The Female Factor in Fertility and Infertility. I. E:ffects of Delayed Fertilization on the Development of the Pronuclei in Rat Ova

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1 The Female Factor in Fertility and Infertility I. E:ffects of Delayed Fertilization on the Development of the Pronuclei in Rat Ova Richard J. Blandau, Ph.D., M.D. THIS PAPER deals with the structural alterations observed with the phase microscope in living rat eggs which have aged for specific periods of time before artificial insemination. Previous investigations in the rat, 5 guinea pig, 10 and rabbit14 have clearly demonstrated that as the interval between ovulation and fertilization is prolonged, within rather narrow limits, rapid and progressive decrease results in the number of animals which become impregnated and complete their pregnancy. Moreover there is a significant increase in the number of abnormal pregnancies which are terminated by death and subsequent resorption or abortion of the conceptus.5 10 Up to the present time no investigations have dealt specifically with the alterations which occur in the living, aged eggs during the period of pronuclei formation and first segmentation division. It is important to describe more completely the basis for failure of normal development in aged eggs. Does the aging process in the egg affect sperm penetration of the zona pellucida and ooplasm? If sperm penetration is successful can the overripe egg be sufficiently stimulated or rejuvenated so that normal development may begin and continue uninterrupted? The rat is a particularly useful animal for a study of this type. In the first From the Department of Anatomy, School of Medicine, University of Washington, Seattle, Washington. This investigation was supported by a research grant G3118 from the National Institutes of Health, Public Health Service. 349

2 350 BLANDAU [Fertility & Sterility place, the time of ovulation may be easily and accurately determined to the nearest half hour/ 1 and the rate of ascent of spermatozoa to the site of fertilization is known Secondly, the lack of an abundant yolk within the living egg makes possible a detailed examination of its morphologic constituents with the phase microscope 2 and permits study of abnormalities apparent at this level of organization. The interpretation of the changes noted in the aged eggs recorded here is based upon earlier observations on a large series of normally fertilized and cleaving ova. 9 ' MATERIALS AND METHODS The observations described in this paper have resulted from the examination of 933 living ova recovered from 78 sexually mature, female, albino rats of the Wistar strain. Because a new colony of rats was established from animals obtained from the Wistar Institute, Philadelphia, it was considered important to determine the time of ovulation in this inbred strain as accurately as possible. It had been previously established that for the rat the onset of behavioral estrus is a suitable reference point for the determination of the time of ovulation. 4 8 In the present study the writer employed the technic of manual manipulation of the pudendal region as described by Blandau, Boling, and Young. Forty sexually mature females, weighing gm., were killed between the seventh and eleventh hours after the onset of heat. Under a binocular dissecting microscope the ovaries were dissected free from the mesovarium and fat and placed in Zenker formol fixative. They were dehydrated in graded ethyl alcohol, embedded in paraffin (56-58 oc), sectioned serially at 10f-t, and stained with hematoxylin and eosin. The oviducts were cut from the cornua and a fine glass pipette filled with Locke's solution was inserted into the fimbriated end of each. The fluid was forced through the oviduct under controlled pressure thereby flushing out any contained ova. Since the technic of artificial insemination described earlier by Blandau and Jordan 6 has been modified considerably the method as used in the present study will be reviewed here: The body of the uterus of all females which were to be artificially inseminated was tied off during the early hours of heat so as to prevent escape of the uterine fluids. This was done by making a short incision just above the pubic symphysis and into the peril -,

3 Vol. 3, No. 5, 1952] FEMALE FACTOR IN FERTILITY: I 351 f toneal cavity. Avoiding the uterine blood vessels as much as possible a ligature was placed about the body of the uterus and tied tightly. This procedure may be completed within 2 or 3 minutes, thus avoiding prolonged ether anesthesia. At the time of insemination an adult male weighing in excess of 200 grams was killed and the caudae epididymides and vasa deferentia were removed. Adherent fat tissue and the deferential blood vessels were quickly cut away with an iridectomy scissors. With fine forceps the epididymus was massaged with gradually increasing pressure toward the vas deferens. Ribbons of compacted sperm can thus be expressed from the distal end of the vas deferens. When the tubules of the epididymus have been emptied of their contents, the massaging is continued along the vas deferens so as to force the remaining spermatozoa from it and into a dish containing 0.1 cc. Locke's solution (another suitable medium for the sperm suspension is uterine fluid aspirated from the donor female just before insemination). The suspension was examined miscroscopically to be certain that the sperm were actively motile. They were then drawn into a "Tuberculin" syringe through a No. 27 hypodermic needle. Sperm from the second epididymus were prepared in the same manner. The female to be inseminated was anesthetized with ether. The previous incision was reopened and the uterine cornua exposed. The sperm suspension was injected into the lumen of the uterus by piercing the antimesometrial muscular wall. The procedure for examining the ova of the inseminated animals was as follows: All of the animals were examined between the fifteenth and seventy-second hours after ovulation. After the animals were decapitated the oviducts were cut just above the uterotubal junction and dissected from the ovaries under a binocular dissecting microscope. A fine glass pipette containing Locke's solution was inserted into the fimbriated end of each oviduct and held in place with a fine forceps. The fluid was forced through the oviduct under controlled pressure, thereby slowly flushing out the ova. The eggs examined during the first few hours after fertilization were surrounded by a varying number of layers of granulosa cells. These cells were removed by the addition of small quantities of an hyaluronidase preparation. 0 Each ovum was taken up separately into a capillary pipette with a small amount of Locke's solution. It was then transferred to a glass slide * The hyaluronidase preparation was supplied by the Ortho Research Foundation, Raritan, New Jersey, through the courtesy of Doctor Carl S. Hartman.

4 352 BLANDAU [Fertility & Sterility and surrounded by a thin vaseline ring about 1 em. in diameter. The vaseline ring was complete except for a 2--3 mm. gap. An 18 mm. round cover glass was lowered carefully upon the vaseline ring. After the cover glass was in position, a small drop of mineral oil was placed at the edge of the cover glass adjacent to the opening in the vaseline ring to complete the seal of the preparation. All the ova were examined with dark contrast-medium phase objectives. Observations were described in detail and recorded photographically on DuPont High Speed Pan Type, number 428 film. OBSERVATIONS Relationship Between Ovulation and Beginning of Heat In a study of this type it is of the utmost importance that the time of ovulation and fertilization be determined as accurately as possible. Table 1 TABLE 1. Progress of Ovulation after the Onset of Heat Ovulation Hours after onset of heat Number of animals Ruptured follicles Unruptured follicles 119* * Not including follicles destined for atresia. summarized the temporal relationship between the onset of heat and ovulation as determined from 40 unmated females which came into heat between 6:00P.M. and 11:00 P.M. Serial sections of the 80 ovaries of these animals were examined to determine the number of ovulatory follicles in which rupture was imminent or had occurred. The follicles which are destined to ovulate may be distinguished easily from those which will undergo atresia by observing the folding of the granulosa cell wall, the secretion of the secondary follicular fluid, the loosening of the cumulus oophorus cells, and the presence of some stage in the formation of the first polar body. An average number of 12.2 ovulatory follicles or early corpora lutea per animal was tabulated in this group. This figure corresponds favorably with the average number of ova recovered ( 11.9) from the oviducts in the in-

5 Vol. 3, No. 5, 1952] FEMALE FACTOR IN FERTILITY: I 353 seminated animals (Table 2). These data are also in agreement with those published earlier 11 for a mixed group of rats and re-emphasize the fact that the onset of the heat response is a reliable and accurate method for the determination of the time of ovulation. Results of Artificial Insemination Before and After Ovulation Table 2 summarizes the results of artificial insemination before and after ovulation in 78 animals. A total of 160 eggs were recovered from 13 females inseminated prior to TABLE 2. Condition ofova Recovered Eggs Eggs Condition of fertilized eggs recovemd fertilized Number Normal Abnormal Time of of Per- Per- Perinsemination animals Total Average Total centage Total centage Total centage Control group inseminated during heat Inseminated 3 through 5 hrs. after ovulation Inseminated 6 through 8 hrs. after ovulation Inseminated 9 through 12 hrs. after ovulation ovulation. With one exception all of the females were successfully inseminated. In this animal 6 of 6 ova had been fertilized in the right oviduct. None of the 8 eggs recovered from the left tube had been penetrated by spermatozoa, nor were spermatozoa observed in the fluids washed from this oviduct. Of the 160 ova mentioned above 92 per cent were fertilized and the pronuclei appeared to be developing normally in 99 per cent of them. When these figures ~re compared with the artificial inseminations of 57 females reported in 1941 by Blandau and Jordan in which 83 per cent of the animals were impregnated, one notes an increase in the fertilization rate in the present experiment which may be attributable to an improvement in the methods of artificial insemination. As the time of artificial insemination was successively advanced after

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7 Vol. 3, No. 5, 1952] FEMALE FACTOR IN FERTILITY: I 355 ovulation there was a decrease in the number of eggs fertilized and a striking increase in the number of abnormally developing ova It is interesting that in the 18 females artificially inseminated between the third and fifth hour after ovulation, 93 per cent of the eggs had been penetrated by spermatozoa, and only 4 per cent of the fertilized eggs showed developmental abnormalities. Of the 178 ova recovered from 15 females inseminated 6 through 8 hours after ovulation, 88 per cent were fertilized, abnormalities were detected in 14 per cent. The greatest decrease in the percent of fertilization occurred in the 375 eggs recovered from 32 animals inseminated between the ninth and twelfth hour after ovulation. The majority of the unfertilized ova were recovered from females in which the eggs had been aged 10 to 12 hours. Of the 266 fertilized ova 43 per cent in this group showed some kind of maldevelopment. The average number of eggs recovered in the aged groups approximates closely that of the control inseminated animals and also the number of ovulatory follicles recorded in the sectioned material (Table 1). Two hundred and sixteen of the fertilized eggs contained 355 accessory spermatozoa. The mean number per ovum was 1.2, range 1 to 13. There was no significant difference in the number of accessory sperm within the Fig. 1. *Normal ovum in which pronuclei are of maximum size. Removed 18 hours after fertilization. X 500. Fig. 2. Control ovum removed 20 hours after fertilization, or a short interval before the first segmentation division. Note accessory spermatozoa in pervitelline space. X 500. Fig. 3. An aged ovum in which the female nuclear mass had not differentiated. The male pronucleus appears normal. Note sperm flagellum. X 500. Fig. 4. Fragmentation of the female pronucleus in an ovum aged 10 hours before insemination. Note accessory spermatozoon in pervitelline space. X 500. Fig. 5. An unfertilized aged ovum showing the vesicular appearance of the female chromosomal mass 20 hours after ovulation. Note aggregation of mitochondria about the vesicles. X 500. Fig. 6. Higher power view of the female chromosomal mass. Note differences in size and contrast between the various nuclear components. X * All the photomicrographs used with this article were made from living specimens photographed under dark contrast-medium objectives of the phase microscope. 9 = female pronucleus or female nuclear mass.?; = male pronucleus. M = middle-piece of flagellum. PB = polar body. AS = accessory sperm. SMS = spiral mitochondrial sheath.

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9 Vol. 3, No. 5, 1952] FEMALE FACTOR IN FERTILITY: I 357 various experimental groups. The accessory sperm were invariably confined to the perivitelline space (Fig. 9). Pronuclear Changes in the Ova Aged Before Fertilization It is significant that from a single oviduct of an experimental animal it is possible to recover both normal appearing and grossly abnormal fertilized ova. The stage of development of the male pronuclear components in both normal and abnormal ova from a single animal was remarkably constant. That an egg has been penetrated by sperm can be easily verified by the presence of the flagellum (Figs. 1, 3, 10, and 11). In the rat the entire flagellum enters the ooplasm at the time of sperm penetration. The appearance of normally developing living ova is shown in Figs. 1 and 2. Both of these ova were removed between 16 and 20 hours after ovulation. The pronuclei are growing rapidly in size and are approaching one another. In Fig. 2 the nucleoli have decreased in number and contrast, indicating that this egg could be expected soon to undergo the first segmentation division.u The representative ova in Figs. 3 through 12 were aged from 9 to 12 hours before fertilization and examined between the sixteenth and twentieth hour after fertilization. Only the male pronucleus is developing normally in the ovum shown in Fig. 3. The female chromosomal mass did not develop into a definite pronucleus, but appeared to be composed of a number of small vesicles of variable size and high contrast. As may be seen from the photographs the appearance of the female chromosomal mass varies considerably in aged ova (Figs. 5, 6, 10, and 12). A common appearance of the female chromosomal mass, in situ is seen in Figs. 5 and 6. The individual units of the aggregation appear globular in shape, vary in size, and the majority of them are of high contrast. Ordinarily these chromosomal aggregations are surrounded by large numbers of rodlike mitochondria (Fig. 5). Whether the vesicles of high contrast are true nucleoli or are the chromosomes which have become globular is being investigated. In approximately half of the abnormal ova examined the Figs. 7, 8, 10, 11, and 12. Appearance of nuclear components of ova aged 9 through 12 hours before insemination and examined between 18 and 20 hours after fertilization. X 484. Fig. 9. An aged ovum containing accessory spermatozoa. The fertilizing spermatozoa had differentiated normally. The female chromosomal mass was vesicular and appeared similar to that shown in Fig. 5. X 484.

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11 Vol. 3, No. 5, 1952] FEMALE FACTOR IN FERTILITY: I 359 female pronucleus had undergone varying degrees of fragmentation and growth. In some ova, although the female nucleus appeared normal in size and nucleolar composition, a single accessory nuclear component was present (Figs. 4, 7, and 10). On the other hand aged, fertilized eggs were found containing many nuclear fragments, each with one or more nucleoli (Figs. 8, 11, and 12). On occasion the normal appearing male pronucleus was surrounded by a halo of smaller female nuclei, all of similar size and appearance (Fig. 12). The globular nuclear vesicles described here are very similar to those diagramed by Charlton for the unfertilized egg in the mouse. In the majority of aged fertilized ova the second polar body is abstricted and appears similar to that described for normally fertilized ova. It is of great interest that even though the female nuclear mass showed relatively little change, the male pronucleus continued to differentiate at a normal rate. The nucleoli vanished and an aggregate of male chromosomes appeared on a metaphase plate as if in preparation for a normal segmentation division. In Fig. 13 the female chromosomal mass is in the vesicular stage previously described. The nucleoli of the male pronucleus are beginning to disappear, heralding the appearance of the definitive chromosomes. Such chromosomes, in the form of a net and embedded in a clear cytoplasm, may be seen in Fig. 15. A further advance in their aggregation on the metaphase plate and the faint outline of a spindle are shown in Fig. 16. The general features of the male chromosomes, in situ, is pictured in a greatly flattened ovum in Fig. 14. Fig. 13. An aged ovum in which the female pronucleus (arrows) did not develop. The nucleoli within the male pronucleus are disappearing. This normally occurs just before the chromosomes become visible. X 484. Fig. 14. A greatly flattened and partially ruptured, aged ovum to show the appearance of the male chromosomal plate. X 484. Fig. 15. An aged egg in which the chromosomes of the male pronucleus are embedded in a clear cytoplasm. The female chromosomal mass has not developed into a pronucleus. X 484. Fig. 16. A lateral view of the male chromosomes arranged on the metaphase plate. A faint outline of a spindle is visible. The female pronucleus within this aged egg had not developed. X 484. Fig. 17. Living chromosomes from the metaphase plate of a normal segmentation division. X 968. Fig. 18. Appearance of only the male chromosomes arranged on a metaphase plate of an aged ovum. X 968. Fig. 19. Male chromosomes from an aged egg removed 48 hours after fertilization. X Fig. 20. Flagellum of a spermatozoon removed from an aged egg just before segmentation division. The spiral mitochondrial sheath had slipped off from the axial filament and lay free in the medium. x 968.

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13 Vol. 3, N9. 5, 1952] FEMALE FACTOR IN FERTILITY: I 361 The appearance of the male chromosomal aggregates varied from egg to egg. On occasion they were elongated and had contours similar to those observed in eggs undergoing normal segmentation. In other eggs the male chromosomes presented the appearance of shortened rods and spheres (Fig. 18 ) ; in yet others they were manifested as a condensed net and obviously in some stage of degeneration (Fig. 19). The phenomenon of the fragmentation of the nuclei in the one-cell stage is continued into the two-cell stage (Figs. 22, 23, and 25). The number of nuclear fragments found within a single blastomere varied widely. In some eggs only one blastomere contained a nuclear fragment (Fig. 25). There is great variation in the size and contrast of the nucleoli within these fragments (compare Figs. 22 and 25). In other eggs each blastomere contained two to six nuclei; each bit of nucleoplasm contained one or more nucleoli (Fig. 23). Frequently two-celled ova were observed in which the nuclear components appeared normal but a third and smaller cytoplasmic unit had separated from the blastomere and may or may not have contained nuclear material (Fig. 24). Still another condition was observed in which the male chromosomes were visible on the metaphase plate; while the female chromosomes were vesicular and the ovum had fragmented into a number of units of varying sizes and shapes (Fig. 26). Observations of the changes in the cytoplasmic components of the flagellum in the aged eggs have revealed that the dissolution of the external membranes and the dispersal of the spiral mitochondrial sheath occurred in the expected manner and was completed during the two-cell stage in both normal and abnormal ova ( Fig. 20). DISCUSSION As studies on the physiology and biochemistry of the gametes are pursued, it is becoming increasingly evident that these cells may be listed among the shortest lived in the body. Since aged eggs may be fertilized and develop Fig. 21. Normal two-cell ovum to show the appearance of the nuclei and the presence of sperm flagellum. Egg removed 30 hours after fertilization. X 484. Figs. 22, 23, 24, 25. Aged fertilized two-cell ova removed between 24 and 28 hours after ovulation. Note the various nuclear fragments and the unequal non-nucleated blastomere in Fig. 24. X 484. Fig. 26. Aged fertilized, fragmenting ovum re- '. moved 36 hours after fertilization. X 484.

14 362 BLANDAU [Fertility & Sterility abnormally, it is imperative that exacting studies be directed toward the determination of the time of ovulation before artificial insemination is performed in any animal. The data presented in this paper give evidence for the first time that the sperm nucleus may undergo apparently normal transformation into the male pronucleus and form a segmentation spindle within fertilized eggs which have undergone degenerative changes sufficiently extensive to preclude normal development. Whether an ovum in which only the male pronucleus has differentiated is capable of division into two blastomeres is unknown at the present time, but there are some observations which indicate that such may be the case. A number of aged, fertilized two-cell ova have been observed in which vesicular aggregations were present closely resembling those described for the undifferentiated female chromosomal mass. Similar aggregations were never seen in normally fertilized eggs. The ability of the unfertilized rat egg to cleave and fragment into units which appear indistinguishable from normally segmenting ova should instill caution into any interpretation of the mechanism or division in rat eggs experimentally treated. The failure of sperm penetration of the zona pellucida in the 109 eggs aged 9 to 12 hours before insemination presents a challenging problem. That adequate numbers of spermatozoa were available to have fertilized them was evidenced by the excess number present at the site of fertilization. Also, many of the fertilized eggs in this group had been penetrated by more than one spermatozoon. Chang suggested that there is a relationship between the loss of the granulosa cells and fertilizability. He demonstrated in the rabbit that there is a precipitous drop in fertilizability at the time of complete denudation of the egg, and attributes this to a physicochemical change in the zona pellucida. In other words, in the rabbit if fertilization is to occur it must happen before the granulosa cells have been dispersed. In rat ova the number of layers of granulosa cells present 10 to 12 hours after ovulation is not noticeably different from that found in ova examined immediately after ovulation. Ordinarily all of the eggs are encompassed within a single cumulus mass and are easily visualized in the greatly dilated loop of the ampulla. Furthermore, the aged egg mass examined several hours after insemination may reveal that some of the eggs had been penetrated by one or more sperm, while eggs lying a millimeter or so away were not fertilized. Thus in our present state of knowledge the loss of fertilizability

15 Vol. 3, No. 5, 1952] FEMALE FACTOR IN FERTILITY: I 363 in the rat cannot be correlated with any detectable change in the condition of the granulosa cells. During an earlier study of fertilization in the rat it was frequently noted that no spermatozoa were present within the fluid of the dilated ampulla. 9 However, when the granulosa cells were removed by the addition of an hyaluronidase preparation frequently no spermatozoa were found within the cumulus mass but all of the zonae pellucidae had been penetrated by one or more sperm. This suggests that the rat ovum may have extended some kind of attractive influence toward the spermatozoa, perhaps chemical in nature, which directs the spermatozoa through the numerous layers of the granulosa cells and into the egg. Such a phenomenon is well known in the invertebrates. 18 It is supposed that those eggs which remain unfertilized have probably died or have lost their ability to attract spermatozoa or otherwise changed so as to become unfertilizable. This problem is being investigated further. The manner in which spermatozoa penetrate the zona pellucida is not known for any mammal and may vary greatly from animal to animal. In the rabbit, guinea pig, and monkey, one frequently finds spermatozoa embedded in the zona pellucida. However, the examination of several thousand fertilized rat ova has failed to show a single spermatozoon adherent to any part of the zona pellucida. Certain conclusions may be drawn by comparing the data in the present study with those obtained earlier, 5 in which rats were also inseminated after ovulation. In the latter study the early stages of fertilization were not examined, since the purpose was to observe the females for normal and abnormal pregnancies and litter size. In females inseminated 6 hours after ovulation only 47 per cent were impregnated and in these, abnormal pregnancies were detected in 48 per cent. In the present study 89 per cent of the eggs recovered from animals inseminated du;ing this period were fertilized, but maldevelopment revealed itself in only 14 per cent up until the two-cell stage. From this comparison one would anticipate a further loss of the normally appearing ova in the latter stages of development. Furthermore, it demonstrates that the method of phase microscope observation used in the present study can detect only the more gross abnormalities as the changes not represented by morphologic defects at the microscopic level of organization would escape notice. Even more striking differences between the two sets of data are observed

16 364 BLANDAU [Fertility & Sterility in the group of eggs aged 12 hours before insemination. In the previous work only 4 per cent of 48 animals were successfully impregnated and of these 100 per cent ended with some abnormality. But in a similar group in the present study, 71 per cent of the eggs were fertilized, yet in only 43 per cent of these were deviations from normal-usually characterized by nuclear fragmentation seen with the phase microscope during early cleavage. SUMMARY 1. Seventy-eight female rats were artificially inseminated in four groups. A control group was inseminated during heat and prior to ovulation. In the three remaining groups insemination was delayed from 3 through 5 hours; 6 through 8 hours; and 9 through 12 hours after ovulation. The inseminated animals were killed between 15 and 72 hours after ovulation. 2. Ninety-two per cent of the total eggs recovered in the control group were fertilized, and 1.4 per cent of the fertilized eggs were abnormal to microscopic examination. 3. Of the 220 eggs aged 3 through 5 hours before insemination, 93 per cent were fertilized and 4 per cent revealed some nuclear abnormality. 4. Eighty-nine per cent of 178 eggs aged 6 through 8 hours before insemination were fertilized; 14 per cent of these showed some nuclear aberration. 5. The greatest decrease in fertilizability and increase in maldevelopment occurred in eggs aged 9 through 12 hours before insemination. Of 375 eggs recovered 71 per cent were fertilized and of these 43 per cent appeared abnormal. 6. If an aged egg is penetrated by a spermatozoon the female chromosomal mass may undergo relatively little pronuclear differentiation. The male element, on the other hand, may be transformed into the male pronucleus and its chromosomes may arrange themselves on the metaphase plate. A definite spindle may form. 7. The most obvious alteration in aged fertilized eggs in the two-cell stage is fragmentation of the nuclei. REFERENCES I. AusTIN, C. R. ]. Endocrinol. 6:104, AusTIN, C. R., and SMILES, J. ]. Roy. Micr. Soc. 68:13, BLANDAU, R. J. Unpublished observations. 4. BLANDAU, R. J., BoLING, J. L., and YoUNG, W. C. Anat. Rec. 79:453, BLANDAU, R. J., and JoRDAN, E. S. Am.]. Anat. 68:215, 1941.

17 Vol. 3, No. 5, 1952] FEMALE FACTOR IN FERTILITY: I BLANDAU, R. J., and JoRDAN, E. S. ]. Lab. & Clin. Med. 26:1361, BLANDAU, R. J., and MoNEY, W. L. Anat. Rec. 86:191, BLANDAU, R. J., and MoNEY, W. L. Anat. Rec. 90:255, BLANDAU, R. J., and OnoR, D. L. Fertil. & Steril. 3:13, BLANDAU, R. J., and YoUNG, W. C. Am.]. Anat. 64:303, BoLING, J. L., BLANDAU, R. J., SonERWALL, A. L., and YouNG, W. C. Anat. Rec. 79:313, CHANG, M. C. Fertil. & Steril. 2:205, IS. CHARLTON, H. H. Biol. Bull. 33:321, HAMMOND, J. ]. Exper. Biol. 11:115, LEONARD, S. L., and PERLMAN, P. L. Anat. Rec. 104:89, ODOR, D. L., and BLANDAU, R. J. Anat. Rec. 110:329, OnoR, D. L., and BLANDAU, R. J. Am.]. Anat. 89:29, TYLER, A. Physiol. Rev. 28:180, 1948.