Stromal-Derived Factor-1 Acutely Upregulates Expression of HAS-2 and TSG-6 in Feline Cumulus Cell Oocyte Complexes (COCs).

YOUNG, KA; ROJO, JL; PELUFFO, MC; LINARI, M

Congreso; 50th SSR Annual Meeting; 2017

Society for the Study of Reproduction (SSR)

The cumulus cell-oocyte complex (COC) is regulated by a number of factors, possibly including signaling by ovarian chemokines. Stromal-derived factor-1 (SDF1; CXCL12), is a chemokine that is expressed in multiple tissue types, including in the ovary, and serves as the ligand to chemokine CXC-motif receptor-4 (CXCR4). While LH upregulates follicular CXCR4, the role that SDF1/CXCR4 signaling might play in the COC was unknown. We hypothesized that the SDF-1/CXCR4 system was present in feline COCs and that culture of feline COCs with SDF-1 would directly upregulate genes expressed in the ovulatory cascade. To address this hypothesis, a COC culture was established. Ovaries (n= 50) were obtained from adult domestic cats during the breeding season from routine spays at unknown stages of the estrous cycle. Antral follicles larger than 0.5 mm were mechanically dissected and COCs were prepared for culture. The culture environment was optimized by empirically testing different media types, multiple concentrations of charcoal stripped/unstripped fetal bovine serum (FBS) in the absence (negative control) or presence of recombinant human FSH + LH (GNT, positive control group). To establish culture conditions, COCs (n=24) were cultured at different time-points (3, 12 and 24 h) with and without GNT. Following culture optimization, the effects of 25ng/ml SDF-1 and the CXCR-4 inhibitor were examined in the COC culture for 3, 12, and 24h (n=48). At each collection time point, COCs were either flash frozen for RNA extraction or fixed for immunoflorescent detection of the CXCR4 and SDF1 protein. Expression of key genes in the ovulatory cascade, HAS-2, TSG-6, AREG, and GDF-9, was assessed by real-time PCR. MEM-hepes with 1% of charcoal stripped-FBS was determined to be the optimum medium for this culture, as observed by the increase/expansion of the COC at 24h. The mRNA expression of HAS-2, TSG-6, and AREG peaked at 3h of culture in the GNT group as compared to GNT treatment in the 12 and 24h groups and, as compared to negative controls at all time points (p0.05). COCs cultured with 25ng/ml SDF-1 showed increased HAS-2 and TSG-6 mRNA expression at 3h as compared to negative controls and COCs treated with the CXCR4 inhibitor. In contrast, no changes were observed in HAS-2 and TSG-6 at other time points, and no changes in AREG and GDF-9 expression were noted between SDF-1 and control treated groups, at any time point. CXCR4 and SDF1 immunostaining was present in both cumulus cells and the oocyte of feline COCs. In summary, these results suggest that this model can be used to examine molecular processes within the COC. In addition, the SDF1-CXCR4 pathway may extend its function beyond chemoattractant, and may play a role in cumulus cells via upregulation of key ovulatory genes concomitant with gonadotropin stimulation. This study was supported by PRESTAMO BID PICT 2014 Nº 666 (MCP), Small Faculty Grants Program CNSM CSULB (KAY), and by the Fogarty International Center, of the National Institutes of Health under Award R01TW009163 (MCP).