be determined. In some cases, sperm heads (nuclei) may fail to decondense even after their entry into the egg cytoplasm. Without seeing swollen sperm nuclei, it would be very difficult to evaluate whether the eggs have been penetrated by spermatozoa. Although examination of the eggs with the electron microscope would solve this problem, we could tell promptly if the eggs were penetrated by examining the density of cortical granules in their cortex. Multiple entry of spermatozoa into one egg is common, particularly when it is inseminated with a high concentration of spermatozoa. This is because the hamster egg has no distinct egg membrane block to polyspermy, as already mentioned. Not all the sperm heads (nuclei) incorporated into the egg cytoplasm decondense (swell). Some may stay condensed for many hours. Others may decondense (swell), but not transform into pronuclei because the amount of egg cytoplasmic factors responsible for development of the decondensed sperm nuclei to the pronuclei seems to be limited Fig. 7. Phasecontrast micrographs of the cortex of hamster eggs. Numerous cortical granules are seen in the cortex of a living unfertilizaed egg (A), but not in an egg penetrated by a spermatozoon (oa) (B). Bar = 10 fim.

10 196 Yanagimachi [Hirao and Yanagimachi, 1979; Yanagimachi, Thus, to visualize human sperm chromosomes at a later time, we must avoid excessive polyspermy. Unless they are heavily polyspermic, the eggs penetrated by human spermatozoa will reach the advanced pronuclear stage, then the twocell stage about 16 h after sperm entry (Fig. 8). No further development occurs under the present experimental conditions. USE OF ZONAFREE HAMSTER EGGS FOR ASSESSMENT OF FERTILIZING CAPACITY OF HUMAN SPERMATOZOA Problems Inherent to the Previously Available Techniques for Assessing Male Fertility Fertility of men depends on the quality of semen they ejaculate. The sperm density in the ejaculate is known to be one of the important indications of male fertility. It is generally accepted that men tend to be infertile if the sperm density in their semen is less than 1020 x lo6/& [eg, MacLeod and Gold, 1951; Zuckerman et al, However, according to Davis et al [1979], a sperm concentration of less than 10 x lo6/& is seen in about 3% of fertile men, and conversely a sperm density of more than 100 x 106/ml occurs in about 25% of infertile men. Obviously, other semen parameters (eg, percentage of motile spermatozoa and quality of sperm movement) must be taken into consideration [MacLeod and Wang, However, even if all conceivable aspects are evaluated by conventional microscopic examination, men with socalled abnormal semen parameters are not necessarily infertile. For example, Rehan et al [1975] reported three men with very poor semen parameters (semen volume, ml; sperm density, X 106/ml; % motile sperm, 1 20%) who were nevertheless fertile on occasion. There are many men whose conventional semen parameters are apparently normal yet are clinically infertile. Husbands in couples with unexplained infertility are examples. Thus, an accurate prediction or evaluation of fertility and infertility of men by conventional semen analysis is very difficult except for such extreme cases as severe oligospermia or azoospermia, which obviously indicate infertility. This is quite understandable because simple microscopic examination of semen samples does not tell us everything about the physiological state of the spermatozoa we are observing. The most certain way of evaluating the fertilizing capacity of human spermatozoa is to deposit them in the genital tract of a woman of proven fertility during the periovulatory period and later determine the success or failure of fertilization or pregnancy. If we could inseminate a group of females of proven fertility (as we do in laboratory and farm animals), we would certainly know whether the men in question are fertile. This approach is impractical for obvious reasons. An alternate approach would be to inseminate living human eggs in vitro. This again would provoke ethical debate. The use of nonliving human eggs [eg, Overstreet and Hembree, 1976; Yanagimachi et al, may be the next best way of assessing fertilizing capacity of human spermatozoa. Unfortunately, human eggs are not readily available for routine fertility assays in most hospitals and laboratories. Hamster Test History and ethics. In 1976 we reported that human spermatozoa penetrate zonafree eggs of the golden hamster after the spermatozoa have undergone both

11 Zonafree Hamster Egg 197 Fig. 8. Development of zonafree hamster eggs penetrated by human spermatozoa. (A) Egg in the pronuclear stage with two pronuclei; the sperm and egg pronuclei appear similar. (B) Egg in the pronuclear stage with four pronuclei, one of which is out of focus in this micrograph; one of the pronuclei is believed to be of egg origin, the remaining three most probably being of sperm origin. (C) Side view of metaphase chromosomes shortly before the first cleavage. (D) Egg in the twocell stage. Bar = 20 pm.

12 198 Yanagimachi capacitation and the acrosome reaction [Yanagimachi et al, As capacitation and the acrosome reaction are two important steps mammalian spermatozoa must undergo before fertilizing eggs and the acrosome reaction is obligatory to successful sperm fusion with the egg, we proposed that zonafree hamster eggs could be used as substitutes for human eggs in assessing the fertilizing capacity of human spermatozoa. We originally submitted the paper to Science, then to the Proceedings ufthe NatiunaE Academy of Science (USA), but the paper was rejected by both. Two referees favored the paper, but two others expressed their concern about adding human spermatozoa to animal eggs. The chief editors recommended that we submit the paper to a more specialized journal, which we did. Biology of Reproduction accepted the paper immediately. In a World Health Organization (WHO) workshop held in Geneva, Switzerland, in 1976 I proposed the use of zonafree hamster eggs for assessing the fertilizing capacity of human spermatozoa. The advisory committee for WHO rejected the proposal in 1977 and It was not until 1982 that the committee approved the use of this technique as a means of assessing the fertilizing capacity of human spermatozoa [M.R.N. Prasad, personal communication]. In the paper published in 1976 [Yanagimachi et all we reported that freshly collected human spermatozoa are incapable of immediate penetration into zonafree hamster eggs, but become capable of doing so when preincubated in BWW medium (a modifed KrebsRinger s solution) for several hours to allow their capacitation and acrosome reaction. In the hamster egg penetration by a human spermatozoon (oa), both sperm (human) and egg (hamster) pronuclei develop [Yanagimachi et al, 19761, sperm and egg chromosomes subsequently appear [Rudak et al, 19781, and the egg may reach the twocell stage [Yanagimachi, 1977, 1978a; Brandriff et al, 19821, but never develops in vitro beyond this stage at the present time. Even if the eggs were to develop further, the hybrid would not grow to term because of enormous differences in the genome of the hamster and human, respectively. As long as we use the eggs of animals (including the hamster) for basic research and clinical diagnosis for the benefit of mankind with maximum precautions, the use of zonafree hamster eggs, for instance, for evaluating the function and genetic makeup of human spermatozoa would seem justified. In the past, the bioassay for assessing the fertilizing capacity of human spermatozoa using zonafree hamster eggs has been referred to in many different ways: (1) hamster egg penetration assay (test), (2) zonafree hamster egg assay (test), (3) egg penetration assay (test), or (4) sperm penetration assay (test). In this review, I will call it the hamster egg test or simply the hamster test. Application of the hamster test for assessing the fertilizing capacity of spermatozoa of fertile and infertile men Overall view ofthe results obtained. Barros et a1 [1978] were the first to use zonafree hamster eggs in assessing the fertilizing capacity of men with normal and abnormal semen parameters. They found that spermatozoa of the majority (76%) of men with normal semen characteristics penetrated at least one of the eggs inseminated, while those of only 34% of the men with abnormal semen parameters could do so. Their subsequent study [Barros et al, revealed that spermatozoa in the semen samples with normal semen parameters penetrated 29 f 29% (mean SD) of the eggs, whereas those in the semen with abnormal parameters penetrated only 8 f 17 % of the eggs. Although these studies clearly demonstrated the existence of some

13 Zonafree Hamster Egg 199 qualitative and quantitative differences in the fertilizing capacity of spermatozoa in normal and abnormal semen, it is not clear from the descriptions in their papers whether the men subjected to the tests were clinically fertile or not. Subsequent invertigators have taken the fertility and infertility of the men into consideration. Unfortunately, however, almost every investigator has used different procedures (see Table 2); thus, direct comparison of the data obtained is difficult. Nevertheless, two points have become clear. 1) Spermatozoa from men of proven fertility almost consistently penetrate more than 10% of the eggs, with an overall average of 57% (Table 3). 2) Spermatozoa of clinically infertile men penetrate a significantly lower percentage of eggs (an overall average of 17%) compared with those of fertile individuals (Table 4). It is clear from Table 3 that although spermatozoa of fertile men are able to penetrate the eggs, this ability varies greatly from man to man. Spermatozoa of some fertile men penetrate 100% of the eggs, while those of a few penetrate only about 10% or less. This may be partly due to variation in the techniques used by different investigators, but the existence of variation in eggpenetrating ability among different fertile men seems to be real. Martin and Taylor [1982] reported one (out of 22 fertile men) whose spermatozoa consistently failed to penetrate even a single egg. Albertsen et al [1983] found one (out of 11 fertile men) whose spermatozoa completely failed to penetrate the eggs. Overstreet et a1 [1980a] reported one (out of six fertile men) whose spermatozoa gave five negative (0 % penetation) and one positive (1 1 %) results. As will be discussed later, because sperm penetration rates can be altered by changing technical procedures, negative results must be evaluated cautiously. Furthermore, the possibility must be considered that some men classified as fertile might not be fertile any longer at the time of the tests. For example, the individual cited by Martin and Taylor [ had fathered a child 7 yr earlier, but not since. Despite the existence of a few such exceptions, it can be said in general that spermatozoa of fertile men penetrate more than 10% of the eggs. One dispute concerns the eggpenetrating ability of spermatozoa of clinically infertile men. While some investigators claim that spermatozoa of all infertile men, regardless of their semen parameters, always penetrate less than 1015% of the eggs and propose that 1015% penetration rate as a threshold value for male fertility and infertility [eg, Rogers et al, 1979; Tyler et al, 1981: Pryor et al, 1981: Karp et al, 1981: Junca et al, 19821, others do not see such a clearcut threshold. According to the latter, the penetration rate can vary from 0% to 100% (see Table 4). Clearly, there is a real tendency for spermatozoa of infertile men with abnormal semen parameters to penetrate lower percentages of eggs (mean value, 10%) than those of infertile men with normal semen parameters (mean value, 24%), but it should be noted that penetration rates greater than 1015 % are commonly achieved by spermatozoa of infertile men regardless of the normality of their semen parameters (see Table 4). Although the definition of fertility and infertility and the normality and abnormality of semen parameters are vague and arbitrary, there seems no doubt that spermatozoa of some clinically infertile men can yield high rates of egg penetration. Figure 9 illustrates the frequency distribution of penetration rates by spermatozoa from men of proven fertility and that by spermatozoa from men in couples with unexplained infertility. Unexplained infertility is defined as a case in which the husband s semen parameters are normal and the partners have no clinically recognizable reproductive dysfunctions. This figure illustrates that spermatozoa of fertile men

14 TABLE 2. Methods Used for Hamster Tests Investigators Yanagimachi et a1 [1976] Barros et a1 [I9791 Menge and Black [ Rogers et a1 [1979] Hall et al [I9801 and Hall [1981] Overstreet et a1 [1980a] Campana et a1 [I9811 Karp et a1 [I9811 Pryer et a1 [1981] Siddiquey [I9811 Tyler et a1 [1981] ZausnerGuelman et a1 [I9811 Aitken et a1 [1982a,b,c] BWW (1.8) Cohen et a1 [1982b] TMPA (3.0) Cohen et a1 [1982c] TMPA (3.0) Hammond et a1 [1982] mt (1.0) Junca et a BWW(3.3) Martin andtaylor [1982] BWW (0.5) Postinsemination Sperm preincubation Insemination incubation Medium Sperm Medium Sperm Hours after (albumin concentration Period Gas (albumin concentration Gas insemination when concentration %) X 106/ml (hr) phase concentration %) x 106/ml phase eggs were examined BWW (0.3) TMPA (3.5) BWW (0.4) BWW (0.3) mt (1.O) TMPA (3.5) BWW (0.4) BWW (?) BWW (?) BWW (0.3) BWW (0.3) ? Albertsen et a1 [I9831 BWW (0.3) Air Margalioth et a1 [1983] Wickings et a1 [I9831 BWW (0.3) % COP BWW (0.3) 527? 20? I ? % co* Air? 5% co2 5% c02 Air Air? Air? Air? 1% coz 5% c02? Air Air 5% co2 5% c02 5% co2 BWW (0.3) TMPA (3.5) BWW (0.4) BWW (0.3) mt (1.0) mww (3.5) TMPA(3.5) BWW (0.4) BWW (?) BWW (?) BWW (0.3) BWW (0.3) BWW (1.8) TMPA (3.0) TMPA (3.0) mt (1.0) BWW (3.3) BWW (0.5) BWW (0.3) mbww (3.5) ? ? 20? ? ? % c02 Air? 5% c02 Air 5% CO2 Air Air? 1% c02 5% c02 5% c02 5% coz Air? Air Air Air? 5% co2 5% co* 5% co* Air 5% co2 5% co7 TMPA and mt, modified Tyrode s solutions; BWW, Biggers, Whitten, and Whittingham s medium [197 I]; mbww, modified BWW medium. Air, pure air; 5% and 1 % C02, 5% CO2, and 1 % CO2 in air, respectively ? ?

15 Zonafree Hamster Egg 201 TABLE 3. Rates of Zonafree Hamster Egg Penetration by Spermatozoa of Men With Proven Fertility Number of Percent eggs penetrated Investigators men Mean + SD (Range) Menge and Black [1979] 3 67i 5 Rogers et a1 [ (14100) Hall et a1 [I9801? 64 (20 100) Overstreet et al [1980a] 6 (11100) Hall [1981] (20100) Karp et al [I (15 96) Pryor et al [1981] Siddiquey [I (55100) Tyler et al [1981] i 20 (24 89) ZausnerGuelman et a1 [1981] (15100) Aitken et a1 [1982a,b] 35 44f 3 (14 90) Cohen et al[1982c] ( 1 100) Cohen et a1 [ 1982b] 5 54 L 13 (20 93) Junca et a1 [ ( 8100) Martin and Taylor [ ( 0 60) Albertson et a1 [ f 27 ( 0 90) Comhaire et a1 [ 19831? 64 (25100) Margalioth et al [1983] 14 (20100) Wickings et a1 [1983] k 16 (15 82) Total number and overall mean (1) almost consistently penetrate > 11 % of the eggs and those of infertile men with normal semen parameters (2) penetrate 0% to 100% of the eggs. It should be noted that spermatozoa of a considerable proportion of infertile men penetrate greater than 1015% of the eggs (in some instances, 100%). Table 5 shows some of the data obtained by Stenchever et al [1982], who tested men from 118 infertile couples in which the wives had no known infertility factors. Some of these men had ejaculated normal semen and others abnormal semen. Accordingly to these investigators, 31 men whose wives had previously been pregnant yielded negative results (sperm penetration rate less than 15%) regardless of their semen quality. They claimed that the hamster test has a sensitivity of 100% in detecting infertility of men. For unknown reasons, they did not include data on 89 men whose wives had never become pregnant yet had no female infertility factors. If these data are compiled as in Table 5, 10 (9%) of a total of 118 men (seven with normal semen and three with abnormal semen) produced spermatozoa that gave > 15% penetration rates. Obviously some men in the couples with unexplained infertility had spermatozoa capable of achieving > 15 % penetration rates. A good example of infertile men whose spermatozoa penetrate eggs in vitro is found in oligospermia. Men whose semen has less than 1020 X lo6 spermatozoa per milliliter generally fall into the infertile category. Spermatozoa of the most oligo spermic men have either no ability or only a poor ability to penetrate hamster eggs [eg, Aitken et al, 1982~1, but according to Cohen et al [1982c], spermatozoa of some oligospermic infertile men (with sperm counts of less than 20 x 106/ml) penetrated as many as 89% of hamster eggs. Although spermatozoa of these men definitely failed to fertilize eggs in vivo, they penetrated eggs well in vitro. It is known that

16 i I I 202 Yanagimachi TABLE 4. Rates of Zonafree Hamster Egg Penetration by Spermatozoa of Infertile Men Whose Partners Have No Clinical Evidence of Reproductive Dysfunction Diagnosis of Number Percent eggs penetrated semen aualitya Investigators of men Mean * SD (Range) Normal Hall et a1 [1980] Overstreet et at [1980a] Campana et a1 [1981] Karp et a1 [1981] Pryor et a1 [1981] Siddiquey et a1 [1981] Tyler et al [1981] ZausnerGuelman et a1 [1981] Aitken et a1 [1982a] Cohen et a1 [1982b] Hammond et a1 [1982] Martin and Taylor et al [1982] Marglioth et a1 [1983] Wickings et a1 [1983] Subtotal number and mean Abnormal Hall et al [1980] Overstreet et a1 [198Oa] Campana et a1 [1981] Karp et a1 [1981] Pryor et a1 [1981] Tyler et al [1981] ZausnerGuelman et a1 [1981] Cohen et a1 [1982c] Hammond et a1 [1982] Aitken et a1 [1982c] Comhaire and Vermeulen [1983] Subtotal number and mean Not specified Rogers et a1 [1979] Hall et a1 [1981] Junca et a1 [1982] Subtotal number and mean Total number and overall mean? ? * * * & (580) (01 1) (090) (09) (0100) (0 > 76) (019) (05 1) (0100) (388 1) (0 loo) (01 1) (07) (045) (089) (0 > 10) (060) (010) (0100) adefinition of normality and abnormality is not clarified by most investigators. Overstreet et a1 [198Oa] and Cohen et a1 [1982b,c] define normal semen as follows: sperm density, >20 X lo6/&; percent motile sperm, >40; percent sperm with normal morphology, >4050. Karp et a1 [1981] define it as >20 x lo6/&, >60, and >50, respectively. Camana et a1 [1981] define it as >20 X 106/ml, >60, and >50, respectively. If one or more of these parameters idare below these values, the semen is considered abnormal at least by these investigators. spermatozoa of some oligospermic men clinically diagnosed as infertile over a period of years can fertilize human eggs in vitro [Takada et al, 1981; Testart et al, and produce normal offspring [Trounson and Wood, 1981; Grobstein et al, Another good example is men with Kartagener s syndrome. Their spermatozoa are completely immotile and the men are definitely infertile clinically, yet the spermatozoa can penetrate as many as 30% of zonafree eggs [Aitken et al, 1983~1. How does the ability of human spermatozoa to penetrate zonafree hamster eggs correlate with the ability to penetrate intact human eggs? According to Wolf et al [1983], spermatozoa of 18 (75%) of 24 men fertilized human eggs in vitro and

17 Zonafree Hamster Egg Egg penetration rate (%) Fig. 9. Frequency distribution of egg penetration rates by spermatozoa from men of proven fertility (A) and men in couples with unexplained infertility (B). Al and B1, Aitken et a1 [1982a]; A2, Hall [1981]; A3, Cohen et al [1982c]; B2, Margalioth et al [1983]; A4 and B3, Martin and Taylor [1982]. TABLE 5. Rates of Zonafree Hamster Egg Penetration by Spermatozoa of 118 Infertile Men Whose Partners Have No Known Female Infertility Factors [from Stenchever et al, Normal semen Number (%) of men Abnormal semen Number < 15% Eggs > 15% Eggs < 15% Eggs > 15% Eggs Category of wives of men penetrated penetrated penetrated penetrated Previously pregnant by different spouses (54%) 0 (0%) 11 (46%) 0 (0%) Previously pregnant by same spouses 9 4 (80%) 0 (0%) 1(20%) 0 (0%) Never pregnant (44%) 7 (8%) 40 (45%) 3 (3%) Total (47%) 7 (6%) 52 (44%) 3 (3%) penetrated zonafree hamster eggs at rates ranging from 6% to loo%, with an average of 56 k 32% (mean & SD); spermatozoa of the remaining six men (25%) failed to fertilize zonaintact human eggs in vitro, but penetrated hamster eggs at rates ranging from 5 % to 69 %, with an average of %. In a preliminary report, Hall et a1 [I9831 reported that human spermatozoa penetrating more than 15% of hamster eggs fertilized the partners eggs in vitro in all cases. Spermatozoa that penetrated less than 15% of hamster eggs failed to fertilize the partners eggs in 80% of the cases. Table 6 presents data from a series of experiments in which nonliving zonaintact human eggs and living zonafree hamster eggs were inseminated in the same dish [Overstreet et al, 1980al. Positive correlations (both human and hamster eggs were penetrated or neither human eggs nor hamster eggs were penetrated) were seen in 23 (85%) of a total 27 men including both fertile and infertile. No correlation was seen in the remaining four (15 %) cases where spermatozoa penetrated hamster eggs, but not the

18 204 Yanagimachi TABLE 6. The Correlation Between the Ability of Human Spermatozoa to Penetrate Zonafree Hamster Eggs and Zonae Pellucidae of Human Eggs [from Overstreet et al, 1980al Number (%) of 27 men whose spermatozoa did penetrate (+) or did not penetrate () Hamster eggs (+) Hamster eggs (+) Hamster eggs () Hamster eggs (1 Human zona (+) Human zona () Human zona (+) Human zona () 17 (63 %) 4 (15%) 0 (0%) 6 (22%) human eggs. Thus, in general, human spermatozoa capable of penetrating zonafree hamster eggs can penetrate zonaintact human eggs. When the spermatozoa are incapable of penetrating hamster eggs, they also cannot penetrate human eggs. However, important exceptions should be noted. In 25% Wolf et al, and 15% [Overstreet et al, 1980al of the cases reported, the spermatozoa penetrated hamster eggs, but failed to penetrate human eggs. This negative correlation is easily understandable and even to be expected because sperm penetration into the human egg with an intact zona pellucida requires optimal motility as well as the acrosome reaction, as already mentioned. The human spermatozoa that penetrated zonafree hamster eggs but failed to penetrate human eggs (zonae) must have had weak motility, although many of them were acrosomereacted. This means that human spermatozoa that are able to penetrate zonafree hamster eggs even at a high rate are not necessarily capable of fertilizing a human egg with an intact zona pellucida. Correlation between the results of the hamster test and fertility and infertility of men. Some investigators claim that fertile men always produce spermatozoa that give egg penetration rates greater than 1015% in the hamster test, and infertile men produce spermatozoa that give less than 1015% penetration rates. They think that the hamster test is the test that can evaluate fertility and infertility of men with 100% accuracy. To me, this is unrealistic because no single test can measure all aspects of sperm function. The hamster test can evaluate only a few sperm functions, ie, the ability of spermatozoa to a) undergo capacitation and the acrosome reaction, b) fuse with the egg plasma membrane, and c) form sperm pronuclei (and possibly participate in the first cleavage). It cannot assess other sperm functions such as penetration of the cervical mucus, surviving in and ascending the female genital tract, and penetrating the egg investments (the cumulus oophorus and zona pellucida), all of which are essential for fertilization in vivo. The data compiled in this review indicate that the chance of men being fertile is high if the egg penetration rate if greater than in the hamster test. However, men with high penetration rates are not necessarily fertile in reality. High egg penetration rates in the test merely mean that the spermatozoa are able to undergo capacitation and acrosome reaction and fuse with the egg plasma membrane. These abilities are, of course, essential for fertilizing eggs but are just a few of the abilities spermatozoa must have in order to fertilize eggs in vivo. Some men who give high egg penetration rates in hamster tests are clinically infertile even when their partners have no recognizable infertility factors (see Table 4). Apparently, spermatozoa of these men are unable to survive in and ascend the female genital tract and/or pass through the egg investments. We must be aware that the failure of fertilization is not the only cause of infertility. Immunological or genetic incompatibility of the couple

19 Zonafree Hamster Egg 205 can cause death of their zygotes even if fertilization proceeds normally. Thus, positive results in the hamster test must be evaluated cautiously. A negative hamster test appears to predict infertility of men with some accuracy. If spermatozoa of certain men yield less than 10% penetration rates, their chance of being fertile is very low because a) a negative result means that the spermatozoa have difficulties in becoming capacitated and acrosomereacted and b) the spermatozoa of virtually all men of proven fertility give penetration rates higher than 10% (Table 3). However, there is no absolute guarantee that the men with negative results in the hamster test are clinically infertile. In fact some men whose spermatozoa gave negative results at the time of the hamster tests became fertile later (Table 7). At least four possibilities may account for such negative correlation between the hamster test and fertility. First, the test cannot predict infertility of men with 100% accuracy. Second, the hamster tests might have been run under suboptimal conditions, causing negative results. As reported by many investigators, spermatozoa of some men give both negative ( < 1015 % hamster egg penetration) and positive (> 1015 %) results depending on the experimental procedure and the day of the test. Unknown or unspecified factors might have contributed to the falsenegative results. Third, men who gave negative results might not be infertile before or after the test. It is conceivable that some men usually ejaculate poor semen, but produce better semen from time to time. Finally, men might in fact remain infertile, but their wives become pregnant for some untold reasons. There is an old saying: We cannot keep people in cages. If the spermatozoa in question consistently fail to penetrate zonafree hamster eggs under every possible in vitro condition, these spermatozoa must have some serious functional defects and are probably infertile in vivo. Some investigators have concluded that particular patients are infertile based on a single test or a single series of tests under fixed experimental conditions. As the functional quality of spermatozoa may vary with time even in the same individual and the rate of egg penetration by spermatozoa may be modified by experimental procedures, we must repeat the test under various conditions if a negative result (egg penetration rate 0 or less than %) is obtained in the first test. If a man produces spermatozoa that give consistently negative results, then the spermatozoa of this man must have serious functional abnormalities. He is very likely to be completely infertile or almost infertile. The correlation between the results of the hamster test and semen parameters. Many investigators have studied whether there are correlations between the results of TABLE 7. List of Men Whose Spermatozoa Penetrated Less Than About 10% of Zonafree Hamster Eggs, yet Fathered Before or After the Test (FalseNegative Results) Number (%) of men who gave false Number of men who negative results among men of proven Percent eggs fathered before (B) or fertility (PF), subfertility (SF) or Investigators penetrated after (A) the test suspected infertility (SI) Overstreet et a1 [ 1980a1 < 11 1 (B2 years) 1/6 (17%) of PF Aitken et a1 [1981] < 10 4 (A2 years) 4/? Cohen et al [ (A20 months) 3/79 (4%) of SF Martin and Taylor [ (A2 months) 2/31 (6%) of SI Rogers et a1 [ (?) (9%) of SI

20 206 Yanagimachi the hamster test and parameters revealed by conventional semen analysis. Although some investigators have claimed to find positive correlations between some parameters and the results of the hamster test, most others have failed to do so (Table 8). This is not surprising because the hamster test and semen analysis (and sperm mucuspenetration test) do not (or do not necessarily) measure the same physiological properties of the spermatozoa. The value of the hamster test. We should not expect there to be a single assay technique that will allow us to evaluate the fertilizing capacity of spermatozoa in vivo. If we are not allowed to perform an in vivo assay (insemination of fertile women with the spermatozoa in question), several tests, each measuring different physiological and functional properties of the spermatozoa, must be conducted to gain comprehensive information on the physiology and function of the spermatozoa. The conventional semen analysis must be done first, of course. This is simple, yet gives us much TABLE 8. Correlation Between Semen (Sperm) Parameters and the Hamster Test Some correlation( +) or no definite correlation Parameter () with the hamster test Referencesa Sperm penetration into + 1 cervical mucus 2,3 Semen volume and total sperm number in semen 435 Sperm density in semen Percent motile sperm in semen Percent sperm progressively motile in semen Percent morphologically normal sperm in semen Number of leucocytes in semen + + 6,8,10,19 4,5,7,9,11,16, ,18 4,5,7,10,11,1618 6,12,13 5 Number of bacteria in semen Fumarase activity in semen 6 14 Prolactin concentration + 15 in semen 'References: (1) Soules et a1 [1982]; (2) Overstreet et a1 [198Oa]; (3) Rogers et a1 [1983b]; (4) Zausner Guelman et a1 [1981]; (5) Albertsen et a1 [1983]; (6) R.E. Berger et a1 [1982]; (7) Cohen et a1 [1982a]; (8) Rogers et a1 [1983a]; (9) Rogers et a1 [1979]; (10) Hall et al [1981]; (11) Aitken et a1 [1982a,b]; (12) Koehler et al [1981]; (13) Riedel et a1 [1981]; (14) Aafjes et a1 [1981]; (15) Sueldo et a1 [1983]; (16) Tyler et a1 [1981]; (17) Hall [1981]; (18) Wickings et a1 [1983].

21 Zonafree Hamster Egg 207 information of the properties of the spermatozoa. Semen analysis alone, however, provides only superficial information on the functional capacity of the spermatozoa. The cervical mucus penetration test tells us whether the spermatozoa can penetrate the mucus, the first barrier the spermatozoa must pass through before ascending the female genital tract. A positive mucus test (successful sperm penetration into and through the mucus), however, does not imply that the spermatozoa would be able to fertilize eggs in vivo. The ability of spermatozoa to fertilize eggs must be tested with eggs. If human eggs are readily available and their use is allowed for routine sperm fertility assay, they are obviously the ideal material for assessing the fertilizing capacity of human spermatozoa. If this is impossible, then the test using zonafree hamster eggs is the second choice. Although this test does not allow us to determine whether the spermatozoa in question are capable of penetrating egg investments (cumulus oophorus and zona pellucida), it at least will inform us of whether the spermatozoa are capable of undergoing capacitation, acrosome reaction, and fusion with the egg plasma membrane, all of which are essential preliminaries to normal fertilization. Substitutions are substitutions. We cannot and should not consider normal human eggs and zonafree hamster eggs as equivalent. All we can say is that zonafree hamster eggs are so far the best substitutes for human eggs. In combination with other tests (eg, conventional semen analysis and cervical mucuspenetration test), the hamster test should be able to provide us with important information about the fertility of human spermatozoa. Application of the hamster test for monitoring fertility of men following surgery or drug treatment. The final diagnosis of male fertility or infertility must be based on the outcome of natural coitus or artificial insemination. However, if from time to time during and after treatment, we could monitor the effectiveness of treatments aimed at either curing or inducing infertility, it would increase the efficiency of the treatments. Some investigators have monitored the quality of semen (spermatozoa) by a combination of both conventional semen analysis and the hamster test. Table 9 represents data obtained by Mygatt et a1 [1982] who monitored the fertility and infertility of men who had undergone varicocele repair. Some men with varicocele were apparently fertile, but many were infertile clinically, achieving < 10% hamster egg penetration rates. The 34 men shown in this table were all clinically infertile at the time of admission to the hospital and achieved < 10% penetration rates. In 28 (82%) of the 34 men, the penetration rate did not increase after surgery and the men remained infertile. In the remaining 6 men (18%) the penetration rate increased to more than lo%, but only 3 of these men became fertile. All of these three men had normal or near normal semen parameters before and after TABLE 9. Hamster Test on 34 Men Who Became Fertile or Remained Infertile after Varicocele Repair [from Mygatt et al, Number (%) Percent hamster eggs penetrated Men who of men Before surgery After surgery Failed to impregnate 20 (59%) 0 0 partners after surgery 8 (23%) (9%) 010 > 10 Succeeded in impregnating 3 (9%) 010 > 10 partners after surgery

22 208 Yanagimachi the surgery, with 38 % hamster egg penetration rates before surgery and 1251 % penetration rates after surgery. Based on this, Mygatt et al [1982] inferred that men whose spermatozoa give a 0% penetration rate before surgery would not benefit from varicocele repair therapy as far as the recovery of fertility is concerned. Treating infertile men with doxycycline makes some of them fertile. Semen analysis fails to predict which men have a better chance of success. According to R.E. Berger et a1 [1983], men who favorably responded to the treatment and became fertile (7 or 22% of 32 men) were in the group of 18 (56%) men whose hamster egg penetration rates improved after treatment (from the original 013 % to the posttreatment 1593 %). Some investigators claim that treating men with kallikrein improves their semen quality. Comhaire and Vermeulen [1983], however, have reported that kallikrein treatment of infertile men with poor semen quality neither improves semen quality drastically nor increases the hamster egg penetration rate. None of the ten men they treated impregnated their partners after 3 mo of the treatment. The use of the hamster test may increase the efficiency of evaluating the effectiveness of male contraceptive drugs. So far, no work utilizing this test has been reported. Side effects of therapeutic drugs may also be detected by the hamster test. Sulfasalazine, which has been used for the cure of inflammatory bowel diseases, may cause infertility in man. Hall et a1 [ reported four men who were infertile during the previous 2 to 5 yr of sulfasalazine treatment. Their partners had no reproductive disorders. Analysis of their semen samples revealed that semen parameters (eg, sperm density, total sperm number, and percentage of morphologically normal spermatozoa) were normal or subnormal, yet in no instances did their spermatozoa penetrate the hamster eggs. Application of the hamster test for studying sperm maturation in the epididymis. Mammalian spermatozoa produced in the testis must mature in the epididymis. The site where the spermatozoa begin to acquire their fertilizing capacity may vary from species to species, but it is generally the corpus epididymis [cf Dacheux and Paquignon, Two groups of investigators used the hamster test to determine in which part of the epididymis human spermatozoa begin to gain fertilizing capacity. Hinrichsen and Blaquier reported that it is the cauda epididymis, while Moore et al [1983] claimed that it is the proximal segment of the corpus epididymis [Table lo]. TABLE 10. The Ability of Maturing and Mature Human Spermatozoa to Penetrate Zonafree Hamster Eggs [from Moore et al, Region of the epididymis where spermatozoa were collected Caput Corpus, proximal Corpus, distal Cauda Percent hamster eggs penetrated Ejaculated sperm 68

23 Zonafree Hamster Egg 209 Application of the hamster test for examining the effects of antisperm antibodies on sperm function including fertilization. Sperm surface components play important roles in capacitation and acrosome reaction of spermatozoa as well as in fusion of spermatozoa with eggs [Yanagimachi, Therefore, it is expected that antibodies against sperm surface components would seriously affect fertilization. In 1020% of couples with unexplained infertility, the presence of antisperm antibodies in the serum or/and genital tract secretions has been reported [Menge and Behrman, The detection of antisperm antibodies in serum and cervical mucus has been investigated extensively using a sperm agglutination assay and indirect immunofluorescence. Unfortunately, these studies have not been conclusive, and no consistent relationship has been identified between fertility status and antibody titer [cited from Dor et al, If the antibodies immobilize and agglutinate spermatozoa extensively and irreversibly, immobilization and agglutination of spermatozoa could be the primary cause of infertility. Some couples with unexplained infertility have no detectable spermimmobilizing or agglutinating antibodies in either partner. In such cases, antibodies that prevent spermatozoa from fertilizing eggs may exist in the genital tracts of these patients. Several investigators applied the hamster test to see if such antibodies really exist. According to Menge and Black [1979], antihuman sperm antibodies raised in rabbits significantly inhibit penetration of human spermatozoa into zonafree hamster eggs. Auto and isoantihuman sperm antibodies in sera of some men and women in infertile couples completely inhibit or markedly disturb sperm penetration into hamster eggs [Haas et al, 1980; Dor et al, 1981; Joseph, 1981; Wolf et al, According to Bronson et a1 [1983], IgG and IgM, which fix complement under certain conditions, block sperm penetration into the eggs, while IgA, which does not fix complement, does not. Autoantisperm antibodies that prevent sperm penetration are also demonstrable in sera of some vasectomized men [Wolf et al, and even in sera of some men who ejaculate apparently normal semen after vasovasostomy (reconnection of the vas deferens) yet remain infertile [Requeda et al, Failure of spermatozoa to penetrate into zonafree hamster eggs means that the spermatozoa are unable to undergo capacitation, acrosome reaction, or fusion with the eggs under the particular experimental conditions. Therefore, it is possible that some patients have antisperm antibodies that prevent or interfere with some functions such as capacitation, acrosome reaction, and fusion with eggs. However, these antibodies cannot be the only antibodies that interfere with fertilization. Other antibodies might block sperm interactions with the egg investments, for instance, and this cannot be assessed using zonafree eggs. In fact, Bronson et al [1981] have reported antisperm antibodies that definitely bind to spermatozoa, yet do not interfere with sperm penetration into the hamster egg. Some antibodies prevent sperm attachment to and penetration into zonae pellucidae of human eggs [Bronson et al, This implies that although the hamster test is very useful for detecting and characterizing antisperm antibodies that interfere with fertilization, this test alone does not provide a complete picture of the antibodies that might be responsible for infertility due to fertilization failure. Its combination with other tests (eg, insemination of human eggs in vitro, if possible) will provide us with better information about the nature of the antibodies. Factors affecting the results of the hamster test. As the percentage of eggs penetrated by spermatozoa has been considered to reflect the fertilizing capacity of

24 210 Yanagimachi spermatozoa, it is important to know if and how the rate of sperm penetration is affected by various factors and conditions. Time of collecting eggs. Most investigators have collected unfertilized eggs from oviducts of superovulated hamsters between 15 and 17 hr after hcg injection according to the protocol described by Yanagimachi et a1 [1976]. Although most investigators obtained eggs from sexually mature females, superovulated eggs collected from immature females [Yanagimachi and Chang, can be used for the hamster test [eg, Tyler et al, Eggs should not be collected from the oviducts long after ovulation. According to T. Berger et al [1983b], eggs collected between 16 and 18 hr after hcg injection (ie, within about 5 hr after ovulation) are more penetrable by spermatozoa than those collected later (2022 hr after hcg injection). Method for removing the zona pellucida from the egg. Almost all investigators have used the method originally described by Yanagimachi et a1 El9761 to free the eggs from the cumulus oophorus and zona pellucida. The eggs are first treated with hyaluronidase (to disperse cumulus cells) then with trypsin (to dissolve zona). Although the plasma membrane of the hamster egg retains its ability to fuse with hamster spermatozoa even after rather harsh treatments with trypsin and most other proteolytic enzymes [Hirao and Yanagimachi, 19781, their excessive exposure to proteolytic enzymes is not recommended. It may reduce the sensitivity of the hamster test. According to Hoshi et al [ 1982a1, the highest rates of egg penetration are obtained when zonae are removed with trypsin and mercaptoethanol followed by diapase, pronase, alphachymotrypsin, and dithiothreitol (Table 11). Thus, use of either trypsin or mercaptoethanol is recommended for zona removal in the hamster test. Frequency of semen collection. Although some young men are capable of ejaculating daily for an amazingly long period of time [cf Jacobs et al, 19791, a minimum of 2 or a maximum of 5 days of prior sexual abstinence is recommended to TABLE 11. Rates of Hamster Egg Penetration by Human Spermatozoa After Removal of Zonae Pellucidae by Various Agents [from Hoshi et al, 1982al Treatment Agent period (min) at Percent hamster (concentration) room temperature eggs penetrated Trypsin (0.1%) Pronase (O.OS%) achymotrypsin (0.1 %) Diapase (300 IU/ml) 3 20 Mercaptoethanol (SO mm) 3 20 Dithiothreitol (25 mm)

25 Zonafree Hamster Egg 211 obtain samples with stable parameters [Mortimer et al, In fact, most investigators using the hamster test have requested men to abstain for at least 2 days. Table 12 shows the results of hamster tests run for a young man of proven fertility, whose semen samples were collected at 24hr intervals for a 4 consecutive days. It can be seen from this table that results of the hamster test were consistent despite steady decline in semen parameters. When two or more tests are run with a man abstaining for sufficient periods, consistent results may be obtained during a relatively short period of time (eg, a few months) [Rogers et al, 1979; Overstreet et al, 1980a; Wolf et al, 19831, but we should not expect that the same man will give consistent results over a long period of time. Portion ofsemen. Traditionally, it has been suggested that the first portion of a split ejaculate is more fertile than the second portion. According to Sokol et al [1983a], the first portion has a higher sperm count (sperm concentration, 147 f 32 X 106/ml, mean SD) than the second portion (61 f 28 X 106/ml) and the former gives a higher egg penetration rate (49 k 5%) than the latter (27 k 5%). As far as the egg penetration rate is concerned, there is no significant difference between spermatozoa in the first portion and those in the whole semen [Sokol et al, 1983bl. Seminal plasma. Ideally, for the hamster test spermatozoa should be isolated from the seminal plasma as soon as the semen is collected. Some men prefer to collect their semen samples at home then bring them to the laboratories. In such cases, the semen may have been exposed to temperature variations and the exact time elapsed between collection and assay might be difficult to control. Prolonged delay between semen collection and any test is not recommended. T. Berger et a1 [1983a] have reported that spermatozoa exposed to seminal plasma for 3 hr give lower egg penetration rates than those exposed to the plasma for 12 hr. According to Rogers et a1 [1983c], exposure of spermatozoa to seminal plasma for longer than 30 min at room temperature may reduce the rate of egg penetration by spermatozoa. According to Cohen et al [1982a], spermatozoa of some men whose semen had high viscocity penetrated hamster eggs better when the semen was diluted with collection medium (TMPA with 3% albumin) upon ejaculation than when the semen was allowed to stand for 1 hr at 37 C before the spermatozoa were isolated from it. Van der Ven et a1 [1983], however, reported opposite results. Whether seminal plasma is deleterious to spermatozoa seems to depend on individual semen donors, the manner of semen handling and the time elapsed after semen collection. TABLE 12. Results of Hamster Tests on Spermatozoa of a Fertile Man Who Donated Semen Samples on Four Consecutive Days (A. Saito and R. Yanagimachi, unpublished data) Semen parameters Semen Sperm Percent Percent volume concentration motile hamster eggs Day (d) (X 106/ml) sperm penetrated" 1st nd rd 1.o th aspermatozoa (5 x 106/ml were preimcubated for 7.5 hr in a modified BWW medium with 3.5% human serum albumin. Eggs were examined 4 hr after insemination with preincubated spermatozoa.

26 212 Yanagimachi Cohen et a1 [ 1982bl studied whether the exposure of spermatozoa from one man to the seminal plasma from another man affected the results of the hamster test. Spermatozoa of infertile men with normal semen parameters were exposed for 2 hr at 37 C to the seminal plasma of fertile men prior to the test. Sdarly, spermatozoa of fertile men were exposed to the seminal plasma of infertile men. In most cases exposure of spermatozoa to heterologous seminal plasma did not significantly change the outcome of the test; however, in two cases spermatozoa of fertile men penetrated lower percentages of egg after having been exposed to the seminal plasma of infertile men. According to Binor et al [1982], spermatozoa of some men became capable of penetrating eggs after exposure to the seminal plasma of control (fertile) men. In some cases, exposure to heterologous seminal plasma has decreased the egg penetration rate, whereas in some other cases it has either increased the rate or not changed it significantly [van der Ven et al, Van der Ven et al [1983] have found that seminal plasma contains a heatlabile factor (MW > 10,000) that renders the spermatozoa more capable of penetrating eggs. Its influence is, however, usually masked by some other factors present in the seminal plasma. Human seminal plasma contains macromolecular substances the constant presence of which prevents capacitation of spermatozoa [Kanwar et al, 1979; Van der Ven, Thus, prior to the hamster test spermatozoa must be washed thoroughly to remove these substances. To isolate spermatozoa from the seminal plasma, most investigators have used the centrifugation method originally used by Yanagimachi et al [ It involves dilution of the original semen sample with an appropriate medium (eg, BWW medium), fitration through tissue paper to remove large debris, and sedimentation of spermatozoa following repeated washings by centrifugation. Inevitably, both motile and immotile spermatozoa are collected and transferred to the sperm incubation medium. Introduction of many immotile or dead spermatozoa into the incubation medium can be reduced by the layering or swimup method [cf Overstreet et al, 1980; Tyler et al, 1981; Wolf and Sokoloski, 1982; Weeda and Cohen, 1982; Hoshi et al, 1983; Gould et al, In this method, an aliquot (eg, 0.5 ml) of liquefied semen is placed under an appropriate medium (eg, 2 ml BWW) in a test tube (eg, 15 x 60 m) and the spermatozoa in the semen allowed to swim into the upper medium. The vast majority of spermatozoa that migrate into the medium are highly motile and morphologically normal. The spermatozoa are then washed once or twice by gentle centrifugation before they are transferred into the incubation medium. Under normal in vivo conditions, immotile or weakly motile spermatozoa are eliminated by cervical mucus and only the very actively motile ones are allowed to ascend to the upper region of the female genital tract. Thus, the spermatozoa collected by the layering method may better represent the population of spermatozoa that will participate in fertilization in vivo than those collected by the simple centrifugation method. The layering method, however, may be difficult to use with oligospermic samples. Another method using a Percoll gradient [Forster et al, may be suitable for such samples. Sperm concentration in preincubation and insemination media. The concentration of spermatozoa in the preincubation and insemination medium affects the results of the hamster test considerably. Most investigators suspended the spermatozoa in the preincubation medium at relatively high concentrations (cf Table 2) and introduced the eggs into this medium. The concentration that gives consistently high egg penetration rates by normal spermatozoa has been reported to be about 5 X 106/ml [eg, Binor et al, 1980; Tyler et al, 1981; Wolf and Sokoloski, 1982; Martin

27 Zonafree Hamster Egg 213 and Taylor, 1983; Rogers et al, 1983~1. Martin and Taylor [1983] obtained the highest egg penetration rate at a sperm concentration of 107/ml after studying concentrations ranging between 5 x lo4 and 10 x 107/ml. The optimum sperm concentration has been reported to be between lo6 and 107/ml [Tyler et al, and to be X 107/ml [Rogers et al, 1983~1. Here, it should be noted that the concentration of spermatozoa does not mean the concentration of live spermatozoa. It means the concentration of all spermatozoa, dead or alive. In some experiments by these authors the majority of spermatozoa may have been dead at the end of preincubation and during insemination. If we assume that 90% of the spermatozoa were dead and only 10% were alive at the end of preincubation, the optimum concentration of live spermatozoa giving the highest penetration rate would be on the order of /ml. This concentration is nevertheless still rather high. Why are so many spermatozoa needed to penetrate hamster eggs? When zonafree hamster eggs are inseminated in vitro with preincubated (capacitated and acrosomereacted) hamster spermatozoa, almost all acrosomereacted spermatozoa penetrate the eggs. If the eggs are left in the medium containing acrosomereacted spermatozoa at the concentration of /ml, countless spermatozoa will penetrate each egg and all the eggs will be disrupted. Investigators who examine hamster eggs inseminated with preincubated human spermatozoa see that most spermatozoa that have collided with the egg surfaces swim away. Only a fraction of the spermatozoa bind to and later penetrate into the eggs. Electron microscopy has revealed that all or almost all the human spermatozoa that have bound to the egg surface are acrosomereacted [Yanagimachi et al, 1976; Barros et al, 1979; Talbot and Chacon, 1982; Gould et al, According to Talbot and Chacon [198la,b], only 1020% of the entire sperm population is physiologically acrosomereacted (alive and acrosomereacted) after having been preincubated for 2 8 hr in TMPA medium with a high albumin concentration. In the experiments performed by other investigators, the proportion of acrosomereacted spermatozoa in the entire population at the time of insemination could be higher [Aitken et al, 1983al or lower than the figure presented by Talbot and Chacon [1981a,b]. Even if we assume that at the time of insemination only 1 % of the spermatozoa were acrosomereacted among the 107/ml spermatozoa per ml, the number of acrosomereacted spermatozoa in the medium was rather high (ie, 105/ml). At this concentration of acrosomereacted hamster spermatozoa, 100 % of zonafree hamster eggs will become polyspermic. It is therefore very likely that not all the acrosomereacted human spermatozoa bind to and penetrate zonafree hamster eggs. Only a fraction of them must do so. Some species specificity operating on/in the plasma membrane of the hamster eggs seem to reject attachment and penetration by human spermatozoa. In the hamster test, we are not concerned about the fertilizing capacity of individual spermatozoa. We are concerned with the fertilizing capacity of the sperm sample as a whole. For instance, when the spermatozoa of man A give a higher egg penetration rate than those of another man, B, under exactly the same experimental conditions, all we can say is that a unit volume of a semen sample from man A has more competent spermatozoa than a comparable sample from the man B. The requirement for very high sperm concentration in the hamster test implies that the test itself is not very sensitive in assessing the fertilizing capacity of individual spermatozoa. However, this insensitivity may be beneficial in assessing the fertilizing capacity of spermatozoa in vivo because the spermatozoa that are able to penetrate

28 214 Yanagimachi hamster eggs may represent a highly selected population of spermatozoa with excellent fertilizing capacity. The spermatozoa potentially capable of fertilizing human eggs in vivo may be among those that are able to penetrate zonafree hamster eggs. Composition of medium and incubation atmosphere. Almost all media used for the hamster test are either modified Tyrode s (eg, TMPA) or KrebsRinger s solutions (eg, BWW) (cf Table 2) containing energy sources (ie, glucose, lactate, and pyruvate) and albumin. The standard Tyrode s and KrebsRinger s solutions have an osmolality near 300 mosmol. According to Aitken et a1 [1983a], the medium TMPA containing 3.5% albumin [Barros et al, 1978, has an osmolality of 356 mosmol. It rapidly renders the spermatozoa capable of penetrating hamster eggs, but it does not well maintain sperm motility as compared with BWW medium with 305 mosmol. After comparing several media with different osmolalities and albumin concentrations, they recommended a hypertonic (410 mosoml) BWW medium containing 0.3% albumin for preincubating (capacitating) spermatozoa. When the spermatozoa were preincubated in this medium for 6 hr then mixed with eggs in the same medium, 60 83% of the eggs were penetrated by spermatozoa within 3 hr. I, however, do not recommend the use of such a hypertonic medium because 410 mosmol is certainly unphysiological and this degree of sperm penetration can be obtained using media with a physiologically normal osmolality near 300 mosmol. The concentration of albumin in media used for the hamster test varies from 0.3 to 3.5 % (Table 2). Most investigators have used human serum albumin, but some have used bovine serum albumin [eg, Menge and Black, 1979; Hall, 1981; Karp et al, 1981; ZausnerGuelman et al, 1981; Cohen et al, 1982~1. Some preparations of bovine serum albumin contain substances that react with human seminal plasma components (possibly spermine and other polyamines) to produce substances toxic to both spermatozoa and eggs [Quinn and Stanger, Not all the preparations of bovine serum albumin contain these potentially harmful substances, but whenever albumin is used, it is recommended that the spermatozoa be washed thoroughly to remove seminal plasma components prior to incubation. Regardless of which albumin (human or bovine) is used, we must realize that the properties of albumin vary from lot to lot even if it is produced by the same manufacturer and sold under the same catalogue number. All investigators must have experienced unexpected difficulties from time to time when they have started to use a new batch of albumin for the hamster test. The reason for this is unknown, but the variation seems to stem partly from variation in the species and the amount of phospholipids and fatty acids present as contaminant in each albumin preparation. It is recommended that several batches of albumin be compared from time to time to select the ones that produce consistently high rates of egg penetration by spermatozoa from men of proven fertility. Whether higher or lower concentrations of albumin are superior in capacitating human spermatozoa is a matter of dispute [eg, Wolf and Sokoloski, 1982; Aitken et al, 1983a1, but at least under the experimental conditions used by Gould et a1 [1983], a medium with a high albumin concentration (3.5%) seems to capacitate the spermatozoa somewhat faster than that with a low albumin concentration (0.3 %). Divalent cations, particularly Ca2+, must be in the medium for penetration of human spermatozoa into hamster eggs [Yanagimachi, 1978bl. The ordinary Ca2 concentration in the medium (about 1.7 mm) is high enough to ensure sperm penetration into the eggs, but a higher concentration (eg, 3.4 mm) may increase the rate [Hoshi et al, 1982bl; (see Table 13).

29 Zonafree Hamster Egg 215 TABLE 13. The Effects of Ca Concentration on the Rate of Human Sperm Penetration into Zonafree Hamster Eggs [from Hoshi et al, 1982b]* Ca2+ concentration (mm) Mean number of in insemination Percent eggs sperm penetrated medium penetrated in one egg I1 *Ejaculated spermatozoa were thoroughly washed in Ca2 and Mg2+free medium with 3.5% human serum albumin. They were transferred to Mg2+free BWW media with various concentrations of Ca2+, in which zonafree hamster eggs had been previously placed. The final concentration of spermatozoa was 12 x 107/ml. The preparations were incubated under pure air (37 C) and examined for evidence of sperm penetration 4 hr later. According to Perreault and Rogers [1981], spermatozoa preincubated 1012 hr then mixed with zonafree hamster eggs in glucosefree BWW medium (with 0.3% albumin) penetrated a significantly lower percentage of eggs (220 %) than those handled in the same manner but in normal medium (1771%). They noted that the quality of sperm motility deteriorated with time in the glucosefree medium. Different results were obtained by Hoshi et al [1982c] who mixed washed, fresh (no preincubation) spermatozoa with hamster eggs in BWW medium (with 3.5 % albumin) without glucose. The rates of egg penetration by spermatozoa (determined 4 hr after the mixing) were not significantly different in the glucosefree medium (74%) and in the normal medium (75%). Even in BWW medium free from glucose, lactate, and pyruvate, the same rate of egg penetration was recorded, indicating that human spermatozoa can utilize endogenous energy substrates as well as some unidentified substances associated with albumin for their survival and fusion with the eggs. When the spermatozoa spend considerable time (eg, 1020 hr) in the medium prior to meeting the eggs [Perreault and Rogers, 19811, ample supplies of exogenous energy sources (including glucose) in the medium must be essential for spermatozoa to retain their functions. During a longterm preincubation, such as 1822 hr, excess amounts of exogenous energy sources (eg, 10 mg/ml glucose instead of the normal amount of 1 mg/ml glucose) in the medium may have adverse effects on the spermatozoa [Munemasa et al, The ph of the medium influences the outcome of the hamster test. Excessively low ph (eg, <7.0) might prevent or greatly retard capacitation of spermatozoa, resulting in the failure of spermatozoa to penetrate the eggs. In the experiments originally reported by Yanagimachi et al [ 19761, we induced capacitation of spermatozoa by placing them in BWW medium under mineral oil. The ph of the medium was when the preparation was kept under 5% C02 in air, but was under pure air. When freshly ejaculated spermatozoa (thoroughly washed) were mixed with eggs from the beginning of incubation, sperm penetration occurred faster under an atmosphere of pure air than under 5% COz, although eventually the same rate (5558%) of egg penetration was reached. Apparently capacitation and acrosome reaction occur faster at higher ph (8.18.3) than at lower ph (7.57.6). According to Tyler et al [1981], who compared pure air and 5% C02 in air atmospheres during an 18hr preincubation of spermatozoa in BWW medium, sperm penetration into the eggs occurred much faster under pure air than under 5 % C02 in

30 216 Yanagimachi air. According to them, the ph of the medium was 8.2 under pure air and was 7.2 under 5% C02 in air. Many investigators have preincubated spermatozoa under a pure air atmosphere. Some of them did not even cover the sperm preincubation medium with mineral oil. Although C02 liberated by the spermatozoa may slow down the rise in ph, CO, can readily escape from the medium. A gradual rise in ph is inevitable. Prolonged exposure to high ph (eg, > 8.3) would be a kind of punishment of the spermatozoa. The ph of the NaHC03buffered media like BWW or TMPA that most investigators have used will remain constant only in a C02 incubator holding the concentration of COz constant. As stated already, commercially available albumin is contaminated by miscellaneous substances including fatty acids and phospholipids. Many of the socalled fatty acidfree albumin preparations are loaded with phospholipids. As it will be influenced by such contaminants, the ph of the media should be carefully checked and adjusted before experiments. Some investigators have included 2040 mm Hepes as an additional phbuffering agent [eg, Karp et al, 1981; Tyler et al, 1981; Junca et al, 1982; Martin and Taylor, An advantage of Hepesbuffered medium is that its ph is maintained well under both C02 and pure air atmospheres. The preparations can be taken in and out of the incubator without worrying about ph drift. Although Hepes is an excellent phbuffered agent, it should not replace NaHC03 completely because HCOy/Hf is the natural buffer for the spermatozoa and eggs. Whatever we use as phbuffering agents, we should keep the ph of the medium between 7.3 and 7.7. Lower ph may retard or block sperm capacitation, reducing the rate of egg penetration by spermatozoa. Higher ph, although it may accelerate sperm capacitation and promote egg penetration by spermatozoa, is certainly unphysiological. Sperm preincubation period. The period the spermatozoa are preincubated before they are mixed with the eggs has varied from investigator to investigator (cf Table 2). Some have preincubated as long as 24 hr, and others have not preincubated at all. Whether longer preincubation is superior to shorter preincubation or vice versa is a matter of dispute. This is understandable because the compositions of the media (including ph and albumin concentration) and incubation atmosphere are different from one investigator to another. Under certain experimental conditions, shorter preincubation (eg, 23 hr) yields higher egg penetration rates than longer preincubation (eg, 1724 hr) [cf ZausnerGuelman et al, 1981; Binor et al, Similar results were obtained by Hirshel and Mixon [ who compared 7 and 18hr preincubation. However, there is a report that a longer preincubation (1824 hr) yielded a higher penetration rate than no preincubation at all [Gould et al, Still there is another report that a shorter preincubation (67 hr) and a longer preincubation (1820 hr) make no difference in the rate of egg penetration mickings et al, Thus, it is difficult to conclude at the moment whether shorter or longer preincubation is preferable. Perhaps, it depends on many factors such as a) composition of the medium, b) type and concentration of albumin, and even c) concentration of spermatozoa. One thing of which we must be aware is the existence of considerable individual variation among men. Under the same experimental conditions, spermatozoa of some men become capable of penetrating eggs much faster than others. In other words, spermatozoa of some men capacitate (and undergo the acrosome reaction) in vitro

31 Zonafree Hamster Egg 217 much faster than those of others [Binor et al, 1982; Perreault and Rogers, 1982b; Wolf and Sokoloski, 1982; Saito et al, 1984; cf Fig This variation has no correlation with the race, age, or blood type of the men; extensive variation is seen among men of proven fertility (Saito et al, unpublished data). Perhaps, for slow capacitators, longer sperm preincubation would be preferable to obtain high rates of egg penetration. The best preincubation period must of course be determined for each individual man. For fast capacitators, on the contrary, shorter preincubation will be preferable because their spermatozoa may not remain fertile very long after they have been capacitated and acrosomereacted. To compensate for the two extremes, a median preincubation period of 56 hr may be proposed as a standard. Regarding longterm sperm preincubation, I should like to remind readers that although preincubated spermatozoa may well penetrate zonafree hamster eggs, they are not necessarily capable of fertilizing human eggs (at least penetrating the zona pellucida). According to Hoshi et al [1982d] and Gould et a1 [1983], spermatozoa preincubated 34 hr [Hoshi et all or 1824 hr [Gould et all commonly fail to penetrate human zonae, while they penetrate zonafree hamster eggs at high rates. This should not confuse readers because the hamster test and the human zona pellucida penetration test measure different sperm functions as mentioned already. Both measure the ability of spermatozoa to undergo capacitation and the acrosome reaction. Additionally, the former can assess the sperm s ability to fuse with the egg plasma membrane. However, it does not allow us to assess the sperm s ability to cross the zona pellucida. Spermatozoa do not need to be actively motile to fuse with the egg plasma membrane Time (h) after insemination Fig. 10. A diagram suggesting the variation in capacitation time of spermatozoa among seven men with normal semen parameters. The men A, F, and G (asterisks) are of proven fertility. Zonafree hamster eggs were mixed with freshly ejaculated (washed) spermatozoa in mbww containing 3.5% human serum albumin, and the rates of egg penetration were determined between 1 and 7 hr after insemination [from Saito et al,

32 218 Yanagimachi (penetrate into zonafree eggs) as long as they have acrosomereacted. Acrosome reaction is needed for zona penetration, but it is not enough. Very strong sperm motility is needed in passing through the zona. When spermatozoa are kept in the medium for an excessively long time, acrosomereacted spermatozoa probably lose their ability to move strongly. They will be able to penetrate zonafree eggs without any trouble but will be unable to cross the zona. When we think of one spermatozoon, this spermatozoon gains the ability to cross the zona and fuse with the egg plasma membrane simultaneously upon its acrosome reaction, but its ability to cross the zona will diminish much faster than its ability to fuse with the egg plasma membrane. Miscellaneous substances added to semen or medium. In the ordinary hamster test, no special substances are added to the semen or spermcontaining medium. Several investigators have incorporated various substances into either the semen or spermcontaining media to see how they affect sperm penetration into the eggs (see Table 14). Although information obtained by these studies may not be of immediate value for the routine hamster test, it is interesting to know how such substances influence the outcome of the hamster test. According to Cohen and Aafjes [1982], addition of proteolytic enzymes to semen before separation of spermatozoa from the seminal plasma increases the rate of egg penetration by spermatozoa. The seminal plasma of freshly ejaculated semen has proteolytic activity [see Mann and LutwakMann, 19811, which might assist entry of spermatozoa into cervical mucus [Overstreet et al, 198Obl. Although the exact roles of the proteolytic activity are unknown, it is tempting to speculate that some proteolytic enzymes alter sperm surface components to facilitate capacitation in the upper regions of the female genital tract. Excessive exposure to seminal enzymes may be detrimental to spermatozoa, but a mild modification of sperm surface components by the enzymes may be beneficial. The trypsin and chrymotrypsin Cohen and Aafjes [ added to the semen may have accelerated the modification of sperm surface components, so that the spermatozoa later separated from the seminal plasma could undergo faster and more efficient capacitation and acrosome reaction in vitro. According to T. Berger et al [1982], egg penetration is inhibited when the proteinase inhibitors TLCK and NPBG are included in both sperm preincubation and insemination media. They have no obvious effect when incorporated only in the insemination medium. This could be explained by assuming that these inhibitors prevent the acrosome reaction, not the fusion of acrosomereacted spermatozoa with egg plasma membranes. According to Blazak and Overstreet , 1,OOO pm Zn2+ incorporated into both sperm preincubation and insemination media impairs both sperm motility and egg penetration, whereas subtoxic concentrations of Zn2+ ( pm) have no obvious effects on either function. This is somewhat unexpected because in the mouse, 250 pm Zn2+ drastically inhibits sperm penetration into zonafree eggs when it is incorporated into both sperm preincubation and insemination media [Aonuma et al, That estradiol increases [Chan et al, 1983b] and ethanol reduces [Cash and Rogers, the rate of egg penetration by spermatozoa requires further confirmation. Caffeine, theophylline, and CAMP are all known to stimulate the motility of spermatozoa. Whether these chemicals promote egg penetration by spermatozoa seems to depend on when the spermatozoa are exposed to them (cf Table 14). Some attempts to improve or simplify the hamster test. In the ordinary hamster test, spermatozoa from different men are separately preincubated then mixed

33 Zonafree Hamster Egg 219 TABLE 14. Effects of Various Substances Added to Semen or Sperm Incubation Media on Human Sperm Penetration Into Zonafree Hamster Eggs Substance Added( +) or not added( ) to Sperm Sperm penetration preincubation Insemination rate as compared Name Concentration Semen medium medium with control' dbcamp 510 mm + up' Caffeine 510 mm + Down' + + No difference3 + up4 + Down2, No difference4 Theophylline 1020 mm + up134 + No difference4 17 0Estradiol 50100ngiml + up5 Trypsin or 5 mglml + up6 chymotrypsin TLCK or NPGB 11.6 mm + + Down7 + No difference7 ZnC1' pm + + No difference' Ethanol 400 mg% + + Down' 'References: (I) Chan et a1 [1983a], (2) Aitken et a1 [1983b], (3) Weeds and Cohen [1982], (4) Perreault and Rogers [ 1982a1, (5) Chan et a1 [1983b], (6) Cohen and Aafjes [ 19821, (7) T. Berger et a1 [ 19821, (8) Blazak and Overstreet [1982], (9) Cash and Rogers [1981]. with the eggs. One way to compare the fertility of spermatozoa from two men, for example, would be to inseminate a group of eggs in one dish with equal numbers of living spermatozoa from the two men. Spermatozoa of the two men must be distinguished somehow. According to Blazak et a1 [ 19821, this can be done by vitally staining spermatozoa with the fluorescence dyes, FITC and TRITC. Spermatozoa of one man are stained with FITC and those of another man with TRITC or left unstained. By examining spermatozoa with an ordinary microscope (eg, phasecontrast) and a UV microscope with proper filters, spermatozoa from up to three men can be identified. According to these investigators, the fluorescence dyes do not impair sperm motility or interfere with sperm penetration into the eggs. This technique will be useful in comparing the behavior and functions of spermatozoa from two or three men simultaneously. For the hamster test, we must prepare egg donors (hamsters) in advance. Collection of semen samples at the desirable time may be difficult in some cases. Some men may not want to collect their semen sample at the time we want. If we can preserve eggs and spermatozoa, it will greatly ease our work. Cryopreservation of semen (spermatozoa) is now a common practice. Unfortunately, currently available techniques are not successful for semen (spermatozoa) from all men. Spermatozoa of some men can well withstand freezethawing procedures, but those of many cannot. According to Hall et a1 [ 19791, cryopreserved spermatozoa of some men regain their motility very well and penetrate hamster eggs almost at the same rate as fresh spermatozoa. However, in most cases, freezethawed spermatozoa have inferior motility and penetrate the eggs at lower rates than fresh spermatozoaa [Binor et al, 1980; Fujita et al, 1980; Frizelle and Cameron, 1982; Heath et al, 1982; Urry et al,

34 220 Yanagimachi If we can greatly improve cryopreservation techniques, it will undoutedly facilitate the hamster test. Recently, Bolanos et al reported an interesting result: spermatozoa in semen diluted with a TesTris yolk buffer and stored at 25 C for 48 hr penetrated hamster eggs at higher rates than those of fresh semen in the majority of cases. The difference was quite impressive. Spermatozoa of one man, for example, penetrated less than 10% of the eggs when isolated from freshly ejaculated semen, while they penetrated almost 100% of the eggs when isolated from stored semen [patient 10 in Fig. 3 of Bolanos et al, The reasons for this are unhown, but it is possible that the lowtemperature preservation alters the properties of sperm membranes in such a way that spermatozoa undergo their acrosome reactions very readily. Whether we should use semen samples stored at low temperature (25 C) for the routine hamster test must be the subject of future study. Cryopreservation of hamster eggs allowing subsequent penetration by human spermatozoa has been successful [Fleming et al, 1979; Quinn et al, 19821, but perfection of the technique is definitely needed. USE OF ZONAFREE HAMSTER EGGS TO VISUALIZE HUMAN SPERM CHROMOSOMES Direct analysis of human sperm chromosomes would greatly facilitate, for example, determining the paternal contribution to the chromosome abnormalities of fetuses and offspring, as well as assessing genetic reproductive damage to men exposed to environmental genotoxic agents. Visualization of human sperm chromosomes should be possible by examining in vivo or in vitro fertilized human eggs shortly before the first cleavage begins. By examining chromosomes of multipronuclear (polyspermic?) human eggs fertilized in vitro, Rudak et a1 [1984] could identify the chromosomes of sperm origin in some cases, but could not identify the gametic origin of the chromosomes in most cases even using heteromorphism analysis. Although this approach is attractive, the availability of human eggs for routine chromosome analysis is obviously very limited. We must use other approaches. So far, no one has ever succeeded in inducing sperm chromosomes to appear in artificial media. All attempts to visualize human sperm chromosomes after fusing spermatozoa with somatic cells have failed [cf Yanagimachi, 1982, for discussion]. Although there is a report that the chromosomes of bull spermatozoa become visible after fusing the spermatozoa with human HeLa cells [Johnson et al, 19701, the results reported are not very convincing and the success rate is very low. Ordinary somatic cells seem to lack cytoplasmic factors that decondense the compact sperm nucleus and allow the appearance of chromosomes. Until we develop special media or find somatic cells that permit the appearance of sperm chromosomes, we must depend on the egg cytoplasm, which is obviously constructed to activate the sperm nucleus among other things. Using zonafree hamster eggs, which allow entry of human spermatozoa, Rudak et a1 [1978] first demonstrated human sperm chromosomes (Fig. 11). The outline of the technique was as follows. Eggs were inseminated with preincubated (capacitated) spermatozoa, allowed to remain in the medium for 1516 hr before they were exposed to colcemid for 67 hr, then processed for chromosome spreading and staining. Using this procedure, the chromosome constitutions of 60 spermatozoa from a healthy young man were analyzed. More extensive studies have been done by Martin et a1

35 Zonafree Hamster Egg 221 Fig. 11. Chromosomes of human spermatozoa as seen in hamster eggs. (A) Stained with acetoorcein; from Rudak et a1 [1978], reprinted by permission from Nature vol. 274, No. 5674, pp , 1978 Macmillan Journals Ltd. (B) Stained with dichloromethoxy acridinespermine. Bar = 5 pm.

36 TABLE 15. Chromosome Analyses of Healthy Men Who Had No History of Radio or Chemotherapy and Ejaculated Semen With Normal Parameters No. of sperm with Total No. % Sperm No. (%) sperm abnormal chromosomes, Men of sperm with: chromosomally numerically(n) and /or Investigators No. (Age) analyzed X Y Normal Abnormal structurally(s) Rudak et a1 [I9781 4(9 1 (23) (95) 3(5) 3(N) Martin et a1 [ ? (91) 229) WN), Martin et al [1983a] 33 (2444) 1, (91) 85(9) 52(~), 3 3 ~ Brandriff et a1 [1984]* 4 (3439) (92) 73w 1409, 59(9 *See Additional References.

37 Zonafree Hamster Egg 223 [1982, 1983a1 with an improved technique [Martin, Some data reported so far are summarized in Table 15. As will be seen in this table, there are more Xbearing spermatozoa than Y bearing spermatozoa, but the number of spermatozoa examined is still not large enough to draw any definite conclusion about the ratio of X and Y sperm in normal semen samples. The incidence of spermatozoa with abnormal chromosome constitutions is 5 to 10% in the entire sperm population. Such a high incidence of chromosomally abnormal spermatozoa may be partly due to the method used for isolating spermatozoa from the seminal plasma. Rudak et a1 and Martin et a1 used the conventional centrifugation method to isolate spermatozoa from the semen so both normal and abnormal spermatozoa were given the chance to met the eggs in vitro. Under normal in vivo conditions, cervical mucus serves as a sieve to exclude weakly motile spermatozoa or spermatozoa with grossly abnormal morphology [cf Elstein, 1978; Hanson and Overstreet, Obviously the mucus cannot eliminate all of the genetically (chromosomally) abnormal spermatozoa, but it will at least reduce the chance of the abnormal spermatozoa meeting eggs. If we analyze the chromosome constitution of spermatozoa that have passed through the cervical mucus, the indicence of chromosomally abnormal spermatozoa may not be as high as reported. Similarly, if we use the swimup method for isolation of spermatozoa from the seminal plasma (see the previous section, Seminal plasma) instead of the conventional centrifugation method, we may obtain sperm populations with a lower incidence of chromosomally abnormal spermatozoa. According to Martin et a1 [ 1983b], the incidence of chromosomal abnormalities is 7% among the spermatozoa of 15 men of proven fertility and is 12% among those of 13 men of unproven fertility. As the spermatozoa of 14 (93 X) of the 15 fertile men penetrated more than 15 % of the hamster eggs and only 8 (61 %) out of the 13 men of unproven fertility did so, these investigators have inferred that the frequency of sperm chromosome abnormalities may have some association with fertility. Balkan and Martin [1983a,b] and Balkan et a1 El9831 have studied sperm chromosomes of men with chromosomal translocations and inversions. These studies represent the opening of a new era of investigations concerning paternal genetic contribution. ACKNOWLEDGMENTS I wish to express my sincere thanks to Mrs. Cherrie A. MahiBrown, Miss Lori Kurashima, and Mrs. Hiroko Yanagimachi for their assistance in the preparation of the manuscript. My grateful thanks are due Dr. J.M. Bedford for his valuable suggestions. I am indebted to Drs. A.C. Menge, A. Trounson, R.A. Bronson, and T.S.K. Chang for providing information on references. The studies by my associates and myself were supported by grants from the National Institute of Child Health and Human Development (HD03402) and the International Parenthood Federation. REFERENCES Aafjes JH, Cohen J, Vreeburg JTM, Ooat HJW (1981): Fumarase activity in human ejaculate: Relationship to spemmotility or penetration of zonafree hamster eggs. Andrologia Aitken RJ, Best F, Rudak E, Richard D, Djahanbakhch 0, Mortimer D, Templeton A, Lees M (1981): Analysis of fertilizing capacity of human spermatozoa. Proceedings of the Society for the Study of Fertility at Edinburgh, 1981, p 16 (abstr 12). Aitken RJ, Best FS, Richardson DW, Djahanbakhch 0, Mortimer D, Templeton AA, Lees M (1982a): An analysis of sperm function in case of unexplained infertility: Conventional criteria, movement characteristic, and fertilizing capacity. Fertil Steril 38:

38 224 Yanagimachi Aitken RJ, Best FSM, Richardson DW, Djahanbakhch 0, Lees M (1982b): The correlates of fertilizing capacity of normal fertile men. Fertil Steril38:6876. Aitken RJ, Best FSM, Richardson DW, Djahanbakhkch 0, Templeton A, Lees MM (1982~): An analysis of semen quality and sperm function in cases of oligozoospermia. Fertil Steril 38: Aitken RJ, Wang YF, Liu J, Best F, Richardson DW (1983a): The influence of medium composition, osmolality and albumin content on the acrosome reaction and fertilizing capacity of human spermatozoa: Development of an improved zonafree hamster egg penetrated test. Int J Androl Aitken RJ, Best F, Richardson DW, Schats R, Smith G (1983b): Influence of caffeine and movement characteristics, fertilizing capacity and ability to penetrate cervical mucus of human spermatozoa. J Reprod Fertil68: Aitken RJ, Ross A, Lees M (1983~): Analysis of sperm function in Kartagener s syndrome. Fertil Steril 40: Albertsen PC, Chang TSK, Vidivich D, Robinson C, Smyth JW (1983): A critical method of evaluating tests for male infertility. J Urol 130: Aonuma S, Okabe M, Kawaguchi M, Kishi Y (1981): Zinc effects on mouse spermatozoa and invitro fertilization. J Reprod Fertil63: Austin CR (1956): Cortical granules in hamster eggs. Exp Cell Res 10: Balkan W. Martin RH (1983a): Chromosome segregation into the spermatozoa of two men heterologous for different reciprocal translocation. Hum Genet 63: Balkan W, Martin RH (1983b): Segregation of chromosomes into the spermatozoa of a man heterologous for a 14; 12 Robertsonian translocation. Am J Med Genet 16: Balkan W, Burns K, Martin RH (1983): Sperm chromosome analysis of a man heterologous for a pericentric inversion of chromosome 3. Cytogenet Cell Genet 35: Barros C, Herrera E (1977): Ultrastructural observation of the incorporation of guinea pig spermatozoa into zonafree hamster oocytes. J Reprod Fertil49:4750. Barros C, Berrios M, Herrera E (1973): Capacitation in vitro of guinea pig spermatozoa in a saline solution. J Reprod Fertil Barros C, Gonzalez J, Herrera E, BustosObregon E (1978): Fertilizating capacity of human spermatozoa evaluated by actual penetration of foreign eggs. Contraception 17: Barros C, Gonzalez J, Herrera E, BustosObregon E (1979): Human sperm penetration into zonafree hamster oocytes as a test to evaluate the sperm fertilizing ability. Andrologia 11: Berger RE, Karp LE, Williamson RA, Koehler J, Moore DE, Holmes KK (1982): The relationship of pyospermia and seminal fluid bacteriology to sperm function as reflected in the sperm penetration assay. Fertil Steril 37: Berger RE, Smith WD, Critchlow CW, Stenchever MA, Moore DE, Spadoni LR, Holmes KK (1983): Improvement in the sperm penetration (hamster ova) assay (SPA) results after doxycycline treatment of infertile men. J Androl4: Berger T, Marrs RP, Kletzky OA, Moyer DL (1982): The effect of inhibition of the acrosome reaction and in vitro sperm aging on the hamster penetration test. Biol Reprod 26 (suppl 1): 148a (abstr 224). Berger T, Saito H, Brown J, Marrs RP (1983a): Effect of media and incubation on the hamster penetration test. Fertil Steril 39:413a. Berger T, Marrs RP, Saito H, Kletzky OA (1983b): Factors affecting human sperm penetration of zonafree hamster ova. Am J Obstet Gynecol 145: Biggers JD, Whitten WK, Whittingham DG (1971): The culture of mouse embryo in vitro. In Daniel JC (ed): Methods in Mammalian Embryology. San Francisco: Freeman, pp (Table 65). Binor Z, Sokoloski JE, Wolf DP (1980): Penetration of the zonafree hamster egg by human sperm. Fertil Steril 33: Binor Z, Rao R, Scommegna A (1982): Further studies on dysfunctional human spermatozoa using zonafree hamster oocyte penetration assay. Fertil Steril 37:300301a. Blazak WF, Overstreet JW (1982): Zinc does not inhibit the capacitation of human spermatozoa in vitro. Gamete Res 5: Blazak WF, Overstreet JW, Katz DF, Hanson FW (1982): A comparative in vitro assay of human sperm fertilizing ability utilizing contrasting fluorescent sperm markers. J Androl 3: Bolanos JR, Overstreet JW, Katz DF (1983): Human sperm penetration of zonafree hamster eggs after storage of semen for 48 hours at 2 C to 5 C. Fertil Steril

39 Zonafree Hamster Egg 225 Bousquet D, Brackett BG (1982): Penetration of zonafree hamster ova as a test to assess fertilizing ability of hull sperm after frozen storage. Theriogenology 17: Brackett BF, Killen DK, Peace MD (1971): Cleavage of rabbit ova inseminated in vitro after removal of follicular cells and zonae pellucidae. Fertil Steril22: Brackett BG, Cofone MA, Boice ML, Bousquet D (1982): Use of zonafree hamster ova to assess fertilizing ability of bull and stallion. Gamete Res 5: Brandiff B, Gordon L, Watchmaker G, Summers L, Wyroheck A (1982): Gamete interaction and developmental progression to the 2cell stage in the human sperm/hamster egg interspecific cross. J Cell Biol 95: 154a. Bronson R. Cooper G, Rosenfeld D (1981): Ability of antibodyhound human sperm to penetrate zonafree hamster ova in vitro. Fertil Steril36: Bronson RA, Cooper GW, Rosenfeld DL (1982): Spermspecific isoantibodies and autoantibodies inhibit the binding of human sperm to the human zona pellucida. Fertil Steril38: Bronson RA, Cooper GW, Rosenfeld DL (1983): Complementmediated effects of sperm headdirected human antibodies on the ability of human spermatozoa to penetrate zonafree hamster eggs. Fertil Steril40:9195. Campana A, Balerna M, Nutini L, Kaplan E, Eppenberger U, Tomasi F (1981): New parameters for selection of A.I.D. patients: Sperm acrosin content and hamster zonafree oocyte penetration. INSERM 103: Cash MKM, Rogers BJ (1981): The effect of alcohol on fertilization by human and hamster spermatozoa. Biol Reprod 24 (Suppl 1): abstr 233. Chan SYW, Tang LCH, Ma HK (1983a): Stimulation of the human spermatozoal fertilizing ability by dibutyryl3,5 camp and theophylline in vitro. Arch Androl 11:1923. Chan SYW, Tang LCH, Ma HK (1983h): Stimulation of the zonafree hamster ova penetration efficiency by human spermatozoa after 17Pestradiol treatment. Fertil Steril 39: Cohen J, Aafjes JH (1982): Proteolytic enzymes stimulate human spermatozoal motility and in vitro hamster egg penetration. Life Sci 30: Cohen J, Mooyaart M, Vreeburg JTM, Zeilmaker GH (1982a): Fertilization of hamster ova by human spermatozoa in relation to other semen parameters. Int J Androl 5: Cohen J, Mooyaart M, Vreeburg JTM, Yanagimachi R, Zeilmaker GH (1982b): Fertilizing ability and motility of spermatozoa from fertile and infertile men after exposure to heterologous seminal plasma. In Hafez ESE, Semm K (eds): Instrumental Insemination. Hague/Boston: Martinus Nijhoff, pp Cohen J, Webber RFA, Van der Vijver JCM, Zeilmaker GH (1982~): In vitro fertilizing capacity of human spermatozoa with the use of zonifree hamster ova: Interassay variation and prognostic value. Fertil Steril 37: Comhaire F, Vermeulen L (1983): Effect of high dose oral kallikrein treatment in men with idiopathic subfertility: Evaluation by means of in vitro penetration test of zonafree hamster ova. Int J Androl 6: Dacheux JL, Paquignon M (1980): Relations between the fertilizing ability, motility and metabolism of epididymal spermatozoa. Reprod Nutr Dev 20: Davis G, Jouannet P, MartinBoyce A, Spira A, Schwartz D (1979): Sperm counts in fertile and infertile men. Fertil Steril 31: Dor J, Rudak E, Aitken RJ (1981): Antisperm antibodies: Their effects on the process of fertilization studied in vitro. Fertil Steril 35: Elstein M (1978): Morphology of cervical mucus and its clinical assessment. In Ludwig H, Tauber PF (eds): Human Fertilization. Herfweg: Gerog Thieme Veral, pp Fleming AD, Yanagimachi R, Yanagimachi H (1979): Fertilizahility of cryopreserved zonafree hamster ova. Gamete Res 2: Fleming AD, Yanagimachi R, Yanagimachi H (1981): Spermatozoa of the atlantic bottlenose dolphin, Tursiops truncatus. J Reprod Fertil63: Forster MS, Smith WD, Lee WI, Berger RE, Karp LE, Stenchever MA (1983): Selection of human spermatozoa according to their relative motility and their interaction with zonafree hamster eggs. Fertil Steril 40: Frizelle FA, Cameron MC (1982): The fertilizing ability of fresh and cryopreserved human spermatozoa in vitro. Proc Univ Otago Med School 60:6162. Fujita JS, van Campen H, Rogers BJ (1980): The effect of freezing on fertilizing capacity of human spermatozoa using zonafree eggs. J Androl 1 :77a.

40 226 Yanagimachi Gordon M, Dandekar PV (1976): Electron microscope assessment of fertilization of rabbit ova treated with concanavalin A and wheat germ agglutinin. J Exp Zool 198: Gould JE, Overstreet JW, Yanagimachi H, Yanagimachi R, Katz DF, Hanson FW (1983): What functions of the sperm cells are measured by in vitro fertilization of zonafree hamster eggs. Fertil Steril 40: Grobstein C, Flower M, Mendeloff J (1983): External human fertilization: An evaluation of policy. Science 222: Haas GG, Sokoloski JE, Wolf DP (1980): The interfering effect of human IgG antisperm antibodies on human sperm penetration of zonafree hamster eggs. Am J Reprod Immunol 1:4043. Hall JL (1981): Relationship between semen quality and human sperm penetration of zonafree hamster ova. Fertil Steril 35: Hall JL, Sloan CS, Williams WD (1979): Evaluation of freeze preserved human sperm fertilizing potential using zonafree animal ova. Biol Reprod 20 (Suppl 1:94a) (abstr 169). Hall JL, Sloan CS, Hammond MG (1980): Correlation of heterologous in vitro fertilization using human sperm and hamster ova with clinical evaluation of male infertility. Fertil Steril33:238a. Hall JL, Sessions JP, Fried FA (1981): Sperm fertilizing deficiency in patients treated with sulfasalazine. Fertil Steril 35:245a. Hall JL, Marik J, Engel D (1983): Screening for human in vitro fertilization success using hamster zonafree ovum test. Fertil Steril40:412a. Hammond MG, Sloan CS, Hall JL (1982): Application of interspecies in vitro fertilization in initial assessment of the infertile couple. Am J Obstet Gynecol 142: Hanada A, Chang MC (1972): Penetration of zonafree eggs by spermatozoa of different species. Biol Reprod 6: Hanada A, Chang MC (1976): Penetration of hamster and rabbit zonafree eggs by rat and mouse spermatozoa with special reference to sperm capacitation. J Reprod Fertil46: Hanada A, Chang MC (1978): Penetration of the zonafree or intact eggs by foreign spermatozoa and the fertilization of deer mouse eggs in vitro. J Exp Zool 203: Hanada A, Nagase H (1981): Effects of sperm preincubation in rabbit uterus and imidazol on the penetration of zonafree hamster eggs by bull and boar spermatozoa in vitro. Jpn J Anim Reprod 27: Hanson FW, Overstreet JW (1981); The interaction of human spermatozoa with cervical mucus in vivo. Am J Obstet Gynecol 140: Heath E, Jeyendran R, PerezPelaez, M (1982): Penetration of hamster oocytes and ultrastructural categorization and acrosomal regional profiles of human spermatozoa cryopreserved in glycerol or testcy. Biol Reprod 26 (Suppl 1): 157a (abstr 242). Hinrichsen MJ, Blaquier JA (1980): Evidence supporting the existence of sperm maturation in the human epididymis. J Reprod Fertil 60: Hirao Y, Yanagimachi R (1978): Effects of various enzymes on the ability of hamster egg plasma membrane to fuse with spermatozoa. Gamete Res 1:312. Hirao Y, Yanagimachi R (1979): Development of pronuclei in polyspermic eggs of the golden hamster: Is there any limit to the number of sperm heads that are capable of developing into male pronuclei? Zool Mag (Tokyo) 88:2433. Hirschel MD, Mixon BA (1983): Comparison of long and short capacitation period in the sperm penetration assay. Biol Reprod 28 (Suppl 1):104a (abstr 144). Hoshi K, Saito A, Suzuki M, Hayashi K, Yanagimachi R (1982a): Effects of agents used for removal of zona pellucida on human sperm penetration into zonafree hamster egg. Acta Obstet Gynecol Jpn , Hoshi K, Saito A, Suzuki M, Hayashi K, Yanagimachi R (1982b): Effects of calcium and magnesium on in vitro fertilization in human. Acta Obstet Gynecol Jpn 34: Hoshi K, Saito A, Suzuki M, Hayashi K, Yanagimachi R (1982~): Effects of substrates on penetration of human spermatozoa into zona pellucica of human eggs and the zonafree hamster eggs. Jpn J Fertil Steril 27: Hoshi K, Saito A, Susuki M, Yanagimachi R (1982d): Effects of preincubation of human spermatozoa on in vitro fertilization. Jpn J Fertil Steril 27: Hoshi K, Nagaike F, Momono K, Kyono K, Tsuiki A, Saito A, Suzuki M, Hayashi K, Yanagimachi R (1983): A layering method to separate a population of good spermatozoa from semen sample. Jpn J Fertil Steril 28:

41 Zonafree Hamster Egg 227 Imai H, Niwa K, Iritani A (1977): Penetration in vitro of zonafree hamster eggs by ejaculated boar spermatozoa. J Reprod Fertil51: Imai H, Niwa K, Iritani A (1979): Time requirement for capacitation of boar spermatozoa assessed by their ability to penetrate the zonafree hamster egg. J Reprod Fertil 56: Imai H, Niwa K, Iritani A (1980): Ultrastructural observations of boar spermatozoa penetrating zonafree hamster eggs. Biol Reprod 23:48I486. Jacobs W, Smith ML, Plymate SR, Cricco CF (1979): Daily variations in human semen quality. Fertil Steril32:493a. Jeyendran RS, Van der Ven HH, Kennedy WP, Heath E, PerezPelaez M, Sobrero AJ, Zanaveld LJD (1983): Biochemical properties and oocyte penetration of acrosomeless human spermatozoa. J Androl4:40a (abstr K12). Johnson RT, Rao PN, Huges HD (1970): Mammalian cell fusion. III. A HeLa cell inducer of premature chromosome decondensation active in cells from a variety of animal species. J Cell Physiol 76: Joseph M (1981): Antisperm antibodies: Effects on sperm penetration. Biol Reprod 24 (Suppl l):(abstr 235). Junca A, Mandelbaum J, Plachot M, De Grouchy J (182): Evaluation de la fkcondance du sperme humain par la fkcondation in vitro interspkcifique (hommehamster). Ann Gynecol 25:9395. Kanwar KC, Yanagimachi R, Lopata A (1979): Effects of human seminal plasma on fertilizing capacity of human spermatozoa. Fertil Steril31: Karp LE, Williamson RA, Moore DE, Shy KK, Plymate R, Smith WD (1981): Sperm penetration assay: Useful test in evaluation of male fertility. Obstet Gynecol 5: Kim CI, Niwa K, Imai H, Iritani A (1980): Penetration of zonafree hamster eggs in vitro by goat spermatozoa preincubated in the reproductive tract isolated from a maturing gilt. J Exp Zoo1 213: Koehler JK, Berger RE, Karp LE, Williamson R, Smith D (1981): Phagocytosis of spermatozoa in human semen: Morphology and some clilnical laboratory correlations. Biol Reprod 24 (Suppl l):(abstr 131). Koehler JK, DeCurtis I, Stenchever MA, Smith D (1982): Interaction of human sperm with zonafree hamster eggs: A freezefracture study. Gamete Res 6: Koehler JK, Smith D, Stenchever MA (1983): Attachment of acrosomeintact sperm to the plasma membrane of zonafree hamster eggs. Fertil Steril39:413414a. Lambert H (1981): Temperature dependence of capacitation in bat sperm monitored by zonafree hamster ova. Gamete Res MacLeod J, Gold RZ (1951): The male factor in fertility and infertility. 11. Spermatozoon counts in 1000 men of known fertility and 1000 cases of infertile marriage. J Urol66: MacLeod J, Wang Y (1979); Male fertility potential in terms of semen quality: A review of the past, a study of the present. Fertil Steril31: Mann T, LutwakMann C (1981): Male Reproductive Function and Semen. BerWHeidelberg: Springer Verlag, pp Margalioth EJ, Laufer N, Navot D, Voss R, Schenker JS (1983): Reduced fertilization ability of zonafree hamster ova by spermatozoa from male partners of normal infertile couples. Arch Androl Martin RH (1983): A detailed method for obtaining preparations of human sperm chromosomes. Cytogenet Cell Genet 35: Martin RH, Taylor PJ (1982): Reliability and accuracy of the zonafree hamster ova assay in the assessment of male fertility. Br J Obstet Gynaecol 89: Martin RH, Taylor PJ (1983): Effect of sperm concentration in the zonafree hamster ova penetration assay. Fertil Steril 39: Martin RH, Lin CC, Balkan W, Burns K (1982): Direct chromosome analysis of human spermatozoa: Preliminary results from 18 normal men. Am J Human Genet 34: Martin RH, Balkan W, Burns K, Rademaker AW, Lin CC, Rudd NL (1983a): The chromosome constitution of 1000 human spermatozoa. Hum Genet 63: Martin RH, Balkan W, Lin CC, Burns K, Taylor PJ, Rudd N (1983b): Sperm chromosome abnormalities in men of proven and unproven fertility. Fertil Steril39:44la. Menge AC, Behrman SJ (1980): Immunological aspects of infertility. In Hafez ESE, Evans TN (eds): Human Reproduction: Conception and Contraception. New York: Harper and Row, pp

42 228 Yanagimachi Menge AC, Black CS (1979): Effects of antisera on human sperm penetration of zonafree hamster ova. Fertil Steril 32: Moore HDM (1981): An assessment of the fertilizing ability of spermatozoa in the epididymis of the marmoset monkey (Callithrix jacchus). Int J Androl4: Moore HDM, Hartman TD, Pryor JP (1983): Development of oocytepenetrating capacity of spermatozoa in the human epididymis. Int J Androl 6: Mortimer D, Templeton AA, Lenton EA, Colemen RA (1982): Influence of abstinence and ejaculationtoanalysis delay on sperm analysis parameters of suspected infertile men. Arch Andol 8: Munemasa KH, Cash MKM, Rogers BJ (1983): Inhibition of human sperm penetration into zonafree hamster eggs by glucose and fructose. Biol Reprod 38 (Suppl 1): (abstr 14). Mygatt GG, Soderdahl DG, Rogers BJ (1982): In vitro fertilization rates after varicocele repair. J Urol 127: Overstreet JW, Hembree WC (1976): Penetration of the zona pellucida of nonliving human oocytes by human spermatozoa in vitro. Fertil Steril 27: Overstreet, JW, Yanagimachi R, Katz DF, Hayashi K, Hanson FW (1980a): Penetration of human spermatozoa into human zona pellucida and the zonafree hamster egg: A study of fertile donors and infertile patients. Fertil Steril 33: Overstreet JW, Coats C, Katz D, Hanson FW (1980b): The importance of seminal plasma for sperm penetration of human cervical mucus. Fertil Steril Pavlok A (1979): Interspecies interaction of zonafree ova with spermatozoa in mouse, rat and hamster. Anim Reprod Sci 2: Pavlok A (1981): Penetration of hamster and pig zonafree eggs by boar ejaculated spermatozoa preincubated in vitro. Int J Fertil 26:lOl106. Pavlok A, Travnik P, KopEcny V, Stastna J (1982): Fusion of hamster and pig zonafree eggs stimulated by boar and guinea pig sperm at fertilization in vitro. Gamete Res 6: Perreault SD, Rogers BJ (1981): Effects of various sugars on the course of human spermatozoal capacitation in vitro. J Androl 2:2223a. Perreault SD, Rogers BJ (1982a): Relationship between fertilizing ability and CAMP in human spermatozoa. J Androl3: Perreault SD, Rogers BJ (1982b): Capacitation pattern of human spermatozoa. Fertil Steril38: Pryor JP, Collins WP, Landon G, Tyler JPP (1981): The clinical application of electron microscopy and the heterologous ova penetration test to the assessment of spermatozoa from infertile men. Br J Urol53: Quinn P (1979): Failure of human spermatozoa to penetrate zona free mouse and rat ova in vitro. J Exp Zoo1 210: Quinn P, Stanger JD (1980): Effect of purification of bovine serum albumin on the interaction of human semen with mouse ova in vitro. Biol Reprod 22: Quinn P, Barros C, Whittingham DG (1982): Preservation of hamster oocytes to assay the fertilizing capacity of human spermatozoa. J Reprod Fertil66: Rehan NE, Sobrero AJ, Fertig JW (1975): The semen of fertile men: Statistical analysis of 1300 men. Fertil Steril26: Requeda E, Charron J, Roberts KD, Chapdelaine A, Bleau G (1983): Fertilizing capacity and sperm antibodies in vasovasostomized men. Fertil Steril39: Riedel HH, Bankloh V, Mettler L (1981): Correlations between sperm quality and the penetration rates of human and zonafree hamster oocytes. INSERM 103: Rogers BJ, Van Campen H, Ueno M, Lambert H, Bronson R, Hale R (1979): Analysis of human spermatozoal fertilizing ability using zonafree ova. Fertil Steril 32: Rogers BJ, McCarville C, Soderdahl D, Hale R (1982): Reevaluation of the zonafree egg test with regard to its use in human fertility assessment. Fertil Steril 37:296a. Rogers BJ, Bentwood BJ, Van Campen H, Helmbrecht G, Soderdahl D, Hale RW (1983a): Sperm morphology assessment as an indicator of human fertilizing capacity. J Androl4: Rogers BJ, Takemoto F, Wiltbank M, Hale R (1983b): Comparison of the penetrating ability of human spermatozoa into bovine cervical mucus and zonafree hamster eggs. Fertil Steril 39:437a. Rogers BJ, Perreault S, Bentwood BJ, McCarville C, Hale RW, Soderdahl DW (1983~): Variability in the humanhamster in vitro assay for fertility evaluation. Fertil Steril 39: Rudak E, Jacobs PA, Yanagimachi R (1978): Direct analysis of the chromosome constitution of human spermatozoa. Nature 274:

43 Zonafree Hamster Egg 229 Rudak E, Dor J, Mashiach S, Nebel L, Goldman B (1984): Chromosome analysis of multipronuclear human oocytes fertilized in vitro. Fertil Steril41: Saito A, Hoshi K, Suzuki M, Hayashi K, Yanagimachi R (1984): Capacitation time of human spermatozoa. Jpn J Fertil Steril29:8896. Siddiquey AKS (1981): Humadhamster invitro fertilization: A useful test for fertility status in male. INSERM 103: Smith M, Peterson RN, Russell L (1983): Penetration of zonafree hamster eggs by boar sperm treated with ionophore A23187 and inhibition of penetration by antiplasma membrane antibodies. J Exp Zool 225: Sokol RZ, Madding C, Swerdloff RS (1983a): The split ejaculate: Assessment of fertility potential using zonafree hamster assay. Fertil Steril 39:412413a. Sokol RZ, Madding C, Swerdloff RS (1983b): Coniparison of the fertility potential of a split ejaculate with the whole ejaculate using the zonafree hamster assay. Fertil Steril40:413414a. Soules MR, Moore DE, Spadoni LR, Stenchever MA (1982): The relationship between the postcoital test and the sperm penetration assay. Fertil Steril 38: Soupart P, Strong PA (1975); Ultrastructural observations on polyspermic penetration of zona pellucidafree human oocytes inseminated in vitro. Fertil Steril Stenchever MA, Spadoni LR, Smith WD, Karp LE, Shy KK, Moore DE, Berger R (1982): Benefits of the sperm (hamster ova) penetration assay in the evaluation of the infertile couple. Am J Obstet Gynecol 143:9194. Sueldo C, Berger T, Kletzky OA, Marrs RP (1983): Seminal prolactin concentration and sperm fertilizing capacity. Fertil Steril40:415416a. Takada Y, Hasgawa A, Koyama K, Isojima S (1981): Studies of fertilizing capacity of spermatozoa from infertile men with oligozoospermia: AIH by washed and concentrated spermatozoa and in vitro fertilizing capacity to zonafree hamster eggs. Acta Obstet Gynecol Jpn 33: Talbot P, Chacon RS (1981a): Observations on the acrosome reaction of human sperm in vitro. Am J Primatol 1 : Talbot P, Chacon RS (1981b): A triplestain technique for evaluating normal acrosome reactions of human sperm. J Exp Zool 215: Talbot P, Chacon RS (1982): Ultrastructural observations on binding and membrane fusion between human sperm and zonafree hamster oocytes. Fertil Steril37: Testart J, Lassalle B, Frydman R, Belaisch JC (1983): A study of factors affecting success of human fertilization in vitro. 11. Influence of semen quality and oocyte maturity on fertilization and cleavage. Biol Reprod 28: Thadani VM (1980): A study of heterospecific spermegg interactions in the rat, mouse and deer mouse using in vitro fertilization and sperm injection. J Exp Zool 212: Toyoda Y, Chang MC (1968): Sperm penetration of rat eggs in vitro after dissolution of zona pellucida by chymotrypsin. Nature 220: Trounson A, Wood C (1981): Extracorporeal fertilization and embryo transfer. Clin Obstet Gynecol 8: Tyler JPP, Pryor JP, Collins WP (1981); Heterologous ovum penetration by human spermatozoa. J Reprod Fertil63: Urry RL, Carrel DT, Hull DB, Middleton RG, Wiltbank MC (1983): Penetration of zonafree hamster ova and bovine cervical mucus by fresh and frozen human spermatozoa. Fertil Steril39: Van der Ven H, Bhattacharyya AK, Binor Z, Let0 S, Zaneveld LJD (1982): Inhibition of human sperm capacitation by high molecular weight factor from the human seminal plasma. Fertil Steril 38~ Van der Ven HH, Binor 2, Zaneveld LJD (1983): Effect of heterologous seminal plasma on the fertilizing capacity of human spermatozoa as assessed by the zonafree hamster egg test. Fertil Steril40: Weeda AJ, Cohen J (1982): Effects of purification or split ejacultation of semen and stimulation of spermatozoa by caffeine on their motility and fertilizing ability with the use of zonafree hamster ova. Fertil Steril37: Wiessenberg R, Eshkol A, Rudak E, Lunenfeld B (1982): Inability of round acrosomeless human spermatozoa to penetrate zonafree hamster ova. Arch Androl 11: Wickings EJ, Freischem CW, Langer K, Nieschlag E (1983): Heterologous ovum penetration test and seminal parameters in fertile and infertile men. J Androl4:

44 230 Yanagimachi Wolf DP, Armstrong PB (1978) Penetration of the zonafree mouse egg by capacitated epididymal sperm: Cinematographic observations. Gamete Res 1 :3946. Wolf DP, Sokoloski JE (1982): Characterization of the sperm penetration bioassay. J Androl3: Wolf DP, Inoue M, Stark RA (1976): Penetration of zonafree mouse ova. Biol Reprod 15: Wolf DP, Sokoloski JE, Haas GG (1981); Human sperm autoantibodies inhibit sperm penetration of zonafree hamster eggs. Fertil Steril 35:246a. Wolf DP, Sokoloski JE, Quigley MM (1983): Correlation of human in vitro fertilization with the hamster egg bioassay. Fertil Steril40:5359. Yanagimachi R (1972a): Penetration of guineapig spermatozoa into hamster eggs in vitro. J Reprod Fertil28: Yanagimachi R (1972b): Fertilization of guinea pig eggs in vitro. Anat Rec 174:920. Yanagimachi R (1977): Specificity of spermegg interaction. In Edidin M, Johnson MH (eds): Immunobiology of Gametes. London: Cambridge University Press, pp Yanagimachi R (1978a): Spermegg association in mammals. In Moscona AA, Monroy A (eds): Current Topics in Developmental Biology. New York: Academic Press, Vol 12, pp Yanagimachi R (1978b): Calciumrequirement for spermegg fusion in mammals. Biol Reprod 19: Yanagimachi R (1981): Mechanisms of fertilization in mammals. In Mastroianni L, Biggers JD (eds): Fertilization and Embryonic Development In Vitro. New York: Plenum Press, pp Yanagimachi R (1982): Potential methods for examining sperm chromosomes. In Amann RP, Seidel GE (eds): Prospects for Sexing Mammalian Sperm. Boulder: Colorado Association University Press, pp Yanagimachi R, Chang MC (1964): In vitro fertilization of golden hamster ova. J Exp Zoo1 156: Yanagimachi R, Noda YD (1970a): Electron microscope studies of sperm incorporation into the golden hamster egg. Am J Anat 128: Yanagimachi R, Noda YD (1970b): Physiological changes in the postnuclear cap region of mammalian spermatozoa: A necessary preliminary to the membrane fusion between sperm and egg cells. J Ultrastruct Res 3 1 : Yanagimachi R, Yanagimachi H, Rogers BJ (1976): The use of zonafree animal ova as a test system for the assessment of the fertilizing capacity of human spermatozoa. Biol Reprod 15: Yanagimachi R, Lopata A, Odom CB, Bronson RA, Mahi Ca, Nicolson GL (1979): Retention of biologic characteristics of zona pellucida in highly concentrated salt solution: The use of saltstored eggs for assessing the fertilizing capacity of spermatozoa. Fertil Steril ZausnerGuelman B, Blasco L, Wolf DP (1981): Zonafree hamster eggs and human sperm penetration capacity: A comparative study of proven fertile donors and infertile patients. Fertil Steril36: Zuckerman 2, RodriguezRigau LJ, Smith KD, Steinberger E (1977): Frequency distribution of sperm counts in fertile and infertile males. Fertil Steril28: ADDITIONAL REFERENCES After the manuscript was sent to the publisher, the following papers dealing with the hamster test and examination of human sperm chromosomes using zonafree hamster eggs appeared. Related to the Hamster Test (Reviews) Aitken RJ (1983): The zonafree hamster egg penetration test. In Hargreve TB (ed): Male Infertility. BerlidHeidelberg: SpringerVerlag, pp Aitken RJ (1983): Attributes and applications of the zonafree hamster egg penetration test. In Crosignani PG, Rubin BL (eds): In Vitro Fertilization and Embryo Transfer. London: Academic Press, pp Aitken RJ, Best FSM, Templeton AA, Richardson DW, Schats R, Djahanbakhch 0, Lees MM (1983): Fertilizing capacity of human spermatozoa: A study of oligospermia and unexplained infertility.

45 Zonafree Hamster Egg 231 In D Agata R, Lipsett MB, Polasa P, Van Molen HJ (eds.): Recent Advances in Male Reproduction (Serono Symposium Publication Series). New York: Raven Press, vol 7, pp Aitken RJ, Templeton A, Schats R, Richardson D, Djahanbakhch 0, Lees M(1983): Methods for assessing the functional capacity of human spermatozoa: Their role in the selection of patients for in vitro fertilization. In Beier HM, Lindner HR (eds): Fertilization of Human Egg In Vitro. BerlinlHeidelberg: SpringerVerlag, pp Blasco L (1984): Clinical tests of sperm fertilizing ability. Fertil Steril41: Prasad MRN (1984): The in vitro sperm penetration test. Int J Androl7:522. Rogers BJ (1983): Hamster egg: Evaluation of human sperm using in vitro fertilization. In Crosignani PG, Rubin BL (eds): In Vitro Fertilization and Embryo Transfer. London: Academic Press, pp (Regular articles) Alexander NJ (1984): Antibodies to human spermatozoa impede sperm penetration of cervical mucus or hamster eggs. Fertil Steril41: Aron R, Johnson BA, Syms AJ, Lipshultz LI, Smith RG (1984): Conditions influencing human sperm capacitation and penetration of zonafree hamster ova. Fertil Steril41: Barros C, Vigil P, Herrera E, Perez A, Guadarrama A, BustosObregon E (1983): In vitro interaction between human spermatozoa and human cervical mucus. Microscop Electronic Biol Celul 7: Campana A, Gatti MY, Ruspa M, Van Kooij R, Buetti G, Penberger U, Balerna M (1983): Relationship between fertility, semen analysis, and human sperm penetration of zonafree hamster eggs. Acta Europ Fertil 14: Chan SYW (1984): Prolactin does not influence the human spermatozoa1 penetration into zonafree hamster ova. IRCS Med Sci (Biochem) 12: Comhaire F, Vermenlen L (1983): Effect of high dose oral kallikrein treatment in men with idiopathic subfertility: Evaluation by means of in vitro penetration test of zonafree hamster ova. Int J Androl6: Isojima S, Koyama K, Hasegawa A, Tsunoda Y, Hanada A (1984): Monoclonal antibodies to porcine zona pellucida antigens and their inhibitory effect on fertilization. J Reprod Immunol 6:7787. Jeyendran RS, Van der Ven HH, Kennedy W, PeresPelaez M, Zaneveld LJD (1984): Comparison of glycerol and a zwitter ion buffer system as cryoprotective media for human spermatozoa: Effect on motility, penetration of zonafree hamster oocytes, and acrosin/proacrosin. J Androl 5: 17. Jeyender RS, Van der Ven HH, PerezPalaez M, Crabo BG, Zaneveld LJD (1984): Development of an assay to assess the functional integrity of the human sperm membrane and its relationship to other semen characteristics. J Reprod Fertil70: Junca AM, Plachot M, Mandelbaum J (1983): Evaluation of human sperm fertility by interspecific (human spermatozoahamster oocytes) in vitro fertilization. Acta Europ Fertil 14: Kennedy WP, Van der Ven HH, Straus JW, Bhattachryya AK, Waller DP, Zaneveld LJD, Polakosk KL (1983): Gossypol inhibition of acrosin and proacrosin, and oocyte penetration by human spermatozoa. Biol Reprod 29: Koehler JK, DeCurtis I, Stenchever MA, Smith D (1983): Freezefracture observations on the interaction of human spermatozoa with zonafree hamster oocytes. In Andre J (ed): The Sperm Cell. Hague: Martinus Nijhoff, pp Koehler JK, Smith D, Karp LE (1984): The attachment of acrosomeintact sperm to the surface of zonafree hamster oocytes. Gamete Res 9: O Rand MG, Irons GP (1984): Monoclonal antibodies to rabbit sperm autoantigens. 11. Inhibition of human sperm penetration of zonafree hamster eggs. Biol Reprod 30: Riedel HH, Baukloh V, Mettler L (1983): Die Bedeutung der SpermaQuantitat fiir die In Vitro Fertilization: Ergebnisse von Untersuchungen im Humansystem und im Zona Pellucidafreien Hamstereizellsystem. Andrologia Riedel HH, Baukloh V, Steinberg R, WaheRohrboach T, Mettler L (1983): Inhibitory effect of antispermantibodies on the penetration rate of human spermatozoa studied in the zonafree hamster egg system. In Schulman S, Dondero F (eds): Immunologic Factors in Human Contraception. Rome: Field Education Italia, pp Swanson RJ, Ackerman S (1983): Zonapellucidafree hamsterlhuman sperm in vitro interaction as an indicator of oolenma permeability. Infertility 6: Tsuiki A, Hoshi K, Saito A, Kyono K, Hoshiai H, Suzuki M, Horiuchi T (1983): Time sequential observation on human sperm penetration into zona pellucidafree hamster oocytes by scanning electron microscopy. Acta Obstet Gynecol Jpn 35:

46 232 Yanagimachi Wagenknecht LV, Lotzin CF, Somnier HJ, Schirren C (1983): The importance of sperm quality for in vitro fertilization: Results of studies in human and the zona pellucidafree hamster egg cell system. Andrologia 15 : (Abstracts) Ausmanas M, Tureck RW, Mastroianni L, Kopf G, Ribas J, Blasco L (1984): The zonafree hamster penetration assay as a prognostic indicator in an in vitro fertilization program. Fertil Steril 41 : 106s. Berger T, Mans RP, Sat0 F, Brown J (1984): Comparison of the hamster penetration test and human in vitro fertilization. Fertil Steril41: 106s. Byrd W, Wolf DP (1984): Evaluation of capacitation in human sperm using monoclonal antibodies against acrosomal cap antigens. Biol Reprod 30 (Suppl 1):75a. Dandekar PV, Wolf DP (1983): Characterization of a human spermassociated factor that enhances sperm fusion with zonafree hamster eggs. J Cell Biol97: 184a. Gelas M, Sele B, Warenbourgh E, Ostorero C, Estrade C, Racinet C, Jalbert P (1983): Fertilizing ability and sperm cytogeneticity: Two application of the in vitro fertilization of zonafree hamster eggs by human spermatozoa. Arch Androl 11:215a. Hall JL, Engel D, Berger GS, Dingfelder JR, Marik J (1984): Use of the hamster zonafree ovum test in human in vitro fertilization program. Fertil Steril41: 105s106s. Inoue M, Kaneko M, Uchimura M, Shinohara M, Fujita A (1983): Analysis of human sperm fertilizing ability using zonafree hamster eggs: Results of 309 infertile patients. Acta Obstet Gynecol 35:2196a. Margalioth EJ, Navot D, Laufer N, MoyYosef S, Rabinowitz R, Yarkoni S, Schenker JG (1983): Zonafree hamster ova sperm penetration assay as a screening procedure for in vitro fertilization. Arch Androl ll:205a. Mayer JF, Jones K, McDowell J (1984): Comparison of short and long term preincubation times in the zonafree hamster ova assay with human fertilization. Fertil Steril41: 106s107s. Menge AC, Kelsey DE, Lee CG, Wong E (1984): Effects of antisperm monoclonal antibodies on human sperm function. Fertil Steril41:9s. Naz RK, Alexander NJ, Isahakia M, Hamilton M (1984): Inhibition of fertilization by a specific monoclonal antibody against human germ cells and isolation of the involved membrane glycoprotein. Biol Reprod 30 (Suppl 1):75a. O Rand MG, Irons GP (1983): Identification of a human sperm antigen complex involved in penetration of the zonafree hamster egg. J Cell Biol97:lSla. Riedel HH, Baukloh V, WaheRohrbach T, Mettler L (1983): Monoclonal antibodies tested in the zonafree hamster oocyte system (human spermatozoa). J Reprod Immunol 5 suppl: 54a. Singer S, Lambert H, Overstreet JW, Yanagimachi R, Hanson AW (1984): The kinetics of human sperm binding to the human zonafree hamster oocytes in vitro. Fertil Steril41:8s. Sutherland PD, Matson PL, Landon GV, Collins WP, Pryor JP (1983): Failure of roundheaded spermatozoa to penetrate zonafree hamster ova: A case report. Proc Soc Study Fertil (London) no. 29. Syms, AJ, Johnson A, Lipshultz LI, Smith RG (1984): Studies on human spermatozoa with round head syndrome. Fertil Steril41: 17s18s. Syms AJ, Johnson A, Lipshultz LI, Smith RG (1984): Reduced ability of motile human spermatozoa obtained from oligospermic males to penetrate zonafree hamster eggs. Fertil Steril41: 105s. Vaid P, Matson PL, Sutherland P, Parsons JH, Goswamy R, Whitehead M, Collins WP, Pryor JP (1983): Errors in assessing the number of spermotozoa during routine semen analysis and before incubation with ova. Proc Soc Study Fertil (London) no. 27. Wiltbank MC, Kosas TS, Rogers J (1984): Treatment of infertile patients by intrauterine insemination of washed spermatozoa. Fertil Steril41: 108s. Wolf DP, Sokoloski JE (1984): Synchronization of human sperm capacitation by low extracellular calcium exposure. Fertil Steril41: 16s17s. Related to Chromosome Examination Bandliff B, Gordon L, Ashworth L, Watchmaker G, Carrano A, Wyrobek A (1984): Chromosomal abnormalities in human sperm: Comparisons among four healthy men. Human Genet 66: Chaudhuri JP, Yanagimachi R (1984): An improved method to visualize human sperm chromosomes using zonefree hamster eggs. Gamete Res 9: in press. Martin RH (1984): Analysis of human sperm chromosome complement from a male heterologous for a reciprocal translocation t( 11:22) (q23:qll). Clin Genet 25: Tomkins PT, Carroll C, Walsh S, Houghton JA (1984): An improved assay for the chromosomes of human sperm. Iri J Med Sci 153:30a.