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Effect of oocyte-sperm co-incubation on acrosome reaction in the goat
ELSEVIER
EFFECT OF OOCYTE-SPERM CO-INCUBATION ON ACROSOME REACTION IN THE GOAT
E. Martin-Lunasl, A. Martino*, M.T. Paramio*, M.J.Palomo*, M.T. Mogas*, M.A. Bielsa3, P. Andolz3 and P. Martinezl ,a
1 lnstituto de Biologia Fundamental and Departamento de Biologia Celular Universidad Autonoma de Barcelona. 08193 Bellaterra, Spain * Departamento de Patologia y Production Animal Universidad Autonoma de Barcelona 3 Laboratorio de lnvestigaciones Seminologicas Barcelona, Spain
Received for publication: Accepted:
~~~~ 13, 1995 February
15,
1996
ABSTRACT The induction of acrosome reaction of goat spermatozoa was investigated. The acrosomal status of spermatozoa was determined by a triple-staining technique. The effect of the presence of goat oocytes on the proportion of acrosome-reacted Ovulated oocytes were obtained from spermatozoa was also determined. superstimulated adult goats. Other sources of oocytes were adult and prepubertal goats; oocytes from both sources were maturated in vitro. There was an increase in the percentage of acrosome-reacted spermatozoa from 4%f 0,98 to 9%+ 1,41 when oocytes from adult females were used. Similar induction rates were measured with prepubertal and adult oocytes maturated in vitro (10,4%f 2.06 and 8.75%+ 1.06, respectively). The influence of several qualities of cumulus oophorus as well as the presence of zona pellucida was also investigated. No significant differences were obtained with any cumulus oophorus or zona pellucida oocyte complexes. Although oocyte quality is important for high fertilization rates, it does not seem to be crucial for the induction of acrosome reaction. Key words: goat spermatozoa, acrosome reaction, sperm-oocyte interaction
Acknowledgments This work was supported by the Fondo de lnvestigaciones (FIS 91/0293). acorrespondence and reprint requests.
Theriogenology 46:321-330, 1996 0 1996 by Elsevier Science Inc
Sanitarias
0093-691X/96/$15.00 PII SOO93-691X(96)00186-7
Theriogenology
322 INTRODUCTION
The acrosome reaction of mammalian spermatozoa is a prerequisite for sperm penetration of the zona pellucida and fusion with the oocyte plasma membrane. It involves fusion and progressive vesiculation between the outer acrosomal membrane and the overlying plasma membrane, with the subsequent release of acrosomal enzymes. Since the acrosome reaction is an essential event in fertilization, information about the acrosomal status is important for semen quality control in artificial insemination and fertilization trials. The production of IVF embryos in domestic animals is important because of the production or transgenic animals that secrete substances of interest. In goats, the interest of the mammary gland secretions has promoted research on in vitro maturation of goat oocytes for in vitro fertilization (15). Several authors have demonstrated that the presence of the cumulus oophorus or components of the extracellular matrix are capable of inducing acrosome reaction in vitro in human (6,19,23), hamster (1) and bovine spermatozoa (13). It is also well known that human follicular fluid components induce acrosome reaction (19,20). Since sperm acrosome reaction is induced near the site of fertilization, oocyte layers, which include the matrix of the cumulus oophorus and the zona pellucida, may promote acrosome reaction (4,10,21). The oocyte-cumulus complex (COC) has been studied for its ability to induce acrosome reaction in human spermatozoa. Several studies have implicated both the cumulus mass (19,21) and the zona pellucida (9), in human sperm acrosome reaction, but little information has been published on induced acrosome reaction in goats. The only references that have been reported in ejaculated goat spermatozoa by Kusunoki et al. (11,12) have shown that acrosome reaction may be induced by in vitro incubation in the presence of uterine fluid. These authors (12) reported a 16% rate of acrosome-reacted cells in samples incubated for 5 h in the presence of rat uterine fluid. Several techniques have been described for the evaluation of the acrosome reaction of mammalian spermatozoa; however, most of them do not differentiate between true acrosome reaction (live spermatoa) and degenerative acrosome reaction (dead spermatozoa). Some of the new methods use specific reagents that bind to the inner acrosomal membrane: fluorescein-isothiocyanate concanavalin A (8) or monoclonal antibodies reacting with internal antigens (2) without distinction of true or degenerative acrosome reaction. The combination with the viability stain Hoechst 33258 allows for distinguishing between true acrosome reaction and degenerative acrosomal loss. An earlier study (5) describes the acrosomal state from viable spermatozoa using fluorescent dye in combination with phase contrast and fluorescence microscopy. Simultaneous determination of acrosomal status and viability of spermatozoa allows for the determination of the true acrosome reaction. Kligman et al. (10) defined several intermediate steps in the mouse zona pellucidainduced acrosome reaction by using the fluorescent probe chlortetracycline (CTC). We have used the triple-stain tecnique of Talbot and Chacon (22), because it is an unexpensive and easy method that allows for the evaluation of the true acrosome reaction of viable cells.
323
73etiogenology
We investigated whether or not cells from the cumulus oophorus or the zona pellucida of goat oocytes can promote acrosome reaction in goat spermatozoa. Previous experiments obtained in our group have shown low rates of in vitro fertilization in the goat. Thus, we decided to determine if the quality of the oocyte envelopes had some effect on the induction of acrosome reaction. To that end, we adapted the triple-stain technique, originally developed for human spermatozoa, to goat spermatozoa, to differentiate between spermatozoa that had undergone the acrosome reaction and the dead spermatozoa with degenerative acrosome reactions.
MATERIALS AND METHODS Semen Collection
and Sperm Capacitation.
Semen was collected by artificial vagina from males. Sperm concentration was determined by a hemocytometer. The ejaculates were processed according to a method described previously (7). The method consisted of diluting the ejaculates in 8 ml of defined medium with Hepes (DMH), and centrifuging at 200x9 for 10 min. The DMH consisted of 129.5 mM NaCI, 4.16 mM NaHCOsand 10 mM Hepes; the pH was adjusted to 7.0. After removal of the supernatant, 70 pl of the pellet was deposited at the bottom of a conical tube with 2 ml of 20% estrous sheep serum (ESS) in DMH, pH 7.3. After 1.5 hour at 38.5”C, the most motile spermatozoa were recovered from the upper part of the tube. The spermatozoa were dlluted in the same medium to a concentration of 10 x 10s sperm/ml and incubated for 4 h at 385°C; the sperm suspension was diluted a 1 x 10s sperm/ml in the fertilization medium, prepared with 20% FSS, 20mM calcium lactate (lactic acid L+; Sigma Chemicals, St. Louis, MO, USA) in DMH, pH 7.7. Oocyte Collection and Maturation. To obtain ovulated oocytes adult goats were treated for 11 d with intravaginal sponges containing 45 mg of fluorogestone acetate (FGA; Chrono-Gest, Intervet, Salamanca, Spain). Then, 24 h before sponge removal, the goat received lml PG F2a (Dynolitic, Upjohn, Sant Cugat, Spain). The goats received a superovulation treatment of 16 mg of p-FSH (Ovaset, Sanofi, Lyon, France) in 6 injections every 12 h beginning 48 h before sponge removal. Oocytes were obtained by flushing the oviducts 57 h after sponge removal. lnmature oocytes were obtained after dissection of follicles of ovaries obtained from a local slaughterhouse or from diestrous goats treated with 7 mg FSH given in decreasing doses in 3 injections every 12 h . A second source of oocytes was obtained after dissection of ovarian follicles from prepubertal female goats from local slaughterhouse. (14). For in vitro maturation, oocytes were cultured 27 h in Medium 199 supplemented with 10% fetal calf serum (FCS), 10 @ml FSH, 10 us/ml LH, and 1 fug/ml of 17 B-estradiol at 385°C.
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Incubation of Spermatozoa with Oocytes After 4 h of capacitation, samples of 1 ml of spermatozoa (final concentration 1xl 06 sperm/ml) were co-incubated with 5 to 10 oocytes for 17 h at 385X. In Experiment 1 (n = 3) oocytes were obtained from several sources: a) ovulated oocytes, b) adult oocytes maturated in vitro and c) prepubertal oocytes maturated in vitro. A control was included in the 3 replicates: spermatozoa without oocytes, spermatozoa under conditions similar to these for a spontaneous acrosome reaction assessment. We evaluated the percentage of true acrosome reacted spermatozoa at 21 h of maturation. Prepubertal oocytes were used in Experiment 2. Before co-incubation, the oocytes were classified as Type I or II with respect of their cumulus oophorus (Type I: 3 or more complete layers of cumulus cells and Type II: 1 or 2 complete layers); sperm concentration was the same as that described above. Experiment 2 was performed with prepubertal oocytes (Type I or II): a) surrounded by a cumulus oophorus (n = 2) and b) denuded (without cumulus oophorus) (n=4). After culture, the oocytes were digested by a short hyaluronidase treatment and pipetting and then they were coincubated with spermatozoa. After 21 h, the acrosome-reacted spermatozoa were examined. Acrosome Staining Method To evaluate the acrosome reaction, the triple-stain technique was used. Using this method the spermatozoa were classified into 4 patterns, with differentiation between true acrosome reaction (live reacted spermatozoa) and total acrosome reaction (live and dead reacted spermatozoa). Briefly, a 200 1.11 aliquot of the sperm suspension co-incubated with or without oocytes was placed into a plastic tube; an equal volume of 2% trypan blue in albumin-free medium was added. The tube was then incubated for 15 min in a 38.5”C water bath. After incubation, the spermatozoa were diluted with 2 ml of albumin-free fertilization medium and then centrifuged at 200xg for 5 min. The supernatant was removed, and the sperm pellet was resuspended in 2 ml of albumin-free fertilization medium and centrifuged as before. Spermatozoa were resuspended in 200 ul of 3% glutaraldehyde in O.lM cacodilate buffer, pH 7.4, and kept for 1 h at 4 X. The spermatozoa were washed twice, and an aliquot of the sperm suspension was placed on a slide and smeared with a second glass slide. The smears were air-dried at room temperature. Spermatozoa on the slides were stained for 50 min with 0.8% brown bismark in distilled water at pH of 1.8. The slides were stained with 0.8% rose bengal in 0.1 M Tris buffer, pH 5.3, for 30 min. A coverslip was mounted, and the spermatozoa were examined with bright field microscopy at x1000. At least 400 spermatozoa were classified according to presence of 4 patterns: 1) light brown postacrosomal regions with pink acrosomes; live spermatozoa with nonreacted acrosome; 2) light brown postacrosomal regions with unstained acrosomal regions; live spermatozoa with a reacted acrosome; 3) dark brown postacrosomal regions with dark pink acrosomes; dead sperm cells with a nonreacted acrosome; 4) dark brown postacrosomal regions with unstained dark blue acrosomal regions; dead sperm cells with a reacted acrosome. These patterns are shown in Figure 1.
Theriogenology
Figure
1. Four patterns spermatozoa with acrosome; c) dead spermatozoa with
325
obtained by the triple-stain method (x1000): a) live non-reacted acrosome; b) live spermatozoa with reacted spermatozoa with a non-reacted acrosome and d) dead reacted acrosome
Theriogenology
326
Statistical Analysis Data were analyzed by Analysis of Variance; comparison using the Student-Newman-Keuls’ Analysis test.
of means was tested
RESULTS Figure 2 shows the percentage of control values and standard deviation (k SD) of live goat spermatozoa undergoing spontaneous acrosome reaction at 0, 4 and 21 h of incubation in DM-H medium supplemented with 20% ESS. Between 0 and 4 h no significant difference was observed, but after 21 h acrosome reaction increased twofold over the initial value (P < 0,001). There was a clear dependence of acrosome reaction percentage on time. In Experiment 1, several sources of goat oocytes were used for the sperm-oocyte co-incubation. The acrosome-reacted control sperm show a small but significant rise from 4 to 21 h. The acrosome reaction observed in presence of adult goat oocytes maturated in vitro was not significantly different from that induced by ovulated oocytes or prepubertal oocytes maturated in vitro at 21 h; but, these induced acrosome reactions were significantly different compared with spontaneous acrosome reaction in the DM-H medium without oocytes (P < O.OOOl), as shown in Figure 2. Thus, very similar responses were observed in samples co-incubated with oocytes from different sources. Experiment 2 was designed to elucidate whether the presence/absence of the cumulus oophorus in prepubertal oocytes maturated in vitro would induce acrosome reaction differently. The cumulus oophorus was classified as Type I or Type II. No differences were detected in the induced acrosome reaction rates when the oocytes were co-incubated with or without the cumulus oophorus. Similarly, Type I or Type II oocytes had no effect on sperm acrosome reaction. These results suggest that the quality or source of oocyte has no influence on acrosome reaction. We evaluated both the true and the total acrosome reaction percentages as it is shown in Table 1. The total acrosome reaction is about 3-4 times that of the true acrosome reaction.
Table 1. Percentages obtained when true acrosome reaction (left) or total (true and degenerative acrosome reaction) (right) are evaluated in experiment. (C.O.= cumulus oophorus). Total Acrosome Reaction (%) True Acrosome Reaction (%)
Oocytes
Type I
with C.O. without C.O.
Type II
with C.O. without C.O.
35.58 f 1.6 37.19 f 8.85
11.16f0.8 10.51 f 2.06
28.3 + 1.6
10.3 f 2.5
35.86 + 9.54
10.25 k 1.84
Theriogenology
327
Time I33 Spermatozoa 0 Spermatozoa H Spermatozoa q Spermatozoa
in DM-H/ESS coincubated coincubated coincubated
(hours) medium with adult goat oocytes maturated in vitro with ovulated oocytes with prepubertal oocytes
Figure 2. Mean &SD) percentage of spermatozoa showing the acrosome reaction when incubated for 17 h in the absence or presence or oocytes after 4 h of capacitation.
DISCUSSION It is well known that the maturational status of the oocyte-cumulus complex can affect the success of in vitro fertilization, but its relationship to the acrosome reaction is not well defined. The induction of spermatozoa by several oocyte-associated factors has been extensively discussed; but very little is known about the induction of goat sperm acrosome reaction. The results of this study show the capability of both mature and immature oocytes to induce the acrosome reaction. The incubation of goat spermatozoa in the presence of goat oocytes significantly increases the proportion of spermatozoa undergoing the acrosome reaction. Acrosome reaction is initiated in vivo in the envelope surrounding the oocyte, namely the zona pellucida, before fusion of oocyte-sperm plasma membranes. There are probably agents that induce acrosome reaction near the oocyte or in the glycoprotein stucture of the zona. Studies on induction of acrosome reaction should be evaluated on viable live spermatozoa (true acrosome reaction), because this reflects the physiological acrosomal loss. But, many authors use techniques that evaluate the biochemical pannel of molecules in membranes of acrosome-reacted spermatozoa, rather than measuring the funcionality of spermatozoa (total acrosome reaction); therefore the information given by a variety of methodologies is different. One of the most extensive staininig techniques for the identification of reacted cells has been the triple-stain technique originally described by Talbot and Chacon (22). The 4 patterns
328
Theriogenology
described for the triple-stain must show high intensity colors that can be easily used to classify the true acrosome-reacted cells (white acrosome, light brown postacrosome) from the remaining cells. The use of the triple staining technique allows us to have both the true and the total acrosome reaction and to compare our data to the results obtained by other authors. Cross et al. (4) reported an increase in acrosome-reaction in human spermatozoa when incubated with intact zona pellucida oocytes and with disaggregated zonae pellucida. The acrosome reaction may be stimulated in vitro by human follicular fluid as previously reported (20). Mot-timer et al. (17) found that follicular fluid promotes human acrosome reaction in a concentration-dependent manner. These authors, detected up to 11 .l% live reacted cells by triple-staining when 6 h precapacitated spermatozoa were incubated for 2 h in a medium containing 100% of follicular fluid. Similar acrosome reaction rate were obtained in human spermatozoa (10.6%) in a Hepes-buffered BWW medium after a long incubation period (20 h), as assessed by the triple-stain technique; however, a shorter incubation period (3 h) yielded only 6.1% acrosome-reacted spermatozoa (18). When total acrosome reaction was evaluated in our experiments (dead + live sperm cells) the mean value obtained for the co-incubated capacitated sperm-oocyte was 35 to 37%. Another study (4) found a higher induced acrosome reaction percentage with human spermatozoa: 46%&l 5 by indirect immunofluorescence with fluoresceinated Pisum sativum agglutinin (FITC-PSA) which determines total acrosome reaction. Hoshi et al. (9) investigated the induction of human acrosome reaction by the zona pellucida and the cumulus oophorus, as evaluated by FITC-PSA. Their results showed that the zona pellucida induces acrosome reaction (35.7 versus 2.8%) but the cumulus oophorus is unable to promote acrosome reaction. Our results show that live spontaneous acrosome reaction was 2.0% f 1.2 after 4 h of incubation, Kusunoki (11) reports similar percentages, evaluated by the triple stain technique, for spontaneous acrosome reaction (1.8 %) in goat spermatozoa after 5 h of incubation. The results of our present study demonstrate that goat oocytes can significantly stimulate acrosome reaction. The source of goat oocytes (adult oocytes maturated in vitro, ovulated oocytes or prepubertal oocytes) did not influence the induction of acrosome reaction, which ranged from 8.7 to 10.5 %. Kusunoki found 16% acrosome-reacted spermatozoa when acrosome reaction was stimulated by coincubation with isolated rat uterine cells. This rate of acrosome reaction was slightly higher than in our study (16% versus lo%), but these differences may be due to the use of different inducers. Although acrosome reaction evaluation by the triple-stain technique is subjective if the parameters are not universally established, the ability of oocyte layers to induce acrosome reaction could be deduced by comparing rates with those of spontaneous acrosome reaction. Thus, Sullivan et al. (21) report high values for acrosome reaction induction in human spermatozoa by cumulus cells: 46% acrosome reaction for live acrosome-reacted spermatozoa, versus 15% of sperm noninduced acrosome reaction in the control. The increase in acrosome reaction induction was 68.8%. In our study, the average acrosome reaction increase was 57.5%.
Theriogenology
329
There were no differences in acrosome reaction rates for the co-incubation of spermatozoa with 3 sources of oocytes used in this study. The zona pellucida of all oocytes has the necessary structures for inducing acrosome reaction. Since the best fertilization rate was obtained with Type I oocytes (results not shown; 15), we tried to investigate whether the zona pellucida from these oocytes could stimulate acrosome reaction better. Two Types of oocytes were used for this purpose (Type I and Type II). Our results are similar to those of Sullivan et al. (21), who investigated how the maturational status or quality of the cumulus has no influence on the percentage of live human acrosome-reacted spermatozoa. Thus, we can conclude that, although the Type of oocyte affects viability in fertilization, the induction of acrosome reaction is not dependent on the quality of the oocyte cumulus. As has been reported (16), both acrosome-reacted and acrosome-intact spermatozoa can be found close to the zona pellucida. Thus, perhaps the acrosomereacted spermatozoa are stimulated by the cumulus oophorus and the acrosomeintact cells may be induced to react by the zona pellucida. Our results indicate that the presence of stimulatory molecules from the oocyte-cumulus complex or oocyte zona pellucida increases spontaneous acrosome reaction and may represent a mechanism for maximizing the fertilizing ability of spermatozoa. High quality semen is essential for a successful artificial insemination program (3). Assessment of semen should include parameters such as volume, number of sperm cells, morphology, and acrosomal status and function. The percentage of acrosome-reacted cells is indicative of sperm fertilizing ability and may be an additional assessment of semen quality to be used in in vitro fertilization of goats, to predict the success of fertilization.
REFERENCES 1. Bavister BD. Evidence for a role of postovulatory cumulus components in supporting fertilizing ability of hamster spermatozoa. J Androl 1982; 3: 365-372. 2. Braun J, Hirsch T, Krause W, Ziegler A. Evaluation of the acrosome reaction using monoclonal antibodies against different acrosomal antigens-comparison with the triple-stain technique. Int J Androl 1991; 14: 431-436. 3. Chandler JE, Painter CL, Adkinson RW, Memon MA, Hoyt PG. Semen quality characteristics of dairy goats. J Dairy Sci 1988; 71: 1638-l 646. 4. Cross NL, Morales P, Overstreet JW, Hanson FW. Induction of acrosome reaction by the human zona pellucida. Biol Reprod 1988; 38: 235-244. 5. De Leeuw AM, Den Daas JHG, Woelders H. The fix vital stain method. Simultaneous determination of viability and acrosomal status of bovine spermatozoa. J Androl 1991; 12: 112-l 18. 6. DeJonge Ch, Rawlins RG, Zaneveld LJD. Induction of the human sperm acrosome reaction by human oocytes. Fertil Steril 1988; 50: 949-953. 7. De Smedt V, Crozet N, Ahmed-Ali N, Martin0 A, Cognie Y. In vitro maturation and fertilization of goat oocytes. Theriogenology 1992; 37: 1049-1060. 8. Holden CA, Trounson AO. Staining of the inner acrosomal membrane of human spermatozoa with concavalin A lectin as an indicator of potential egg penetration ability. Fertil Steril 1991; 56: 967-974.
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9. Hoshi K, Sugano T, Endo Ch, Yoshimatu N, Yanagida K, Sato A. Induction of the acrosome reaction in human sperm by the human zona pellucida and effect of cervical mucus on zona induced acrosome reaction. Fertil Steril 1993; 60: 149153. 10. Kligman I, Glassner M, Storey BT, Kopf GS. ZP-mediated acrosomal exocitosys in mouse spermatozoa: Characterization of an intermediate stage prior to the completion of the acrosome reaction. Dev Biol 1991; 145: 344-355. 11. Kusunoki H, Yasu T, Kato S, Kand S. Identification of acrosome-reacted goat spermatozoa by a simplified triple-stain technique. Jpn J Zootech Sci. 1984; 55: 832-837. 12. Kusunoki H, Sakakue M, Kato S, Kauda S. Induction of the acrosome reaction in ejaculated goat and boar spermatozoa by preincubation in isolated rat uterus and elucidation of possible inducing factor(s). Jpn J Anim Reprod 1988; 34: 225236. 13. Lenz RW, Ax RL, Grimek HJ, First NL. Proteoglycan from bovine follicular fluid enhances an acrosome reaction in bovine spermatozoa. Biochem Biophys Res Comm 1982; 106: 1092-1098. 14. Martin0 A, Mogas T, Palomo MJ, Paramio MT. Meiotic competence of prepubertal goat oocytes. Theriogenology 1994; 41: 969-980. 15. Martin0 A, Mogas T, Palomo MJ, Paramio MT. In vitro maturation and fertilization of prepubertal goat oocytes. Theriogenology, 1995; 43: 473-486. 16. Morales P, Cross NL, Overstreet JW, Hanson FW. Acrosome intact and acrosome-reacted human sperm can initiate binding to the zona pellucida. Dev Biol 1989; 133: 385-392. 17. Mortimer D, Camenzind AR. The role of follicular fluid in inducing the acrosome reaction of human spermatozoa incubated in vitro. Human Reprod 1989; 4: 169174. 18. Sanchez R, Schill WB. Influence of incubation time/temperature on acrosome reaction and sperm penetration assay. Arch Androl 1991; 27: 35-42. 19. Siiteri JE, Dandekar P, Meizel S. Human sperm acrosome reaction-inducing activity associated with the human cumulus oophorus and mural granulosa cells. J Exp Zool 1988; 246: 71-80. 20. Suarez SS, Wolf DP, Meizel S. Induction of the acrosome reaction in human spermatozoa by a fraction of human follicular fluid. Gamete Res 1986; 14: 107121. 21. Sullivan R, Duchesne C, Fahmy N, Morin M, Dione P. Protein synthesis and acrosome reaction-inducing activity of human cumulus cells. Human Reprod 1990; 5: 830-834. 22. Talbot P, Chacon RS. A triple-stain technique for evaluating normal acrosome reaction of human sperm. J Exp Zool 1981; 215: 201-208. 23. Tesarik J. Comparison of acrosome reaction-inducing activities of human cumulus oophorus, follicular fluid and ionophore A23187 in human sperm populations of proven fertilizing ability in vitro. J Reprod Fertil 1985; 74: 383-388.
EFFECT OF OOCYTE-SPERM CO-INCUBATION ON ACROSOME REACTION IN THE GOAT
E. Martin-Lunasl, A. Martino*, M.T. Paramio*, M.J.Palomo*, M.T. Mogas*, M.A. Bielsa3, P. Andolz3 and P. Martinezl ,a
1 lnstituto de Biologia Fundamental and Departamento de Biologia Celular Universidad Autonoma de Barcelona. 08193 Bellaterra, Spain * Departamento de Patologia y Production Animal Universidad Autonoma de Barcelona 3 Laboratorio de lnvestigaciones Seminologicas Barcelona, Spain
Received for publication: Accepted:
~~~~ 13, 1995 February
15,
1996
ABSTRACT The induction of acrosome reaction of goat spermatozoa was investigated. The acrosomal status of spermatozoa was determined by a triple-staining technique. The effect of the presence of goat oocytes on the proportion of acrosome-reacted Ovulated oocytes were obtained from spermatozoa was also determined. superstimulated adult goats. Other sources of oocytes were adult and prepubertal goats; oocytes from both sources were maturated in vitro. There was an increase in the percentage of acrosome-reacted spermatozoa from 4%f 0,98 to 9%+ 1,41 when oocytes from adult females were used. Similar induction rates were measured with prepubertal and adult oocytes maturated in vitro (10,4%f 2.06 and 8.75%+ 1.06, respectively). The influence of several qualities of cumulus oophorus as well as the presence of zona pellucida was also investigated. No significant differences were obtained with any cumulus oophorus or zona pellucida oocyte complexes. Although oocyte quality is important for high fertilization rates, it does not seem to be crucial for the induction of acrosome reaction. Key words: goat spermatozoa, acrosome reaction, sperm-oocyte interaction
Acknowledgments This work was supported by the Fondo de lnvestigaciones (FIS 91/0293). acorrespondence and reprint requests.
Theriogenology 46:321-330, 1996 0 1996 by Elsevier Science Inc
Sanitarias
0093-691X/96/$15.00 PII SOO93-691X(96)00186-7
Theriogenology
322 INTRODUCTION
The acrosome reaction of mammalian spermatozoa is a prerequisite for sperm penetration of the zona pellucida and fusion with the oocyte plasma membrane. It involves fusion and progressive vesiculation between the outer acrosomal membrane and the overlying plasma membrane, with the subsequent release of acrosomal enzymes. Since the acrosome reaction is an essential event in fertilization, information about the acrosomal status is important for semen quality control in artificial insemination and fertilization trials. The production of IVF embryos in domestic animals is important because of the production or transgenic animals that secrete substances of interest. In goats, the interest of the mammary gland secretions has promoted research on in vitro maturation of goat oocytes for in vitro fertilization (15). Several authors have demonstrated that the presence of the cumulus oophorus or components of the extracellular matrix are capable of inducing acrosome reaction in vitro in human (6,19,23), hamster (1) and bovine spermatozoa (13). It is also well known that human follicular fluid components induce acrosome reaction (19,20). Since sperm acrosome reaction is induced near the site of fertilization, oocyte layers, which include the matrix of the cumulus oophorus and the zona pellucida, may promote acrosome reaction (4,10,21). The oocyte-cumulus complex (COC) has been studied for its ability to induce acrosome reaction in human spermatozoa. Several studies have implicated both the cumulus mass (19,21) and the zona pellucida (9), in human sperm acrosome reaction, but little information has been published on induced acrosome reaction in goats. The only references that have been reported in ejaculated goat spermatozoa by Kusunoki et al. (11,12) have shown that acrosome reaction may be induced by in vitro incubation in the presence of uterine fluid. These authors (12) reported a 16% rate of acrosome-reacted cells in samples incubated for 5 h in the presence of rat uterine fluid. Several techniques have been described for the evaluation of the acrosome reaction of mammalian spermatozoa; however, most of them do not differentiate between true acrosome reaction (live spermatoa) and degenerative acrosome reaction (dead spermatozoa). Some of the new methods use specific reagents that bind to the inner acrosomal membrane: fluorescein-isothiocyanate concanavalin A (8) or monoclonal antibodies reacting with internal antigens (2) without distinction of true or degenerative acrosome reaction. The combination with the viability stain Hoechst 33258 allows for distinguishing between true acrosome reaction and degenerative acrosomal loss. An earlier study (5) describes the acrosomal state from viable spermatozoa using fluorescent dye in combination with phase contrast and fluorescence microscopy. Simultaneous determination of acrosomal status and viability of spermatozoa allows for the determination of the true acrosome reaction. Kligman et al. (10) defined several intermediate steps in the mouse zona pellucidainduced acrosome reaction by using the fluorescent probe chlortetracycline (CTC). We have used the triple-stain tecnique of Talbot and Chacon (22), because it is an unexpensive and easy method that allows for the evaluation of the true acrosome reaction of viable cells.
323
73etiogenology
We investigated whether or not cells from the cumulus oophorus or the zona pellucida of goat oocytes can promote acrosome reaction in goat spermatozoa. Previous experiments obtained in our group have shown low rates of in vitro fertilization in the goat. Thus, we decided to determine if the quality of the oocyte envelopes had some effect on the induction of acrosome reaction. To that end, we adapted the triple-stain technique, originally developed for human spermatozoa, to goat spermatozoa, to differentiate between spermatozoa that had undergone the acrosome reaction and the dead spermatozoa with degenerative acrosome reactions.
MATERIALS AND METHODS Semen Collection
and Sperm Capacitation.
Semen was collected by artificial vagina from males. Sperm concentration was determined by a hemocytometer. The ejaculates were processed according to a method described previously (7). The method consisted of diluting the ejaculates in 8 ml of defined medium with Hepes (DMH), and centrifuging at 200x9 for 10 min. The DMH consisted of 129.5 mM NaCI, 4.16 mM NaHCOsand 10 mM Hepes; the pH was adjusted to 7.0. After removal of the supernatant, 70 pl of the pellet was deposited at the bottom of a conical tube with 2 ml of 20% estrous sheep serum (ESS) in DMH, pH 7.3. After 1.5 hour at 38.5”C, the most motile spermatozoa were recovered from the upper part of the tube. The spermatozoa were dlluted in the same medium to a concentration of 10 x 10s sperm/ml and incubated for 4 h at 385°C; the sperm suspension was diluted a 1 x 10s sperm/ml in the fertilization medium, prepared with 20% FSS, 20mM calcium lactate (lactic acid L+; Sigma Chemicals, St. Louis, MO, USA) in DMH, pH 7.7. Oocyte Collection and Maturation. To obtain ovulated oocytes adult goats were treated for 11 d with intravaginal sponges containing 45 mg of fluorogestone acetate (FGA; Chrono-Gest, Intervet, Salamanca, Spain). Then, 24 h before sponge removal, the goat received lml PG F2a (Dynolitic, Upjohn, Sant Cugat, Spain). The goats received a superovulation treatment of 16 mg of p-FSH (Ovaset, Sanofi, Lyon, France) in 6 injections every 12 h beginning 48 h before sponge removal. Oocytes were obtained by flushing the oviducts 57 h after sponge removal. lnmature oocytes were obtained after dissection of follicles of ovaries obtained from a local slaughterhouse or from diestrous goats treated with 7 mg FSH given in decreasing doses in 3 injections every 12 h . A second source of oocytes was obtained after dissection of ovarian follicles from prepubertal female goats from local slaughterhouse. (14). For in vitro maturation, oocytes were cultured 27 h in Medium 199 supplemented with 10% fetal calf serum (FCS), 10 @ml FSH, 10 us/ml LH, and 1 fug/ml of 17 B-estradiol at 385°C.
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Incubation of Spermatozoa with Oocytes After 4 h of capacitation, samples of 1 ml of spermatozoa (final concentration 1xl 06 sperm/ml) were co-incubated with 5 to 10 oocytes for 17 h at 385X. In Experiment 1 (n = 3) oocytes were obtained from several sources: a) ovulated oocytes, b) adult oocytes maturated in vitro and c) prepubertal oocytes maturated in vitro. A control was included in the 3 replicates: spermatozoa without oocytes, spermatozoa under conditions similar to these for a spontaneous acrosome reaction assessment. We evaluated the percentage of true acrosome reacted spermatozoa at 21 h of maturation. Prepubertal oocytes were used in Experiment 2. Before co-incubation, the oocytes were classified as Type I or II with respect of their cumulus oophorus (Type I: 3 or more complete layers of cumulus cells and Type II: 1 or 2 complete layers); sperm concentration was the same as that described above. Experiment 2 was performed with prepubertal oocytes (Type I or II): a) surrounded by a cumulus oophorus (n = 2) and b) denuded (without cumulus oophorus) (n=4). After culture, the oocytes were digested by a short hyaluronidase treatment and pipetting and then they were coincubated with spermatozoa. After 21 h, the acrosome-reacted spermatozoa were examined. Acrosome Staining Method To evaluate the acrosome reaction, the triple-stain technique was used. Using this method the spermatozoa were classified into 4 patterns, with differentiation between true acrosome reaction (live reacted spermatozoa) and total acrosome reaction (live and dead reacted spermatozoa). Briefly, a 200 1.11 aliquot of the sperm suspension co-incubated with or without oocytes was placed into a plastic tube; an equal volume of 2% trypan blue in albumin-free medium was added. The tube was then incubated for 15 min in a 38.5”C water bath. After incubation, the spermatozoa were diluted with 2 ml of albumin-free fertilization medium and then centrifuged at 200xg for 5 min. The supernatant was removed, and the sperm pellet was resuspended in 2 ml of albumin-free fertilization medium and centrifuged as before. Spermatozoa were resuspended in 200 ul of 3% glutaraldehyde in O.lM cacodilate buffer, pH 7.4, and kept for 1 h at 4 X. The spermatozoa were washed twice, and an aliquot of the sperm suspension was placed on a slide and smeared with a second glass slide. The smears were air-dried at room temperature. Spermatozoa on the slides were stained for 50 min with 0.8% brown bismark in distilled water at pH of 1.8. The slides were stained with 0.8% rose bengal in 0.1 M Tris buffer, pH 5.3, for 30 min. A coverslip was mounted, and the spermatozoa were examined with bright field microscopy at x1000. At least 400 spermatozoa were classified according to presence of 4 patterns: 1) light brown postacrosomal regions with pink acrosomes; live spermatozoa with nonreacted acrosome; 2) light brown postacrosomal regions with unstained acrosomal regions; live spermatozoa with a reacted acrosome; 3) dark brown postacrosomal regions with dark pink acrosomes; dead sperm cells with a nonreacted acrosome; 4) dark brown postacrosomal regions with unstained dark blue acrosomal regions; dead sperm cells with a reacted acrosome. These patterns are shown in Figure 1.
Theriogenology
Figure
1. Four patterns spermatozoa with acrosome; c) dead spermatozoa with
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obtained by the triple-stain method (x1000): a) live non-reacted acrosome; b) live spermatozoa with reacted spermatozoa with a non-reacted acrosome and d) dead reacted acrosome
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Statistical Analysis Data were analyzed by Analysis of Variance; comparison using the Student-Newman-Keuls’ Analysis test.
of means was tested
RESULTS Figure 2 shows the percentage of control values and standard deviation (k SD) of live goat spermatozoa undergoing spontaneous acrosome reaction at 0, 4 and 21 h of incubation in DM-H medium supplemented with 20% ESS. Between 0 and 4 h no significant difference was observed, but after 21 h acrosome reaction increased twofold over the initial value (P < 0,001). There was a clear dependence of acrosome reaction percentage on time. In Experiment 1, several sources of goat oocytes were used for the sperm-oocyte co-incubation. The acrosome-reacted control sperm show a small but significant rise from 4 to 21 h. The acrosome reaction observed in presence of adult goat oocytes maturated in vitro was not significantly different from that induced by ovulated oocytes or prepubertal oocytes maturated in vitro at 21 h; but, these induced acrosome reactions were significantly different compared with spontaneous acrosome reaction in the DM-H medium without oocytes (P < O.OOOl), as shown in Figure 2. Thus, very similar responses were observed in samples co-incubated with oocytes from different sources. Experiment 2 was designed to elucidate whether the presence/absence of the cumulus oophorus in prepubertal oocytes maturated in vitro would induce acrosome reaction differently. The cumulus oophorus was classified as Type I or Type II. No differences were detected in the induced acrosome reaction rates when the oocytes were co-incubated with or without the cumulus oophorus. Similarly, Type I or Type II oocytes had no effect on sperm acrosome reaction. These results suggest that the quality or source of oocyte has no influence on acrosome reaction. We evaluated both the true and the total acrosome reaction percentages as it is shown in Table 1. The total acrosome reaction is about 3-4 times that of the true acrosome reaction.
Table 1. Percentages obtained when true acrosome reaction (left) or total (true and degenerative acrosome reaction) (right) are evaluated in experiment. (C.O.= cumulus oophorus). Total Acrosome Reaction (%) True Acrosome Reaction (%)
Oocytes
Type I
with C.O. without C.O.
Type II
with C.O. without C.O.
35.58 f 1.6 37.19 f 8.85
11.16f0.8 10.51 f 2.06
28.3 + 1.6
10.3 f 2.5
35.86 + 9.54
10.25 k 1.84
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Time I33 Spermatozoa 0 Spermatozoa H Spermatozoa q Spermatozoa
in DM-H/ESS coincubated coincubated coincubated
(hours) medium with adult goat oocytes maturated in vitro with ovulated oocytes with prepubertal oocytes
Figure 2. Mean &SD) percentage of spermatozoa showing the acrosome reaction when incubated for 17 h in the absence or presence or oocytes after 4 h of capacitation.
DISCUSSION It is well known that the maturational status of the oocyte-cumulus complex can affect the success of in vitro fertilization, but its relationship to the acrosome reaction is not well defined. The induction of spermatozoa by several oocyte-associated factors has been extensively discussed; but very little is known about the induction of goat sperm acrosome reaction. The results of this study show the capability of both mature and immature oocytes to induce the acrosome reaction. The incubation of goat spermatozoa in the presence of goat oocytes significantly increases the proportion of spermatozoa undergoing the acrosome reaction. Acrosome reaction is initiated in vivo in the envelope surrounding the oocyte, namely the zona pellucida, before fusion of oocyte-sperm plasma membranes. There are probably agents that induce acrosome reaction near the oocyte or in the glycoprotein stucture of the zona. Studies on induction of acrosome reaction should be evaluated on viable live spermatozoa (true acrosome reaction), because this reflects the physiological acrosomal loss. But, many authors use techniques that evaluate the biochemical pannel of molecules in membranes of acrosome-reacted spermatozoa, rather than measuring the funcionality of spermatozoa (total acrosome reaction); therefore the information given by a variety of methodologies is different. One of the most extensive staininig techniques for the identification of reacted cells has been the triple-stain technique originally described by Talbot and Chacon (22). The 4 patterns
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described for the triple-stain must show high intensity colors that can be easily used to classify the true acrosome-reacted cells (white acrosome, light brown postacrosome) from the remaining cells. The use of the triple staining technique allows us to have both the true and the total acrosome reaction and to compare our data to the results obtained by other authors. Cross et al. (4) reported an increase in acrosome-reaction in human spermatozoa when incubated with intact zona pellucida oocytes and with disaggregated zonae pellucida. The acrosome reaction may be stimulated in vitro by human follicular fluid as previously reported (20). Mot-timer et al. (17) found that follicular fluid promotes human acrosome reaction in a concentration-dependent manner. These authors, detected up to 11 .l% live reacted cells by triple-staining when 6 h precapacitated spermatozoa were incubated for 2 h in a medium containing 100% of follicular fluid. Similar acrosome reaction rate were obtained in human spermatozoa (10.6%) in a Hepes-buffered BWW medium after a long incubation period (20 h), as assessed by the triple-stain technique; however, a shorter incubation period (3 h) yielded only 6.1% acrosome-reacted spermatozoa (18). When total acrosome reaction was evaluated in our experiments (dead + live sperm cells) the mean value obtained for the co-incubated capacitated sperm-oocyte was 35 to 37%. Another study (4) found a higher induced acrosome reaction percentage with human spermatozoa: 46%&l 5 by indirect immunofluorescence with fluoresceinated Pisum sativum agglutinin (FITC-PSA) which determines total acrosome reaction. Hoshi et al. (9) investigated the induction of human acrosome reaction by the zona pellucida and the cumulus oophorus, as evaluated by FITC-PSA. Their results showed that the zona pellucida induces acrosome reaction (35.7 versus 2.8%) but the cumulus oophorus is unable to promote acrosome reaction. Our results show that live spontaneous acrosome reaction was 2.0% f 1.2 after 4 h of incubation, Kusunoki (11) reports similar percentages, evaluated by the triple stain technique, for spontaneous acrosome reaction (1.8 %) in goat spermatozoa after 5 h of incubation. The results of our present study demonstrate that goat oocytes can significantly stimulate acrosome reaction. The source of goat oocytes (adult oocytes maturated in vitro, ovulated oocytes or prepubertal oocytes) did not influence the induction of acrosome reaction, which ranged from 8.7 to 10.5 %. Kusunoki found 16% acrosome-reacted spermatozoa when acrosome reaction was stimulated by coincubation with isolated rat uterine cells. This rate of acrosome reaction was slightly higher than in our study (16% versus lo%), but these differences may be due to the use of different inducers. Although acrosome reaction evaluation by the triple-stain technique is subjective if the parameters are not universally established, the ability of oocyte layers to induce acrosome reaction could be deduced by comparing rates with those of spontaneous acrosome reaction. Thus, Sullivan et al. (21) report high values for acrosome reaction induction in human spermatozoa by cumulus cells: 46% acrosome reaction for live acrosome-reacted spermatozoa, versus 15% of sperm noninduced acrosome reaction in the control. The increase in acrosome reaction induction was 68.8%. In our study, the average acrosome reaction increase was 57.5%.
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There were no differences in acrosome reaction rates for the co-incubation of spermatozoa with 3 sources of oocytes used in this study. The zona pellucida of all oocytes has the necessary structures for inducing acrosome reaction. Since the best fertilization rate was obtained with Type I oocytes (results not shown; 15), we tried to investigate whether the zona pellucida from these oocytes could stimulate acrosome reaction better. Two Types of oocytes were used for this purpose (Type I and Type II). Our results are similar to those of Sullivan et al. (21), who investigated how the maturational status or quality of the cumulus has no influence on the percentage of live human acrosome-reacted spermatozoa. Thus, we can conclude that, although the Type of oocyte affects viability in fertilization, the induction of acrosome reaction is not dependent on the quality of the oocyte cumulus. As has been reported (16), both acrosome-reacted and acrosome-intact spermatozoa can be found close to the zona pellucida. Thus, perhaps the acrosomereacted spermatozoa are stimulated by the cumulus oophorus and the acrosomeintact cells may be induced to react by the zona pellucida. Our results indicate that the presence of stimulatory molecules from the oocyte-cumulus complex or oocyte zona pellucida increases spontaneous acrosome reaction and may represent a mechanism for maximizing the fertilizing ability of spermatozoa. High quality semen is essential for a successful artificial insemination program (3). Assessment of semen should include parameters such as volume, number of sperm cells, morphology, and acrosomal status and function. The percentage of acrosome-reacted cells is indicative of sperm fertilizing ability and may be an additional assessment of semen quality to be used in in vitro fertilization of goats, to predict the success of fertilization.
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