The gene expression pattern induced by high plating density in cultured bovine and buffalo granulosa cells might be regulated by specific miRNA species

Abstract

Precise regulation of cell type-specific gene expression profiles precedes the profound morphological reorganization of somatic cell layers during folliculogenesis, ovulation and luteinization. Cell culture models are essential to the study of corresponding molecular mechanisms of gene regulation. In a recent study, it was shown that an increased cell plating density can largely change gene expression profiles of cultured bovine granulosa cells. In our present study, we comparatively analyzed cell plating density effects on cultured bovine and buffalo granulosa cells. Cells were isolated from small- to medium-sized follicles (2-6 mm) and cultured under serum-free conditions at different plating densities. The abundance of selected marker transcripts and associated miRNA candidates was determined by quantitative real-time RT-PCR. We found in both species that the abundance of CYP19A1, CCNE1 and PCNA transcripts was remarkably lower at a high plating density, whereas VNN2 and RGS2 transcripts significantly increased. In contrast, putative regulators of CYP19A1, miR-378, miR-106a and let-7f were significantly higher in both species or only in buffalo, respectively. Also miR-15a, a regulator of CCNE1, was upregulated in both species. Thus, increased plating density induced similar changes of mRNA and miRNA expression in granulosa cells from buffalo and cattle. From these data, we conclude that specific miRNA species might be involved in the observed density-induced gene regulation.

Fig. 1.

Bovine and buffalo granulosa cells after 4 days in culture. Bovine granulosa cells cultured at low (0.5 × 10³ cells/mm²) and high density (5 × 10³ cells/mm²) are shown in A and B, respectively, whereas granulosa cells from buffalo are shown in C at a low plating density low (1 × 10³ cells/mm²) and in D at a high plating density (5 × 10³ cells/mm²). Photomicrographs of cultured bovine cells were taken with a Nikon TMS- F inverted microscope, whereas those of buffalo cells were taken with a Nikon eclipse Ti microscope.

Fig. 2.

Effects of cell plating density on the abundance of selected transcripts in cultured granulosa cells from buffalo and cattle. Whereas the abundance of CYP19A1 (A), PCNA (B) and CCNE1 (C) transcripts was considerably lower at high (filled columns) compared with low (empty columns) plating density, VNN2 (D) and RGS2 (E) transcripts were significant increased. Transcript abundance was determined by real-time RT-PCR in granulosa cells plated at high (buffalo and cattle: 5 × 10³ cells/mm²) and low density (buffalo, 1 × 10³ cells/mm²; cattle, 0.5 × 10³ cells/mm², respectively) after 4 days in culture. The data are indicated as transcript abundance relative to RPLP0 (mean ± SEM) of three independent experiments. Different letters indicate significant differences at P < 0.05 levels.

Fig. 3.

Abundance of different miRNAs in cultured granulosa cells from buffalo and cattle at different plating densities. miR-378 (A), miR-106 (B) and miR-15a (D) showed significantly higher abundance in high- (filled columns) compared with low-density (empty columns) cultures, whereas let-7f (C) was only increased in buffalo. miRNA abundance is indicated relative to U6 transcripts in granulosa cells plated at high (buffalo and cattle, 5 × 10³ cells/mm²) and low density (buffalo, 1 × 10³ cells/mm²; cattle, 0.5 × 10³ cells/mm², respectively) after 4 days in culture. The data are indicated as mean ± SEM of three independent experiments. Different letters indicate significant difference at P < 0.05 levels.