The effects of LH inhibition with cetrorelix on cumulus cell gene expression during the luteal phase under ovarian coasting stimulation in cattle.

Cumulus cells have an important role to play in the final preparation of the oocyte before ovulation. During the final phase of follicular differentiation, FSH levels are low and LH maintains follicular growth; however, it is not known if at that time LH has an influence on cumulus cells inside the follicle. In humans, LH is often inhibited to avoid a premature ovulatory LH surge. This procedure provides a tool to investigate the role of LH in follicular development. In this study, we investigated the impact of suppressing LH using the GnRH antagonist cetrorelix during an ovarian coasting stimulation protocol on the transcriptome of bovine cumulus cells (CC). Oocytes were collected twice from 6 dairy cows. For the first collection, the cows received FSH twice daily for 3 d, followed by FSH withdrawal for 68 h as a control protocol. For the second collection, the same stimulation protocol was used, but the cows also received, starting on day 2 of FSH stimulation, a GnRH antagonist once a day until recovery of the cumulus-oocyte complexes (COC). Half of the COC were subjected to in vitro maturation, fertilization, and culture to assess blastocyst rates. The other half of the COC underwent microarray analysis (n = 3 cows, 2 treatments, 6 oocyte collections) and qRT-PCR (n = 6 cows: 3 microarray cows +3 other cows, 2 treatments, 12 oocyte collections). The differential expression of specific genes was confirmed by RT-qPCR: decrease of ATP6AP2, SC4MOL, and OSTC and increase of PTGDS in the LH-inhibited condition. The global transcriptomic analysis of cumulus cells demonstrated that the inhibition of LH secretion may decrease survival and growth of the follicle. Moreover, the results suggested that LH may be important to cumulus for the maintenance of cellular mechanisms such as global RNA expression, protein and nucleic acid metabolism, and energy production. These results support the hypothesis that LH support is important during the final part of follicle maturation through its influence on the cumulus cells.

Transcriptional characteristics of different sized follicles in relation to embryo transferability: potential role of hepatocyte growth factor signalling.

STUDY HYPOTHESIS: We hypothesized that a better discrimination between follicles containing oocytes with high developmental competence and those containing oocytes with low competence, based on a combination of a follicle's size and transcriptomic signature, will provide a reliable method to predict embryonic outcome of IVF. STUDY FINDING: This study provides new insights on the impact of follicular size on oocyte quality as measured by embryonic development and demonstrates that medium follicles yield a better percentage of transferable embryos. WHAT IS KNOWN ALREADY: Although it is generally accepted that large ovarian follicles contain better eggs, other studies report that a better follicular size subdivision and a better characterization are needed. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: Individual follicles (n = 136), from a total of 33 women undergoing IVF, were aspirated and categorized on the basis of their follicular liquid volume (small, medium or large) and the embryonic outcome of the enclosed oocyte: poor or good development. Comprehensive gene expression analysis between cells from the different sized follicles was performed using microarrays and quantitative RT-PCR to find molecular markers associated with follicular maturity and oocyte developmental competence. MAIN RESULTS AND THE ROLE OF CHANCE: The analysis of embryonic outcome in relation to follicular size indicates that the medium-sized follicles category yielded more transferable embryos (35%) compared with the largest follicles (30%) (NS). Gene expression analysis revealed expression markers with significant (P < 0.05) discrimination between the poor development groups for all three follicle sizes, and good development medium-size follicles, including up-regulation of thrombomodulin, transforming growth factor, beta receptor II and chondrolecti, and those associated with hyaluronan synthesis, coagulation and hepatocyte growth factor signalling. LIMITATIONS, REASONS FOR CAUTION: These analyses were performed in a single cohort of patients coming from a single clinic and the biomarkers generated will require validation in different geographical and biological contexts to ensure their global applicability. WIDER IMPLICATIONS OF THE FINDINGS: Medium-size follicles seem to be the optimal size for a positive embryonic outcome and are associated with competence markers that may help in understanding the ideal differentiation status during late folliculogenesis. LARGE SCALE DATA: The data discussed in this publication have been deposited in The National Center for Biotechnology Information Gene Expression Omnibus database and are accessible through GEO Series accession number GSE52851. STUDY FUNDING AND COMPETING INTERESTS: This study was supported by Canadian Institutes of Health Research (CIHR) and Natural Sciences and Engineering Research Council of Canada (NSERC) to M.A.S. There are no competing interests to declare.

Cumulus cell gene expression associated with pre-ovulatory acquisition of developmental competence in bovine oocytes.

The final days before ovulation impact significantly on follicular function and oocyte quality. This study investigated the cumulus cell (CC) transcriptomic changes during the oocyte developmental competence acquisition period. Six dairy cows were used for 24 oocyte collections and received FSH twice daily over 3 days, followed by FSH withdrawal for 20, 44, 68 and 92 h in four different oestrous cycles for each of the six cows. Half of the cumulus-oocyte complexes were subjected to in vitro maturation, fertilisation and culture to assess blastocyst rate. The other half of the CC underwent microarray analysis (n=3 cows, 12 oocyte collections) and qRT-PCR (n=3 other cows, 12 oocyte collections). According to blastocyst rates, 20 h of FSH withdrawal led to under-differentiated follicles (49%), 44 and 68 h to the most competent follicles (71% and 61%) and 92 h to over-differentiated ones (51%). Ten genes, from the gene lists corresponding to the three different follicular states, were subjected to qRT-PCR. Interestingly, CYP11A1 and NSDHL gene expression profiles reflected the blastocyst rate. However most genes were associated with the over-differentiated status: GATM, MAN1A1, VNN1 and NRP1. The early period of FSH withdrawal has a minimal effect on cumulus gene expression, whereas the longest period has a very significant one and indicates the beginning of the atresia process.