FSHR and LHR functional compensation reveals the mechanism and treatment of Ovarian Hyperstimulation Syndrome

Domain: biology
Published: 2026-04-01
Editorial outcome: minor_revision

Abstract
Gain-of-function mutations in the human follicle-stimulating hormone receptor (FSHR) cause spontaneous ovarian hyperstimulation syndrome (OHSS), a serious reproductive disorder. However, the molecular physiology and treatment options for OHSS remain elusive. Notably, estrildid finches naturally carry an FSHR variant (Thr449Ala) analogous to the pathogenic mutation in humans yet are resistant to OHSS. Here we show that this resistance stems from significantly reduced luteinizing hormone receptor expression in estrildid ovarian granulosa cells. Furthermore, treatment with the luteinizing hormone receptor antagonist alleviates OHSS symptoms in mouse models. Single-cell RNA transcriptomic reveals functional compensation of the two receptors to regulate estrogen production and vascular permeability, resembling the adaptive mechanisms observed in estrildid finches. Our study unravels the molecular mechanism underlying the physiological adaptation of estrildid ovaries to high FSHR constitutive activity and is a example of how the concept of Darwinian Medicine could be exploited to identify novel drug targets for ovarian hyperstimulation syndrome treatment.

Introduction
Ovarian hyperstimulation syndrome (OHSS) is a serious complication of ovarian stimulation, typically triggered by the excessive development of multiple follicles1. The syndrome was first recognized in 1943, when ovulation was induced using exogenous gonadotropins2. These early therapeutic agents were derived from pregnant mare serum, sheep pituitary glands, and the urine of pregnant women2, 3. Mild OHSS occurs in approximately 20-30% of in vitro fertilization (IVF) cycles, whereas intermediate and severe forms are observed in 3-6% and 0.1-2% of cases, respectively4. Clinically, OHSS manifests as abdominal distension and discomfort is manifested by, for example, bilateral, multiple ovarian cysts, ascites, and low circulating protein levels5. Although the exact mechanism remains incompletely understood, its pathophysiology is thought to proceed as follows: pharmacologically stimulation induces multi-follicular development during the follicular phase, followed by extensive luteinization of the retrieval follicles, leading to the formation of multiple corpora lutea. This, in turn, causes ovarian enlargement and an exaggerated release of ovarian hormones and vasoactive mediators, including cytokines, angiotensin, estrogen, and vascular endothelial growth factor (VEGF)6, 7. These factors collectively increase vascular permeability and promote fluid extravasation.

In female mammals, follicle development is tightly regulated by two pituitary gonadotropins: follicle-stimulating hormone (FSH) and luteinizing hormone (LH)8. Folliculogenesis typically culminates in the selection of a single dominant follicle, and the process is conventionally divided into gonadotropin-independent and gonadotropin-dependent phases. Early follicle development, encompassing the primordial, primary, and secondary stages, occurs independently of gonadotropins. The transition from the pre-antral to the early antral stage marks the onset of the gonadotropin-dependent phase, during which further growth and maturation require both FSH and LH8, 9. LH and FSH act through their specific receptors-FSH receptor (FSHR) and LH receptor (LHR)-both members of the G-protein coupled receptor (GPCR) family characterized by large extracellular domains responsible for hormone bindings10. In females, FSHR expression is restricted to granulosa cells throughout preovulatory follicular development, whereas LHR mRNAs is localized exclusively in the thecal cells of immature follicles and in both thecal and granulosa cells of mature antral follicles. Since LH and FSH cooperatively promote follicles growth, ovulation, and corpus luteum formation, they are routinely used during ovarian stimulation for in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI). FSH derives follicular growth and maturation and contributes to LH-induced ovulation and luteinization11. Human chorionic gonadotropin (hCG), a natural analogue of LH that shares the same receptor but exhibits greater biological activity and a longer half-life, remains the gold standard for triggering ovulation12.

OHSS typically arises following the administration of exogenous gonadotropins, such as FSH or human menopausal gonadotropin (hMG), in combination with hCG for ovulation induction, particularly when estradiol levels are elevated13. In rare instances, OHSS can occur in the absence of exogenous hormonal stimulation, a condition referred to as spontaneous OHSS (sOHSS)14. sOHSS is thought to result from unusually high circulating levels of endogenous hCG or thyroid-stimulating hormone (TSH), both of which can mimic gonadotropin activity and aberrantly activate FSHR on granulosa cells59. Up to now, seven distinct heterozygous activating FSHR variations have been identified: p.Asp567Asn/Gly, p.Thr449Ile/Ala, p.Ile545Thr, and p.Ser128Tyr/Thr, which can potentially trigger the development of severe sOHSS15, 16, 17, 18, 19, 20, 21, 22. The FSHR variants have a markedly increased sensitivity to normal TSH and/or hCG levels. Except for the p.Ser128Tyr/Thr mutation, the other amino acid exchanges are also known to induce constitutive activity of FSHR23. Laboratory studies using a genetically engineered mouse model harboring a constitutively active FSHR mutation (FSHRD580H) demonstrate that this mutation leads to a range of ovarian abnormalities including irregular ovarian structure, hemorrhagic cysts, accelerated deletion of primordial, primary, and secondary follicles, increased estradiol synthesis, and elevated prolactin (PRL) levels in adolescent mice24. It is anticipated that humans with similar mutations may exhibit a comparable phenotype24. Additionally, a positive correlation between the degree of constitutive activity and the severity of ovarian abnormalities has been suggested24. Mice with the FHSRD580Y mutation exhibit a milder ovarian phenotype, primarily characterized by hemorrhagic cysts24.

The prevailing models on the functioning of GPCRs stipulate that they require the binding of at least one endogenous ligand to activate heterotrimeric G proteins, which regulate various physiological functions24. However, research has shown that GPCR variants with constitutive activity are associated with different diseases25, 26 . This indicates that the constitutive activities stimulated by these GPCRs are intricately linked to their corresponding physiological functions27. In clinical patients, numerous gain-of-function variations have been discovered in GPCRs over the past few decades26, 28, 29. Specifically, functionally activated variations in FSHR, accompanied by constitutive activation of Gs signaling, can facilitate OHSS30. However, the molecular physiology and treatment of OHSS remain elusive for a long time.

Here, the evolutionary analysis of vertebrate FSHR unexpectedly revealed that the gain-of-function variation T449A, previously discovered in clinical OHSS patients31, naturally occurs in the FSHR of estrildid finches examined. The Estrildidae family belongs to the songbird suborder Passeri, including species such as the zebra finch (Taeniopygia guttata, tg), a well-established model organism for birds, as well as the Gouldian finch (Erythrura gouldiae, eg), Bengalese finch (Lonchura striata domestica, ls), and white Java sparrow (Padda oryzivora, po), all examined in this study32. In the present study, we elucidated the physiological adaptation of the estrildid ovary due to this natural FSHR variant and proposed an alternative therapeutic approach for treating OHSS. For a comprehensive list of abbreviations and compilation of animal species (Latin and common names) and their abbreviations, see Supplementary Data 1a (sheet1) and 1b (sheet2), respectively.

Results
Natural p.Thr449Ala variant confers enhanced activity to estrildid FSHR
To evaluate whether FSHR underwent positive selection in estrildids, we retrieved the coding sequence of 57 vertebrate species from the NCBI database and used PCR amplification and sequencing to obtain the sequence information of E. gouldiae and P. oryzivora (Supplementary Data 2), resulting in a dataset of 59 species for branch-site selection analysis. A phylogenetic tree was generated using TimeTree to represent the evolutionary relationships among these 59 species, and two additional species, namely common wombats (Vombatus ursinus, vu) and Megaleporinus macrocephalus (a headstander fish), were included in the tree (Fig. 1a) because they also carry the p.Thr449Ala variant, thus providing a broader visual context for this rare substitution. The branch‑site test showed that only estrildid FSHR orthologs had a large nonsynonymous (dN)/synonymous (dS) substitution rate ratio (branch-site dN/dS of ω >> 1) that was highly significant (likelihood ratio tests [LRTs], P < 0.05) (Fig. 1b). Two amino acid sites (167 and 449) underwent positive selection in estrildid birds, while the ancestral positions 167 and 449 were conserved in most vertebrates (Fig. 1a-c). Notably, at site 449, the amino acid threonine (T) is ubiquitously observed across nearly 3000 vertebrate species, with the exceptions of estrildid finches, Vombatus ursinus, and headstanders (Anostomidae), which exhibit a T to alanine (A) variation at this site (Supplementary Fig. 1). Additionally, species in the Mormyridae family and Greenland flounder (Reinhardtius hippoglossoides, rh) exhibit a T to isoleucine (I) variation at the same site (Supplementary Fig. 1). To explore the functional ramifications of this variation, the 449 sites were swapped between human and estrildid FSHR. The constitutive activity was evaluated using a CRE (cAMP response element)-luciferase assay in HEK293 cells. Our findings revealed a consistent enhancement in the constitutive activity of FSHR across a diverse array of species including mammals, birds, amphibians, reptiles, and fish when the 449th amino acid was replaced from T to A (Fig. 1d and Supplementary Fig. 2). Similarly, a substitution from T to I at this position enhanced FSHR constitutive activity as well (Supplementary Fig. 3). Consistently, in species like the estrildid finches, which naturally possess the 449A variant, swapping to T at this position resulted in diminished FSHR constitutive activity and cellular cAMP concentration (Fig. 1d and Supplementary Fig. 2b, 4a,b). Notably, in V. ursinus, this variation led to an abolition of constitutive activity (Fig. 1d and Supplementary Fig. 2). Estrildid finch FSHR exhibited vigorous constitutive activity between 2.9-14.1 times higher than that observed in the non-estrildid Serinus canaria (sc) and chicken (Gallus gallus, gg) when expression was adjusted to levels similar to endogenous FSHR in estrildid ovaries (Supplementary Fig. 4c,d). These results also indicated that physiological levels of FSHR expression are sufficient to elicit constitutive activity (Supplementary Fig. 4c,d). Hence, the T449A variation universally augmented the Gs pathway of all vertebrate FSHRs and was specific to the Gs pathway (Supplementary Fig. 5).

T449A substitution enables FSHR activation by LHR ligands
The FSHRT449A mutation was initially identified in patients with OHSS, and experiments have confirmed that it expands the ligand range of FSHR31. Based on this finding, we assessed the impact of the T449A variation on the ligand-induced response of avian and mammalian FSHR in HEK293 cells. Our results showed that mammalian FSHR and LHR (including those from humans, rats, mice, and V. ursinus) exhibited a high specificity for the FSH and LH ligands (Supplementary Fig. 6-8). In contrast, receptor-ligand specificity was markedly weaker in birds. While mammalian systems maintain strict FSH-FSHR and LH-LHR pairing, we found that in several avian species (such as T. guttata, L. striata domestica, G. gallus, and S. canaria), FSH can cross-activate LHR (Supplementary Fig. 8b). Moreover, both mammalian and avian FSHR and LHR could be activated by hCG (Fig. 1e and Supplementary Fig. 8a, b). The T449A variation increased FSHR sensitivity to hCG in both mammalian and avian species, consistent with previous findings31 (Fig. 1e). Notably, LH, which originally could only specifically activate LHR but not FSHR in humans and non-estrildid birds, was found to non-specifically activate FSHR in estrildid finches, as well as non-estrildid FSHR carrying the p.Thr449Ala variant (Supplementary Fig. 7). This result indicated that T449A enhanced the response of FSHR to LHR ligands (LH and hCG) and thus broadened its ligand range. Furthermore, the differential sensitivity of the 449T and 449A variant receptors to FSH varied among vertebrates. E.g., in the estrildid birds, T. guttata and L. striata domestica, the T449A mutation enhanced the stimulation by both recombinant human FSH (r-hFSH) and chicken FSH (ggFSH) (Supplementary Fig. 6). In contrast, amino acid exchanges at position 167 had no impact on the response of FSHR, nor did it affect the constitutive activity of the receptors, except for a slight decrease in T. guttata (tg)FSHR (Supplementary Fig. 9). These findings revealed that during the evolutionary process, estrildid finch FSHR had gained enhanced constitutive activity and a broader ligand range due to the T449A variation, similar to the Homo sapiens (hs)FSHRT449A mutation, making it a valuable animal model for studying the mechanistic impacts of the natural FSHRT449A variant.

FSHRT449A knock-in mice, unlike estrildids, are susceptible to OHSS
To investigate the impact of the FSHRT449A variation on ovarian function, we generated a knock-in mouse model harboring the T449A mutation (FSHRT449A-KI) (Fig. 2a, Supplementary Fig. 10a). Our findings revealed that the T449A mutation did not affect ovarian histological architecture, ovary weight, ovarian cAMP levels, and the mice remained fertile (Supplementary Fig. 10). However, serum estradiol and FSH concentrations were significantly elevated in the homozygous FSHRT449A-KI (HO) mice (Supplementary Fig. 10). Excessive doses of pregnant mare serum gonadotropin (PMSG), followed by administration of 5-30 IU hCG, are commonly used to induce OHSS in rodent6, 33. In the control group (standard superovulation protocol), animals received a single injection of PMSG followed by 5 IU of hCG 48 hours later. Because the FSHRT449A mutation enhances FSHR responsiveness to both FSH and hCG and expands its ligand spectrum, we applied a milder OHSS-induction protocol using a reduced hCG dose (1 IU) to assess whether FSHRT449A-KI mice are predisposed to OHSS (Fig. 2b). We found that following induction with the same doses of PMSG and hCG, homozygous KI mice exhibited more severe OHSS symptoms compared to wild-type (WT) mice, as evidenced by a significantly greater increase in ovary size, serum and ovarian VEGF levels, serum estradiol concentrations and elevated numbers of corpora lutea (Fig. 2c-g, Supplementary Fig.11g). Additionally, ovarian cAMP levels and genes related to the estrogen pathway, such as steroidogenic acute regulatory protein (Star), cytochrome P450 family 19 subfamily a member 1 (Cyp19a1), as well as genes regulating vascular permeability, including bone morphogenetic protein 2 (Bmp2)34 and early growth response 1 (Egr1)35 were significantly elevated in homozygous OHSS mice (Fig. 2h,i). Consistent with these findings, under the standard superovulation protocol, homozygous KI mice displayed clear OHSS-related phenotypes compared to WT mice. The KI animals showed significantly elevated ovarian cAMP levels, ovarian enlargement, and increased estrogen and VEGF production (Supplementary Fig. 11a-f), indicating an inherent susceptibility to OHSS even during routine superovulation procedures. Since the ovary/body weight ratio, serum estradiol and VEGF levels were related to the dose of hCG in WT mice (Supplementary Fig. 12), and higher hCG easily triggered OHSS, we hypothesized that the difference might be due to the enhanced responsiveness of FSHR to hCG induced by the T449A mutation, as detected at the cellular level (Fig. 1e).

Similar to mammals, avian FSHR was mainly expressed in ovary tissue, followed by the lung, and at low levels in non-gonadal tissues36, 37 (Supplementary Fig. 13). Despite the gain-of-function variation in estrildid FSHR, no significant differences were observed in ovary weight or serum hormone concentrations (estradiol and FSH) between estrildid finches and other bird species without the FSHRT449A variation (Supplementary Fig. 13c, e). Further histological analysis revealed no abnormal ovarian structures or hemorrhagic cysts, unlike those observed in FSHRD580H-KI mice24 (Supplementary Fig. 13d). Estrildid finches serve as natural models for the FSHRT449A variation, yet it remained uncertain whether they would be susceptible at all to OHSS, as seen in mice and patients. To further investigate, we attempted to trigger OHSS in birds using PMSG and hCG or hFSH, since hCG does not naturally occur in birds (Fig. 2j). We found that hCG/hFSH could increase ovary weight ratio and estrogen levels in non-estrildid birds and induce symptoms similar to OHSS, but had a negligible effect on estrildid finches (Fig. 2k-o). In addition, histological examination revealed a greater number of enlarged follicles after PMSG/hFSH stimulation, especially in non-estrildid birds (Fig. 2p). Expression of VEGFA significantly increased in all four species (Supplementary Fig. 14). However, EGR1, BMP2, adhesion G Protein-Coupled Receptor G1 (ADGRG1, a gene that can regulate VEGF secretion38), and CCAAT/enhancer-binding protein beta (CEBPB, which increased in OHSS mice (Fig. 5)) showed significant increases in non-estrildid species but not in estrildid finches (Fig. 2q, Supplementary Fig. 14). These results indicated that although estrildid finches harbor the FSHRT449A variation, they did not appear to be more susceptible to OHSS mice, suggesting that Estrildidae had evolved physiological adaptations throughout evolution to protect against OHSS.

LHR significantly decreased in FSHRT449A-bearing estrildid ovaries
To further investigate the intrinsic adjustment mechanisms of estrildid finches to avoid OHSS, we performed single-cell RNA sequencing (scRNA-seq) on the ovaries of two estrildid species (T. guttata, tg, and L. striata domestica, ls) as well as one non-estrildid bird (S. canaria, sc) under steady-state physiological conditions without exogenous stimulation This experimental design enables the identification of baseline transcriptional adaptations in estrildid finches. After integration based on the canonical correlation analysis (CCA) strategy39, all high-quality cells were divided into ten main homologous cell types based on the canonical cell markers (mesenchymal cells: DCN, MMP2, COL6A1; granulosa cells: FSHR, FOXL2, EMX2; endothelial cells: S1PR1, ADGRL4, KDR; immune cells: IL16, PTPRC, SPI1; glial cells: HEBP2, FRMD4A; HSD11B2+ cells: HSD11B2, SPP1; oocytes: DAZL, DDX4, ZP2; red blood cells: AK2, TAL1, CREG1, epithelial cells: KRT18, KRT6, MIOX+ cells: HSD17B4, PSPH) (Fig. 3a-c, Supplementary Fig.15a). By analyzing the distribution of each population in all species, the granulosa cells, immune cells, and mesenchymal cells constitute a relatively substantial proportion across ovarian atlases in all species (Fig. 3b). Principal component analysis (PCA) was performed to explore cell-type relationships across species. Correlation analysis and PCA showed tight clustering of cell types in each species, indicating that the inter-species difference was more significant than intra-species. The L. striata domestica was phylogenetically closest to T. guttata, and S. canaria was distant to both, reflecting their evolutionary relationships (Fig. 3d,e). Feature plots demonstrated that FSHR expression was primarily localized to granulosa and pre-granulosa cells across all three species, with additional expression observed in mesenchymal and epithelial cells (Fig. 3f). A cross-species comparison of granulosa cells revealed a significant downregulation in estrildid finches of pathways related to cell cycle regulation, blood vessel development and morphogenesis, and steroid metabolic process (Fig. 3g, Supplementary Fig.15b). These findings were based on the intersection of differentially expressed genes (DEGs) from pairwise comparisons between T. guttata and S. canaria, as well as L. striata domestica and S. canaria, representing consistent transcriptional changes in estrildids relative to the non‑estrildid control. The expression of VEGFA and ADGRG1, which enriched in angiogenesis/vasculature development significantly increased in estrildid finches. In contrast, the expression of EGR1, BMP2, angiotensin I converting enzyme (ACE)40, and inhibitor of DNA binding 1 (ID1)41, 42 in the angiogenesis/vasculature development pathways that regulate vascular permeability were significantly decreased (Fig. 3g and Supplementary Data 3). Additionally, the expression of STAR and 17β-hydroxysteroid dehydrogenase type 7 (HSD17B7), which is enriched in the estrogen signaling pathway, was significantly decreased (Fig. 3g, Supplementary Data 3). These differentially expressed genes identified in scRNA-seq were further validated by quantitative PCR analysis (Fig. 3h-j). Among these genes, EGR1 has been reported to promote the development of OHSS43 and ACE inhibition can mitigate OHSS symptoms44. This suggests that the observed changes in gene expression within granulosa cells reflect an adaptive evolutionary mechanism in estrildid finches, potentially serving as a strategy to mitigate the risk of OHSS.

In addition to scRNA-seq, a total of 12 ovaries were collected from two estrildid species (T. guttata, n=3; L. striata domestica, n=3), as well as two non-estrildid species (S. canaria, n=3; G. gallus, n=3), and their RNA was deep-sequenced (with 78.5 ± 17.6 SD million reads per sample) (Supplementary Data 3). Comparative analysis of transcriptomes between estrildid and non-estrildid ovaries revealed that 291 genes were expressed at higher and 284 genes at lower levels (Supplementary Data 4). Notably, the LHR gene, which was significantly decreased in granulosa cells of estrildid finches according to scRNA-seq analysis of avian ovaries, also consistently exhibited lower expression in estrildid ovaries as confirmed by RNA-seq analysis (Fig. 3g, Supplementary Fig. 16). scRNA-seq revealed that LHR was highly expressed in the granulosa and theca cells of S. canaria but was nearly undetectable in the granulosa cells of the T. guttata and L. striata domestica (Fig. 3k,l). Real-time PCR (qRT-PCR) further confirmed the lower expression of LHR in the ovaries of Estrildidae species (Fig. 3m). This suggested that the diminished LHR expression could be a crucial mechanism by which estrildids adapted to the FSHR variation.

LHR as a therapeutic target for OHSS intervention
To investigate whether the reduction of LHR signaling is a key adaptive mechanism for estrildid finches to avoid OHSS, we induced OHSS in C57BL/6 mice using PMSG and hCG, and administered the allosteric LHR inhibitor LUF577145 to investigate the effects (Fig. 4a). RU486 was used as a positive control, as it has been shown to reduce LHR expression and OHSS symptoms46. As expected, PMSG and hCG significantly induced ovary enlargement, increased levels of estradiol and VEGF, and elevated numbers of corpora lutea (Fig. 4b-h, Supplementary Fig. 17a). After treatment with LUF5771, there was a significant reduction in cAMP concentration in the mouse ovaries, as well as a decrease in ovary weight, estradiol, and VEGF levels (Fig. 4b-h, Supplementary Fig. 17a), indicating that LUF5771 could ameliorate the OHSS phenotype in mice. Additionally, we explored the therapeutic effects of two newly discovered LHR antagonists, BAY-298 and BAY-899, on rat models of OHSS, as these inhibitors selectively antagonize LHR in humans, rhesus macaques, and rats47 (Fig. 4i). Compared to the untreated OHSS group, ovaries treated with BAY-298/899 exhibited significantly lower cAMP concentrations, reduced ovary weight, and decreased levels of estradiol and VEGF, effectively treating OHSS in the rat model (Fig. 4j-p, Supplementary Fig. 17b). These findings suggested that inhibition of LHR signaling is a physiological adaptation used by estrildid finches in response to the FSHRT449A variation, which could also serve as a therapeutic strategy for OHSS.

Functional compensation of evolutionarily related FSHR and LHR
LHR and FSHR are evolutionarily related and conserved GPCRs across the entire vertebrate spectrum (Supplementary Fig. 18). As members of the glycoprotein hormone GPCR family, they are predominantly expressed in the ovary tissue of females (Supplementary Fig. 19). To elucidate, why estrildid finches have evolved to reduce LHR as an adaptation to the FSHRT449A variation, we treated homozygous FSHRT449A- KI mice with LHR and FSHR antagonists after administration of PMSG and hCG, followed by comparative analyses to investigate the downstream signaling pathways of LHR and FSHR in the OHSS model (Fig. 5a). As expected, LHR antagonist LUF5771 and FSHR antagonists hFSH-β-(33-53)48 significantly countered the OHSS-induced ovary weight increase, serum estradiol and serum and ovarian VEGF levels (Fig. 5b-e, Supplementary Fig. 17c). Additionally, these antagonists reduced cAMP concentrations and the number of corpora lutea in FSHRT449A-KI mice (Fig. 5f-h). The therapeutic effect of LUF5771 was superior, with all indicators almost returning to the control levels, in OHSS model of FSHRT449A-KI mice, comparable to OHSS model of WT mice (Fig. 5c-f and Fig. 4c-g). Ovaries from the OHSS and antagonists treated groups were collected for scRNA-seq analysis. After integration using CCA, all high-quality cells were classified into eight distinct cell types based on canonical markers (Fig. 5i-j, Supplementary Fig. 20). Granulosa cells represented a relatively substantial proportion of cells in the OHSS ovaries (Fig. 5k). Both Fshr and Lhr were expressed in granulosa and theca cells of mouse ovaries (Fig. 5j). After treatment with LUF5771 and hFSH-β-(33-53), the differentially expressed genes in the ovaries when compared to those in OHSS ovaries were predominantly found in theca cells, followed by granulosa cells (Fig. 5l). Furthermore, the ratio of granulosa to theca cell numbers was significantly reduced after treatment of LUF5771 or hFSH-β-(33-53) (Fig. 5k).

By comparing gene expression in granulosa cells between the treatment groups and the untreated OHSS group, we identified 482 differentially expressed genes in the LUF5771-treated mice and 460 DEGs in the hFSH-β-(33-53)-treated mice (Supplementary Fig. 20 and Supplementary Data 5). Venn analysis revealed 227 shared DEGs following treatment with either antagonist, including genes involved in the cAMP pathway, such as AKT serine/threonine kinase 3 (Akt3), proto-oncogene (Jun and Fos), ATPase, Na+/K+ transporting subunit beta 2 (Atp1b2), Lhr, etc (Supplementary Data 5). The decreased expression of Akt3, Jun and Fos may influence several signaling pathways, including the VEGF, MAPK, TNF, and estrogen signaling pathway (Fig. 5m, Supplementary Data 5). In addition, the Hippo and TGF-β pathways were also downregulated (Fig. 5m). Using the R package GSEABase and the AddModuleScore function, we further assessed signature scores for angiogenesis and estrogen synthesis pathways. LUF5771 treatment significantly lowered the estrogen synthesis pathway score in both granulosa and theca cells, and reduced the angiogenesis pathway score in granulosa cells. Similarly, TFA treatment decreased the estrogen synthesis pathway score in granulosa cells (Fig. 5n). In the OHSS mouse model, the estrogen synthesis-related gene Star was highly expressed, and treatment with LUF5771 and hFSH-β-(33-53) did not alter its expression (Fig. 5o, Supplementary Data 5). However, several other genes related to estrogen signaling, such as Akt3, Fos, heparin-binding epidermal growth factor-like growth factor (Hbegf), heat shock protein family A member 1A (Hspa1a), and Jun, were significantly downregulated following treatment (Fig. 5o, Supplementary Data 5). Among VEGF pathway genes, the expression of Akt3 and protein phosphatase 3 catalytic A (Ppp3ca) was reduced (Fig. 5p, Supplementary Data 5). Genes associated with vascular permeability, such as Bmp2 and its receptor Bmpr2, Id1, and Klf649, were all downregulated (Fig. 5q, Supplementary Data 5). Additionally, the TNF pathway gene Cebpb and the cAMP signaling-related gene Nfkbia showed decreased expression (Fig. 5r,s and Supplementary Data 5). In theca cells, treatment with LUF5771 and hFSH-β-(33-53) resulted in 462 overlapping DEGs, which were mainly enriched in the metabolism of xenobiotics by cytochrome P450, cAMP signaling and HIF-1 pathways, and more (Fig. 5m, Supplementary Data 5). This suggests that FSHR and LHR share similarities in their downstream signaling pathways.

To assess changes in granulosa cell and theca cell subtypes, we performed re-clustering and annotation using high-quality marker genes reported in the literature. Granulosa cells were classified into distinct subtypes, including an Antral-Mural state (Supplementary Fig. 21a,b). Compared with the OHSS group, treatment with LUF5771 and hFSH-β-(33-53) reduced the number of granulosa cells in atretic follicles, while cells in the preantral follicle state showed signs of recovery (Supplementary Fig. 21c), suggesting that these treatments may help restore normal granulosa cell composition and limit follicle atresia. The expression patterns of differentially expressed genes related to estrogen signaling and vascular permeability further pointed to their potential role in this process (Supplementary Fig. 21d). Theca cells were primarily classified into early-stage cells (involved in androgen synthesis) and late-stage cells (associated with signal transduction, chemotaxis, and cell growth) (Supplementary Fig. 21e-g). Following treatment with LUF5771 or hFSH‑β‑(33‑53), the proportion of early‑stage theca cells decreased relative to the OHSS group (Supplementary Fig. 21f), indicating a possible downregulation of the androgen synthesis pathway that serves as the precursor for estrogen production. Moreover, expression of Bmp2 and Bmpr2 was reduced in both early‑ and late‑stage theca cells after treatment (Supplementary Fig. 21h).

Together, these findings suggest that enhanced function caused by the FSHR variation could be balanced by reducing the activity of the LHR pathway. This represents an important adaptive mechanism in estrildid finches in response to the FSHRT449A variation.

Methods
Animals
zebra finches (Taeniopygia guttata), Bengalese finches (Lonchura striata domestica), Gouldian finches (Erythrura gouldiae), common canaries (Serinus canaria), budgerigars (Melopsittacus undulatus), Japanese white-eyes (Zosterops japonicus), white Java sparrows (Padda oryzivora) and chickens (Gallus gallus) were bought from Flower and Bird Market (Nanjing, China). Birds were maintained in a temperature-controlled (21-23 °C) and humidity-controlled (48% average) vivarium under a 12/12 h light/dark cycle. Animals were housed at a density of five per cage (160 × 50 × 40 cm). Female C57BL/6 mice were obtained from GemPharmatech Co., Ltd., Jiangsu, China. FSHRT449A-KI mice (The hsFSHR 449 site corresponds to the homologous site 448 in mice, for consistency, we refer to it as 449A throughout the article) were generated on a C57BL/6 background using CRISPR-Cas9-mediated gene editing. Two pairs of gRNAs were designed to induce double-strand breaks, and a donor vector containing the "KI region-p.T448A (ACT to GCT)" cassette, along with Cas9 mRNA, was co-injected into fertilized mouse zygotes to produce the desired knock-in offspring. Founder (F0) animals were identified through PCR and confirmed by sequence analysis. These F0 mice were then bred with wild-type mice to evaluate germline transmission and generate F1 offspring. Details of the gRNA sequences and PCR primers used for sequencing are provided in Supplementary Data 6. The homozygous and WT littermates used in this study were produced by breeding heterozygous parents. Mice were housed in plastic cages in specific pathogen free (SPF) grade animal rooms. They were kept under a 12h light/dark cycle-controlled atmosphere, with ambient temperature of 22 ± 2°C and relative humidity of 50 ± 10%. Standard chow and water were available ad libitum. All animal experimental and care procedures were performed in accordance with the guidelines of Animal Management Regulations of the Ministry of Health, China (Document No. 55, 2001) and were approved by the Nanjing Normal University Animal Faculty [IACUC- 20210233].

Reagents
The in vivo and in vitro experiments utilized hCG (Anhui Fengyuan Pharmaceutical Co., Ltd), recombinant human FSH (Gonal-f, Serono Pharma S.p.A., Bari, Italy) and recombinant human LH (Luveris, Serono Pharma S.p.A., Bari, Italy) were generously provided as gifts by Dr. Chen Li of Jinling Hospital (Nanjing, China). PMSG were purchased from Ningbo Second Hormone Factory (Ningbo, China). Chicken FSH and estradiol (JL15972) ELISA kit was purchased from Shanghai Jianglai Biotechnology Co., Ltd, China. Mouse and rat VEGF (YJ002076, YJ064294) and estradiol ELISA (YJ001962, YJ002871) kit were purchased from Shanghai Enzyme-linked Biotechnology Co., Ltd, China. LHR allosteric inhibitor LUF5771, and antagonist BAY-298 was synthesized by Yanshen Technology Co., Ltd., (Jilin, China) and WuXi AppTec Co., Ltd., (Wuxi, China), respectively. Progesterone antagonist RU486 (84371-65-3), LHR antagonist BAY-899 (2471967-92-5) were purchased from Sigma. FSHR antagonist hFSH-β-(33-53) (HY-P3343A) was obtained from MedChemExpress (MCE, Shanghai, China).

Phylogenetic Analysis of FSHR in Vertebrates
The FSHR coding sequences were downloaded from NCBI, translated into amino acid (aa) sequences, and aligned with MAFFT (v7.515) using default settings. A maximum-likelihood (ML) phylogenetic tree was constructed by IQ-TREE (v2.2.2.7) based on multiple sequence alignments with the options -m MFP -B 1000 --bnni -T AUTO. Positive selection amino acid positions were detected using a branch-site model of PAML (Phylogenetic Analysis by Maximum Likelihood) version 4.9. The estrildid branch was set as the foreground branch, and other species were set as the background branch. Specifically, we used Model A to test for positive selection on the foreground branch. This analysis compares the alternative hypothesis, in which sites on the foreground branch are allowed to evolve with ω > 1, against the null hypothesis, in which ω is fixed at 1. Sites under positive selection were identified using the Bayes Empirical Bayes (BEB) method with a posterior probability ≥ 0.8. For the FSHR coding sequences of E. gouldiae and P. oryzivora, we performed PCR and tblastn on the RNAseq de novo assembled using trinity.
To investigate the distribution of amino acid at the FSHR 449 sites across all species with genomes cataloged in the NCBI database, we utilized the Human FSHR protein sequence as a query. Subsequently, we performed both blastp and tblastn. From the blast results, we manually curated and confirmed the orthologous FSHR genes. Further, we conducted sequence alignments using the MAFFT (v7.515) software, extracting the corresponding amino acid sequences for alignment. The construction of the phylogenetic tree encompassing all species was carried out using the NCBI taxonomy database (https://www.ncbi.nlm.nih.gov/taxonomy/), and then visualized by iTOL (https://itol.embl.de/). All animal silhouettes were sourced from phylopic.org.

Prediction of N-linked Glycosylation Sites on FSH and LH
The amino-acid sequences of the target ligands (CGA, FSHB, and LHB) were used to generate three-dimensional structural models with the AlphaFold 3 server (https://alphafoldserver.com/), resulting in high-confidence protein structures. These models were subsequently analyzed using the GlycoShape online platform (https://glycoshape.org/). Following the methodology described in the associated article69, automated glycosylation analysis was performed using its core Re-Glyco algorithm. To cross-validate and complement the structure-based predictions, we additionally employed the sequence-based prediction tool NetNGlyc 1.0 (https://services.healthtech.dtu.dk/services/NetNGlyc-1.0/). Amino acid sequences in FASTA format were submitted to the NetNGlyc server using default parameters to independently assess potential N-glycosylation sites based on primary-sequence features.

Plasmids used for luciferase assay
Full-length FSHR (including point-mutations) and LHR of various species, G. gallus (gg)CGA, ggLHB and ggFSHB, were synthesized by General Bio Co., Ltd (Anhui, China) based on NCBI sequences, and then cloned into pcDNA3.1-V5/His vector with C-terminal V5-His tag. The serum response element (SRE), cAMP response element (CRE), T-lymphocyte activation of nuclear factor-responsive element (NFAT-RE) and serum response factor response element (SRF-RE) luciferase reporter plasmids were obtained from Promega.

Cell culture
HEK293 cells were obtained from Fuxiang biotechnology (Shanghai, China) and maintained in Dulbecco’s modified Eagle’s medium (DMEM, Thermo-Fisher, 11965092), supplemented with 10% fetal bovine serum (FBS, Gibco, A5256701), 1% penicillin-streptomycin (Gibco, 15140122), in a 37 °C incubator in 5% CO2 atmosphere.

Luciferase assay
HEK293 cells were placed into 24-well dishes and transfected with the indicated amounts of receptors together with 50 ng of CRE-luciferase reporter plasmid using lipofectamine 6000 regent (Beyotime, C0526, Shanghai, China). Six hours after transfection, the medium was replaced with fresh serum-containing medium and the constitutive activity was detected using the assay kit (Beyotime, RG005, Shanghai, China) 48 hours after transfection by an EnVision™ Multilabel Plate Reader (PerkinElmer, USA). CRE, NFAT-RE, SRF-RE and SRE were utilized for testing Gs, Gq, G12 and potential Gi pathway respectively.
For ligand stimulation (hCG, hLH and hFSH), the culture medium was replaced with serum-free medium 36 hours after transfection and, at the same time, cells were given the corresponding doses of ligands. Luciferase activity was measured 12 hours after ligand stimulation.
For ggFSH and ggLH stimulation assays, HEK293 cells were seeded into 48-well plates and co-transfected in serum-free medium using a lipofection reagent (Yeasen, 40802ES03, Shanghai, China). Equal amounts (50 ng each) of receptor plasmid and cAMP response element (CRE)-luciferase reporter plasmid were used, with the empty vector pcDNA3.1 serving as a negative control. Six hours after transfection, the medium was replaced with fresh serum-containing medium, and the cells were further cultured. At 24 hours post-transfection, ggFSHB/ggLHB and ggCGA plasmids were co-transfected at a 1:1 ratio in serum-free medium at gradient concentrations (2, 5, 10, 20, 50, 100, and 200 ng) to assemble and stimulate the receptors. After 6 hours, the medium was once more replaced with fresh medium, and the cells were cultured for an additional 18 hours. Cells were then collected, and the supernatant was discarded. Luciferase activity was measured using a luciferase assay kit (Beyotime, RG005, Shanghai, China). All experiments were performed independently at least three times.

cAMP detection
For in vitro studies, HEK293 cells were seeded into 24-well plates and transfected with indicated amount of plasmid. After 48 hours of transfection, the culture medium was changed to 200 μl serum-free medium and incubated for 30 min, followed by washing with 100 μl of PBS and digestion with 50 μl trypsin. Cells were resuspended in serum-free medium containing 0.3% BSA and 3-isobutyl-1-methylxanthine (IBMX, MCE, HY-12318, Shanghai, China), and 5 μl of cell suspension transferred to 384-well plate for analysis.
For in vivo studies, 0.1 g of ovary tissue was homogenized on ice in RIPA buffer containing IBMX, followed by centrifugation at 4°C, 4700 ×g for 30 seconds. The supernatant was placed on ice for 5 min, followed by centrifugation at 4°C, 20100 ×g for 10 min. Five μl of the supernatant was taken for analysis in a 384-well plate. The detection procedure was done in accordance with the instructions of the cAMP Gs dynamic kit (CSBIO, 62AM4PEB).

Rat OHSS induction and pharmacological intervention
All animal protocols were approved by the committee on the Ethics of Animal Experiments at Nanjing Normal University. To induce OHSS, three-week-old female Wistar rats were housed under a 12-hour light/dark cycle at 22 °C with free access to food and water. The OHSS group was treated with daily intraperitoneal injections of 50 IU PMSG for four consecutive days, followed by a single injection of 20 IU hCG on the fifth day. The test compounds (BAY-298 and BAY-899) were dissolved in a vehicle consisting of 15% DMSO, 15% Tween-80, and 70% saline, and administered intraperitoneally at doses of 4.5 mg/kg and 14 mg/kg, respectively. Drug treatment began 24 hours after hCG injection and continued for two consecutive days. Rats receiving an equal volume of the vehicle alone served as the solvent control group. An additional conventional superovulation group (non-OHSS control) received a single intraperitoneal injection of 10 IU PMSG, followed 48 hours later by 10 IU hCG. All rats were humanely euthanized by decapitation under isoflurane anesthesia 24 hours after the last drug or solvent administration.

Mouse OHSS induction and pharmacological intervention
The OHSS model in C57BL/6 mice was established using a protocol similar to that for rats. Five-week-old female C57BL/6 mice were used. The OHSS group received daily intraperitoneal injections of 20 IU PMSG for four consecutive days, followed by a single injection of 5 IU hCG on the fifth day. For adult (8-week-old) female mice, the induction protocol was modified by increasing the PMSG dose to 30 IU per day to account for increased body weight, while the hCG dose remained unchanged at 5 IU. LUF5771 and RU486 were dissolved in a vehicle consisting of 15% DMSO, 15% Tween-80, and 70% saline, and administered intraperitoneally at doses of 20 mg/kg. Drug treatment began 24 hours after hCG injection and continued for two consecutive days. Mice receiving an equal volume of the vehicle alone served as the solvent control group. An additional conventional superovulation group (non-OHSS control) received a single intraperitoneal injection of 5 IU PMSG, followed 48 hours later by 5 IU hCG70. All mice were humanely euthanized by decapitation under isoflurane anesthesia 24 hours after the last drug or solvent administration.
For FSHRT449A-KI mice, which have an elevated risk of developing OHSS due to the point mutation, the hormone dosages were adjusted accordingly. The OHSS group (five-week-old) was injected with 20 IU of PMSG for four consecutive days, followed by a single injection of 1 IU hCG on the fifth day. For adult (8-week-old) female mice, the induction protocol was modified by increasing the PMSG dose to 30 IU per day, while the hCG dose remained unchanged at 1 IU. LUF5771 and hFSH-β-(33-53) were administered intraperitoneally at doses of 20 mg/kg. Drug treatment began 24 hours after hCG injection and continued for two consecutive days. Mice receiving an equal volume of the vehicle alone served as the solvent control group. An additional conventional superovulation group (non-OHSS control) received a single intraperitoneal injection of 5 IU PMSG, followed 48 hours later by 1 IU hCG. All mice were humanely euthanized by decapitation under isoflurane anesthesia 24 hours after the last drug or solvent administration.

Avian ovarian hyperstimulation induction
Adult female birds were intraperitoneally injected with PMSG at a dose of 20 IU for four consecutive days, followed by a single intraperitoneal injection of 5 IU hCG or hFSH on the fifth day to induce OHSS. The control birds were treated with 5 IU PMSG once and intraperitoneally injected with 5 IU hCG or FSH 48 hours later. All birds were humanely euthanized by decapitation under isoflurane anesthesia 24 hours after hCG administration.

Hematoxylin-eosin staining
The ovaries were fixed overnight in 4% paraformaldehyde (PFA) and subsequently embedded in paraffin. Serial sections (5 μm in thickness) were obtained from the largest cross-sectional region, yielding nine consecutive sections per ovary. The sections were stained with haematoxylin and eosin (H&E) for morphological assessment and quantification of corpora lutea. For corpus luteum counting, the nine consecutive H&E-stained sections were analysed as a continuous series. Each section was examined in reference to its preceding and succeeding section to trace the structural continuity of individual corpora lutea and to prevent redundant counting. The average number of corpora lutea per ovary was used for comparison among different treatment groups.

ELISA
For hormone detection, blood was collected and centrifuged at 1260 ×g for 10 min. Serum concentrations of LH, FSH, and estradiol were determined using the enzyme-linked immunosorbent assay (ELISA) following the instructions of the suppliers.
For total FSHR protein expression detection, HEK293 cells were placed into 24-wells dishes and transfected with the indicated amounts of receptors using lipofectamine 6000 reagent. The supernatant was removed 48 h after transfection and the cells fixed with 150 μl of 4% PFA for 15 min, and then treated with 150 μl of 0.1% Triton-X-100 for 15 min. After PBS washing, cells were blocked with 1% BSA for 1 hour, incubated with V5 antibody (CST, 13202S, 1:1000 dilution, USA) for 2 hours and then with HRP- conjugated secondary antibody (CST, 7076, 1:1000 dilution, USA) for 1 hour, and finally 200 μl of TMB (Beyotime, ST746, Shanghai, China) were added. After incubation for 30 min at room temperature, the absorbance value was measured at 370 nm using BioTek Synergy H1.

Real-time PCR (qRT-PCR)
Total RNA was extracted using TRIzon reagent (CWBIO, CW0580S, Beijing, China) and 1 μg RNA was reverse-transcribed into cDNA using HiScript IV All-in-One Ultra RT SuperMix for qPCR (Vazyme, R433-01, Nanjing, China) as per manufacturer's instructions. For real-time PCR, cDNAs were amplified using SYBR Green mixture (Vazyme, Q312-02, Nanjing, China), and each primer pair was amplified in an ABI 7500 real-time PCR system (Applied Biosciences). Results were semi-quantified with GAPDH gene expression levels as an internal reference. Primers used in this study are shown in Supplementary Data 6.

Single-cell capture, library construction and next generation sequencing
For avian ovaries, scRNA‑seq samples were processed as single organs (one ovary per bird). The T. guttata (tg) and S. canaria (sc) groups each contain n=3 biological replicates, while the L. striata domestica (ls) group contains n=2 biological replicates. Due to the limited cell yield from a single mouse ovary, ovaries were pooled prior to single‑cell suspension preparation. Specifically, the OHSS group consisted of n = 1 biological replicate, generated by pooling both ovaries from one mouse. The LUF5771‑treated and hFSH‑β‑(33-53)-treated groups each contained n = 2 biological replicates. Each replicate was prepared by pooling both ovaries from each of two mice, resulting in a total of four ovaries per replicate. All animal ovaries were dissected and trimmed of excess impurities in culture dishes containing DPBS (Gibco, 14287080). The digestion protocol was similar in all species. The single-cell suspension of ovaries was generated by 2 mg/ml collagenase Type II/IV (1:1) digestion for 30 min at 37 °C. The supernatant was removed by centrifugation at 11300 ×g for 10 seconds at 4°C and immediately placed on ice, then 0.04% BSA (Sigma-Aldrich, 9048-46-8)-DPBS was added for resuspension. Finally, the single-cell suspension was stained with trypan blue to assess cell number and viability (>80%) and used to construct scRNA-seq libraries with the Chromium Single Cell 3ʹ Gel Bead-in-Emulsion (GEM), Library & Gel Bead Kit v3 (10× Genomics, Cat:1000121), according to the manufacturer’s instructions as briefly described: The single-cell suspension was loaded onto a 10× Chromium system (10× Genomics). Following gel bead-in-emulsion generation, cDNA synthesis proceeded using the Single Cell 3’ Reagent Kit v3 (10× Genomics). The cDNA libraries were PCR amplified followed by fragmenting and sequencing on the Illumina NovaSeq 6000 platform.

Processing of scRNA-seq data
CellRanger software (v7.0.1) was used to process, align, and summarize unique molecular identifier (UMI) counts against bTaeGut1.4 (T. guttata), cibio_Scana_2019 (S. canaria), lonStrDom2 (L. striata domestica) and GRCm39 (M. musculus). Ovary libraries were processed with SoupX (v0.3.1)71 to remove ambient RNA. Filtered UMI count matrices were imported into R for downstream analysis. To obtain high-quality data for all species, different quality control parameters have been set: S. canaria (sc), cells with over 20% UMIs of mitochondrial RNA, below 500 total UMIs and above 4000 total UMIs were removed; T. guttata (tg): cells with over 20% UMIs of mitochondrial RNA, below 200 total UMIs and above 7000 total UMIs were removed; L. striata domestica (ls): cells with over 10% UMIs of mitochondrial RNA, below 200 total UMIs and above 7000 total UMIs were removed; M. musculus (mm): cells with over 15% UMIs of mitochondrial RNA, below 200 total UMIs and above 7500 total UMIs were removed. Variable features were identified in Seurat before scaling data and running PCA. Gene counts were normalized using the NormalizeData method and 2000 highly variable genes were selected for downstream analysis. We applied the Canonical Correlation Analysis (CCA) method for dataset integration to correct batch effects. The union of the top 2000 genes in each dataset with the highest dispersion was taken, and anchors were determined using the FindIntegrationAnchors function. Then we applied the IntegrateData function to generate an integrated expression matrix. We scaled the data and applied the RunPCA function for dimensional reduction. Cells were clustered using the official Seurat pipeline and visualized using uniform manifold approximation and projection (UMAP). Cluster markers were found using FindAllMarkers and FindMarkers with the Wilcoxon rank sum test.
For the re-clustering of ovarian granulosa and theca cells, respective cell populations were extracted from corresponding clusters across the OHSS, LUF5771-, and hFSH-β-(33-53)-treated groups. Datasets were grouped by condition, re-normalized, and integrated using the FindIntegrationAnchors and IntegrateData functions in Seurat. Following integration, cells were re-clustered and visualized in UMAP using the top 2000 highly variable genes and the first 20 principal components. Positively expressed differentially expressed genes (DEGs) for each cluster were identified with the FindAllMarkers function. Pathway activity for estrogen response and angiogenesis was scored and visualized using the AddModuleScore function from Seurat, with gene sets obtained via the R package GSEABase (v.1.68) and referenced against the mouse database.

Bulk RNA-seq and analysis
The avian ovaries were processed and analysed as single organs (one ovary per bird). No pooling was performed. For the comparative analysis presented in Supplementary Figure 16, each group contains n = 3 biological replicates. Total RNA was extracted from each sample using RNAiso Plus reagent according to the manufacturer’s instructions. We estimated the integrity and quality of the total RNA using a Bioanalyzer 2100 system (Agilent Technologies, Palo Alto, CA, USA) and an RNA 6000 Nano kit. Sixty poly (A) RNA-seq libraries were constructed and sequenced using the BGISEQ DNBSEQ-T7C platform (BGI lnc., Shenzhen, China) with a paired-end sequencing length of 150 bp (PE150) at Novogene Bioinformatics Technology Co., Ltd (Beijing, China). The quality of the raw data was determined using the FastQC software72 and the adapters were trimmed using Trimmomatic73. The clean reads were aligned to corresponding reference genomes from Ensembl database (tg: bTaeGut1_v1; ls: LonStrDom1; sc: SCA1.107; gg: GRCg7b) by HISAT2 (v2.2.1) with default parameters by HISAT2 (v2.2.1)74. SAMTools was used to sort the bam files of the aligned reads74. The transcripts were assembled by StringTie (v1.3.6) with parameters to assess gene expression based on the TPM (Transcripts Per Million) values of each mRNA75.
To compare transcription rates across species, we downloaded the homologous gene lists among three species from Ensemble BioMart (http://www.ensembl.org/biomart/martview/8031c97af5aea7bfb70ce3f47e39e344). As a Lonchura striata domestica dataset was not available in Ensembl Biomart, we performed reciprocal blast of the two proteomes (T. guttata and L. striata domestica) to identify orthologous genes. We combined the data with the homologous gene lists from Ensembl Biomart to form a one-to-one homologous gene set shared by the four bird species. The DEGs based on the read count data from these orthologous gene pairs were identified using DESeq2 package76. The significant DEGs were screened with a false discovery rate <0.05 and |log2 fold change|>1 as cutoffs. To investigate the expression patterns of DEGs across species, we used transcripts per million 10K (TPM10K), a metric which normalizes TPM to account for different sequencing depths among species77.
For the RNA-seq de novo assembly, we used trimmomatic filters raw readings to remove adapters and low-quality sequences. The de novo assembly was performed by Trinity (v2.15.1) using the default parameters. Then BLAST software (v2.13.0+) was used to generate P. oryzivora FSHR sequences from the assembly results as a database, and L. striata domestica FSHR sequence as a reference.
Functional enrichment analyses were performed using Metascape (http://metascape.org) with default parameters78. All genes were converted to human orthologs by Ensembl Biomart, and the target gene lists were uploaded as inputs for enrichment. We chose H. sapiens as the target species, and enrichment analysis was performed against all genes in the genome as the background set, with the biological process (BP) of Gene Ontology (GO) as the functional test set. Only GO terms with a P value < 0.01 and annotated to ≥3 genes were considered significant.

Statistics
All experiments were repeated independently at least three times. The data were analyzed using GraphPad Prism 8.3 and represented as the mean ± SD from at least three independent experiments, performed in triplicates. No statistical method was used to predetermine sample size. No data were excluded from the analyses. The experiments were randomized. The investigators were not blinded to allocation during experiments and outcome assessment. Differences between the two groups were compared using two-tailed Student's t-test. Comparisons between multiple groups were performed using one-way ANOVA. Statistical significance was defined as a p < 0.05.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript entitled ‘Physiological Adaptation of Estrildid Ovaries Illuminates the Molecular Physiology and Therapeutic Strategies for Ovarian Hyperstimulation Syndrome (OHSS)’ describes the evolutionary adaptation of estrildid finches for a FSHR variant with high basal activity.\nThe work elegantly breaks down the question using a variety of methods, including in vivo experiments in birds and mice, and molecular analyses of FSHRs of different species, the use of inhibitors and scRNA-analyses.\nThe MS is well-written and illustrated, as well as logically explained.\nI have a few queries.\nThe first is the title. While this is the authors’ choice, it is too ‘anthropocentric’ as OHSS is a human complication that occurs usually during hyperstimulation or, in very rare cases, due to the presence of a FSHR mutations – mostly promiscuous the hCG. As hCG and pregnancy do not occur in birds, this ‘humanisation’ of their physiology is questionable. While the authors should discuss this issue, one should be carefully to miss the forest by the trees.\nOne of the big questions that remains open is whether the ligands (Fsh and Lh) are also different (evolved somehow) in estrildid finches. This should be analysed at the sequence level (amino acids) and, if such information exists, glycosylation level. This is important as it has been shown that in other species, such as horses (equid family), the chorionic LH – also known as PMSG or equid CG – is LH with different glycosylation pattern than pituitary LH. This PMSG is able to activate LHR and FSHR in most mammals but not in equids where it remains specific to LHR. The authors do show some experiments using chicken (Gallus, gg) hormones and the results are clearly different than using human LH/hCG or FSH. In Fig Suppl 7, the human LH versus the chicken (gg) LH result in very different responses. While response to hLH is relatively low in both FSHR versions, when ggLH is used the sensitivity of avian receptors increases dramatically. Further sensitivity by the FSHR449A. This suggests that the receptor-ligand evolved hand-to-hand.\nThe ligand selectivity the authors claim to be reduced in the 449A variant, is not really clear for hLH and hFSH stimulations. The hCG stimulation (Fig 1e) is questionable as it is artificial (no such hormone exists in birds). Why this has not been done with ggLh? And ggFsh?\nIn fact, Fig 1e shows that the response to hCG in tg, ls and gg (and likely vu and rn) is almost identical (once removing the basal level) whether it is FSHR449A or FSHR449T.\nLine 125 - In contrast, avian species (such as the Taeniopygia guttata, Lonchura striata domestica, Gallus gallus, and Serinus canaria) demonstrated weaker specificity for these ligands, with FSH able to non-specifically activate LHR (Supplementary Fig. 8b).\nThis is not completely true in the sense that the human ligands might have more promiscuous effect than the avian (or species-specific) ligands.\nAlso, as the authors correctly state, in (some) fish such as zebrafish, the promiscuous activities of LHR and FSHR have been carefully dissected. This is something that should be discussed in more detail.\nFigure 1c (and there forth): the receptor presented is a good representation of GPCR but not FSHR (and later LHR) as it is missing the large extracellular domain\nLine 128 - The T449A variation increased FSHR sensitivity to hCG, consistent with previous findings20 (Fig. 1e).\nThis needs clarification. Is this the human or mammalian or avian or all?\nLine 132 - Furthermore, the differential sensitivity of the 449T and 449A variant receptors to FSH varied among vertebrates.\nE.g., in the estrildid birds, Taeniopygia guttata and Lonchura striata domestica, the T449A mutation enhanced the stimulation by human FSH (Supplementary Fig. 6). This indicated that T449A enhanced the response of FSHR to LHR ligands and thus broadened its ligand range.\nThis shows higher response to Fsh in Taeniopygia guttata and Lonchura striata domestica but nothing about LH and hCG!\nAlso, the activation of Fsh in Lonchura striata is identical to FSHR449T if one deduces the baseline. In Taeniopygia guttata the difference is only at high doses in fact.\nSuppl Fig c – the expression of FSHR-449A version is expressed less than the 449T version.\nThis is obviously just speculation as it is done in HEK cells but this could also explain that even at higher basal level, having a lower expression of Fshr would also result in low(er) baseline – close to what we know in other species. Since the authors have performed scRNA-Seq, then they could assess the expression of FSHR in different species – as compared to several other genes.\nAs LHR is diminished in estrildids birds, so seem to be the genes downstream of LHR, including those involved in steroidogenesis. Suppl Fig 16 shows that there is a reduction in ‘hormone levels’ as well as in ‘reproductive structure development’ what is a bit unclear what it means.\nA bit more discussion in the interplay of FSHR and LHR during the ovulation cycle would be helpful. These 2 receptors are expressed, at least in mammals, not always at the exact same time (at least in number of receptors) and their co-expression in the same cell type changes the activation duration. One both wonders whether such thing occurs in birds which have also a different regulation of their ovaries. As far as this reviewer knows, most birds use a single ovary per cycle – meaning that the other one is quiescent somehow. Some birds have strict breeding seasons, slightly unlike mice or humans that their cycles are regular. This suggest that avian ovaries are more regulated than mammalian ones. This should be discussed as well.\nThere is some evidence in Suppl Fig14 that genes downstream of the LHR such as Cebpd respond less to the overstimulation of PMSG and hCG. While FSH/LH downstream genes such as Vegf4 are still responding to the stimulation.\nThe other option is that the G proteins responsible for downstream signalling are ‘less responsive’.\nThis, together with the shown reduction in Lhr expression in estrildid finches, could explain the evolutionary adaptation.\nObviously, one option is that the entire gene expression system downstream receptor activation, AKA the transcriptional activators and/or promoters of downstream genes, are adapted to an overreactive Fshr.\nFinally, I suggest to ‘tune down’ the statements to a more open question style. While a good part of the evidence suggests that one of the mechanisms of action of the reduction of LHR, for example, many other unknown mechanisms might play a role as well, in some case even more important. Thus, the discussion shall contain other possibilities unexplored in this pioneering work.\nIn Fig 2, it is not defined whether the hormone (Fsh) is human – it is assumed it is as there is also hCG treatment but using the same abbreviations would be helpful (rhFSH for example)", "reviewer_2": "In this study, the authors describe a range of analyses to investigate the activity of follicle-stimulating hormone receptor and its effects in estrildid finches. My review of this submission is limited to the taxonomic and phylogenetic aspects of the study.\nLine 78. Italic font should be used only for genus and species names, but not names of higher taxa (such as the families Estrildidae, Anostomidae, and Mormyridae).\nLine 78. The songbird suborder is Passeri, not Passeridae. Note that higher taxon names should be in roman font and not italic font.\nLine 79. Common names of birds should either be capitalised (e.g., “Zebra Finch”, “Gouldian Finch”) or have capital letters only for proper nouns (e.g., “zebra finch”, “Gouldian finch”).\nLine 727. Please provide more detail about the branch-site models that were used in PAML, as there are about 8 different models implemented in this program. The specific models are listed in Figure 1b but require further explanation. Given the importance of these results, I would also be more assured if additional methods were used to test for selection and to identify the codons under selection.\nLine 736. The phylogenetic tree is not really constructed in this approach. The NCBI taxonomy database simply draws a tree based on the taxonomic classification of the chosen species. This is adequate for the present study, but the authors should check that the taxonomic classifications are consistent with the latest (genomic) estimates of the phylogenetic relationships. In particular, the relationships among the included bird species should be examined more carefully, and perhaps some of the lineages (Passeriformes, Passeri, and Estrildidae) should be labeled to aid interpretation.", "reviewer_3": "Review of Manuscript: Physiological Adaptation of Estralid Ovaries Illuminates the Molecular Physiology and Therapeutic Strategies for Ovarian Hyperstimulation Syndrome (OHSS)\nGeneral Comments\nThe manuscript addresses an important topic in ovarian biology and OHSS. However, the overall presentation lacks clarity regarding the target audience, rationale, and clinical relevance. As currently written, it is unclear who the intended readership is: basic biomedical researchers, comparative biologists, evolutionary biologists, reproductive clinicians, or all. If the goal is to appeal to all groups, greater clarity and detail is needed regarding the ovarian biology, clinical significance of the work and its applicability to OHSS patients.\nThe authors aim to make a case for comparative biology and evolution to solve medical problems (i.e. Darwinian medicine). However, this case is not strongly or compellingly presented. The gain-of-function mutation had already been identified in some women with OHSS, meaning that the outcomes of this study (confirmation of the role of the mutation in OHSS and testing of potential therapeutics) could have been reached without the comparative biology approach. While the science is interesting, the translational argument is weak.\nSpecific Comments\n1. Introduction\n• The introduction is superficial and does not clearly define the cell biological, clinical or scientific gap being addressed.\n• The authors need to provide a clear description of normal antral follicle development and the physiological roles of FSH and LH in the ovary, including their functions in follicle selection, antral follicle growth, survival, maturation, and ovulation. This should be linked to their known roles in the pathogenesis of OHSS. In addition, the manuscript should outline the standard ovarian stimulation protocols used clinically and explain how these can trigger OHSS, to provide essential context for interpreting the study.\n• It is unclear whether the work applies to all OHSS patients or only those with FSHR mutations. The manuscript should specify how broadly relevant these findings are. HOw many women have these mutations? How often does OHSS occur?\n• Second paragraph (line 47): The relevance of FSH to primordial follicles is not explained in the context of OHSS. Primordial follicles in humans and mice are gonadotropin-independent. The species in the study cited should be clarified, and differences between multi-ovular mice and mono-ovular humans should be made clear.\n• Line 64: “Small follicles” what are they?\n• The paragraph beginning at line 67 is vague and unclear; the main point should be explicitly stated.\n2. Mouse Model Validity and Relevance\n• The knock-in mouse model demonstrates only slight sensitivity to supraphysiologic hormone doses. Normal hormone stimulation did not induce an OHSS phenotype; only extreme conditions caused some OHSS markers to increase. The clinical relevance of this model is therefore questionable.\n• The model would be more meaningful if standard stimulation protocols were sufficient to induce OHSS in the mutated mice.\n3. Figures\nFigure 2\n• Histological images of ovaries are too small. Larger, clearer images are required in the main figure.\n• Ovarian enlargement should be quantified (e.g., number of antral follicles, corpora lutea, cysts).\n• Finch ovarian histology should also be included in the main figure.\n• Ovarian weight normalized to body weight can be moved to supplemental information. Raw weight should remain in the main figure.\nFigure 4\n• CL counts in controls are unusually low. The methodology for counting CLs should be described and accuracy of counting method fully validated.\n• Other treatment groups are missing from Figure 4D.\n• Fluid accumulation, an important OHSS hallmark, should be reported, particularly in the T-A variant model.\nFigure 5\n• The value of scRNAseq analysis (Fig. 5i–r) is unclear; it largely confirms expected overlap in downstream signaling of LHR and FSHR.\n• The assignment of the proportions of cell populations is uncertain. It seems highly unlikely that the proportion of immune cells in the ovary would be similar to granulosa cells (as shown in Fig. 5K, hFSH group), even after an inflammatory event. Even after macrophage/T-cell infiltration or expansion, they should not make up a third of the ovarian cellular composition.\n• GAPDH is used as a housekeeping gene. Did the authors test multiple housekeeping genes and confirm GAPDH stability across treatments? GAPDH is often an unreliable housekeeper in ovarian tissue (it changes with treatments).\n4. Drugs and Methods\n• LUF5771 can partially activate LHR; this should be acknowledged.\n• The rationale for changing the OHSS-inducing hormone regimen between Figures 2B and 4A is not explained.\n• The use of Bay298 and Bay899 in rats is unclear. Can these compounds be used in mice, and what justifies the species change?\n• Catalogue numbers should be provided for all kits and reagents.\n• Five-week-old female mice were used for the OHSS model; these animals are sexually immature. The study should have been conducted in sexually mature mice.\n• Methods are generally okay but it is very difficult to determine which methods apply to which experiments. Greater detail is required to clarify exactly what was done in each experiment.\n5. Discussion\n• Much of the discussion simply repeats results without adding new insight or clinical context. More emphasis is needed on the significance, limitations, and potential translational relevance of the findings. As it is written, the discussion adds little to the manuscript.\n• In addition, a lot of the background information included in the discussion (lines 300–341) belongs in the introduction.\nSummary\nThe manuscript addresses an important and clinically relevant topic, but, there are significant deficiencies. The rationale and overall approach lack clarity, and essential background on ovarian biology, gonadotropin function, and the clinical features and mechanisms of OHSS is missing. Without these details, it is difficult to place the findings in context or evaluate their broader significance."}, "author_response": ""}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have addressed all my queries.\nAt most the drawings of the FSHR and LHR require a little better extracellular domains *ECD) (see attached image) as these are essential for hormone binding. The ECD is nicely organised not like the one the authors have generated. It would be a shame to have so beautiful figures as they have provided and have a deformed LHR/FSHR.", "reviewer_2": "The authors have addressed most of the comments in my previous review.\nI have one remaining comment about the phylogenetic tree in Figure 1. The caption mentions that the tree was constructed using TimeTree, but this is not mentioned in the main text (which instead mentions that the tree is based on the NCBI Taxonomy Database). Please clarify the approach used to generate the tree in Figure 1. TimeTree can take a list of species and produce a time-scaled phylogeny, so it is unclear whether the tree is based on this approach.", "reviewer_3": "I thank the authors for addressing all of my comments. Could the authors please clarify in the methods section if ovaries were pooled for bulk and scRNA seq, or if they were processed and analysed as single organs, and how many ovaries were analysed along with technical/biological replicates.", "reviewer_4": "In this manuscript, the authors combine evolutionary analysis, cross-species scRNA-seq, genetic mouse models and pharmacological perturbations to investigate functional compensation between FSHR and LHR in the context of OHSS. The comments below focus specifically on the scRNA-seq analyses, which form a central component of the mechanistic argument.\nThe authors state that “Among all cell types, granulosa cells from T. guttata and L. striata domestica shared the most differentially transcribed genes.” it will be helpful to include plots or summaries to explicitly support this statement.\nThe figures referenced with around “Since FSHR is primarily expressed in the granulosa cells of all three species, we conducted a comparative analysis of the granulosa cells across species” are confusing as they don’t specifically compare granulosa cells across species.\nThe following claim is primarily supported by qualitative feature plots “scRNA seq revealed that LHR was highly expressed in the granulosa and theca cells of G.s gallus but was nearly undetectable in the granulosa cells of the T. guttata and L. striata domestica (Fig. 3k)”. Complementary quantitative representations and statistical tests (e.g., violin plots) would strengthen this claim.\nIn Fig. 3g, it is unclear whether the GO enrichment reflects tg vs sc, ls vs sc, or a combined estrildid vs non-estrildid comparison. Clarifying this comparison would aid interpretation.\nGiven the focus on OHSS, it may be informative to score OHSS-related pathway signatures (e.g., angiogenesis, vascular permeability, estrogen signaling) across conditions or species, rather than relying primarily on individual genes.\nThe avian scRNA-seq data are collected under steady-state physiological conditions, rather than under stimulation. The inferred compensatory mechanisms likely reflect baseline adaptations rather than inducible responses. The authors could more explicitly acknowledge this distinction.\nThe authors report remarkable cell composition changes between OHSS and agonist treated groups in the mouse scRNA-seq data. It would be helpful to clarify whether the reported DEGs are influenced by shifts in cell states with the major cell types. For example, the authors could subset to only Granulosa/Theca cells and re-cluster to assess emergence or suppression of specific cell states.\nAs a supplementary analysis, CellChat/NicheNet could be used to test whether OHSS induces, and LHR antagonism suppresses, granulosa/theca to endothelial signaling associated with vascular permeability."}, "author_response": "A: We thank the reviewer for this insightful comment. We have revised the title to: “Functional compensation between FSHR and LHR illuminates the molecular mechanism and treatment of Ovarian Hyperstimulation Syndrome (OHSS)”. This revised title more accurately reflects the focus of our study: it emphasizes the mechanistic discovery of FSHR/LHR functional compensation, while situating OHSS in its appropriate human clinical context. Importantly, our work uses avian models to reveal conserved molecular principles underlying gonadotropin receptor function, without implying that avian physiology directly models human OHSS.\n\nA: We thank the reviewer for raising this important question regarding potential co-evolution of the ligands. To address this, we performed a comprehensive sequence analysis of glycoprotein hormone alpha subunit (CGA), follicle-stimulating hormone beta subunit (FSHB), and luteinizing hormone beta subunit (LHB) in estrildid finches. Maximum-likelihood phylogenetic trees were constructed using IQ-TREE (v2.2.2.7) with the parameters “-m MFP -B 1000 --bnni -T AUTO” based on multiple sequence alignments. Positive selection was assessed using a branch-site model in PAML (v4.9), designating the estrildid finch lineage as the foreground branch and other species as the background. These analyses show no significant evidence of positive selection in the ligand (see Response figure 1).\nWe further analyzed potential glycosylation differences by predicting N-glycosylation sites across vertebrate sequences, and found no notable changes in glycosylation patterns or sites in estrildid finch ligands (Supplementary figure 21, experimental methods described in the revised manuscript, lines 764-773). Together, these results indicate that, in contrast to FSHR, the FSH and LH ligands have remained largely conserved in estrildid finches. This analysis has now been incorporated into the Discussion (line 366-371) and Supplementary figure 21.\n\nA: We agree that testing native ligands is important. As shown in Supplementary figures 6b and 7b (experimental methods described in the revised manuscript, lines 800–810), we conducted key experiments using ggLH and ggFSH. These results are consistent with those obtained using human hormones: the T449A mutation enhances the receptor’s responsiveness to ggFSH (Supplementary figure 6b). Although wild-type avian FSHR (449T) is not activated by ggLH, the T449A variant exhibits substantial responsiveness to this ligand (Supplementary figure 7b). These experiments using native avian ligands directly demonstrate that the T449A mutation reduces FSHR ligand selectivity, supporting our conclusion regarding the functional impact of this variant.\n\nA: We thank the reviewer for this observation. It is correct that the hCG response curves in tg, ls, and gg FSHR appear similar in shape. However, the key functional difference lies in the markedly elevated basal (constitutive) activity of the FSHRT449A variants compared with FSHRT449T (Figure. 1d). Although ligand-induced curves may appear parallel after baseline subtraction, the receptor’s total signaling output combines both constitutive and ligand-stimulated activity. The T449A mutation substantially increases constitutive activity across species, thereby augmenting overall signaling capacity. This concept-that constitutive receptor activity is biologically meaningful is supported by recent study published in Nature (PMID: 40031956), which demonstrated that a naturally occurring constitutively active GCGR mutation in birds contributes to physiological regulation of blood glucose.\n\nA: We thank the reviewer for highlighting this potential ambiguity. The intended interpretation is that the reduced ligand specificity is inherent to the avian system, rather than reflecting greater promiscuity of human ligands. To clarify, we have revised the sentence (revised MS, Lines 138-141) as follows:“In contrast, receptor-ligand specificity was markedly weaker in birds. While mammalian systems maintain strict FSH-FSHR and LH-LHR pairing, we found that in several avian species (such as T. guttata, L. striata domestica, G. gallus, and S. canaria), FSH can cross-activate LHR.” This revision removes any ambiguity and accurately reflects our observation of inherent receptor-ligand promiscuity in birds.\n\nA: We thank the reviewer for this valuable suggestion. In response, we have expanded the Discussion (lines 350-361) to include a more detailed treatment of the promiscuous activities of LHR and FSHR in fish. We integrated key references demonstrating ligand-receptor cross-activation, overlapping expression patterns, and functional compensation between these hormonal pathways in species such as goldfish, tilapia, and salmon. These evolutionary parallels in fish provide additional support for our hypothesis that the FSHR-T449A substitution in estrildid finches increases receptor promiscuity.\n\nA: In accordance with the reviewer’s suggestion, we have revised the schematics of FSHR and LHR in Figures 1C and 6 to include the N-terminal extracellular domains. These updated diagrams now more accurately represent the structural features of FSHR and LHR, while maintaining the clarity of the overall receptor depiction.\n\nA: Our results demonstrate that the T449A variation increases FSHR sensitivity to hCG in both mammalian and avian species. Accordingly, we have clarified the sentence in the revised manuscript (Line 143-144) to: \"The T449A variation increased FSHR sensitivity to hCG in both mammalian and avian species, consistent with previous findings.\"\n\nA: Regarding the statement \"This indicated that T449A enhanced the response of FSHR to LHR ligands and thus broadened its ligand range,\" we agree that its original placement was suboptimal. We have reorganized this section of the text to improve the logical flow and have moved this concluding statement to a more appropriate position (MS, line 147-148).\nConcerning the response to FSH in Lonchura striata domestica, we have now performed additional experiments using the native avian ligand, Chicken FSH (ggFSH). The new data presented in Supplementary figure 6b clearly show that the 449A variant exhibits a stronger response to ggFSH stimulation compared to the 449T variant in these species, even after baseline deduction. This confirms that the T449A mutation enhances the receptor's sensitivity to its cognate ligand, FSH, in estrildid finches. (Supplementary figure 6b).\n\nA: In Suppl Fig 4c and d, we intend to demonstrate that physiological levels of FSHR expression are sufficient to elicit constitutive activity and to compare basal signaling across avian species at near‑native expression levels.\nWe thank the reviewer for raising this interesting possibility. To directly address your question, we analyzed our scRNA-seq data to compare FSHR expression across species. As shown in Response figure 2a, FSHR expression in granulosa cells of estrildid finches was higher than in canaries. At the whole-ovary level, FSHR expression did not differ significantly between estrildid and non-estrildid birds, with comparable levels observed in species such as Serinus canaria and Lonchura striata (Response figure 2b). These scRNA-seq results indicate that the FSHR-449A variant is not expressed at lower levels than FSHR-449T.\nThe apparent discrepancy between the HEK293 experiments and the scRNA‑seq results may stem from technical and biological variables, including cell‑type specificity, age, seasonal factors, and experimental systems. Importantly, the functional differences we report are more likely attributable to intrinsic signaling properties of the T449A variant rather than to differential expression. This conclusion is strengthened by the primary ovarian cell data, which extend beyond the heterologous HEK293 system.\n\nA：In bulk ovarian transcriptome analysis, differentially expressed genes (DEGs) in estrildid finches were enriched in the “regulation of hormone levels” and “reproductive structure development” pathways. This differs from the single-cell analysis, where DEGs were derived specifically from granulosa cells; the different cell populations analyzed lead to differences in pathway enrichment. Genes enriched in the “reproductive structure development” pathway may influence follicle formation and development. e.g., FOX2 knockout leads to female infertility (PMID: 16647286); In the ovary, the TGFB2 null-mutant mice had an increase in the number of germ cells at P0 (PMID: 18790002); while genes in the “regulation of hormone levels” pathway, such as BMP5 and LHR, can affect steroid biosynthesis (PMID: 16079308). To align with our single-cell results and better illustrate our conclusions, we have revised the heatmap in Supplementary figure 16 to display only genes within the “regulation of hormone levels” pathway.\n\nA: In response, we have expanded our discussion of FSHR and LHR interplay during the ovulation cycle in both the Introduction (lines 43–58) and Discussion (lines 377–389) of the revised manuscript. Specifically, we address differences in the timing and co-expression patterns of these receptors between mammals and birds.\nWe also discuss the unique physiology of the avian ovary, including the predominance of a single functional ovary per cycle and the influence of seasonal breeding patterns, which contrasts with the more regular cycles observed in mice and humans. Despite these differences, the FSH-LH system maintains analogous functional roles in follicle development, selection, and ovulation across birds and mammals.\n\nA:We thank the reviewer for these insightful suggestions. We agree that the first hypothesis-reduced responsiveness of downstream signaling due to lower LHR expression-is most consistent with our observations.\nTo evaluate the alternative possibility of G-protein adaptation, we performed evolutionary analyses on all G-protein genes in estrildid finches. These genes were found to be highly conserved, showing no evidence of positive selection. This indicates that the reduced signaling likely reflects lower LHR expression rather than adaptations in G-proteins.\n\nA: According to your suggestion, we have added the following statement in the Discussion: “While current evidence supports reduced LHR expression as one mechanism conferring protection against OHSS, additional, yet unidentified mechanisms may also contribute-potentially even more significantly in some contexts. These possibilities warrant further investigation in future studies.” (Revised MS, line 399-402)\n\nA: Thank you for the comment. As indicated in the revised manuscript (lines 151 and 568) and the Methods section (lines 707-709), the FSH and LH used in our study are indeed the recombinant human forms.\nTo maintain consistency with the “hCG” nomenclature, we have now adopted the same prefix for these hormones, revising all instances in the MS, figures, and legends to “hFSH” and “hLH”. The manuscript has been amended throughout.\n\nA: Thank you for pointing this out; we have now corrected the formatting by removing the italic font from the family names Estrildidae (line 24, 91, 208, 254, 395, 408, 439, 502, 620-622, 626-627), Anostomidae (line 112), and Mormyridae (line 113) in the revised MS.\n\nA: We changed \" Passeridae” to “Passeri” in revised MS (line 91).\n\nA: In the revised manuscript, we have followed the style of capitalizing only proper nouns (e.g., “zebra finch,” “Gouldian finch”). All common names of birds have been updated accordingly in the text (lines 92–94, 113, 721–724) and in Supplementary Table 1 (sheet 2).\n\nA: In response to the request for more detail on the branch-site models, we have expanded the Methods section (lines 748-752). We explicitly state that we used the branch-site model Model A to test for positive selection on the foreground branches (estrildid finches). The analysis compares the alternative hypothesis (Model A, which allows sites to evolve with ω > 1 on the foreground branch) against the null hypothesis (the same model but with ω fixed at 1). Sites under positive selection were identified with a posterior probability (PP) ≥ 0.8 using the Bayes Empirical Bayes (BEB) method.\nTo further validate our evolutionary findings, we performed complementary analyses using the MEME, FEL, and aBSREL methods on the Datamonkey server. While our initial PAML branch-site analysis robustly identified the T449 site under positive selection, this specific signal was not detected by the other methods. This difference likely stems from their distinct statistical designs.\nThe PAML branch-site model is specifically optimized to detect site-specific positive selection concentrated on a pre-defined short lineage-precisely matching our hypothesis of a rare, lineage-restricted adaptation at T449 in estrildid finches. In contrast, MEME and FEL are designed to identify pervasive or episodic selection across the entire phylogeny, and aBSREL tests for branch-wide (not site-specific) selection events. The extremely localized nature of the T449A substitution-present only in estrildid finches, common wombats, and headstanders among ~3000 species analyzed (Supplementary Fig. 1)-explains why it was strongly captured by the tailored PAML model but fell below the detection threshold of these more generalized methods.\nCritically, the lack of detection by MEME/FEL/aBSREL does not undermine the central conclusion that T449 is a positively selected site driving functional change. This evolutionary inference is strongly corroborated by our extensive functional data:\n1.The T449A mutation enhances receptor constitutive activity (Fig. 1d and Supplementary Figs. 2, 4).\n2.It increases FSHR responsiveness to hCG and FSH stimulation (Fig. 1e and Supplementary Fig. 6).\n3.It enables FSHR activation by LH (Supplementary Fig. 7).\n4.FSHRT449A-KI mice exhibit heightened susceptibility to OHSS (Fig. 2 and Supplementary Fig. 11).\nThus, the convergent evidence from evolutionarily informed statistical modeling and direct experimental validation consistently supports T449 as a key adaptive residue that alters receptor function and influences OHSS risk.\n\nA: As the reviewer rightly noted, our species tree was not reconstructed through sequence analysis. Our primary objective was to utilize the vertebrate taxonomic tree from the NCBI Taxonomy database to provide readers with a clear visualization of the phylogenetic distribution of species carrying the FSHR449A variant. This approach was adopted because currently available FSHR sequences for many species in NCBI are incomplete, which would render a sequence-based phylogenetic reconstruction problematic. Although comprehensively verifying the entire vertebrate tree structure in the NCBI Taxonomy database is challenging, we have carefully validated the placement of all major avian orders within the tree based on the literature (PMID: 38560995) (Supplementary figure 1).\n\nA: We thank the reviewer for this valuable feedback. Our study is intended for a broad readership, including basic biomedical researchers, comparative and evolutionary biologists, and reproductive clinicians. In response, we have revised the manuscript to provide more detailed explanations of ovarian biology, the clinical relevance of our findings, and their applicability to OHSS patients, thereby enhancing clarity and accessibility across disciplines.\n\nA: We thank the reviewer for this comment. While the gain-of-function FSHR mutation had been previously identified, our comparative biology approach was crucial in revealing novel therapeutic strategies. We searched for corresponding variations in the animal kingdom to then investigate how these species “deal with it”, i.e., what are the underlying molecular mechanisms to ameliorate negative effects. Specifically, we found that ovarian LHR expression is downregulated in estrildid finches. Guided by this evolutionary insight, we applied the LHR antagonist LUF5771, which effectively alleviated OHSS symptoms in both FSHR T449A mutant and wild-type mice. These results demonstrate that the targets and therapeutic strategies identified through a comparative biology framework are relevant not only to patients carrying FSHR mutations but also to conventional IVF-induced OHSS. This underscores the unique translational relevance and added value of our evolutionary perspective.\n\nA: We thank the reviewer for these constructive suggestions. In response, we have thoroughly revised the Introduction (lines 43-79) to provide a comprehensive overview of antral follicle development and the physiological roles of FSH and LH, including their functions in follicle selection, growth, survival, maturation, and ovulation. We also describe the standard clinical ovarian stimulation protocols and explain how these interventions can trigger OHSS. These additions provide a clearer mechanistic background and highlight the scientific questions addressed by our study.\n\nA: Our findings are relevant to both spontaneous OHSS (caused by rare FSHR mutations like T449A) and the much more common iatrogenic OHSS. The prevalence of activating FSHR mutations is very low, and the exact carrier rate for T449A remains unreported. In contrast, clinically observed OHSS is far more common, occurring in approximately 20-30% (mild) and 0.1-6% (moderate to severe) of IVF cycles (line 32-33 in Revised MS). The broad relevance of our work is demonstrated by the efficacy of the LHR-targeting inhibitor in alleviating OHSS in both the constitutive model (FSHR-T449A KI mice) and the standard wild-type OHSS model. We have clarified this broad translational potential in the Discussion (lines 396-399).\n\nA: We thank the reviewer for this insightful comment. We agree that the role of FSH in primordial follicle formation is highly species-specific, and its relevance to the pathophysiology of OHSS was not clearly established in that particular paragraph. The cited statement“FSH injection during the early stage of primordial follicle formation significantly increases the number of primordial” was primarily based on studies in chickens (PMID: 30837955) and rodents (PMID: 19641178, 14585807). However, to maintain a sharper focus on the gonadotropin-dependent stages of folliculogenesis that are directly relevant to human OHSS, we have removed this paragraph from the revised introduction.\n\nA: In the cited reference (PMID: 20172968), the authors collectively refer to primordial, primary, and secondary follicles as \"small follicles.\" While we followed this terminology in our original manuscript, we agree that greater clarity is beneficial. As suggested, we have revised the manuscript to explicitly state \"primordial, primary, and secondary follicles\" instead of using the term \"small follicles\" to avoid any potential ambiguity (MS, line 75).\n\nA：In response to the reviewer’s comment, we have thoroughly reviewed and revised the manuscript. The paragraph originally at line 67 (“The prevailing models … regulate various physiological functions”) was intended to illustrate the ligand-dependent activation mechanism of GPCRs under classical theory, thereby contrasting with the subsequent discussion of ligand-independent functions. To improve logical coherence and clarity in the Introduction, this paragraph has been removed in the revised version. We have instead introduced additional background information on OHSS, the process of antral follicle development, and the physiological roles of FSH and LH in the ovary. These additions provide a more comprehensive research context, highlight the main focus of the study, and more effectively lead into the key issues addressed in our work.\n\nA: We thank the reviewer for this insightful comment. We would like to clarify that our study employed multiple ovarian stimulation protocols to assess the model's response.\nThe conventional mouse OHSS model typically involves four consecutive days of PMSG (20 IU) priming, followed by ovulation triggering with 5 IU of hCG. In contrast, the control group received a standard superovulation regimen: a single injection of PMSG, followed by 5 IU of hCG 48 hours later (as outlined in Figure 4a). When applying these protocols to the FSHRT449A knock-in mice, we observed more severe OHSS-related symptoms in the mutants under two different conditions:\nThe control (standard superovulation) protocol (as shown in Supplementary figure 11a) and a milder OHSS-induction protocol with a reduced hCG dose (1 IU) (as shown in Figure 2b).\nTherefore, our key finding is that the FSHRT449A mutant mice exhibit heightened sensitivity and develop a pronounced OHSS phenotype not only under a milder OHSS-specific regimen but, crucially, under a standard ovulation induction protocol. This demonstrates that the model has clinical relevance, as the mutation predisposes the animals to OHSS under conditions that are normal in a clinical setting, thereby supporting the link between the T449A mutation and OHSS risk.\n\nA：As suggested, we have now provided larger and clearer histological images of the ovaries in the main figures to improve visualization.\n\nA: We thank the reviewer for this suggestion. Ovarian enlargement has now been quantified through multiple parameters, including ovary weight and the number of corpora lutea. To better quantify ovarian enlargement in both wild-type and FSHR-T449A KI, we did ovarian H&E staining and quantitative analysis in Figure 2h. These data provide clear morphological and quantitative evidence of the ovarian response.\n\nA: We have now included histological sections of finch ovaries in the main figures. The new panel (Revised Figure 2p) shows that, compared to controls, ovaries from PMSG/hCG-induced birds display a marked increase in overall size and contain a greater number of enlarged follicles, consistent with an OHSS-like phenotype and Palmer’s reports (PMID: 1623425).\n\nA: As recommended, ovarian weight normalized to body weight (formerly shown in Figures 2d, 4c, 4k, and 5c) has been moved to Supplementary figure 11g and Supplementary figure 17a-c. The raw ovarian weight measurements remain in the main figures.\n\nA: We have now included a detailed description of the corpus luteum (CL) counting procedure in the Methods section (lines 869-875) of the revised manuscript, as follows: “Serial sections (5 μm in thickness) were obtained from the largest cross-sectional region, yielding nine consecutive sections per ovary. The sections were stained with haematoxylin and eosin (H&E) for morphological assessment and quantification of corpora lutea. For corpus luteum counting, the nine consecutive H&E-stained sections were analysed as a continuous series. Each section was examined in reference to its preceding and succeeding sections to trace the structural continuity of individual corpora lutea and to prevent redundant counting. The average number of corpora lutea per ovary was used for comparison among different treatment groups.”\nThe control group CL counts in our study align with values reported in well-established OHSS models in both mice (PMIDs: 38573397, 35221672) and rats (PMID: 36159336), supporting the appropriateness of our counting methodology and the biological validity of our control data.\n\nA: The RU486 treatment group data for ovarian cAMP levels has now been added to Figure 4d as suggested. This addition provides a more complete representation of the experimental results.\n\nA: We thank the reviewer for highlighting the importance of fluid accumulation in OHSS. In response, we carefully evaluated the presence of ascites in our models, including the T449A variant. No significant or measurable ascites was observed across experimental groups. This is likely because our induction protocol models the early to moderate stages of OHSS, focusing on ovarian-level changes such as ovarian enlargement, VEGF upregulation, and follicular hyperstimulation, rather than the severe systemic form characterized by massive fluid extravasation. This feature is consistent with established OHSS models, where primary readouts typically include ovarian weight, histopathology, and angiogenic factor expression without reported ascites. Therefore, the absence of fluid accumulation does not diminish the validity of our model for investigating the core mechanisms of ovarian hyperstimulation.\n\nA: We thank the reviewer for this comment. The primary goal of our scRNA-seq analysis (Fig. 5i–r) was to provide molecular evidence for the overlap in downstream signaling pathways of FSHR and LHR. Based on our studies, the FSHR in estrildid finches carries a naturally occurring T449A mutation, which confers high constitutive activity and responsiveness to ligands such as LH, hCG, and FSH, increasing the risk of ovarian hyperstimulation. Interestingly, when hormones are administered experimentally, estrildid finches do not readily develop OHSS-like phenotypes, suggesting that their ovaries have evolved a compensatory mechanism.\nOur single-cell analysis revealed that LHR expression is downregulated in estrildid finches. Given the evolutionary relationship between FSHR and LHR, and prior observations in fish suggesting potential compensation between these receptors, we sought to investigate whether a similar compensatory mechanism exists at the molecular level in birds. The scRNA-seq results demonstrate that FSHR and LHR share overlapping downstream pathways under OHSS conditions, supporting the hypothesis that LHR downregulation represents an adaptive response to the high constitutive activity of FSHR in estrildid finches. This analysis thus provides mechanistic insight into how estrildid finch ovaries resist hyperstimulation despite the presence of a gain-of-function FSHR mutation.\n\nA: Our cell type annotation was based on well-established marker genes from the CellMarker 2.0 database and published references (e.g., Mary E. Morris et al., PMID: 36205477). The observed proportional shift in immune cells may be related to the inflammatory response induced by OHSS. Several studies have reported similar shifts in ovarian cellular composition under both physiological and pathological conditions. For example, in mouse ovaries, the proportion of immune cells increases from approximately 9% at 3 months of age to 21% at 9 months, while granulosa cells decrease from 28% to 17%, making immune cells comparable in proportion to granulosa cells in aged mice (Response figure 4a; PMID: 38200272). Similarly, single-cell analyses of control and obese mouse ovaries show that immune cells can reach nearly one-third of total ovarian cells, comparable to granulosa cell proportions (Response figure 4b; PMID: 38956667). These observations demonstrate that ovarian cellular composition can vary substantially depending on physiological or pathological states.\nFurthermore, in our separate study using a rat OHSS model, single-cell analysis revealed a high proportion of immune cells, particularly macrophages, in ovarian tissue (Response figure 5a). We validated this finding through CD68 immunofluorescence staining, confirming abundant macrophage infiltration in OHSS ovaries (Response figure 5b). This independent validation supports the biological accuracy of our cell type classification and reflects the genuine inflammatory state of the ovary under OHSS conditions.\n\nA: We sincerely thank the reviewer for this important comment. To assess the stability of GAPDH in our OHSS model, we compared the CT values of GAPDH with two additional housekeeping genes, β-actin and Hprt1. As shown in Response figure 6, the CT values of all three genes remained consistent across control, stimulation, and drug-treated groups. Moreover, we normalized the expression of Jun to each of these three reference genes and observed consistent expression trends. These results confirm that GAPDH is a suitable reference gene for quantitative analysis in our experimental system.\n\nA: We acknowledge the reviewer's point that LUF5771 can act as a partial agonist of LHR. In our study, however, we utilized LUF5771 primarily for its antagonistic properties to inhibit LHR activity in both wild-type and FSHR-mutant mice. This treatment partially alleviated OHSS symptoms, supporting the notion that the strategy observed in estrildid finches-where reduced LHR expression compensates for FSHR overactivation-serves as a mechanism to mitigate the risk of ovarian overstimulation.\n\nA: The change in protocol between Figures 2b (1 IU hCG) and 4a (5 IU hCG) was deliberate and based on the following rationale:\n1. Demonstrating enhanced hCG sensitivity: The FSHR-T449A mutation increases receptor responsiveness to hCG. We therefore used a lower hCG dose (1 IU) in Figure 2b to specifically challenge the mice and reveal inherent phenotypic differences between KI and WT mice. Under this low-dose challenge, KI mice developed a more pronounced OHSS phenotype, consistent with the heightened sensitivity observed at the cellular level.\n2. Modeling the clinical context and confirming risk: Since OHSS in patients typically occurs after standard ovulation induction protocols, we also subjected KI and WT mice to a conventional superovulation regimen (5 IU PMSG followed by 5 IU hCG 48 hours later; Supplementary fig. 9). KI mice consistently exhibited more pronounced OHSS markers, including increased ovarian size, VEGF, and estradiol levels, confirming their higher susceptibility under standard clinical-like conditions.\nThus, the regimen in Figure 2b was optimized to probe mechanistic sensitivity, while the standard protocol in Supplementary Fig. 9 validates the clinical relevance of our findings. We have clarified these rationales in the revised manuscript (lines 169–172, 180-184).\n\nA: We thank the reviewer for this comment. The use of rats was necessary for testing BAY-899 and BAY-298 due to their species-specific efficacy (revised MS, line 269). As reported by Wortmann et al. (PMID: 35670515), these compounds act on human, rat, and cynomolgus monkey LHCGR, but are inactive at the mouse receptor, making them unsuitable for use in mice.\nImportantly, the consistent therapeutic effects observed in rats further support the broader relevance of the mechanism we identified. The finding that dampening LHR function alleviates OHSS-demonstrated across wild-type mice, FSHR-KI mice, and rats-provides robust cross-species evidence for the evolutionary insight gained from estrildid finches.\n\nA: We thank the reviewer for this suggestion. As requested, the catalog numbers for all kits and reagents have now been added to the Materials and Methods section.\n\nA：We appreciate the reviewer's insightful comment regarding the use of sexually mature animals. We acknowledge that the clinical condition occurs in adult women, and we agree that validation in mature animals strengthens the study.\nThe use of immature mice or rat for the OHSS model is an established approach in the field (PMIDs: 31329230, 23348396, 38573397, 18937946, 19443574, 35221672). A key methodological advantage is the synchronization of their hormonal status, which minimizes confounding variables from the estrous cycle and provides a more uniform baseline for induction.\nImportantly, to directly address the reviewer's concern and to enhance the clinical relevance of our findings, we have now performed additional experiments using sexually mature female mice. As shown in the Supplementary figure 17d-m, LUF5771 treatment effectively suppressed ovarian enlargement and the associated increases in VEGF and estradiol synthesis in both wild-type and FSHR-mutant adult mice. These results confirm that our therapeutic findings are consistent and applicable in mature animals, thereby broadening the potential clinical significance of our proposed mechanism.\n\nA: To enhance clarity, we have now reorganized the Methods section to explicitly detail the procedures specific to each experiment. This includes providing distinct subsections for the induction protocols in mice, rats, and birds, as well as separately describing the specific steps for constitutive activity assays, ligand stimulation, and avian ligand stimulation experiments.\n\nA: We thank the reviewer for this suggestion. We have completely restructured the Discussion to provide deeper biological insight and clinical context. The revised version now emphasizes the evolutionary trade-off hypothesis, the compensatory role of LHR downregulation, and the translational potential of targeting LHR for OHSS treatment, while explicitly acknowledging the study's limitations. These changes significantly enhance the significance and interpretation of our findings.\n\nA: Accordingly, we have deleted or moved the indicated paragraph from the Discussion to the Introduction. This adjustment significantly improves the structural logic of our manuscript.\n\nWe sincerely thank you for acknowledging the clinical importance of our work and for providing constructive feedback to strengthen the manuscript. We have thoroughly revised the manuscript to provide a clear and detailed background of ovarian biology, the clinical significance of our findings, and their applicability to OHSS patients, thereby improving the clarity and cross-disciplinary accessibility of the paper. We hope our revisions meet with your approval.\n\nA: We thank the reviewer for this constructive suggestion. We have accordingly revised the schematic drawings of FSHR and LHR in the manuscript to better represent the organization and structure of their extracellular domains (ECDs). The updated illustrations are now included in the revised Figures (Figure 1c and Figure 6).\n\nA: Thank you for your careful observation. To clarify: the phylogenetic tree in Figure 1a was indeed generated using TimeTree to establish the evolutionary relationships among species included in our analysis. We have updated the main text accordingly (lines 106-110 in the revised manuscript) to explicitly state that the tree was constructed with TimeTree.\n\nA: Thank you for your comment. We have clarified these points in the Methods section as requested (revised MS, lines 943-950, 995-997), as well as figure legends (lines 705-707). Please find the details below.\nFor the bulk RNA-seq of avian ovaries (related to Figure 3 and Supplementary Fig. 16): The ovaries were processed as single organs (one ovary per bird per sample). No pooling was performed. In Figure 3, the T. guttata (tg) and S. canaria (sc) groups each contain n = 3 biological replicates, while the L. striata domestica (ls) group contains n = 3 biological replicates. In Supplementary Fig. 16, each group includes n = 3 biological replicates (ovaries from 3 individual birds). No technical replicates were included in these experiments.\nFor the single-cell RNA-seq of mouse ovaries (related to Figure 5): Due to the limited cell yield from a single mouse ovary, ovaries were pooled prior to single‑cell suspension preparation.\nSpecifically, the OHSS group consisted of n = 1 biological replicate, generated by pooling both ovaries from one mouse. The LUF5771‑treated and hFSH‑β‑(33–53)-treated groups each contained n = 2 biological replicates. Each replicate was prepared by pooling both ovaries from each of two mice, resulting in a total of four ovaries per replicate.\nWe have updated the Methods to explicitly state the sample handling and replicate design for both experiments.\n\nA: Thank you for raising this point. As summarized in response Figure 1, we compared the number of shared differentially expressed genes (relative to the S. canaria (sc)) across cell types between T. guttata (tg) and L. striata domestica (ls) and ranked them accordingly. This analysis confirmed that granulosa cells share the highest number of differentially transcribed genes among all cell types examined. To avoid duplication with a companion paper where these data are presented in full, we have removed the corresponding statement from the revised manuscript. We appreciate your careful attention to this detail.\n\nA: We agree that the original statement could be misleading regarding cross‑species comparison. Accordingly, we have revised the relevant sentence in the manuscript (lines 232–234) to more accurately reflect the data presented in the figures. The updated text now reads: “Feature plots demonstrated that FSHR expression was primarily localized to granulosa and pre‑granulosa cells across all three species, with additional expression observed in mesenchymal and epithelial cells.”\n\nA: We thank the reviewer for this suggestion. To provide quantitative support, we have added a violin plot with statistical analysis to the revised manuscript (revised Figure 3l). The plot confirms that LHR expression is significantly higher in S. canaria granulosa cells than in those of T. guttata and L. striata domestica (p < 0.0001, Wilcoxon rank-sum test), robustly supporting our original observation that LHR is nearly undetectable in estrildid finch granulosa cells. This related information is also included in the Figure legend (lines 486-488).\n\nA. In Figure 3g, the GO enrichment analysis reflects a combined comparison between estrildid finches and the non‑estrildid species. Specifically, it is based on (1) the intersection of upregulated differentially expressed genes (DEGs) from both L. striata domestica vs S. canaria and T. guttata vs S. canaria comparisons, and (2) the intersection of downregulated DEGs from the same two pairwise comparisons. This approach identifies biological processes that are consistently altered in both estrildid species relative to the non‑estrildid control. We have added this clarification to the revised Manuscript (lines 234–239).\n\nA: We sincerely thank the reviewer for raising this insightful and constructive point. Utilizing the R package GSEABase and the AddModuleScore function, we further evaluated the signature scores of the angiogenesis and estrogen synthesis pathways. As shown in response Figure2, LUF5771 treatment resulted in a significant reduction in the estrogen synthesis pathway score in both granulosa and theca cells, as well as a significant decrease in the angiogenesis pathway score in granulosa cells. In addition, TFA treatment led to a reduced estrogen synthesis pathway score in granulosa cells. This analysis has been incorporated into revised Figure 5n. We have added the corresponding methodological description, results, and figure legends to the revised manuscript (Methods: lines 990–992; Results: lines 314–319; Figure legend: lines 520–522).\n\nA: We thank the reviewer for this suggestion. We have revised the manuscript to explicitly clarify this important distinction (revised MS, lines 217-219). The relevant text now states: “To further investigate the intrinsic adjustment mechanisms of estrildid finches to avoid OHSS, we performed single-cell RNA sequencing (scRNA-seq) on the ovaries of two estrildid species (T. guttata, tg, and L. striata domestica, ls) as well as one non-estrildid bird (S. canaria, sc) under steady-state physiological conditions without exogenous stimulation This experimental design enables the identification of baseline transcriptional adaptations in estrildid finches.”\n\nA: To assess changes in granulosa cell and theca cell subtypes, we performed re-clustering and annotation using high-quality marker genes reported in the literature. Granulosa cells were classified into distinct subtypes, including an Antral-Mural state (response Figure. 3a,b). Compared with the OHSS group, treatment with LUF5771 and hFSH-β-(33-53) reduced the number of granulosa cells in atretic follicles, while cells in the preantral follicle state showed signs of recovery (response Figure. 3c), suggesting that these treatments may help restore normal granulosa cell composition and limit follicle atresia. The expression patterns of differentially expressed genes related to estrogen signaling and vascular permeability further pointed to their potential role in this process (response Figure. 3d). Theca cells were primarily classified into early-stage cells (involved in androgen synthesis) and late-stage cells (associated with signal transduction, chemotaxis, and cell growth) (response Figure. 3d e-g). Following treatment with LUF5771 or hFSH‑β‑(33‑53), the proportion of early‑stage theca cells decreased relative to the OHSS group (response Figure. 3f), indicating a possible downregulation of the androgen synthesis pathway that serves as the precursor for estrogen production. Moreover, expression of Bmp2 and Bmpr2 was reduced in both early‑ and late‑stage theca cells after treatment (response Figure. 3h). We have incorporated this graph and the corresponding image into the revised manuscript (lines 332–346, 711-719, 984-990) and as Supplementary Figure 21, as suggested.\n\nA: We thank the reviewer for this constructive suggestion. The proposed analysis using CellChat/NicheNet could indeed provide valuable insight into how vascular permeability‑related signaling between granulosa/theca cells and endothelial cells is altered in OHSS and modulated by LHR antagonism. However, our current scRNA‑seq dataset lacks a corresponding control group, which precludes a direct comparison of cell‑cell communication before and after OHSS induction. The primary objective of the present study was to identify the shared pathways through which FSHR and LHR antagonism suppress OHSS progression and to elucidate the compensatory adaptation mechanism in estrildid finches. We therefore focused on comparative transcriptomic and functional analyses that align with these aims.\nWe acknowledge that exploring intercellular signaling networks represents an important future direction, and we plan to incorporate such analyses in subsequent studies to further dissect the cellular communication dynamics underlying OHSS pathophysiology."}]

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