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The co-culture of cumulus-enclosed bovine oocytes and hemi-sections of follicles: Effects on meiotic resumption
Theriogenology
40:933-942,
1993
THE CO-CULTURE OF CUMULUS-ENCLOSED BOVINE OOCYTES AND HEMISECTIONS OF FOLLICLES: EFFECTS ON MEIOTIC RESUMPTION M.A. Sirard 1 and K. Coenen Departement de Zootechnie and Unite d’ontogenie et Reproduction Centre de Recherche du CHUL, Universite Laval, Quebec, GlK 7P4 Received for publication: Accepted:
October
27,
JUZY 23,
1992
1993
ABSTRACT To prolong the culture of oocytes, it is essential to know how the follicle maintains meiotic arrest. This study was undertaken to evaluate the short-term effects (24 h) of the co-culture of follicular hemi-sections, including theta and granulosa cells, with cumulus-enclosed primary oocytes on meiotic resumption. Bovine oocytes were collected from 1 to 5-mm follicles from ovaries kept at 35°C. Follicular hemi-sections were prepared by careful dissection of another group of follicles of the same size but from ovaries transported on ice. Following 24 h of coculture, the oocytes were either fixed for determination of nuclear maturation or matured for an additional 24 h to evaluate reversibility of inhibition. The inhibitory action of the hemi-sections on meiotic resumption of oocytes was directly related to the amount of tissue and did not require direct physical contact between the cumulus and the follicular wall. The inhibition was reversible after 24 h of coculture. Therefore, follicular tissue can be used to maintain meiotic arrest for at least 24 h, thus allowing for the study of changes in developmental competence during late folliculogenesis. Key words: meiosis, oocyte, follicle, cattle, culture INTRODUCTION In mammals, the removal of the cumulus-enclosed oocyte from the ovarian follicle induces the resumption of meiotic division (14). This natural phenomenon has lead to the possibility of culturing immature oocytes from growing follicles. In cattle, in vitro maturation of immature oocytes recovered after slaughter, followed by in vitro fertilization has lead to embryonic development and the birth of calves (10,23). Acknowledgments: This work was supported by the Natural Research Council of Canada and Semex Canada. lCorrespondence and reprint requests.
Copyright 0 1993 Buttetwot-th-Heinemann
Sciences
and
Engineering
934
Since most oocytes selected for maturation based on the healthy appearance of their cumulus do not express developmental competence, it is suspected that their cytoplasmic maturation has not been completed. This is supported by a recent study showing that large follicles contain oocytes with a high developmental capacity (13). Therefore, most oocytes would benefit from the influence of large follicles if the maturation conditions could be reproduced in vitro. To achieve this purpose it is essential to maintain the imma~re status of the nucleus. Nuclear transformation is believed to result from the removal of an inhibitory component present in the follicle (2, 26). In mice, it has been demonstrated that hypoxanthine, a component of follicular fluid, -could maintain cumulus-enclosed oocytes in a germinal vesicle configuration for extended periods (3). It was later shown that the inhibitory activity of various purines was directly related to their phosphodiesterase inhibitory activity, thus explaining their synergism with CAMP (4. In cattle, the purines fadenosine and hypoxanthine) have a transient effect on meiotic resumption and therefore can not be used for prolonged culture in the germinal vesicle configuration (21). The replacement of purines with other potent phosphodiesterase inhibitors stimulates cumulus expansion and results in transient meiotic inhibition unless a toxic amount of dibutiryl-CAMP or forskolin is added (7, 21). Other factors reported to inhibit bovine oocyte meiotic resumption in vitro are protein synthesis inhibitors (12,22), phosphorylation inhibitors such as 6-DMAP (6), protease and RNA synthesis inhibitors (ll), and sodium fluoride (17). Most of these products are not compatible with a long-term survival of the oocyte and therefore can not be used to enhance its developmental competence in culture. More than 20 years ago, Foote and Thibault (5) asked if it were possible to use the natural meiotic i~ibitory function of the follicfe in vitro. The answer is still unclear today. Using bovine oocytes and a portion of the follicular wall, they concluded that a contact was required between the granulosa, but not the theta and the cumulus to maintain the dyctiate stage in the oocyte nucleus. More recently, an experiment in the hamster showed that the surgical transfer of oocytes from follicle to follicle was successful in maintaining the meiotic arrest only if close contact was re-established between the granulosa and the cumulus (15). Does the contact act as a signal or the close proximity the only requirement for meiotic arrest? Cumulus-enclosed bovine oocytes are sensitive to the inhibitory influence of granulosa cells either in suspension (18) or in a monolayer (191, but a large number of cells is required to achieve this inhibition, and direct contact is important in this process. Is the signal metabolized by the cumulus or does it accumulate in the follicular fluid? Follicular fluid alone has a dose-dependant transient inhibitory effect on meiotic resumption on the oocyte (201. To understand the mechanism by with the follicle controls meiotic resumption, it is necessary to reconstitute its components in vitro. Therefore, this study was designed to examine the effect of the dissected follicular wall in co-culture with cumulus enclosed oocytes on meiotic resumption.
935
Theriogenology
MATERIALS
AND METHODS
Recovery of Oocytes Ovaries from non-pregnant or pregnant heifers or cows were removed within 30 min after slaughter and transported to the laboratory at about 35’C in 0.9% NaCl aqueous solution containing 100,000 IIJ/l penicillin, 100 mg/l streptomycin and 250 pg/f amphoteri~in B (Sigma, St-Louis, MO, USA). Briefly, the cumulus-oo~te complexes (COC) were aspirated from 1 to 5-mm follicles with an 18-g needle, pooled, and selected for a complete or compact cumulus (8). The COC were washed time rapidly in Hepes buffered Tyrode’s medium (TLH) (1) supplemented with 10% heat-treated fetal calf serum (FCS; Flow lab., Missisauga, Canada), 0.2 mM pyruvic acid (Sigma), and 50 pg/ml gentamicin (Sigma). The oocytes were then immediately transferred to their respective treatments described below. Preparation
of Half-Follicles
and the Culture of Oocytes
Bovine follicles were obtained from ovaries collected at random at an abattoir and transported in saline as described above but on ice. Follicles were dissected free of the interstitial tissue and were selected according to size (1 to 5-mm), transparency and a nearly perfect spherical shape. The selected follicles (mainly 2 to 3-mm diameters) were then halved with a scalpel before being washed in TCM-199 medium. Follicles with loosely attached granulosa layers were discarded before incubation; 0 (control), 1,2 or 5 hemi-sections were used to assess a dose response to follicular influence. Care was taken not to use 2 hemi-sections from the same follicle when 2 hemi-sections were used together, to minimize follicIe to follicle variations. Each experiment was replicated 2 to 5 times with a minimum of IO oocytes per treatment, for a total number of 769 individual measures. In the first experiment, to evaluate the effect of the follicles on meiotic resumption, increasing numbers (1,2 5) of follicular hemi-sections were co-incubated in 50-uL droplets under oil (Aldrich Chemical, Milwaukee, WI, USA) with 10 COC for 24 h in a media similar to that generally used for in vitro maturation: TCM-199 with Earle’s salt (Gibco Laboratories, Burlington, Ontario, Canada) and bicarbonate (Sigma) supplemented with 10% heat-treated FCS (Flow Laboratories); 0.2 mM pyruvic acid; 50 pg/ml gentamicin sulfate; 1 pg/ml FSH (National Institute of Diabetes and Digestive and Kidney Diseases); and 1 l.tg/ml estradiol 1713 (Sigma). The droplets were pre-exposed to the culture conditions for at least 2 h (38.5”C, 5% COZ, 95% air atmosphere with 100% humidity) before being used. The oocytes were added to the medium after the follicles but not inside the small cups formed by the hemisections of the follicules. In a second experiment, to determine if the conditioning of the same medium by hemi-sections would enhance inhibit.3ry capacity, the hemi-sections, one ,two or five were preincubated 24 h before the oocytes were added to the medium as above.
Theriogenology
936
In a third experiment media conditioned for 24 h was use alone with 10 COC to determine if the inhibitory products or their effect would accumulate in the media. And in the fourth experiment, to assess the reversibility of meiotic inhibition, oocytes preincubated with either 1, 2 or 5 hemi-sections for a first 24-h period like in first experiment, were further cultured without hemi-sections in a maturation medium for a subsequent 24 h. The maturation medium used for the second 24 h consisted of the same media as above but with different hormonal supplements (5 pg/ml LH [NIDDKI and 1 pg/ml progesterone). Fixation of Oocytes At the end of the incubation, the oocytes were transferred into small centrifuge tubes and were vortex-agitated for 5 min. Completely denuded oocytes were recovered and transferred to glass slides in small drops. Vaseline and paraffin were used to maintain the coverslip in contact with the oocytes without excessive pressure. The coverslip was then fixed with epoxy glue, and the slide was immersed in a fixative solution (ethanol: acetic acid, 3:ll for a minimum of 24 h. The slides were stained with 1% aceto-orcein and examined with phase contrast microscope at x 100 and x 400 magnification. Oocytes were classified as germinal vesicles (GV); intermediate stage (from germinal vesicle breakdown to Metaphase I); mature stage (from Anaphase I to Metaphase II) and degenerated (abnormal change in the cytoplasm or if chromatin impossible to assess after fixation). The significance of individual comparisons between treatments were evaluated using Chi-square analysis with correction for continuity (24). RESULTS The first figure illustrates status of the nucleus of oocytes following 24-h incubation with an increase in the number of hemi-sections in the culture drops. In all treatments, most of the oocytes were found to be at the germinal vesicle stage, but co-incubation with 5 hemi-sections resulted in the highest number of meiotic-arrest oocytes P
Theriogenology
937
1
lStemi-sections5
control
Nuclear status of bovine cumulus-enclosed oocytes (n=230) co-cultured with 0 (control), 1, 2 or 5 follicular hemi-sections for 24 hours. The oocytes were classified as germinal vesicle (GV), intermediary or mature. Compared to the control, all treatments are different (Chi-square; P
100
60
1, 24
h
2, 24 h
5, 24 h
control
Hemi-sections Nuclear status of bovine cumulus-enclosed oocytes co-cultured (n=249) for 24 hours with 0 (control), 1, 2 or 5 follicular hemi-sections that were put in culture 24 hours before the addition of oocytes. The oocytes were classified as germinal vesicle (GV), intermediary or mature. Compared to the control, all treatments are different (Chi-square; P
Theriogenology
938 g
100 -
Sk :
H GV
!
al
inrnmeediary
80 -
0 1
2
5
Hemi-section
supernatant
control
Figure 3. Nuclear status of cumulus-enclosed oocytes (n=246) cultured for 24 hours in conditioned medium obtained from the culture of 1, 2 or 5 follicular hemi-sections in small droplets compared with that of culture in fresh medium (control). The oocytes were classified as germinal vesicle (GV), intermediary or mature. Compared to the control, all treatments are different for intermediary and mature-stages (Chi-square; P
Theriogenology
939
GV intermediary mature
80
60
f 40
El
b
20
0 1
2
Number of hemi-sections
5
used during the inhibition period
Figure 4. Nuclear status of bovine cumulus-enclosed oocytes (n=164) cultured in maturation media (cf methods) without hemi-sections for 24 hours following an initial 24-ho.ur period of co-culture with 1, 2 or 5 follicular hemi-sections. The oocytes were classified as germinal vesicle (GV), intermediary or mature. The letters a, b, c, d, e and f indicate a difference between treatments (Chi-square; P
940
Theriogenoiogy
data were obtained from a limited number of oocytes (n=8), the conclusion of the above authors was that contact between granulosa and cumulus cells is essential to prevent cumulus expansion and meiotic resumption. The difference between the above results and those in our study may be explained by 2 factors: 1) The volume of culture medium in the earlier experiment did not allow for the accumulation of a sufficient amount of putative inhibitor to inhibit all oocytes. This conclusion is further supported by the fact that meiotic resumption in oocytes cultivated inside the dome formed by the hemi-sections but without complete contact was also inhibited in 7 of 8 instances (5). 2) The closer contact of the cultured oocytes with the inner layer of the follicle (granulosa) in earlier study resulted in a contact signal which prevented meiotic resumption. This effect was not observed when the oocytes were positioned on the outer layer (interstitial and theca) as in our study. Leibfried and First (9) investigated the effect of the hem§ion co-culture on porcine oocytes. Their results showed that the presence of the follicular wall can maintain the dyctiate stage in nearly 50% of the oocytes. The incubation conditions averaged 1 hemi-section and 2 to 3 oocytes per ml of TCM-199 in culture dishes. Porcine oocytes are also sensitive to granulosa cells alone, since meiotic inhibition could be observed by co-culturing cumulus-enclosed oocytes in direct or indirect contact with 10 million granulosa cell from medium-sized follicles or a portion of the follicular wall (25). In contrast, Sato and Ishibashi (161, using porcine follicles, showed that the prevention of direct contact between the oocytes and the granulosa cells eliminated most of the inhibitory effect. The in~bitory effect of direct granulosa-cumulus contact was also observed in bovine oocytes using agar embedding to separate (200 to 300 urn) cumulus-enclosed oocytes from a suspension of granulosa cells (19). Since close proximity between the 2 types of cells results in a higher gradient of any possible factor, it is quite possible that close contact by itself is not the principal inhibitory pathway. The results presented here provide new information on the possible mechanism of meiotic inhibition. The dose-response inhibitory influence of the follicular wall without direct contact between cum~us-granulosa cells supports the presence of a putative factor produced by the follicle and which acts on a population of oocytes which respond heterogenously to a given amount of this factor. The action of the hemi-sections does not seems to depend on the metabolism or removal of products present in the maturation medium (TCM 199) since conditioned medium is not as inhibitory for the resumption of meiosis as the hemi-sections themselves. The bovine foiiicular fluid-like effect of the conditioned medium would support the hypothesis of a labile factor with either a short half-life or rapid degradation by the cumulus cells. The in~bition obtained with use of the follicular wall is reversible unless a large amount of tissue is used in the culture medium, which may injure the oocyte.
941
Theriogenology
REFERENCES 1. 2. 3.
4.
5. 6. 7.
8. 9. 10. 11. 12. 13. 14. 15.
16.
Bavister BD, Leibfried L.M, Lieberman G. Development of preimplantation embryos of the golden hamster in a defined culture medium. Biol Reprod 1983; 28: 235-247. Buccione R, Schroeder AC, Eppig JJ. Interactions between somatic cells and germ cells throughout mammalian oogenesis. Biol Reprod 1990; 43: 543-547. Downs SM, Coleman DL, Ward-Bailey PF, Eppig JJ. Hypoxanthine is the principal inhibitor of murine oocyte maturation in a low molecular weight fraction of porcine follicular fluid. Proc Nat1 Acad Sci USA 1985; 82: 454-458. Downs SM, Daniel SA, Bornslaeger EA, Hoppe PC, Eppig JJ. Maintenance of meiotic arrest in mouse oocytes by purines: modulation of CAMP levels and CAMP phosphodiesterase activity. Gamete Res 1989; 23: 323-334. Foote WD, Thibault C. Recherches experimentales sur la maturation in vitro des ovocytes de truie et de veau. Am Biol anim Bioch Biophys 1969; 3: 329-349. Fulka JJr, Leibfried-Rutledge ML, First NL. Effect of Dimethylaminopurine on germinal vesicle breakdown in bovine oocytes. Molec Reprod Develop 1991; 29: 379-384. Homa ST. Effects of cyclic AMP on the spontaneous meiotic maturation of cumulus-free bovine oocytes cultured in chemically defined medium. J Exp Zoo1 1988; 248: 222-231. Leibfried ML, First NL. Characterization of bovine follicular oocytes and their ability to mature in vitro. J Anim Sci 1979; 48: 76-86. Leibfried ML, First NL. Effect of bovine and porcine follicular fluid and granulosa cells on maturation of oocytes in vitro. Biol Reprod 1980; 23: 699704. Lu KH, Gordon I, Gallagher M, McGovern H. Pregnancy established in cattle by transfer of embryos derived from in vitro fertilisation of oocytes matured in vitro. Vet Ret 1987; 121: 259-260. Motlik J. Cytoplasmic aspects of oocyte growth and maturation in mammals. J Reprod Fertil 1989; suppl38: 17-25. Motlik J, Lie BY, Shioya Y. Sensitivity levels of cattle oocytes to puromycin. Biol Reprod 1990; 43: 994-998. Pavlok A, Lucas-Hahn A, Niemann H. Fertilization and developmental competence of bovine oocytes derived from different categories of antral follicles. Mol Reprod Develop 1992; 31: 63-67. Pincus G, Enzmann EV. The comparative behavior of mammalian eggs in vitro and in vivo. J Exp Med 1935; 62: 665-675. Racowsky C, Baldwin KV. In vitro and in vivo studies reveal that hamster oocyte meiotic arrest is maintained only transiently by follicular fluid, but persistently by membrane/cumulus granulosa cell contact. Dev Biol 1989; 134: 297-306. Sato E, Ishibashi T. Meiotic arresting action of the substance obtained from cell surface of porcine ovarian granulosa cells. Jap J Zootech Sci 1977; 48: 22-26.
942 17. 18. 19. 20, 21. 22. 23. 24. 25. 26.
Theriogenology
Sirard M.A. Temporary inhibition of in vitro meiotic resumption by adenylate cyclase stimulation in immature bovine oocytes. Theriogenology 1990; 33: 757-767. Sirard MA, Bilodeau S. Effects of granulosa cell co-culture on in-vitro meiotic resumption of bovine oocytes. J Reprod Fertil 1990; 89: 459-465. Sirard MA, Bilodeau S. Granulosa cells inhibit the resumption of meiosis in bovine oocytes in vitro. Biol Reprod 1990; 43: 777-783. Sirard MA, Coenen K, Bilodeau S. Effect of fresh or cultured follicular fractions on meiotic resumption in bovine oocytes. Theriogenology 1992; 37: 39-57. Sirard MA, First NL. In vitro inhibition of oocytes nuclear maturation in the bovine. BioI Reprod 1988; 39: 229-234. Sirard MA, Florman HM, L~bfried-Rutledge ML, Barnes FL, Sims ML, First NL. Timing of nuclear progression and protein synthesis necessary for meiotic maturation of bovine oocytes. Biol Reprod 1989; 40: 1257-1263. Sirard MA, Parrish JJ, Ware CB, Leibfried-Rutledge ML, First NL. The culture of bovine oocytes to obtain developmentally competent embryos. Biol Reprod 1988; 39: 546-552. Snedecor GW, Cochran WG. Statistical Methods. Iowa State University Press, Ames, IA 1980. Tsafriri A, Channing C. An inhibitory influence of granulosa cells and follicular fluid upon porcine oocyte meiosis in vitro. Endo 1975; 96: 922-927. Tsafriri A, Dekel N, Bar-Ami S. The role of oocyte maturation inhibition in follicular regulation of oocyte maturation. J Reprod Fertil 1982; 64: 541-551.
40:933-942,
1993
THE CO-CULTURE OF CUMULUS-ENCLOSED BOVINE OOCYTES AND HEMISECTIONS OF FOLLICLES: EFFECTS ON MEIOTIC RESUMPTION M.A. Sirard 1 and K. Coenen Departement de Zootechnie and Unite d’ontogenie et Reproduction Centre de Recherche du CHUL, Universite Laval, Quebec, GlK 7P4 Received for publication: Accepted:
October
27,
JUZY 23,
1992
1993
ABSTRACT To prolong the culture of oocytes, it is essential to know how the follicle maintains meiotic arrest. This study was undertaken to evaluate the short-term effects (24 h) of the co-culture of follicular hemi-sections, including theta and granulosa cells, with cumulus-enclosed primary oocytes on meiotic resumption. Bovine oocytes were collected from 1 to 5-mm follicles from ovaries kept at 35°C. Follicular hemi-sections were prepared by careful dissection of another group of follicles of the same size but from ovaries transported on ice. Following 24 h of coculture, the oocytes were either fixed for determination of nuclear maturation or matured for an additional 24 h to evaluate reversibility of inhibition. The inhibitory action of the hemi-sections on meiotic resumption of oocytes was directly related to the amount of tissue and did not require direct physical contact between the cumulus and the follicular wall. The inhibition was reversible after 24 h of coculture. Therefore, follicular tissue can be used to maintain meiotic arrest for at least 24 h, thus allowing for the study of changes in developmental competence during late folliculogenesis. Key words: meiosis, oocyte, follicle, cattle, culture INTRODUCTION In mammals, the removal of the cumulus-enclosed oocyte from the ovarian follicle induces the resumption of meiotic division (14). This natural phenomenon has lead to the possibility of culturing immature oocytes from growing follicles. In cattle, in vitro maturation of immature oocytes recovered after slaughter, followed by in vitro fertilization has lead to embryonic development and the birth of calves (10,23). Acknowledgments: This work was supported by the Natural Research Council of Canada and Semex Canada. lCorrespondence and reprint requests.
Copyright 0 1993 Buttetwot-th-Heinemann
Sciences
and
Engineering
934
Since most oocytes selected for maturation based on the healthy appearance of their cumulus do not express developmental competence, it is suspected that their cytoplasmic maturation has not been completed. This is supported by a recent study showing that large follicles contain oocytes with a high developmental capacity (13). Therefore, most oocytes would benefit from the influence of large follicles if the maturation conditions could be reproduced in vitro. To achieve this purpose it is essential to maintain the imma~re status of the nucleus. Nuclear transformation is believed to result from the removal of an inhibitory component present in the follicle (2, 26). In mice, it has been demonstrated that hypoxanthine, a component of follicular fluid, -could maintain cumulus-enclosed oocytes in a germinal vesicle configuration for extended periods (3). It was later shown that the inhibitory activity of various purines was directly related to their phosphodiesterase inhibitory activity, thus explaining their synergism with CAMP (4. In cattle, the purines fadenosine and hypoxanthine) have a transient effect on meiotic resumption and therefore can not be used for prolonged culture in the germinal vesicle configuration (21). The replacement of purines with other potent phosphodiesterase inhibitors stimulates cumulus expansion and results in transient meiotic inhibition unless a toxic amount of dibutiryl-CAMP or forskolin is added (7, 21). Other factors reported to inhibit bovine oocyte meiotic resumption in vitro are protein synthesis inhibitors (12,22), phosphorylation inhibitors such as 6-DMAP (6), protease and RNA synthesis inhibitors (ll), and sodium fluoride (17). Most of these products are not compatible with a long-term survival of the oocyte and therefore can not be used to enhance its developmental competence in culture. More than 20 years ago, Foote and Thibault (5) asked if it were possible to use the natural meiotic i~ibitory function of the follicfe in vitro. The answer is still unclear today. Using bovine oocytes and a portion of the follicular wall, they concluded that a contact was required between the granulosa, but not the theta and the cumulus to maintain the dyctiate stage in the oocyte nucleus. More recently, an experiment in the hamster showed that the surgical transfer of oocytes from follicle to follicle was successful in maintaining the meiotic arrest only if close contact was re-established between the granulosa and the cumulus (15). Does the contact act as a signal or the close proximity the only requirement for meiotic arrest? Cumulus-enclosed bovine oocytes are sensitive to the inhibitory influence of granulosa cells either in suspension (18) or in a monolayer (191, but a large number of cells is required to achieve this inhibition, and direct contact is important in this process. Is the signal metabolized by the cumulus or does it accumulate in the follicular fluid? Follicular fluid alone has a dose-dependant transient inhibitory effect on meiotic resumption on the oocyte (201. To understand the mechanism by with the follicle controls meiotic resumption, it is necessary to reconstitute its components in vitro. Therefore, this study was designed to examine the effect of the dissected follicular wall in co-culture with cumulus enclosed oocytes on meiotic resumption.
935
Theriogenology
MATERIALS
AND METHODS
Recovery of Oocytes Ovaries from non-pregnant or pregnant heifers or cows were removed within 30 min after slaughter and transported to the laboratory at about 35’C in 0.9% NaCl aqueous solution containing 100,000 IIJ/l penicillin, 100 mg/l streptomycin and 250 pg/f amphoteri~in B (Sigma, St-Louis, MO, USA). Briefly, the cumulus-oo~te complexes (COC) were aspirated from 1 to 5-mm follicles with an 18-g needle, pooled, and selected for a complete or compact cumulus (8). The COC were washed time rapidly in Hepes buffered Tyrode’s medium (TLH) (1) supplemented with 10% heat-treated fetal calf serum (FCS; Flow lab., Missisauga, Canada), 0.2 mM pyruvic acid (Sigma), and 50 pg/ml gentamicin (Sigma). The oocytes were then immediately transferred to their respective treatments described below. Preparation
of Half-Follicles
and the Culture of Oocytes
Bovine follicles were obtained from ovaries collected at random at an abattoir and transported in saline as described above but on ice. Follicles were dissected free of the interstitial tissue and were selected according to size (1 to 5-mm), transparency and a nearly perfect spherical shape. The selected follicles (mainly 2 to 3-mm diameters) were then halved with a scalpel before being washed in TCM-199 medium. Follicles with loosely attached granulosa layers were discarded before incubation; 0 (control), 1,2 or 5 hemi-sections were used to assess a dose response to follicular influence. Care was taken not to use 2 hemi-sections from the same follicle when 2 hemi-sections were used together, to minimize follicIe to follicle variations. Each experiment was replicated 2 to 5 times with a minimum of IO oocytes per treatment, for a total number of 769 individual measures. In the first experiment, to evaluate the effect of the follicles on meiotic resumption, increasing numbers (1,2 5) of follicular hemi-sections were co-incubated in 50-uL droplets under oil (Aldrich Chemical, Milwaukee, WI, USA) with 10 COC for 24 h in a media similar to that generally used for in vitro maturation: TCM-199 with Earle’s salt (Gibco Laboratories, Burlington, Ontario, Canada) and bicarbonate (Sigma) supplemented with 10% heat-treated FCS (Flow Laboratories); 0.2 mM pyruvic acid; 50 pg/ml gentamicin sulfate; 1 pg/ml FSH (National Institute of Diabetes and Digestive and Kidney Diseases); and 1 l.tg/ml estradiol 1713 (Sigma). The droplets were pre-exposed to the culture conditions for at least 2 h (38.5”C, 5% COZ, 95% air atmosphere with 100% humidity) before being used. The oocytes were added to the medium after the follicles but not inside the small cups formed by the hemisections of the follicules. In a second experiment, to determine if the conditioning of the same medium by hemi-sections would enhance inhibit.3ry capacity, the hemi-sections, one ,two or five were preincubated 24 h before the oocytes were added to the medium as above.
Theriogenology
936
In a third experiment media conditioned for 24 h was use alone with 10 COC to determine if the inhibitory products or their effect would accumulate in the media. And in the fourth experiment, to assess the reversibility of meiotic inhibition, oocytes preincubated with either 1, 2 or 5 hemi-sections for a first 24-h period like in first experiment, were further cultured without hemi-sections in a maturation medium for a subsequent 24 h. The maturation medium used for the second 24 h consisted of the same media as above but with different hormonal supplements (5 pg/ml LH [NIDDKI and 1 pg/ml progesterone). Fixation of Oocytes At the end of the incubation, the oocytes were transferred into small centrifuge tubes and were vortex-agitated for 5 min. Completely denuded oocytes were recovered and transferred to glass slides in small drops. Vaseline and paraffin were used to maintain the coverslip in contact with the oocytes without excessive pressure. The coverslip was then fixed with epoxy glue, and the slide was immersed in a fixative solution (ethanol: acetic acid, 3:ll for a minimum of 24 h. The slides were stained with 1% aceto-orcein and examined with phase contrast microscope at x 100 and x 400 magnification. Oocytes were classified as germinal vesicles (GV); intermediate stage (from germinal vesicle breakdown to Metaphase I); mature stage (from Anaphase I to Metaphase II) and degenerated (abnormal change in the cytoplasm or if chromatin impossible to assess after fixation). The significance of individual comparisons between treatments were evaluated using Chi-square analysis with correction for continuity (24). RESULTS The first figure illustrates status of the nucleus of oocytes following 24-h incubation with an increase in the number of hemi-sections in the culture drops. In all treatments, most of the oocytes were found to be at the germinal vesicle stage, but co-incubation with 5 hemi-sections resulted in the highest number of meiotic-arrest oocytes P
Theriogenology
937
1
lStemi-sections5
control
Nuclear status of bovine cumulus-enclosed oocytes (n=230) co-cultured with 0 (control), 1, 2 or 5 follicular hemi-sections for 24 hours. The oocytes were classified as germinal vesicle (GV), intermediary or mature. Compared to the control, all treatments are different (Chi-square; P
100
60
1, 24
h
2, 24 h
5, 24 h
control
Hemi-sections Nuclear status of bovine cumulus-enclosed oocytes co-cultured (n=249) for 24 hours with 0 (control), 1, 2 or 5 follicular hemi-sections that were put in culture 24 hours before the addition of oocytes. The oocytes were classified as germinal vesicle (GV), intermediary or mature. Compared to the control, all treatments are different (Chi-square; P
Theriogenology
938 g
100 -
Sk :
H GV
!
al
inrnmeediary
80 -
0 1
2
5
Hemi-section
supernatant
control
Figure 3. Nuclear status of cumulus-enclosed oocytes (n=246) cultured for 24 hours in conditioned medium obtained from the culture of 1, 2 or 5 follicular hemi-sections in small droplets compared with that of culture in fresh medium (control). The oocytes were classified as germinal vesicle (GV), intermediary or mature. Compared to the control, all treatments are different for intermediary and mature-stages (Chi-square; P
Theriogenology
939
GV intermediary mature
80
60
f 40
El
b
20
0 1
2
Number of hemi-sections
5
used during the inhibition period
Figure 4. Nuclear status of bovine cumulus-enclosed oocytes (n=164) cultured in maturation media (cf methods) without hemi-sections for 24 hours following an initial 24-ho.ur period of co-culture with 1, 2 or 5 follicular hemi-sections. The oocytes were classified as germinal vesicle (GV), intermediary or mature. The letters a, b, c, d, e and f indicate a difference between treatments (Chi-square; P
940
Theriogenoiogy
data were obtained from a limited number of oocytes (n=8), the conclusion of the above authors was that contact between granulosa and cumulus cells is essential to prevent cumulus expansion and meiotic resumption. The difference between the above results and those in our study may be explained by 2 factors: 1) The volume of culture medium in the earlier experiment did not allow for the accumulation of a sufficient amount of putative inhibitor to inhibit all oocytes. This conclusion is further supported by the fact that meiotic resumption in oocytes cultivated inside the dome formed by the hemi-sections but without complete contact was also inhibited in 7 of 8 instances (5). 2) The closer contact of the cultured oocytes with the inner layer of the follicle (granulosa) in earlier study resulted in a contact signal which prevented meiotic resumption. This effect was not observed when the oocytes were positioned on the outer layer (interstitial and theca) as in our study. Leibfried and First (9) investigated the effect of the hem§ion co-culture on porcine oocytes. Their results showed that the presence of the follicular wall can maintain the dyctiate stage in nearly 50% of the oocytes. The incubation conditions averaged 1 hemi-section and 2 to 3 oocytes per ml of TCM-199 in culture dishes. Porcine oocytes are also sensitive to granulosa cells alone, since meiotic inhibition could be observed by co-culturing cumulus-enclosed oocytes in direct or indirect contact with 10 million granulosa cell from medium-sized follicles or a portion of the follicular wall (25). In contrast, Sato and Ishibashi (161, using porcine follicles, showed that the prevention of direct contact between the oocytes and the granulosa cells eliminated most of the inhibitory effect. The in~bitory effect of direct granulosa-cumulus contact was also observed in bovine oocytes using agar embedding to separate (200 to 300 urn) cumulus-enclosed oocytes from a suspension of granulosa cells (19). Since close proximity between the 2 types of cells results in a higher gradient of any possible factor, it is quite possible that close contact by itself is not the principal inhibitory pathway. The results presented here provide new information on the possible mechanism of meiotic inhibition. The dose-response inhibitory influence of the follicular wall without direct contact between cum~us-granulosa cells supports the presence of a putative factor produced by the follicle and which acts on a population of oocytes which respond heterogenously to a given amount of this factor. The action of the hemi-sections does not seems to depend on the metabolism or removal of products present in the maturation medium (TCM 199) since conditioned medium is not as inhibitory for the resumption of meiosis as the hemi-sections themselves. The bovine foiiicular fluid-like effect of the conditioned medium would support the hypothesis of a labile factor with either a short half-life or rapid degradation by the cumulus cells. The in~bition obtained with use of the follicular wall is reversible unless a large amount of tissue is used in the culture medium, which may injure the oocyte.
941
Theriogenology
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Sirard M.A. Temporary inhibition of in vitro meiotic resumption by adenylate cyclase stimulation in immature bovine oocytes. Theriogenology 1990; 33: 757-767. Sirard MA, Bilodeau S. Effects of granulosa cell co-culture on in-vitro meiotic resumption of bovine oocytes. J Reprod Fertil 1990; 89: 459-465. Sirard MA, Bilodeau S. Granulosa cells inhibit the resumption of meiosis in bovine oocytes in vitro. Biol Reprod 1990; 43: 777-783. Sirard MA, Coenen K, Bilodeau S. Effect of fresh or cultured follicular fractions on meiotic resumption in bovine oocytes. Theriogenology 1992; 37: 39-57. Sirard MA, First NL. In vitro inhibition of oocytes nuclear maturation in the bovine. BioI Reprod 1988; 39: 229-234. Sirard MA, Florman HM, L~bfried-Rutledge ML, Barnes FL, Sims ML, First NL. Timing of nuclear progression and protein synthesis necessary for meiotic maturation of bovine oocytes. Biol Reprod 1989; 40: 1257-1263. Sirard MA, Parrish JJ, Ware CB, Leibfried-Rutledge ML, First NL. The culture of bovine oocytes to obtain developmentally competent embryos. Biol Reprod 1988; 39: 546-552. Snedecor GW, Cochran WG. Statistical Methods. Iowa State University Press, Ames, IA 1980. Tsafriri A, Channing C. An inhibitory influence of granulosa cells and follicular fluid upon porcine oocyte meiosis in vitro. Endo 1975; 96: 922-927. Tsafriri A, Dekel N, Bar-Ami S. The role of oocyte maturation inhibition in follicular regulation of oocyte maturation. J Reprod Fertil 1982; 64: 541-551.