Evaluation of heat stress effects on cellular and transcriptional adaptation of bovine granulosa cells

Abstract

Background: Heat stress is known to affect follicular dynamics, oocyte maturation, and fertilization by impairing steroidogenic ability and viability of bovine granulosa cell (bGCs). The present study explored the physiological and molecular response of bGCs to different heat stress intensities in-vitro. We exposed the primary bGCs to heat stress (HS) at 39 °C, 40 °C and 41 °C along with control samples (38 °C) for 2 h. To evaluate the impact of heat stress on bGCs, several in vitro cellular parameters including cell apoptosis, intracellular reactive oxygen species (ROS) accumulation and HSP70 kinetics were assessed by flow cytometry, florescence microscopy and western blot, respectively. Furthermore, the ELISA was performed to confirm the 17β-estradiol (E₂) and progesterone (P₄) levels. In addition, the RNA sequencing (RNA-Seq) method was used to get the molecular based response of bGCs to different heat treatments.

Results: Our findings revealed that the HS significantly decreased the cell viability, E₂ and P₄ levels in bGCs, whereas, increased the cellular apoptosis and ROS. Moreover, the RNA-Seq experiments showed that all the treatments (39 °C, 40 °C and 41 °C) significantly regulated many differentially expressed genes (DEGs) i.e. BCL2L1, STAR, CYP11A1, CASP3, SOD2, HSPA13, and MAPK8IP1 and pathways associated with heat stress, apoptosis, steroidogenesis, and oxidative stress. Conclusively, our data demonstrated that the impact of 40 °C treatment was comparatively detrimental for cell viability, apoptosis and ROS accumulation. Notably, a similar trend of gene expression was reported by RT-qPCR for RNA-seq data.

Conclusions: Our study presented a worthy strategy for the first time to characterize the cellular and transcriptomic adaptation of bGCs to heat stress (39, 40 and 41 °C) in-vitro. The results infer that these genes and pathways reported in present study could be useful candidates/indicators for heat stress research in dairy cattle. Moreover, the established model of bGCs to heat stress in the current study provides an appropriate platform to understand the mechanism of how heat-stressed bGCs can affect the quality of oocytes and developing embryo.

Keywords: Bovine granulosa cells; Differentially expressed genes; Follicles; Heat stress; RNA-Seq.

Fig. 1

Temperature humidity index can affect body rectal temperature: Evaluation of change in rectal body temperature (RT) with increase in percent temperature humidity index (%THI)

Fig. 2

Heat stress induces HSP70 expression in bovine granulosa cells: mRNA as well as protein expression of HSP70 (a, b) in bovine granulosa cells cultured under heat stress (39, 40 and 41 °C) and corresponding control (38 °C). β-ACTIN was used to normalize the expression of target protein expression of HSP70. The results are expressed as the mean ± SEM of n = 3. Superscripts (a, b, c) show significant difference, P < 0.05

Fig. 3

Heat stress exposure elevates bovine granulosa cell apoptosis and decreases viability: Flow cytometric analysis of bovine granulosa cells (bGCs) cultured under heat stress (39, 40 and 41 °C) and corresponding control (38 °C) (a, b). The analyzed cell counts for apoptosis and viability are indicated on the Y-axis and the temperature treatments are indicated on the X-axis. Data shown as means ± SEM, n = 3, P < 0.05. Fluorescent photomicrographs of bovine granulosa cells (bGCs) stained with 2′,7′-dichlorofluorescin diacetate (H₂DCFDA) were shown control (38 °C) (c) and heat stress (39, 40 and 41 °C) (d, e, f, respectively). The images shown are representative of the three independent image acquisitions. g Quantitative analysis of relative fluorescence emission. Values are expressed as mean ± SEM of n = 3. Superscripts (a, b, c) show significant difference, P < 0.05

Fig. 4

Effects of heat stress on E₂ and P₄ secretion by bovine granulosa cells: Concentration of E₂ (a) and P₄ (b) in culture media of bovine granulosa cells (bGCs) cultured under heat stress (39, 40 and 41 °C) and corresponding control (38 °C). Values are expressed as mean ± SEM of n = 3. Superscripts (a, b, c) show significant difference, P < 0.05

Fig. 5

Heat stress enhanced intracellular ROS accumulation in bovine granulosa cells: Fluorescent photomicrographs of bovine granulosa cells (bGCs) stained with 2′,7′-dichlorofluorescin diacetate (H₂DCFDA) were shown control (38 °C) (a) and heat stress (39, 40 and 41 °C) (b, c, d, respectively). The images shown are representative of the three independent image acquisitions. e Quantitative analysis of relative fluorescence emission. Values are expressed as mean ± SEM of n = 3. Superscripts (a, b, c) show significant difference, P < 0.05

Fig. 6

RNA sequencing data Analysis for identifying differentially expressed genes among three groups (Control vs. 39 °C, Control vs. 40 °C and Control vs. 41 °C): DEGs in different comparisons in bGCs. a Graphical representation of significant DEGs disclosed among three comparison groups of bovine granulosa cells cultured under different intensities of heat stress. b Venn diagrams shows overlapping DEGs after heat stress among three comparisons. c. Heatmap of top 45 differentially expressed granulosa cell genes in heat stressed groups with FC > 2, P < 0.05. Red corresponds to up-regulated gene product, and green corresponds to down-regulated gene product. Each differentially expressed gene is represented by a single row, and each heat treatment group is represented by a single column

Fig. 7

Pathway analysis of differentially expressed genes among three groups in response to heat stress: Enriched gene pathways in granulosa cells along all comparison of Control vs. 39, 40 and 41 °C cultured groups. Only significantly (P < 0.05) regulated pathways with up and down genes were shown (a, b, c)

Fig. 8

Regulation of signaling pathways under heat stress affecting bGCs functions: A network map of pathways significantly (P < 0.05) enriched after heat stress. The nodes are the pathways, and edges connect the genes involved in the pathway

Fig. 9

Protein-protein interaction (PPI) networks of DEGs significantly enriched pathways associated with bGCs functions under heat stress: Protein-protein interaction (PPI) networks in the comparison of Control vs. 39 °C (a) Control vs. 40 °C (b) and Control vs. 41 °C (c). Various color lines represent seven types of evidence used in predicting associations. Red line: fusion evidence; blue line: co-occurrence evidence; yellow line: text mining evidence; green line: neighborhood evidence; purple line: experimental evidence; light blue line: database evidence; and the black line: co-expression evidence

Fig. 10

Research review: Mechanisms of regulating heat stress response related to follicular function within bovine ovary. Upregulated genes caspase-3, SOD, BCL-2, BAX, and HSPs (HSP70, HSPA13, HMOX1) were involved in the regulating mechanism of bGCs via induced or inhibited cell apoptosis. Under heat stress, Down-regulated genes CAT, FOXO3 were involved in production of reactive oxygen species (ROS). Likewise, down-regulation of STAR, and CYP11A1 were involved in the secretion of E₂ and P₄. Moreover, the decline of E₂, and enhancing of ROS in turn, might enhance the possibility of GC apoptosis and follicle function