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PubMed1998

The effect of follicle stimulating hormone and epidermal growth factor on the developmental capacity of in-vitro matured mouse oocytes.

This study investigates the effects of follicle stimulating hormone (FSH) or epidermal growth factor (EGF) on the development of mouse oocytes matured in vitro. The data show that addition of FSH or EGF does not significantly increase the proportion of oocytes maturing to metaphase II but does increase the ability of these oocytes to cleave to the 2-cell stage after fertilization. After transfer of 2-cell embryos to pseudopregnant recipients, 64-78% of the embryos implanted, which was significantly reduced compared to embryos from in-vivo matured controls (89%). Fewer fetuses at day 14 of gestation were produced from embryos derived from oocytes matured in basal conditions (26%), or in the presence of EGF (32%), compared to oocytes matured in vivo (64%) or in the presence of FSH (58%). Examination of polar bodies and pronuclei of oocytes matured in vitro suggests that an increase in the rate of triploidy may be partly responsible for the increased fetal loss after maturation in the absence of FSH. This study shows that the fertilization rate after in-vitro maturation can be improved by FSH and EGF and that subsequent embryonic development is improved specifically by FSH.

Animals↗

PubMed1996

Developmental competence of in vivo and in vitro matured porcine oocytes after subzonal sperm injection.

In vivo and in vitro matured porcine oocytes were fertilized by subzonal sperm injection (SUZI), and their subsequent development in vitro was examined to determine whether ooplasmic incompetence is the major cause of limited developmental ability of in vitro matured/ fertilized porcine oocytes (Experiment 1). There was no significant difference in rates of fertilization (61% vs. 70%), monospermy (37% vs. 45%), and male pronuclear formation (77% vs. 61%) between in vivo and in vitro matured oocytes. Blastocyst formation rate was significantly lower for in vitro matured oocytes (11% vs. 42%; P < 0.001). Forty-six percent of in vivo matured oocytes cleaved to the 2-4 cell stage by 24 hr in culture after SUZI, compared with 3% of in vitro matured oocytes (P < 0.01). In experiment 2, in vitro development of in vitro matured oocytes with evenly and unevenly granulated cytoplasm were compared after SUZI to examine whether developmentally competent in vitro matured oocytes can be identified on the basis of morphological appearance. Most of the blastocysts obtained developed from oocytes with unevenly granulated cytoplasm (7/56 vs. 1/45; P > 0.05). Experiment 3 revealed that the proportion of oocytes with evenly granulated cytoplasm was originally low (11%) in the population of oocytes used for in vitro maturation, and it increased approximately 3-fold (36%; P < 0.001) after maturation. These results suggest that ooplasmic incompetence in porcine in vitro matured oocytes is the major cause of their limited developmental competence. Cytoplasmic maturation measured by male pronucleus formation does not directly reflect developmental competence of the oocytes. It was also shown that evenness of granulation of the cytoplasm is not a useful morphological indicator of developmental competence.

Animals↗

PubMed1995

Activation of in vitro matured mouse oocytes arrested at first or second meiotic metaphase.

Some mammalian oocytes fail to complete maturation in vitro and arrest development at the first metaphase stage. The response of such blocked oocytes to sperm penetration was investigated. Ovarian mouse oocytes from two inbred strains, CBA/Kw and KE, were cultured in vitro for 20 h. Both oocytes arrested at the first metaphase (MI oocytes) and second metaphase (MII oocytes) were then inseminated. The majority of MII and MI oocytes reinitiated meiosis in response to sperm penetration, although those from the CBA strain did with higher frequency. Moreover, a high proportion of unpenetrated oocytes from CBA, but not the KE strain, resumed meiosis (33% for MII and 48% for MI oocytes, respectively). Parthenogenetic activation of MI-arrested oocytes was demonstrated in (CBAxKE)F1 mice; ovarian oocytes matured in vitro and then treated by electric shock were activated with a similar total frequency of 52.4% for MI and 47.8% for MII oocytes. The rate of activation increased equivalently for both MI and MII oocytes as the length of maturation prolonged. This demonstrates that mouse oocytes arrested at MI during their maturation in vitro continue cytoplasmic maturation and become capable of undergoing activation in a way similar to those maturing to MII. Additionally, in MII oocytes cultured for an equal time in vitro the rate of activation increased with the time lapse after first polar body (PB1) extrusion. This indicates that after PB1 extrusion, the oocyte requires some resting time before it may be activated, perhaps to restore the proper balance between elements of the cell cycle controlling the mechanism involved in first meiotic division.

Animals↗

PubMed2006

Pregnancy and delivery after in vitro maturation of naked ICSI-GV oocytes with GH and transfer of a frozen thawed blastocyst: case report.

PURPOSE: To determine if GV oocytes, collected at the time of ICSI, can be matured in vitro and rescued for therapeutic treatment. A patient for whom all the collected oocytes at the GV stage after a classical COH protocol were matured in vitro with GH. METHOD: All the naked oocytes were matured in a culture medium (ISM2) containing 15% patient serum +1.6 units of GH (Saizen) per millilitre. Oocytes were incubated overnight at 37 degrees C. The MII oocytes obtained were micro-injected. A fresh transfer was performed and a supernumerary blastocyst was frozen. RESULTS: The patient was pregnant and delivered a healthy girl after transfer of the frozen/thawed blastocyst. The baby girl is now 2 years old. CONCLUSION: In vitro maturation with GH allows rescuing naked GV oocytes collected at the time of ICSI. GH action does not pass through the cumulus cells. According to the possible lack of synchrony between the embryo and the uterus, we recommend to freeze the embryos obtained and to replace them in a controlled cycle.

Adult↗

PubMed1978

Method for the preparation of active maturation promoting factor (MPF) from in vitro matured oocytes of Xenopus laevis.

A method for the large scale extraction of Maturation Promoting Factor (MPF) from in vitro matured oocytes of Xenopus laevis is described. MPF has been previously described only as a component(s) of hormone-matured cytoplasm within amphibian oocytes (or eggs) which is able to induce the reinitiation of the meiotic process from late diplotene stage until second metaphase arrest, when microinjected into diplotene arrested (fully grown) recipient oocytes. Standard biochemical methods for the extraction and purification of this factor(s) haven been unsuccessful due to its extreme instability and sensitivity to dilution. The procedure is dependent upon the inclusion of sodium fluoride (NaF) in the extraction medium with its effect presumably due to its ability to inhibit phosphorprotein phosphatases. The successful preservation of MPF activity described in this report permits further attempts to be made to isolate and characterize this, to date, elusive cytoplasmic factor, which plays a key role in the complex cellular processes involved in the hormone-dependent differentiation of an oocyte into an egg.

Animals↗

PubMed2002

Fertilization of mouse oocytes from in vitro-matured preantral follicles using classical in vitro fertilization or intracytoplasmic sperm injection.

Early preantral mouse follicles with a diameter of 110-160 microm were cultured in vitro for 10 or 12 days. Mature oocytes were retrieved following hCG, and fertilization was attempted either by in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). Two-cell and blastocyst formation rates and blastocyst cell numbers were compared between 10-day and 12-day in vitro-matured oocytes versus in vivo-matured oocytes. Uncleaved IVF oocytes were subjected to chromosome analysis. The 2-cell formation rate was significantly improved by ICSI compared with IVF both in 10-day (72.1% versus 56.1%; P = 0.03) and 12-day cultures (74.1% versus 54.5%; P = 0.028). Cytogenetic analysis of uncleaved MII oocytes following IVF showed that about 30% of MII oocytes showed no sign of sperm penetration. The blastocyst formation rate was significantly lower in 12-day versus 10-day cultures, whether fertilization was by IVF (40.7% versus 62.4%, P = 0.016) or by ICSI (32.5% versus 57.1%, P = 0.035). Blastocyst cell numbers from IVF and ICSI 10-day groups were similar and both significantly higher (P < 0.001) than from IVF 12-day cultures. All above expressed values were significantly higher for in vivo-matured oocytes. In conclusion, fertilization of oocytes from in vitro-matured mouse preantral follicles can be optimized with ICSI, giving significantly higher 2-cell formation rates than IVF. Blastocyst formation rate was not influenced by the technique of fertilization but rather by the extent of the in vitro culture period. Best results on preimplantation development of oocytes for in vitro-matured preantral follicles were obtained with ICSI on oocytes from 10-day in vitro cultures.

Animals↗

PubMed1999

Embryo development and pregnancies from in-vitro matured and fertilized human oocytes.

There is an increasing interest in retrieving immature oocytes in the absence of or with limited gonadotrophin exposure, with the aim of maturing them in vitro for embryo transfer purposes. The aim of this report is to present our experience of fertilization, embryonic development and pregnancies from in-vitro maturation cycles. A total of 18 patients underwent 21 cycles in which an average of 8.1 immature oocytes was retrieved after limited exposure to human menopausal gonadotrophin (HMG) and no exposure to human chorionic gonadotrophin (HCG). In one cycle, no oocytes were recovered. The oocytes were cultured for 44 h and 121 oocytes which reached MII were injected with a single spermatozoon. A total of 71 oocytes showed two pronuclei and 53 zygotes cleaved. Forty-four embryos were transferred in 17 cycles. Five weeks after embryo transfer, ultrasound examination indicated the presence of one gestational sac and one fetal heart beat in two patients. The results suggest that in-vitro matured oocytes can undergo fertilization and the resulting embryos may result in pregnancies. However, the success rate was not sufficient to recommend widespread use of the technique without further research.

Cytoplasm↗

PubMed2004

Trichlorfon predisposes to aneuploidy and interferes with spindle formation in in vitro maturing mouse oocytes.

The pesticide trichlorfon (TCF) has been implicated in human trisomy 21, and in errors in chromosome segregation at male meiosis II in the mouse. We previously provided evidence that TCF interferes with spindle integrity and cell-cycle control during murine oogenesis. To assess the aneugenic activity of TCF in oogenesis, we presently analysed maturation, spindle assembly, and chromosome constitution in mouse oocytes maturing in vitro in the presence of 50 or 100 microg/ml TCF for 16 h or in pulse-chase experiments. TCF stimulated maturation to meiosis II at 50 microg/ml, but arrested meiosis in some oocytes at 100 microg/ml. TCF at 100 microg/ml was aneugenic causing non-disjunction of homologous chromosomes at meiosis I, a significant increase of the hyperploidy rate at metaphase II, and a significant rise in the numbers of oocytes that contained a 'diploid' set of metaphase II chromosomes (dyads). TCF elevated the rate of precocious chromatid segregation (predivision) at 50 and 100 microg/ml. Pulse-chase experiments with 100 microg/ml TCF present during the first 7 h or the last 9 h of maturation in vitro did not affect meiotic progression and induced intermediate levels of hyperploidy at metaphase II. Exposure to > or =50 microg/ml TCF throughout maturation in vitro induced severe spindle aberrations at metaphase II, and over one-third of the oocytes failed to align all chromosomes at the spindle equator (congression failure). These observations suggest that exposure to high concentrations of TCF induces non-disjunction at meiosis I of oogenesis, while lower doses may preferentially cause errors in chromosome segregation at meiosis II due to disturbances in spindle function, and chromosome congression as well as precocious separation of chromatids prior to anaphase II. The data support evidence from other studies that TCF has to be regarded as a germ cell aneugen.

Aneuploidy↗

PubMed1983

[Separate groups in the oocyte population of cows, their cytogenetic analysis and capacity for in vitro maturation].

Heterogenic population of the follicular oocytes of bovine ovaries has been divided into 5 groups on the basis of morphological characteristics. Chromatin structure, frequency of degenerative changes and the ability to maturation in vitro of different oocyte groups have been studied. The correlation of morphological characteristics of native oocytes and their ability to mature in vitro have been shown. The bovine follicular oocyte classification allows to pick out more homogeneous oocyte groups in comparison with the population as a whole and the oocyte group which is the best for maturation in vitro.

Animals↗

PubMed2005

Fertilization of in vitro matured human oocytes by intracytoplasmic sperm injection (ICSI) using ejaculated and testicular spermatozoa.

AIM: To evaluate the fertilization competence of spermatozoa from ejaculates and testicle when the oocytes were matured in vitro following intracytoplasmic sperm injection (ICSI). METHODS: Fifty-six completed cycles in 46 women with polycystic ovarian syndrome were grouped according to the semen parameters of their male partners. Group 1 was 47 cycles that presented motile and normal morphology spermatozoa in ejaculates and Group 2 was the other nine cycles where male partners were diagnosed as obstructive azoospermia and spermatozoa could only be found in testicular tissue fragment. All female patients received minimal stimulation with gonadotropin. Immature oocytes were matured in vitro and inseminated by ICSI. The spermatozoa from testes were retrieved by testicular fine needle aspiration. RESULTS: A total of 449 and 78 immature oocytes were collected and cultured for 48 hours, 75.5 % (339/449) and 84.6 % (66/78) oocytes were matured in Groups 1 and 2, respectively. The percentage of oocytes achieving normal fertilization was significantly higher in Group 1 than that in Group 2 (72.9 % vs. 54.5 %, P 0.05). There were no significant differences in the rates of oocytes cleavage and clinical pregnancies in these two groups [87.4 % (216/247) vs. 88.9 % (32/36); 21.3 % (10/47) vs. 44.4 % (4/9)]. A total of 15 babies in the two groups were healthy delivered at term. CONCLUSION: It appears that IVM combined with ICSI using testicular spermatozoa can produce healthy infants, while the normal fertilization rate of in vitro matured oocytes after ICSI using testicular spermatozoa was significantly lower than using the ejaculated spermatozoa.

Adult↗

PubMed1997

Dynamics of maturation-promoting factor and its constituent proteins during in vitro maturation of bovine oocytes.

Maturation-promoting factor (MPF) is known to be a key regulator of both mitotic and meiotic cell cycles. MPF is a complex of a B cyclin and the cyclin-dependent kinase cdkl (p34cdc2). Oocyte maturation and its arrest at metaphase of meiosis II (MII) are regulated by changes in MPF activity. In this study, experiments were conducted to examine the dynamics of MPF activity and its constituent proteins during in vitro maturation of bovine oocytes. Bovine oocytes displayed relatively low levels of MPF (histone H1 kinase) activity at the germinal vesicle stage during the first 8 h of maturation. MPF activity increased gradually thereafter, and its first peak of activity occurred at 12-14 h of maturation (presumptive metaphase I), which was followed by an abrupt reduction in activity at 16-18 h, during presumptive anaphase and telophase. MPF activity then increased, reaching a plateau at 20-24 h of maturation (MII stage). This high level of MPF activity was maintained for several hours but decreased gradually after 30 h of maturation and became barely detectable by 48 h of in vitro maturation (IVM) culture. At each time point, there was a significant variation among individual oocytes in histone H1 kinase activity, which was probably due to asynchronous maturation. Abundance of cdk1 increased gradually during the first 8 h and then remained relatively constant except for an apparent reduction at 18-22 h of IVM. The level of cyclin B2 increased quickly during the initial 2 h of culture, and this high level was maintained until 16 h, after which a significant reduction was observed between 18 and 22 h of IVM. The de novo synthesis of cyclin B2, however, exhibited a biphasic oscillation during maturation, with peaks before the onset of MI and of MII. These results have defined the profiles of MPF activity and its individual components during bovine oocyte maturation in vitro. We conclude that active MPF regulates bovine oocyte maturation and that de novo synthesis of cyclin B2 occurs during the process of maturation.

Animals↗

PubMed1993

Quantitative cytochemical study of some enzymatic activities in preovulatory bovine oocytes after in vitro maturation.

In the present study quantitative cytochemical assays were used to measure some enzymatic activities in situ in bovine meiotically immature oocytes and oocytes matured in vitro, since the special metabolic activity of the growing oocytes may be a pivotal factor in stabilizing the meiotically arrested oocytes. Modifications of this particular metabolism might destabilize the arrested meiosis. Preovulatory oocytes, mostly at the germinal vesicle stage, were obtained by puncturing follicles ranging from 2 to 6 mm in diameter with a hypodermic needle. A group of collected oocytes was incubated in maturation medium CRML 1066 to obtain metaphase II oocytes. Succinate, lactate and glucose-6-phosphate dehydrogenase activities in just collected meiotically immature and in vitro matured oocytes were assayed cytochemically. Microdensitometric measurements were made with a Vickers M85a scanning microdensitometer. Our findings show that: 1) succinate dehydrogenase activity was significantly increased in matured oocytes; 2) lactate dehydrogenase activity was present and very strong in immature oocytes but was detectable in only about 50% of matured oocytes, with significantly lower integrated optical density values; 3) glucose-6-phosphate dehydrogenase activity was very high in immature oocytes but significantly decreased after in vitro maturation; 4) there was no linear correlation between the integrated optical densities of the three enzymatic activities and the diameters of the oocytes. We suggest that the ability to utilize glucose may appear earlier in bovine oocytes than in other species and takes place at the time of maturation.

Animals↗

PubMed2005

Fertilization, cleavage and blastocyst development according to the maturation timing of oocytes in in vitro maturation cycles.

BACKGROUND: This study was to examine the developmental capacity of oocytes collected from an in vitro maturation (IVM) programme according to their maturation time. METHODS: The study included 47 IVM cycles that underwent blastocyst transfer. The patients (n = 38) were primed with 10 000 IU HCG 36 h before their oocyte retrieval. The oocytes were classified into three groups: group 1 (n = 139) where oocytes were matured on day of oocyte collection; group 2 (n = 627) where oocytes were matured on day 1 after IVM; group 3 (n = 163) where oocytes matured on day 2 after IVM. Fertilization, cleavage and blastocyst formation were compared between three groups. RESULTS: Rates of cleavage and blastocyst development in group 3 (72.2%, 96/133; 19.0%, 15/133) were significantly lower than those of group 1 (100%, 108/108; 58.3%, 63/108) and group 2 (91.5%, 487/532; 50.4%, 268/532) respectively (P < 0.01). The number of freezable good quality blastocysts among blastocysts developed from group 1 (52.4%, 33/63) was significantly higher than those from group 2 (35.4%, 95/268) and group 3 (6.7%, 1/15) (P < 0.01). There were 24 clinical pregnancies (51.1%, 24/47) after transfer of the blastocysts and 29 healthy babies were delivered. CONCLUSION: These results suggest that oocytes reaching metaphase II faster in an IVM programme have better embryonic developmental competence.

Adult↗

PubMed1993

In vitro maturation of oocytes from a marsupial, the tammar wallaby (Macropus eugenii).

This study examined the competence of oocytes from the tammar wallaby, Macropus eugenii to mature in vitro. Oocytes were collected from follicles > 1 mm diameter 24 h after pregnant mare serum gonadotrophin (PMSG) treatment and incubated in Eagle's minimum essential medium supplemented with 10% fetal calf serum, at 35 degrees C in 5% CO2 in air for 24, 36, or 48 h. Oocytes were incubated either granulosa cell-intact or granulosa cell-free or in the presence of 10 IU ml-1 PMSG or 10 micrograms ml-1 porcine luteinizing hormone (LH) + 10 micrograms ml-1 porcine follicle stimulating hormone (FSH). The ability of oocytes recovered from small (< 1.5-mm-diameter) and large (> or = 1.5 mm-diameter) follicles to mature in vitro was also examined. The nuclear status of oocytes was assessed using the DNA-specific dye Hoechst 33342. Initially, all oocytes examined contained a germinal vesicle. After 24 h of culture, 60% of oocytes had progressed to metaphase I or anaphase I. After 36 h, approximately 20% of oocytes possessed metaphase II chromosomes, and 20% of oocytes were at metaphase I or anaphase I. At the completion of the 48 h culture period, 40% of oocytes had completed maturation to the metaphase II stage. In vitro oocyte maturation after 48 h was not affected by the presence of granulosa cells, PMSG, or LH and FSH. More oocytes from large follicles (55%) completed maturation by 48 h than from small follicles (20%). Approximately 50% of oocytes remained at the GV stage at all times under all conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed2000

Nuclear status and cytogenetics of embryos derived from in vitro-matured oocytes.

OBJECTIVE: To analyze embryos after in vitro maturation by investigating their nuclear status and cytogenetic constitution. DESIGN: Prospective randomized laboratory study. SETTING: Reproductive medicine unit in an academic hospital. PATIENT(S): Patients with male and tubal factor infertility undergoing fertility treatment. INTERVENTION(S): Denuded immature oocytes (n = 75) were matured in vitro for 24-30 hours, and intracytoplasmic sperm injection was performed 30 hours after oocyte retrieval. Fluorescence in situ hybridization was performed on the produced embryos. MAIN OUTCOME MEASURE(S): Blastomere content of the total embryo. RESULT(S): The in vitro-matured oocytes showed a similar fertilization rate as the in vivo-matured oocytes, but with a higher incidence of noncleavage (21.0%). In addition, 26.7% of these embryos arrested at the first mitotic division. Thirty embryos were processed for fluorescence in situ hybridization; only 6.7% had all mononuclear blastomeres, 30.0% had at least one binuclear blastomere, 43.3% had at least one multinuclear blastomere, and 56.6% contained anuclear cells. The chromosomal constitution was analyzed in 14 embryos, and chromosomal anomalies were found in 11 (78.5%). CONCLUSION(S): Germinal vesicle oocytes retrieved from superovulated patients and cultured in vitro for a short time had the ability to resume meiosis and achieve fertilization. However, arrest of embryo development was common. These embryos showed a high incidence of multinuclear blastomeres and aneuploidy, suggesting abnormal cytokinesis or genetic abnormalities.

Adult↗

PubMed2006

Glutathione content and glutathione peroxidase expression in in vivo and in vitro matured equine oocytes.

The in vitro developmental competence of equine oocytes is still low in comparison with other domestic mammals. A major factor affecting the viability of cells during in vitro culture is the increased oxidative stress. Oxidative modifications could be responsible for oocyte incompetence for in vitro maturation (IVM). Cysteamine, a glutathione (GSH) synthesis enhancer, has been shown to increase intracellular GSH content and to improve embryo development when added during IVM of bovine, porcine, and ovine oocytes. The aim of the present study was (1) to determine whether equine cumulus-oocyte complexes (COCs) benefit from the addition of cysteamine during IVM, (2) to compare the GSH content of oocytes after in vivo maturation and IVM, (3) to assess whether cysteamine administration during IVM of equine oocyte enhances early embryonic developmental capability following ICSI, (4) to study the glutathione peroxidase (GPX) mRNA level in COCs. In vivo matured COCs were collected by aspiration from preovulatory follicles, and analyzed at collection. Immature COCs were collected in vivo or from slaughterhouse ovaries and matured in culture media supplemented or not with 100 microM cysteamine. After nuclear stage assessment, oocytes were analyzed for GSH concentration and both oocytes and cumulus cells were analyzed for GPX and GAPDH mRNA. Our data showed that the maturation capability was similar in both in vivo aspirated oocytes and in those isolated from slaughterhouse ovaries. Moreover, the addition of cysteamine during IVM affected neither GSH content nor maturation rate. At the time of collection, intra-oocyte GSH content was not influenced by the chromatin status. GSH concentration was similar in in vivo and in vitro matured metaphase II (MII) stage oocytes, and was significantly higher in MII than immature germinal vesicle stage oocytes. Moreover, the presence of serum inhibited whereas its absence stimulated the accumulation of GSH within oocytes during IVM. After ICSI, a similar proportion of zygotes in each group developed beyond the two-cell stage after 72 hr of culture. Cumulus cells expressed GPX mRNA, while GPX transcript was absent in both immature and mature oocytes. Cumulus expression of GPX mRNA was significantly higher when analyzed at collection than after IVM. Taken together, our results demonstrate that in equine oocytes, GSH increases during IVM but the relative intra-oocyte content of this thiol does not affect maturation and early development efficiency after fertilization. We hypothesize that factor(s) other than GSH/GPX are responsible for the limited in vitro early developmental capability of equine oocytes.

Animals↗

PubMed2004

Relationship between the morphological changes of somatic compartment and the kinetics of nuclear and cytoplasmic maturation of oocytes during in vitro maturation of porcine follicular oocytes.

Based on the morphology and expansion of the cumulus cells, several different classes of porcine cumulus-oocyte complexes (COCs) can be distinguished, during their maturation in vitro. The goal of the present study was to find out the rate of each morphologic category in case of COCs and granulosa-cumulus-oocyte complexes (GCOCs), the characteristics of their nuclear progression, cytoplasmic maturation, and the frequency of monospermy after IVF. It was found that the frequency of cumulus expansion is higher in case of GCOCs than that of COCs. Nuclear progression of COCs was more accelerated than that of GCOCs. Oocytes attached to the bottom of culture dish with dark, compact cumulus underwent nuclear and acquired their ability to be activated earlier than that of oocytes showing normal cumulus expansion. The rate of monospermic fertilization after IVF of normal COCs showing normal cumulus expansion was higher than that of COCs attached to the dish. These results suggest that diverse behavior of cumulus cells during in vitro culture affects nuclear and cytoplasmic maturation of porcine oocytes, which also affects IVF results. It can be concluded that granulosa cells promote normal cumulus expansion thus decrease heterogeneity in nuclear and cytoplasmic maturation amongst oocytes.

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↗