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. 2015 Mar;22(3):377-84.
doi: 10.1177/1933719114529375. Epub 2014 Apr 7.

CYP51A1 induced by growth differentiation factor 9 and follicle-stimulating hormone in granulosa cells is a possible predictor for unfertilization

Affiliations

CYP51A1 induced by growth differentiation factor 9 and follicle-stimulating hormone in granulosa cells is a possible predictor for unfertilization

Tomoko Nakamura et al. Reprod Sci. 2015 Mar.

Abstract

Growth differentiation factor 9 (GDF9), an oocyte-secreted factor, whose receptors exist in granulosa cells, is involved in follicle progression. Therefore, GDF9 is considered to potentially mediate signals necessary for follicular growth. However, the effect of GDF9 on human granulosa cells is not fully understood. Human immortalized nonluteinized granulosa cell line (HGrC1) which we have previously reported was stimulated with GDF9 and/or follicle-stimulating hormone (FSH). Granulosa cells obtained from in vitro fertilization (IVF) patients were also evaluated with quantitative reverse transcription polymerase chain reaction (RT-PCR). Real-time RT-PCR showed that GDF9 increased messenger RNA (mRNA) levels of enzymes required for cholesterol biosynthesis, such as 3-hydroxy-3-methylglutanyl-CoA synthase 1 (HMGCS1), farnesyl-diphosphate farnesyltransferase 1, squalene epoxidase, lanosterol synthase, and cytochrome P450, family 51, subfamily A, polypeptide 1 (CYP51A1). A greater increase in mRNA levels of HMGCS1 and CYP51A1 was observed by combined treatment with GDF9 and FSH. Clinical samples showed a significant increase in CYP51A1 mRNA in the group of granulosa cells connected with unfertilized oocytes. Our results suggest that GDF9, possibly with FSH, may play significant roles in the regulation of cholesterol biosynthesis and the expression of CYP51A1 which might be a predictor for unfertilization.

Keywords: CYP51A1; GDF9; cholesterol synthesis pathway; fertilization; granulosa cell.

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Conflict of interest statement

Declaration of Conflicting Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Figures

Figure 1.
Figure 1.
Real-time RT-PCR analysis of mRNA transcripts of HGrC1. HGrC1 was stimulated for 48 hours with 5 IU/mL FSH, 2 μg/mL GDF9, or both. Combined effects on upregulation were seen in HMGCS and CYP51A1. The data are presented as the mean ± SD of 3 independent replicates. Different letters show significant difference (P < .05). RT-PCR indicates reverse transcription polymerase chain reaction; mRNA, messenger RNA; FSH, follicle-stimulating hormone; HGrC1, human immortalized nonluteinized granulosa cell line; GDF9, growth differentiation factor 9; HMGCS, 3-hydroxy-3-methylglutanyl-CoA synthase 1; CYP51A1, cytochrome P450, family 51, subfamily A, polypeptide 1; SD, standard deviation.
Figure 2.
Figure 2.
Immunoblotting for CYP51A1 and HMGCS. Representative images of immunoblotting against CYP51A1 (57 kDa), HMGCS (57 kDa), and β-actin (42 kDa) expressed by HGrC1. CYP51A1 was significantly induced by stimulation of cells with GDF9, FSH, or both for 48 hours in HGrC1 cells (upper panel). HMGCS was similarly induced by stimulation of cells with GDF9 or GDF-9 + FSH for 48 hours in HGrC1 cells (middle panel). HMGCS indicates 3-hydroxy-3-methylglutanyl-CoA synthase 1; CYP51A1, cytochrome P450, family 51, subfamily A, polypeptide 1; HGrC1, human immortalized nonluteinized granulosa cell line; GDF9, growth differentiation factor 9; FSH, follicle-stimulating hormone.
Figure 3.
Figure 3.
The cholesterol biosynthesis pathway. Stimulation of HGrC1 cells with GDF9, FSH, or GDF9 + FSH resulted in elevated mRNA levels in 6 enzymes along the cholesterol biosynthesis pathway. Solid line boxes show the enzymes of which mRNA levels were upregulated more than 2.0-fold in the microarray analysis. The enzymes in gray boxes were elevated with GDF9, FSH, or GDF9 + FSH stimulation, while those in white boxes were not changed or analyzed. The enzymes in dotted lined boxes were not evaluated either by microarray or RT-PCR. HGrC1 indicates human immortalized nonluteinized granulosa cell line; GDF9, growth differentiation factor 9; FSH, follicle-stimulating hormone; mRNA, messenger RNA; RT-PCR, reverse transcription polymerase chain reaction.
Figure 4.
Figure 4.
Relative expression levels of CYP51A1 and SC4MOL in granulosa cells. THE x-axis shows each patient (a-t). Fold increase of CYP51A1 and SC4MOL in the granulosa cells connected with unfertilized oocytes compared to the granulosa cells connected with fertilized oocytes was represented by white bars and black bars, respectively. Increase of CYP51A1 in the granulosa cells connected with unfertilized oocytes, not SC4MOL, was statistically significant (P = .004). CYP51A1 indicates cytochrome P450, family 51, subfamily A, polypeptide 1; SC4MOL, sterol-C4-methyl oxidase-like.

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