Effects of Follicle Cells on the Chymotrypsin Resistance of Mouse Oocytes / 대한불임학회지

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.

Differential Expression of Glucose Transporter Gene in Mouse Early Embryos / 대한불임학회지

The uptake of glucose for metabolism and growth is essential to most animal cells and is mediated by glucose-transporter (GLUT) proteins. The aim of this study was to determine which class of glucose transporter molecules was responsible for uptake of glucose in the mouse early embryo and at which stage the corresponding genes were expressed. In addition, co-culture system with vero cell was used to investigate the effect of the system on GLUT expression. Two-cell stage embryos were collected from the superovulated ICR female and divided into 3 groups. As a control, embryos were cultured in 0.4% BSA-T6 medium which includes glucose. For the experimental groups, embryos were cultured in either co-culture system with vero cells or glucose-free 76 medium supplemented with 0.4% BSA and pyruvate as an energy substrate. 2-cell to blastocyst stage embryos in those groups were respectively collected into microtubes (50 embryos/tube). Total RNA was extracted and RT-PCR was performed. The products were analysed after staining ethidium bromide by 2% agarose gel electrophoresis. Blastocysts were collected from each group at 120hr after hCG injection. They were fixed in 2.5% glutaraldehyde, stained with hoechst, and mounted for observation. In control, GLUT1 was expressed from 4-cell to blastocyst. GLUT2 and GLUT3 were expressed in morula and blastocyst. GLUT4 was expressed in all stages. When embryos were cultured in glucose-free medium, no significant difference was shown in the expression of GLUTI1, 2 and 3, compared to control. However GLUT4 was not expressed until morular stage. When embryos were co-cultured with vero cell, there was no significant difference in the expression of GLUT1, 2, 3 and 4 compared to control. To determine cell growth of embryos, the average cell number of blastocyst was counted. The cell number of co-culture (93.8+/-3.1, n=35) is significantly higher than that of control and glucose-free group (76.6 +/- 3.8, n=35 and 68.2+/-4.3, n=30). This study shows that the GLUT genes are expressed differently according to embryo stage. GLUTs were detectable throughout mouse preimplantation development in control and co-culture groups. However, GLUT4 was not detected from 2- to 8-cell stage but detected from morula stage in glucose-free medium, suggested that GLUT genes are expressed autocrinally in the embryo regardless of the presence of glucose as an energy substrate. In addition, co-culture system can increase the cell count of blastocyst but not improve the expression of GLUT. In conclusion, expression of GLUT is dependent on embryo stage in preimplantation embryo development.

Zona Hardening of Mouse Oocytes Undergone Meiotic Resumption In Vivo / 대한불임학회지

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.

Studies on the lectin binding patterns of the oviduct luminal epithelium and oocytes in mouse / 대한불임학회지

No abstract available.

Mouse oviduct induces the changes of egg structure / 대한불임학회지

No abstract available.