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PubMed1994

Ooplasmectomy has no influence on FSH-induced expansion of bovine cumulus investment in vitro.

Bovine cumulus-oocyte complexes with or without attached pieces of the mural stratum granulosum were matured with or without FSH in vitro for 24 h. Some complexes were ooplasmectomized (removal of ooplasm) at the onset of culture or after 9 h of culture, and in others the complete oocyte, including the zona pellucida, was removed. At the end of culture, the degree and morphology of corona and cumulus expansion were determined by a subjective scoring system, transmission electron microscopy followed by computerized image analysis, or scanning electron microscopy. In the absence of FSH no expansion was seen. In the presence of FSH, hyaluronidase-sensitive cumulus expansion was observed in intact complexes and complexes ooplasmectomized after 9 h of culture. In complexes ooplasmectomized at the start of culture, hyaluronidase-insensitive expansion of the corona compartment was seen as well. A similar reaction was found in free corona and cumulus cell masses cultured in the presence or absence of oocytes. Mural granulosa in intact and ooplasmectomized complexes expanded only slightly under the influence of FSH and exhibited no hyaluronidase sensitivity. Ooplasmectomy at the start of culture increased the frequency of ruffled cell membranes and decreased the occurrence of intercellular bridges as compared with unmanipulated complexes cultured under corresponding conditions. It is concluded that FSH-induced cumulus expansion and hyaluronidase-sensitive extracellular mucus production of bovine cumulus investment in vitro are not dependent on the oocyte, except for minor changes in the surface morphology of the cumulus cells.

Animals↗
PubMed1996

Prolonged dominance of follicles and reduced viability of bovine oocytes.

Prolonging the lifespan of bovine follicles is known to result in reduced fertility after ovulation and insemination. In this study, the effect of prolonged development of follicles on oocyte viability was examined. In Expt 1, six cows in the aspirated-prolonged-follicle group received a vaginal progesterone releasing device on day 4 of the oestrous cycle (day 1 = ovulation) and prostaglandin F2 alpha (40 mg) on day 6. Ultrasound-guided follicular aspiration was performed on day 13. Six cows in the aspirated-growing-follicle group received prostaglandin F2 alpha on day 6, and follicular aspiration on day 7. In Expt 2, all cows were stimulated with 36 mg FSH-P to develop multiple large follicles for study. Three cows in the prolonged-multiple-follicle group received the same treatment as did cows in Expt 1, but were ovariectomized on day 13. Three cows in the growing-multiple-follicle group received prostaglandin F2 alpha on day 6 and were ovariectomized on day 7. Oocytes recovered in both experiments were stained to reveal the stage of nuclear development. All oocytes from aspirated-prolonged and prolonged-multiple follicles showed expanded cumulus cells and condensed chromatin dispersed in the ooplasm, with possible germinal vesicle breakdown. Oocytes from aspirated-growing and growing-multiple follicles showed compact cumulus cells and an intact germinal vesicle. Plasma concentrations of oestradiol in both growing follicle groups increased until oocyte recovery. Oestradiol in the aspirated-prolonged-follicle group increased after luteolysis on day 6 and remained high until follicular aspiration. In contrast, in the FSH-stimulated prolonged-multiple-follicle group, oestradiol fell to trace amounts on day 8 and remained low. Oestradiol concentrations in follicular fluid were consistent with plasma concentrations for all groups. Bovine oocytes from prolonged dominant follicles undergo premature maturation in vivo, which perhaps accounts for the poor fertility observed in other studies when oestrous synchronization with progestins prolongs follicle lifespan.

Animals↗
PubMed1987

Morphology and fertilizability of frozen human oocytes.

Human oocytes were frozen and thawed by four methods previously used for cryopreservation of human embryos. Most of these oocytes were inseminated after thawing to assess their capacity to fertilize and form pronuclear ova. Their morphology was assessed by phase-contrast microscopy used in routine IVF. Twenty-three oocytes were examined by electron microscopy to critically evaluate the effects of cooling and cryopreservation and to confirm fertilization. Morphological survival was observed in more than 60% of the oocytes examined after freeze-thawing. The main features of cryoinjury were cracks in the zona pellucida, disruption of the plasma membrane and extensive disorganization of the ooplasm. Subtle changes in the cytosol of cumulus cells was also observed. Cooling to 0 degrees C or -6 degrees C had little effect on cytoplasmic structure. Spindles were damaged in two frozen oocytes. Cumulus cell activity, sperm binding to the zona, sperm penetration of the zona seem to be largely unaffected by freeze-thawing. Fertilization was observed in eight oocytes after postthaw insemination and three embryos (8-cell to morula stages) were developed from pronuclear ova on further culture. Both monospermic and polyspermic fertilization were confirmed by electron microscopy and micronuclei were detected in three pronuclear ova. The genetic implications of these nuclear aberrations are discussed. These preliminary studies indicate that oocyte freezing needs to be integrated cautiously with clinical IVF by further assessment of embryos developed from frozen oocytes.

Female↗
PubMed2006

Localization and quantitative expression of the calcium-sensing receptor protein in human oocytes.

OBJECTIVE: To investigate the expression of the calcium-sensing receptor (CaSR) protein in human oocytes at the germinal vesicle (GV) and metaphase I (MI) and II (MII) stages. DESIGN: Prospective study. SETTING: Academic basic research laboratory and hospital-based fertility center. PATIENT(S): Immature and supernumerary mature oocytes (n = 118) excluded from intracytoplasmic sperm injection treatment. INTERVENTION(S): Immunofluorescence and Western blot with a primary antibody against human CaSR. Confocal laser scanning microscopy (CLSM) together with quantitative image analysis used to measure the fluorescence intensity variations in oocytes at GV, MI, and MII stages. MAIN OUTCOME MEASURE(S): The CaSR expression pattern as evaluated by immunostaining in denuded oocytes and cumulus cells, CLSM, and three-dimensional image reconstructions; quantitative analysis at the equatorial plane of the oocyte. RESULT(S): We identified CaSR in human oocytes and cumulus cells. The fluorescence intensity within the oocyte varied with the developmental stage, with the greatest increase at the MI stage. CONCLUSION(S): The present study demonstrates for the first time the expression and localization of CaSR protein in human oocytes. Increased CaSR protein expression in the MI stage suggests that it may be involved in the regulation of human oocyte development and maturation.

Cells, Cultured↗
PubMed1992

Structural aspects of bovine oocyte maturation in vitro.

Bovine cumulus-oocyte complexes (COCs) were collected from 4-8 mm follicles and graded into four categories on their morphological characteristics. These four categories were matured in vitro and processed for transmission electron microscopy at 24 h after the onset of culture. The morphology of the four groups of oocytes was analysed and compared with that of oocytes that had matured in vivo and were collected 20-23 h after the preovulatory luteinizing hormone peak. After in vivo maturation, oocytes formed a homogeneous group with respect to their morphological characteristics. After in vitro maturation, the oocytes formed a heterogeneous group with respect to their morphology between as well as within the four categories of oocytes. Oocytes from the first three categories showed the same morphology after in vitro maturation. The fourth category showed some specific characteristics: 1) vacuolization, 2) flattening of cumulus cells, and 3) almost complete lack of cortical granules in some category 4 oocytes. These characteristics are interpreted as signs of degeneration. Besides these aspects of degeneration, other deviations from normal development were seen: 1) retraction of cumulus cell process endings from the oocyte without the breaking down of these processes, 2) retardation of some aspects of the cytoplasmic maturation, and 3) incomplete cumulus expansion. It is concluded that oocytes capable of development in vitro show a large morphological variability before the onset of culture. In vitro maturation systems can support normal development, but many oocytes show signs of degeneration and deviant development after in vitro maturation.

Animals↗
PubMed1997

Correlations between chemical parameters, mitogenic activity and embryotrophic activity of bovine oviduct-conditioned medium.

To establish parameters predicting the quality of bovine oviduct epithelial cell-conditioned media, we compared media conditioned by oviduct cells from cows at Day 2 (n = 3) and Day 15 (n = 3) of the estrous cycle. In addition, we tested the influence of time of conditioning. Media were evaluated for their embryotrophic activity using a cumulus cell co-culture system as a control. The same media were tested for their mitogenic activity on NIH 3T3 cells and for chemical parameters, including total protein, and de novo synthesized protein as well as for concentrations of glucose, lactate and ammonium. Analysis of variance did not reveal a significant effect by stage of the estrous cycle on the embryotrophic activity of conditioned media. However, there was a significant effect by time of conditioning on the proportion of 5- to 8-cell embryos (P < 0.01) and of blastocysts and hatched blastocysts (P < 0.05). None of the conditioned media (19 to 31% blastocysts) was superior to the cumulus cell co-culture system (32% blastocysts). In the conditioned media, the proportion of 5- to 8-cell embryos correlated positively with mitogenic activity on 3T3 cells (r = 0.64; P < 0.05), whereas the proportion of blastocysts was not significantly correlated with this parameter. In summary, our results provide evidence for an effect of time of conditioning on embryotrophic activity of oviduct epithelial cell-conditioned media. The fact that mitogens for NIH 3T3 cells affect the proportion of 5- to 8-cell embryos but not of blastocysts suggests different culture requirements for early and late preimplantation stage development of bovine embryos.

Journal Article↗
PubMed1990

Structural aspects of oocyte maturation in the blue fox (Alopex lagopus).

Blood samples were taken weekly from seventeen mature blue fox vixens (average age five years), from late anoestrus until pro-oestrus, and then taken daily. The vixens were sacrificed at various stages of oestrus, and oocytes were collected from ovarian follicles by aspiration, and/or from oviducts by flushing. The structural features of oocyte maturation were related to the time of the luteinizing hormone (LH) peak. On days 1-2 after the LH peak the oocyte nucleus migrated from a central to a peripheral position in the ooplasm and assumed a flattened appearance. The cumulus investment expanded simultaneously and ovulation took place around day 2. On days 2-3 the oocyte nuclear envelope broke down, the nucleoli disappeared, the metaphase of the first meiotic division was reached, the Golgi complexes decreased in size, the perivitelline space enlarged, and all junctional contact between cumulus cell projections and oocyte was disrupted. On days 3-5 the first polar body was extruded, the metaphase of the second meiotic division was reached, and the cumulus cells degenerated. On day 5 the release of cortical granule content was occasionally seen, and from day 6 the oocytes showed signs of degeneration. In a few animals deviant oocyte maturation was noticed.

Animals↗
PubMed1999

Ultrasound-guided follicle aspiration: the collection of bovine cumulus-oocyte complexes from ovaries of slaughtered or live cows.

To increase the collection efficiency of bovine cumulus-oocyte-complexes (COCs) by transvaginal aspiration, the effects of aspiration pressure and needle diameter on bovine follicular oocyte collection were assessed. Oocytes were aspirated from ovaries of slaughtered cows using 2 different diameter needles (18- or 21-gauge) with 4 different aspiration pressures (40, 80, 120 or 160 mmHg) and of live cows using 18-gauge needles with 40 or 80 mmHg, or using 21-gauge needles with 80 or 120 mmHg. The recovered oocytes were divided into 4 categories according to the surrounding cumulus cells and quality of oocytes: 1) 4 or more layers, 2) between 1 and 3 layers, 3) completely or partially denuded and 4) all others, including expanded cumulus cells and degenerated oocytes. The highest oocyte recovery rates from Categories 1 and 2 were obtained using 18-gauge needles with 40 mmHg pressure and 21-gauge needles with 120 mmHg pressure, respectively, from the ovaries of slaughtered cows. When oocytes were collected from live cows, the highest recovery rates for Categories 1 and 2 were obtained using an 18-gauge needle and 40 mmHg pressure, and 21-gauge needle and 80 mmHg, respectively. In addition, the proportion of oocytes in each category were compared between ovaries from slaughtered and live cows. The proportion of Category 1 oocytes collected from live cows was lower than from slaughtered cows when 18-gauge needles at 80 mmHg (P<0.05). The results show that the combination of aspiration pressure and needle diameter is crucial for COC collection, and they suggest that optimal aspiration conditions for ovaries of slaughtered cows are not necessarily applicable to live cows.

Animals↗
PubMed1993

Effect of cyclic AMP on the isolated human oocyte--cumulus complex.

Cyclic AMP (cAMP) is one of the key regulators of the resumption of oocyte meiosis (oocyte maturation) in several mammalian species. In this study we have examined the role for cAMP in regulation of spontaneous maturation of human oocytes. Oocytes were obtained in connection with gynaecological operations and cultured for 24 or 48 h. A significantly larger proportion (77%) of oocytes cultured in the presence of 1 mM dibuturyl-cAMP (dbcAMP) for 24 h were arrested in meiosis compared to oocytes cultured in control medium (25%). In addition, dbcAMP stimulated progesterone accumulation during 48 h of incubation and inhibited monolayer formation of the cumulus cells attached to the oocyte. The data presented in this study demonstrate that dbcAMP delayed the spontaneous maturation of human oocytes and stimulated progesterone production by the cumulus cells. These results indicate that cAMP may be important also for regulation of meiosis in human oocytes and confirm results obtained in other species.

Adult↗
PubMed1993

[Electron microscopy immunolocalization of ribosomal ribonucleoproteins using autoantibodies from patients with systemic lupus erythematosus].

Serum of patients against rRNP was tested by Western blot method. Four ribonucleoproteins (15, 16, 36 and 38 kDa) specifically reacted with the antiserum used. The autoantibodies were used for immunolocalization of antigenic structures by immunogold method on ultrathin cryosections of hepatocytes, cumulus cells of antral follicle and spermatocytes. Mice and hamsters were used. The cytoplasm appeared to be labelled most intensively. Gold particles were loosely arranged in the cytoplasm (cumulus cells) or they were associated with endoplasmatic reticulum (hepatocytes). Nucleoli were labelled by a lower incidence of the marker. Gold particles were found over the dense fibrillar component bordering on the granular component. In the cytoplasm individual gold particles were observed. (Fig. 7, Ref. 31.).

Animals↗
PubMed2004

Methylation reprogramming and chromosomal aneuploidy in in vivo fertilized and cloned rabbit preimplantation embryos.

Active demethylation of the paternal genome but not of the maternal genome occurs in fertilized mouse, rat, pig, and bovine zygotes. To study whether this early demethylation wave is important for embryonic development, we have analyzed the global methylation patterns of both in vivo-fertilized and cloned rabbit embryos. Anti-5-methylcytosine immunofluorescence of in vivo-fertilized rabbit embryos revealed that the equally high methylation levels of the paternal and maternal genomes are largely maintained from the zygote up to the 16-cell stage. The lack of detectable methylation changes in rabbit preimplantation embryos suggests that genome-wide demethylation is not an obligatory requirement for epigenetic reprogramming. The methylation patterns of embryos derived from fibroblast and cumulus cell nuclear transfer were similar to those of in vivo-fertilized rabbit embryos. Fluorescence in situ hybridization with chromosome-specific BACs demonstrated significantly increased chromosomal aneuploidy rates in cumulus cell nuclear transfer rabbit embryos and embryos derived from nuclear transfer of rabbit fibroblasts into bovine oocytes compared with in vivo-fertilized rabbit embryos. The incidence of chromosomal abnormalities was correlated with subsequent developmental failure. We propose that postzygotic mitotic errors are one important explanation of why mammalian cloning often fails.

Aneuploidy↗
PubMed1992

Investigation of means to improve rates of fertilization in in vitro matured/in vitro fertilized bovine oocytes.

Experiments were conducted with 5,979 oocytes to determine whether detaching some of the cumulus cells from oocytes either before or after maturation would improve the fertilization rate and proportion of oocytes that developed to expanded blastocysts. Oocytes were aspirated from ovaries of slaughtered cows and matured, fertilized and cultured in vitro. Pipetting immature oocytes before maturation to detach some of the cumulus, with all cumulus cells left in the maturation wells, significantly increased fertilization rates, especially of oocytes that initially had a full cumulus investment. In further experiments, pipetting oocytes either before or after maturation to detach most of the cumulus, or treating with hyaluronidase after maturation to disperse the cumulus, significantly increased fertilization rates and proportions of oocytes developing to expanded blastocysts.

Journal Article↗
PubMed1992

Mouse oocytes promote proliferation of granulosa cells from preantral and antral follicles in vitro.

Evidence is now emerging that the oocyte plays a role in the development and function of granulosa cells. This study focuses on the role of the oocyte in the proliferation of (1) undifferentiated granulosa cells from preantral follicles and (2) more differentiated mural granulosa cells and cumulus granulosa cells from antral follicles. Preantral follicles were isolated from 12-day-old mice, and mural granulosa cells and oocyte-cumulus complexes were obtained from gonadotropin-primed 22-day-old mice. Cell proliferation was quantified by autoradiographic determination of the 3H-thymidine labeling index. To determine the role of the oocyte in granulosa cell proliferation, oocyte-cumulus cell complexes and preantral follicles were oocytectomized (OOX), oocytectomy being a microsurgical procedure that removes the oocyte while retaining the three-dimensional structure of the complex or follicle. Mural granulosa cells as well as intact and OOX complexes and follicles were cultured with or without FSH in unconditioned medium or oocyte-conditioned medium (1 oocyte/microliter of medium). Preantral follicles were cultured for 4 days, after which 3H-thymidine was added to each group for a further 24 h. Mural granulosa cells were cultured as monolayers for an equilibration period of 24 h and then treated for a 48-h period, with 3H-thymidine added for the last 24 h. Oocyte-cumulus cell complexes were incubated for 4 h and then 3H-thymidine was added to each group for an additional 3-h period. FSH and/or oocyte-conditioned medium caused an increase in the labeling index of mural granulosa cells in monolayer culture; however, no differences were found among treatment groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2001

Oocyte developmental competence after in vitro maturation depends on the persistence of cumulus-oocyte comunications which are linked to the intracellular concentration of cAMP.

Oocyte and cumulus cells are interconnected by an extensive network of gap junctions (GJ) formed by connexin 43. In the present study the functional and morphological status of GJ was investigated during in vitro maturation of bovine oocytes using media known to induce different rates of maturation and developmental competence as well as different levels of cumulus expansion and intracellular concentrations of cAMP. GJ functional condition was studied by microinjection of the fluorescent dye Lucyfer Yellow in cumulus-enclosed oocytes and the intracellular distribution of connexin 43 was examined by immunofluorescence immunocytochemistry. Both functional and morphological analysis of GJ between oocytes and cumulus cells indicated that high maturation and development rates are accompanied by the prolonged persistence of permeable communications, which, however, are independent from cumulus expansion. On the contrary, the premature interruption of such communications was linked to low maturation and development. When these results were correlated to the measurements of intracellular levels of cAMP it was observed that cumulus expansion, GJ permeability and good quality maturation require the highest levels of cAMP. Intermediate cAMP levels determine GJ permeability and good quality maturation in the absence of cumulus expansion while below a threshold level of cAMP, oocyte maturation is defective with a premature interruption of communications through GJ and lack of cumulus expansion which lead to poor development.

Animals↗
PubMed1994

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 diameter 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.

Journal Article↗
PubMed2004

Possible function of the ADAM1a/ADAM2 Fertilin complex in the appearance of ADAM3 on the sperm surface.

In mouse, two different isoforms of ADAM1 (fertilin alpha), ADAM1a and ADAM1b, are produced in the testis. ADAM1a is localized within the endoplasmic reticulum of testicular germ cells, whereas epididymal sperm contain only ADAM1b on the plasma membrane. In this study, we show that the loss of ADAM1a results in the male infertility because of the severely impaired ability of sperm to migrate from the uterus into the oviduct through the uterotubal junction. However, epididymal sperm of ADAM1a-deficient mice were capable of fertilizing cumulus-intact, zona pellucida-intact eggs in vitro despite the delayed dispersal of cumulus cells and the reduced adhesion/binding to the zona pellucida. Among testis (sperm)-specific proteins examined, only the level of ADAM3 (cyritestin) was strongly reduced in ADAM1a-deficient mouse sperm. Moreover, the appearance of ADAM3 on the sperm surface was dependent on the formation of a fertilin protein complex between ADAM1a and ADAM2 (fertilin beta) in testicular germ cells, although no direct interaction between the fertilin complex and ADAM3 was found. These results suggest that ADAM1a/ADAM2 fertilin may be implicated in the selective transport of specific sperm proteins including ADAM3 from the endoplasmic reticulum of testicular germ cells onto the cell surface. These proteins then can participate in sperm migration into the oviduct, the dispersal of cumulus cells, and sperm binding to the zona pellucida.

ADAM Proteins↗
PubMed1999

Effect of lactate dehydrogenase activity and isoenzyme localization in bovine oocytes and utilization of oxidative substrates on in vitro maturation.

Oocyte nutritional metabolism changes during maturation in order to increase the energy available to support metabolic requirements. The aim of this work was to study pyruvate and lactate utilization as oxidative substrates on IVM and lactate dehydrogenase (LDH) activity and localization of their isoenzymes in bovine oocytes. Immature cumulus-oocyte complexes (COCs) were recovered by aspiration of antral follicles in ovaries obtained from slaughtered cows. The COCs and denuded oocytes were separately cultured in TCM-199 with steer serum (controls) and were supplemented with pyruvate, lactate or lactate plus NAD for 24 h at 39 degrees C in 5% CO2:95% humidified air. No significant differences were found in IVM rates of COCs matured according to the various treatments (P>0.05). The IVM rate in denuded oocytes without supplementation was 47.8%. The presence of pyruvate in the culture medium resulted in an increased number of matured denuded oocytes (59.4%; P<0.05), but the addition of lactate failed to improve the IVM rate of matured denuded oocytes (47.6%, P>0.05). When the medium was supplemented with lactate plus NAD, the IVM rate of denuded oocytes likewise failed to differ from that obtained with the addition of pyruvate (59.9%, P>0.05). The LDH activity in immature and matured COCs and denuded oocytes was (3.1+/-1.6) 10(-3), (3.3+/-1.6) 10(-3) U/COC, (5.2+/-2.0) 10(-5), (5.4+/-3.5) 10(-5) U/oocyte with pyruvate as substrate, and (1.2+/-0.5) 10(-3), (1.0+/-0.5) 10(-3) U/COC, (2.2+/-0.1) 10(-5), (2.5+/-1.4) 10(-5) U/oocyte respectively, with lactate; no significant differences due to maturation status were observed (P>0.05; n = 9 for each LDH activity). Electrophoresis disclosed that the principal band corresponded to the LDH-1 isoenzyme in oocytes, while there was no predominance of any isoenzyme in cumulus cells. Due to the fact that LDH-1 is the main oocyte isoenzyme, the pyruvate used during oocyte maturation could be partly produced from lactate when the NAD supply is adequate. Cumulus cells would be responsible for providing pyruvate and/or lactate as oxidative substrates to be used by the bovine oocyte and this supply would be regulated by the LDH activity in these cells.

Animals↗
PubMed2001

Influence of different stages of the oestrous cycle on cumulus-oocyte communications in canine oocytes: effects on the efficiency of in vitro maturation.

The aim of this study was to investigate the effects of different stages of the oestrous cycle on the communications between dog oocytes and cumulus cells, as well as their possible relationships with the meiotic competence of oocytes matured in vitro. Ovaries were harvested from bitches undergoing anoestrus or late pro-oestrus. Maturation of cumulus-oocyte complexes (COCs) was performed in TCM-199 with 0.3% (w/v) BSA, 0.5 iu FSH ml-1 and 0.5 iu LH ml-1 for 72 h. At the time of collection (0 h), and after 24, 48 or 72 h of culture, some oocytes were stained with Lacmoid to evaluate the nuclear stage of maturation. At 0, 24 or 48 h of in vitro maturation (IVM), oocytes were microinjected (n = 84) with a 3% (w/v) Lucifer yellow solution to evaluate the presence of cumulus-oocyte communications through gap junctions. At t = 0, communications were open in 89% of COCs collected during late pro-oestrus, whereas such communications were not evident in any of the oocytes collected during anoestrus. Communications remained permeable for the first 24 h of maturation in many late pro-oestrous oocytes (67%), whereas no fluorescence diffusion was observed at 48 h of maturation. The presence of cumulus-oocyte communications in late pro-oestrous oocytes was accompanied by their ability to resume meiosis and reach telophase I or metaphase II at higher rates than in oocytes collected during anoestrus (11.1% versus 0%, respectively; P < 0.01). These results indicate that, during anoestrus, there are no permeable gap junctions between oocytes and surrounding cumulus cells at the time of isolation from the ovary. This finding indicates that such oocytes are unsuitable for IVM, as confirmed by their inability to reach telophase I or metaphase II in culture. In contrast, a positive correlation was observed between the presence of permeable gap junctions and meiotic competence in COCs isolated at late pro-oestrus. However, the small proportion of oocytes reaching telophase I and metaphase II indicates that further efforts to define better culture conditions are still required.

Anestrus↗