Abstract

Cyclooxygenase2 (COX-2), encoded by the Cox-2 gene within the periovulatory follicles is a 26 critical mediator of oocyte development. Growth differentiation factor 9 (GDF-9) and bone 27 morphogenetic protein 15 (BMP-15) participate in the modulation of certain target genes in the 28 ovary, possibly influencing the Cox-2 gene expression. However, this relationship has not been 29 characterized in canines. This study aimed to examine the possible relationships among BMP- 30 15, GDF-9, progesterone and Cox-2 gene expression in granulosa-cumulus cells in dogs. 31 Granulosa cells from antral follicles and their corresponding cumulus-oocyte complexes 32 (COCs) and follicular fluid (FF) were separately obtained from 56 ovaries collected from adult 33 bitches at estrus (n= 15) and proestrus (n=13) following ovariohysterectomy. Total RNA 34 extraction was performed in follicular cells, and Cox-2 gene expression was assessed by q-PCR 35 analysis. Progesterone, BMP-15 and GDF-9 were determined in the FF samples using ELISA 36 assays. COCs were subjected to IVM with or without (control) recombinant GDF-9 and BMP- 37 15. After 72 h of culture, Cox-2 transcript analyses were performed in cumulus cells via q-PCR. 38 Data were evaluated by ANOVA. An increased (P<0.05) in Cox-2 mRNA levels was observed 39 in granulosa/theca cells from follicles at estrus with respect to those at proestrus. However, the 40 levels of BMP-15 and GDF-9 in FF decreased (P<0.05), whereas progesterone increased 41 (P<0.05) from the proestrus phase to the estrus phase. Cox-2 gene expression in cumulus cells 42 was 4-fold greater (P<0.01) than that in the control when both growth factors were added to the 43 IVM culture. In conclusion, although BMP-15-GDF-9 appear to upregulate the levels of Cox-2 44 transcripts during in vitro maturation, the inverse relationship of these paracrine factors with 45 Cox2 gene expression and the positive correlation of progesterone with Cox2 transcripts, 46 suggest that the high progesterone levels could be more relevant in the local mechanisms 47 regulating the Cox-2 gene expression.