Long Non-Coding RNA GDAR Regulates Ovine Granulosa Cells Apoptosis by Affecting the Expression of Apoptosis-Related Genes

Abstract

Short-term dietary supplementation of ewes during the luteal phase can increase fertility, most probably by stimulating glucose uptake by the follicles. However, the molecular mechanism of glucose regulation of follicular development has not yet been clarified, especially the further study of long non-coding RNA (lncRNA) in determining fertility during follicular development. We generated granulosa cell (GC) models of different doses of glucose (0, 2.1, 4.2, 8.4, 16.8 and 33.6 mM), and observed that the highest cell viability was recorded in the 8.4 mM group and the highest apoptosis rates were recorded in the 33.6 mM group. Therefore, a control group (n = 3, 0 mM glucose), a low glucose group (n = 3, add 8.4 mM glucose), and a high glucose group (n = 3, add 33.6 mM glucose) of GCs were created for next whole genomic RNA sequencing. In total, 18,172 novel lncRNAs and 510 annotated lncRNAs were identified in the GCs samples. Gene Ontology indicated that differentially expressed lncRNAs associated with cell apoptosis were highly enriched. Kyoto Encyclopedia of Genes and Genomes enrichment analysis of lncRNA target genes found that the apoptosis pathway and the p53 signaling pathway were both enriched. Furthermore, we focused on the function of a lncGDAR and verified that lncGDAR could influence cell apoptosis in GC development through affecting the mRNA and protein expression of apoptosis-related markers. These results provide the basis for further study of the lncRNA regulation mechanism in nutrition on female fertility.

LncGSAR Controls Ovarian Granulosa Cell Steroidogenesis via Sponging MiR-125b to Activate SCAP/SREBP Pathway

“…Previous studies confirm that short-term dietary supplementation of ewes during the luteal phase can increase fertility due to elevated glucose and steroid hormone concentrations in follicles [ 19 , 28 , 29 ], whereas glucose has deleterious effects on ovary structure and function at higher concentrations [ 5 , 30 , 31 ]. Our previous study also demonstrated that glucose dose had a significant effect on GCs function, with 8.4 mM representing the optimal glucose concentration for GCs to secrete steroid hormones in vitro and a higher concentration of glucose (33.6 mM) inhibiting GCs proliferation and steroidogenesis [ 6 , 7 ]. Therefore, there is a potential regulatory relationship between glucose metabolism and steroid hormone synthesis, and further investigation is required to validate the molecular mechanism of glucose-induced GCs differentiation and steroid hormone secretion.…”

“…When cell confluence reached 80% medium, the original medium is removed. Cells were washed with 1 × PBS, and then, all treatments were cultured in DMEM, which was prepared with no serum, glucose, pyruvate, and phenol red (Solarbio, Beijing, China) for 8 h. Subsequently, the cells were supplemented with 8.4 mM and 33.6 mM of glucose cultured for an additional 24 h. The 8.4 mM represented an optimum glucose concentration for the secretion of steroid hormones by ovine GCs in vitro [ 6 , 7 ], and the 33.6 mM group represents 30 times the physiological concentration of glucose in follicular fluid and was used to detect changes in steroid hormones at super-physiological concentrations [ 48 , 49 , 50 ]. This culture system was developed so that GC retains hormonally responsive aromatase activity and does not luteinize with time in culture [ 51 , 52 , 53 , 54 ].…”

“…RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 System (Agilent Technologies, Santa Rosa, CA, USA). Library preparation and Illumina sequencing analysis were performed as previously described, and putative lncRNAs were screened using unknown transcripts [ 7 ].…”

“…These complex cellular and development processes depend on the precise spatiotemporal expression of regulatory factors. Our previous studies found that glucose dose had a direct effect on GCs proliferation, apoptosis, and steroid hormone secretion, and glycolytic metabolites were positively correlated with steroid hormone secretions [ 6 , 7 ]. This confirms the connection between GCs steroidogenesis and glucose availability, but the molecular mechanisms underlying these changes require further study.…”

“…In this study, to explore the role of lncRNAs in regulating glucose-stimulated ovarian GCs steroidogenesis, ovarian GCs cultured with different glucose doses (optimum glucose concentration (8.4 mM) and high glucose concentration (33.6 mM)) [ 6 , 7 ] were used for RNA sequencing (RNA-seq). Based on this result, we investigated the function of a novel lncRNA named granulosa cell steroidogenesis-associated RNA (GSAR).…”