MOLECULAR REPRODUCTION AND DEVELOPMENT 2Z345-347 (1990)
Penetration of Intact Bovine Ova With Ram Sperm In Vitro T. SLAVfK, A. PAVLOK, AND J. FULKA Czechoslovak Academy of Sciences, Institute of Animal Physiology and Genetics, LibCchov, Czechoslovakia In culture, mature bovine ovaABSTRACT rian oocytes were fertilized in vitro with freshly ejaculated ram spermatozoa treated with heparin. The zona pellucida does not prevent penetration of ram spermatozoa. The penetration rate varied between 10 and 84%, and in most instances, after 24 hr of culture, two normal-looking pronuclei and sperm tail were present in the cytoplasm. These results suggest that the zona pellucida of bovine oocytes does not represent a barrier for the penetration of ram spermatozoa.
All these experiments were carried out under natural conditions, i.e., females were inseminated by alien spermatozoa by a n intrauterine or intratubal route. In our experiment, a completely different approach was chosen: sheep and cattle oocytes were matured in culture and fertilization was carried out in vitro.
MATERIALS AND METHODS
Culture medium (CM) was the same as described by Pavlok et al. (1988) and composed of TC 199 (Sevac Praha) supplemented with 2.92 mmol Ca-lactate, 2 mmol Na-pyruvate, 33.9 mmol HEPES (Serva HeidelKey Words: Bovine oocytes, Cross-fertilization in berg), antibiotics, and 15% calf serum (Bioveta Ivanovvitro, Cattle, Sheep, lnterspecies fertilization ice). For in vitro maturation, FSH (Calbiochem; 0.2 i.u./ml CM) was added to culture medium (CM-FSH). For in vitro fertilization, it was supplemented with INTRODUCTION heparin sodium (Spofa Praha; 50 i.u./ml CM; CMIt has generally been accepted that mammalian HEP). oocyte is protected against foreign sperm penetration Bovine oocytes were obtained from the ovary of by a barrier, localized on the level of zona pellucida, of slaughtered hormonally non-stimulated animals by asooplasm or on both of them (Yanagimachi, 1981). How- piration of follicles 4-6 mm in diameter. The source of ever, the sperm penetration through the zona pellucida sheep oocytes was identical; only the diameter of reand sperm egg fusion may occur following cross-insem- lated follicles was smaller (2-5 mm). The aspiration ination between closely related species. This interspe- was carried out in the laboratory within 2-3 h r after cies barrier seems to be weak between ferret eggs and killing. The ovaries were kept before aspiration in a mink sperm, but after insemination of mink eggs by thermobox at approximately 20°C. For further cultivaferret spermatozoa, the fertilization rate is very low tion, only oocytes with compact cumulus were chosen. (Chang, 1965, 1968). Domestic rabbit eggs can easily The cumulus-oocyte complexes were placed in a dropbe fertilized by hare and snowhare spermatozoa (Ad- let (0.1 ml) of CM-FSH under paraffin oil and cultured ams, 1957; Chang and Adams, 1962; Chang et al., under atmosphere 5% CO,, 10% O,, and 85% N2 a t 1964). In contrast, the fertilization of snowhare eggs 385°C. In each droplet were placed from 25 to 40 cuwith rabbit spermatozoa can be observed only excep- mulus-oocyte complexes. tionally (Chang, 1965). The oocytes in this medium were matured for 24-26 Several attempts have been made to clarify the ex- hr. At the beginning and the end of this period, some istence of the fertilization barrier between sheep and oocytes were fixed for control of nuclear maturation. goat. These experiments show that goat eggs are ferFor fertilization, 500 pl of freshly ejaculated ram setilized by sheep sperm under natural conditions in a men was diluted with 5 ml of culture medium (CM) and high frequency, whereas the fertilization is very low, if centrifuged a t approximately 600g for 10 min. After any, after insemination of sheep with goat semen. In discarding the supernatant, this procedure was resuch a combination, Hancock (1964) observed only 5% peated once more. Approximately 100 p1 of the sedifertilized eggs. McGovern (1973) showed, in this case, that the eggs do not posses a n intrinsic barrier to the entry of the goat spermatozoa, and for low fertilization, there are factors responsible for affecting the transport, survival, and capacitation of goat spermatozoa in the Received July 20, 1989; accepted October 24, 1989. ewe oviduct. This observation was fully confirmed by Address reprint requests to TomaS Slavik, Institute of Animal Physiology and Genetics, 277 21 Libechov, Czechoslovakia. Eppleston and Moore (1977).
O 1990 WILEY-LISS, INC.
346
T. SLAVIK ET AL.
Fig. 1. Ovine oocyte fertilized with ram spermatozoa. Two normally developed pronuclei with the sperm tail (arrow) in cytoplasm. Magnification 600 x .
TABLE 1. Parallel Estimation of Penetration of Ram Spermatozoa Into Sheep and Cow Oocytes In Vitro
Fig. 2. Bovine oocyte fertilized with ram spermatozoa. Both pronuclei at similar stage of development as in Figure 1 and the sperm tail (arrow) is present. One pronucleus is slightly out of focus. Magnification 600 x .
TABLE 2. penetration Rate of R~~ s~~~~~~~~~~ Into Bovine Oocvtes In Vitro
No. (%) Experiment Oocyte Total no. Experiment oocytes penetrated Polyspermya no. no. origin 14 5(35.7) 1.00 1 Bovine 1 35 27(77.1) 1.15 Ovine 2 2
Bovine
Ovine
27 24
21(77.8) 22(91.7)
1.19 1.23
aAverage number of spermatozoa in one penetrated egg.
ment was diluted with 1.5 ml of CM, and spermatozoa were allowed to swim-up for 30-45 min. The spermatozoa from the upper layer were added at the concentration of 1.0-2.0 x 106/ml to the matured bovine or ovine oocytes. Before mixing with the sperm suspension, the oocytes were transferred to the CM-HEP. Under these conditions, oocytes and spermatozoa remained together in the atmosphere and temperature described above for 16-20 hr. After this culture, the oocytes were washed, mounted on slides between vaseline strips under a coverslide, fixed with acetic acidalcohol (1:3) overnight, stained with 2% acetic orcein, and examined under a phase-contrast microscope. The presence or absence of spermatozoa and pronuclear formation in each oocyte was determined. Only those oocytes with the sperm tails localized in the vicinity of pronuclei or decondensing sperm head chromatin were considered as penetrated.
RESULTS From the samples taken at the beginning of culture, ovine and bovine oocytes were in the germinal vesicle stage. In this stage, a distinct nuclear membrane and finely granulated nucleoplasm with few orcein-positive zones lying alongside nuclear membrane are charac-
3 4 5 6 7 8 9 10
Total no. oocytes
penetrated
Polyspermy
19 31 12 30 20 21 23 12 12 19
5 (26.3) 3 (9.7) 4 (33.3) 9 (30.0) 5 (25.0) 17 (80.9) 15 (65.0) 6 (50.0) 2 (16.7) 16 (84.2)
1.00 1.00 1.00 1.00 1.00 1.35 1.13 1.00 1.oo 1.38
No. (%)
teristic. Using this technique, the nucleolus is rarely visible. After 24-26 hr of culture, 87% ovine and 89% bovine oocytes had completed meiosis to metaphase I1 and had extruded the first polar body. The rest of the oocytes remained in less advanced stages. In two preliminary experiments, 77.1 and 91.7% ovine oocytes were fertilized with ram spermatozoa, and both pronuclei and sperm tail were present (Fig.1). The penetration rate of bovine oocytes by ram spermatozoa after estimation in parallel experiments was lower, 35.7 and 77.8% (Table 1). To prove the previous results, a n additional ten experiments were carried out with bovine oocytes only. In all of them, the penetration of bovine oocytes withram spermatozoa was found. The penetration rate varied in individual experiments from 9.7 to 84.2% (Table 2). The pronuclei originating from ram spermatozoa were in most cases normally developed (Fig. 2). Polyspermy occurred occasionally and in the experiments with the higher penetration rate.
IN VITRO CROSS-FERTILIZATION DISCUSSION In her review, Gray (1972) summarized the results of hybridization between different mammalian species. In this review, attempts at hybridization between Bos taurus and Ovis aries are also mentioned. Attention was, however, focused on the characterization of progeny originated from such experiments. Unfortunately, in no instance were convincing proofs offered. Moreover, until now, i t is not known with certainty if ram spermatozoa penetrate the zona pellucida of bovine oocytes naturally. Some previous experiments have revealed that not only a n interspecies barrier represented by zona pellucida, but also environmental conditions in Fallopian tubes, can lead to the failure of fertilization (ferret x mink, Chang, 1968; snowhare x domestic rabbit, Chang, 1965; sheep x goat, Eppleston and Moore, 1977). In our experimental conditions, it was demonstrated that ram spermatozoa penetrate intact bovine oocytes on a very similar level as do bovine spermatozoa (Pavlok et al., 1988; present results). It means that zona pellucida of bovine oocytes does not represent a barrier preventing penetration of ram spermatozoa and that the treatment of ram ejaculates including procedures during fertilization are compatible with the penetration of the zona pellucida and vitelline membrane. According to the stage of pronuclei development in both, i.e., ovine and bovine oocytes, i t can also be assumed that the sequence of events realized on the surface membranes leading to acrosome reaction are identical in bull and ram spermatozoa. The present results support the idea that a n in vitro system can serve as a convenient tool for elucidation of events that are difficult to study under natural conditions in farm animals. A similar approach, using zonafree oocytes (see Yanagimachi, 1984, 19881, gave very valuable knowledge about the specificity of cytoplasmic membrane in fertilization processes. It cannot be
347
excluded that artificial conditions in vitro influence the interaction between spermatozoa and zona pellucida of much less distant species.
ACKNOWLEDGMENTS The authors wish to thank to Mrs. JiFina Zelenkova for excellent technical help.
REFERENCES A d a m CE (1957): An attempt to cross the domestic rabbit (Oryctolugus cuniculus) and hare (Lepus europaeus). Nature 180(4591):853. Chang MC (1965): Implantation of ferret ova fertilized by mink sperm. J Exp Zoo1 160:67-80. Chang MC (1968):Reciprocal insemination and egg transfer between ferrets and mink. J Exp Zoo1 168:49-60. Chang MC, Adams CE (1962): Fate of rabbit ova fertilized by hare spermatozoa. In “XXV. anno della fondazione dello Istituto sperimentale italiano ‘L. Spallanzani’ per la fecondazione artificiale pp 186-191. Chang MC, Marston J H , Hunt DM (1964): Reciprocal fertilization between the domestic rabbit and snowhare with special reference to insemination of rabbits with equal number of hare and rabbit spermatozoa. J Exp Zoo1 155:437-446. Eppleston J, Moore NW (1977): Fertilization between sheep and goats and survival of hybrid embryos (Abstract).Theriogenology 8(4):165. Gray AP (1972):“Mammalian Hybrids.” Commonwealth Agricultural Bureaux, pp 114-147. Hancock J L (1964): Attempted hybridization of sheep and goat. In “Proceedings of the International Congress on Animal Reproduction and Artificial Insemination, Trento,” Vol 3, pp 445-450. McGovern PT (1973):The effect of maternal immunity on the survival of goat x sheep hybrid embryos. J Reprod Fert 34:215-220. Pavlok A, Torner H, Motlik J , Fulka J, Kauffold P, Duschinski U (1988): Fertilization of bovine oocytes in vitro: Effect of different sources of gametes on fertilization rate and frequency of fertilization anomalies. Anim Reprod Sci 16:207-213. Yanagimachi R (1981): Mechanism of fertilization in mammals. In Mastroiani L, Biggers J D J r (eds): “Fertilization and Embryonic Development In Vitro.” New York Plenum, pp 81-182. Yanagimachi R (1984):Zona-free hamster eggs: Their use in assessing fertilizing capacity and examining chromosomes of human spermatozoa. Gamete Res 10:187-232. Yanagimachi R (1988):Mammalian fertilization. In Knobil E, Neil1 J et al. (eds): “The Physiology of Reproduction.” New York: Raven Press, pp 135-185.
Penetration of Intact Bovine Ova With Ram Sperm In Vitro T. SLAVfK, A. PAVLOK, AND J. FULKA Czechoslovak Academy of Sciences, Institute of Animal Physiology and Genetics, LibCchov, Czechoslovakia In culture, mature bovine ovaABSTRACT rian oocytes were fertilized in vitro with freshly ejaculated ram spermatozoa treated with heparin. The zona pellucida does not prevent penetration of ram spermatozoa. The penetration rate varied between 10 and 84%, and in most instances, after 24 hr of culture, two normal-looking pronuclei and sperm tail were present in the cytoplasm. These results suggest that the zona pellucida of bovine oocytes does not represent a barrier for the penetration of ram spermatozoa.
All these experiments were carried out under natural conditions, i.e., females were inseminated by alien spermatozoa by a n intrauterine or intratubal route. In our experiment, a completely different approach was chosen: sheep and cattle oocytes were matured in culture and fertilization was carried out in vitro.
MATERIALS AND METHODS
Culture medium (CM) was the same as described by Pavlok et al. (1988) and composed of TC 199 (Sevac Praha) supplemented with 2.92 mmol Ca-lactate, 2 mmol Na-pyruvate, 33.9 mmol HEPES (Serva HeidelKey Words: Bovine oocytes, Cross-fertilization in berg), antibiotics, and 15% calf serum (Bioveta Ivanovvitro, Cattle, Sheep, lnterspecies fertilization ice). For in vitro maturation, FSH (Calbiochem; 0.2 i.u./ml CM) was added to culture medium (CM-FSH). For in vitro fertilization, it was supplemented with INTRODUCTION heparin sodium (Spofa Praha; 50 i.u./ml CM; CMIt has generally been accepted that mammalian HEP). oocyte is protected against foreign sperm penetration Bovine oocytes were obtained from the ovary of by a barrier, localized on the level of zona pellucida, of slaughtered hormonally non-stimulated animals by asooplasm or on both of them (Yanagimachi, 1981). How- piration of follicles 4-6 mm in diameter. The source of ever, the sperm penetration through the zona pellucida sheep oocytes was identical; only the diameter of reand sperm egg fusion may occur following cross-insem- lated follicles was smaller (2-5 mm). The aspiration ination between closely related species. This interspe- was carried out in the laboratory within 2-3 h r after cies barrier seems to be weak between ferret eggs and killing. The ovaries were kept before aspiration in a mink sperm, but after insemination of mink eggs by thermobox at approximately 20°C. For further cultivaferret spermatozoa, the fertilization rate is very low tion, only oocytes with compact cumulus were chosen. (Chang, 1965, 1968). Domestic rabbit eggs can easily The cumulus-oocyte complexes were placed in a dropbe fertilized by hare and snowhare spermatozoa (Ad- let (0.1 ml) of CM-FSH under paraffin oil and cultured ams, 1957; Chang and Adams, 1962; Chang et al., under atmosphere 5% CO,, 10% O,, and 85% N2 a t 1964). In contrast, the fertilization of snowhare eggs 385°C. In each droplet were placed from 25 to 40 cuwith rabbit spermatozoa can be observed only excep- mulus-oocyte complexes. tionally (Chang, 1965). The oocytes in this medium were matured for 24-26 Several attempts have been made to clarify the ex- hr. At the beginning and the end of this period, some istence of the fertilization barrier between sheep and oocytes were fixed for control of nuclear maturation. goat. These experiments show that goat eggs are ferFor fertilization, 500 pl of freshly ejaculated ram setilized by sheep sperm under natural conditions in a men was diluted with 5 ml of culture medium (CM) and high frequency, whereas the fertilization is very low, if centrifuged a t approximately 600g for 10 min. After any, after insemination of sheep with goat semen. In discarding the supernatant, this procedure was resuch a combination, Hancock (1964) observed only 5% peated once more. Approximately 100 p1 of the sedifertilized eggs. McGovern (1973) showed, in this case, that the eggs do not posses a n intrinsic barrier to the entry of the goat spermatozoa, and for low fertilization, there are factors responsible for affecting the transport, survival, and capacitation of goat spermatozoa in the Received July 20, 1989; accepted October 24, 1989. ewe oviduct. This observation was fully confirmed by Address reprint requests to TomaS Slavik, Institute of Animal Physiology and Genetics, 277 21 Libechov, Czechoslovakia. Eppleston and Moore (1977).
O 1990 WILEY-LISS, INC.
346
T. SLAVIK ET AL.
Fig. 1. Ovine oocyte fertilized with ram spermatozoa. Two normally developed pronuclei with the sperm tail (arrow) in cytoplasm. Magnification 600 x .
TABLE 1. Parallel Estimation of Penetration of Ram Spermatozoa Into Sheep and Cow Oocytes In Vitro
Fig. 2. Bovine oocyte fertilized with ram spermatozoa. Both pronuclei at similar stage of development as in Figure 1 and the sperm tail (arrow) is present. One pronucleus is slightly out of focus. Magnification 600 x .
TABLE 2. penetration Rate of R~~ s~~~~~~~~~~ Into Bovine Oocvtes In Vitro
No. (%) Experiment Oocyte Total no. Experiment oocytes penetrated Polyspermya no. no. origin 14 5(35.7) 1.00 1 Bovine 1 35 27(77.1) 1.15 Ovine 2 2
Bovine
Ovine
27 24
21(77.8) 22(91.7)
1.19 1.23
aAverage number of spermatozoa in one penetrated egg.
ment was diluted with 1.5 ml of CM, and spermatozoa were allowed to swim-up for 30-45 min. The spermatozoa from the upper layer were added at the concentration of 1.0-2.0 x 106/ml to the matured bovine or ovine oocytes. Before mixing with the sperm suspension, the oocytes were transferred to the CM-HEP. Under these conditions, oocytes and spermatozoa remained together in the atmosphere and temperature described above for 16-20 hr. After this culture, the oocytes were washed, mounted on slides between vaseline strips under a coverslide, fixed with acetic acidalcohol (1:3) overnight, stained with 2% acetic orcein, and examined under a phase-contrast microscope. The presence or absence of spermatozoa and pronuclear formation in each oocyte was determined. Only those oocytes with the sperm tails localized in the vicinity of pronuclei or decondensing sperm head chromatin were considered as penetrated.
RESULTS From the samples taken at the beginning of culture, ovine and bovine oocytes were in the germinal vesicle stage. In this stage, a distinct nuclear membrane and finely granulated nucleoplasm with few orcein-positive zones lying alongside nuclear membrane are charac-
3 4 5 6 7 8 9 10
Total no. oocytes
penetrated
Polyspermy
19 31 12 30 20 21 23 12 12 19
5 (26.3) 3 (9.7) 4 (33.3) 9 (30.0) 5 (25.0) 17 (80.9) 15 (65.0) 6 (50.0) 2 (16.7) 16 (84.2)
1.00 1.00 1.00 1.00 1.00 1.35 1.13 1.00 1.oo 1.38
No. (%)
teristic. Using this technique, the nucleolus is rarely visible. After 24-26 hr of culture, 87% ovine and 89% bovine oocytes had completed meiosis to metaphase I1 and had extruded the first polar body. The rest of the oocytes remained in less advanced stages. In two preliminary experiments, 77.1 and 91.7% ovine oocytes were fertilized with ram spermatozoa, and both pronuclei and sperm tail were present (Fig.1). The penetration rate of bovine oocytes by ram spermatozoa after estimation in parallel experiments was lower, 35.7 and 77.8% (Table 1). To prove the previous results, a n additional ten experiments were carried out with bovine oocytes only. In all of them, the penetration of bovine oocytes withram spermatozoa was found. The penetration rate varied in individual experiments from 9.7 to 84.2% (Table 2). The pronuclei originating from ram spermatozoa were in most cases normally developed (Fig. 2). Polyspermy occurred occasionally and in the experiments with the higher penetration rate.
IN VITRO CROSS-FERTILIZATION DISCUSSION In her review, Gray (1972) summarized the results of hybridization between different mammalian species. In this review, attempts at hybridization between Bos taurus and Ovis aries are also mentioned. Attention was, however, focused on the characterization of progeny originated from such experiments. Unfortunately, in no instance were convincing proofs offered. Moreover, until now, i t is not known with certainty if ram spermatozoa penetrate the zona pellucida of bovine oocytes naturally. Some previous experiments have revealed that not only a n interspecies barrier represented by zona pellucida, but also environmental conditions in Fallopian tubes, can lead to the failure of fertilization (ferret x mink, Chang, 1968; snowhare x domestic rabbit, Chang, 1965; sheep x goat, Eppleston and Moore, 1977). In our experimental conditions, it was demonstrated that ram spermatozoa penetrate intact bovine oocytes on a very similar level as do bovine spermatozoa (Pavlok et al., 1988; present results). It means that zona pellucida of bovine oocytes does not represent a barrier preventing penetration of ram spermatozoa and that the treatment of ram ejaculates including procedures during fertilization are compatible with the penetration of the zona pellucida and vitelline membrane. According to the stage of pronuclei development in both, i.e., ovine and bovine oocytes, i t can also be assumed that the sequence of events realized on the surface membranes leading to acrosome reaction are identical in bull and ram spermatozoa. The present results support the idea that a n in vitro system can serve as a convenient tool for elucidation of events that are difficult to study under natural conditions in farm animals. A similar approach, using zonafree oocytes (see Yanagimachi, 1984, 19881, gave very valuable knowledge about the specificity of cytoplasmic membrane in fertilization processes. It cannot be
347
excluded that artificial conditions in vitro influence the interaction between spermatozoa and zona pellucida of much less distant species.
ACKNOWLEDGMENTS The authors wish to thank to Mrs. JiFina Zelenkova for excellent technical help.
REFERENCES A d a m CE (1957): An attempt to cross the domestic rabbit (Oryctolugus cuniculus) and hare (Lepus europaeus). Nature 180(4591):853. Chang MC (1965): Implantation of ferret ova fertilized by mink sperm. J Exp Zoo1 160:67-80. Chang MC (1968):Reciprocal insemination and egg transfer between ferrets and mink. J Exp Zoo1 168:49-60. Chang MC, Adams CE (1962): Fate of rabbit ova fertilized by hare spermatozoa. In “XXV. anno della fondazione dello Istituto sperimentale italiano ‘L. Spallanzani’ per la fecondazione artificiale pp 186-191. Chang MC, Marston J H , Hunt DM (1964): Reciprocal fertilization between the domestic rabbit and snowhare with special reference to insemination of rabbits with equal number of hare and rabbit spermatozoa. J Exp Zoo1 155:437-446. Eppleston J, Moore NW (1977): Fertilization between sheep and goats and survival of hybrid embryos (Abstract).Theriogenology 8(4):165. Gray AP (1972):“Mammalian Hybrids.” Commonwealth Agricultural Bureaux, pp 114-147. Hancock J L (1964): Attempted hybridization of sheep and goat. In “Proceedings of the International Congress on Animal Reproduction and Artificial Insemination, Trento,” Vol 3, pp 445-450. McGovern PT (1973):The effect of maternal immunity on the survival of goat x sheep hybrid embryos. J Reprod Fert 34:215-220. Pavlok A, Torner H, Motlik J , Fulka J, Kauffold P, Duschinski U (1988): Fertilization of bovine oocytes in vitro: Effect of different sources of gametes on fertilization rate and frequency of fertilization anomalies. Anim Reprod Sci 16:207-213. Yanagimachi R (1981): Mechanism of fertilization in mammals. In Mastroiani L, Biggers J D J r (eds): “Fertilization and Embryonic Development In Vitro.” New York Plenum, pp 81-182. Yanagimachi R (1984):Zona-free hamster eggs: Their use in assessing fertilizing capacity and examining chromosomes of human spermatozoa. Gamete Res 10:187-232. Yanagimachi R (1988):Mammalian fertilization. In Knobil E, Neil1 J et al. (eds): “The Physiology of Reproduction.” New York: Raven Press, pp 135-185.
