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

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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.

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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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Acknowledgments

We thank Veronica Schreiter for excellent technical assistance. This study was supported by grants of the Deutsche Forschungsgemeinschaft (DFG: VA135/5-2).

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  1. Reproductive Biology, Leibniz Institute for Farm Animal Biology (FBN), Wilhelm-Stahl-Allee 2, 18196, Dummerstorf, Germany

    Anja Baufeld & Jens Vanselow

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  1. Anja Baufeld
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  2. Jens Vanselow
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Correspondence to Jens Vanselow.

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Baufeld, A., Vanselow, J. Increasing cell plating density mimics an early post-LH stage in cultured bovine granulosa cells. Cell Tissue Res 354, 869–880 (2013). https://doi.org/10.1007/s00441-013-1712-9

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  • DOI: https://doi.org/10.1007/s00441-013-1712-9

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