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Leptin is an important hormone influencing reproductive function. However, the mechanisms underpinning the role of leptin in the regulation of reproduction remain to be completely deciphered. In this study, our objective is to understand the mechanisms regulating the expression of leptin receptor (Lepr) and its role in ovarian granulosa cells during ovulation. First, granulosa cells were collected from superovulated mice to profile mRNA expression of Lepr isoforms (LeprA and LeprB) throughout follicular development. Expression of LeprA and LeprB was dramatically induced in the granulosa cells of ovulating follicles at 4 h after human chorionic gonadotropin (hCG) treatment. Relative abundance of both mRNA and protein of CCAAT/enhancer-binding protein b (Cebpb) increased in granulosa cells from 1 to 7 h post-hCG. Furthermore, chromatin immunoprecipitation assay confirmed the recruitment of Cebpb to Lepr promoter. Thus, hCG-induced transcription of Lepr appears to be regulated by Cebpb, which led us to hypothesise that Lepr may play a role during ovulation. To test this hypothesis, we used a recently developed pegylated superactive mouse leptin antagonist (PEG-SMLA) to inhibit Lepr signalling during ovulation. I.p. administration of PEG-SMLA (10 mg/g) to superovulated mice reduced ovulation rate by 65% compared with control treatment. Although the maturation stage of the ovulated oocytes remained unaltered, ovulation genes Ptgs2 and Has2 were downregulated in PEG-SMLA-treated mice compared with control mice. These results demonstrate that Lepr is dramatically induced in the granulosa cells of ovulating follicles and this induction of Lepr expression requires the transcription factor Cebpb. Lepr plays a critical role in the process of ovulation by regulating, at least in part, the expression of the important genes involved in the preovulatory maturation of follicles.

Ovulation is triggered by gonadotropin surge-induced signaling cascades. To study the role of extracellular signal-regulated kinase 1/2 (ERK1/2) in bovine ovulation, we administered the pharmacological inhibitor, PD0325901, into the preovulatory dominant follicle by intrafollicular injection. Four of five cows treated with 50 µM PD0325901 failed to ovulate. To uncover the molecular basis of anovulation in ERK1/2-inhibited cows, we collected granulosa and theca cells from Vehicle and PD0325901 treated follicles. Next-generation sequencing of granulosa cell RNA revealed 285 differentially expressed genes between Vehicle and PD0325901-treated granulosa cells at 6 h post-GnRH. Multiple inflammation-related pathways were enriched among the differentially expressed genes. The ERK1/2 dependent LH-induced genes in granulosa cells included EGR1, ADAMTS1, STAT3 and TNFAIP6. Surprisingly, PD0325901 treatment did not affect STAR expression in granulosa cells at 6 h post-GnRH. Granulosa cells had higher STAR protein and theca cells had higher levels of STAR mRNA in ERK1/2-inhibited follicles. Further, both granulosa and theca cells of ERK1/2-inhibited follicles had higher expression of SLC16A1, a monocarboxylate transporter, transporting substances including β-hydroxybutyrate across the plasma membrane. Taken together, ERK1/2 plays a significant role in mediating LH surge-induced gene expression in granulosa and theca cells of the ovulating follicle in cattle.

Oocytes collected from prepubertal animals are known to be less developmentally competent than those from adult animals. There is evidence suggesting that acquisition of developmental competence in bovine oocytes may be linked to the expression profile of genes in the granulosa cells (GCs). Cumulus–oocyte complexes (COC) and GCs were collected from 12 Holstein heifers between 2 and 6 months of age (nine follicle‐stimulating hormone [FSH] treated and three untreated) and eight FSH‐treated cows. The COCs from prepubertal animals were matured, fertilized, and cultured in vitro to assess development to the blastocyst stage. The relative messenger RNA (mRNA) abundance of FSHR, StAR, CYP19A1, HSD3B1, CX43, FOXO1, and XIAP in GCs were quantified by real‐time quantitative polymerase chain reaction. Results from this study revealed that GCs of prepubertal animals respond to FSH treatment by increasing mRNA levels of genes promoting estradiol synthesis and follicular growth ( FSHR and CYP19A1), and preventing cell apoptosis ( XIAP), and by decreasing mRNA levels of genes promoting progesterone production ( StAR and HSD3B1). This study also revealed that the relative mRNA abundance of FOXO1 in GCs is associated with oocyte competence to support embryo development to the blastocyst stage in prepubertal Holstein heifers.

The fatty acid binding protein 6 (Fabp6) is commonly regarded as a bile acid binding protein found in the distal portion of the small intestine and has been shown to be important in maintaining bile acid homeostasis. Previous studies have also reported the presence of Fabp6 in human, rat and fish ovaries, but the significance of Fabp6 in this organ is largely unknown. Therefore, we surveyed murine ovaries for Fabp6 gene expression and evaluated its role in ovarian function using mice with whole body Fabp6 deficiency. Here we show that the Fabp6 gene is expressed in granulosa and luteal cells of the mouse ovary. Treatment with gonadotropins stimulated Fabp6 gene expression in large antral follicles. The ovulation rate in response to superovulatory treatment in Fabp6-deficient mice was markedly decreased compared to wildtype (C57BL/6) mice. The results of this study suggest that expression of Fabp6 gene in granulosa cells serves an important and previously unrecognized function in fertility.

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