Ciesiółka S, Bryja A, Budna J, Kranc W, Chachuła A, Bukowska D, Piotrowska H, Porowski L, Antosik P, Bruska M, Brüssow K P, Nowicki M, Zabel M, Kempisty B
Department of Histology and Embryology, Poznan University of Medical Sciences, Poznan, Poland.
Department of Anatomy, Poznan University of Medical Sciences, Poznan, Poland.
J Biol Regul Homeost Agents. 2016 Jul-Sep;30(3):693-702.
The process of oocyte growth and development takes place during long stages of folliculogenesis and oogenesis. This is accompanied by biochemical and morphological changes, occurring from the preantral to antral stages during ovarian follicle differentiation. It is well known that the process of follicle growth is associated with morphological modifications of theca (TCs) and granulosa cells (GCs). However, the relationship between proliferation and/or differentiation of porcine GCs during long-term in vitro culture requires further investigation. Moreover, the expression of cytokeratins and vimentin in porcine GCs, in relation to real-time cell proliferation, has yet to be explored. Utilizing confocal microscopy, we analyzed cytokeratin 18 (CK18), cytokeratin 8 + 18 + 19 (panCK), and vimentin (Vim) expression, as well as their protein distribution, within GCs isolated from slaughtered ovarian follicles. The cells were cultured for 168 h with protein expression and cell proliferation index analyzed at 24-h intervals. We found the highest expression of CK18, panCK, and Vim occurred at 120 h of in vitro culture (IVC) as compared with other experimental time intervals. All of the investigated proteins displayed cytoplasmic distribution. Analysis of real-time cell proliferation revealed an increased cell index after the first 24 h of IVC. Additionally, during each period between 24-168 h of IVC, a significant difference in the proliferation profile, expressed as the cell index, was also observed. We concluded that higher expression of vimentin at 120 h of in vitro proliferation might explain the culmination of the stromalization process associated with growth and domination of stromal cells in GC culture. Cytokeratin expression within GC cytoplasm confirms the presence of epithelial cells as well as epithelial-related GC development during IVC. Moreover, expression of both cytokeratins and vimentin during short-term culture suggests that the process of GC proliferation is also highly associated with porcine ovarian follicular granulosa cell differentiation in vitro.
卵母细胞的生长和发育过程发生在卵泡发生和卵子发生的漫长阶段。这伴随着从卵巢卵泡分化的窦前期到窦期出现的生化和形态学变化。众所周知，卵泡生长过程与卵泡膜细胞（TCs）和颗粒细胞（GCs）的形态学改变有关。然而，猪颗粒细胞在长期体外培养过程中的增殖和/或分化之间的关系需要进一步研究。此外，猪颗粒细胞中细胞角蛋白和波形蛋白的表达与实时细胞增殖的关系尚未得到探索。利用共聚焦显微镜，我们分析了从屠宰后的卵巢卵泡中分离出的颗粒细胞中细胞角蛋白18（CK18）、细胞角蛋白8 + 18 + 19（泛CK）和波形蛋白（Vim）的表达及其蛋白质分布。将细胞培养168小时，每隔24小时分析蛋白质表达和细胞增殖指数。我们发现，与其他实验时间间隔相比，CK18、泛CK和Vim的最高表达出现在体外培养（IVC）的120小时。所有研究的蛋白质均呈细胞质分布。实时细胞增殖分析显示，IVC的前24小时后细胞指数增加。此外，在IVC的24 - 168小时的每个时间段内，以细胞指数表示的增殖曲线也存在显著差异。我们得出结论，体外增殖120小时时波形蛋白的高表达可能解释了与颗粒细胞培养中基质细胞的生长和主导相关的基质化过程的 culmination（此处原文可能有误，推测是culmination，意为顶点、高潮）。颗粒细胞胞质内的细胞角蛋白表达证实了上皮细胞以及IVC期间与上皮相关的颗粒细胞发育的存在。此外，短期培养期间细胞角蛋白和波形蛋白的表达表明，颗粒细胞增殖过程也与体外猪卵巢卵泡颗粒细胞分化高度相关。
