Transcriptome Analysis of Porcine Granulosa Cells in Healthy and Atretic Follicles: Role of Steroidogenesis and Oxidative Stress

Abstract

One of the main causes of female infertility is a deregulated antral follicular atresia, a process of which the underlying molecular mechanisms are largely unknown. Our objective was therefore to characterize the complex transcriptome changes in porcine granulosa cells of healthy antral (HA) and advanced antral atretic (AA) follicles, using ELISA and RNA-Seq followed by qRT-PCR and immunohistochemistry. Granulosa cell RNA-Seq data revealed 2160 differentially expressed genes, 1483 with higher and 677 with lower mRNA concentrations in AA follicles. Bioinformatic analysis showed that the upregulated genes in AA follicles were highly enriched in inflammation and apoptosis processes, while the downregulated transcripts were mainly highlighted in the steroid biosynthesis pathway and response to oxidative stress processes including antioxidant genes (e.g., GSTA1, GCLC, GCLM, IDH1, GPX8) involved in the glutathione metabolism pathway and other redox-related genes (e.g., RRM2B, NDUFS4). These observations were confirmed by RT-qPCR and immunohistochemistry. Additionally, the granulosa cells of AA follicles express significantly stronger 8-OHdG immunostaining, a marker of oxidative DNA damage, implicating that oxidative stress may participate in follicular atresia. We hypothesize that the decrease in anti-apoptotic factors and steroid hormones coincides with increased oxidative stress markers and the expression of pro-apoptotic factors, all contributing to antral follicular atresia.

Characterization of Long Non-Coding RNA Profiles in Porcine Granulosa Cells of Healthy and Atretic Antral Follicles: Implications for a Potential Role in Apoptosis

“…We show here that CYP19A1 is a downstream potential mRNA target of NOVEL_00001850, the lncRNA that is highly downregulated in granulosa cells of AA follicles. This association between NOVEL_00001850 and reduced CYP19A1 gene expression is further confirmed by a lower aromatase protein expression in granulosa cells of AA follicles compared with HA follicles [20], and is presumably responsible for the previously observed lower estradiol follicular fluid concentrations in AA follicles [17]. We therefore hypothesize that NOVEL_00001850 might promote apoptosis of granulosa cells possibly by binding ssc-miR-125a, resulting in down-regulation of the CYP19A1 gene.…”

“…These results point out that circRNAs, like lncRNAs, may be involved in granulosa cells apoptosis possibly by regulating the expression of their targeted protein-coding genes. To obtain a more complete overview of the genes involved in antral follicular atresia, we further analyzed the mRNA transcriptome profiles changes in porcine granulosa cells of advanced AA follicles [20]. Granulosa cell RNA-seq data revealed 2160 differentially expressed genes (DEGs), 1483 with higher and 677 with lower mRNA concentrations in AA follicles.…”

“…Briefly, paired-end reads were harvested from the Illumina HiSeq 4000 sequencer; quality control was performed by Q30 after 3' adaptor-trimming and the removal of low-quality reads by Fastp software (v1.9.3). The high-quality trimmed reads were used to analyze circRNAs [17], mRNAs [20] and lncRNAs, respectively.…”

Oxidative stress in oocyte aging and female reproduction

“…Follicle atresia is a strictly regulated process, characterized by the initial rapid loss of granulosa cells and the slow loss of thecal cells (F. Matsuda et al, 2012). Oxidative stress may be one of the initiating factors for selective atretic follicles (Meng et al, 2020). The deletion of antioxidant genes in the ovaries can cause follicular growth abnormally.…”

“…Furthermore, N‐acetylcysteine‐fed (continuously) GGT ‐deficient t male and female mice were fertile and produced normal numbers of offspring when mated to wild‐type control mice (Kumar et al, 2000). Bioinformatic analysis showed that the upregulated genes in porcine atretic antral follicles were highly enriched in inflammation and apoptosis processes, while the downregulated transcripts were mainly highlighted in the steroid biosynthesis pathway and response to oxidative stress processes including antioxidant genes ( GSTA1, GCLC, GCLM, IDH1, GPX8 ) involved in the glutathione metabolism pathway and other redox‐related genes ( RRM2B, NDUFS4 ) compare with porcine healthy antral follicles (Meng et al, 2020). In addition, increased atretic follicles and reduced growing follicles appeared in the ovarian oxidative stress mouse model established by intraperitoneal injections of arsenic sodium, and the SOD and GSH‐Px levels in the ovarian homogenate significantly decreased compared with the normal mice (X. Wang et al, 2012).…”