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BimEL protein is involved in follicular atresia by regulating granulosa cell apoptosis, but the dynamic changes of BimEL phosphorylation during follicular atresia are poorly understood. The aim of this study was to explore the changes of key BimEL phosphorylation sites and their upstream regulatory pathways. First, the levels of BimEL-Ser65 and BimEL-Thr112 phosphorylation (p-BimEL-S65, p-BimEL-T112) in granulosa cells (GC) from healthy (H), slightly-atretic (SA), and atretic (A) follicles and in cultured GC after different treatments were detected by Western blotting. Next, the effects of the corresponding site mutations of BIM on apoptosis of GC were investigated. Finally, the pathways of two phosphorylation sites were investigated by kinase inhibitors. The results revealed that p-BimEL-S65 levels were higher in GC from H than SA and A, whereas p-BimEL-T112 was reversed. The prosurvival factors like FSH and IGF-1 upregulated the level of p-BimEL-S65, while the proapoptotic factor, heat stress, increased the level of p-BimEL-T112 in cultured GC. Compared with the overexpression of wild BimEL, the apoptotic rate of the GC overexpressed BimEL-S65A (replace Ser65 with Ala) mutant was significantly higher, but the apoptotic rate of the cells overexpressing BimEL-T112A did not differ. In addition, inhibition of the ERK1/2 or JNK pathway by specific inhibitors reduced the levels of p-BimEL-S65 and p-BimEL-T112. In conclusion, the levels of p-BimEL-S65 and p-BimEL-T112 were reversed during follicular atresia. Prosurvival factors promote p-BimEL-S65 levels via ERK1/2 to inhibit GC apoptosis, whereas proapoptotic factor upregulates the level of p-BimEL-T112 via JNK to induce GC apoptosis.

Insulin-like growth factor-1 (IGF-1) plays a crucial role during folliculogenesis, which has been demonstrated by previous research. However, the optimal IGF-1 dosage in the three-dimensional (3D) culture system is unknown. Mouse secondary follicles (140–150 µm) were cultured for 6 days within an alginate bead in a medium supplemented with 0 (G0), 5 ng/mL (G5), 10 ng/mL (G10), or 50 ng/mL IGF-1 (G50). Secretions of 17β-estradiol and progesterone were significantly increased in G10 and G50 (p < 0.05). However, G50 significantly inhibited follicular growth (p < 0.05), while G10 showed a higher oocyte maturation rate. Thus, the 10 ng/mL IGF-1 was used in subsequent experiments. IGF-1 enhanced the function of granulosa cells (GCs) by upregulating expressions of Star, Cyp19a1, Hsd3b1, Fshr, and Lhcgr. Oocyte secretory function was promoted by upregulating expressions of Bmp-15, Gdf-9, and Fgf-8. Addition of IGF-1 showed anti-apoptotic effect. However, G10 did not improve fertilization rate of MII oocytes compared to G0. In an intraperitoneal injection experiment in mice, IGF-1 significantly increased the number of ovulated oocytes (p < 0.05). In conclusion, 10 ng/mL IGF-1 can promote the production of mature oocytes in the 3D culture medium and injection of IGF-1 before superovulation increases the number of ovulated oocytes.

Vitamins and microelements play essential roles in mammalian ovarian physiology, including follicle development, ovulation, and synthesis and secretion of hormones and growth factors. However, it is nevertheless elusive to what extent exogenous supplementation with mixtures of vitamins ADE, zinc (Zn), and selenium (Se) affects follicular growth and granulosa cells (GCs) molecular function. We herein investigated their effect on follicular growth and GCs physiological function. We showed that follicular growth and ovulation time was accelerated and shortened with the increases of vitamins ADE, Zn, and Se doses by continually monitoring and recording (one estrus cycle of about 21 days) with an ultrasound scanner. Integrated omics analysis showed that there was a sophisticated network relationship, correlation expression, and enrichment pathways of the genes and metabolites highly related to organic acids and their derivatives and lipid-like molecules. Quantitative real-time PCR (qPCR) results showed that vitamin D receptor (VDR), transient receptor potential cation channel subfamily m member 6 (TRPM6), transient receptor potential cation channel subfamily v member 6 (TRPV6), solute carrier family 5 member 1 (SLC5A1), arachidonate 5-lipoxygenase (ALOX5), steroidogenic acute regulatory protein (STAR), prostaglandin-endoperoxide synthase 2 (PTGS2), and insulin like growth factor 1 (IGF-1) had a strong correlation between the transcriptome data. Combined multi-omics analysis revealed that the protein digestion and absorption, ABC transporters, biosynthesis of amino acids, aminoacyl-tRNA biosynthesis, mineral absorption, alanine, aspartate and glutamate metabolism, glycine, serine and threonine metabolism, arginine biosynthesis, and ovarian steroidogenesis were significantly enriched. We focused on the gene-metabolite interactions in ovarian steroidogenesis, founding that insulin receptor (INSR), phospholipase a2 group IVA (PLA2G4A), adenylate cyclase 6 (ADCY6), cytochrome p450 family 1 subfamily b member 1 (CYP1B1), protein kinase camp-activated catalytic subunit beta (PRKACB), cytochrome p450 family 17 subfamily a member 1 (CYP17A1), and phospholipase a2 group IVF (PLA2G4F) were negatively correlated with β-estradiol (E2), progesterone (P4), and testosterone (T) (P &lt; 0.05). while ALOX5 was a positive correlation with E2, P4, and T (P &lt; 0.05); cytochrome p450 family 19 subfamily a member 1 (CYP19A1) was a negative correlation with cholesterol (P &lt; 0.01). In mineral absorption, our findings further demonstrated that there was a positive correlation between solute carrier family 26 member 6 (SLC26A6), SLC5A1, and solute carrier family 6 member 19 (SLC6A19) with Glycine and L-methionine. Solute carrier family 40 member 1 (SLC40A1) was a negative correlation with Glycine and L-methionine (P &lt; 0.01). TRPV6 and ATPase Na+/K+ transporting subunit alpha 1 (ATP1A1) were positively associated with Glycine (P &lt; 0.05); while ATPase Na+/K+ transporting subunit beta 3 (ATP1B3) and cytochrome b reductase 1 (CYBRD1) were negatively related to L-methionine (P &lt; 0.05). These outcomes suggested that the vitamins ADE, Zn, and Se of mixtures play an important role in the synthesis and secretion of steroid hormones and mineral absorption metabolism pathway through effects on the expression of the key genes and metabolites in GCs. Meanwhile, these also are required for physiological function and metabolism of GCs. Collectively, our outcomes shed new light on the underlying mechanisms of their effect on follicular growth and GCs molecular physiological function, helping explore valuable biomarkers.