Cell and Tissue Research

, Volume 354, Issue 3, pp 869–880 | Cite as

Increasing cell plating density mimics an early post-LH stage in cultured bovine granulosa cells

Regular article

Abstract

Cultured ovarian granulosa cells are essential models to study molecular mechanisms of gene regulation during folliculogenesis. Here, we characterize primary tissue culture models for bovine granulosa cells by morphological and physiological parameters and by novel molecular luteinization markers, as transcript abundance and DNA methylation levels. The data show that: (1) collagen substrate increased the number of attached, viable cells; (2) the expression of the key transcripts of estrogen synthesis, CYP19A1, could be induced and maintained in granulosa cells from small to medium but not from large follicles, whereas (3) only granulosa cells from large but not from smaller follicles were responsive to LH; (4) serum supplementation unfavorably transformed the cellular phenotype, induced proliferation and PCNA expression, reduced or abolished the transcript abundance of steroidogenic key genes and of gonadotropin receptor genes, CYP11A1, CYP19A1, FSHR and LHCGR but, however, did not increase the abundance of the luteinization-specific marker transcripts PTGS2, PTX3, RGS2 and VNN2; but (5) by increasing the plating density, estradiol production and the abundance of CYP19A1 transcripts, in particular those derived from the main ovarian promoter P2, were decreased concurrently leaving P2-specific DNA methylation levels unchanged, whereas progesterone secretion was stimulated and the expression of both luteinization-specific marker transcripts, RGS2 and VNN2, was significantly induced. From these data, we conclude that increasing the plating density induces a different, partly complementary, physiological and gene expression profile in cultured bovine granulosa cells and drives the cells towards an early post-LH stage of luteinization, even in the absence of luteinizing agents.

Keywords

Tissue culture Gene expression DNA methylation Luteinization Steroid hormones 

Abbreviations

FCS

Fetal calf serum

FSH

Follicle stimulating hormone

GC

Granulosa cells

IGF-1

Insulin-like growth factor

LH

Luteinizing hormone

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Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  1. 1.Reproductive Biology, Leibniz Institute for Farm Animal Biology (FBN)DummerstorfGermany

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