Expression of PCOS candidate genes in bovine fetal and adult ovarian somatic cells

Abstract

Polycystic ovary syndrome (PCOS) is an endocrine metabolic disorder that appears to have a genetic predisposition and a fetal origin. The fetal ovary has two major somatic cell types shown previously to be of different cellular origins, different morphologies and to differentially express 15 genes. We isolated the somatic gonadal ridge epithelial-like (GREL) cells (n = 7) and ovarian fetal fibroblasts (n = 6) by clonal expansion. Using qRT-PCR, we compared the gene expression levels of PCOS candidate genes with previous data on the expression levels in whole fetal ovaries across gestation. We also compared these levels with those in bovine adult ovarian cells including fibroblasts (n = 4), granulosa cells (n = 5) and surface epithelial cells (n = 5). Adult cell types exhibited clear differences in the expression of most genes. In fetal ovarian cells, DENND1A and ERBB3 had significantly higher expression in GREL cells. HMGA2 and TGFB1I1 tended to have higher expression in fetal fibroblasts than GREL cells. Another 19 genes did not exhibit differences between GREL cells and fetal fibroblasts and FBN3, FSHB, LHCGR, FSHR and ZBTB16 were very lowly expressed in GREL cells and fibroblasts. The culture of fetal fibroblasts in EGF-containing medium resulted in lower expression of NEIL2, but higher expression of MAPRE1 compared to culture in the absence of EGF. Thus, the two fetal ovarian somatic cell types mostly lacked differential expression of PCOS candidate genes.

Figure 1

mRNA expression levels of genes expressed during early gestation. Scatter plots on the left in each subfigure show gene expression levels in fetal ovaries (n  = 27) from our previous publications (Hartanti et al. 2020b, Liu et al. 2020). Gene expression levels in cultured GREL cells (n  = 7) and fetal fibroblast (n  = 6) are presented in the middle graph of each subfigure. Data of gene expression in adult fibroblasts (n  = 4), granulosa cells (GC; n  = 5) and ovarian surface epithelial cells (OSE; n  = 5) are presented as mean ± s.e.m. (normalised to PPIA and RPL32) in the graph on the right side of each subfigure. Significant differences between the three adult cell types were determined by one-way ANOVA with Tukey’s post hoc test. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.

Figure 2

mRNA expression levels of genes expressed during late gestation. Scatter plots on the left in each subfigure show gene expression levels in fetal ovaries (n  = 27) from our previous publications (Hartanti et al. 2020b, Liu et al. 2020). Gene expression levels in cultured GREL cells (n  = 7) and fetal fibroblast (n  = 6) are presented in the middle graph of each subfigure. Data of gene expression in adult fibroblasts (n  = 4), granulosa cells (GC; n  = 5) and ovarian surface epithelial cells (OSE; n  = 5) are presented as mean ± s.e.m. (normalised to PPIA and RPL32). Significant differences between the three adult cell types were determined by one-way ANOVA with Tukey’s post hoc test. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.

Figure 3

mRNA expression levels of genes expressed throughout gestation. Scatter plots left in each subfigure of gene expression levels in fetal ovaries (n  = 27) are from our previous publications (Hartanti et al. 2020b, Liu et al. 2020). Gene expression levels in cultured GREL cells (n  = 7) and fetal fibroblast (n  = 6) are presented in the middle graph of each subfigure. Data of gene expression in adult fibroblasts (n  = 4), granulosa cells (GC; n  = 5) and ovarian surface epithelial cells (OSE; n  = 5) are presented as mean ± s.e.m. (normalised to PPIA and RPL32). Significant differences between the three adult cell types were determined by one-way ANOVA with Tukey’s post hoc test. ^*P < 0.05, ^***P < 0.001.

Figure 4

mRNA expression levels of genes expressed throughout gestation (continued). Scatter plots on the left in each subfigure show gene expression levels in fetal ovaries (n  = 27) from our previous publications (Hartanti et al. 2020b, Liu et al. 2020). Gene expression levels in cultured GREL cells (n  = 7) and fetal fibroblasts (n  = 6) are presented in the middle graph of each subfigure. Data of gene expression in adult fibroblasts (n  = 4), granulosa cells (GC; n  = 5) and ovarian surface epithelial cells (OSE; n  = 5) are presented as mean ± s.e.m. (normalised to PPIA and RPL32). Significant differences between the three adult cell types were determined by one-way ANOVA with Tukey’s post hoc test. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.

Figure 5

mRNA expression levels of eight newly examined PCOS candidate genes in fetal fibroblasts cultured in the presence or absence of EGF. Columns in white colour and black colours show gene expression in fetal fibroblasts cultured in the absence of EGF (n  = 11) and in the presence of EGF (n  = 6), respectively. Data of gene expression are presented as mean ± s.e.m. (normalised to PPIA and RPL32). Significantly differences between groups were determined by Student’s t-test. ^* P < 0.05.