The effect of heat stress on gene expression, synthesis of steroids, and apoptosis in bovine granulosa cells

Abstract

Summer heat stress (HS) is a major contributing factor in low fertility in lactating dairy cows in hot environments. Heat stress inhibits ovarian follicular development leading to diminished reproductive efficiency of dairy cows during summer. Ovarian follicle development is a complex process. During follicle development, granulosa cells (GCs) replicate, secrete hormones, and support the growth of the oocyte. To obtain an overview of the effects of heat stress on GCs, digital gene expression profiling was employed to screen and identify differentially expressed genes (DEGs; false discovery rate (FDR) ≤ 0.001, fold change ≥2) of cultured GCs during heat stress. A total of 1211 DEGs including 175 upregulated and 1036 downregulated ones were identified, of which DEGs can be classified into Gene Ontology (GO) categories and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The results suggested that heat stress triggers a dramatic and complex program of altered gene expression in GCs. We hypothesized that heat stress could induce the apoptosis and dysfunction of GCs. Real-time reverse transcription-polymerase chain reaction (RT-PCR) was used to evaluate the expression of steroidogenic genes (steroidogenic acute regulatory protein (Star), cytochrome P-450 (CYP11A1), CYP19A1, and steroidogenic factor 1 (SF-1)) and apoptosis-related genes (caspase-3, BCL-2, and BAX). Radio immunoassay (RIA) was used to analyze the level of 17β-estradiol (E2) and progesterone (P4). We also assessed the apoptosis of GCs by flow cytometry. Our data suggested that heat stress induced GC apoptosis through the BAX/BCL-2 pathway and reduced the steroidogenic gene messenger RNA (mRNA) expression and E2 synthesis. These results suggest that the decreased function of GCs may cause ovarian dysfunction and offer an improved understanding of the molecular mechanism responsible for the low fertility in cattle in summer.

Keywords: Bovine; Follicles; Granulosa cells; Heat.

Fig. 1

Highly enriched GO terms analyzed of upregulated (a) and downregulated (b) genes that changed fold ≥2 by Gene Ontology analysis (P values <0.05). Only the most specific subcategories of GO terms under biological process which was considered significant by the Benjamini-Yuketeli test (P < 0.05) were ranked. Biological process analyzed (c) of regulated genes (up and down genes) that changed fold ≥2 folds by Gene KEGG analysis

Fig. 2

a Validation of differentially expressed genes by real-time qPCR. Relative quantification of seven representative genes was performed. qRT-PCR values were determined from the ^ΔΔCt for the target genes relative to 18S. Significantly different results for treatments used one-way ANOVA with Fisher’s LSD post hoc test; P < 0.05. A single asterisk indicates a statistical difference of P < 0.05, and a double asterisk indicates a statistical difference of P < 0.01. b. Comparison of RT-PCR findings to DGE profiling results

Fig. 3

Effect of heat stress on E₂ (a) and P₄ (b) in culture media. A single asterisk indicates a statistical difference of P < 0.05, and a double asterisk indicates a statistical difference of P < 0.01

Fig. 4

The effect of heat treatment on cell apoptosis. After cells were treated with heat stress, the relative number of cells undergoing apoptosis was determined by flow cytometry after annexin V/propidium iodide staining and the apoptosis rates were analyzed (a, d). The ratio of BAX/BCL-2 (b) and the activity of caspase-3 in GCs (c). A single asterisk indicates a statistical difference of P < 0.05, and a double asterisk indicates a statistical difference of P < 0.01

Fig. 5

Proposed mechanisms of regulating heat stress response related to follicular function within bovine ovary. Upregulated genes—BCL-2, BAX, and HSP—were involved in the regulating mechanism of GCs via induced or inhibited cell apoptosis. Downregulated genes—SF-1, CYP19A1, STAR, and CYP11A1—were involved in the secretion of E₂ and P₄. Moreover, the decline of E₂, in turn, might enhance the possibility of GC apoptosis and follicle function. Black line arrows indicate a directly stimulating effect (→); black dashed arrows indicate an indirectly stimulating effect (→); black line from HSP protein indicates an inhibiting effect (⊣)