Kempisty B, Ziółkowska A, Ciesiółka S, Piotrowska H, Antosik P, Bukowska D, Nowicki M, Brüssow K P, Zabel M
Department of Histology and Embryology, Poznań University of Medical Sciences, Poznań, Poland.
Department of Toxicology, Poznań University of Medical Sciences, Poznań, Poland.
J Biol Regul Homeost Agents. 2014 Oct-Dec;28(4):625-35.
Granulosa cells (GCs) play an important role during follicle growth and development in preovulatory stage. Moreover, the proteins such as connexins are responsible for formation of protein channel between follicular-cumulus cells and oocyte. This study was aimed to investigate the role of connexin expression in porcine GCs in relation to their cellular distribution and real-time cell proliferation. In the present study, porcine GCs were isolated from the follicles of puberal gilts and then cultured in a real-time cellular analyzer (RTCA) system for 168 h. The expression levels of connexins (Cxs) Cx36, Cx37, Cx40 and Cx43 mRNA were measured by RQ-PCR analysis, and differences in the expression and distribution of Cx30, Cx31, Cx37, Cx43 and Cx45 proteins were analyzed by confocal microscopic visualization. We found higher level of Cx36, Cx37, and Cx43 mRNA expression in GCs at recovery (at 0 h of in vitro culture, IVC) compared to all analyzed time periods of IVC (24, 48, 72, 96, 120, 144 and 168 h; P<0.001). On the other hand, the expression level of Cx40 transcripts was higher after 24 h of IVC compared to 0 h and the other times of IVC (P<0.001). Similarly to mRNAs, the expression levels of Cx31, Cx37 and Cx45 proteins were higher before (0 h) compared to after 168 h of IVC. The expression of Cx30 and Cx43, however, did not vary between the groups. In all, the proteins were distributed throughout the cell membrane rather than in the cytoplasm both before and after IVC. After 24 h of IVC, we observed a significant increase in the proliferation of GCs (log phase). We found differences in the proliferation index between 72-96 and 96- 140 h within the same population of GCs. In conclusion, the decrease in the expression of Cx mRNAs and proteins following IVC could be associated with a breakdown in gap-junction connections (GJCs), and leads to the decreased of their activity, which may be a reason of non-functional existence of connexon in follicular granulosa cells. These data indicated that the differentiation and proliferation of GCs and lutein cells are regulated by distinct mechanisms in pigs.
颗粒细胞（GCs）在排卵前阶段的卵泡生长和发育过程中发挥着重要作用。此外，连接蛋白等蛋白质负责在卵泡-卵丘细胞与卵母细胞之间形成蛋白质通道。本研究旨在探讨连接蛋白表达在猪颗粒细胞中的作用及其细胞分布和实时细胞增殖情况。在本研究中，从青春期母猪的卵泡中分离出猪颗粒细胞，然后在实时细胞分析仪（RTCA）系统中培养168小时。通过RQ-PCR分析测定连接蛋白（Cxs）Cx36、Cx37、Cx40和Cx43 mRNA的表达水平，并通过共聚焦显微镜可视化分析Cx30、Cx31、Cx37、Cx43和Cx45蛋白表达和分布的差异。我们发现，与体外培养（IVC）的所有分析时间段（24、48、72、96、120、144和168小时）相比，恢复时（体外培养0小时）颗粒细胞中Cx36、Cx37和Cx43 mRNA表达水平更高（P<0.001）。另一方面，与0小时及其他体外培养时间相比，体外培养24小时后Cx40转录本的表达水平更高（P<0.001）。与mRNA类似，与体外培养168小时后相比，Cx31、Cx37和Cx45蛋白的表达水平在（0小时）之前更高。然而，Cx30和Cx43的表达在各组之间没有差异。总体而言，无论在体外培养前还是培养后，这些蛋白均分布于整个细胞膜而非细胞质中。体外培养24小时后，我们观察到颗粒细胞增殖显著增加（对数期）。我们发现在同一群体的颗粒细胞中，72 - 96小时和96 - 140小时之间的增殖指数存在差异。总之，体外培养后连接蛋白mRNA和蛋白表达的降低可能与间隙连接连接（GJCs）的破坏有关，并导致其活性降低，这可能是卵泡颗粒细胞中连接子无功能存在的一个原因。这些数据表明，猪颗粒细胞和黄体细胞的分化和增殖受不同机制调控。
