Staszkiewicz Jaroslaw, Skowronski Mariusz T, Kaminski Tadeusz, Siawrys Gabriela, Krazinski Bartlomiej E, Kusmider Maciej, Przala Jadwiga, Okrasa Stanislaw
Department of Animal Physiology, University of Warmia and Mazury in Olsztyn, Oczapowskiego 1A, 10-719 Olsztyn, Poland.
Anim Reprod Sci. 2007 Sep;101(1-2):97-112. doi: 10.1016/j.anireprosci.2006.09.003. Epub 2006 Sep 9.
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 24h-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 (beta-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.
以往的研究已证实卵巢中存在内源性阿片肽（EOP），并提示其参与卵巢结构内的局部相互作用。然而，关于卵巢细胞中编码阿片肽前体的基因表达的数据仍然很初步，且尚无猪的相关数据。本研究旨在检测阿片肽前体基因——阿黑皮素原（POMC）、脑啡肽原（PENK）和强啡肽原（PDYN）——在未处理及促性腺激素处理的、从猪卵巢卵泡分离出的卵泡膜细胞和颗粒细胞中是否表达。细胞从小（发情周期第15 - 16天）、大（第19 - 20天）猪卵泡中分离。分散的细胞在含95%空气和5% CO₂的水饱和气氛下，于Eagle培养基中培养，添加或不添加相应促性腺激素；卵泡膜细胞添加LH（100 ng/ml），颗粒细胞添加FSH（100 ng/ml）。孵育24小时后，收获细胞并提取总RNA。通过半定量RT-PCR技术估计编码阿片肽前体的基因表达，该技术涉及靶标cDNA（POMC、PENK或PDYN）与对照cDNA（β-肌动蛋白或18S rRNA）的共扩增。PCR产物的特异性通过Southern分析和测序得以证实。在卵泡膜细胞中，除了从大卵泡分离的细胞中的PENK外，阿片肽前体的表达似乎依赖于促性腺激素。相反，颗粒细胞在FSH处理下独立表现出POMC和PENK基因的表达。这种促性腺激素诱导从小卵泡和大卵泡分离的颗粒细胞中PDYN基因的表达，并显著增加大卵泡细胞中POMC mRNA的含量。本研究表明猪卵泡细胞（尤其是颗粒细胞）可能产生阿片肽，且促性腺激素可能调节这些细胞中其前体的基因表达。此外，我们的结果支持阿片肽参与卵巢卵泡内的局部调节。
