Expression of proopiomelanocortin, proenkephalin and prodynorphin genes in porcine theca and granulosa cells

Abstract

Previous studies have demonstrated the presence of endogenous opioid peptides (EOP) in the ovary and suggested their implication in local interactions within ovarian structures. Nevertheless, data pertaining to the expression of genes, coding for the opioid precursors, in ovarian cells are still rudimentary and not available for the pig. The study was undertaken to test whether genes of the opioid precursors – proopiomelanocortin (POMC), proenkephalin (PENK) and prodynorphin (PDYN) – are expressed in non-treated and gonadotropin-treated theca and granulosa cells isolated from ovarian follicles of the pig. The cells were isolated from small (days 15–16 of the estrous cycle) and large (days 19–20) porcine follicles. Dispersed cells were cultured in Eagle's medium under the water saturated atmosphere of 95% air and 5% CO 2, in the presence or absence of respective gonadotropin; theca cells with LH (100 ng/ml) and granulosa cells with FSH (100 ng/ml). Following 24 h-incubation, the cells were harvested and the total RNA was isolated. The expression of genes coding for opioid precursors was estimated by the semi-quantitative RT-PCR technique involving co-amplification of the target cDNA (POMC, PENK or PDYN) and control cDNA (β-actin or 18S rRNA). Specificities of PCR products were confirmed by Southern analysis and sequencing. In theca cells the expression of opioid precursors appeared to be gonadotropin-dependent except for PENK in the cells isolated from large follicles. In turn, granulosa cells exhibited the expression of POMC and PENK genes independently on treatment with FSH. This gonadotropin induced the expression of PDYN gene in granulosa cells isolated from small and large follicles and significantly increased POMC mRNA content in the cells from the large ones. The present studies indicate that porcine follicular cells (especially granulosa cells) may produce opioid peptides and that gonadotropins may modulate gene expression of their precursors in these cells. Moreover, our results support a participation of opioid peptides in the local regulations within ovarian follicle.