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 (E 2 ) and progesterone (P 4 ) 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, E 2 and P 4 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.

RNAi-Mediated Silencing of Catalase Gene Promotes Apoptosis and Impairs Proliferation of Bovine Granulosa Cells under Heat Stress

“…It is well-known that HS is responsible for inducing ROS production, resulting in oxidative stress [51,52] and cell apoptosis [53], which afterwards compromise fertility [54,55]. In our previous work, we confirmed that HS at 40 • C significantly increased intracellular ROS accumulation and apoptosis in GCs [52]. Some other studies found that pro-apoptotic genes (BAX and Caspase 3) were also up-regulated under HS, through the apoptosis signaling pathway [21,56,57].…”

“…In addition, HS has influenced the regulation of genes, i.e., STAR and Cyp11A1, associated with steroidogenesis (E2 and P4) [52]. It was suggested that E2 biosynthesis in the ovarian follicles is promoted by the positive regulation of Cyp11A1 [69].…”

“…After successful knockdown, GCs were exposed to different treatment groups (40 °C + NC, 40 °C + CAT si RNA) and corresponding NC group. It is well-known that HS is responsible for inducing ROS production, resulting in oxidative stress [ 51 , 52 ] and cell apoptosis [ 53 ], which afterwards compromise fertility [ 54 , 55 ]. In our previous work, we confirmed that HS at 40 °C significantly increased intracellular ROS accumulation and apoptosis in GCs [ 52 ].…”

Influence of long-term thermal stress on the in vitro maturation on embryo development and Heat Shock Protein abundance in zebu cattle

“…The abundance of HSP70 in cumulus cells increased, as expected, after heat stress. Khan et al (2020) reported increased mRNA and protein expression of HSP70 after acute heat stress during the culture of bovine granulosa cells. The increase in apoptosis, decrease in cell viability, reduction of estradiol and progesterone secretion, and accumulation of intracellular reactive oxygen species (ROS), were also observed.…”

“…The abundance of HSP70 in cumulus cells increased, as expected, after heat stress. Khan et al. (2020) reported increased mRNA and protein expression of HSP70 after acute heat stress during the culture of bovine granulosa cells.…”