Synergistic Effect of Human Chorionic Gonadotropin and Extracellular Matrix on in Vitro Differentiation of Human Granulosa Cells: Progesterone Production and Gap Junction Formation*

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Abstract
The effects of extracellular matrix (ECM) in the form of a native basement membrane and of hCG on progesterone production, cellular morphology and gap junction formation in cultured human granulosa cells (GCs) were examined. GCs were harvested from human preovulatory follicles in the course of oocyte retrieval for in vitro fertilization. Cells grown on ECM secreted by bovine corneal endothelial cells (BCE) produced significantly higher amounts of progesterone (up to 3-fold) than cells on uncoated dishes. Culturing of cells on BCE-ECM in the presence of hCG resulted in a further increase in progesterone production in the first 2 days an additive effect was noted, and on days 3, 4, and 5 a synergistic effect because evident (1.2- to 1.8-fold above the sum of the individual treatments). LH/hAG receptor content on day 3 of culture was 4-fold higher in GCs grown on BCE-ECM compared to that in uncoated dishes. A similar elevation was found when cells were grown on ECM secreted by PFHR-9 mouse endodermal cells. Cells grown on both types of ECM maintained their epitheloid shape but, in addition, formed cytoplasmic processes interconnected with neighboring cells by gap junctions. Morphometric analysis revealed that the area of cell membrane occupied by gap junctions increased by 3- to 4-fold on ECM compared to that in cells grown on plastic. Enlargement of endoplasmic reticulum was evident on ECM along with clustering of numerous mitochondria, lipid droplets, and lysosomes characteristic of highly luteinized cells. Cells grown in the presence of hCG on ECM showed morphological modulation similar to that of cells cultured on ECM alone. These observations suggest that growing of human GCs on a ECM similar to a naturally produced basement membrane enhances morphological and biochemical differentiation, and that ECM and gonadotropin affect synergistically the extent of luteinization in these cells.

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