Abstract
Prolactin (PRL) plays a key role in the growth and ovulation of animal follicles, but its impact on follicular recruitment in ewes remains uncertain. In this study, a total of sixteen healthy ewes (Hu sheep, aged 2-3 years, with continuous reproduction and housed separately), matched for parity and weight (52.98 ± 0.96 kg), were randomly assigned to two groups: a control group (C) and a treatment group (T, PRL inhibition). Ovaries were collected in vivo after anesthesia during the estrus stage, and tissue morphology was observed using hematoxylin-eosin (HE) staining. By using RNA sequencing on the ovaries of C and T groups and conducting bioinformatics analysis, the essential genes and pathways involved in the regulation of PRL inhibition were pinpointed. Subcellular localization of key genes in ovarian tissue was determined using a fluorescence in situ hybridization (FISH) assay and immunohistochemistry. The function of key genes was validated using knockout and overexpression techniques. During the estrus phase, we noted a marked rise in the count of large follicles within ovarian tissue following the inhibition of prolactin. In total, 328 differentially expressed genes (DEGs) were detected, with 162 upregulated and 166 downregulated. The results indicated that inhibiting PRL primarily influences follicle recruitment by acting on the target gene PIKfyve. Following the inhibition of PRL during the estrus phase, there was an increase in the expression of PIKfyve. PIKfyve was primarily localized in the ovarian granulosa cells (GCs) and cumulus cells (CCs) in the ovarian tissue of ewes. The overexpression of PIKfyve decreased cell apoptosis and enhanced steroid hormone release, whereas knockout of PIKfyve had the reverse effect. In conclusion, PRL inhibition promoted follicle recruitment in ewes by upregulating PIKfyve during the estrus stage.