SAT-192 Chemokine Receptor CCR2 as a Novel Mediator of LH Signaling

Shortly before ovulation the LH surge induces processes critical for fertility, including cumulus-oocyte expansion (C-OE) and resumption of meiosis. While some of the paracrine-acting factors important for these events have been identified, the molecular mechanisms responsible for initiating such complex processes are not fully understood. Recent work from our laboratory showed a direct effect of the monocyte chemoattractant protein 1 (MCP1)/ C-C motif chemokine receptor 2 (CCR2) system, by increasing the mRNA levels of key genes involved in the ovulatory cascade in vitro. The aim of the present study was to determine whether inhibition of CCR2 signaling in the COC interfere with the expression of periovulatory genes and/or oocyte maturation. To test this, ovaries were surgically removed from adult female domestic cats (Felis catus, n=33) at unknown stages of the estrous cycle during the breeding season. Then, COCs were isolated from antral follicles and cultured for 3 hr (time when periovulatory genes peak in our culture system) or 28 hr [in vitro maturation (IVM)] with known inducers of C-OE and/or oocyte maturation [Gonadotropins (GNTs), amphiregulin (AREG) and PGE2)] in the presence or absence of a highly selective CCR2 antagonist (1 µM; RS 504393). At the end of culture (3 hr), total RNA from each COC (n=40) was individually extracted and the mRNA expression of periovulatory genes (HAS2, AREG, TSG6, PTX3) was analyzed by quantitative real-time PCR using specific TaqMan probes. In a second experiment, after IVM (28 hr) oocytes (n=161) were denuded and fixed to subsequent assess their nuclear oocyte maturation by immunofluorescence, due to the dark appearance of the feline oocytes. Expression results showed that RS 504393 was able to prevent or interfere (p< 0.05) with the stimulation of genes induced by either GNTs, AREG or PGE2. When combined with GNTs, RS 504393 decreased the expression of AREG, HAS2 and TSG6 mRNA within the COC (p<0.05). In contrast, the addition of RS 504393 together with the GNTs increased PTX3 mRNA levels within the COC (p<0.05). Also, RS 504393 significantly decreased the mRNA levels of AREG and TSG6 induced by AREG and PGE2 treatments. However, RS 504393 was not able to inhibit the stimulation of PTX3 mRNA levels induced by AREG (p> 0.05). Regarding oocyte maturation, RS 504393 did not provoke a significant reduction of the proportion of MII oocytes stimulated by GNT or AREG. No significant effect was observed in the presence of PGE2 under our IVM culture conditions. In conclusion, our results demonstrated that the GNT stimulation of AREG, HAS2 and TSG6 mRNA levels occurs, at least in part, through the CCR2/MCP1 pathway, proposing CCR2 receptor as a novel mediator of the ovulatory LH signaling. This study was supported by PRESTAMO BID PICT 2014 Nº 666 and by the Fogarty International Center, of the National Institutes of Health under Award Number R01TW009163.