Bovine oocytes from early antral follicles grow to meiotic competence in vitro: effect of FSH and hypoxanthine

A large number of oocytes are contained in the mammalian ovary. A very small number of these oocytes grow to the final size, mature, and are ovulated. In the ovary there are more early antral follicles than late antral or preovulatory follicles, offering a large pool of oocytes for IVM and IVF if appropriate culture conditions could be devised. In the present study, early antral follicles containing oocytes 90 to 99 microm in diameter were isolated from bovine ovaries. Cumulus-oocyte complexes (COC) with pieces of parietal granulosa (COCG) were then dissected from the follicles. The COCGs were embedded in collagen gels and cultured in Medium 199 with 10% fetal calf serum (FCS) for 8 d. In Experiment 1, the effect of hypoxanthine and FSH on the growth of bovine oocytes was examined. When hypoxanthine (2 and 4 mM) and FSH (10 ng/ml) were added to the culture medium, the number of granulosa cell-enclosed oocytes increased significantly (P < 0.05). All of the oocytes surrounded by granulosa cells showed a normal morphology and were at the germinal vesicle stage, while 75 to 94% of the denuded oocytes were degenerated and had resumed meiosis. The mean diameter of the oocytes showing normal morphology was significantly higher than that measured before culture (P < 0.05). In Experiment 2, the maturational competence of in vitro-grown bovine oocytes was examined. Oocytes which were 90 to 99 microm in diameter before culture did not have meiotic competence. After being in a growth culture of 4 mM hypoxanthine- and 10 ng/ml FSH-supplemented medium for 7 or 11 d, granulosa cell-enclosed oocytes were recovered from the COCGs. No significant difference (P < 0.05) in the diameters of the oocytes was observed between 7 and 11 d of culture (7 d: 107.5 +/- 6.1 microm, n = 30; 11 d: 108.0 +/- 5.3 microm, n = 35). After a subsequent 24 h in a maturation free of hypoxanthine and FSH medium, only 17% of the oocytes cultured for 7 d underwent germinal vesicle breakdown. On the other hand, 89% of the oocytes cultured for 11 d underwent germinal vesicle breakdown, and 11% of the oocytes emitted the first polar body and reached metaphase II. These results demonstrate for the first time that bovine oocytes harvested from early antral follicles can grow, and acquire meiotic competence in vitro.     

Meiotic competence of in vitro grown goat oocytes

The objective of the present study was to grow meiotically incompetent goat oocytes from early antral follicles in vitro and to render them competent to undergo germinal vesicle breakdown. Cumulus-oocyte complexes with pieces of parietal granulosa cells were isolated from follicles 0.35-0.45 mm in diameter using both mechanical and enzymatic methods. The cumulus-oocyte complexes were divided into two groups according to oocyte diameter (group A: < 95 microm; group B: > 95 microm) and cultured for 8 or 9 days on granulosa cell monolayers. Within 8 days of culture, the mean oocyte diameter increased from 86 +/- 0.4 microm to 95 +/- 0.7 microm in group Aand from 106 +/- 0.2 microm to 109 +/- 0.5 microm in group B. After 9 days of culture, the mean diameter of oocytes from groups A and B were 99 +/- 0.5 microm and 112 +/- 0.4 microm, respectively. The meiotic competence of oocytes grown in vitro was evaluated by in vitro maturation. Within 8 days of culture, only 3% of oocytes from group A and 6% of oocytes from group B acquired the ability to undergo germinal vesicle breakdown. After 9 days of culture, 7% of group A oocytes and 42% of group B oocytes were competent to resume meiosis. The expression of p34(cdc2) in oocytes grown in vitro was analysed by the western blot technique. During 9 days of culture, p34(cdc2) accumulated in both groups of growing oocytes, but its concentration was lower than in fully grown oocytes used as controls. The results showed for the first time that goat oocytes from early antral follicles can grow, accumulate p34(cdc2) and acquire the ability to resume meiosis, when cultured for 9 days on granulosa cell monolayers.     

In vitro growth and development of bovine oocyte-granulosa cell complexes on the flat substratum: effects of high polyvinylpyrrolidone concentration in culture medium

The aim of this study was to establish a culture system to support the growth of bovine oocytes as enclosed in granulosa cell complexes that extend on a flat substratum. Such systems have been established for mouse oocytes but are not applicable to larger animals because it is difficult to maintain an appropriate association between the oocyte and companion somatic cells. Growing bovine oocytes with a mean diameter of 95 microm were isolated from early antral follicles: the growing stage corresponds to that of oocytes in preantral follicles of 12-day-old mice. Oocyte-granulosa cell complexes were cultured for 14 days in modified TCM199 medium supplemented with 5% fetal bovine serum, 4 mM hypoxanthine, and 0.1 microg/ml estradiol. The novel modification made for this medium was a high concentration, 4% (w/v), of polyvinylpyrrolidone (PVP; molecular weight of 360000). The flat substratum used was either an insert membrane fit in the culture plate or the bottom surface of the wells of 96-well culture plates. PVP influenced the organization of complexes, resulting in a firm association between the oocyte and the innermost layer of surrounding cells. More oocytes enclosed by a complete cell layer were recovered from the medium supplemented with 4% PVP than from the control medium. Similarly, of the oocytes initially introduced into the growth culture, a significantly larger proportion developed to the blastocyst stage from medium containing 4% PVP than from medium without PVP. When PVP medium was used, the overall yield of blastocysts was similar between the system with the insert membranes (12%) and that with the 96-well culture plates (9%). A calf was produced from one of four embryos derived from oocytes grown in 96-well culture plates, matured, and fertilized in vitro and then transferred to a recipient cow.     

Nuclear configuration of bovine oocytes derived from fresh and in vitro-cultured preantral and early antral ovarian follicles

The aim of this experiment was to characterize the growth and nuclear configuration of oocytes isolated from late preantral and early antral bovine ovarian follicles immediately after recovery and after the in vitro culture. Individual follicles were isolated by microdissection from slices of the ovarian cortex. Follicles were sorted by diameter into 175 to 224, 225 to 274 and 275 to 325 microm-size classes. The follicles selected for in vitro culture were placed singly into 40 microL droplets of medium (TCM 199 enriched with FCS, insulin, transferrin, sodium selenite, sodium pyruvate, 1-glutamine, hypoxanthine, FSH and estradiol-17beta) and cultured for 6, 8, 11, 14 or 17 d. The sizes of follicles and oocytes were related to the duration of culture and gradually increased as culture duration was prolonged. The analysis of the relationship between mean diameters of oocytes at the time of recovery and after the in vitro culture, has shown significant differences after culture lasting 8 d (76.9+/-9.9 vs. 86.1+/-11.1 microm; P < 0.05), 11 d (77.0+/-9.9 vs. 91.9+/-17.5 microm; P < 0.01), 14 d (80.0+/-9.5 vs. 97.9+/-16.5 microm; P < 0.01) and 17 d (82.6+/-6.6 vs. 97.2+/-11.5 microm; P< 0.01). No statistical differences were shown among oocytes in the 5 pre-culture groups (79.5+/-8.8; 76.9+/-9.9; 77.1+/-9.9; 80.1+/-9.5 and 82.6+/-6.6 microm). Meiotic arrest was preserved in 71.9% of oocytes in our culture system up to 14 d. Frequency of the germinal vesicle (GV) stage did not significantly differ among oocytes evaluated "fresh" or cultured for 6, 8, 11 or 14 d. No relationship was observed between the size class of follicles and the frequency of the GV-stage. Prolonging the culture period to 17 d drastically decreased the percentage of oocytes in the GV-stage (18.7%) and increased the percentage of oocytes having premature initiation of meiosis (GVBD; 46.3%) and degeneration (25.0%). These results suggest that out of all culture periods used in our experiment, Day 14 was found to be the longest culture time allowing for both oocyte growth and maintenance of nuclear configuration at the GV-stage.     

Comparison of embryonic developmental competence of mouse oocytes grown with and without serum.

The first objective of this study was to determine whether oocyte growth in serum-free medium affects the solubility of the zona pellucida to alpha-chymotrypsin digestion, which is an index of zona pellucida "hardening" and reflects the potential penetrability of the zona pellucida by sperm. Oocyte-granulosa cell complexes were isolated from the preantral follicles of 12-day-old mice and cultured for 10 days in medium containing 5% fetal bovine serum (FBS) or in serum-free medium. The zonae pellucidae of oocytes grown in serum-free medium were four times as hard as freshly isolated germinal vesicle (GV)-stage oocytes grown in vivo or oocytes grown in vitro in FBS-containing medium. The hardening of the zonae pellucidae of oocytes grown in serum-free medium was prevented by addition of fetuin. The second objective was to compare the competence to undergo embryogenesis of oocytes that grew in serum-free vs. FBS-containing medium. Approximately 70% of the oocytes underwent maturation regardless of whether the medium was serum-free or contained FBS. Of the mature ova grown in medium containing FBS, 53% cleaved to the two-cell stage after insemination compared with only 6% of the ova grown in serum-free medium. Addition of fetuin to the serum-free medium used for oocyte growth increased the frequency of cleavage to the two-cell stage. Of the embryos derived from oocytes that grew in FBS-containing medium, 70% completed the two-cell stage to blastocyst transition.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of FSH and hCG on the resumption of meiosis of bovine oocytes surrounded by cumulus cells connected to membrana granulosa

Cumulus oocyte complexes (COCs) and cumulus oocyte complexes connected to a piece of the membrane granulosa (COCGs) were isolated from bovine antral follicles with a diameter of 2 to 8 mm. After culture of COCGs without gonadotrophic hormones for 22 hr approximately 50% of the oocytes were still in the germinal vesicle (GV) stage Histology of the COCGs showed that the pieces of the membrana granulosa were free of thecal cells and parts of the basal membrane. This indicates that the membrana granulosa solely inhibits the progression of meiosis. To investigate the effect of gonadotropins on the resumption of meiosis of oocytes from small and medium sized antral follicles, COCs and COCGs were cultured with or without rec-hFSH or hCG. Addition of 0.05 IU rec-hFSH to the culture medium of COCGs resulted in germinal vesicle breakdown in 97.8% of the oocytes compared to 46% in the control group, and an increase of the diameter of the COCs (479 μm vs. 240 μm in the control group). Addition of 0.05 IU hCG to the culture medium had no effect on nuclear maturation (47.2% GV vs. 48.5% GV in the control group nor on cumulus expansion (246 μm vs. 240 μm in the control group). RT-PCR on cDNA of the follicular wall, cumulus cells, granulosa cells, COCs, and oocytes revealed that mRNA for FSH receptor was present in all cell types except oocytes. mRNA of the LH receptor was detected exclusively in thecal cells. Nucleotide sequence analysis and alignment of the cloned PCR products showed the presence of two isoforms of the FSH receptor mRNA and two isoforms of the LH receptor mRNA. It is concluded that, in vitro, resumption of meiosis of oocytes, originating from small and medium sized antral follicles and meiotically arrested by the membrana granulosa, is triggered by FSH and not by LH. This is supported by the fact that receptors for FSH, but not for LH, are transcribed in the cumulus and granulosa cells of these follicles. © 1996 Wiley-Liss, Inc.     

Effect of dibutyryl cyclic adenosine monophosphate on granulosa cell proliferation, oocyte growth and meiotic maturation in isolated mouse primary ovarian follicles cultured in collagen gels.

Isolated primary follicles from 10-day-old mice were cultured in a collagen gel matrix for 6 days in Minimum Essential Medium + foetal calf serum, followed by culture in unsupplemented medium (control) or in medium containing hypoxanthine (2 mM) or dibutyryl cyclic adenosine monophosphate (dbcAMP, 0.25 mM) for a further 3 or 6 days. Less than 10% of oocytes resumed meiosis during the culture period in all groups. At recovery, the diameter of oocytes at the germinal vesicle stage was recorded and their ability to resume meiosis was determined. Hypoxanthine had little effect on oocyte growth and meiotic competence, but culture in dbcAMP resulted in oocytes that were larger (60.2 +/- 0.6 microns) than those of controls (55.8 +/- 0.5 microns) and more competent to resume meiosis than were controls (42.9% and 10.8%, respectively). The addition of dbcAMP to the culture medium induced a 4-5-fold increase in the number of granulosa cells oocyte compared with controls (3757 +/- 423 and 838 +/- 93, respectively). These results indicate that increased oocyte growth and meiotic competence is primarily mediated via dbcAMP effects on the granulosa cells.     

Effects of cycloheximide on chromatin condensations and germinal vesicle breakdown (GVBD) of cumulus-enclosed and denuded oocytes in cattle

To determine if newly synthesized protein is imperative for the resumption of meiosis in bovine follicular oocytes collected from small antral follicles, cumulus-enclosed and denuded oocytes were cultured in TCM-199 both with and without various concentrations of the protein synthesis inhibitor, cycloheximide. After 11 h of culture in inhibitor-free medium, all oocytes had undergone germinal vesicle breakdown (GVBD). However, when concentrations of more than 1.0 mug/ml cycloheximide were added to the medium, the meiotic resumption of bovine oocytes was completely blocked. This inhibitory effect of cycloheximide was fully reversible after removal of the inhibitor from maturation media. Germinal vesicle breakdown following removal of cycloheximide occurred twice as fast as in the control medium. Nevertheless, when oocytes were arrested at the germinal vesicle (GV) stage by cycloheximide, a significantly higher proportion of chromatin condensation (40 to 57%) was observed in denuded oocytes than in cumulus-enclosed oocytes (11 to 22%). Thus the cycloheximide treatment could not prevent the chromatin condensation in only denuded oocytes. We conclude that protein synthesis is a prerequisite for GVBD in bovine follicular oocytes and that cumulus cells are responsible for the complementary regulation of the chromatin condensation at the GV stage, regardless of protein synthesis in the oocytes.     

Survival and meiotic competence of bovine oocytes originating from early antral ovarian follicles

The aim of the present study was to examine the growth and survival in culture, and the subsequent meiotic competence, of bovine oocytes recovered from early antral ovarian follicles. Follicles isolated by microdissection of the ovarian slices were sorted into two size groups: (I) 0.2-0.5 mm diameter; and (II) 0.4-0.7 mm diameter. Group I follicles were cultured intact while in Group II, cumulus-oocyte complexes with pieces of parietal granulosa were dissected from the follicles and cultured. Follicles or cumulus-oocyte complexes with parietal granulose were embedded in collagen gel and cultured in TCM 199 supplemented with 3% BSA and 4 mM hypoxanthine for 14 days (Group I) or 7-10 days (Group II). After this, cumulus-oocyte complexes were recovered from the gel. Oocytes that had lost the majority of the cumulus were fixed immediately after recovery. Cumulus-oocyte complexes showing normal morphology were either fixed immediately or were subjected to IVM for an additional 24h, and then were fixed. At the end of the growth culture, 57.6% of the compact COCs in Group I follicles were preserved in the GV configuration, 16.7% had resumed meiosis, and 25.8% were degenerated or did not show detectable chromatin. After IVM, the proportion of oocytes resuming meiosis increased significantly (from 16.7% versus 42.7%; P < 0.05), and 9.1% of all oocytes had reached TI or MII. The isolated cumulus-oocyte complexes in Group II began creating follicle-like structures following 24 h of growth culture (7.1%). The proportion of these structures reached 50.8% on days 2-3, and then gradually decreased due to degeneration. On day 10 only 5.8% of cumulus-oocyte complexes were classified as intact. Of the cumulus intact oocytes recovered from the newly created follicle-like structures at 7-10 days, 54.7% were in the germinal vesicle stage, 31.0% underwent germinal vesicle breakdown, 14.3% were degenerated or the chromatin configuration was not detectable. After 24 h of IVM, 67.6% of oocytes had resumed meiosis, and 21.6% of all oocytes had reached TI and MII. These results show that isolated early follicles and cumulus-oocyte complexes from intact early antral follicles can grow in culture and can develop meiotic competence.     

Hormone-controlled culture of secondary follicles of domestic cats

Ovaries were obtained from domestic cats during ovariohysterectomy. Large, preantral follicles were freed by dissection and mechanical crushing, and were cultured in TCM 199 + 10% FCS medium in the presence or absence of hormones (FSH, hydrocortisone and Insulin-Transferrin-Selenite) as well as in hypoxanthine. A decline in growth potential along with increasing follicle size were observed after one week, with no FSH added. Hormone-supplemented medium was found to induce growth to 2 or 3 times the original volume in more than 80% of follicles of all sizes. Oocyte diameters were continuously increasing, depending on follicle size, and reached 90 mum (80 %) at the point of antrum formation. Nuclear configuration of oocytes from follicles which had been cultured without addition of hormones up to the antral stage indicated a high rate of degeneration which, however, could be reduced by gonadotrophic stimulation. Meiosis at the germinal vesicle stage was found to be inhibited by hypoxanthine. For some oocytes, evidence was provided to meiotic maturation up to extrusion of the first polar body.     

Chromatin condensation and histone H1 kinase activity during growth and maturation of rabbit oocytes

Fully grown rabbit oocytes, isolated from preovulatory follicles, exhibit highly condensed bivalents within an intact germinal vesicle while a very low level of histone H1 kinase activity could be detected in their extracts. Chromatin condensation started in growing oocytes isolated from antral follicles presenting a diameter of 0.5 mm. This event was accompanied by a transient rise in histone H1 kinase activity which culminated in large antral follicles measuring 0.75 to 1 mm in diameter. However, the extent of histone H1 kinase activity observed in these growing oocytes remained far less important than that recorded in extracts prepared from in vitro cultured metaphase I and metaphase II oocytes. Moreover, this activity was insufficient to induce germinal vesicle breakdown which will only occur with an increasing efficiency, following in vitro culture of medium, large, and fully grown antral follicles. © 1994 Wiley-Liss, Inc.     

Effects of gonadotrophins, growth hormone, and activin A on enzymatically isolated follicle growth, oocyte chromatin organization, and steroid secretion

So far, standard follicle culture systems can produce blastocyst from less than 40% of the in vitro matured oocytes compared to over 70% in the in vivo counterpart. Because the capacity for embryonic development is strictly associated with the terminal stage of oocyte growth, the nuclear maturity status of the in vitro grown oocyte was the subject of this study. Mouse early preantral follicles (100-130 microm) and early antral follicles (170-200 microm) isolated enzymatically were cultured for 12 and 4 days, respectively, in a collagen-free dish. The serum-based media were supplemented with either 100 mIU/ml FSH (FSH only); 100 mIU/ml FSH + 10 mIU/ml LH (FSH-LH); 100 mIU/ml FSH + 1 mIU/ml GH (FSH-GH) or 100 mIU/ml FSH + 100 ng/ml activin A (FSH-AA). Follicle survival was highest in follicle stimulating hormone (FSH)-AA group in both cultured preantral (91.8%) and antral follicles (82.7%). Survival rates in the other groups ranged between 48% (FSH only, preantral follicle culture) and 78.7% (FSH only, antral follicle culture). Estradiol and progesterone were undetectable in medium lacking gonadotrophins while AA supplementation in synergy with FSH caused increased estradiol secretion and a simultaneously lowered progesterone secretion. Chromatin configuration of oocytes from surviving follicles at the end of culture revealed that there were twice more developmentally incompetent non-surrounded nucleolus (NSN) oocytes (>65%) than the competent surrounded nucleolus (SN) oocytes (<34%). We conclude that the present standard follicle culture system does not produce optimum proportion of developmentally competent oocytes.     

Effect of androstenedione on the growth and meiotic competence of bovine oocytes from early antral follicles

Summary Medium that contains 17β-estradiol has been reported to support in vitro growth of bovine oocytes, isolated from early antral follicles, until the final stage. The aim of this study was to determine the effects of androstenedione in medium on such growing bovine oocytes. Oocyte-granulosa cell complexes were collected from early antral follicles and cultured for 14 days in medium supplemented with 17β-estradiol (0, 10 and 100 ng/ml) or androstenedione (0, 10 and 100 ng/ml). The mean diameter of oocytes measured after seeding on the culture substrate was 96.9 μm (n = 191). Either steroid was necessary for maintainance of the organization of oocyte-granulosa cell complexes over the 14-day culture period. In the 17β-estradiol- or the androstenedione-supplemented medium about 80% or 65%, respectively, of viable oocytes were recovered. In both groups the increase in oocyte size was significant after 14 days. The in vitro grown oocytes were cultured for a further 22-24 h for oocyte maturation; 13% and 30% of oocytes grown in the 10 and 100 ng/ml 17β-estradiol-supplemented medium reached metaphase II, respectively; more than 64% of oocytes grown in the androstenedione-supplemented medium matured to metaphase II. These results show that androstenedione, as 17β-estradiol, can maintain the viability of bovine oocyte-granulosa cell complexes and support the growth of oocytes, and that androstenedione promotes the acquisition of oocyte meiotic competence efficiently at a low dose.     

Activation of p34(cdc2) kinase around the meiotic resumption in bovine oocytes cultured in vitro

The p34(cdc2) kinase has been identified as a protein factor that is a regulator of meiotic maturation in mammalian oocytes. To investigate the regulatory function of the meiotic resumption in bovine oocytes cultured in vitro, the changes in the phosphorylation states of p34(cdc2) kinase and the histone H1 kinase activity were examined around germinal vesicle breakdown (GVBD). All bovine oocytes just after isolation from their follicles were arrested at the germinal vesicle (GV) stage, and these extracts exhibited two (upper and lower) bands of p34(cdc2) kinase on SDS-PAGE followed by immunoblotting with an antibody against C-terminal peptide of p34(cdc2). When these oocytes were cultured for 24 h in a medium supplemented with 100 microg/ml genistein, tyrosine phosphorylation inhibitor, GVBD was induced in 85% of oocytes, indicating that the upper band of p34(cdc2) kinase in bovine oocytes at the GV stage was already fully phosphorylated tyrosine residue prior to culture. Another (middle) band of p34(cdc2) kinase between the upper and lower bands appeared in the extracts of the oocytes cultured for 4 h, and significant activation of the histone H1 kinase was found in these oocytes (67 +/- 18 fmol/h/oocyte) as compared to that in oocytes cultured for 0 h (46 +/- 11 fmol/h/oocyte). The staining intensity of the middle band and the activity of the histone H1 kinase were further increased after the initiation of GVBD at 6 h of culture, but the quantitative changes of upper and lower bands were not detected throughout the 12 h of culture. Thus, it is concluded that the dephosphorylation of p34(cdc2) kinase followed by activation of the histone H1 kinase after the onset of culture plays a key role in the resumption of meiosis in bovine oocytes.     

In vitro growth and differentiation of primary follicles isolated from cryopreserved sheep ovarian tissue

Achieving full in vitro growth of oocytes of both domestic animals and humans remains a major challenge. The objective of this study was to examine the in vitro development of primary follicles isolated enzymatically from cryopreserved sheep ovarian tissue. In Experiment 1, isolated primary follicles (mean diameter 60.1+/-0.78microm) were cultured in serum-free medium on fibronectin-coated wells for 42 days. Initially follicular structure was lost as granulosa cells plated down, but by Day 7 two distinct morphologies began to emerge. Nineteen out of 36 oocytes were gradually re-surrounded by granulosa cells, forming follicle-like units (reorganized follicles), and the remaining 17 were not (non-reorganized follicles). On Day 2, there was no difference in diameter of oocytes between reorganized and non-reorganized follicles. The diameter (mean+/-S.E.M.) of oocytes of reorganized follicles increased (P<0.05) from 47.1+/-2.2microm to 65.3+/-2.6microm between Day 2 and Day 42, respectively, but that of oocytes of non-reorganized follicles showed no change. In Experiment 2, oocyte growth and granulosa cell differentiation during long-term culture of primary follicles (>42 days) were examined. Oocytes of reorganized follicles reached a maximum diameter of 75.4+/-2.0microm, a size equivalent to that of oocytes of ovine secondary follicles. Using RT-PCR, mRNA for follicle stimulating hormone receptor was detected in granulosa cells of freshly isolated secondary follicles and of long-term cultured reorganized follicles, but not of non-reorganized follicles. In Experiment 3, we tested if the culture conditions could support further oocyte growth in secondary follicles. The oocytes from enzymatically isolated secondary follicles increased in diameter from 77.7+/-1.6microm to 98.8+/-2.1microm (P<0.05) during 28 days in culture. The changes in oocyte size and in gene expression by granulosa cells support the conclusion that isolated ovine primary follicles developed in vitro to reach the secondary follicle stage.     

Developmental competence of bovine oocytes after specific inhibition of MPF kinase activity: effect of estradiol supplementation and follicle size

In the bovine, the concentration of 17beta-estradiol (E2) in the follicular fluid of the dominant follicle is high, indicating a possible role of E2 on the cytoplasmic maturation that occurs before the LH surge. The aim of this study was to investigate the role of E2 on the developmental competence of bovine oocytes originating from different sized follicles and temporarily maintained at the germinal vesicle stage with roscovitine (ROS). First, the efficiency of ROS to inhibit germinal vesicle breakdown (GVBD) in oocytes harvested from small (3-4 mm diameter) and medium (5-8 mm diameter) sized follicles was demonstrated. Next, the effect of E2 during temporary inhibition of GVBD by ROS on the subsequent nuclear maturation was evaluated. Oocytes from small and medium sized follicles were cultured in the presence of ROS, FSH and with or without E2 for 24 h. After this period, oocytes were cultured for another 24 h with FSH but without ROS and E2, after which the nuclear stages and the developmental competence of oocytes were assessed. In conclusion, it is demonstrated that exposure to E2, during temporary inhibition of the GVBD with ROS, affected neither nuclear nor cytoplasmic maturation of oocytes originating from small and medium sized follicles. It might be that in vivo, the increase of E2 during follicle growth is more related to selection of the dominant follicle than to the cytoplamsic maturation of the oocyte as such.     

Meiotic state of bovine oocytes is regulated by interactions between cAMP,cumulus, and granulosa

Bovine oocytes are arrested at the prophase of first meiotic cell cycle. Meiosis resumes in oocytes of pre-ovulatory follicles upon LH surge. However, oocytes from secondary follicles spontaneously resume meiosis in the absence of hormones if removed from the follicle and cultured in vitro. The nature of meiotic arrestor in bovine follicles is poorly understood. In this study we investigated the role of cell-cell interactions between granulosa and cumulus cells and the oocyte in mediating maintenance of meiotic arrest by cAMP. We sorted oocytes as granulosa-cumulus oocyte complexes (GCOC) if surrounded with cumulus cells attached to a large granulosa investment or cumulus oocytes complexes (COC) if surrounded with cumulus cells only and investigated the role cAMP in maintenance of meiotic arrest in these oocytes under various conditions. In hormone- and serum-free medium both GCOC and COC enclosed oocytes resumed meiosis. When [cAMP](i) was elevated with addition of invasive adenylate cyclase (iAC) GCOC enclosed oocytes were maintained in the prophase with intact germinal vesicle (GV) while COC enclosed oocytes underwent GV breakdown (GVBD). iAC elevated [cAMP](i) in both types of oocytes to the same level. If oocytes were liberated from the cumulus and granulosa cells, they re-initiated meiosis in serum and hormone free medium, but remained in the GV stage if iAC was added to the medium. Untreated GCOC and COC enclosed oocytes extruded first polar body at the same frequency in hormone-supplemented media. GCOC and COC enclosed oocytes but not denuded oocytes (DO) cultured without somatic cells acquired developmental competence if cultured in hormone-containing medium. It is concluded that maintenance of meiotic arrest is regulated by the interplay of [cAMP](i), and cumulus and granulosa cells.     

Mouse antral oocytes regulate preantral granulosa cell ability to stimulate oocyte growth in vitro

In this study we evaluated whether mouse oocytes derived from early antral or preovulatory follicles could affect the ability of preantral granulosa cells to sustain oocyte growth in vitro. We found that early antral oocytes with a diameter > or =75 microm did not grow any further during 3 days of culture on preantral granulosa cell monolayers in vitro, while most of the oocytes with a smaller diameter increased significantly in size. Similarly, about 65% of growing oocytes isolated from preantral follicles grew when cultured on preantral granulosa cells. By coculturing with growing oocytes fully grown early antral or preovulatory oocytes, a small proportion (about 10%) of growing oocytes increased in diameter, and changes in granulosa cell morphology were observed. Such effects occurred as a function of the fully grown oocyte number seeded and were not associated with a decrease in coupling index values. By avoiding physical contact between antral oocytes and granulosa cells, the proportion of growing oocytes undergoing a significant increase in diameter was about 36%. These results indicate that fully grown mouse oocytes can control preantral granulosa cell growth-promoting activity through the production of a soluble factor(s) and the maintenance of functional communications with surrounding granulosa cells.     

小鼠年龄及卵丘—卵母细胞间联接的解离对卵母细胞体外成熟的影响

实验研究了小鼠卵母细胞体外过程中卵丘－卵母细胞间的相互作用。实验小鼠为雌性Ｂ６Ｄ２杂交一代。激素处理４８小时后分离出卵后天和卵母细胞复合体,并培养在含有次黄嘌呤的培养液中。２４小时后检查卵母细胞核成熟情况。