Fahrenkrug Jan, Georg Birgitte, Hannibal Jens, Hindersson Peter, Gräs Søren
Department of Clinical Biochemistry, Bispebjerg Hospital, DK-2400 Copenhagen NV, Denmark.
Endocrinology. 2006 Aug;147(8):3769-76. doi: 10.1210/en.2006-0305. Epub 2006 May 4.
Circadian rhythms are generated by endogenous clocks in the central brain oscillator, the suprachiasmatic nucleus, and peripheral tissues. The molecular basis for the circadian clock consists of a number of genes and proteins that form transcriptional/translational feedback loops. In the mammalian gonads, clock genes have been reported in the testes, but the expression pattern is developmental rather than circadian. Here we investigated the daily expression of the two core clock genes, Per1 and Per2, in the rat ovary using real-time RT-PCR, in situ hybridization histochemistry, and immunohistochemistry. Both Per1 and Per2 mRNA displayed a statistically significant rhythmic oscillation in the ovary with a period of 24 h in: 1) a group of rats during proestrus and estrus under 12-h light,12-h dark cycles; 2) a second group of rats representing a mixture of all 4 d of the estrous cycle under 12-h light,12-h dark conditions; and 3) a third group of rats representing a mixture of all 4 d of estrous cycle during continuous darkness. Per1 mRNA was low at Zeitgeber time 0-2 and peaked at Zeitgeber time 12-14, whereas Per2 mRNA was delayed by approximately 4 h relative to Per1. By in situ hybridization histochemistry, Per mRNAs were localized to steroidogenic cells in preantral, antral, and preovulatory follicles; corpora lutea; and interstitial glandular tissue. With newly developed antisera, we substantiated the expression of Per1 and Per2 in these cells by single/double immunohistochemistry. Furthermore, we visualized the temporal intracellular movements of PER1 and PER2 proteins. These findings suggest the existence of an ovarian circadian clock, which may play a role both locally and in the hypothalamo-pituitary-ovarian axis.
昼夜节律由位于中枢脑振荡器视交叉上核以及外周组织中的内源性生物钟产生。生物钟的分子基础由许多形成转录/翻译反馈环的基因和蛋白质组成。在哺乳动物性腺中，睾丸中已报道有生物钟基因，但其表达模式是发育性的而非昼夜节律性的。在此，我们运用实时逆转录聚合酶链反应、原位杂交组织化学和免疫组织化学方法，研究了大鼠卵巢中两个核心生物钟基因Per1和Per2的每日表达情况。在以下三组大鼠中，Per1和Per2 mRNA在卵巢中均呈现出具有统计学意义的24小时周期性节律振荡：1）一组处于动情前期和发情期的大鼠，饲养于12小时光照、12小时黑暗周期环境中；2）第二组大鼠代表动情周期所有4天的混合情况，饲养于12小时光照、12小时黑暗条件下；3）第三组大鼠代表动情周期所有4天的混合情况，饲养于持续黑暗环境中。Per1 mRNA在授时因子时间0 - 2时较低，在授时因子时间12 - 14时达到峰值，而Per2 mRNA相对于Per1延迟约4小时。通过原位杂交组织化学方法，Per mRNA定位于窦前卵泡、窦卵泡和排卵前卵泡、黄体以及间质腺组织中的类固醇生成细胞。利用新研制的抗血清，我们通过单/双免疫组织化学证实了Per1和Per2在这些细胞中的表达。此外，我们还观察到了PER1和PER2蛋白在细胞内的时间性移动。这些发现提示卵巢中存在昼夜生物钟，其可能在局部以及下丘脑 - 垂体 - 卵巢轴中发挥作用。
