Cumulus Expansion Of Cumulus-oocyte Complexes Research Articles

The Calcium-Sensing Receptor Is Involved in Follicle-Stimulating Hormone-Induced Cumulus Expansion in in vitro Cultured Porcine Cumulus-Oocyte Complexes.

The Calcium-Sensing Receptor (CASR) is a G protein-coupled receptor of the C family that reportedly promotes maturation of porcine oocytes. However, its role in cumulus expansion of cumulus-oocyte complexes (COCs) is not well known. This study was conducted to determine the role of CASR and potential mechanisms involved during in vitro maturation (IVM) of porcine COCs. After culture of COCs in follicle-stimulating hormone (FSH)-supplement maturation medium for 24 h, the time of breakdown of the germinal vesicle (GVBD), indicative of initiation of meiotic maturation, resulted in an increased (p < 0.05) CASR mRNA expression level in cumulus cells. Moreover, IVM of COCs in 10 μM of the CASR agonist NPS R-568 promoted (p < 0.05) cumulus expansion but only in FSH-containing medium. Conversely, 20 μM of the CASR inhibitor NPS2390 precluded cumulus expansion. We next tested the effect of the CASR agonist/inhibitor on the expression of cumulus expansion-related genes. The CASR agonist significantly upregulated the expression of hyaluronan acid synthase 2 (HAS2), whereas the CASR inhibitor downregulated the expression of all HAS2, prostaglandin-endoperoxide synthase 2 (PTGS2), and tumor necrosis factor a-induced protein 6 (TNFAIP6). Altogether, these results suggest that CASR activity is involved in FSH-stimulated porcine cumulus expansion.

Synergistic effect between LH and estrogen in the acceleration of cumulus expansion via GPR30 and EGFR pathways.

The estrogen membrane receptor GPR30 (also known as G-protein coupled receptor 30) has recently been shown to be involved in the regulation of oocyte maturation and cumulus expansion. However, whether GPR30 expression is regulated by gonadotropin stimulation and how it participates in the regulation of the maturation process is still not clear. In this study, we explored the mechanism underlying the synergy between luteinizing hormone and 17β-estradiol (17β-E2) to improve the epidermal growth factor (EGF) response in cumulus oocyte complexes (COCs) during oocyte maturation in mice. The expression and distribution of GPR30, EGFR, and EGF-like growth factors were examined by real-time quantitative PCR, western blot, and immunofluorescence staining. Lyso-Tracker Red labeling was performed to detect the lysosomal activity in follicle granular cells (FGCs). Cumulus expansion of COCs was evaluated after in vitro maturation for 16 h. We found that EGF-like growth factors transmit LH signals to increase GRP30 levels by inhibiting protein degradation in lysosomes. Meanwhile, 17β-E2 stimulates the GPR30 signaling pathway to increase EGF receptor levels, enhancing the response ability of EGF signaling in COCs and thus promoting cumulus expansion. In conclusion, our study reveals the synergistic mechanism between LH and estrogen in the regulation of cumulus expansion during oocyte maturation process.

Estrogen improves the development of yak (Bos grunniens) oocytes by targeting cumulus expansion and levels of oocyte-secreted factors during in vitro maturation.

The estrogen-signalling pathway is critical for normal follicular development; however, little is known about its importance during in vitro maturation (IVM) in large animals, particularly yaks (Bos grunniens). Through the present study, we aimed to determine the mechanisms underlying estrogen involvement in cumulus expansion and the subsequent development of cumulus-oocyte complexes (COCs). COCs were cultured in the maturation medium supplemented with different concentrations (10−6–10−3 mM) of 17β-estradiol (E2) or its receptor antagonist, fulvestrant, and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and western blot were performed to determine the expression of cumulus-expansion related factors and oocyte-secreted factors (OSFs). The cumulus expansion of COCs was observed using an inverted microscope, and COCs developmental ability were judged by the evaluation of cleavage and blastulation rates per inseminated oocytes by IVF, and the number of cells in the blastocyst. Cumulus expansion increased with 10−6–10−3 mM E2, but decreased with fulvestrant. HAS2, PTGS2, PTX3 and OSFs expression increased in the 10−6–10−3 mM E2 groups. Significantly higher cleavage and blastocyst rates were observed in the 10−4 mM E2 group than in the fulvestrant and 0 mM E2 groups. Moreover, in the 10−4 mM group, blastocysts at 7 days had higher cell counts than the other groups. In conclusion, the increase in cumulus expansion and subsequent oocyte development after the addition of E2 to IVM medium may have resulted from increased cumulus-expansion-related factor expression and OSF levels.

265 HYALURONAN SYNTHESIS ABILITY OF PORCINE CUMULUS - OOCYTE COMPLEXES DERIVED FROM SMALL FOLLICLES

The degree of cumulus expansion, an important step in oocyte maturation, of porcine cumulus–oocyte complexes (COC) derived from small follicles (SF: 1 to 2 mm in diameter) is known to be lower than those derived from middle follicles (MF: 3 to 6 mm in diameter). The objective of this study was to compare the abilities of hyaluronan (HA) synthesis of COC from SF and MF. Furthermore, the effect of oestradiol during pre-incubation of COC on proliferation of the cumulus cells was examined. Cumulus–oocyte complexes from SF and MF of porcine ovaries were cultured for in vitro maturation [IVM, in modified porcine oocyte medium (Yoshioka et al. 2008 J. Reprod. Dev. 54, 208–213) supplemented with 50 µM β-mercaptoethanol, 10 IU mL–1 of eCG, 10 IU mL–1 of hCG, and 1 mM dbcAMP for 20 h and then in the fresh medium without those supplements for another 24 h]. Hyaluronan production was quantified at 20 h after the start of IVM with a commercial HA-ELISA kit (20 COC/tube × 4 times). The number of cumulus cells was assessed 0 and 20 h after the start of IVM (50 COC × 4 times). Furthermore, proliferation of cumulus cells was examined after pre-culture of COC (n = 40 COC × 5 times) in modified porcine oocyte medium with various concentrations of oestradiol (0, 0.1, 1, and 10 ng mL–1) for 6 h. Statistical analyses of results from 4 to 5 replicated trials were performed by ANOVA with a Bonferroni-Dunn post-hoc test (significance, P &lt; 0.05). The degree of cumulus expansion of COC from MF (n = 152) was higher than that of COC from SF (n = 156). The incidence of metaphase-II oocytes was significantly lower in COC from SF (n = 133; 48.9%) than in COC from MF (n = 148; 74.7%). The HA content of COC was higher in those from MF (20.8 µg/COC) than in those from SF (10.8 µg/COC), whereas the content per cumulus cell was not different because the numbers of cumulus cells at 0 and 20 h were also higher in COC (n = 200 in each group) from MF (3.0 × 103 and 3.3 × 103 cells, respectively) than from SF (2.0 × 103 and 2.5 × 103 cells, respectively). Cumulus cells proliferated significantly in the presence of oestradiol, regardless of the concentration, during pre-incubation for 6 h (2.5 to 2.8 × 103 cells), as compared with the oestradiol-free controls (2.2 × 103 cells). These results demonstrate that the different abilities of cumulus expansion between COC (n = 200 in each group) from SF and MF may be due to the number of cumulus cells per COC. Pre-incubation in the presence of oestradiol stimulates the proliferation of cumulus cells and may improve the oocyte maturation of COC derived from SF.