Successful fertilization in vitro of fresh intact oocytes by perivitelline (acrosome-reacted) spermatozoa of the rabbit*

Transcription

1 FERTILITY AND STERILITY Copyright 1984 The American Fertility Society Vol. 41, 5, May 1984 Printed in U.8A. Successful fertilization in vitro of fresh intact oocytes by perivitelline (acrosome-reacted) spermatozoa of the rabbit* Frank B. Kuzan, Ph.D. t Alan D. Fleming, Ph.D.+ George E. Seidel, Jr., Ph.D. Animal Reproduction Laboratory, Colorado State University, Fort Collins, Colorado The ability of supplementary rabbit spermatozoa recovered from the perivitelline space of fertilized eggs to penetrate and fertilize fresh oocytes was determined. Onecell embryos were recovered from superovulated does - 4 hours after ovulation. The perivitelline spermatozoa were released by rupturing the zona pellucida of mechanically and enzymatically denuded I-cell embryos and then fresh cumulusintact oocytes were added. Fertilization of fresh oocytes was evaluated by pronuclei and second polar body formation. Thirty-three of 143 oocytes (4%) were fertilized by perivitelline spermatozoa. Of these, eight (4%) progressed to normal -cell embryos in vitro. These data suggest that some completely acrosome-reacted perivitelline rabbit spermatozoa retain the ability to penetrate the cumulus, bind and penetrate the zona pellucida, and fuse with the vitellus of fresh intact unfertilized oocytes to initiate apparently normal embryonic development. Fertil SteriI41:766, 1984 Mammalian spermatozoa must have undergone the acrosome reaction in order to fuse with the vitelline membrane and initiate the events of fertilization.1-4 The specific site of the acrosome reaction and particularly where in relation to sperm penetration of the egg's investments is an area of some disagreement.1,, 4-6 This may be a consequence of species differences or of the in vitro conditions, such as the use of cumulus-free oocytes, employed in many studies as suggested by Yanagimachi4 and Bavister.5 Received November 1,1983; revised and accepted January 13,1984. *Supported by National Institutes of Health grant HD awarded to G. E. S. tpresent address and reprint requests: Dr. Frank B. Kuzan, Department of Obstetrics and Gynecology, University of Washington School of Medicine, Seattle, Washington tpresent address: Dr. Alan D. Fleming, Perinatal Biology Program, Southwest Foundation for Biomedical Research, P.O. Box 8147, San Antonio, Texas Kuzan et ai. Fertilization by perivitelline sperm Evidence for acrosome-intact spermatozoa binding to the zona pellucida exists for the mouse6-9 and pig. 1-1 By contrast, only acrosomereacted spermatozoa can bind to and then penetrate the zona in the guinea pig.13, 14 The acrosomal status of hamster spermatozoa at the time of binding to the zona pellucida is still in question.3-5, 15, 16 An appreciation of the potential for distinct or even unique characteristics of sperm/zona interaction in the different species is essential to the elucidation of gamete interactions in each species as' well as for the successful development of in vitro capacitation and fertilization systems. For example, a system that induces the acrosome reaction in (all) free-swimming spermatozoa may not support sperm binding to the zona and subsequent penetration in species requiring an intact acrosome at the time of zona binding. Conversely, a zona-free egg penetration assay,17 which does require acrosome-reacted spermatozoa, may give

2 erroneous results as to the fertilizing ability of spermatozoa if the acrosome reaction of freeswimming spermatozoa is not inducible under the conditions employed. Finally, a system that promotes the acrosome reaction of free-swimming spermatozoa while maintaining their vigorous motility would be a requirement for species in which it is the reacted spermatozoa that can bind and then penetrate the zona pellucida. The rabbit provides an excellent model for the study of the ability of acrosome-reacted spermatozoa to bind and penetrate the zona pellucida, because a number of supplementary spermatozoa penetrate the zona but do not fuse with the vitelline membrane.18 These spermatozoa are acrosome-reacted1,, 19 and highly motile,18,, 1 thus providing a pure population of motile, acrosome-reacted spermatozoa. This study was undertaken to assess the fertilizing capability of supplementary perivitelline rabbit spermatozoa with respect to fresh intact oocytes in vitro. MATERIALS AND METHODS Female Dutch Belted rabbits (6 months of age) were obtained from the Colorado State University breeding colony. Does were superovulated, and does to be used as perivitelline sperm donors were artificially inseminated with semen from bucks of proven fertility as previously described. Ova were collected at laparotomy 13 hours (for fresh unfertilized intact oocytes) or 16 hours (for fertilized oocytes with perivitelline sperm) after luteinizing hormone (.5 mg; Rheis) injection. Briefly, the oviducts were cannulated through the ostium; medium was introduced through the uterotubal junction in a retrograde manner; and ova were collected in a Petri dish (1 x 35 mm, Falcon Plastics, Oxnard, CA). The medium used for all ova collections and gamete incubations was a modified Brackett's medium composed of 4. mm KCI,. mm CaCI,.9 mm NaHP4, 36.9 mm NaHC 3, 1.7 mm NaCI, 5.6 mm glucose,.5 mm MgCI, 5.4 mm Na-Iactate, 1. mm Napyruvate, and.3% bovine serum albumin (Fraction V; Calbiochem, La Jolla, CA) with 1 Vlml of penicillin and 1 j.lg/ml streptomycin. One-cell embryos were mechanically denuded of corona cells and observed at a magnification of x to detect motile spermatozoa in the perivitelline space. Only embryos containing> 5 highly motile spermatozoa were used for fertilization experiments; embryos with ~ 5 perivitelline spermato- Vol. 41, 5, May 1984 zoa served as controls for cleavage to -cell embryos after 4 hours of incubation. For each experiment, 3 to 6 1-cell embryos were used as perivitelline sperm donors. Embryos were pretreated with.1% trypsin (Sigma Chemical Company, St. Louis, MO) for 1 minute, washed four times (3 to 4 mllwash) with medium, and transferred to.1 ml of medium under paraffin oil (saybolt viscosity 15/135; Fisher, Englewood, CO). The trypsin treatment was necessary to remove spermatozoa on the surface of the zona and to prevent perivitelline spermatozoa from rebinding to the external surface of the zona after the zona was ruptured. To release perivitelline spermatozoa, the zonae pellucidae were ruptured with a finely drawn pipette; the vitelli (embryos) were destroyed by this process. Fresh cumulus-intact oocytes were added to the sperm suspension, in as small a volume as possible, and incubated at 37 C in a humidified atmosphere of 5% CO in air for 6 to 8 hours. Oocytes were then treated with.1% hyaluronidase (Sigma) to remove cumulus cells, mechanically denuded of corona cells (but with the zonae left intact), washed four times and placed individually intoo.5-ml drops of medium under oil. Oocytes were observed at 1 to 1 hours for the presence of two pronuclei (PN) and two polar bodies (PB; 3 x) and at 4 hours for the presence of -cell embryos. Only those oocytes with two PN visible within the vitellus and two PB visible in the perivitelline space ( PN + PB) were considered fertilized. RESULTS Initially, perivitelline spermatozoa were released from donor embryos after complete removal of cumulus and corona cells and extensive washing, but without any trypsin treatment. This procedure proved unacceptable, because when the zona was ruptured, most of the perivitelline spermatozoa were seen to rebind to the external surface of the zona from which they had just been released and initiate repenetration of it. While this provided promising evidence that perivitelline spermatozoa were capable of binding and penetrating the zona a second time, too often few or no perivitelline spermatozoa remained available to the fresh intact oocytes in which fertilization could be assessed. Therefore, a brief trypsin treatment was introduced after removal of corona cells and prior to extensive washing. As well as preventing the perivitelline spermatozoa from Kuzan et al. Fertilization by perivitelline sperm 767

3 Table 1. Fertility of Perivitelline (PV) Spermatozoa Exposed to Fresh Intact Oocytes Replicates oocytes PN PN + PB with PV sperm -cell at 4 hours (%)" degeneration at 4 hours b (1.4) (6.) (19.4) (8.) 3 (1) 5 (45.5) 1 11 Total Control1-cell embryos Control unfertilized oocytes (3.8) 56 (87.5) 8 (4.) 34 (6.7) (%) anumber (%) of fertilized oocytes ( PN + PB) that continued to apparently normal -cell embryos at 4 hours. bnumber of oocytes among all oocytes exposed to PV spermatozoa (column ) which had degenerated by 4 hours. These were recognized and recorded in order to distinguish them from normal cleaving embryos. rebinding to the original zona, this procedure also ensured that any spermatozoa at the surface of the zona or which had penetrated only very slightly into the zona were removed. After 6 to 8 hours of exposure to perivitelline spermatozoa (1 to 13/m I), there was no alteration in the appearance or extent of the cumulus investment. All oocytes were still in the common cumulus clot, where they were when removed from the females' oviducts. Fertilization results are presented in Table 1. Only ova with two PN and two PB (Fig. 1) were considered fertilized, while ova with only two PN were considered parthenogenetically activated. With these criteria, 33 of 143 ova (4%) were fertilized, were parthenotes, and the remainder appeared to be normal secondary oocytes. In comparison, none of 33 control ova with no exposure to spermatozoa were activated by similar hyaluronidase treatment and incubation. A high percentage of oocytes, fertilized by perivitelline spermatozoa, developed into -cell embryos in two of the four replicates (Table 1). Overall, 8 of 33 embryos (4%) derived from in vitro fertilization by perivitelline spermatozoa progressed to the -cell stage (Fig. ), compared with 34 of 56 embryos (61%) derived from normal in vivo fertilization. No embryo fertilized in vitro ( PN + PB) fragmented or degenerated during the 4-hour incubation period. the observations of Bedford1. on rabbit ova fertilized in vivo, but in contrast to sperm-zona interactions of the mouse6. 7 and pig. 1-1 Clearly, further investigation is required as to the nature of the sperm membrane requirement for successful zona binding in other species, so that the appropriate model systems can be utilized in the study of mechanisms of fertilization in vivo and in vitro. It is also apparent from this study that at least some members of a population of spermatozoa, which have completely penetrated the investments of one oocyte and been exposed to cortical granule material, are capable of penetrating the DISCUSSION These results demonstrate that the rabbit is a species in which the plasmalemma over the acrosomal region is not required for sperm binding to the zona pellucida prior to zona penetration. This is in agreement with studies in species such as the guinea pig and hamster, and with 768 Kuzan et al. Fertilization by perivitelline sperm Figure 1 One-cell embryo observed 1 to 1 hours after exposure to perivitelline spermatozoa. Two pronuclei (PM and two polar bodies (PR) are visible. Live whole mount (original magnification, x 79).

4 Figure Two-cell embryo observed 4 hours after exposure to perivitelline spermatozoa. The embryo is compressed between the slide and the coverslip (original magnification, x 79). investments of and fusing with another oocyte. Thus, the requirement for a zona-lysin may be minimal, as suggested previously.14, 3 The failure of the perivitelline spermatozoa to alter or disperse the cumulus clot suggests that hyaluronidase and possibly other acrosomal enzymes were absent or present only in very low concentrations. That fertilization still occurred raises a question as to the necessity of these enzymes in the fertilization process. However, acrosomal enzymes which may facilitate cumulus and zona penetration could remain associated with the inner acrosomal membrane or within the equatorial segment and exert only a very local effect at the leading edge of the penetrating spermatozoon. How long after the occurrence of the acrosome reaction and initial zona penetration a perivitelline spermatozoon retains its ability to fertilize a second intact oocyte is not known. However, the fertile life of perivitelline spermatozoa must be at least 3 minutes, based on the time required for collection, processing, and mixing of gametes. This is in agreement with the extended fertile life of guinea pig spermatozoa after completion of their acrosome reactions. 14 The concentration of rabbit perivitelline spermatozoa when exposed to fresh intact oocytes was in the range of 1 to 13 /ml. Successful fertilization at low sperm/egg ratios has also been revol. 41, 5, May 1984 ported in the guinea pig14 and the hamster. 4 The success in the guinea pig and rabbit at least is probably due to the selection of a homogenous population of acrosome-reacted spermatozoa, some of which still possess the vigorous (hyperactivated) motility characteristic of the species. Successful fertilization by perivitelline spermatozoa suggests that the zona pellucida may be used as a "trap" to collect demonstrably fertile spermatozoa for subsequent mixing with fresh, intact oocytes. In species without a block to polyspermy at the level of the vitelline membrane, the native oocytes in "trap" zonae could be destroyed prior to exposure to spermatozoa. 5, 6 The accumulation of perivitelline spermatozoa could be done in vitro or even in vivo, particularly in species where an efficient in vitro fertilization system is not available. This procedure would, of course, be effective only in species in which acrosome-reacted spermatozoa are capable of binding and penetrating the zona pellucida. This may include the human. Soupart7 and Soupart and Strong8 have presented evidence that human spermatozoa undergo their acrosome reactions prior to or during passage through the cumulus matrix (Le., prior to zona binding) based upon transmission electron microscopic examination of cumulus-oocyte complexes fertilized in vitro. The capability of these perivitelline spermatozoa to support complete embryonic development remains uncertain. Better culture conditions, including medium optimized for rabbit embryo culture,9 coupled with embryo transfer should resolve this question. Acknowledgment. The authors gratefully acknowledge the expert technical assistance of Mr. Mark Barone. REFERENCES 1. Bedford JM: Ultrastructural changes in the sperm head during fertilization in the rabbit. Am J Anat 13:39, 1968 Bedford JM: An electron microscopic study of sperm penetration into the rabbit egg after natural mating. Am J Anat 133:13, 197 Shalgi R, Phillips DM: Mechanics of in vitro fertilization in the hamster. BioI Reprod 3:433, 198 Yanagimachi R: Mechanisms of fertilization in mammals. In Fertilization and Embryonic Development In Vitro, Edited by L Mastroianni, JD Biggers. New York, Plenum Press, 1981, p 81 Bavister BD: Evidence for a role of post-ovulatory cumulus components in supporting fertilizing ability of hamster spermatozoa. J Androl 3:365, 198 Kuzan et a1. Fertilization by perivitelline sperm 769

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