Meiotically incompetent and competent goat oocytes: timing of nuclear events and protein phosphorylation

The ability of mammalian oocytes to resume meiosis and to complete the first meiotic division is acquired sequentially during their growth phase. The acquisition of meiotic competence in goat oocytes has been previously correlated with follicular size (9). Since protein phosphorylation/dephosphorylation play a key role in oocyte maturation, it could be that in meiotically incompetent oocytes, such post-translational modifications are inadequate. The aim of this study was to analyze whether changes in oocyte proteins phosphorylation occurred during the acquisition of meiotic competence. For this propose, goat oocytes were divided into 4 classes according to follicular size and meiotic competence: Class A oocytes from follicles < 0.5 mm in diameter: Class B oocytes from follicles 0.5-0.8 mm; Class C oocytes from follicles 1-1.8 mm and class D oocytes from follicles > 3 mm. The protein phosphorylation patterns of these classes of oocytes were studied at different times of in vitro maturation. After 4h of culture, when all oocytes were in the germinal vesicle stage, only the oocytes from Class D displayed the phosphoproteins at 110 kD, 31 kD and around 63 kD. In contrast to Class D oocytes Classes B and C oocytes were partially competent to mature, they underwent germinal vesicle breakdown later than fully competent Class D oocytes and remained in early prometaphase I or in metaphase I, respectively. They exhibited the phosphoprotein changes that are associated with commitment to resume meiosis; but the changes occurred later than in Class D oocytes, which were fully competent to reach metaphase II. After 27 h of culture, the phosphorylation patterns of Class B, C and D oocytes were identical, whereas the meiotic stages reached were quite different. The phosphoprotein changes associated with oocyte maturation did not occur in meiotically incompetent Class A oocytes, which were blocked at the germinal vesicle stage. From these results it can be concluded that, at the GV stage, meiotically incompetent and competent goat oocytes display different patterns of protein phosphorylation. Once oocytes are able to resume meiosis they undergo specific phosphorylation changes, but whether these changes are markers or regulators of maturation events remains to be determined.     

Extracellular and intracellular factors affecting nuclear and cytoplasmic maturation of porcine oocytes collected from different sizes of follicles

Nuclear and cytoplasmic maturation of porcine oocytes collected from different sizes of follicles were examined. Oocyte-cumulus complexes were collected from small (1-2 mm in diameter), medium (3-6 in diameter) and large (7-8 mm in diameter) follicles and cultured in a modified tissue culture medium 199 for 44 h. Nuclear maturation was evaluated after orcein staining, and cytoplasmic maturation was evaluated by intracellular glutathione (GSH) assay. Oocyte diameter, cumulus morphology, steroid hormones and glutathione in the follicular fluid (FF), were also examined. Significantly higher proportions of oocytes collected from large and medium follicles reached metaphase II than did oocytes from small follicles. Oocytes from small follicles also had a smaller size. GSH content was significantly higher (p < 0.05) in oocytes from large (14.24 +/- 2.1 pmol/oocyte) and medium (13.69 +/- 1.5 pmol/oocyte) follicles than in oocytes from small (9.44 +/- 1.28 pmol/oocyte) follicles just after collection. After maturation, oocytes from medium follicles had a higher GSH concentration than oocytes from small follicles. It was found that between 49.7 +/- 5.18 nM and 52.25 +/- 0.78 nM GSH was present in FF but there was no statistical difference between follicle sizes. A significantly higher (p < 0.001) estradiol level was present in FF from large follicles (299.2 +/- 68.6 ng/ml) than from medium (40.0 +/- 6.4 ng/ml) and small (41.2 +/- 3.7 ng/ml) follicles. Progesterone concentrations in FF from large (281.6 +/- 45.9 ng/ml) and medium (267.5 +/- 38.6 ng/ml) follicles were significantly higher than that (174.7 +/- 22.0 ng/ml) from small follicles. These results indicate that the oocyte's ability to accumulate intracellular GSH during maturation, and extracellular steroid hormones and cumulus cells, affect the competence of porcine oocytes to undergo nuclear and cytoplasmic maturation.     

Relationship among the status of the human oocyte, the 17 beta-estradiol concentration in the antral fluid and the follicular size

We studied the relationship among the status of the human oocytes, the E2 concentration in the antral fluid and the follicular size in the different phases of the menstrual cycle, in order to determine the microenvironment of the follicles with healthy or degenerative oocytes in the human ovary. In the follicular phase of the menstrual cycle, follicles which contained a healthy but not degenerative oocyte had a significantly higher level of 17 beta-estradiol (E2). In the late follicular phase, the larger follicles (greater than or equal to 13 mm, in diameter) had only health oocytes. It seems that the follicle containing a degenerative oocyte does not develop physiologically until maturation of the preovulatory follicle. In the luteal phase, there were no relationships among the status of the oocyte, E2 concentration in the antral fluid and the follicular size. However, the E2 levels of the antral follicles with healthy oocytes in an ovary with corpus luteum were significantly lower than those in the contralateral ovary. The results suggest that the corpus luteum may exert an influence on the adjacent follicles.     

In vitro development of individually matured bovine oocytes in relation to follicular wall atresia

Morphologically good-quality cumulus oocyte complexes (COCs) can originate from slightly atretic follicles. Biochemical and ultrastructural investigations reveal that a very high percentage of bovine antral follicles express some degree of atresia. The aim of the present study was to determine the developmental competence of good quality COCs in relation to their biochemically estimated follicular wall apoptosis. For experimental design a single oocyte maturation system was established, followed by group culture processing oocytes together according to their level of follicular wall atresia estimated by an ELISA for apoptotic cell death. Single oocyte culture during maturation reduced the developmental capacity of oocytes significantly (P < 0.01), with 5% blastocysts versus 25% after common group culture. Blastocyst formation for single oocyte maturation was found exclusively in oocytes isolated from luteal stage ovaries with low degree of apoptosis. The level of follicular wall apoptosis in luteal stage follicles (0.79 +/- 0.05 units/mg protein, n = 198) was lower than in follicular stage follicles (1.14 +/- 0.05 units/mg protein, n = 208). This was caused by significant higher levels in small (< 3.5 mm diameter) and large (> 5.5 mm diameter) follicles of the latter group. In conclusion, despite reduced developmental capacity after single oocyte maturation, we were able to reveal some functional relationship between oocyte origin and quality. It was shown that morphologically good quality COCs isolated from follicles with higher degree of apoptosis lose their developmental capacity.     

Effect of follicular size on meiotic and developmental competence of porcine oocytes

In several species, the developmental competence of the oocyte is acquired progressively during late follicular growth, after the acquisition of the competence to resume and complete meiosis. In the pig, full meiotic competence of the oocyte is reached in ovarian follicles with a diameter of 3 mm or more. However, there is no information about developmental competence acquisition. We analyzed the ability of oocytes from three foll icular size classes to resume and complete meiosis, to be fertilized, and to develop in vitro to the blastocyst stage. A total of 941 follicles were dissected from slaughterhouse gilt ovaries and classified as small (<3 mm, n = 330), medium (3-5 mm, n = 373), or large (>5 mm, n = 238). The cumulus-oocyte complexes recovered from these follicles were submitted to in vitro maturation for 44 h in TCM199 supplemented with 10 ng/ml EGF, 400 ng/ml pFSH and 570 microM cysteamine; in vitro fertilized for 18 h in mTBM with 10(5) frozen-thawed percoll-selected sperms/ml; and developed for 7 days in mSOF. Samples of oocytes or presumptive zygotes were fixed and stained at the end of maturation and fertilization. Groups of oocytes were cultured for 3 h in the presence of 35S-methionine before or after maturation for SDS-PAGE analysis of protein neosynthesis. More oocytes originating from medium and large follicles were competent for maturation than oocytes from small follicles (77 and 86% of metaphase II, respectively, versus 44%, P < 0.05). More oocytes from medium and large follicles werepenetratedby spermatozoa during in vitro fertilization, resulting in significantly more oocytes presenting two or more pronuclei at the end of fertilization (73 and 77% for medium and large follicles, respectively, versus 53% for small follicles, P < 0.05). More oocytes from medium and large follicles developed to the blastocyst stage (14 and 23%, respectively) than those from small follicles (3%, P < 0.05), even if the development rates were corrected by the maturation or fertilization rates. It is concluded that a high proportion of oocytes harvested from follicles of less than 3 mm in the pig are not fully competent for meiosis and are cytoplasmically deficient for development.     

Meiotic competence of prepubertal goat oocytes

The object of this work was to evaluate in vitro maturation of follicular oocytes from the ovaries of prepubertal goats obtained from the slaughterhouse. To obtain the oocytes, follicles were dissected and classified according to their diameters. In the first experiment, oocytes were matured in vitro with granulosa cells. No significant differences were detected in the percentages of maturation between adult and prepubertal goat oocytes recovered from follicles of 2.5 to 6.0 mm in diameter (81.82 vs 72.47%, respectively). The percentage of maturation increased to 88.0% in prepubertal goat oocytes from 3.0 to 6.0-mm follicles. In the second experiment, the percentage of maturation of prepubertal goat oocytes was greater after 27 than after 24 h. In the third experiment, the maturational capacity of prepubertal goat oocytes according to follicular diameter was evaluated. The percentages of maturation after 27 h of culture with no granulosa cells were 24.14, 56.60 and 74.78%, respectively, for follicles 1.0 to 1.9 mm, 2.0 to 2.9 mm, and 3.0 to 6.0 mm in diameter. As the follicular diameter increased, growth of the oocyte as well as a greater number of oocytes with more cumulus cell layers were observed. A correlation between the diamter of the oocyte and its competence to complete in vitro maturation was also observed. Oocytes with more cumulus cell layers showed only a slight superiority in their capacity for maturation in large-size follicles (3.0 to 6.0 mm), but the difference was not significant. In conclusion, oocytes from prepubertal goats complete their growth and reach meiotic competence in follicles larger than 3.0 mm. With these oocytes it is possible to obtain in vitro maturation results similar to those from adult goats.     

Effects of bovine follicular fluid on maturation of bovine oocytes

Three experiments were conducted to determine the effects of follicular fluid and media on bovine oocyte maturation. Experiments 1 and 3 test the effects of follicular fluid obtained at different times after the LH surge on bovine oocyte maturation in vitro, while Experiment 2 was designed to compare TALP and Medium 199 as serum-free maturation media. Bovine follicular fluid (BFF) was obtained from preovulatory follicles either before (0 h BFF) or at 4, 8, 12 or 20 h after a GnRH-induced LH surge. Oocytes were obtained from follicles 1 to 6 mm in diameter from ovaries retrieved from a slaughterhouse. In Experiment 1, both 0 h and 4 h BFF inhibited resumption of meiosis, whereas BFF collected at 8, 12 and 20 h did not. When oocytes were cultured in media that contained equal portions of 0 and 8 h BFF, meiosis was not inhibited. In Experiment 2, Medium 199 supplemented with bovine serum albumin (BSA) was superior to Tyrode's medium with albumin, lactate and pyruvate for oocyte maturation. In Experiment 3, a higher percentage (P<0.05) of oocytes cultured for 18 h in 40% 20 h BFF in Medium 199 reached Metaphase-II (64%) than those cultured in 0 h BFF (41%) or control medium (39%). There was a transient meiotic arrest due to 0 h BFF as evidenced by the higher percentage of oocytes with germinal vesicles at 8 h of incubation (35% with 0 h vs 20% with 20 h; P<0.05). Furthermore, expansion of cumulus cells was induced in 8 and 20 h BFF, but not 0 h BFF.     

Developmental stage of the oocyte during antral follicle growth and cumulus investment determines in vitro embryo development of sow oocytes

The aim of the study was to determine the contribution of cumulus cells on the developmental competence of porcine oocytes during follicle growth. Oocytes from large (5-8mm) and small (2-3mm) follicles were cultured with or without follicle stimulating hormone (FSH), subsequently examined for nuclear stage and spindle morphology, or fertilized and cultured for embryo development, or analyzed for glutathione content. Additionally, the significance of cumulus investment, corona radiata cells, cumulus cell number and origin of cumulus cells for oocyte maturation were investigated. Small follicle oocytes cultured without FSH exhibited the highest incidence of spindle aberrations. Oocytes cultured without FSH exhibited reduced sperm penetration and blastocyst rates, and a higher proportion monospermic oocytes developed to the blastocyst stage when derived from large follicles. The glutathione content in oocytes increased during follicle growth and oocyte maturation, but no direct correlation between oocyte glutathione content and oocyte developmental capacity was observed. Oocytes with a bigger cumulus investment exhibited better embryo development. Oocytes with a single corona radiata cell layer (CROs) exhibited similar progression through meiosis to oocytes with more cumulus cell layers, but showed reduced embryo development. More blastocysts were observed when CROs were cultured with disconnected cumulus cells during IVM, but no blastocyst increase was observed when CROs were cocultured with a higher number of cumulus cells or with cumulus cells from large follicles. We conclude that increased developmental capacity of oocytes during follicle growth is intrinsic and whether cumulus cells originate from large or small follicles, their contribution to oocyte maturation remains unchanged. Further, cumulus investment can be used as a variable to predict oocyte developmental capacity.     

Effects of ovarian stimulation, with and without human chorionic gonadotrophin, on oocyte meiotic and developmental competence in the marmoset monkey (Callithrix jacchus)

A reliable ovarian stimulation protocol for marmosets is needed to enhance their use as a model for studying human and non-human primate oocyte biology. In this species, a standard dose of hCG did not effectively induce oocyte maturation in vivo. The objectives of this study were to characterize ovarian response to an FSH priming regimen in marmosets, given without or with a high dose of hCG, and to determine the meiotic and developmental competence of the oocytes isolated. Ovaries were removed from synchronized marmosets treated with FSH alone (50 IU/d for 6 d) or the same FSH treatment combined with a single injection of hCG (500 IU). Cumulus-oocyte complexes (COCs) were isolated from large (>1.5mm) and small (0.7-1.5mm) antral follicles. In vivo-matured oocytes were subsequently activated parthenogenetically or fertilized in vitro. Immature oocytes were subjected to in vitro maturation and then activated parthenogenetically. Treatment with FSH and hCG combined increased the number of expanded COCs from large antral follicles compared with FSH alone (23.5 +/- 9.3 versus 6.4 +/- 2.7, mean +/- S.E.M.). Approximately 90% of oocytes surrounded by expanded cumulus cells at the time of isolation were meiotically mature. A blastocyst formation rate of 47% was achieved following fertilization of in vivo-matured oocytes, whereas parthenogenetic activation failed to induce development to the blastocyst stage. The capacity of oocytes to complete meiosis in vitro and cleave was positively correlated with follicle diameter. A dramatic effect of follicle size on spindle formation was observed in oocytes that failed to complete meiosis in vitro. Using the combined FSH and hCG regimen described in this study, large numbers of in vivo matured marmoset oocytes could be reliably collected in a single cycle, making the marmoset a valuable model for studying oocyte maturation in human and non-human primates.     

Effect of administering a crude equine gonadotrophin preparation to mares on follicular development, oocyte recovery rate and oocyte maturation in vivo

In mares, the shortage of oocytes and the variability in nuclear maturation at a certain time of the oestrous cycle hinders the optimization of methods for in vitro maturation and in vitro fertilization. Increasing the number of small-to-medium-sized follicles available for aspiration in vivo may increase the overall oocyte yield. The aims of the present study were to investigate whether administration of crude equine gonadotrophins affects follicular development, oocyte recovery rate, in vivo oocyte maturation and follicular concentrations of meiosis-activating sterols. During oestrus, all follicles >/= 4 mm were aspirated from 19 pony mares (first aspiration: A1). Over the next 8 days, the mares were treated daily with either 25 mg crude equine gonadotrophins (n = 10) or physiological saline (n = 9). Between day 1 and day 8, follicular growth was monitored by ultrasonography. On day 8, all follicles >/= 4 mm were evacuated (second aspiration: A2) and nuclear maturation of the recovered oocytes was assessed after orcein staining. Follicular growth between A1 and A2, as well as the number and size of follicles at A2 were similar for control mares and mares treated with crude equine gonadotrophins. The oocyte recovery rates at A1 and A2 were similar. At A2, the oocyte recovery rate and oocyte maturation in vivo were not affected by treatment with crude equine gonadotrophins. The number of expanded cumulus oophorus complexes recovered from follicles 相似文献   

Effect of follicular cells,IGF-I and tyrosine kinase blockers on oocyte maturation

The aim of this study was to test the following hypotheses: (i) that oocyte maturation is controlled by surrounding follicular cells; (ii) that a meiosis-regulating factor of follicular origin is not species-specific; (iii) that one of the follicular regulators of oocyte maturation is IGF-I; and, (iv) that Cumulus oophorus and tyrosine kinase-dependent intracellular mechanisms do not mediate IGF-I action on oocytes. It was found that co-culture of cumulus-enclosed bovine oocytes with isolated bovine ovarian follicles or with isolated porcine ovarian follicles significantly increased the proportion of matured oocytes (at metaphase II of meiosis) after culture. Porcine oocytes without cumulus investments had lower maturation rates than cumulus-enclosed oocytes. Co-culture with isolated porcine ovarian follicles resulted in stimulation of maturation of both cumulus-free and cumulus-enclosed porcine oocytes. These observations suggest that follicular cells (whole follicles or Cumulus oophorus) support bovine and porcine oocyte maturation, and that follicular maturation-promoting factor is not species-specific. The release of significant amounts of IGF-I by cultured bovine and porcine isolated follicles and granulosa cells was demonstrated. Addition of IGF-I to culture medium at 10 or 100 (but not 1000) ng/ml stimulated meiotic maturation of both cumulus-enclosed and cumulus-free porcine oocytes. Neither of the tyrosine kinase blockers, genistein or lavendustin (100 ng/ml medium), changed the stimulating effect of IGF-I on porcine oocytes. The present data suggest that at least one of the follicular stimulators of oocyte nuclear maturation is IGF-I, and that its effect is probably not mediated by cumulus investment or by tyrosine kinase-dependent intracellular mechanisms.     

In vivo changes in oocyte germinal vesicle related to follicular quality and size at mid-follicular phase during stimulated cycles in the cynomolgus monkey

Histological examination of gonadotrophin stimulated Macaca fascicularis ovaries removed at mid-follicular phase showed that germinal vesicles (GV) could exhibit different configurations in follicles greater than 1000 microns in diameter. We describe 3 types of nuclear organization called GV1 (dispersed and filamentous chromatin), GV2 (clumped and filamentous chromatin) and GV3 (perinucleolar chromatin condensation). Gonadotrophin stimulation and follicular atresia induced modifications in GV chromatin dispersion. Such modifications were of a higher degree in the case of atresia which could even induce in vivo germinal vesicle breakdown (GVBD). Our findings were as follows. The frequency of GV1 oocytes was always low, but was higher in healthy than in atretic follicles, whereas GV3 oocytes were more frequent in atretic compared to healthy follicles; the oocytes which resumed meiosis in vitro were most probably those which were at the GV3 stage at the time of recovery; GV nuclear changes were related to follicle size and quality, but not to oocyte size. The mean follicular size increased from GV1 to GV3 oocyte stages whatever the follicle quality; the nucleus was often observed in a peripheral position even in GV1 oocytes; zona pellucida appearance was related to GV stage and follicle quality and was more often observed to be abnormal or absent in case of GV3 oocytes included in atretic follicles. Oocyte nuclear modifications therefore appear to be a prerequisite to resumption of meiosis.     

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.     

Relationship between antral follicle size,oocyte diameters and nuclear maturation of immature oocytes in pigs

We designed the present study to examine the possible relationship between oocyte, antral follicle size and the nuclear heterogeneity of immature pig oocytes, in order to study the heterogeneity of oocyte populations in ovaries obtained from slaughterhouses. Previously, we carried out an initial experiment to determine, by histological analysis, the effectiveness of the macroscopic criteria (MC) used to screen atretic and nonatretic antral follicles. We recovered 239 follicles by mechanical dissection, measured them with a computerized image analysis system, and classified them into five size categories according to their diameter (FD): Group 1 (0.40-0.99 mm), Group 2 (1.00-2.19 mm), Group 3 (2.20-2.79 mm), Group 4 (2.80-3.59 mm) and Group 5 (3.60-6.50 mm). In relation to histological analysis, the results showed that MC is an effective method to select atretic and nonatretic antral follicles from 0.40 to 6.50 mm in diameter (overall accuracy was 80.75%, with sensitivity and specificity rates of 79.33 and 82.20%, respectively). In a second experiment, we recovered 454 nonatretic follicles, then measured and classified them as mentioned above. We removed oocytes individually from follicles and measured their size (oocyte diameter without and with zona pellucida, OD and TOD, respectively). Finally, we evaluated the relationship between OD, FD and nuclear maturation of immature oocytes (germinal vesicles (GV) Stages 0, I, II, III and IV; diakinesis, prophase I, and metaphase I). Overall OD was 101.77 +/- 0.65, 109.19 +/- 0.45, 113.55 +/- 0.50, 116.92 +/- 0.46 and 117.13 +/- 0.47 microm (Groups 1, 2, 3, 4, and 5, respectively). Differences in OD between groups were significant (P < 0.01), although from 2.80 to 6.50 mm follicles, the oocytes were not different in size. There was a certain heterogeneity in OD within each follicular group. Although we observed a certain degree of nuclear variability, regardless of FD or OD, the present study showed a clear progression in GV when FD increased from 0.40 to 6.50 mm. A positive correlation (r2 = 0.4248; P > 0.05) was established mainly between the nuclear stage and oocyte diameter.     

Human oocytes reversibly arrested in prophase I by phosphodiesterase type 3 inhibitor in vitro

This study addresses the role of cAMP hydrolytic isoenzyme phosphodiesterase type 3 (PDE 3) modulation on human oocyte maturation in vitro. Presence of phosphodiesterase type 3 A (PDE 3A) mRNA was confirmed in human germinal vesicle-stage (GV) oocytes. Making use of a selective PDE 3 inhibitor, Org 9935 (10 microM), oocytes retrieved from immature follicles were arrested in prophase I with a high efficiency for up to 72 h. Cumulus oocyte complexes (COCs) were retrieved in the follicular phase of the cycle before or after exposure to endogenous LH or hCG administration in vivo and randomly distributed into maturation medium with or without the PDE 3 inhibitor. Previous exposure of small follicles to LH activity in vivo had no influence on the arresting capacity of the PDE 3 inhibitor. Reversal from pharmacological arrest leads to a progression through meiosis in a normal time frame with formation of a well-aligned metaphase plate. Ultrastructure analysis of COC derived from follicles between 8 and 12 mm showed that the induced extension of prophase I arrest in vitro resulted in cytoplasm changes but not in apparent nuclear changes during culture.     

Size of the donor follicle, but not stage of reproductive cycle or seasonality, influences meiotic competency of selected domestic dog oocytes

Ability of ovarian oocytes from the domestic dog to complete nuclear maturation in vitro (IVM) varies markedly among donors and generally is 20% or less of all oocytes cultured. To identify the cause(s) underlying these significant variations in meiotic maturation (to metaphase II; MII), we retrospectively analyzed data from 1,643 oocytes recovered from 90 bitches for which stage of reproduction and season of year were known. Neither stage of reproduction (proestrus/estrus, diestrus, anestrus, or prepuberty) nor season (P > 0.05) influenced the ability of oocytes to achieve nuclear maturation in vitro. A second study was conducted to examine the impact of follicular size on meiotic maturation. Populations of large oocytes were recovered from four categories of follicles (ranging from <0.5 to > 2 mm in diameter) and cultured in TCM 199 for 48 hr. Follicular size influenced (P < 0.05) meiotic competence. Mean percentages of MII oocytes were 16.9 +/- 9.2, 26.1 +/- 7.6, 38.4 +/- 9.2, and 79.5 +/- 10.9 for oocytes recovered from < 0.5 mm, > or = 0.5-< 1 mm, 1-2 mm, and > 2 mm diameter follicles, respectively. In summary, stage of reproduction and season have no impact on the ability of dog oocytes to achieve nuclear maturation in vitro. However, we demonstrated for the first time that dog oocytes acquire meiotic competency during follicular development. IVM success of selected oocytes from large size follicles (almost 80%) is about 60% higher than measured in most previous studies involving randomly collected oocytes.     

The stage-dependent inhibitory effect of porcine follicular cells on the development of preantral follicles

The objective of this study was to examine the effects of follicular cells on the in vitro development of porcine preantral follicles. In Experiment 1, one preantral follicle alone (Trt 1) was cocultured with a follicle of the same size with oocytes (Trt 2) or without oocytes (Trt 3). Preantral follicles cultured alone in vitro for 12 days had greater follicle diameters (1017 +/- 96 microm versus 706 +/- 69 or 793 +/- 72 microm, P < 0.05), growth rates (201 +/- 0.3 versus 103 +/- 0.2 or 128 +/- 0.2, P < 0.05) and oocyte survival rates (73% versus 48, or 25%, P < 0.05) than other groups. The inhibitory effects of follicle cells on the growth of preantral follicles and oocyte survival rates were not enhanced by the addition of oocytectomized preantral follicles (Experiment 2). Follicles were cocultured with different sources of follicular cells in other experiments. Coculture with cumulus cells enhanced oocyte survival compared to the control (without coculture) and mural follicular cell groups (Experiment 3). The growth and survival rates of oocytes collected from the group of follicles cocultured with cumulus cells from large antral follicles (>3 mm) were greater (P < 0.05) than those from small antral follicles (<3 mm), or than the control group (without cumulus cells, experiment 4). No significant differences in the follicular diameters (674 +/- 30 microm versus 638 +/- 33 and 655 +/- 28 microm) and growth rate (105% versus 94 and 105%) were observed among the preantral follicles of the different treatments (P > 0.05). Taken together, coculture with the cells from large antral follicles (>3 mm) exerted a significant positive effect on oocyte survival. The growth and oocyte survival of preantral follicle cocultured with the same size of follicles (with or without oocyte) were inhibited. Growth and survival rates of preantral follicles and oocytes are improved by coculturing them with the cumulus cells derived from larger antral follicles.     

In vitro maturation and early developmental capacity of bovine oocytes cultured in pure follicular fluid and supplementation with follicular wall

Mammalian oocytes mature in follicular fluid (FF), surrounded by follicular cells. In the present study, in vitro maturation of bovine oocytes cultured in FF from dominant follicles 15-17mm in diameter (with various forms of heat pretreatment) and supplementation with follicular wall from follicles 3-5mm in diameter (FW1) were examined. Heat pretreatment of FF was as follows: (1) no treatment (FF1); (2) 56 degrees C for 30min (FF2); and (3) 100 degrees C for 20s (FF3). After IVM in FF1, oocytes underwent IVF and IVC and embryo development was assessed (up to the morula stage). The rate of oocyte maturation was decreased in pure FF1 versus control (44.5% versus 62.8%, P<0.001). In the control medium, FW1 did not significantly affect nuclear maturation. By contrast, the addition of FW1 to FF1 increased the rate of matured oocytes approximately two-fold (85.9% versus 45.6%, P<0.001). Furthermore, the maturation rate in the FF+FW1 system declined (from 85.9 to 71.0%, P<0.001), whereas that in the FF system increased (from 45.6 to 71.6%, P<0.001) with increased temperature of the FF treatment. Supplementation of the control medium with FW1 increased the yield of morulae (42.6% versus 13.7%, P<0.001). However, the stimulatory effect of FW1 on the morula rate was much higher in pure FF1 (72.5% versus 31.7%, P<0.001). These findings indicated, for the first time, the stimulatory impact of FW1 on in vitro maturation and early developmental capacity of bovine oocytes cultured in pure FF from dominant follicles. We also inferred that bovine FF constituents affecting bovine oocyte maturation and the meiosis-promoting ability of the FW were heat-labile.