Volonte, Y.; Heredia, F.; Zanini, R.; Menezes, J.; Perez, M. S.; Gualdino, M.; Lage, L.; Cruz, L. d. S.; Casimiro, A. P.; Garelli, A.; Gontijo, A. M.

Ovulation allows mature oocytes to exit the ovary for potential fertilization. In Drosophila, ovulation can be induced by mating or occur spontaneously in lower amounts in virgin females. Virgin ovulation rates show high populational variation, with short oocyte retention times being ancestral, and long retention being selected for in colder climates. The molecular mechanisms controlling virgin ovulation rates are poorly understood. Here, we show that reduced activity of the Drosophila insulin-like peptide 8 (Dilp8), a relaxin-like peptide secreted from terminal follicle cells of the ovary, slows virgin ovulation and ovulated egg genital tract transit by a factor of ~5-6. These phenotypes are largely rescued by mating, suggesting other mechanisms override the Dilp8-Lgr3 pathway post-mating. Knockdown of the Dilp8 receptor, Lgr3--an orthologue of vertebrate relaxin RXFP1/2 receptors--in at least two subsets of reproductive-tract-innervating neurons, one of which are octopaminergic, produces similar phenotypes. Dilp8-Lgr3 signaling in virgin females ensures high oocyte quality by promoting the elimination of aging oocytes and by systemically antagonizing insulin signaling, which is critical for oogenesis progression beyond previtellogenic stages. We hypothesize this latter Dilp8 activity factors in oocyte stock size in the nutrient-availability-based decision to invest in producing more oocytes or not, ensuring oocyte overstocking in virgins only occurs in highly favorable nutritional conditions. Our findings provide a molecular basis for oocyte retention time regulation in Drosophila virgins. Our work brings further support for an ancient, conserved role for relaxin-like signaling in regulating ovulation and overall female reproductive physiology.