IN VITRO FERTILIZING ABILITY OF TESTICULAR, EPIDIDYMAL, AND EJACULATED RABBIT SPERMATOZOA*t

Transcription

1 FERTILITY AND STERILITY Copyright 1978 The American Fertility Society Vol. 29, No.5, May 1978 Printed in U.SA. IN VITRO FERTILIZING ABILITY OF TESTICULAR, EPIDIDYMAL, AND EJACULATED RABBIT SPERMATOZOA*t BENJAMIN G. BRACKET!', D.V.M., PH.D.:j: JERRY L. HALL, PH.D. YON-KAK OH, D.V.M., M.S. Section of Reproductive Studies, Department of Clinical Studies, School of Veterinary Medicine, and Division of Reproductive Biology, Department of Obstetrics and Gynecology, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania The fertilizing ability of testicular, epididymal, and ejaculated rabbit spermatozoa was evaluated in vitro following in vitro capacitation by high ionic strength treatment. Fewer than 11% of inseminated ova were apparently fertilized (i.e., in pronuclear, two-, and four-cell stages as determined by light microscopy) when testicular sperm treated with caffeine, caput epididymal, or corpus epididymal sperm samples were tested. A greater fertilizing ability, reflected by the percentage of ova fertilized and more normal progression of embryonic development, was exhibited by cauda epididymal sperm. Of93 ova, 68 (73.1%) were fertilized by cauda sperm, whereas ejaculated sperm from the same 10 bucks fertilized 34 (36.6%) of93 ova (P < 0.005). Ultrastructural examination of selected ova apparently fertilized by sperm from levels of the male reproductive tract proximal to the cauda epididymidis revealed abnormal activation. Authentic fertilization occurred when ova were inseminated with cauda epididymal and ejaculated sperm. An unusual and infrequent form of activation involving failure of cortical granule breakdown in ova penetrated by cauda epididymal and ejaculated sperm was seen. A comparison of fertilizing ability of sperm from first, second, and third ejaculates revealed a significant decrease with the third ejaculate (P < 0.01). Definition of the location within the male reproductive tract from which sperm that are competent to undergo capacitation and fertilization can be obtained is of great interest in approaches to male contraception involving targets distal to the process of spermatogenesis. Initial studies in the rabbit pointed to the first appearance of sperm fertilizing ability in the lower half of the corpus epididymidis.1. 2 A variation in the site from Received November 2,1977; accepted December 6,1977. *Supported by Grant HD and Career Development Award HD from the National Institute of Child Health and Human Development, N ationallnstitutes of Health. tpresented at the Ninth World Congress on Fertility and Sterility and the Thirty-Third Annual Meeting of The American Fertility Society, April 12 to 16, 1977, Miami Beach, Fla. :j:reprint requests: Benjamin G. Brackett, D.V.M., Ph.D., University of Pennsylvania School of Veterinary Medicine, New Bolton Center, R.D. #1, Kennett Square, Pa which epididymal sperm, competent to fertilize, could be obtained was discovered when individual rabbit bucks were closely observed by Orgebin Crist and Tichenor.3 The most recent and most comprehensive investigation revealed 23% and 77% of rabbit bucks to contain the first fertile sperm in the proximal corpus epididymidis and the distal corpus epididymidis, respectively.4 In previous studies the methodology included a requirement for the sperm cell to reach the site of fertilization within the female reproductive tract prior to fertilizing the ovum. Sperm from the male reproductive tract were introduced into test females and, upon recovery of ova from the females, assessment of fertilizing ability was determined by light microscopic observations. The possibility of abnormal activation by immature sperm has been suggested by reports of increased incidences of polyploidy, 1, 2 delayed fer-

2 572 BRACKETT ET AL. May 1978 tilization,4 and increased preimplantational embryonic mortality.2 The present report includes initial data on the fertilizing ability of rabbit sperm from various levels of the male reproductive tract assessed by in vitro fertilization following in vitro sperm capacitation.5 The quality of gamete interaction and the resulting ovum activation was assessed by light and electron microscopy. CAPUT 11 CORPUS VAS DEFERENS MATERIALS AND METHODS Animals. Sexually mature male and female Dutch Belted rabbits obtained from a local supplier (Skippack Farms, Skippack, Pa.) were used in these experiments. Males and females were housed in separate rooms and individually caged..lights were on in the room housing the males for 10 hours and in the room housing the females for 14 hours of each 24-hour period. All animals were maintained for at least 3 weeks prior to the experiment. Animals were fed Purina rabbit chow and provided with water ad libitum. Recovery of Sperm. Figure 1 depicts the anatomical regions from which sperm were obtained from bucks following sacrifice by cervical dislocation. For testicular, caput epididymal, and corpus epididymal sperm recovery, tissue was removed and minced with irridectomy scissors while submerged in warm medium; following an interval of approximately 5 minutes, the supernatant fluid containing sperm cells was aspirated, and sperm suspensions in 5-mlvolumes were subjected to initial centrifugation for 5 minutes at 734 x g. The medium used throughout this study was the defined medium for in vitro fertilization reported previously5 prepared with sodium pyruvate and essentially fatty acidfree bovine albumin (Sigma Chemical Co., St. Louis, Mo.) Cauda sperm were obtained by incising the cauda epididymidis following its distention with fluid introduced retrograde via the freshly cut stump of the vas deferens. Ejaculated sperm were obtained from semen collected by use of an artificial vagina 6 and a teaser doe. In each instance the collections were diluted to approximately 5 ml for initial centrifugation as mentioned. Sperm recovery, centrifugation, and manipulations prior to in vitro insemination were carried out at room temperature (22 C) except as otherwise indicated. Sperm Treatment. Following collection and initial centrifugation of sperm cells, supernatant fluid was discarded in each case. Sperm cells were then routinely resuspended in 2 ml of high FIG. 1. Diagrammatic representation ofthe sites from which sperm cells were obtained for testing their fertilizing ability in vitro. ionic strength medium consisting of the defined medium supplemented with NaCI to a measured osmolality of 380 to 390 mosmoleslkg. A 5- minute incubation in this medium at 38 C was routinely used for in vitro capacitation. Following this treatment, sperm were recentrifuged as before, the supernatant fluid was discarded, and the sperm cells were resuspended to 2 ml with defined medium; such suspensions comprised the sperm source for in vitro insemination. Osmolalities were determined by a vaporpressure osmometer (model 5130, Wescor Inc., Logan, Utah). When testicular sperm were used, in all but an initial experiment, the sperm cells were treated for an additional 5-minute interval with 6 mm caffeine at 38 C followed by an additional centrifugation prior to resuspension for insemination. This step was necessary to initiate motility of sperm cells from this source. In Vitro Fertilization. Ovum donors were primed with pregnant mare serum gonadotropin (Gestyl, Organon, West Orange, N. J.), and injected with human chorionic gonadotropin (HCG) (APL, Ayerst Laboratories, New York, N. Y.) and ova were collected from the ovarian surfaces under sterile saline beginning 12 hours after the HCG injection.5. 7 Ovum recovery was completed within 30 minutes, by which time all ova with surrounding cellular investments were pooled in 3 to 4 ml of defined medium and covered with silicone oil (Dow Corning "200 dielectric fluid, 50 cs viscosity," Dow Corning Corporation, Midland, Mich.) Insemination was carried out by the addition of appropriate volumes (0.01 to 1.0 ml) of defined medium containing sperm (in the order of 10 6 cells) to ova, already pooled, resulting in a total volume of approximately 4.0 ml. Sperm concentrations were

3 Vol. 29, No.5 IN VITRO FERTILIZING ABILITY OF RABBIT SPERMATOZOA 573 in the range of 2 to 8 x 10 5 /ml. Sperm counts were estimated by hemocytometer counting of resuspended samples just prior to insemination. Evaluation of sperm motility was also estimated at this time, and some motility was observed in all cases comprising this report. Following insemination, the medium in each small, glass, tissue culture dish (20 mm diameter and 12 mm deep) was overlaid with silicone oil to fill the dish completely, then covered with the dish top, wrapped in foil, and incubated at 38 C under 8% O2, 5% CO2, balance N2 with high relative humidity resulting from passage of the warm gas mixture through 38 C distilled water. The bicarbonate-containing medium and silicone oil were equilibrated with this atmosphere just prior to use. The time interval between initiation of ovum recovery and incubation of ova with sperm was not more than 35 minutes, and the ova were handled under fluid at 38 C during this entire interval. All vessels, culture media, pipettes, syringes, etc. that might be a potential source of bacterial contamination were sterilized prior to use. Examination of Ova for Evidence of Fertilization. Ova were routinely examined microscopically (x20 to x70) approximately 27 hours after in vitro insemination and were reported to be apparently fertilized (or activated) if in pronuclear or early cleavage stages. The latter criteria were considered presumptive evidence for fertilization. Ova with any degree of cytoplasmic fragmentation were noted. Representative ova were examined in detail by high-resolution light and electron microscopy. For electron microscopy ova were fixed with 2.0% glutaraldehyde in 0.1 M phosphate buffer, postfixed with 1.0% osmium tetroxide, dehydrated, and embedded in Epon 812 prior to serial sectioning at 10-6 m and staining with toluidine blue for more detailed light microscopic study and orientation purposes. Thin sections were stained with uranyl acetate and lead citrate and examined with a Hitachi HU-12A electron microscope. In this study abnormal activation refers to apparently fertilized ova in. which cortical granules were retained as determined by electron microscopy. Morphologic variations of sperm from various regions of the male reproductive tract were observed after incubations with ova. Experiments Designed To Compare Fertilizing Ability of Sperm Samples. Proportions of ova fertilized by ejaculated sperm were compared with those resulting from in vitro insemination with cauda epididymal sperm. In these experiments cauda epididymal sperm were recovered from the same animals just after collection of ejaculated sperm. In three other experiments, proportions offertilized ova resulting from insemination with sperm from first, second, and third ejaculates of two males were compared. Data from these experiments were treated by x. 2 analysis. Experiments for Assessment of Sperm Capacitating Treatment. Several experiments were carried out to explore the possibility that less formidible pretreatment of epididymal sperm was necessary for their capacitation prior to in vitro insemination. In one experiment the fertilizing ability of caput epididymal sperm after an initial washing procedure was compared with that following the routinely employed high ionic strength treatment (described above). In two additional experiments cauda epididymal sperm were used for insemination immediately after their recovery in an unwashed state, after initial washing, after incubation as described above for high ionic strength treatment but in isotonic (295 to 305 mosmoleslkg) defined medium, and following the routinely employed high ionic strength treatment (described above). RESULTS A summary of presumptive evidence for the occurrence of fertilization following in vitro insemination of freshly ovulated rabbit ova with sperm from various regions of the male reproductive tract is shown in Table 1. In these experiments, testicular sperm were treated with 6 mm caffeine to initiate progressive motility in addition to the high ionic strength treatment which was used for sperm capacitation in all experiments summarized in Table 1. Approximately 10% to 20% of the testicular sperm could be rendered progressively motile. Without the caffeine treatment none of 19 ova were activated by testicular sperm with vibration motility in an initial experiment. With this exception, some progressively motile sperm cells were present in all samples used for in vitro insemination in this study. Low levels of apparent fertilization were also observed following insemination with sperm from the caput and corpus epididymides. A striking result was the relatively high proportion of ova that were fertilized by cauda epididymal sperm and the remarkable advantage in fertilization as assessed by the proportion of ova fertilized when

4 C11 TABLE 1. Presumptive in Vitro Fertilizing Ability of Testicular, Caput, Corpus, and Cauda Epididymal and Ejaculated Rabbit Sperm Capacitated in Vitro A: Summary of Proportions of Ova Showing Evidence for Fertilization Assessed by Light Microscopy 27 Hours Postinsemination Testicular sperm Caput sperm Corpus sperm Cauda sperm Ejaculated sperm No. of experiments Ova Ova % Ova inseminated fertilized fertilized Ova Ova % Ova inseminated fertilized fertilized Ova Ova % Ova inseminated fertilized fertilized Ova Ova % Ova inseminated fertilized fertilized Ova Ova % Ova inseminated fertilized fertilized Total B: Summary of Developmental Stages Comprising Light Microscopic Evidence for Fertilization 27 Hours Postinsemination tl:l No. of Testicular sperm Caput sperm Corpus sperm Cauda sperm experiments Pronuclear 2 Cell 4-Cell Pronuclear 2 Cell 4 Cell Pronuclear 2 Cell 4-Cell Pronuclear 2-Cell 4-Cell 2 1a 2a a 3 a a 1a 4 d 1 7c 14b a 2 4b 17b a 20c Total 3 a 2a 6 d 3 a 3 2a 3a 2a 38e 63 e 32 c Ejaculated sperm Pronuclear 2-CeU 4-Cell 5 d 3 a 2a 9 d 14' 1 14d 17" 3a alncludes one abnormal (fragmented and/or distorted) ovum. Footnotes b, c, d, e, f, g, and h include two, three, four, five, six, seven, and eight abnormal ova, respectively. '

5 Vol. 29, No.5 IN VITRO FERTILIZING ABILITY OF RABBIT SPERMATOZOA 575 FIG. 2. Pronuclear-appearing ovum 27 hours after in vitro insemination with testicular sperm pretreated with high ionic strength medium and caffeine. Note severe fragmentation (arrow). cauda sperm were compared with ejaculated sperm in these experiments (Table 1A). A comparison of fertilizing ability of cauda epididymal sperm versus ejaculated sperm from the same bucks in the ten experiments of the last two series in Table 1 reveals a highly significant difference (P < 0.005). Additionally, a greater proportion of the ova exhibiting evidence of fertilization developed to the four-cell stage within the incubation period following insemination with cauda epididymal sperm <P < 0.05) in these 10 experiments (Table 1B). Throughout these experiments the development ofthe fertilization process was generally retarded, but cleavage stages were observed even after insemination of ova with testicular and caput epididymal sperm. None of 28 ova showed signs of activation after incubation in five of these experiments when the insemination step was omitted. Ovum activation was not observed in every dish following incubation of ova with any one of the sperm types studied. Attention was paid at the light microscopic level to fragmentation and abnormalities of activated ova. Total numbers of abnormal ova with evidence of fertilization are indicated in Table lb. Abnormalities included fragmentation which ranged from severe involvement (Fig. 2)-which, although less common over-all, was more common in ova that were pronuclear or at the two-cell stage-to (more commonly seen) subtle fragmentation detectable only after proper focusing at high magnification (Fig. 3). Abnormalities less frequently seen included distorted or swollen zonae pellucidae and irregular or unequal blastomeres. The proportion of ova with evidence of fertilization classified as abnormal following light microscopic examination ranged from 40.0% to 42.9% following insemination with testicular, caput, and corpus sperm, whereas 9.8% and 35.3% of fertilized ova inseminated with cauda and ejaculated sperm, respectively, were considered somewhat abnormal. A highly significant increase in the incidence of abnormal ova with evidence of

6 576 BRACKE'IT ET AL. May 1978 FIG. 3. Four-cell stage ovum (three blastomeres in focus) 27 hours after in vitro insemination with corpus epididymal sperm. Note minor degree of fragmentation (arrow). fertilization was observed following in vitro insemination with sperm obtained above, or beyond the cauda epididymidis (P < 0.01). Abnormalities of unfertilized ova, usually of slight degree, were noted at the end of the incubation interval. Among these irregularities were fragmentation, misshapen or distorted appearance of the vitellus, and, rarely, dissolution of the zona pellucida. Following insemination with testicular, caput, corpus, cauda, and ejaculated sperm such irregularities were noted in 14%, 28%, 31%, 32%, and 37%, respectively, of the totals of ova not fertilized. Ejaculated rabbit sperm capacitated by treatment with high ionic strength medium were observed to undergo the acrosome reaction and to penetrate through the zona pellucida following in vitro insemination (Fig. 4). Cortical granules located at the periphery of the ooplasm of recently ovulated ova normally disappeared rapidly as normal activation of the ovum was observed following sperm penetration. Sperm remnants were identified within the ooplasm during development of the normal fertilization process (Fig. 5). These ultrastructural features, i.e., cortical granule breakdown and identification of sperm remnants within ova, were accepted as major criteria upon which ultrastructural evaluation of normal fertilization was based. Ultrastructural assessment of normalcy of fertilization in ova selected as representative or as questionable in appearance as judged by light microscopy is summarized in Table 2. Abnormal activation was characterized by the persistence of cortical granules in ova exhibiting other features of fertilization. Degeneration denotes loss of structural integrity, and this occurred in a way to resemble a two-cell ovum upon observation at low magnification (Table 2). Since selection of ova was necessary because of the time and expense involved in ultrastructural studies, the proportions of abnormal development indicated are not an accurate reflection of expected incidences under the present conditions. With the exception of three of the two-cell ova (from an unmatched experiment) resulting from insemination with corpus sperm, all ova described in Table 2 were included in the summaries of Table 1. It was concluded from this study that activation of ova by

7 Vol. 29, No.5 IN VITRO FERTll..IZING ABILITY OF RABBIT SPERMATOZOA 577 FIG. 4. Sperm within the perivitelline space (PVS) of an activated ovum showing clearly the inner acrosomal membrane (iam) and loss of the outer acrosomal remnants (x20,ooo). immature sperm was a frequent occurrence and that the trend to normal fertilization increases as sperm gain maturity; abnormal activation was infrequent following insemination with ejaculated sperm capacitated in vitro. An incidental observation was the striking heterogeneity of sperm forms obtained from the caput epididyrnidis and found associated with in vitro-inseminated ova (Figs. 6 and 7). These varied from undeveloped forms (Fig. 6) to those resembling normal ejaculated sperm and exhibiting evidence of having undergone the acrosome reaction (Fig. 7). A progressive facility for sperm-ovum interaction was suggested from increasing numbers of sperm cells associated with zonae pellucidae, from few when testicular sperm were used to many when cauda epididymal or ejaculated sperm were used. FIG. 5. Sperm remnant in close apposition to male pronucleus (mpn). Sperm fibrils (SF) and sperm mitochondria (8m) are clearly seen (x24,ooo).

8 578 BRACKETT ET AL. May 1978 Source of sperm for insemination No.ofava studied TABLE 2. Ultrastructural Assessment of Fertilization in Selected Ova Stage of development ll Character of development assessed by electron microscopy Pronuclear 2-Cell 4-Cell Normal fertilization Abnormal fertilization Degeneration Epididymidis Caput Corpus Cauda Ejaculate a Assessed by light microscopy 27 hours after in vitro insemination. Ova showing signs of activation, Le., pronuclear and/or cleavage stages, following insemination with testicular sperm and sperm from the caput and midcorpus regions of the epididymis were uniformly found to retain cortical granules at the periphery of the ooplasm and/or blastomericcytoplasm. A four-cell stage ovum with cortical granules present and no sperm remnants within the perivitelline space following insemination with caput sperm and a similarly activated twocell stage ovum resulting from insemination with corpus epididymal sperm are shown in Figures 8 and 9, respectively. No distinction could be made between these abnormally activated ova and the appearance of normally fertilized rabbit ova at the light microscopic level. Sperm remnants were not seen within the perivitelline spaces of ova inseminated by sperm obtained from the male reproductive tract proximal to the cauda epididymidis in this study. Normally, 5 to 22 cauda epididymal or ejaculated sperm cells could be found within perivitelline spaces of inseminated ova, and on occasion these supplementary sperm were too numerous to count (i.e., more than 40). An unusual finding was the presence of cauda epididymal or ejaculated sperm within the perivitelline spaces of cleaved ova in which cortical granules remained present (Fig. 10). This was observed in one pronuclear ovum and in one two-cell stage ovum following insemination with cauda epididymal sperm and in the single abnormally activated ovum, which was in the two-cell stage, resulting from insemination with ejaculated sperm (Table 2). The additional four ova abnormally activated by cauda epididymal sperm (Table 2) were in the two-cell stage. The discrepancy in fertilizing ability between ejaculated sperm and cauda epididymal sperm raised the question of whether sperm from more distal regions of the male reproductive tract might, upon successive ejaculation, provide better proportions of fertilized ova in our system. The results of three experiments to test this idea are summarized in Table 3. The bucks used were known to be good semen donors, and it is apparent that they provided sperm superior in fertilizing ability to the average first ejaculates employed in experiments shown in Table 1A. The second ejaculate seemed to provide somewhat better fertilization results, but the only difference in fertilization rates reaching statistical significance followed comparison of those afforded by second and third ejaculates. The increased proportions of more immature sperm forms in the third ejaculate seems an obvious explanation for lowered fertilizing ability (Table 3). FIG. 6. Transverse section of immature caput epididymal sperm head found on the surface of the zona pellucida 27 hours after in vitro insemination. Note the variable shape of electron-dense acrosome materials surrounding the sperm nucleus (SN) (x40,ooo).

9 Vol. 29, No.5 IN VITRO FERTILIZING ABILITY OF RABBIT SPERMATOZOA 579 FIG. 7. Longitudinal section of caput epididymal sperm head found on the surface of the zona pellucida 27 hours after in vitro insemination. Note the loss of plasma membrane and acrosomal contents (x20,ooo). Several experiments were carried out for assessment of variations in pretreatment of epididymal sperm. Once-washed caput epididymal sperm activated one of five inseminated ova, whereas high ionic strength-treated caput sperm activated one of six ova inseminated. Activation in both cases led to pronuclear development during the incubation interval. Two additional experiments were carried out to compare the fertilizing ability of cauda epididymal sperm unwashed, oncewashed, treated with isotonic defined medium, and treated with high ionic strength medium as routinely practiced. None of four ova were activated by cauda epididymal sperm used for insemination directly after their recovery, i.e., unwashed. With the use of once-washed cauda epididymal sperm, 4 of 17 ova were activated; 3 reached the pronuclear stage and 1 reached the two-cell stage within approximately 27 hours after in vitro insemination. Following treatment with the isotonic defined medium, 10 of 10 inseminated ova were activated; of these, 2 were pronuclear and 8 reached the two-cell stage. Following treatment of cauda epididymal sperm with the routinely employed in vitro capacitation treatment (i.e., high ionic strength treatment), 12 of 14 inseminated ova were activated; of these, 3 were pronuclear, 7 reached the two-cell stage, and 2 reached the 4-ceU stage during the incubation interval. DISCUSSION An in vitro approach was employed for assessment of the ability of rabbit sperm recovered from various levels of the male reproductive tract to undergo capacitation and fertilization under defined conditions. The high ionic strength treatment found effective for in vitro capacitation of ejaculated rabbit sperms proved to be even more FIG. 8. Four-cell stage ovum activated by caput epididymal sperm, 27 hours after in vitro insemination. Note the presence of cortical granules (eg) at the periphery of the blastomeric cytoplasm (x5,ooo).

10 580 BRACKETT ET AL. May 1978 FIG. 9. Two-cell stage ovum activated by corpus epididymal sperm, 27 hours after in vitro insemination. Note the presence of cortical granules (eg) at the periphery of each blastomere (x8,000). effective in preparing cauda epididymal sperm for fertilization in vitro. By using this approach, ovum activation followed in vitro insemination with immature sperm but ultrastructural examination verified normal fertilization only after insemination with cauda epididymal or ejaculated sperm. Cauda epididymal sperm exhibited a marked advantage in fertilizing ability as reflected by increased proportions of ova fertilized and by initiation of early embryonic development more closely approaching that expected following sperm-ovum contact in vivo. The marked advantage in the fertilizing ability of cauda epididymal sperm was not surprising in light of the apparent ease with which capacitation of cauda epididymal sperm of various species takes place in vitro. A high incidence of ovum abnormalities was noted in both activated and unactivated ova, FIG. 10. An unusual two-cell stage ovum activated by cauda epididymal sperm, 27 hours after in vitro insemination. Note the presence of cortical granules (eg) and a sperm head (sp) in the perivitelline space (PV8) (x5,000)..

11 Vol. 29, No.5 IN VITRO FERTll..IZING ABILITY OF RABBIT SPERMATOZOA 581 TABLE.3. In Vitro Fertilizing Ability of Rabbit Sperm from First, Second, and Third Ejaculates Developmental stage Sperm from Ova Ova fertilized 27 hours postinsemination ejaculate inseminated (%) -::--:----= Pronuclear 2-Cell 4-Cell (69.2) 22 (81.5)" 7 (39.9)a asignificantly different (P < 0.01) although the degree oreach abnormality, in general, was considered to be minor. Of the apparently fertilized ova, those developing to the four-cell stage following insemination with cauda epididymal sperm displayed the lowest. incidence of irregularities as observed by light microscopy. Possible explanations for morphologic aberrations of ova throughout this study might include a predisposition of ova to certain changes by their in vivo. development or by their in vitro handling prior to insemination. The uniform absence of any degenerative or parthenogenetic changes in ova not inseminated implicates the sperm or substances associated with them as initiators of the ovum changes observed. Furthermore, a suggestion that progressively motile sperm might be required for ovum activation follows the observation that vibrating testicular sperm were not effective whereas testicular sperm rendered progressively motile by caffeine treatment were effective in activating some ova. The initiation of progressive motility of testicular sperm attributed here to treatment with the phosphodiesterase inhibitor. caffeine, was probably also dependent on interaction with the albumin or with a componentof testicular origin.8 Ultrastructural events of the normal fertilization process9 were observed following in vitro insemination with in vitro-capacitated ejacu.lated and cauda epididymal sperm. Fraser et al.10 reported cortical granules to be lost from ooplasm as early as 1 hour after insemination with opti-. mally capacitated uterine sperm under conditions: similar to those: used in the present study. No detectable parthenogenetic activation of ova was found in the system by Fraser et aly Parthenogenetically activated rabbit ova retain -their cortical granules. 12 Pincus and Shapiro13 and Chang14 artificially activated rabbit ova by subjecting them to 10 C for 25 hours, followed by culture at 37 C. By using this approach, Longo15 reported cleavage of 5 of 19 unfertilized ova and published photographs of a nucleated onecell stage ovum and a two-cell -stage ovum that were similar in appearance to those presently o 4 o reported to follow insemination with immature sperm, being indistinguishable from normally.fertilized rabbit ova under Nomarski optics. An alteration inthe incidence of activation and in the types of mouse parthenogenones has been shown to follow variations in physicochemical treatment, but the genetic influences are also of great importance in predisposition to activation16 and also in the resulting type of parthenogenone. 17, 18 In our study, ova exhibiting evidence of abnormal activation may have been those earliest ovulated and hence more easily stimulated, as a result of their age, by the presence of immature sperm cells. The factors (e.g.; hyaluronidase) from the sperm responsible for abnormal activation in. our experiments are open to speculation. The. postovulatory deterioration of ova. is a wellrecognized cause of abnormalitiesoffertilization. 19 ThibauWo reported a high incidence of digyny following in vitro insemination with uterine sperm when ova were recovered about 8.5 hours after ovulation, in contrast to no abnormalities found following insemination of ova recovered around 1.5 hours after ovulation, a time comparable to that when ovum recovery was carried out in the present study. According to Austin,21 certain fish ova are activated by sperm cells that penetrate them but do not form male pronuclei. The activated fish ovum retains the second polar body and develops diploid offspring. This process has been termed gynogenesis and is distinct from parthenogenesis, in which process sperm cell involvement is absent. The abnormal ovum activation observed throughout this study might lie between these two processes,. since our data might be interpreted as initiation of activation by substances from immature sperm cells without ovum penetration while activating substances from more mature cauda epididymal-and eja<:ulated sperm cells were, at least on occasion, associated with sperm penetrationthrough the zona pellucida. Theimpression follows from present data that a progressive development of activating ability develops concurrently with functional sperm maturation. The most convincing demonstration of normal fertilizing ability by caput epididymal sperm was the birth of one offspring following vaginal insemination of six does.22 It was further reported that 25%, 14.3%, and 50.3%. of does delivered following insemination with corpus, proximal cauda, and distal cauda epididymal sperm, respec-. tively. In an extensive survey of fine structure of epididymal rabbit sperm, Bedford23 reported a few

12 582 BRACKE'IT ET AL. May 1978 sperm cells possessing apparently mature acrosomes in all segments of the caput epididymis. As judged by our ultrastructural observations on the heterogeneity of maturity of caput epididymal sperm, it seems reasonable to expect some fertilization to occur upon their exposure to ova. Orgebin-Crist 2 reported 77.6% and 65A% of all fertilized ova -to be lost when does were inseminated with sperm from the lower corpus epididymis and the distal cauda epididymis, respectively, as compared with 38.9% when ejaculated sperm were used; and that the losses occurred before implantation. More recent work reported.by Orgebin-Crist and Jahad 4 confirmed the previously' reported -delay in the fertilization process effected by immature sperm, thereby reenforcing the -idea that this might account for the preimplantational embryonic wastage reported earlier. Overstreet and Bedford did not observe differences in cleavage rates after contralateral inseminations into oviducts or uterine horns of sperm from the distal corpus and the ejaculate. From the present study the possibility that immature sperm mightinadequately activate the ova would appear to be a reasonable.explanation for increased preimplantational embryonic losses observed in the earlier experiments of Orgebin Crist. 2 Although total proportions of ova undergoing fertilization did not differ significantly when fertilizing abilities of first and second ejaculates were compared, some development to the four-cell stage followed insemination with sperm from the second ejaculate, suggesting a possible advantage in speed of ovum penetration. This idea appears to be compatible with the advantage of cauda epididymal sperm, since the second ejaculate might more closely represent this sperm type. In addition to appropriate aging, the association off ewer fertilization inhibitors and/or the association of fertilization inhibitors in a more easily dissociated manner might account for the apparent advantage of cauda epididymal sperm, and perhaps of sperm from second ejaculates, seen in the present study. Acknowledgment. The authors gratefully acknowledge the expert technical assistance provided by Ms. Carolyn Mathias. REFERENCES 1. Bedford JM: Development of the fertilizing ability of spermatozoa in the epididymis of the rabbit. J Exp Zool 163:319, Orgebin-Crist MC: Maturation of spermatozoa in the rabbit epididymis: fertilizing ability and. embryonic mortality in does inseminated with epididymal spermatozqa. Ann BioI Anim Biochim Biophys 7:373, Orgebin-Crist MC, Tichenor P: A technique for studying sperm maturation in vitro.. Nature 239:227, Orgebin-Crist Me, Jahad N: Delayed cleavage of rabbit ova after fertilization by young epididymal spermatozoa. 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