Mao Dagan, Hou Xiaoying, Talbott Heather, Cushman Robert, Cupp Andrea, Davis John S
Olson Center for Women's Health, Department of Obstetrics/Gynecology, Nebraska Medical Center, Omaha, NE 68198.
Mol Endocrinol. 2013 Dec;27(12):2066-79. doi: 10.1210/me.2013-1274. Epub 2013 Nov 6.
The present study investigated the induction and possible role of activating transcription factor 3 (ATF3) in the corpus luteum. Postpubertal cattle were treated at midcycle with prostaglandin F2α(PGF) for 0-4 hours. Luteal tissue was processed for immunohistochemistry, in situ hybridization, and isolation of protein and RNA. Ovaries were also collected from midluteal phase and first-trimester pregnant cows. Luteal cells were prepared and sorted by centrifugal elutriation to obtain purified small (SLCs) and large luteal cells (LLCs). Real-time PCR and in situ hybridization showed that ATF3 mRNA increased within 1 hour of PGF treatment in vivo. Western blot and immunohistochemistry demonstrated that ATF3 protein was expressed in the nuclei of LLC within 1 hour and was maintained for at least 4 hours. PGF treatment in vitro increased ATF3 expression only in LLC, whereas TNF induced ATF3 in both SLCs and LLCs. PGF stimulated concentration- and time-dependent increases in ATF3 and phosphorylation of MAPKs in LLCs. Combinations of MAPK inhibitors suppressed ATF3 expression in LLCs. Adenoviral-mediated expression of ATF3 inhibited LH-stimulated cAMP response element reporter luciferase activity and progesterone production in LLCs and SLCs but did not alter cell viability or change the expression or activity of key regulators of progesterone synthesis. In conclusion, the action of PGF in LLCs is associated with the rapid activation of stress-activated protein kinases and the induction of ATF3, which may contribute to the reduction in steroid synthesis during luteal regression. ATF3 appears to affect gonadotropin-stimulated progesterone secretion at a step or steps downstream of PKA signaling and before cholesterol conversion to progesterone.
本研究调查了激活转录因子3（ATF3）在黄体中的诱导情况及其可能的作用。对青春期后的母牛在发情周期中期用前列腺素F2α（PGF）处理0至4小时。对黄体组织进行免疫组织化学、原位杂交以及蛋白质和RNA的分离处理。还从黄体中期和妊娠早期的母牛收集卵巢。通过离心淘洗制备并分选黄体细胞，以获得纯化的小黄体细胞（SLCs）和大黄体细胞（LLCs）。实时PCR和原位杂交显示，体内PGF处理1小时内ATF3 mRNA增加。蛋白质印迹和免疫组织化学表明，ATF3蛋白在LLCs细胞核中1小时内表达，并持续至少4小时。体外PGF处理仅在LLCs中增加ATF3表达，而TNF在SLCs和LLCs中均诱导ATF3表达。PGF刺激LLCs中ATF3浓度和时间依赖性增加以及MAPKs磷酸化。MAPK抑制剂组合抑制LLCs中ATF3表达。腺病毒介导的ATF3表达抑制LLCs和SLCs中LH刺激的cAMP反应元件报告荧光素酶活性和孕酮产生，但不改变细胞活力，也不改变孕酮合成关键调节因子的表达或活性。总之，PGF在LLCs中的作用与应激激活蛋白激酶的快速激活和ATF3的诱导有关，这可能有助于黄体退化期间类固醇合成的减少。ATF3似乎在PKA信号下游且在胆固醇转化为孕酮之前的一个或多个步骤影响促性腺激素刺激的孕酮分泌。
