Lara Lage

Ovulation enables mature oocytes to exit the ovary for potential fertilization. In Drosophila, ovulation is induced by mating but also occurs spontaneously in virgins, with rates varying widely in natural populations: short oocyte retention is ancestral, while longer retention is favored in colder climates. The molecular regulation of spontaneous ovulation remains unclear. Here, we show that disrupting the relaxin/insulin-like peptide Dilp8 or its receptor Lgr3 (an ortholog of vertebrate RXFP1/2) in follicle cells or specific neurons, respectively, delays ovulation, slows average egg transit time in the reproductive tract, and facilitates oogenesis progression beyond approximately two mature oocytes per ovariole, leading to mature follicle accumulation in the ovary. Mating largely rescues these defects, suggesting the pathway is dispensable post-mating. Dilp8-Lgr3 signaling ensures high oocyte quality by promoting elimination of lower-quality aging oocytes and by antagonizing oogenesis progression via an undefined mechanism downstream of Lgr3-positive neurons. Our findings provide a molecular basis for oocyte retention time regulation in Drosophila involving ovarian-nervous system cross-talk, and bring further support for an ancient, conserved role for relaxin-like signaling in regulating ovulation and overall female reproductive physiology.

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, and bring further support for an ancient, conserved role for relaxin-like signaling in regulating ovulation and overall female reproductive physiology. Significance statement: Ovulation enables mature oocytes to exit the ovary for potential fertilization. In Drosophila , ovulation is induced by mating but also occurs spontaneously in virgins, with rates varying widely in natural populations: short oocyte retention is ancestral, while longer retention is favored in colder climates. The molecular regulation of virgin ovulation remains unclear. Here, we show that disrupting the Drosophila relaxin/insulin-like peptide Dilp8 or its receptor Lgr3—an orthologue of vertebrate RXFP1/2—in follicle cells or specific neurons delays ovulation, slows egg transit, and reduces oocyte quality. Mating largely rescues these defects, suggesting the pathway is dispensable post-mating. Our findings reveal a molecular mechanism for oocyte retention in Drosophila virgins and support an ancient, conserved role for relaxin-like signaling in ovulation.

The ability to achieve a species-specific size and proportion despite developmental or environmental perturbations is termed developmental stability. The molecular and cellular processes behind this are best understood in insects. In Drosophila, a peripheral-tissue stress signal, the relaxin/insulin-like peptide Dilp8, promotes developmental stability during larval development via its neuronal receptor, Lgr3, an ortholog of vertebrate relaxin receptors. Lgr3 signaling is widely accepted to occur in–and to activate (depolarize)–the central brain growth-coordinating interneurons (PIL/GCL neurons). Here, using neurogenetic approaches, we confirm the requirement of Lgr3 in PIL/GCL neurons, but unexpectedly find that they require both silenced (hyperpolarized) and active (depolarized) states for an appropriate response to Dilp8. These results are most simply explained if Lgr3 activation by Dilp8 triggers PIL/GCL-neuron oscillatory activity, and such oscillations promote developmental stability. PIL/GCL neurons express and require Cyclin A–which can form cell-cycle oscillator complexes with cyclin-dependent kinases–for their response to Dilp8, independently of Rca1 (regulator of CycA)/Emi1 (early mitotic inhibitor). This opens the possibility that cell-cycle machinery can be co-opted for postmitotic neuron oscillations, adding to an increasing list of postmitotic roles for cyclins. Neuroanatomically, we show that PIL/GCL neurons form reciprocally-innervating loops, which are common architectures in oscillating circuits and central pattern generators. The role of PIL/GCL neurons in developmental stability mirrors other homeostasis-regulating, peptide-driven oscillatory circuits found in the vertebrate hypothalamus, a developmentally-homologous region to the one occupied by PIL/GCL neurons in the fly brain.