MATURATION IN WTRO AND SUBSEQUENT PENETRABILITY OF BOVINE
FOLLICULAR
OOCYTES 1
B. F. Shea 2 , J. P. A. Latour 3 , K. N. Bedirian4 and R. D. Baker
SUMMARY To investigate optimum conditions for the resumption of meiosis in vitro, 450 bovine follicular oocytes were incubated for 0 to 36 hr in Ham's medium F-10 with 10% fetal calf serum. At recovery one oocyte was binucleate, 4% were degenerative and 8% had undergone germinal vesicle breakdown. After 28 hr in culture, 60% of the oocytes reached metaphase II and further incubation did not increase the proportion that matured. No significant difference was observed in the proportion of oocytes that matured in vitro from follicles flushed either 1 hr (78%) or 2 hr (72%) after the death of the donors. Oocytes matured in media with pH's ranging from 6.70 to 7.59. The highest proportion (69%) reached metaphase II in media at pH 7.00 to 7.29. Attempts to obtain sperm penetration of bovine oocytes incubated in vitro were successful in the pig oviduct (17%) but not in the sheep oviduct. In conclusion, under conditions which yielded a large proportion of metaphase II oocytes, these oocytes were able to be penetrated and form pronuclei. (Key Words: Oocytes, Bovine, Maturation, Penetration.)
pool to upgrade world livestock, are slaughtered daily, for reasons other than ovarian dysfunction. Recovery and fertilization of follicular oocytes would make available additional sources of desirable genes. However, before fertilization can occur, the oocytes must resume meiosis to metaphase II or mature. Edwards (1965) demonstrated that cow follicular oocytes undergo the maturation division when incubated in vitro. This observation has since been confirmed by others (Foote and Thibault, 1969; Hunter et al., 1972; Jagiello et al., 1974). After an oocyte matures in vitro or in vivo, it must be fertilized or it soon degenerates. One of the first structures to be visibly affected is the second metaphase spindle and associated chromatin. Sufficient time must be allowed for oocytes to mature in vitro and yet not too much or degeneration occurs. Experiments were undertaken to determine the time interval required for bovine oocytes to reach metaphase II in vitro, to determine the optimum conditions for culture and to investigate the penetrability of oocytes matured in vitro by bull spermatozoa.
INTRODUCTION
MATERIALS AND METHODS
Follicular oocytes comprise a large group of genetic material which has not been utilized to its fullest extent. Superior females, whose ovaries might contribute to the available gene
Ovaries from cows and heifers of various ages and breeds were obtained at a local abattoir. Approximately 20 min elapsed between death of the animal and removal of the ovaries. A further 20 min was required for transport to the laboratory. No precautions were taken to maintain body temperature. The ovaries were washed with saline to remove adhering blood. Visible follicles were then punctured and the contents expelled into a disposable petri dish (100 • 15 ram, Falcon Plastic Company, Los Angeles) containing 10 ml culture medium (table 1), supplemented with 500 units heparin to prevent clotting of the follicular fluid. The oocytes were then trans-
1This study was supported financially by Quebec Agriculture Research Council grant No. McA-72-491 and Medical Research Council grant No. 3711. 2Present address: University of Calgary, Division of Medical Biochemistry, Calgary, Alberta. 3Department of Obstetrics and Gynaecology, Royal Victoria Hospital, Montreal. 4present address: Via Pax Corporation Ltd., 7531 Pine Valley Drive, Woodbridge, Ontario. s Department of Animal Science.
8O9 JOURNAL OF ANIMAL SCIENCE, Vol. 43, No. 4 (1976)
Downloaded from https://academic.oup.com/jas/article-abstract/43/4/809/4697570 by 04860000 user on 14 January 2019
Macdonald Campus of McGill University s, Ste-Anne-de-Bellevue, Quebec HOA 1CO
810
SHEA ET AL.
TABLE 1. MEDIUM FOR INCUBATINGBOVINE OOCYTES Tissue culture medium Ham's F-IOa
90 mt
Fetal calf seruma Penicillina Streptomycin a Sodium bicarbonate (10%)a
10ml 10 91 per ml 10 ~g per ml to yield pH 7.1 when gassed with 5% CO2 in air
ferred to petri dishes (60 • 15 mm, Falcon) containing 10 to 15 ml fresh Ham's F-10 culture medium without heparin. The medium had been passed through a filter (.22/a, MiNipore Filter Corporation, Bedford) and was placed under the conditions of incubation for a minimum of 2 hr before starting the incubation to allow equilibrium of the temperature (37 C) and gases (5% CO2 in air). After incubation (0 to 30 hr) the oocytes were removed from culture and denuded of surrounding follicular cells by mechanical agitation in saline for 5 to 10 minutes. The oocytes were then mounted and fixed (three parts ethanohone part glacial acetic acid) for 24 hr, stained with aceto-orcein and examined to determine the stage of maturation. If examination revealed an intact nuclear membrane with the chromatin pattern characteristic of the meiotically inactive primary oocyte, it was designated as being in the germinal vesicle or vesicular nucleus (VN) stage. If the nuclear membrane had broken down and a chromatin pattern characteristic of an oocyte resuming meiosis was present, it was designated as having undergone g e r m i n a l vesicle breakdown (GVBD), which included those stages from diakinesis to metaphase II. If a polar body was present within the perivitelline space and the maternal chromatin complement was identified, the oocyte was designated as being mature, that is, having completed meiosis to the ovulatory stage. If an oocyte had undergone obvious degenerative changes, such as an ooplasm which was vacuolated, shrunken or fragmented or a chromatin complement which was scattered or very highly condensed, then it was classed as degenerate. The first experiment was designed to define the timing of the meiotic division in vitro under our culture conditions. All oocytes from one set of ovaries were placed into a culture dish. About 30% of the oocytes were randomly selected as controls and were removed from the
culture for fixation. The remaining oocytes were incubated for 22 to 30 hours. The second experiment was set up to study the effect of increasing the time from slaughter to incubation from the usual 1-hr interval to 2 hours. Oocytes from one ovary of each female were recovered and placed in culture within 1 hr after death as in experiment 1. The remaining ovary was held at room temperature until 2 hr elapsed between death of the female and recovery of the oocytes, which were placed into culture. The third experiment was designed to investigate the influence of pH of the medium on oocyte maturation. Equal numbers of oocytes from each female were randomly placed into culture dishes with medium adjusted with glacial acetic acid or bicarbonate to yield pH ranges of 6.70 to 6.99, 7.00 to 7.29 or 7.30 to 7.59. The fourth experiment was concerned with the fertilization of oocytes matured in vitro. Several attempts in our lab to fertilize bovine oocytes in vitro were not successful (Baker and Polge, 1976). Ten immature pigs (70 to 90 kg) injected intramuscularly with 2 ml of saline containing 400 IU of Pregnant Mare's Serum Gonadotropin (PMSG-Equinex, Ayerst Laboratories, Montreal) plus 200 IU of Human Chorionic Gonadotropin (HCG--ALP, Ayerst Laboratories, Montreal) and five cycling ewes were used as recipients for incubated bovine oocytes. Gilts and ewes were used rather than heifers due to the availability of females and the costs involved. Bovine oocytes were shown to be penetrable in sheep oviducts (Sreenan, 1970) and porcine oviducts (Bedirian et al., 1975). The oocytes used in this study were incubated under optimal conditions (28 hr in Ham F-10 at pH 7.2 and 37 C) and were transferred to the infundibulum of the oviducts of estrous ewes and gilts 96 hr after the PMSG:HCG injection or approximately 20 hr prior to ovulation. Bull semen diluted 1:3 with Tyrode's solution was
Downloaded from https://academic.oup.com/jas/article-abstract/43/4/809/4697570 by 04860000 user on 14 January 2019
aSupplied by Difco Laboratories, Detroit.
MATURATION OF BOVINE OOCYTES
811
Downloaded from https://academic.oup.com/jas/article-abstract/43/4/809/4697570 by 04860000 user on 14 January 2019
Figure 1. Non-denuded bovine follicular oocyte showing surrounding cells of the corona radiata (CR), zona pellucida (Z) and the ooplasm (O). (Unstained, phase contrast, • Figure 2. Vesicular nucleus of follicular oocyte with nucleus and chromatin threads discernible (stained with aceto-orcein, phase contrast, X 1000). Figure 3. Binucleate bovine follicular oocyte. The two vesicular nuclei were totally distinct from one another and were not fused (stained with aceto-orcein, phase contrast, • 500).
SHEA ET AL.
812
TABLE 2. TIMING OF MATURATION OF BOVINE OOC'YTES I N V I T R O Time in culture (hours) 0
22-25
26-28
29-30
No. of ooeytes Germinal vesicle, % Metaphase I, % Anaphase I or Telophase 1, % Metaphase II, % Degenerate, %
54 89 4 0 . 4a 4
38 21 24 18 34 b 3
48 8 17 6 60 c 8
26 4 8 0 73 c 15
a'b'CValues with different superscripts are significantly (P
FOLLICULAR
OOCYTES 1
B. F. Shea 2 , J. P. A. Latour 3 , K. N. Bedirian4 and R. D. Baker
SUMMARY To investigate optimum conditions for the resumption of meiosis in vitro, 450 bovine follicular oocytes were incubated for 0 to 36 hr in Ham's medium F-10 with 10% fetal calf serum. At recovery one oocyte was binucleate, 4% were degenerative and 8% had undergone germinal vesicle breakdown. After 28 hr in culture, 60% of the oocytes reached metaphase II and further incubation did not increase the proportion that matured. No significant difference was observed in the proportion of oocytes that matured in vitro from follicles flushed either 1 hr (78%) or 2 hr (72%) after the death of the donors. Oocytes matured in media with pH's ranging from 6.70 to 7.59. The highest proportion (69%) reached metaphase II in media at pH 7.00 to 7.29. Attempts to obtain sperm penetration of bovine oocytes incubated in vitro were successful in the pig oviduct (17%) but not in the sheep oviduct. In conclusion, under conditions which yielded a large proportion of metaphase II oocytes, these oocytes were able to be penetrated and form pronuclei. (Key Words: Oocytes, Bovine, Maturation, Penetration.)
pool to upgrade world livestock, are slaughtered daily, for reasons other than ovarian dysfunction. Recovery and fertilization of follicular oocytes would make available additional sources of desirable genes. However, before fertilization can occur, the oocytes must resume meiosis to metaphase II or mature. Edwards (1965) demonstrated that cow follicular oocytes undergo the maturation division when incubated in vitro. This observation has since been confirmed by others (Foote and Thibault, 1969; Hunter et al., 1972; Jagiello et al., 1974). After an oocyte matures in vitro or in vivo, it must be fertilized or it soon degenerates. One of the first structures to be visibly affected is the second metaphase spindle and associated chromatin. Sufficient time must be allowed for oocytes to mature in vitro and yet not too much or degeneration occurs. Experiments were undertaken to determine the time interval required for bovine oocytes to reach metaphase II in vitro, to determine the optimum conditions for culture and to investigate the penetrability of oocytes matured in vitro by bull spermatozoa.
INTRODUCTION
MATERIALS AND METHODS
Follicular oocytes comprise a large group of genetic material which has not been utilized to its fullest extent. Superior females, whose ovaries might contribute to the available gene
Ovaries from cows and heifers of various ages and breeds were obtained at a local abattoir. Approximately 20 min elapsed between death of the animal and removal of the ovaries. A further 20 min was required for transport to the laboratory. No precautions were taken to maintain body temperature. The ovaries were washed with saline to remove adhering blood. Visible follicles were then punctured and the contents expelled into a disposable petri dish (100 • 15 ram, Falcon Plastic Company, Los Angeles) containing 10 ml culture medium (table 1), supplemented with 500 units heparin to prevent clotting of the follicular fluid. The oocytes were then trans-
1This study was supported financially by Quebec Agriculture Research Council grant No. McA-72-491 and Medical Research Council grant No. 3711. 2Present address: University of Calgary, Division of Medical Biochemistry, Calgary, Alberta. 3Department of Obstetrics and Gynaecology, Royal Victoria Hospital, Montreal. 4present address: Via Pax Corporation Ltd., 7531 Pine Valley Drive, Woodbridge, Ontario. s Department of Animal Science.
8O9 JOURNAL OF ANIMAL SCIENCE, Vol. 43, No. 4 (1976)
Downloaded from https://academic.oup.com/jas/article-abstract/43/4/809/4697570 by 04860000 user on 14 January 2019
Macdonald Campus of McGill University s, Ste-Anne-de-Bellevue, Quebec HOA 1CO
810
SHEA ET AL.
TABLE 1. MEDIUM FOR INCUBATINGBOVINE OOCYTES Tissue culture medium Ham's F-IOa
90 mt
Fetal calf seruma Penicillina Streptomycin a Sodium bicarbonate (10%)a
10ml 10 91 per ml 10 ~g per ml to yield pH 7.1 when gassed with 5% CO2 in air
ferred to petri dishes (60 • 15 mm, Falcon) containing 10 to 15 ml fresh Ham's F-10 culture medium without heparin. The medium had been passed through a filter (.22/a, MiNipore Filter Corporation, Bedford) and was placed under the conditions of incubation for a minimum of 2 hr before starting the incubation to allow equilibrium of the temperature (37 C) and gases (5% CO2 in air). After incubation (0 to 30 hr) the oocytes were removed from culture and denuded of surrounding follicular cells by mechanical agitation in saline for 5 to 10 minutes. The oocytes were then mounted and fixed (three parts ethanohone part glacial acetic acid) for 24 hr, stained with aceto-orcein and examined to determine the stage of maturation. If examination revealed an intact nuclear membrane with the chromatin pattern characteristic of the meiotically inactive primary oocyte, it was designated as being in the germinal vesicle or vesicular nucleus (VN) stage. If the nuclear membrane had broken down and a chromatin pattern characteristic of an oocyte resuming meiosis was present, it was designated as having undergone g e r m i n a l vesicle breakdown (GVBD), which included those stages from diakinesis to metaphase II. If a polar body was present within the perivitelline space and the maternal chromatin complement was identified, the oocyte was designated as being mature, that is, having completed meiosis to the ovulatory stage. If an oocyte had undergone obvious degenerative changes, such as an ooplasm which was vacuolated, shrunken or fragmented or a chromatin complement which was scattered or very highly condensed, then it was classed as degenerate. The first experiment was designed to define the timing of the meiotic division in vitro under our culture conditions. All oocytes from one set of ovaries were placed into a culture dish. About 30% of the oocytes were randomly selected as controls and were removed from the
culture for fixation. The remaining oocytes were incubated for 22 to 30 hours. The second experiment was set up to study the effect of increasing the time from slaughter to incubation from the usual 1-hr interval to 2 hours. Oocytes from one ovary of each female were recovered and placed in culture within 1 hr after death as in experiment 1. The remaining ovary was held at room temperature until 2 hr elapsed between death of the female and recovery of the oocytes, which were placed into culture. The third experiment was designed to investigate the influence of pH of the medium on oocyte maturation. Equal numbers of oocytes from each female were randomly placed into culture dishes with medium adjusted with glacial acetic acid or bicarbonate to yield pH ranges of 6.70 to 6.99, 7.00 to 7.29 or 7.30 to 7.59. The fourth experiment was concerned with the fertilization of oocytes matured in vitro. Several attempts in our lab to fertilize bovine oocytes in vitro were not successful (Baker and Polge, 1976). Ten immature pigs (70 to 90 kg) injected intramuscularly with 2 ml of saline containing 400 IU of Pregnant Mare's Serum Gonadotropin (PMSG-Equinex, Ayerst Laboratories, Montreal) plus 200 IU of Human Chorionic Gonadotropin (HCG--ALP, Ayerst Laboratories, Montreal) and five cycling ewes were used as recipients for incubated bovine oocytes. Gilts and ewes were used rather than heifers due to the availability of females and the costs involved. Bovine oocytes were shown to be penetrable in sheep oviducts (Sreenan, 1970) and porcine oviducts (Bedirian et al., 1975). The oocytes used in this study were incubated under optimal conditions (28 hr in Ham F-10 at pH 7.2 and 37 C) and were transferred to the infundibulum of the oviducts of estrous ewes and gilts 96 hr after the PMSG:HCG injection or approximately 20 hr prior to ovulation. Bull semen diluted 1:3 with Tyrode's solution was
Downloaded from https://academic.oup.com/jas/article-abstract/43/4/809/4697570 by 04860000 user on 14 January 2019
aSupplied by Difco Laboratories, Detroit.
MATURATION OF BOVINE OOCYTES
811
Downloaded from https://academic.oup.com/jas/article-abstract/43/4/809/4697570 by 04860000 user on 14 January 2019
Figure 1. Non-denuded bovine follicular oocyte showing surrounding cells of the corona radiata (CR), zona pellucida (Z) and the ooplasm (O). (Unstained, phase contrast, • Figure 2. Vesicular nucleus of follicular oocyte with nucleus and chromatin threads discernible (stained with aceto-orcein, phase contrast, X 1000). Figure 3. Binucleate bovine follicular oocyte. The two vesicular nuclei were totally distinct from one another and were not fused (stained with aceto-orcein, phase contrast, • 500).
SHEA ET AL.
812
TABLE 2. TIMING OF MATURATION OF BOVINE OOC'YTES I N V I T R O Time in culture (hours) 0
22-25
26-28
29-30
No. of ooeytes Germinal vesicle, % Metaphase I, % Anaphase I or Telophase 1, % Metaphase II, % Degenerate, %
54 89 4 0 . 4a 4
38 21 24 18 34 b 3
48 8 17 6 60 c 8
26 4 8 0 73 c 15
a'b'CValues with different superscripts are significantly (P
