No abstract available.
Animals ; Cumulus Cells* ; Mice* ; Zygote*

OBJECTIVE: Mammalian follicle cells are the most important somatic cells which help oocytes grow, mature and ovulate and thus are believed to provide oocytes with various functional and structural components. In the present study we have examined whether cumulus or granulosa cells might play a role in establishing the plasma membrane structure of mouse oocytes during meiotic maturation. DESIGN: In particular the differential resistances of mouse oocytes against chymotrypsin treatment were examined following culture with or without cumulus or granulosa cells, or in these cell-conditioned media. RESULTS: When mouse denuded oocytes, freed from their surrounding cumulus cells, were cultured in vitro for 17~18 hr and then treated with 1% chymotrypsin, half of the oocytes underwent degeneration within 37.5 min (t50=37.5+/-7.5 min) after the treatment. In contrast cumulus-enclosed oocytes showed t50=207.0. Similarly, when oocytes were co-cultured with cumulus cells which were not associated with the oocytes but present in the same medium, the t50 of co-cultured oocytes was 177.5+/-13.1 min. Furthermore, when oocytes were cultured in the cumulus cell-conditioned medium, t50 of these oocytes was 190.0+/-10.8 min whereas t50 of the oocytes cultured in M16 alone was 25.5+/-2.9 min. Granulosa cell-conditioned medium also increased the resistance of oocytes against chymotrypsin treatment such that t50 of oocytes cultured in granulosa cell-conditioned medium was 152.5+/-19.0 min while that of oocytes cultured in M16 alone was 70.0+/-8.2 min. To see what molecular components of follicle cell-conditioned medium are involved in the above effects, the granulosa cell-conditioned medium was separated into two factions by using Microcon-10 membrane filter having a 10 kDa cut-off range. When denuded oocytes were cultured in medium containing the retentate, t50 of the oocytes was 70.0+/-10.5 min. In contrast, t50 of the denuded oocytes cultured in medium containing the filtrate was 142.0+/-26.5 min. T50 of denuded oocytes cultured in medium containing both retentate and filtrate was 188.0+/- 13.6 min. However, t50 of denuded oocytes cultured in M16 alone was 70.0 +/-11.0 min and that of oocytes cultured in whole granulosa cell-conditioned medium was 156.0+/-27.9 min. When surface membrane proteins of oocytes were electrophoretically analyzed, no difference was found between the protein profiles of oocytes cultured in M16 alone and of those cultured in the filtrate. CONCLUSIONS: Based upon these results, it is concluded that mouse follicle cells secrete a factor(s) which enhance the resistance of mouse oocytes against a proteolytic enzyme treatment. The factor appears to be a small molecules having a molecular weight less than 10 kDa.
Animals ; Cell Membrane ; Chymotrypsin* ; Cumulus Cells ; Female ; Granulosa Cells ; Membrane Proteins ; Membranes ; Mice* ; Molecular Weight ; Oocytes*

It is well known that the bona pellucidae of mouse oocytes become 'hardened' when they are allowed to mature in vitro in the absence of serum components. To see if oocytes already undergone meiotic resumption in vivo exhibit similar zona hardening, hardening of ZP of cumulus-enclosed oocytes(CEOs) was examined after culture in vitro since their release from follicles various hours after hCG injection. When CEOs matured in vivo for 3h or longer were subjected to culture in vitro for 14h with BSA alone, zona hardening was significantly reduced compared to those cultured in vitro from the begining of maturation. However, when CEOs matured in vivo for 5h were freed from cumulus cells and then cultured in vitro with BSA alone, little reduction of zona hardening was observed. Preincubation of CEOs for 5h with fetuin, one of the well known inhibitor of in vitro zone hardening, did not prevent bona hardening during its subsequent culture of CEOs for 14h without fetuin. However, when CEOs precultured with both fetuin and PMSG for 5h and then further cultured with BSA alone for 14h, zona hardening was dramatically reduced. Under these conditions, the expansion of cumulus cell was observed. In addition, CEOs cultured with both BSA and dbcAMP to prevent their meiotic resumption showed a significant increase of zona hardening. Whether the observed zona hardening was correlated with the conversion of ZP2 to ZP2f was examined. Zona pellucida, isolated from CEOs matured for 5h in vivo and then further cultured with BSA alone was subjected to SDS-PAGE. Most of ZP2 molecules from these CEOs did not undergo conversion from ZP2 to ZP2f. From these results, it is concluded that CEOs undergone meiotic resumption in vivo do not exhibit bona hardening when they were subsequently cultured in vitro without serum components. It appears that cumulus cells play an important role in this phenomenon.
Animals ; Bucladesine ; Cumulus Cells ; Electrophoresis, Polyacrylamide Gel ; Fetuins ; Herpes Zoster* ; Mice* ; Oocytes* ; Zona Pellucida

Despite the rapid development of assisted reproductive technologies (ART) in recent years, implantation rates after replacement of embryos into the uterine cavity remains low. Several techniques such as culture conditions based on formulations of human tubal fluid and various ART techniques as GIFT, ZIFT, TET have been adopted in recent years to improve embryo viability in vitro and implantation rates. Also, coculture of human IVF-derived embryos have been used in an effort to increase the number of viable embryos following IVF and to improve synchrony between the developing embryo and the uterine environment. The aim of this study was to evaluate whether the use of coculture with autologous cumulus cells has a significant beneficial effect on the development of embryos in vitro and its relation to the pregnancy rates in 120 patients with previous failed IVF-ET from September, 1995 to January 1998. We obtained the results from which significant improvement in the quality of viable embryos were observed using a coculture system with autologous cumulus cells, but pregnancy rates in this group of patients did not differ from the rate in the standard IVF group during the same period. Our study shows that a simplified short-term coculture system with autologous cumulus cells may help rescue moderate quality embryos to cleave regularly.
Coculture Techniques* ; Cumulus Cells* ; Embryonic Structures* ; Humans ; Pregnancy Rate* ; Pregnancy* ; Reproductive Techniques, Assisted ; Zygote Intrafallopian Transfer

This study was performed to investigate the stimulating effect on oocyte maturation and cumulus cell expansion in TC199 media by human cord serum (HCS) supplementation. Immature mouse oocyte cumulus complexes (OCCs) were cultured in TC199 media supplemented with bovine serum albumin (BSA), HCS and human chorionic gonadotropin (hCG) instead of luteinizing hormone (LH) respectively, and the expression of cumulus expansion and oocyte maturation were observed. After 4hr and 24hr culture with or without OCCs, media containing 0.4% BSA, 10% HCS and 10 lU hCG respectively were collected and analyzed for changing concentrations of estradiol (E2), progesterone(P4), testosterone(T), and PGF2. There were no elevation of E2, T, and PGF2 by OCCs culture, but minute elevation of P4 level by 24hr OCCs culture in hCG supplementation (p=0.048). The stimulating pattern of cumulus expansion of OCCs by HCS and hCG supplementation was similar to our previously report using Ham's F-10 media, however oocyte maturation rates after 24hr OCCs culture in all media were increased by 20~30% compared to Ham's F-10 media. These results suggest that LH in HCS induce cumulus expansion probably by P4 secretion of OCCs, and TC199 is efficient media for immature mouse oocyte maturation.
Animals ; Chorionic Gonadotropin ; Cumulus Cells* ; Dinoprost ; Estradiol ; Humans* ; Luteinizing Hormone ; Mice ; Oocytes* ; Serum Albumin, Bovine

Mammalian oocytes within Graafian follicles are arrested at prophase I of meiosis. C-type natriuretic peptide (NPPC), secreted by mural granulosa cells (MGCs), maintains oocyte meiotic arrest via binding to its cognate receptor natriuretic peptide receptor 2 (NPR2) and producing cyclic guanosine monophosphate (cGMP). NPR2 is most concentrated in the cumulus cells. In addition, cAMP, gap junction, inosine monophosphate dehydrogenase (IMPDH) and other important regulatory factors are also involved in meiotic arrest. Luteinizing hormone (LH) then rapidly decreases cGMP and induces oocyte meiotic resumption. In this paper, advances in the molecular mechanisms of meiotic arrest and LH-induced meiotic resumption were reviewed. This paper may provide new ideas for the prevention, diagnosis and treatment of related reproductive diseases.
Animals ; Cumulus Cells ; Female ; Luteinizing Hormone ; Meiosis ; Natriuretic Peptide, C-Type ; genetics ; Oocytes

OBJECTIVE: To establish the evaluation system of the quality of oocytes on the basis of the incidence of cumulus cells apoptosis, to investigate the relationships between the incidence of cumulus cells and the outcomes of IVF-ET. METHOD: Thirth-four cycles undergoing controlled ovarian hypersimulation for IVF-ET with tubal infertility (23 cycles) or unexplained infertility (11 cycles) were included in this study. Cumulus cell masses surrounding mature oocyte and co-culture of embryos with autologous cumulus cells during IVF-ET process. The incidence of apoptosis in cumulus cells was assessed by apoptosis detection kit fluorescein. The effect of co-culture using cumulus cells and the incidence of cumulus cells apoptosis. RESULTS: The results were as follows: 1. The incidence of apoptosis in cumulus cells markedly increased in patients aged 40 or over, while the fertilization rate was greatly decreased in those age group. 2. Apoptosis in cumulus cells was found in both the fertilized oocytes and unfertilized oocytes, but the incidence of apoptosis was higher in unfertilized oocytes. 3. There is no clear correlation between apoptosis in cumulus cells and the number of oocytes retrieved. However, the incidence of apoptosis was increased when the number of oocytes retrieved was 5 and fewer in comparison with 6~10. 4. Embryo grade was significantly affected by the incidence of apoptosis in cumulus cells. 5. Pregnancy rate of IVF-ET per cycle was 29.4%, and the pregnant group had the higher fertilization rate and a significantly lower incidence of apoptosis in cumulus cells compared with the nonpregnant group. 6. When cumulus cells were used as helper cells in the co-culture of the embryo, in vitro activity of cumulus cells based on morphological change and proliferation did not influence the quality of embryo, but was closely associated with the implantation rate and pregnancy rate, which was enhanced when morphological changes and proliferation of cumulus cells was more active. 7. This difference in the outcome of IVF-ET according to in vitro activity of cumulus cells used for co-cultue was not associated with the incidence of apoptosis in cumulus cells, but rather had likely relations with the different secretion pattern of protein, which may be an embryotrophic factor by cumulus cells. CONCLUSION: These results suggest that the incidence of apoptosis in cumulus cells can be used in predicting oocyte qualities and the outcomes of IVF-ET. And the effect of co-culture largely depends on the in vitro activity of cumulus cells as well.
Apoptosis ; Coculture Techniques ; Cumulus Cells* ; Embryonic Structures ; Fertilization ; Fluorescein ; Humans ; Incidence ; Infertility ; Oocytes ; Pregnancy Rate ; T-Lymphocytes, Helper-Inducer

OBJECTIVE: To evaluate whether the rate of early mouse embryonal development could be enhanced by cumulus cell coculture in vitro. METHODS: Ham's F-10 culture media supplemented with 0.4% bovine serum albumin were used. Two-cell F1 mouse embryos were cultured in media with or without cumulus cells of female ICR mouse embryo for 96 hours, and the rates of embryonal development were observed and compared. RESULTS: The percentage of hatched blastocyst in the coculture group was significantly higher than that in the control group by 87.3% vs 64.8% respectively (p< 0.05). CONCLUSION: This study provides confirmative information that cumulus cell coculture will be useful in enhancing early mouse embryonal development.
Animals ; Blastocyst ; Coculture Techniques* ; Culture Media ; Cumulus Cells* ; Embryonic Structures ; Female ; Humans ; Mice* ; Mice, Inbred ICR ; Serum Albumin, Bovine

OBJECTIVE: This study was conducted to compare the effects of static culture, dynamic culture, and the combination of dynamic culture with specialized surfaces involving co-culture on human embryonic development. Embryos cultured using conventional static culture (SC) techniques served as a control group. We compared dynamic culture using micro-vibration culture (MVC) and micro-vibration with co-culture (MCoC), in which autologous cumulus cells were used as a specialized surface. METHODS: We conducted a chart review of patients who were treated between January 2011 and November 2014 in order to compare embryonic development rates and pregnancy rates among the groups. Zygotes were cultured in micro-droplets, and embryos were subsequently selected for transfer. Some surplus embryos were cryopreserved, and the others were cultured for blastocyst development. A micro-vibrator was set at the frequency of 42 Hz for duration of 5 seconds per 60 minutes to facilitate embryo development. RESULTS: No significant differences among the groups were present in patient's characteristics. However, the clinical pregnancy rates were significantly higher in the MVC group and the MCoC group than in the SC group. No significant differences were found in the blastocyst development rate between the SC group and the MVC group, but the blastocyst development rate in the MCoC group was significantly higher than in the SC and MVC groups. CONCLUSION: The clinical pregnancy rate was significantly increased by the application of micro-vibration to the embryonic cultures of poor responders. The blastocyst development rate was significantly increased by the application of MCoC to surplus embryos.
Blastocyst ; Coculture Techniques* ; Cumulus Cells ; Embryo Culture Techniques ; Embryonic Development ; Embryonic Structures ; Female ; Humans ; Pregnancy ; Pregnancy Rate ; Zygote

OBJECTIVE: The goal of this study was to evaluate the pregnancy potential of immature (metaphase I or germinal vesicle stage) oocytes retrieved in intracytoplasmic sperm injection (ICSI) cycles. METHODS: A total of 1,871 couples with infertility underwent 2,984 ICSI cycles. Cycles in which three or fewer oocytes were retrieved were included in this study in order to evaluate the pregnancy potential of immature oocytes. Cycles were divided into five groups (group I-V), according to the maturation status of the oocytes at the time of cumulus cell removal and ICSI. The fertilization and pregnancy rates after ICSI were analyzed and compared among the study groups based on the maturation status of the retrieved oocytes. RESULTS: The retrieval of only immature oocytes was associated with a significant decrease in the fertilization rate (76.1%+/-37.3% vs. 49.0%+/-49.1%, 66.7%+/-48.7%; group I vs. group II, group III, respectively) and the average number of transferred embryos (1.5+/-0.7 vs. 1.1+/-0.4, 1.1+/-0.6). The cycle cancellation rate was significantly higher when only immature oocytes were retrieved. The clinical pregnancy rate decreased significantly when the transferred embryos had originated from immature oocytes (16.9% vs. 10.3%, 1.2%). CONCLUSION: In ICSI cycles, the fertilization potential and pregnancy potential of the immature oocytes retrieved in ICSI cycles were inferior to those of mature oocytes. Therefore, increasing the number of injectable oocytes and transferrable embryos by using immature oocytes after their spontaneous in vitro maturation does not necessarily improve pregnancy outcomes.
Cumulus Cells ; Embryonic Structures ; Family Characteristics ; Female ; Fertilization* ; Infertility ; Oocytes* ; Pregnancy Outcome ; Pregnancy Rate ; Pregnancy* ; Sperm Injections, Intracytoplasmic*