CircSHOC2 regulates steroid hormone synthesis in ovarian granulosa cells through the mir-130b-5p/ASH1L pathway

Estrus is a critical phase in the reproductive cycle of sows and depends on normal ovarian development and steroid hormone secretion. Granulosa cells (GCs) play crucial roles in maintaining normal ovarian physiological functions in sows by secreting estradiol (E2) and progesterone (P4). CircRNAs influence steroid hormone synthesis, but their involvement in regulating gilt estrous remains unclear. In this study, circRNA sequencing was performed on the ovaries of estrus (ES) and nonestrus (NES) gilts, leading to the identification of a novel circRNA, termed circSHOC2, which is highly expressed in ES ovaries. The overexpression of circSHOC2 promoted the synthesis of E2 and P4 and increased the protein levels of key steroidogenic enzymes. Further investigation revealed that circSHOC2 acts as a sponge for miR-130b-5p. Silencing miR-130b-5p significantly enhanced E2 and P4 production, along with the upregulation of steroidogenic proteins. Additionally, miR-130b-5p was found to target ASH1 like histone lysine methyltransferase (ASH1L), with miR-130b-5p overexpression significantly inhibiting ASH1L expression. Cotransfection experiments revealed that ASH1L mitigated the inhibitory effects of miR-130b-5p on E2 and P4 synthesis in GCs. This study, through circRNA sequencing of ovaries from ES and NES gilts, revealed that circSHOC2 regulates steroid hormone synthesis in porcine GCs via the miR-130b-5p/ASH1L axis, shedding light on a potential molecular pathway involved in gilts estrus. Our results will provide molecular targets for the selection of excellent estrus sows, thereby improving the utilization rate of gilts in estrus. Overall design: At a pig farm in Shaanxi Province, 643 healthy, lineage-verified New French Yorkshire sows with at least three parities, an average litter size of 12.5 or more piglets, and a minimum of seven pairs of teats were selected as the maternal stock to establish the core breeding population (C0 generation) for purebred production. We selected 102 litters (290 gilts) of C1 generation gilts and began estrus detection from 165 days of age. Gilts with two consecutive estrus cycles were recorded as ES, those with only one estrus cycle were recorded as irregular estrus, and those without estrus were recorded as NES. Ultimately, 24 litters were fully estrus, 12 litters were completely nonestrus, 32 litters had irregular estrus, and in 34 litters, oth ES and NES gilts were present. To study the differences between full-sibling individuals with estrus and nonestrus, we excluded 4 litters with inconsistent body condition from the 34 litters that met the experimental requirements, ultimately selecting 30 litters. From the same litter, one ES gilt and one NES gilt were selected, and the ES gilt was slaughtered during its third estrus period. A total of 6 groups were slaughtered, including 6 ES gilts and their corresponding full-sibling NES gilts. The right ovaries were frozen and stored at -80 °C for subsequent sequencing result verification, while the left ovaries were used for circRNA sequencing.