In the mouse ovary, the growing oocyte secretes R-spondin2 (RSPO2), a secreted enhancer of canonical WNT/β-catenin (CTNNB1) signaling, which acts in a paracrine manner on neighboring granulosa cells to sustain their proliferation and permit the primary-to-secondary follicle transition (De Cian et al., 2020). Four facets of this signaling axis — three mechanistic and one translational — were left unresolved by that study and remain incompletely settled: (1) the oocyte-intrinsic regulation of Rspo2 expression, since the assignment to NOBOX is confounded by oocyte degeneration in Nobox-null ovaries and FOXL2 cannot directly regulate an oocyte-expressed gene in mouse; (2) the physical route by which secreted RSPO2 crosses the zona pellucida to reach its granulosa-cell targets (transzonal projections, oocyte microvilli, or diffusion); (3) the molecular mechanism by which RSPO2/CTNNB1 and GDF9:BMP15/SMAD2 signaling are integrated in granulosa cells — functional cooperation has now been demonstrated (Wang et al., 2025), but the crosstalk mechanism and the specific hypothesis that GDF9-dependent transzonal projections deliver RSPO2 after zona pellucida formation are untested; and (4) whether RSPO2 or its pathway components contribute to premature ovarian insufficiency in women, where human genetic evidence remains limited to a negative 100-patient screen.

Background

Mammalian ovarian folliculogenesis begins when a primordial follicle — an oocyte surrounded by flattened pre-granulosa cells — activates: granulosa cells become cuboidal, divide, and form layers, and the oocyte grows. During this period the oocyte secretes glycoproteins ZP1–3 that assemble into the zona pellucida, an extracellular coat that separates the oocyte membrane from the granulosa cells. Communication across this barrier is bidirectional: granulosa cells extend transzonal projections (TZPs), actin-rich filopodia that traverse the zona pellucida and form gap junctions with the oocyte, and the oocyte secretes paracrine growth factors. Two oocyte-secreted factors (OSFs) are classical: GDF9 and BMP15, TGF-β superfamily members that signal through SMAD2/3 in granulosa cells; GDF9 is also required for TZP formation. The oocyte-derived mushroom-like microvilli (Oo-Mvi) described by Zhang et al. (2021) constitute a second oocyte-side structure implicated in releasing OSFs.

R-spondins (RSPO1–4) are secreted agonists of the canonical WNT pathway: RSPO binds the LGR4/5/6 receptors and the transmembrane E3 ubiquitin ligases ZNRF3/RNF43, stabilizing Frizzled receptors and potentiating β-catenin (CTNNB1)-dependent transcription. In vitro work had suggested oocyte-derived RSPO2 promotes follicle development (Cheng et al., 2013), but results were conflicting. De Cian et al. (2020) established the in vivo function: RSPO2 expression is restricted to the oocyte of developing follicles in mouse and human ovaries; Rspo2-null follicles arrest at the primary stage with granulosa-cell cycle arrest and downregulated granulosa WNT/CTNNB1 signaling, a phenotype independent of GDF9 and recapitulated by postnatal granulosa-specific Ctnnb1 deletion. RSPO2 is therefore an oocyte-to-granulosa paracrine signal required for early follicular growth.

Three mechanistic issues in this system were left open by the source study, and a fourth concerns translation. First, upstream regulation: the oocyte-specific homeobox transcription factor NOBOX was assigned to regulate Rspo2 because Rspo2 is downregulated in Nobox-null newborn ovaries (Choi et al., 2007) and NOBOX transactivates the RSPO2 promoter in vitro (Bouilly et al., 2017); however, Nobox ablation triggers rapid perinatal oocyte degeneration, which secondarily suppresses many oocyte genes, and FOXL2, a transcription factor that controls RSPO2 expression in the goat ovary (Kocer et al., 2008), is expressed in granulosa cells in mouse and so cannot directly regulate the oocyte-expressed Rspo2. Second, delivery: how secreted RSPO2 physically reaches granulosa-cell receptors across the zona pellucida is unknown; WNT ligands can be transported along filopodia (Stanganello et al., 2015), so RSPO2 might use an analogous route, but this has not been tested for RSPO2 in the follicle. Third, integration with GDF9: because GDF9 is required for TZP formation (El-Hayek et al., 2018) and both GDF9 and RSPO2 drive granulosa-cell proliferation, the source study proposed — without proof — that GDF9 and RSPO2 cooperate, possibly by GDF9-dependent TZPs delivering RSPO2 once the zona pellucida has formed. Fourth, human relevance: heterozygous Rspo2-mutant mice show fertility defects and RSPO2 expression is conserved in the human ovary, making RSPO2 a candidate gene for premature ovarian insufficiency (POI, loss of ovarian function before age 40), yet a screen of 100 POI patients found no clearly causative RSPO2 variant (Bouilly et al., 2017) and homozygous human RSPO2 loss causes lethal tetra-amelia syndrome (absence of all four limbs with lung hypoplasia) with no available folliculogenesis data. Granulosa-cell WNT signaling is independently essential for folliculogenesis (Habara et al., 2021), underscoring that the CTNNB1 branch the oocyte RSPO2 signal feeds into is a core growth pathway of the follicle.

Problem Statement

Achieve a complete mechanistic account of the oocyte-derived RSPO2 paracrine signaling axis that activates WNT/CTNNB1 signaling in granulosa cells and drives the primary-to-secondary follicle transition, together with the translational question of whether that axis contributes to premature ovarian insufficiency in women. Within this single objective, the unresolved facets — which are scientifically linked, since each concerns how the oocyte RSPO2 signal is produced, delivered, integrated with the co-secreted GDF9/BMP15 system, or translates to disease — are all jointly required for a full resolution: (1) identify the oocyte-intrinsic mechanism(s) that regulate Rspo2 expression in the postnatal oocyte in vivo — specifically, at least one such mechanism and the cis-regulatory element(s) through which it acts — resolving the confounded NOBOX assignment (in vitro promoter transactivation versus oocyte degeneration in Nobox-null ovaries) and the FOXL2 cell-type mismatch; (2) establish the physical route by which oocyte-secreted RSPO2 protein crosses the zona pellucida to reach and activate its LGR4/WNT–CTNNB1 targets on granulosa cells — granulosa-cell transzonal projections, oocyte-derived microvilli, or free diffusion — including whether GDF9-dependent transzonal projections are required for RSPO2 delivery once the zona pellucida has formed; (3) determine the molecular mechanism by which RSPO2/CTNNB1 and GDF9:BMP15/SMAD2 signals are integrated in granulosa cells, given that gene-specific synergistic and antagonistic cooperation is now demonstrated but its crosstalk mechanism is unexplained; and (4) establish whether loss-of-function variation in RSPO2 or other components of its ovarian pathway contributes to premature ovarian insufficiency in women, going beyond the currently negative single-gene screening data. The complete mechanistic account is defined by these four facets at the thresholds stated: a solution that establishes all four to that bar is a full resolution of the objective.

References

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