Reproductive Biology and Endocrinology Open Access Bovine Cumulus-granulosa Cells Contain Biologically Active Retinoid Receptors That Can Respond to Retinoic Acid
Reproductive Biology and Endocrinology Open Access Bovine Cumulus-granulosa Cells Contain Biologically Active Retinoid Receptors That Can Respond to Retinoic Acid
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生殖生物学和内分泌学开放获取牛卵丘颗粒细胞含有可对视黄酸做出反应的生物活性类视黄醇受体
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通讯作者：
Nagaraja Ramavadhani
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作者：
M. Mohan;N. Thirumalapura;J. Malayer;Nagaraja Ramavadhani
维甲酸是一类包括视黄醇及其代谢物维甲酸的化合物，对卵巢类固醇的产生、卵母细胞成熟和早期胚胎发育是绝对必要的。以前的研究已经在牛的大卵泡中检测到高浓度的视黄醇。此外，在体内注射视黄醇和在体外成熟过程中补充维甲酸可以促进胚胎发育。在目前的研究中，我们假设以前在体内或在体外给予维甲酸可以在卵丘颗粒细胞中发挥受体介导的作用。从体外培养的卵丘颗粒细胞中提取总RNA，逆转录-聚合酶链式反应(RT-PCR)检测视黄醇结合蛋白(RBP)、视黄酸受体α(RARpha)、视黄酸受体β(RARbeta)、视黄酸受体γ(RARGamma)、维甲酸X受体α(RXRpha)、维甲酸X受体β(RXRbeta)、视黄醛脱氢酶-2(RALDH-2)和过氧化物酶体增殖物激活受体γ(PARGamma)的mRNA表达。在卵丘颗粒细胞中未检测到。用免疫印迹法检测到牛卵丘颗粒细胞中也有RARpha和RXRbeta蛋白的表达。利用pAAV-MCS-betaRARE-Luc载体的瞬时报告基因检测这些内源性视黄酸受体的生物活性。与缺乏维甲酸反应元件的对照报告细胞相比，加入0.5和1微摩尔的全反式维甲酸显著提高了pAAV-MCS-betaRARE-Luc报告基因的活性(P<；0.05)。加入5或10微摩尔的全反式视黄醇可使报告蛋白的活性略有增加，但差异无统计学意义。根据这些结果，我们得出结论，卵丘细胞含有内源性活性的维甲酸受体，也可能有能力利用前体视黄醇合成维甲酸。这些结果还间接地提供了证据，表明以前在体内或在体外给药的维甲酸可能对卵丘颗粒细胞发挥了受体介导的作用。
Retinoids, a class of compounds that include retinol and its metabolite, retinoic acid, are absolutely essential for ovarian steroid production, oocyte maturation, and early embryogenesis. Previous studies have detected high concentrations of retinol in bovine large follicles. Further, administration of retinol in vivo and supplementation of retinoic acid during in vitro maturation results in enhanced embryonic development. In the present study, we hypothesized that retinoids administered either in vivo previously or in vitro can exert receptor-mediated effects in cumulus-granulosa cells. Total RNA extracted from in vitro cultured cumulus-granulosa cells was subjected to reverse transcription polymerase chain reaction (RT-PCR) and mRNA expression for retinol binding protein (RBP), retinoic acid receptor alpha (RARalpha), retinoic acid receptor beta (RARbeta), retinoic acid receptor gamma (RARgamma), retinoid X receptor alpha (RXRalpha), retinoid X receptor beta (RXRbeta), retinaldehyde dehydrogenase-2 (RALDH-2), and peroxisome proliferator activated receptor gamma (PPARgamma). was not detected in cumulus-granulosa cells. Using western blotting, immunoreactive RARalpha, and RXRbeta protein was also detected in bovine cumulus-granulosa cells. The biological activity of these endogenous retinoid receptors was tested using a transient reporter assay using the pAAV-MCS-betaRARE-Luc vector. Addition of 0.5 and 1 micro molar all-trans retinoic acid significantly (P < 0.05) increased the activity of the pAAV-MCS-betaRARE-Luc reporter compared to cells transfected with the control reporter lacking a retinoic acid response element. Addition of 5 or 10 micro molar all-trans retinol stimulated a mild increase in reporter activity, however, the increase was not statistically significant. Based on these results we conclude that cumulus cells contain endogenously active retinoid receptors and may also be competent to synthesize retinoic acid using the precursor, retinol. These results also indirectly provide evidence that retinoids administered either in vivo previously or in vitro may have exerted a receptor-mediated effect on cumulus-granulosa cells.