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.

Circular RNAs (circRNAs) are thought to play essential roles in multiple biological processes, including apoptosis, an important process in antral follicle atresia. We aimed to investigate the potential involvement of circRNAs in granulosa cell apoptosis and thus antral follicle atresia. CircRNA expression profiles were generated from porcine granulosa cells isolated from healthy antral (HA) and atretic antral (AA) follicles. Over 9632 circRNAs were identified, of which 62 circRNAs were differentially expressed (DE-circRNAs). Back-splicing, RNase R resistance, and stability of DE-circRNAs were validated, and miRNA binding sites and related target genes were predicted. Two exonic circRNAs with low false discovery rate (FDR) high fold change, miRNA binding sites, and relevant biological functions—circ_CBFA2T2 and circ_KIF16B—were selected for further characterization. qRT-PCR and linear regression analysis confirmed expression and correlation of the targeted genes—the antioxidant gene GCLC (potential target of circ_CBFA2T2) and the apoptotic gene TP53 (potential target of circ_KIF16B). Increased mRNA content of TP53 in granulosa cells of AA follicles was further confirmed by strong immunostaining of both p53 and its downstream target pleckstrin homology like domain family a member 3 (PHLDA3) in AA follicles compared to negligible staining in granulosa cells of HA follicles. Therefore, we concluded that aberrantly expressed circRNAs presumably play a potential role in antral follicular atresia.

Long non-coding RNAs (lncRNAs) play important roles in multiple biological processes including ovarian follicular development. Here we aimed to gain novel information regarding lncRNAs transcriptome profiles in porcine granulosa cells of advanced atretic antral (AA) and healthy antral (HA) follicles using RNA-seq. A total of 11,321 lncRNAs including 10,813 novel and 508 annotated lncRNAs were identified, of which 173 lncRNAs were differentially expressed (DE-lncRNAs); ten of these were confirmed by qRT-PCR. Gene Ontology indicated that DE-lncRNAs associated with developmental processes were highly enriched. Pathway analysis demonstrated predicted cis- and trans-targets of DE-lncRNAs. Potential mRNA targets of up-regulated DE-lncRNAs were mainly enriched in apoptosis related pathways, while targeted genes of downregulated DE-lncRNAs were primarily enriched in metabolism and ovarian steroidogenesis pathways. Linear regression analyses showed that expression of upregulated DE-lncRNAs was significantly associated with apoptosis related genes. NOVEL_00001850 is the most-downregulated DE-lncRNA (FDR = 0.04, FC = −6.53), of which miRNA binding sites were predicted. KEGG analysis of its downregulated target genes revealed that ovarian steroidogenesis was the second most highlighted pathway. qRT-PCR and linear regression analysis confirmed the expression and correlation of its potential targeted gene, CYP19A1, a key gene involved in estradiol synthesis. Our results indicate that lncRNAs may participate in granulosa cells apoptosis and thus antral follicular atresia.

In mammals, β-defensins have been reported to play pivotal roles in sperm protection and fertilization. However, the function and mechanism of porcine β-defensin 129 (pBD129) in the sperm remain unclear. Here, we demonstrate that pBD129 is a glycosylated protein and broadly exists in accessory sex glands and coats the sperm surface. We inhibited the pBD129 protein on the sperm surface with an anti-pBD129 antibody and found that sperm motility was not significantly affected; however, sperm acrosome integrity and tyrosine phosphorylation levels increased significantly with time (p < 0.05) during capacitation. These changes were accompanied by an increase in sperm Ca²⁺ influx, resulting in a significantly reduced in vitro fertilization cleavage rate (p < 0.05). Further investigation revealed that treatment with recombinant pBD129 markedly restored the sperm motility in semen contaminated with Escherichia coli. The results suggest that pBD129 is not only associated with poor sperm motility after genital tract infection but can also protect the spermatozoa from premature capacitation, which may be beneficial for semen preservation.

In the present investigation, we examined whether a change in whole body energy fluxes could affect ovarian follicular development, employing mice ectopically expressing uncoupling protein 1 in skeletal muscle (UCP1-TG). Female UCP1-TG and wild-type (WT) mice were dissected at the age of 12 weeks. Energy intake and expenditure, activity, body weight and length, and body composition were measured. Plasma insulin, glucose, leptin, plasma fibroblast growth factor 21 (FGF21) and plasma insulin-like growth factor 1 (IGF1) levels were analyzed and ovarian follicle and corpus luteum numbers were counted. IGF1 signaling was analyzed by immunohistochemical staining for the activation of insulin receptor substrate 1/2 (IRS1/2) and AKT. UCP1-TG female mice had increased energy expenditure, reduced body size, maintained adiposity, and decreased IGF1 concentrations compared to their WT littermates, while preantral and antral follicle numbers were reduced by 40% and 60%, respectively. Corpora lutea were absent in 40% of the ovaries of UCP1-TG mice. Phospho-IRS1, phospho-AKT -Ser473 and -Thr308 immunostaining was present in the granulosa cells of antral follicles in WT ovaries, but faint to absent in the antral follicles of UCP1-TG mice. In conclusion, the reduction in circulating IGF1 levels due to the ectopic expression of UCP1 is associated with reduced immunostaining of the IRS1-PI3/AKT pathway, which may negatively affect ovarian follicle development and ovulation.