long non-coding RNA GDAR regulate ovine granulosa cells apoptosis by affecting the expression of apoptosis-related genes

Short-term dietary supplementation of female sheep during the luteal phase can increase fertility and most probably by stimulating glucose uptake by the follicles. However, the molecular mechanism of glucose regulates 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 GCs 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 groups. Therefore, the control (n = 3, 0 mM glucose group), low glucose (n = 3, add 8.4 mM glucose concentration group), and high glucose (n = 3, add 33.6 mM glucose concentration group) of GCs were created for the next whole genomic RNA sequencing (RNA-seq). In total, 11,221 novel lncRNAs and 510 annotated lncRNAs were identified in the GCs samples. Gene ontology term enrichment indicates the obvious enrichment in apoptosis and its related pathways, which suggests that cell apoptosis could play a critical role in the process of glucose-induced GCs differentiation. Furthermore, we focused on the function of a lncGDAR and verified that lncGDAR could involve cell apoptosis by effecting the expression of Bcl-2, BAX, Caspase-3, and Caspase-7 genes. These results provide the basis for further study of LncRNA regulation mechanism in nutrition on female fertility. Fresh ewe ovaries from Thin-tailed Han sheep aged one year were collected from the local abattoir and transported to the laboratory within 3 h in a buffered saline solution supplemented with a streptomycin/penicillin mixture (1%) and maintained at 37 ℃. Ovaries were washed three times with prewarmed normal saline before isolation of follicles. The follicles (the diameter of 3-5 mm) were isolated and punctured with a disposable syringe to collect follicular fluid. The follicle suspensions were pooled, and GCs were harvested immediately after centrifuging at 1000×g for 10 min. GCs were counted with a hemocytometer and their viability was confirmed by trypan blue exclusion. Then, the GCs were seeded in cell culture plates (Thermo Fisher Scientific, USA) at a density of 1×105/well and cultured in Dulbecco’s Modified Eagle Medium (DMEM/F12, Gibco, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, Grand Island, NY, USA) and 1% streptomycin/penicillin mixture in a humidified atmosphere at 37 ℃ and 5% CO2 for 48 h with the medium changed every 24 h. The GCs were allocated to six treatments and cultured in 6-well plates at a density of 1×106/well using DMEM/F12 medium supplemented with 10% FBS. All treatments were cultured without glucose and serum but containing streptomycin/penicillin mixture (1%) for 8 h; then, the treatments received various solutions of glucose and were cultured for an additional 24 h as follows: 0 mM (i.e., zero glucose), 2.1 mM (378.3 μg/ml), 4.2 mM (756.6 μg/ml), 8.4 mM (1513.2 μg/ml), 16.8 mM (3026.4 μg/ml), 33.6 mM (6052.8 μg/ml). These doses were designed to span the normal physiological ranges of follicles (1.1- 2.1 mM) [27-31]. The 16.7 mM glucose concentration that represented the normal rate of glucose supplementation in commercial preparations of these media, and the maximum glucose concentration of 33.6 mM represents 30 times the super-physiological concentration, which is used to detect the changes in cell apoptosis and proliferation at the super-physiological concentration of glucose. This culture system has been developed so that GC retain hormonally responsive aromatase activity and do not luteinize with time in culture [32-35]. The media and GCs were collected for subsequent measurements after the 24 h treatment period. Sequencing data were deposited with the NCBI. The procedures regarding sequencing, data processing, and further bioinformatics analyses can be found in detail in the Supplemental Materials and Methods. Total RNAs from ovine granulosa cells subjected to control (n = 3, 0 mM glucose groups), low glucose (n = 3, add 8.4 mM glucose concentration groups), and high glucose (n = 3, add 33.6 mM glucose concentration groups) were isolated and quality controlled. The preparation of whole-transcriptome libraries and next-generation sequencing were conducted by Novogene Bioinformatics Technology (Beijing, China). RNA-seq was performed on an Illumina Hiseq platform, and 150-bp-paired single-end reads were generated according to Illumina’s protocol. All the downstream bioinformatics analyses were based on clean data of high quality.