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.

Papaverine-sensitive phosphodiesterase activity is measured in bovine spermatozoa.

Cyclic adenosine monophosphate (cAMP) plays a crucial role as a signaling molecule for capacitation, motility, and acrosome reaction in mammalian spermatozoa. It is well-known that cAMP degradation by phosphodiesterase (PDE) enzyme has a major impact on sperm functions. This study was undertaken to characterize cAMP-PDE activity in bovine spermatozoa. Total cAMP-PDE activity in cauda epididymal and ejaculated spermatozoa was 543.2 ± 49.5 and 1252.6 ± 86.5 fmoles/min/106 spermatozoa, respectively. Using different family-specific PDE inhibitors, we showed that in cauda epididymal and ejaculated spermatozoa, the major cAMP-PDE activity was papaverine-sensitive (44.5% and 57.5%, respectively, at 400 nm, papaverine is a specific inhibitor of the PDE10 family). These data are supporting the functional presence of PDE10 in bovine spermatozoa and were further confirmed by western blot to be PDE10A. Using immunocytochemistry, we showed immunoreactive signal for PDE10A present on the post-acrosomal region of the head and on the flagella of ejaculated spermatozoa. Using papaverine, we showed that it promotes tyrosine phosphorylation of sperm proteins, phosphorylation of Erk1 and Erk2, and Ca2+ release from Ca2+ store. These results suggest that PDE10 is functionally present in bovine spermatozoa and is affecting different molecular events involved in capacitation, most probably by cAMP local regulation.

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.