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In vitro fertilization of cattle and sheep follicular oocytes by goat spermatozoa
Small Ruminant Research ELSEVIER
Small Ruminant Research 15 (1994) 55-58
In vitro fertilization of cattle and sheep follicular oocytes by goat spermatozoa J.F. Cox*, A. Catal~in, F. Saravia, J. Avila, A. Santa Maria Department of Animal Production, Facultyof Veterinary Medicine Universityof Concepci6n, Casilla537, Chill6n, Chile Accepted 25 December 1993
Abstract
The study compared efficiency of goat spermatozoa to penetrate cattle and sheep intact follicular oocytes, and evaluated effect of cumulus cells on the ability of sperm to penetrate heterologous oocytes. Ruminant cumulus-oocyte complexes (COC) were obtained by aspiration of ovarian follicles and were matured by culturing them in medium 199 plus goat serum in microdrops under paraffin oil for 24 h. Fresh semen was collected by artificial vagina, incubated for 4 h at room temperature, washed, and spermatozoa were then suspended in Talp medium plus goat serum and treated for capacitation by using incubation at 39°C and heparin. In the first experiment, goat, cattle and sheep matured oocytes were incubated in goat sperm preparation, whereas in the second experiment, cattle and sheep oocytes either partially (cumulus enclosed oocytes = + ) or totally denuded from granulosa cells (denuded oocytes = - ) were added to the prepared spermatozoa. Fertilization rates obtained in the first experiment (goat sperm-goat oocytes: 91.3%; goat-cattle: 82.7%; and goat-sheep: 79.8%, P > 0.05), showed that goat sperm were highly efficient in penetrating cattle and sheep oocytes as compared with homologous fertilization, and that goat sperm penetrated equally well cattle and sheep oocytes. Fertilization rates from the second experiment (cattle oocytes: + 74.0%, -88.89%; sheep oocytes: + 68.42%, -78.85%, P > 0.05), showed that cumulus cells did not modify the ability of goat spermatozoa to penetrate heterologous oocytes. Keywords: Heterologous fertilization; Cumulus cell; Fertilization, in vivo; Goat; Fertilization, in vitro
1. Introduction Fertilization is an event whose species specificity is controlled at several stages, including mating behaviour, sperm transport and sperm--egg interaction (Yanagimachi, 1988). It is thought that the barrier which limits heterologous gamete interaction at the oocyte level is the zona pellucida (O'Rand, 1988). Depending on the species, zonae pellucidae may modulate binding, *Corresponding author. 0921-4488/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved
SSD10921-4488(94)00003-P
acrosome reaction induction and/or zona penetration by foreign spermatozoa (Cox, 1992). However, the phylogenetic relationship between gametes may reduce the efficiency of the zona pellucida to act as a barrier. Cox (1992) showed that bull spermatozoa were able to penetrate sheep oocytes but unable to penetrate pig or mouse oocytes. On the other hand, S l a v ~ et al. (1990) and Cox (1992) showed that ram spermatozoa easily penetrated cattle oocytes. It is unclear why the zona pellucida is unable to recognize spermatozoa of related species but the sharing of common molecules
56
J. F. Cox et al. /Small RuminantResearch 15 (1994) 55-58
at the sperm zona interface may be a possibility (O'Rand, 1988). Heterologous fertilization would not only contribute to elucidate mechanisms related to fertilization and embryo development, but also would facilitate the development of procedures for semen evaluation. By using in vitro fertilization techniques, Cox et al. (1992) and Slavik and Fulka (1992) have shown that goat spermatozoa may also penetrate cattle and sheep follicular oocytes. The aims of the present study were (1) to complete these earlier reports by having a homologous system as a reference, and (2) to evaluate the effect of the cumulus on heterologous fertilization using goat spermatozoa.
2. Materials and methods
2.1. Oocyte collection and culture
Ovaries were obtained from cows, ewes and does killed at a local abattoir and were transported at 3035°C in insulated containers to the laboratory within 2 h. Cumulus-oocyte complexes (COC) were collected by aspiration from follicles between 2-5 nun in diameter with an 18-gauge needle attached to a 10-ml syringe. Oocytes with compact cumulus and evenly granulated cytoplasm were cultured in 100/xl drops of culture medium under paraffin oil (Sigma Chemical Co., St. Louis, MO, USA) in 60-mm plastic dish (Falcon, Becton Dickinson, Lincoln Park, NJ, USA). Maturation medium (Hepes-199) contained TCM 199 (with Earle's salts, NaHCO3, L-glutamine and 10 mM Hepes; pH 7.4; Sigma Chemical Co.), plus 20% v/v heat-inactivated oestrus goat serum (OGS) and 20/xg/ ml of insulin (Sigma Chemical Co. ). 10-20 COC were cultured per drop for 24-26 h at 39°C, 5% CO2 in air, in a static system under a humidified atmosphere (Gordon and Lu, 1990). 2.2. Sperm preparation
Semen from four Anglo Nubian bucks was collected by artificial vagina, washed twice (300×g, 5 min) with Dulbecco's PBS and incubated at about 20°C for 3-4 h (Cheng, 1985). After one more wash, spermatozoa were resuspended in TALP IVF (Parrish et al.,
1988) plus 10% v/v OGS and 2.5/zg/ml of heparin (Sigma Chemical Co.; pH 7.5) at a concentration of 108 sperm/ml and incubated at 39°C and 5% CO2 in air for 40 min. Then, the sperm suspension was further diluted to 10 6 sperm/ml using TALP IVF (plus serum and heparin) and 50-/zi microdrops under paraffin oil were prepared and then incubated at 39°C and 5% CO2 in air until incubation with the eggs (usually about 20 min). 2.3. In vitro fertilization
Only oocytes with clear and well expanded cumulus cells were used in these experiments. After maturation, oocytes were partially (cumulus enclosed) or completely denuded from granulosa cells by passing them in and out of a pulled-glass pipette. The oocytes were washed in Herpes-199 supplemented with 0.4 mg/ml BSA (Fraction V, Sigma Chemical Co.) instead of OGS, and incubated in the already prepared sperm suspensions (five to eight oocytes/drop) at 39°C and 5% CO2 in air. In experiment No. 1, cattle, sheep and goat cumulus-enclosed oocytes were allocated at random to goat sperm preparations and left to interact for 16 h. In experiment No. 2, denuded and cumulus-enclosed cattle and sheep oocytes were incubated in sperm suspensions and left for fertilization to occur. 2.4. Evaluation o f sperm penetration
Sperm penetration in the cytoplasm was evaluated under phase-contrast microscopy by fixing wholemounted completely denuded oocytes in a solution of aceto-alcohol (1:3, v/v) and staining them with a 1% solution of aceto-lacmoid. Only oocytes with pronuclear development and sperm tail(s) were considered to be penetrated. 2.5. Statistical analysis
The analysis of the efficiency of sperm penetration was done by calculating the fertilization rate of each treatment, and comparing them using either an ANOVA and Newman-Keuls test or by the Student's t-test with arc sin-transformed data, using the Statview ® program. A P < 0.05 was considered to be significant.
J.F. Cox et al. / Small Ruminant Research 15 (1994) 55-58
3. Results
The efficiency of sperm penetration in foreign oocytes was evaluated using homologous penetration as a point of reference. Germinal-vesicle staged and ruptured oocytes were not considered (less than 5%) (Table 1). Sperm concentrations used here were not adjusted to establish differences between homologous and heterologous sperm penetration, but to establish differences between foreign oocytes in their ability to interact with goat spermatozoa. The results obtained showed that goat spermatozoa penetrated cattle and sheep oocytes equally well, with similar ranges between replicates. To evaluate whether cumulus cells would modify the ability of goat spermatozoa to penetrate foreign oocytes, denuded and cumulus-enclosed cattle and Table 1 Fertilization rate of goat, cattle and sheep matured oocytes using goat spermatozoa capacitated in vitro Gamete origin
Replicates
Sperm penetration in oocytes
(n) sperm
Goat Goat Goat
oocytes
goat cattle sheep
4 4 4
penetration rate
range
(%)
(%)
84/92 (91.30) ~j 67/81 (82.72) a 67/84 (79.76) a
78.9-100 52.6-100 68.2-100
Similar superscripts indicate non-significant differences (P > 0.05 ). Table 2 Penetration rates of goat spermatozoa in cattle and sheep oocytes as influenced by the presence of cumulus cells Origin of Presence of Replicates Sperm penetration in oocytes oocytes cumulus (n) penetration rate Range
(%)
(%)
Cattle
+1 Pooled
4 4
37/50 40/45 77/95
Sheep
+
4 4
39/57 (68.42) a 41/52 (78.85) a 80/109 (73.39) a
Pooled
(74.00) az 54.5-100 (88.89) a 78.6-100 (81.05)" 55.6-100 50.0-100
IBetter sperm activity was seen after incubation in presence of cumulus cells. 2Similar superscripts indicate non-significant differences (P > 0.05 ).
57
sheep oocytes were incubated in suspensions of goat spermatozoa (Table 2). Results show that the heterologous sperm-egg interaction is not influenced by the presence of the cumulus in terms of penetration rate. Although not different in statistical terms ( P > 0 . 0 5 ) , the absence of cumulus cells tended to facilitate sperm penetration in both species, in spite of the better movement of sperm suspensions observed after incubation in presence of somatic cells.
4. Discussion
Results indicate that goat spermatozoa penetrate cattle and sheep matured follicular oocytes very efficiently compared with a homologous system. This observation confirms the earlier report of this laboratory (Cox et al., 1992), and may suggest that the reduced fertilization rate obtained by Slav~ and Fulka (1992), also using cattle and sheep follicular oocytes, could have been due to the low efficiency of the sperm capacitation system. It is thought that the main barrier against heterologous fertilization at oocyte level is the zona pellucida (O'Rand, 1988). The close phylogenetic relationship between domestic ruminants seems to facilitate the sperm-zona peilucida interaction between these species compared with less related species (Cox, 1992). The affinity of goat spermatozoa for cattle and sheep oocytes, measured in terms of fertilization rate, seems similar, confirming the observations of Slav~ and Fulka (1992). Although important advances in the understanding of the mechanisms underlying the zona pellucida induction of the acrosome reaction have been made in recent years in the mouse (Wassarman et al., 1989; Saling, 1989; Kopf et al., 1990), this event is still unclear in livestock ruminants. However, based on information generated from laboratory animals, it seems reasonable to expect that cattle, sheep and goat gametes share common molecules at the gamete recognition interface responsible for triggering the acrosome reaction. Lack of cumulus cell effect on improving fertilization rate of heterologous oocytes was an unexpected finding. We have shown previously that cumulus cells improved sperm penetration rate in heterologous fertilization systems using cattle and sheep gametes (Cox,
58
J.F. Cox et al. / Small Ruminant Research 15 (1994) 55-58
1991; Cox et ai., 1993). The viscous nature of the cumulus matrix would facilitate forward displacement of hyperactivated spermatozoa (Suarez et al., 1991). However, our results suggest that sperm-cumulus interaction is not as simple. Sperm penetration rate should be seen as an expression of the balance of stimulatory and inhibitory components of the cumulus to sperm advance towards the zona pellucida (Katz et al., 1989). In this study, cumulus cells were not able to modify the ability of goat spermatozoa to penetrate in cattle and sheep oocytes, in spite of keeping sperm alive for longer periods; they tended to reduce the fertilization ef~ciency. In conclusion, goat spermatozoa were able to penetrate cattle and sheep follicular oocytes equally well. The presence of the cumulus did not modify the efficiency of this sperm-egg interaction. At present, we are using this information to develop heterologous fertilization systems to overcome the reduced availability of goat oocytes.
Acknowledgments The authors thank Dr. M. Cox and Mrs. K Jackson for the critical review of the manuscript. The study was funded by Fondecyt (Grant 91-318).
References Cheng, W.T.K., 1985. In vitro fertilization in farm animals. PhD Thesis, University of Cambridge, UK. Cox, J.F., 1991. Effect of the cumulus on heterologous fertilization using ruminant gametes. XXIV World Veterinary Congress. Rio de Janeiro, Brazil. p. 2.27.
Cox, J.F., 1992. Heterologous fertilization using livestock gametes. Arch. Med. Vet., 24: 25-31. Cox, J.F., Saravia, F., Avila, J., Catalfm, A. and Santa Maria, A., 1992. Heterologous fertilization of intact sheep and cattle oocytes by goat spermatozoa. Proc. XVII Ann. Meeting of Chilean Society of Animal Production, Chillfm, October 20-22nd. Ref. 63. Cox, J.F., Hormaz~tbal, J. and Santa Mafia, A., 1993. Effect of the cumulus on in vitro fertilization of bovine matured oocytes. Theriogenology, in press. Gordon, I. and Lu, K.H., 1989. Production of embryos in vitro and its impact on livestock production. Theriogenology, 33: 77-87. Katz, D.F., Drobnis, E.Z. and Overstreet, J.W., 1989. Factors regulating mammalian sperm migration through the female reproductive tract and oocyte vestments. Gamete Res., 22: 443--469, Kopf, G.S., Noland, T.D., Glassner, M. and Wilde, M.W., 1990. The zona pellucida-induced acrosome reaction: a model for sperm signal transduction. In: B.D. Bavister, J. Cummins and E.R.S. Rold~tn (Eds.), Fertilization in Mammals. Serono Symposia, Newton, MA. pp. 253-266. O'Rand, M.G. 1988. Sperm recognition and barriers to inter-species fertilization. Gamete Res., 19: 315-328. Parrish, J.J., Susko-Parrish, J., Weiner, M.A. and First, N.L., 1988. Capacitation of bovine sperm by heparin. Biol. Reprod., 38: 145153. Saling, P., 1989. Mammalian sperm interaction with extracellular matrices of the egg. Oxford Rev. Reprod. Biol., 11: 339-388. Slav~, T. and Fulka, J., 1992. In vitro fertilization of intact sheep and cattle oocytes with goat spermatozoa. Theriogenology, 38: 721-726. Slav~, T., Pavlok, A. and Fulka, J., 1990. Penetration of intact bovine ova with ram sperm in vitro. Molec. Reprod. Dev., 25: 345-347. Suarez, S.S., Katz, D.F., Owen, D.H.. Andrew, J.B. and Powell, R.L., 1991. Evidence for the function of hyperactivated motility in sperm. Biol Reprod., 44:375-381. Wassarman, P., Bleil, J., Fimiani, C., Florman, H., Greve, J., Kinloch, R,, Moiler, C., Mortillo, S., Roller, R., Salzmann, G. and Vasquez, M., 1989. The mouse-egg receptor for sperm: a multifunctional zona pellucida glycoprotein. In: J. Dietl (Ed.), The Mammalian Egg Coat. Springer Verlag, Berlin, pp. 18-37. Yanagimachi, R., 1988. Mammalian fertilization. In: E. Knobil et al. (Eds.), The Physiology of Reproduction. Raven Press, New York, pp. 135-185.
Small Ruminant Research 15 (1994) 55-58
In vitro fertilization of cattle and sheep follicular oocytes by goat spermatozoa J.F. Cox*, A. Catal~in, F. Saravia, J. Avila, A. Santa Maria Department of Animal Production, Facultyof Veterinary Medicine Universityof Concepci6n, Casilla537, Chill6n, Chile Accepted 25 December 1993
Abstract
The study compared efficiency of goat spermatozoa to penetrate cattle and sheep intact follicular oocytes, and evaluated effect of cumulus cells on the ability of sperm to penetrate heterologous oocytes. Ruminant cumulus-oocyte complexes (COC) were obtained by aspiration of ovarian follicles and were matured by culturing them in medium 199 plus goat serum in microdrops under paraffin oil for 24 h. Fresh semen was collected by artificial vagina, incubated for 4 h at room temperature, washed, and spermatozoa were then suspended in Talp medium plus goat serum and treated for capacitation by using incubation at 39°C and heparin. In the first experiment, goat, cattle and sheep matured oocytes were incubated in goat sperm preparation, whereas in the second experiment, cattle and sheep oocytes either partially (cumulus enclosed oocytes = + ) or totally denuded from granulosa cells (denuded oocytes = - ) were added to the prepared spermatozoa. Fertilization rates obtained in the first experiment (goat sperm-goat oocytes: 91.3%; goat-cattle: 82.7%; and goat-sheep: 79.8%, P > 0.05), showed that goat sperm were highly efficient in penetrating cattle and sheep oocytes as compared with homologous fertilization, and that goat sperm penetrated equally well cattle and sheep oocytes. Fertilization rates from the second experiment (cattle oocytes: + 74.0%, -88.89%; sheep oocytes: + 68.42%, -78.85%, P > 0.05), showed that cumulus cells did not modify the ability of goat spermatozoa to penetrate heterologous oocytes. Keywords: Heterologous fertilization; Cumulus cell; Fertilization, in vivo; Goat; Fertilization, in vitro
1. Introduction Fertilization is an event whose species specificity is controlled at several stages, including mating behaviour, sperm transport and sperm--egg interaction (Yanagimachi, 1988). It is thought that the barrier which limits heterologous gamete interaction at the oocyte level is the zona pellucida (O'Rand, 1988). Depending on the species, zonae pellucidae may modulate binding, *Corresponding author. 0921-4488/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved
SSD10921-4488(94)00003-P
acrosome reaction induction and/or zona penetration by foreign spermatozoa (Cox, 1992). However, the phylogenetic relationship between gametes may reduce the efficiency of the zona pellucida to act as a barrier. Cox (1992) showed that bull spermatozoa were able to penetrate sheep oocytes but unable to penetrate pig or mouse oocytes. On the other hand, S l a v ~ et al. (1990) and Cox (1992) showed that ram spermatozoa easily penetrated cattle oocytes. It is unclear why the zona pellucida is unable to recognize spermatozoa of related species but the sharing of common molecules
56
J. F. Cox et al. /Small RuminantResearch 15 (1994) 55-58
at the sperm zona interface may be a possibility (O'Rand, 1988). Heterologous fertilization would not only contribute to elucidate mechanisms related to fertilization and embryo development, but also would facilitate the development of procedures for semen evaluation. By using in vitro fertilization techniques, Cox et al. (1992) and Slavik and Fulka (1992) have shown that goat spermatozoa may also penetrate cattle and sheep follicular oocytes. The aims of the present study were (1) to complete these earlier reports by having a homologous system as a reference, and (2) to evaluate the effect of the cumulus on heterologous fertilization using goat spermatozoa.
2. Materials and methods
2.1. Oocyte collection and culture
Ovaries were obtained from cows, ewes and does killed at a local abattoir and were transported at 3035°C in insulated containers to the laboratory within 2 h. Cumulus-oocyte complexes (COC) were collected by aspiration from follicles between 2-5 nun in diameter with an 18-gauge needle attached to a 10-ml syringe. Oocytes with compact cumulus and evenly granulated cytoplasm were cultured in 100/xl drops of culture medium under paraffin oil (Sigma Chemical Co., St. Louis, MO, USA) in 60-mm plastic dish (Falcon, Becton Dickinson, Lincoln Park, NJ, USA). Maturation medium (Hepes-199) contained TCM 199 (with Earle's salts, NaHCO3, L-glutamine and 10 mM Hepes; pH 7.4; Sigma Chemical Co.), plus 20% v/v heat-inactivated oestrus goat serum (OGS) and 20/xg/ ml of insulin (Sigma Chemical Co. ). 10-20 COC were cultured per drop for 24-26 h at 39°C, 5% CO2 in air, in a static system under a humidified atmosphere (Gordon and Lu, 1990). 2.2. Sperm preparation
Semen from four Anglo Nubian bucks was collected by artificial vagina, washed twice (300×g, 5 min) with Dulbecco's PBS and incubated at about 20°C for 3-4 h (Cheng, 1985). After one more wash, spermatozoa were resuspended in TALP IVF (Parrish et al.,
1988) plus 10% v/v OGS and 2.5/zg/ml of heparin (Sigma Chemical Co.; pH 7.5) at a concentration of 108 sperm/ml and incubated at 39°C and 5% CO2 in air for 40 min. Then, the sperm suspension was further diluted to 10 6 sperm/ml using TALP IVF (plus serum and heparin) and 50-/zi microdrops under paraffin oil were prepared and then incubated at 39°C and 5% CO2 in air until incubation with the eggs (usually about 20 min). 2.3. In vitro fertilization
Only oocytes with clear and well expanded cumulus cells were used in these experiments. After maturation, oocytes were partially (cumulus enclosed) or completely denuded from granulosa cells by passing them in and out of a pulled-glass pipette. The oocytes were washed in Herpes-199 supplemented with 0.4 mg/ml BSA (Fraction V, Sigma Chemical Co.) instead of OGS, and incubated in the already prepared sperm suspensions (five to eight oocytes/drop) at 39°C and 5% CO2 in air. In experiment No. 1, cattle, sheep and goat cumulus-enclosed oocytes were allocated at random to goat sperm preparations and left to interact for 16 h. In experiment No. 2, denuded and cumulus-enclosed cattle and sheep oocytes were incubated in sperm suspensions and left for fertilization to occur. 2.4. Evaluation o f sperm penetration
Sperm penetration in the cytoplasm was evaluated under phase-contrast microscopy by fixing wholemounted completely denuded oocytes in a solution of aceto-alcohol (1:3, v/v) and staining them with a 1% solution of aceto-lacmoid. Only oocytes with pronuclear development and sperm tail(s) were considered to be penetrated. 2.5. Statistical analysis
The analysis of the efficiency of sperm penetration was done by calculating the fertilization rate of each treatment, and comparing them using either an ANOVA and Newman-Keuls test or by the Student's t-test with arc sin-transformed data, using the Statview ® program. A P < 0.05 was considered to be significant.
J.F. Cox et al. / Small Ruminant Research 15 (1994) 55-58
3. Results
The efficiency of sperm penetration in foreign oocytes was evaluated using homologous penetration as a point of reference. Germinal-vesicle staged and ruptured oocytes were not considered (less than 5%) (Table 1). Sperm concentrations used here were not adjusted to establish differences between homologous and heterologous sperm penetration, but to establish differences between foreign oocytes in their ability to interact with goat spermatozoa. The results obtained showed that goat spermatozoa penetrated cattle and sheep oocytes equally well, with similar ranges between replicates. To evaluate whether cumulus cells would modify the ability of goat spermatozoa to penetrate foreign oocytes, denuded and cumulus-enclosed cattle and Table 1 Fertilization rate of goat, cattle and sheep matured oocytes using goat spermatozoa capacitated in vitro Gamete origin
Replicates
Sperm penetration in oocytes
(n) sperm
Goat Goat Goat
oocytes
goat cattle sheep
4 4 4
penetration rate
range
(%)
(%)
84/92 (91.30) ~j 67/81 (82.72) a 67/84 (79.76) a
78.9-100 52.6-100 68.2-100
Similar superscripts indicate non-significant differences (P > 0.05 ). Table 2 Penetration rates of goat spermatozoa in cattle and sheep oocytes as influenced by the presence of cumulus cells Origin of Presence of Replicates Sperm penetration in oocytes oocytes cumulus (n) penetration rate Range
(%)
(%)
Cattle
+1 Pooled
4 4
37/50 40/45 77/95
Sheep
+
4 4
39/57 (68.42) a 41/52 (78.85) a 80/109 (73.39) a
Pooled
(74.00) az 54.5-100 (88.89) a 78.6-100 (81.05)" 55.6-100 50.0-100
IBetter sperm activity was seen after incubation in presence of cumulus cells. 2Similar superscripts indicate non-significant differences (P > 0.05 ).
57
sheep oocytes were incubated in suspensions of goat spermatozoa (Table 2). Results show that the heterologous sperm-egg interaction is not influenced by the presence of the cumulus in terms of penetration rate. Although not different in statistical terms ( P > 0 . 0 5 ) , the absence of cumulus cells tended to facilitate sperm penetration in both species, in spite of the better movement of sperm suspensions observed after incubation in presence of somatic cells.
4. Discussion
Results indicate that goat spermatozoa penetrate cattle and sheep matured follicular oocytes very efficiently compared with a homologous system. This observation confirms the earlier report of this laboratory (Cox et al., 1992), and may suggest that the reduced fertilization rate obtained by Slav~ and Fulka (1992), also using cattle and sheep follicular oocytes, could have been due to the low efficiency of the sperm capacitation system. It is thought that the main barrier against heterologous fertilization at oocyte level is the zona pellucida (O'Rand, 1988). The close phylogenetic relationship between domestic ruminants seems to facilitate the sperm-zona peilucida interaction between these species compared with less related species (Cox, 1992). The affinity of goat spermatozoa for cattle and sheep oocytes, measured in terms of fertilization rate, seems similar, confirming the observations of Slav~ and Fulka (1992). Although important advances in the understanding of the mechanisms underlying the zona pellucida induction of the acrosome reaction have been made in recent years in the mouse (Wassarman et al., 1989; Saling, 1989; Kopf et al., 1990), this event is still unclear in livestock ruminants. However, based on information generated from laboratory animals, it seems reasonable to expect that cattle, sheep and goat gametes share common molecules at the gamete recognition interface responsible for triggering the acrosome reaction. Lack of cumulus cell effect on improving fertilization rate of heterologous oocytes was an unexpected finding. We have shown previously that cumulus cells improved sperm penetration rate in heterologous fertilization systems using cattle and sheep gametes (Cox,
58
J.F. Cox et al. / Small Ruminant Research 15 (1994) 55-58
1991; Cox et ai., 1993). The viscous nature of the cumulus matrix would facilitate forward displacement of hyperactivated spermatozoa (Suarez et al., 1991). However, our results suggest that sperm-cumulus interaction is not as simple. Sperm penetration rate should be seen as an expression of the balance of stimulatory and inhibitory components of the cumulus to sperm advance towards the zona pellucida (Katz et al., 1989). In this study, cumulus cells were not able to modify the ability of goat spermatozoa to penetrate in cattle and sheep oocytes, in spite of keeping sperm alive for longer periods; they tended to reduce the fertilization ef~ciency. In conclusion, goat spermatozoa were able to penetrate cattle and sheep follicular oocytes equally well. The presence of the cumulus did not modify the efficiency of this sperm-egg interaction. At present, we are using this information to develop heterologous fertilization systems to overcome the reduced availability of goat oocytes.
Acknowledgments The authors thank Dr. M. Cox and Mrs. K Jackson for the critical review of the manuscript. The study was funded by Fondecyt (Grant 91-318).
References Cheng, W.T.K., 1985. In vitro fertilization in farm animals. PhD Thesis, University of Cambridge, UK. Cox, J.F., 1991. Effect of the cumulus on heterologous fertilization using ruminant gametes. XXIV World Veterinary Congress. Rio de Janeiro, Brazil. p. 2.27.
Cox, J.F., 1992. Heterologous fertilization using livestock gametes. Arch. Med. Vet., 24: 25-31. Cox, J.F., Saravia, F., Avila, J., Catalfm, A. and Santa Maria, A., 1992. Heterologous fertilization of intact sheep and cattle oocytes by goat spermatozoa. Proc. XVII Ann. Meeting of Chilean Society of Animal Production, Chillfm, October 20-22nd. Ref. 63. Cox, J.F., Hormaz~tbal, J. and Santa Mafia, A., 1993. Effect of the cumulus on in vitro fertilization of bovine matured oocytes. Theriogenology, in press. Gordon, I. and Lu, K.H., 1989. Production of embryos in vitro and its impact on livestock production. Theriogenology, 33: 77-87. Katz, D.F., Drobnis, E.Z. and Overstreet, J.W., 1989. Factors regulating mammalian sperm migration through the female reproductive tract and oocyte vestments. Gamete Res., 22: 443--469, Kopf, G.S., Noland, T.D., Glassner, M. and Wilde, M.W., 1990. The zona pellucida-induced acrosome reaction: a model for sperm signal transduction. In: B.D. Bavister, J. Cummins and E.R.S. Rold~tn (Eds.), Fertilization in Mammals. Serono Symposia, Newton, MA. pp. 253-266. O'Rand, M.G. 1988. Sperm recognition and barriers to inter-species fertilization. Gamete Res., 19: 315-328. Parrish, J.J., Susko-Parrish, J., Weiner, M.A. and First, N.L., 1988. Capacitation of bovine sperm by heparin. Biol. Reprod., 38: 145153. Saling, P., 1989. Mammalian sperm interaction with extracellular matrices of the egg. Oxford Rev. Reprod. Biol., 11: 339-388. Slav~, T. and Fulka, J., 1992. In vitro fertilization of intact sheep and cattle oocytes with goat spermatozoa. Theriogenology, 38: 721-726. Slav~, T., Pavlok, A. and Fulka, J., 1990. Penetration of intact bovine ova with ram sperm in vitro. Molec. Reprod. Dev., 25: 345-347. Suarez, S.S., Katz, D.F., Owen, D.H.. Andrew, J.B. and Powell, R.L., 1991. Evidence for the function of hyperactivated motility in sperm. Biol Reprod., 44:375-381. Wassarman, P., Bleil, J., Fimiani, C., Florman, H., Greve, J., Kinloch, R,, Moiler, C., Mortillo, S., Roller, R., Salzmann, G. and Vasquez, M., 1989. The mouse-egg receptor for sperm: a multifunctional zona pellucida glycoprotein. In: J. Dietl (Ed.), The Mammalian Egg Coat. Springer Verlag, Berlin, pp. 18-37. Yanagimachi, R., 1988. Mammalian fertilization. In: E. Knobil et al. (Eds.), The Physiology of Reproduction. Raven Press, New York, pp. 135-185.