CONICET | Buscador de Institutos y Recursos Humanos

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