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In vitro maturation and fertilization of goat oocytes
Therr’ogenology
37:1049-1060,
1992
IN VITRO MATURATION AND FERTILIZATION OF GOAT OOCYTES V. De Smedt,’
N. Crozet,’
M. Ahmed-Ali ’ A. Martin0 2 and Y. Cognie 3
’ Unite de Biologie de la Fecondation-Station de Physiologie animale I.N.R.A. 78352 Jouy-en-Josas cedex, France 2 Departament de Patologia i Productions animales Facultat de Veterinaria Universitat Autonoma de Barcelona 08193 Bellaterra, Barcelona, Spain 3 Unite de Physiologie integree I.N.R.A. B.P. 1, 37380 Nouzilly, France
Received for publication: Accepted:
October
January
8, 1991 28, 1992
ABSTRACT Follicular cumulus-enclosed goat oocytes were matured in vitro in the presence of granulosa ceils, follicle stimulating hormone (FSH), luteinizing hormone (LH) and estradiol-178. While 86% of the oocytes from follicles 2 to 6 mm in diameter achieved meiotic maturation, only 24% of the oocytes from follicles 1 to 2 mm in diameter progressed to Metaphase II. Exposure of follicle-enclosed cumulus-oocyte complexes to 20°C prior to culture resulted in 11.5% of the oocytes exhibiting abnormal meiotic spindle. This indicated that immature goat oocytes are particularly sensitive to temperature. Ejaculated spermatozoa were capacitated according to the technique previously proposed for ram sperm (1). The fertilization rates of ovulated and mechanically denuded in vitro-matured oocytes were 85 and 82.8%, respectively; 59.7% of ovulated and 57.1% of in vitro-matured oocytes were normally fertilized, as shown by the presence of both the fsemale and the male pronucleus as well as by the remnants of the sperm tail in the ooplasm, 17 hours after insemination. Polyspermy was the main abnormality detected, and it affected almost 20% of the inseminated oocytes. The cleavage rate (two to fourcell istage) 41 hours after insemination of in vitro-matured and fertilized oocytes was 58%. Key words: goat oocyte, maturation, fertilization
Acknowledgments The authors thank Y. Lavergne for technical assistance. A. Martin0 was supported by a CIRIT grant, Generalitat de Catalunya, Spain.
Copyright
0 1992 Butterworth-Heinemann
1050
Theriogenology
INTRODUCTION Efficient in vitro procedures for oocyte maturation and fertilization in large domestic species are important for the development of new biotechnologies such as gene transfer and in vitro multiplication of identical embryos. Bovine eggs are particularly in demand; however, goat zygotes may also be valuable for biotechnology applications. For example, transgenic milk goats could be used for producing large quantities of foreign proteins. Although the in vitro-maturation and fertilization of oocytes for two other domestic ruminant species (cattle and sheep) have been extensively investigated (2), very few data have been reported for goats. It was observed that cumulus-enclosed oocytes from both hormonally treated and untreated immature caprine females matured at a higher frequency (61-63%) than naked oocytes (12% to 17%; 3). In vitro fertilization of such in vitro-matured oocytes by epididymal spermatozoa capacitated in a chemicallydefined medium leads to variable results. The highest fertilization rates (57%) were obtained when caprine cumulus-oocyte complexes were cultured for 25 hours prior to the addition of spermatozoa (4). On the other hand, xenogenous fertilization of ovulated goat oocytes by goat spermatozoa was achieved in rabbit oviducts (5). To our knowledge, only one report is available on embryonic development to term after in vitro fertilization in this species. Twelve two-cell-stage embryos resulting from the in vitro fertilization of tubal oocytes were transferred to five recipients; one became pregnant and gave birth to one kid (6). The objective of our present study was to develop reliable in vitro systems for goat oocyte maturation and fertilization which could regularly provide large numbers of competent oocytes and normally fertilized embryos for further biotechnological applications. MATERIALS AND METHODS Animals Goats from French Alpine and Saanen breeds were used. To obtain ovulated oocytes, the goats were synchronized for estrus by a lo-day-long treatment with intravaginal sponges impregnated with 45 mg of fluorogestone acetate (Intervet, Angers, France) and by injection of 100 ug of a prostaglandin analog (Cloprostenol, Estrumate, Pitman-Moore, Meaux, France) 34 hours before sponge removal. Superovulation was obtained by injection of porcine follicle-stimulating hormone (p-FSH, Sanofi, France) in decreasing dosages (60, 60, 30, 30, 30 and 30 ug). In the last four injections the FSH/LH ratio was decreased from 8 to 1.5 and 0.5 by addition of purified porcine LH (Combarnous, INRA-Nouzilly, France). The total dose of pFSH employed was equivalent to 16 mg of the Armour Standart. The goats were slaughtered 30 hours after intramuscular injection of 100 pg of LHRH (U.C.B., Bioproducts, Belgium) given 41 hours after sponge removal. To obtain ovarian oocytes, the goats were synchronized for estrus as described above. Behavioral estrus was monitored, and follicular growth was stimulated by three injections of p-FSH (3,2 and 2 mg) starting on Day 14 or 15 of the estrous Cycle at 48, 40 and 24 hours before slaughter.
Theriogenology
The genital tracts and ovaries were removed at the slaughterhouse transported to the laboratory in a warm chamber (30°C) within 5 minutes.
1051
and
Oocyte Treatments Ovulated oocytes were immediately recovered by retrograde flushing of the oviducts with a defined medium buffered with HEPES (DM-H, pH 7.8) (1) at 30 to 35°C. The ovaries were dissected and nonatretic follicles were selected and distributed into two groups according to size: 1 to 2 mm and 2 to 6 mm in diameter. Dissection of the follicles occurred either at room temperature or at 30 to 35°C. The cumulus-oocyte complexes and granulosa cells were released from the follicles and were washed in warm TC199 medium (Flow Laboratories, France) containing 4 mM NaHCO, and buffered with 20 mM HEPES (pH 7.35). The same medium enriched with 10% heatinactivated calf serum, 1 Fg/ml estradiol-178 and 10 pg/ml FSH and LH was used for the culture. The cumulus-oocyte complexes were co-cultured with granulosa cells (Ix106/ml) at 38.5”C with gentle agitation (7) for 27 hours. Prior to in vitro fertilization, the oocytes were mechanically denuded from the cumulus cells in TC199 medium containing 150 Ul/ml of hyaluronidase (Serva, Heidelberg, Germany) and were extensively washed in TC199 medium. Extrusion of the first polar body was assessed by examination with the dissecting microscope, and only matured oocytes were selected for fertilization. Sperm Capacitation Fresh ejaculates were obtained from two males of proven fertility. The ejaculates were diluted with 8 ml of DM-H medium, a modified Bracket& defined medium (8) buffered with 10 mM HEPES and containing 4.16 mM Na H CO, and 129.5 mM NaCl (pH 7). After centrifugation for 10 minutes at 200 g, the supernatants were discarded and 50 ~1 of the pellet was overlaid with 2 ml of DM-H enriched with 20% of heat-inactivated estrc’us sheep serum (ESS). The spermatozoa were allowed to swim-up for 2 hours at 38.5”C under air. The supernatants containing highly motile spermatozoa were recovered. Sperm motility was evaluated by light microscopy, and sperm concentration was measured with a Thoma cell. Spermatozoa were diluted to 1x107/ml with DM-H + 20% ESS (pH 7.3) and incubated for 5 to 6 hours at 38.5”C in stoppered tubes. After assessment of motility, the spermatozoa were diluted for fertilization to the final concentration of 1~10~ cells/ml in DM-H + 20% ESS. The pH of the medium was adjusted to 7.7 by addition of NaOH. The DM-H medium was supplemented with 7.75 mM ‘calcium lactate (L(+)-lactic acid, hemicalcium salt; Sigma; 9). In viiro Fertilization Five to ten ovulated or mechanically denuded, in vitro-matured oocytes were added to each test tube containing 1 ml of the sperm suspension from an individual male and Incubated for 17 hours at 38.5’C in stoppered tubes. In some experiments, in vitromatured and fertilized oocytes were then transferred to 400 ul of B, medium (I.N.R.A. MenlSzo) + 20% ESS and incubated for 24 hours at the same temperature under 5% CO, in air. At the end of the culture period, the oocytes were examined for cleavage with an inverted microscope. The gametes were manipulated in a room warmed to 30 to 35°C.
1052
Theriogenology
Cytological Procedures Denuded oocytes were fixed after 27 hours of culture and at 17 hours post insemination for the control of maturation and fertilization, respectively. They were fixed in acetic acid (go%)-ethanol (l/3, v/v), for 24 hours at 4°C and stained with lacmoid before nuclear examination by light microscopy. RESULTS Oocyte Maturation Follicular size. Cumulus-oocyte complexes from small (1 to 2 mm in diameter) and medium-sized (2 to 6 mm in diameter) follicles were cultured for 27 hours, and the meiotic stages reached were evaluated in both groups (Table 1). While 86% of the oocytes from medium-sized follicles achieved meiotic maturation, only 17% of the oocytes from small follicles progressed to Metaphase II while 67% remained in Metaphase I. Additional culture for 24 hours did not increase the percentage of oocytes reaching Metaphase II (data not shown), suggesting that the oocytes from small follicles were blocked in Metaphase I and were unable to complete meiotic maturation in vitro. Consequently, only cumulus-oocyte complexes from 2 to 6 mm follicles were used in the subsequent parts of the present study. Influence of temperature. _~_. ___Follicles were dissected from the ovaries either at room temperature (20°C) or at 30 to 35°C. The cumulus-oocvte complexes were cultured for 27 hours and were fixed for nuclear evaluation. Oocytes from both groups reached Anaphase I to Metaphase II stages at the same rate. However, when the follicles were dissected at room temperature, 11.5% of the oocytes exhibited Me&phase I (33%) or Metaphase II (6%) spindle abnormalities. As shown in Figure I, the chromosomes were split into two groups or several chromosomes were located outside the metaphase plate, whose shape was disturbed, or the chromosomes were distributed at random within the cytoplasm. By contrast, when the follicles were collected at 30 to 35°C such abnormalities of the meiotic spindles were never detected (Figure 2). Sperm Capacitation In the present experiment, the semen of two males of proven fertility was used. 80% and 85 to 90% of spermatozoa were highly motile in washed ejaculates and in the supernatant after the swim-up procedure, respectively. Sperm motility was well maintained throughout the capacitation period. At the moment of insemination approximately 80% of the spermatozoa exhibited progressive motility, while a small portion (5 to 10%) exhibited hyperactivated movement. The estrous sheep serum present in the medium caused slight head-to-head agglutination. At the end of capacitation, most of the spermatozoa exhibiting progressive motility were clustered in groups of three to five. In vitro Fertilization The fertilization rates of ovulated and in vitro-matured oocytes were 85 and 82.8%, respectively; 60% and 57% of in vivo and in vitro matured oocytes were normally fertilized containing both the male and female pronucleus (Tables 2,3). Polyspermy,
50
2-6
1 (2)
GV
7 (12)
Prometaphase
I
5 (10)
40 (67)
Metaphase I
2 (4)
1 (2)
Anaphase ITelophase I
41
67
2
5
7
A
B
Total
57 (85)
34
23
total
40 (60)
26
14
2 PN
14 (21)
5
9
3 PN
3 (4)
3
Decond. sp.
No. fertilized 17 hours post insemination (%)
PN: Pronuclei; Decond. sp.: Decondensed spermatozoa.
26
No. of experiments
Male
No. of oocytes
1 (1.5)
1
No. fragmented
Table 2. In vitro fertilization of ovulated goat oocytes inseminated with ejaculated sperm capacitated in vitro
59
No. of oocytes
l-2
(mm)
Follicular size
Meiotic stages reached after 27 hours of culture No. of oocytes (%)
Table 1. Influence of follicular size on goat oocyte in vitro maturation
II
9 (13.5)
7
2
No. unfertilized (%)
43 (86)
10 (17)
Metaphase
?
5 2 5
3
3
6
A
B
Total 35
15
20
No. of oocytes
29 (83)
14
15
Total
8 (23)
1 (3)
1
decond. sp.
1 (3)
1
No. fragmented
2 to 4 cell stage 47 (58)
81
1 (1)
5 to 6 cell stage
30 (37)
Uncleaved
No. of eggs and stages reached 41 hours post insemination
Total
No. of oocytes inseminated
3
5
> 3 PN
Table 4. Cleavage of in vitro matured and in vitro fertilized goat oocytes
20 (57)
IO
10
2 PN
No. fertilized 17 hours post insemination (%)
3 (4)
Fragmented
(%)
5 (14)
1
4
No. unfertilized (%)
goat oocytes inseminated with ejaculated sperm capacitated in vitro
PN: Pronuclei; Decond. sp.: Decondensed spermatozoa.
No. of experiments
Male
Table 3. In vitro fertilization of in vitro-matured
?
2 0‘
4
51 (t, 2. n
The,riogenology
Figure 1.
The follicles were handled at room temperature. The oocytes were cultured for 27 hours and were stained with lacmoid. a) Metaphase I spindle split into two parts (1850). b) Metaphase I spindle and isolated chromosomes (1850x). c) Dispersed chromosomes from Metaphase II stage (1750x).
1056
Figure 2.
Theriogenology
Follicles were handled at 30 to 35°C. The oocytes were cultured for 27 hours and were stained with lacmoid. a) Metaphase I spindle (1500x). b) Anaphase I (1500x). c) Metaphase II spindle (2000x).
1057
Ther-iogenology
primarily dispermy, which affected about 20% of the inseminated oocytes, was the main abnormality. Decondensing sperm heads in the ooplasm, at 17 hours post insemination wen? observed in 4.4 and 2.8% of in vivo and in vitro-matured oocytes, respectively. At 41 hours post insemination, 58% of the in vitro matured inseminated oocytes cleaved and reached the two to four-cell stage in B, medium supplemented with 20% ESS (Table 4). In one preliminary experiment in vitro-matured and fertilized oocytes were transferred to the oviducts of synchronized ewes, and several blastocysts were recovered after 7 days (data not shown). DISCUSSION Almost 90% of the oocytes from follicles larger than 2 mm in diameter matured in vitro within 27 hours. The percentage of oocytes reaching Metaphase II was higher after 27 hours than after 24 hours in culture (Le Gal et al., 10). These maturation rates are comparable to those reported for caprine oocytes (11). Most of the oocytes (67%) from l- to 2-mm follicles resumed meiosis but remained arrested in Metaphase I, indicating that the ability for goat oocytes to achieve meiotic maturation was acquired in follicles larger than 2 mm in diameter. These results werf? comparable to those reported in the pig (12). By contrast, in cattle there is no eviclence that oocytes from small antral follicles fail to complete meiotic maturation in vitro (13). Handling goat follicle-enclosed oocytes at room temperature may induce further abnormalities during meiotic maturation. Although oocytes recovered at room temperature were able to progress up to the Metaphase II stage at the same frequency than the oocytes isolated at 30 to 35”C, 11.5% of them exhibited abnormal meiotic spindles. Exposure of immature goat oocytes to a subnormal temperature may affect further formation of the metaphase plate, resulting in aberrant chromosome distribution. In sheep, handling the follicle-enclosed oocytes and keeping them at room temperature for !j hours (14) did not induce such abnormalities. However, a decrease in temperature during germinal vesicle breakdown may result in further spindle disorganization (15). Addition of estrous sheep serum to the medium was previously proposed for ram sperm capacitation (1). The high capacity-binding protein, albumin, which is present in the serum, may facilitate sperm capacitation by contributing to the depletion of the sperm cholesterol membrane (16). The in vitro fertilization procedure proposed for sheep (1.9) led to a high level of fertilization and to a rapid sperm-oocyte penetration (17). Moreover, the oocytes fertilized under such conditions were able to support further embryonic development, as attested by the birth of 33 lambs (1, 18). In the present study, the same procedure was applied to goats, resulting in fertilization rates comparable to those obtained in sheep. Approximately 60% of ovulated and in vitro-matured oocytes were normally fertilized, as attested by the presence of both the female and male pronucleus ancl by remnants of the sperm tail in the ooplasm 17 hours after insemination. In all the fertilized oocytes the sperm tail was detected close to the male pronucleus, indicating thaI they were not parthenogenetically activated. In previously reported experiments, variable results were obtained according to the capacitation procedure. A short treatment with ionophore A23187 led to 35.3% of fertilized tubal oocytes (6). Capacitation in different culture media in the presence of heparin yielded fertilization rates ranging from
1058
Theriogenology
19.2 to 45.5% for in vivo-matured oocytes (11). Although a fertilization rate of 57% for in vitro-matured oocytes was reported by Song and lritani (4), their finding was based on normally fertilized zygotes as well as on polyspermic eggs and oocytes which failed to decondense the sperm chromatin. More recently, fertilization rates of 56.7%, as attested to by the presence of the male and female pronucleus or by cleavage, were achieved for in vitro-matured oocytes (11). However, the rate of cleavage (33.3%) ova within 48 hours of culture was lower than in the present experiment (58% within 41 hours). In our study, the incidence of polyspermy was relatively high, affecting nearly 20% of the inseminated oocytes. This was probably due to the simultaneous presence of several capacitated spermatozoa in close vicinity of the oocytes. The sperm concentration used for in vitro fertilization led to a much higher sperm-egg ratio than that observed for in vivo fertilization (19). However, extreme dilution of spermatozoa in the culture medium usually results in a dramatic decrease in sperm motility, which may be detrimental to oocyte penetration. The in vitro-matured oocytes underwent fertilization at the same rate than the ovulated oocytes. Moreover, the percentage of normally fertilized oocytes containing two pronuclei and remnants of the sperm tail was identical in both groups. Incomplete cytoplasmic maturation which may occur in vitro in suboptimal conditions results in oocytes that are incompetent to support normal fertilization and further embryonic development (20,21). Failure of sperm chromatin decondensation and of male pronucleus formation are the main abnormalities found during fertilization of such incompletely matured oocytes. The present results indicate that goat oocytes matured in vitro in the presence of granulosa cells and gonadotropins were fully Competent for supporting fertilization. Furthermore, several blastocysts were obtained from in vitro matured and fertilized oocytes. Experiments are in progress for evaluating the full developmental capacity of such in vitro produced zygotes after transfer to recipient goats. Ovaries collected at slaughter or by ovariectomy may provide an abundant source of immature oocytes at a low cost. The present results indicate that it is possible to obtain high rates of matured (90%) and normally fertilized (60%) goat oocytes in vitro. This production could be used in the near future for further research on gamete maturation, fertilization and early development as well as for biotechnological applications such as gene transfer in this domestic species.
REFERENCES 1.
Crozet, N., Huneau, D., De Smedt, V., Theron, M.C., SzBIIBsi, D., Torres, S. and Sevellec, C. In vitro fertilization with normal development in the sheep. Gamete Res. 16: 159-l 70 (1987).
2.
Crozet, N. Manipulation of oocytes and in vitro fertilization. J. Reprod. Fertil. 43 (Suppl.): 235243 (1991).
3.
Song, H.B., Kasai, M., Niwa, K. and Iritani, A. Maturation culture of follicular oocytes from ovaries of immature goats treated with or without gonadotropins. Proc. 3rd AAAP Anim. Sci. Cong. Seoul (Korea) 1: 438440 (1985).
1059
Theriogenology
4.
Song, H.B. and Iritani, A. In vitro fertilization of goat foflicular oocytes with epididymal spermatozoa capacitated in a chemically defined medium. Proc. 3rd AAAP Anim. Sci. Cong. Seoul (Korea) 1: 463-465 (1985).
5.
Duan, E.K., Wang, G.Y., Ma, B.H. and Wang, J.C. Xenogenous fertilization of goat ovulated oocytes in the rabbit oviduct. Theriogenologya: 219 abstr. (1990).
6.
Hanada, A. In vitro fertilization in goats. Jpn. J. Anim. Reprod. 31: 21-26 (1985).
7.
Staigmiller, R.B. and Moor, R.M. Effect of follicle cells on the maturation and developmental competence of ovine oocytes matured outside the follicle. Gamete Res. 9: 211-229 (1984).
8.
Brackett, B.G. and Oliphant, G. Capacitation of rabbit spermatozoa Reprod. 2: 260-274 (1975).
9.
Huneau, D. and Crozet, N. In vitro fertilization in the sheep: effect of elevated calcium concentration at insemination. Gamete Res. 3: 119-l 25 (1989).
10.
Le Gal, F., Gall, L., De Smedt, V. Changes in protein synthesis pattern during in vitro maturation of goat oocytes. Mol. Reprod. Dev. (in press).
11.
Younis, A.I., Zuelke, K.A., Harper, K.M., Oliveira, M.A.L. and Brackett, B.G. In vitro fertilization of goat oocytes. Biol. Reprod. 44, 1177-1182 (1991).
12.
Motlik, J., Crozet, N. and Fulka, J. Meiotic competence in vitro of pig OOCytes isolated from early antral follicles. J. Reprod. Fertil. 72: 323-328 (1984).
13.
Leibfried, L. and First, N.L. Characterization of bovine follicular oocytes and their ability to mature in vitro. J. Anim. Sci. 3: 76-86 (1979).
14.
Staigmiller, R.B. In vitro method for production of viable oocytes. J. Anim. Sci. @ (suppl. 2): 54-64 (1988).
15.
Moor, R.M. and Crosby, I.M. Temperature induced abnormalities OOCytesduring maturation. J. Reprod. Fertil. 3: 467-473 (1985).
16.
Langlais, J., Kan, F.W.K., Granger, L., Raymond, L., Bleau, G. and Roberts, R.O. Identification of sterol acceptors that stimulate cholesterol efflux from human spermatozoa during in vitro capacitation. Gamete Res. 20: 185-201 (1988).
17.
Crozet, N. Fine structure of sheep fertilization in vitro. Gamete Res. 9: (1988).
18.
CogniB, Y., G&in, Y., Guyader, C., Poulin, N. and Crozet, N. In vitro fertilization of sheep oocytes matured in vitro. Theriogenology 35: 393-400 (1990).
19.
Cummins, J.M. and Yanagimachi, R. Sperm-egg ratios and the site of acrosome reaction during in vivo fertilization in the hamster. Gamete Res. 2: 239-256 (1982).
in vitro. Biol.
in sheep
291-303
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Theriogenology
20.
Thibault, C. and Gerard, M. Facteur cytoplasmique necessaire a la formation du pronucleus male dans I’ovocyte de lapine. CR Acad. Sci. (Paris)m: 20252026 (1970).
21.
Moor, R.M. and Trounson, A.O. Hormonal and follicular factors affecting maturation of sheep oocytes in vitro and their subsequent developmental capacity. J. Reprod. Fert. 2: 101-109 (1977).
37:1049-1060,
1992
IN VITRO MATURATION AND FERTILIZATION OF GOAT OOCYTES V. De Smedt,’
N. Crozet,’
M. Ahmed-Ali ’ A. Martin0 2 and Y. Cognie 3
’ Unite de Biologie de la Fecondation-Station de Physiologie animale I.N.R.A. 78352 Jouy-en-Josas cedex, France 2 Departament de Patologia i Productions animales Facultat de Veterinaria Universitat Autonoma de Barcelona 08193 Bellaterra, Barcelona, Spain 3 Unite de Physiologie integree I.N.R.A. B.P. 1, 37380 Nouzilly, France
Received for publication: Accepted:
October
January
8, 1991 28, 1992
ABSTRACT Follicular cumulus-enclosed goat oocytes were matured in vitro in the presence of granulosa ceils, follicle stimulating hormone (FSH), luteinizing hormone (LH) and estradiol-178. While 86% of the oocytes from follicles 2 to 6 mm in diameter achieved meiotic maturation, only 24% of the oocytes from follicles 1 to 2 mm in diameter progressed to Metaphase II. Exposure of follicle-enclosed cumulus-oocyte complexes to 20°C prior to culture resulted in 11.5% of the oocytes exhibiting abnormal meiotic spindle. This indicated that immature goat oocytes are particularly sensitive to temperature. Ejaculated spermatozoa were capacitated according to the technique previously proposed for ram sperm (1). The fertilization rates of ovulated and mechanically denuded in vitro-matured oocytes were 85 and 82.8%, respectively; 59.7% of ovulated and 57.1% of in vitro-matured oocytes were normally fertilized, as shown by the presence of both the fsemale and the male pronucleus as well as by the remnants of the sperm tail in the ooplasm, 17 hours after insemination. Polyspermy was the main abnormality detected, and it affected almost 20% of the inseminated oocytes. The cleavage rate (two to fourcell istage) 41 hours after insemination of in vitro-matured and fertilized oocytes was 58%. Key words: goat oocyte, maturation, fertilization
Acknowledgments The authors thank Y. Lavergne for technical assistance. A. Martin0 was supported by a CIRIT grant, Generalitat de Catalunya, Spain.
Copyright
0 1992 Butterworth-Heinemann
1050
Theriogenology
INTRODUCTION Efficient in vitro procedures for oocyte maturation and fertilization in large domestic species are important for the development of new biotechnologies such as gene transfer and in vitro multiplication of identical embryos. Bovine eggs are particularly in demand; however, goat zygotes may also be valuable for biotechnology applications. For example, transgenic milk goats could be used for producing large quantities of foreign proteins. Although the in vitro-maturation and fertilization of oocytes for two other domestic ruminant species (cattle and sheep) have been extensively investigated (2), very few data have been reported for goats. It was observed that cumulus-enclosed oocytes from both hormonally treated and untreated immature caprine females matured at a higher frequency (61-63%) than naked oocytes (12% to 17%; 3). In vitro fertilization of such in vitro-matured oocytes by epididymal spermatozoa capacitated in a chemicallydefined medium leads to variable results. The highest fertilization rates (57%) were obtained when caprine cumulus-oocyte complexes were cultured for 25 hours prior to the addition of spermatozoa (4). On the other hand, xenogenous fertilization of ovulated goat oocytes by goat spermatozoa was achieved in rabbit oviducts (5). To our knowledge, only one report is available on embryonic development to term after in vitro fertilization in this species. Twelve two-cell-stage embryos resulting from the in vitro fertilization of tubal oocytes were transferred to five recipients; one became pregnant and gave birth to one kid (6). The objective of our present study was to develop reliable in vitro systems for goat oocyte maturation and fertilization which could regularly provide large numbers of competent oocytes and normally fertilized embryos for further biotechnological applications. MATERIALS AND METHODS Animals Goats from French Alpine and Saanen breeds were used. To obtain ovulated oocytes, the goats were synchronized for estrus by a lo-day-long treatment with intravaginal sponges impregnated with 45 mg of fluorogestone acetate (Intervet, Angers, France) and by injection of 100 ug of a prostaglandin analog (Cloprostenol, Estrumate, Pitman-Moore, Meaux, France) 34 hours before sponge removal. Superovulation was obtained by injection of porcine follicle-stimulating hormone (p-FSH, Sanofi, France) in decreasing dosages (60, 60, 30, 30, 30 and 30 ug). In the last four injections the FSH/LH ratio was decreased from 8 to 1.5 and 0.5 by addition of purified porcine LH (Combarnous, INRA-Nouzilly, France). The total dose of pFSH employed was equivalent to 16 mg of the Armour Standart. The goats were slaughtered 30 hours after intramuscular injection of 100 pg of LHRH (U.C.B., Bioproducts, Belgium) given 41 hours after sponge removal. To obtain ovarian oocytes, the goats were synchronized for estrus as described above. Behavioral estrus was monitored, and follicular growth was stimulated by three injections of p-FSH (3,2 and 2 mg) starting on Day 14 or 15 of the estrous Cycle at 48, 40 and 24 hours before slaughter.
Theriogenology
The genital tracts and ovaries were removed at the slaughterhouse transported to the laboratory in a warm chamber (30°C) within 5 minutes.
1051
and
Oocyte Treatments Ovulated oocytes were immediately recovered by retrograde flushing of the oviducts with a defined medium buffered with HEPES (DM-H, pH 7.8) (1) at 30 to 35°C. The ovaries were dissected and nonatretic follicles were selected and distributed into two groups according to size: 1 to 2 mm and 2 to 6 mm in diameter. Dissection of the follicles occurred either at room temperature or at 30 to 35°C. The cumulus-oocyte complexes and granulosa cells were released from the follicles and were washed in warm TC199 medium (Flow Laboratories, France) containing 4 mM NaHCO, and buffered with 20 mM HEPES (pH 7.35). The same medium enriched with 10% heatinactivated calf serum, 1 Fg/ml estradiol-178 and 10 pg/ml FSH and LH was used for the culture. The cumulus-oocyte complexes were co-cultured with granulosa cells (Ix106/ml) at 38.5”C with gentle agitation (7) for 27 hours. Prior to in vitro fertilization, the oocytes were mechanically denuded from the cumulus cells in TC199 medium containing 150 Ul/ml of hyaluronidase (Serva, Heidelberg, Germany) and were extensively washed in TC199 medium. Extrusion of the first polar body was assessed by examination with the dissecting microscope, and only matured oocytes were selected for fertilization. Sperm Capacitation Fresh ejaculates were obtained from two males of proven fertility. The ejaculates were diluted with 8 ml of DM-H medium, a modified Bracket& defined medium (8) buffered with 10 mM HEPES and containing 4.16 mM Na H CO, and 129.5 mM NaCl (pH 7). After centrifugation for 10 minutes at 200 g, the supernatants were discarded and 50 ~1 of the pellet was overlaid with 2 ml of DM-H enriched with 20% of heat-inactivated estrc’us sheep serum (ESS). The spermatozoa were allowed to swim-up for 2 hours at 38.5”C under air. The supernatants containing highly motile spermatozoa were recovered. Sperm motility was evaluated by light microscopy, and sperm concentration was measured with a Thoma cell. Spermatozoa were diluted to 1x107/ml with DM-H + 20% ESS (pH 7.3) and incubated for 5 to 6 hours at 38.5”C in stoppered tubes. After assessment of motility, the spermatozoa were diluted for fertilization to the final concentration of 1~10~ cells/ml in DM-H + 20% ESS. The pH of the medium was adjusted to 7.7 by addition of NaOH. The DM-H medium was supplemented with 7.75 mM ‘calcium lactate (L(+)-lactic acid, hemicalcium salt; Sigma; 9). In viiro Fertilization Five to ten ovulated or mechanically denuded, in vitro-matured oocytes were added to each test tube containing 1 ml of the sperm suspension from an individual male and Incubated for 17 hours at 38.5’C in stoppered tubes. In some experiments, in vitromatured and fertilized oocytes were then transferred to 400 ul of B, medium (I.N.R.A. MenlSzo) + 20% ESS and incubated for 24 hours at the same temperature under 5% CO, in air. At the end of the culture period, the oocytes were examined for cleavage with an inverted microscope. The gametes were manipulated in a room warmed to 30 to 35°C.
1052
Theriogenology
Cytological Procedures Denuded oocytes were fixed after 27 hours of culture and at 17 hours post insemination for the control of maturation and fertilization, respectively. They were fixed in acetic acid (go%)-ethanol (l/3, v/v), for 24 hours at 4°C and stained with lacmoid before nuclear examination by light microscopy. RESULTS Oocyte Maturation Follicular size. Cumulus-oocyte complexes from small (1 to 2 mm in diameter) and medium-sized (2 to 6 mm in diameter) follicles were cultured for 27 hours, and the meiotic stages reached were evaluated in both groups (Table 1). While 86% of the oocytes from medium-sized follicles achieved meiotic maturation, only 17% of the oocytes from small follicles progressed to Metaphase II while 67% remained in Metaphase I. Additional culture for 24 hours did not increase the percentage of oocytes reaching Metaphase II (data not shown), suggesting that the oocytes from small follicles were blocked in Metaphase I and were unable to complete meiotic maturation in vitro. Consequently, only cumulus-oocyte complexes from 2 to 6 mm follicles were used in the subsequent parts of the present study. Influence of temperature. _~_. ___Follicles were dissected from the ovaries either at room temperature (20°C) or at 30 to 35°C. The cumulus-oocvte complexes were cultured for 27 hours and were fixed for nuclear evaluation. Oocytes from both groups reached Anaphase I to Metaphase II stages at the same rate. However, when the follicles were dissected at room temperature, 11.5% of the oocytes exhibited Me&phase I (33%) or Metaphase II (6%) spindle abnormalities. As shown in Figure I, the chromosomes were split into two groups or several chromosomes were located outside the metaphase plate, whose shape was disturbed, or the chromosomes were distributed at random within the cytoplasm. By contrast, when the follicles were collected at 30 to 35°C such abnormalities of the meiotic spindles were never detected (Figure 2). Sperm Capacitation In the present experiment, the semen of two males of proven fertility was used. 80% and 85 to 90% of spermatozoa were highly motile in washed ejaculates and in the supernatant after the swim-up procedure, respectively. Sperm motility was well maintained throughout the capacitation period. At the moment of insemination approximately 80% of the spermatozoa exhibited progressive motility, while a small portion (5 to 10%) exhibited hyperactivated movement. The estrous sheep serum present in the medium caused slight head-to-head agglutination. At the end of capacitation, most of the spermatozoa exhibiting progressive motility were clustered in groups of three to five. In vitro Fertilization The fertilization rates of ovulated and in vitro-matured oocytes were 85 and 82.8%, respectively; 60% and 57% of in vivo and in vitro matured oocytes were normally fertilized containing both the male and female pronucleus (Tables 2,3). Polyspermy,
50
2-6
1 (2)
GV
7 (12)
Prometaphase
I
5 (10)
40 (67)
Metaphase I
2 (4)
1 (2)
Anaphase ITelophase I
41
67
2
5
7
A
B
Total
57 (85)
34
23
total
40 (60)
26
14
2 PN
14 (21)
5
9
3 PN
3 (4)
3
Decond. sp.
No. fertilized 17 hours post insemination (%)
PN: Pronuclei; Decond. sp.: Decondensed spermatozoa.
26
No. of experiments
Male
No. of oocytes
1 (1.5)
1
No. fragmented
Table 2. In vitro fertilization of ovulated goat oocytes inseminated with ejaculated sperm capacitated in vitro
59
No. of oocytes
l-2
(mm)
Follicular size
Meiotic stages reached after 27 hours of culture No. of oocytes (%)
Table 1. Influence of follicular size on goat oocyte in vitro maturation
II
9 (13.5)
7
2
No. unfertilized (%)
43 (86)
10 (17)
Metaphase
?
5 2 5
3
3
6
A
B
Total 35
15
20
No. of oocytes
29 (83)
14
15
Total
8 (23)
1 (3)
1
decond. sp.
1 (3)
1
No. fragmented
2 to 4 cell stage 47 (58)
81
1 (1)
5 to 6 cell stage
30 (37)
Uncleaved
No. of eggs and stages reached 41 hours post insemination
Total
No. of oocytes inseminated
3
5
> 3 PN
Table 4. Cleavage of in vitro matured and in vitro fertilized goat oocytes
20 (57)
IO
10
2 PN
No. fertilized 17 hours post insemination (%)
3 (4)
Fragmented
(%)
5 (14)
1
4
No. unfertilized (%)
goat oocytes inseminated with ejaculated sperm capacitated in vitro
PN: Pronuclei; Decond. sp.: Decondensed spermatozoa.
No. of experiments
Male
Table 3. In vitro fertilization of in vitro-matured
?
2 0‘
4
51 (t, 2. n
The,riogenology
Figure 1.
The follicles were handled at room temperature. The oocytes were cultured for 27 hours and were stained with lacmoid. a) Metaphase I spindle split into two parts (1850). b) Metaphase I spindle and isolated chromosomes (1850x). c) Dispersed chromosomes from Metaphase II stage (1750x).
1056
Figure 2.
Theriogenology
Follicles were handled at 30 to 35°C. The oocytes were cultured for 27 hours and were stained with lacmoid. a) Metaphase I spindle (1500x). b) Anaphase I (1500x). c) Metaphase II spindle (2000x).
1057
Ther-iogenology
primarily dispermy, which affected about 20% of the inseminated oocytes, was the main abnormality. Decondensing sperm heads in the ooplasm, at 17 hours post insemination wen? observed in 4.4 and 2.8% of in vivo and in vitro-matured oocytes, respectively. At 41 hours post insemination, 58% of the in vitro matured inseminated oocytes cleaved and reached the two to four-cell stage in B, medium supplemented with 20% ESS (Table 4). In one preliminary experiment in vitro-matured and fertilized oocytes were transferred to the oviducts of synchronized ewes, and several blastocysts were recovered after 7 days (data not shown). DISCUSSION Almost 90% of the oocytes from follicles larger than 2 mm in diameter matured in vitro within 27 hours. The percentage of oocytes reaching Metaphase II was higher after 27 hours than after 24 hours in culture (Le Gal et al., 10). These maturation rates are comparable to those reported for caprine oocytes (11). Most of the oocytes (67%) from l- to 2-mm follicles resumed meiosis but remained arrested in Metaphase I, indicating that the ability for goat oocytes to achieve meiotic maturation was acquired in follicles larger than 2 mm in diameter. These results werf? comparable to those reported in the pig (12). By contrast, in cattle there is no eviclence that oocytes from small antral follicles fail to complete meiotic maturation in vitro (13). Handling goat follicle-enclosed oocytes at room temperature may induce further abnormalities during meiotic maturation. Although oocytes recovered at room temperature were able to progress up to the Metaphase II stage at the same frequency than the oocytes isolated at 30 to 35”C, 11.5% of them exhibited abnormal meiotic spindles. Exposure of immature goat oocytes to a subnormal temperature may affect further formation of the metaphase plate, resulting in aberrant chromosome distribution. In sheep, handling the follicle-enclosed oocytes and keeping them at room temperature for !j hours (14) did not induce such abnormalities. However, a decrease in temperature during germinal vesicle breakdown may result in further spindle disorganization (15). Addition of estrous sheep serum to the medium was previously proposed for ram sperm capacitation (1). The high capacity-binding protein, albumin, which is present in the serum, may facilitate sperm capacitation by contributing to the depletion of the sperm cholesterol membrane (16). The in vitro fertilization procedure proposed for sheep (1.9) led to a high level of fertilization and to a rapid sperm-oocyte penetration (17). Moreover, the oocytes fertilized under such conditions were able to support further embryonic development, as attested by the birth of 33 lambs (1, 18). In the present study, the same procedure was applied to goats, resulting in fertilization rates comparable to those obtained in sheep. Approximately 60% of ovulated and in vitro-matured oocytes were normally fertilized, as attested by the presence of both the female and male pronucleus ancl by remnants of the sperm tail in the ooplasm 17 hours after insemination. In all the fertilized oocytes the sperm tail was detected close to the male pronucleus, indicating thaI they were not parthenogenetically activated. In previously reported experiments, variable results were obtained according to the capacitation procedure. A short treatment with ionophore A23187 led to 35.3% of fertilized tubal oocytes (6). Capacitation in different culture media in the presence of heparin yielded fertilization rates ranging from
1058
Theriogenology
19.2 to 45.5% for in vivo-matured oocytes (11). Although a fertilization rate of 57% for in vitro-matured oocytes was reported by Song and lritani (4), their finding was based on normally fertilized zygotes as well as on polyspermic eggs and oocytes which failed to decondense the sperm chromatin. More recently, fertilization rates of 56.7%, as attested to by the presence of the male and female pronucleus or by cleavage, were achieved for in vitro-matured oocytes (11). However, the rate of cleavage (33.3%) ova within 48 hours of culture was lower than in the present experiment (58% within 41 hours). In our study, the incidence of polyspermy was relatively high, affecting nearly 20% of the inseminated oocytes. This was probably due to the simultaneous presence of several capacitated spermatozoa in close vicinity of the oocytes. The sperm concentration used for in vitro fertilization led to a much higher sperm-egg ratio than that observed for in vivo fertilization (19). However, extreme dilution of spermatozoa in the culture medium usually results in a dramatic decrease in sperm motility, which may be detrimental to oocyte penetration. The in vitro-matured oocytes underwent fertilization at the same rate than the ovulated oocytes. Moreover, the percentage of normally fertilized oocytes containing two pronuclei and remnants of the sperm tail was identical in both groups. Incomplete cytoplasmic maturation which may occur in vitro in suboptimal conditions results in oocytes that are incompetent to support normal fertilization and further embryonic development (20,21). Failure of sperm chromatin decondensation and of male pronucleus formation are the main abnormalities found during fertilization of such incompletely matured oocytes. The present results indicate that goat oocytes matured in vitro in the presence of granulosa cells and gonadotropins were fully Competent for supporting fertilization. Furthermore, several blastocysts were obtained from in vitro matured and fertilized oocytes. Experiments are in progress for evaluating the full developmental capacity of such in vitro produced zygotes after transfer to recipient goats. Ovaries collected at slaughter or by ovariectomy may provide an abundant source of immature oocytes at a low cost. The present results indicate that it is possible to obtain high rates of matured (90%) and normally fertilized (60%) goat oocytes in vitro. This production could be used in the near future for further research on gamete maturation, fertilization and early development as well as for biotechnological applications such as gene transfer in this domestic species.
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Crozet, N., Huneau, D., De Smedt, V., Theron, M.C., SzBIIBsi, D., Torres, S. and Sevellec, C. In vitro fertilization with normal development in the sheep. Gamete Res. 16: 159-l 70 (1987).
2.
Crozet, N. Manipulation of oocytes and in vitro fertilization. J. Reprod. Fertil. 43 (Suppl.): 235243 (1991).
3.
Song, H.B., Kasai, M., Niwa, K. and Iritani, A. Maturation culture of follicular oocytes from ovaries of immature goats treated with or without gonadotropins. Proc. 3rd AAAP Anim. Sci. Cong. Seoul (Korea) 1: 438440 (1985).
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Theriogenology
4.
Song, H.B. and Iritani, A. In vitro fertilization of goat foflicular oocytes with epididymal spermatozoa capacitated in a chemically defined medium. Proc. 3rd AAAP Anim. Sci. Cong. Seoul (Korea) 1: 463-465 (1985).
5.
Duan, E.K., Wang, G.Y., Ma, B.H. and Wang, J.C. Xenogenous fertilization of goat ovulated oocytes in the rabbit oviduct. Theriogenologya: 219 abstr. (1990).
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Hanada, A. In vitro fertilization in goats. Jpn. J. Anim. Reprod. 31: 21-26 (1985).
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Staigmiller, R.B. and Moor, R.M. Effect of follicle cells on the maturation and developmental competence of ovine oocytes matured outside the follicle. Gamete Res. 9: 211-229 (1984).
8.
Brackett, B.G. and Oliphant, G. Capacitation of rabbit spermatozoa Reprod. 2: 260-274 (1975).
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Huneau, D. and Crozet, N. In vitro fertilization in the sheep: effect of elevated calcium concentration at insemination. Gamete Res. 3: 119-l 25 (1989).
10.
Le Gal, F., Gall, L., De Smedt, V. Changes in protein synthesis pattern during in vitro maturation of goat oocytes. Mol. Reprod. Dev. (in press).
11.
Younis, A.I., Zuelke, K.A., Harper, K.M., Oliveira, M.A.L. and Brackett, B.G. In vitro fertilization of goat oocytes. Biol. Reprod. 44, 1177-1182 (1991).
12.
Motlik, J., Crozet, N. and Fulka, J. Meiotic competence in vitro of pig OOCytes isolated from early antral follicles. J. Reprod. Fertil. 72: 323-328 (1984).
13.
Leibfried, L. and First, N.L. Characterization of bovine follicular oocytes and their ability to mature in vitro. J. Anim. Sci. 3: 76-86 (1979).
14.
Staigmiller, R.B. In vitro method for production of viable oocytes. J. Anim. Sci. @ (suppl. 2): 54-64 (1988).
15.
Moor, R.M. and Crosby, I.M. Temperature induced abnormalities OOCytesduring maturation. J. Reprod. Fertil. 3: 467-473 (1985).
16.
Langlais, J., Kan, F.W.K., Granger, L., Raymond, L., Bleau, G. and Roberts, R.O. Identification of sterol acceptors that stimulate cholesterol efflux from human spermatozoa during in vitro capacitation. Gamete Res. 20: 185-201 (1988).
17.
Crozet, N. Fine structure of sheep fertilization in vitro. Gamete Res. 9: (1988).
18.
CogniB, Y., G&in, Y., Guyader, C., Poulin, N. and Crozet, N. In vitro fertilization of sheep oocytes matured in vitro. Theriogenology 35: 393-400 (1990).
19.
Cummins, J.M. and Yanagimachi, R. Sperm-egg ratios and the site of acrosome reaction during in vivo fertilization in the hamster. Gamete Res. 2: 239-256 (1982).
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in sheep
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Theriogenology
20.
Thibault, C. and Gerard, M. Facteur cytoplasmique necessaire a la formation du pronucleus male dans I’ovocyte de lapine. CR Acad. Sci. (Paris)m: 20252026 (1970).
21.
Moor, R.M. and Trounson, A.O. Hormonal and follicular factors affecting maturation of sheep oocytes in vitro and their subsequent developmental capacity. J. Reprod. Fert. 2: 101-109 (1977).