Neuromedin B Drives Goat Granulosa Cell Proliferation via NMBR-Mediated Calcium Homeostasis

Neuromedin B Drives Goat Granulosa Cell Proliferation via NMBR-Mediated Calcium Homeostasis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Neuromedin B Drives Goat Granulosa Cell Proliferation via NMBR-Mediated Calcium Homeostasis Rongxin Xia, Qi Zhang, Junhui Shao, Yuan Wang, Xinyi Lv, Rui Chen, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6811713/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 19 Nov, 2025 Read the published version in Journal of Ovarian Research → Version 1 posted 9 You are reading this latest preprint version Abstract Background Neuromedin B (NMB) has been implicated in the regulation of female reproductive functions, yet its precise role and underlying mechanisms in ovarian follicular development remain undefined. Granulosa cells (GCs), the principal functional cells within ovarian follicles, directly govern follicular growth and maturation through their proliferation and differentiation. In this study, we explored the regulatory effects and molecular mechanisms of NMB and its receptor (NMBR) on goat GC proliferation. Results We documented dynamic expression patterns of NMB and NMBR throughout ovarian and follicular development. Exogenous NMB treatment markedly enhanced GC proliferation, as evidenced by an increased fraction of S-phase cells and upregulation of CCNE1 and CDK1/2/6. Mechanistically, NMB bound to NMBR to activate phospholipase C β1 (PLCβ1), triggering endoplasmic reticulum (ER) Ca²⁺ release and significantly raising cytosolic Ca²⁺ levels while alleviating ER stress. Further analyses revealed that NMB strengthened mitochondria-associated ER membranes (MAMs) formation via the IRE1α–IP3R–VDAC1 axis, facilitating Ca²⁺ transfer into mitochondria. This led to enhanced mitochondrial function, including increased mitochondrial membrane potential, elevated respiratory chain complex activities, augmented ATP production, and promotion of mitochondrial network fusion. Importantly, these effects were abolished by an NMBR antagonist. Conclusions The molecular mechanism by which NMB-mediated activation of NMBR enhances mitochondrial metabolism through modulation of intracellular calcium homeostasis, thereby promoting proliferation of goat GCs. These findings shed new light on the regulation of follicular development and may inform strategies to improve reproductive efficiency in goats. Neuromedin B granulosa cells cell proliferation endoplasmic reticulum mitochondria goat Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction The ovary, as the central organ of the female reproductive system, determines the reproductive potential of females through the developmental status of its functional unit-the ovarian follicle [ 1 ]. Follicular development is coordinately regulated by endocrine signals (e.g., gonadotropins) and local ovarian factors (e.g., growth factors and cytokines) [ 2 ]. Granulosa cells (GCs), the core functional cells within follicles, critically govern key reproductive events including follicular growth, ovulation efficiency, luteal function, and steroidogenesis through their proliferation and differentiation states [ 3 ]. Therefore, elucidating the regulatory mechanisms of GC proliferation is pivotal for understanding ovarian physiology. Neuromedin B (NMB), a member of the mammalian bombesin like peptide family, is a decapeptide composed of 10 amino acids [ 4 ]. NMB is widely distributed in the central nervous system and peripheral tissues (e.g., gastrointestinal tract and gonads) [ 5 ], mediating diverse biological functions by binding to specific receptors and activating downstream signaling pathways [ 6 ]. Notably, NMB and its receptor exhibit high expression in the reproductive systems of model animals such as humans and mice [ 7 , 8 ], suggesting their potential regulatory roles in reproductive function. Moreover, studies show that NMB enhances the excitability of gonadotropin-releasing hormone (GnRH) neurons in female mice [ 9 ], promoting hypothalamic GnRH secretion to regulate pituitary luteinizing hormone (LH) release [ 10 ]. In addition, NMB directly stimulates pituitary prolactin secretion [ 11 ], indicating its involvement in follicular development via the hypothalamic–pituitary–ovarian axis. Together, these findings indicate that NMB-mediated regulation of reproductive function involves multiple pathways, genes, and factors, yet the precise mechanisms remain to be elucidated. Mitochondria and the endoplasmic reticulum (ER) are core organelles regulating cellular biological functions. Approximately 5–20% of the mitochondrial outer membrane forms highly dynamic physical contacts with the ER, known as mitochondria-associated ER membranes (MAMs) [ 12 , 13 ]. MAMs formation is not merely membrane contact between organelles but a complex, highly regulated process. MAMs mediate the transfer of calcium ion (Ca²⁺), phospholipids, and metabolites between the two organelles [ 13 ], thereby regulating lipid metabolism [ 14 ], mitochondrial dynamics [ 15 ], ER stress responses [ 16 ], calcium homeostasis [ 12 , 17 ], and cell fate [ 18 ]. Notably, MAMs formation is essential for optimal cholesterol transfer from the ER to mitochondria and serves as a central hub for initiating mitochondrial steroidogenesis [ 19 ], suggesting a critical regulatory role of MAMs in reproductive regulation. Meanwhile, a high-fat diet can increase the levels of MAMs related proteins in mouse germ cells, elevate mitochondrial calcium levels, and promote apoptosis [ 20 ]. Exposure to endocrine-disrupting chemicals induces MAMs alterations that disrupt ovarian function in mice [ 21 ]. These observations underscore the potential importance of MAMs in follicular development. Using goat GCs as a model, this study combined NMB treatment and Neuromedin B receptor (NMBR) antagonism experiments to reveal that NMB regulates ER and intracellular Ca²⁺ homeostasis through binding to NMBR, thereby influencing goat GCs proliferation and cell cycle progression. We further investigated how NMB-NMBR interaction modulates the structural coupling efficiency of MAMs and its impact on mitochondrial function and dynamics. The results confirmed that NMB promotes goat GC proliferation via a "Ca²⁺ homeostasis-MAMs-mitochondrial metabolism" cascading signaling axis. This study systematically elucidates a novel mechanism of NMB/NMBR in ovarian function regulation, providing a theoretical breakthrough for reproductive biology research. 2. Materials and methods 2.1 Goat Ovaries and Follicles Collection Goat ovary tissues were collected from the slaughterhouse and stored in a thermos containing sterilized normal saline (30-35 ℃, including 100 IU/L penicillin and 50 mg/L streptomycin) and transported to the laboratory within 1 h after collection. After washing five times with normal saline, the connective tissue on the ovary and the attached fallopian tube were removed. Part of the collected ovarian samples were fixed with Bouin's fixative for immunohistochemical detection; One part was immediately frozen in liquid nitrogen for subsequent extraction of total RNA and protein. According to previous methods [22]and others, healthy follicles were isolated from the ovary and divided into three diameters (≤ 2 mm, 2-5 mm and ≥ 5 mm, with at least 80, 60 and 40 follicles in each diameter group). The isolated follicles were rapidly cryopreserved in liquid nitrogen for subsequent analysis of gene and protein expression. 2.2 Cell Culture and Treatments GCs were isolated from healthy follicles (2-7 mm in diameter) using a 24-gauge needle connected to a 5 mL syringe according to established laboratory protocols after ovarian collection from the slaughterhouse [22]. The GCs were cultured in DMEM/F12 (1:1) medium containing 10% fetal bovine serum (FBS), 100 IU/mL penicillin, and 100 IU/mL streptomycin, and incubated at 37 °C in a 5% CO 2 environment. Goat NMB (RP10507, purity > 95% by HPLC analysis) was synthesized by GenScript Biotech. GCs were cultured in 6-well plates until reaching 50% - 60% confluence, then treated with either different NMB concentrations (10 -10 , 10 -9 , 10 -8 , 10 -7 , 10 -6 M) for 48 h or 10 -7 M NMB for varying durations (3, 6, 12, 24, 48 h), followed by an additional 24 h treatment with 10 -7 M NMB. The NMBR antagonist PD168368 (MCE, HY-116216) was dissolved in dimethyl sulfoxide (DMSO) and stored at a stock concentration of 50 mM. For treatment, 1 μL of the stock solution or DMSO (vehicle control) was added per milliliter of medium, yielding final concentrations of 50 μM and 0.1% DMSO, respectively. 2.3 RNA extraction, cDNA synthesis and gene clone The total RNA was extracted from goat ovaries using TRIzol reagent (Vazyme, R401-01). RNA concentration was determined using the ND1000 spectrophotometer (Thermo Scientifific). RNA was reverse-transcribed into cDNA via the Primer Script RT reagent kit with gDNA eraser (TaKaRa Biotechnology). The cDNA samples were stored at -20 ℃ until use. Cloning method conducted according to the former research with minor modifications [23]. Nucleotide sequence of PCR primers were listed in Table S1. Positive clones were randomly chosen and commercially sequenced through General Biological Technology (Beijing qingke biotechnology Ltd, china). The obtained nucleotide sequences of NMB and its two receptors were uploaded to NCBI. The similarity of amplified sequences and NCBI reference sequences was performed using BLAST (http://www.ncbi.nlm.nih.gov/blast). The phylogenetic tree analysis and homology assessments were performed using the neighbor joining and dislocation comparison method in MEGA-X software. Amino acid sequences were predicted with the Expasy Proteomics Server (http:// www.expasy.ch/tools/dna.html) and then aligned with other species using GenomeNet (http:// www.genome.jp/tools/clustalw). The accession numbers of NMB and its receptors for five species were listed in Table S2. The prediction of transmembrane domains was performed using TMHMM (http://www. cbs.dtu.dk/services/TMHMM-2.0). 2.4 Quantitative real-time polymerase chain reaction Total RNA was extracted from the samples using TRIzol reagent (Vazyme, R401-01) according to the manufacturer’s instructions, and 1 μg of total RNA from each sample was reverse-transcribed into first-strand cDNA using a reverse transcription kit (Vazyme, R323). Real-time PCR (qRT-PCR) with SYBR Green was conducted on the ABI 7500 Real-Time PCR system (Applied Biosystems) using 1 μL cDNA in a total reaction volume of 20 μL, following the manufacturer’s protocol. Amplification specificity was confirmed by melting curve analysis, and the relative mRNA expression levels of target genes were quantified using the 2 −ΔΔCt method, normalized against glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) mRNA expression levels. All experiments were independently performed in triplicate, and the primer sequences for target gene amplification are provided in Table S3. 2.5 Western Blot Assay Proteins were extracted from the samples using RIPA lysis buffer (Thermo Scientific, 89901), and the total protein concentration was determined with a BCA protein assay kit (Beyotime, P0010). Equal amounts of protein (20 μg) were separated by 4%-20% polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, ISEQ00010), and blocked with 5% skimmed milk. After overnight incubation with primary antibodies at 4 °C, the membranes were treated with secondary antibodies for 1 h at room temperature. Protein bands were visualized using the Western Bright ECL kit (Biosharp, No. BL520B-2) on an Image Quant LAS 400 system (Fiji film), and target protein quantification was performed with Image J software (Wayne Rasband), normalized to the internal control (GAPDH). Detailed information on antibodies used in this study is provided in Table S4. 2.6 Immunohistochemistry analysis (IHC) Goat ovaries were fixed, processed, and paraffin-embedded for sectioning as previously described [24]. Following dewaxing, antigen retrieval was performed on the sections according to the protocol of the M&R HRP/DAB Detection IHC Kit (Vazyme, HC301-01). After blocking endogenous peroxidase activity, the sections were incubated with primary antibodies at 4 °C overnight, followed by incubation with secondary antibodies at room temperature for 2 h. Subsequently, DAB chromogenic staining and counterstaining were carried out, and the sections were dehydrated and mounted. Negative controls were run by substituting 5% rabbit serum for the primary antibody. Brown-colored positive signals were visualized, and images were captured using a Nikon microscope (T300). Antibody details are listed in Table S4. 2.7 Immunofluorescence analysis (IF) After treatment of goat GCs, the cells were fixed in 4% paraformaldehyde (PFA) (Beyotime, No. P0099), permeabilized with 0.5% Triton X-100, blocked with 5% bovine serum albumin (BSA), and then incubated with primary antibodies overnight at 4 °C. The next day, cells were incubated with secondary antibodies for 1 h, followed by nuclear staining with Hoechst 33258 (Yeasen, 40730ES03) for 10 min. Finally, coverslips were mounted onto glass slides, and images were captured using a confocal laser scanning microscope (Zeiss, LSM900). Positive signals appeared green, and negative controls were prepared by replacing the primary antibody with 1% rabbit serum. Details of antibodies for this study are provided in Table S4. Live-cell fluorescence staining of goat GCs was performed using either a mitochondrial green fluorescent probe (Yeasen, 40742ES50) diluted to 500 nM working solution and incubated at 37 °C in the dark for 30 min, or an ER red fluorescent probe (Yeasen, 40764ES20) diluted to 1 µM working solution and incubated at 37 °C in the dark for 20 min, followed by nuclear staining with Hoechst 33258 for 10 min. For MAMs structure analysis, cells were first incubated with the ER red fluorescent probe working solution at 37 °C in the dark for 20 min, the solution was then removed, followed by incubation with the mitochondrial green fluorescent probe working solution at 37 °C in the dark for 30 min, and finally nuclear staining with Hoechst for 10 min. Images were observed and captured using a laser confocal microscope (Zeiss, LSM900), and quantitative assessment was performed with Image J software (Wayne Rasband). 2.8 Cell proliferation assay Cell proliferation was assessed using the BeyoClick™ EdU-594 Cell Proliferation Detection Kit (Beyotime, C0078S). Briefly, cells were seeded into 24-well plates pre-placed with sterile cell slides and treated for the indicated durations. Subsequently, cells were incubated with 50 nM EdU working solution for 2 h, fixed with 4% PFA, washed with PBS containing 3% BSA, and permeabilized with PBS containing 0.3% Triton X-100. The Click reaction solution was then applied to the cells and incubated in the dark for 30 min. After washing, nuclei were counterstained with 5 μg/mL Hoechst. Finally, cells were mounted with anti-fade medium, sealed with coverslips, and imaged using a confocal laser scanning microscope (Zeiss, LSM900). Quantification of results was performed using ImageJ software (Wayne Rasband). 2.9 Cell cycle distribution analysis Cell cycle analysis was performed via flow cytometry as follows: harvested cells were fixed overnight in ice-cold 70% ethanol, washed once with ice-cold DPBS, and stained with propidium iodide (PI) staining solution (Beyotime, C1052) prepared according to the manufacturer’s instructions, followed by incubation at 37 °C in the dark for 30 min. Red fluorescence emitted by PI-DNA complexes was detected at an excitation wavelength of 488 nm using a flow cytometer (Becton, Dickinson and Company). 2.10 Intracellular Ca 2+ content determination Intracellular Ca 2+ levels were measured using a Calcium Colorimetric Assay Kit (Beyotime, S1063S). Briefly, after cell lysis, the supernatant was incubated with the working solution at room temperature in the dark for 10 min, while a standard curve was prepared in parallel using the provided standards. Absorbance was measured with a multimode microplate reader (Thermo Scientific), and Ca 2+ concentrations in each sample were calculated based on the standard curve. Intracellular ER Ca²⁺ levels were measured using a commercial kit (BestBio, BB-481159). Briefly, after standard treatments, the culture medium was removed, and cells were washed three times with HBSS. Subsequently, cells were incubated with BBcellProbe® C93 staining working solution at 37 °C for 45 min, followed by three additional HBSS washes. Cells were then covered with HBSS and incubated again at 37 °C for 30 min. Mitochondrial Ca²⁺ levels were assessed using another kit (Beyotime, S1062S). In brief, after treatments, the medium was discarded, cells were washed three times with HBSS, and Rhod-2 staining solution was added. Cells were incubated at 37 °C for 20 min, after which the supernatant was removed, and cells were washed three times. Fluorescence images were captured using a laser scanning confocal microscope (Zeiss, LSM900), and the average fluorescence intensity of each image was quantified with Image J software (Wayne Rasband). 2.11 Small interfering RNA transfection siNC and siXBP1S-1/2/3/4 were purchased from Genepharma and transfected into GCs using Lipofectamine™ 3000 (Invitrogen, L3000150). The sequence information of the siRNAs is listed in Table S5. 2.12 Transmission Electron Microscopy Analysis GCs were fixed with 2.5% glutaraldehyde (in 0.1 M PBS, pH 7.4) at 4°C for 4 h, followed by dehydration through a graded ethanol series. Samples were embedded in resin, sectioned into ultrathin slices, and dual-stained with uranyl acetate-lead citrate. Mitochondrial morphology, ultrastructural changes, and MAMs integrity were observed using TEM. Image J quantified mitochondrial aspect ratio and MAMs contact points. The fraction of mitochondria in contact with ER within a 50 nm range was calculated and normalized to the mitochondria perimeter, as previously described [25]. 2.13 Co-immunoprecipitation (CO-IP) Immunoprecipitation was performed using an immunoprecipitation kit (Proteintech, PK10008). Cultured GCs were lysed with lysis buffer according to the manufacturer’s protocol, followed by incubation with specific antibodies under rotation at 4 °C overnight. Subsequently, pre-resuspended Protein A/G beads slurry was added to precipitate the immune complexes, which were further incubated under rotation at 4 °C for 4 h. After washing, the complexes were eluted, and the eluate was mixed with alkali neutralization buffer and 5× sample buffer, heated in boiling water for 5 min, and analyzed by western blotting for the input lysates. 2.14 Mitochondrial Membrane Potential Assay （ JC-1 ） According to the manufacturer’s protocol (Solarbio, M8650), treated GCs were incubated with JC-1 staining working solution and thoroughly mixed. The cells were then maintained in a cell culture incubator at 37 °C for 20 min. After incubation, the liquid was aspirated, and the cells were washed twice with JC-1 staining buffer (1×). Images were observed and captured using a laser confocal microscope (Zeiss, LSM900). 2.15 Reactive Oxygen Species Assay （ ROS ） According to the manufacturer’s protocol (Beyotime, S0033S), treated GCs were incubated with DCFH-DA diluted to a final concentration of 10 µM and thoroughly mixed. The cells were then maintained in a cell culture incubator at 37 °C for 20 min. After incubation, the liquid was aspirated, and the cells were washed twice with DPBS. Images were observed and captured using a laser confocal microscope (Zeiss, LSM900). 2.16 Mitochondrial complex reductase activity Assay (MRC) According to the manufacturer’s protocols for mitochondrial respiratory chain complexes I, II, III, IV, and V (Solarbio, BC0515, BC3235, BC3245, BC0945, BC1445), the complexes were extracted from collected cells, incubated with their respective working solutions, and the absorbance was measured using a multifunctional microplate reader (Thermo Scientific). The activity levels of the complexes in the samples were subsequently calculated. 2.17 Statistical analysis All experiments were repeated at least three times. Statistical analyses were analyzed with SPSS 24.0 (SPSS Inc., IL, USA). Shapiro-Wilk test was used to determine the normality of data. F test (two groups) and Brown-Forsythe test (three or more groups) were used to test the homogeneity of variance. Continuous variables with normal distribution were presents as mean ± SEM. Data with normal distribution were compared using Student’s t-test for two groups, and one-way analysis of variance (ANOVA) with Turkey’s or Dunnett’s post-hoc analyses were employed for comparisons involving three groups or more. A value of p < 0.05 are considered as statistically significant. 3. Results 3.1 Dynamic Expression of NMB and Its Receptors in Ovarian Follicle Development of Goats To explore the roles of NMB and its receptors in ovarian follicular development, we first cloned and performed structural analyses of NMB and its receptors, revealing that goat NMB and receptor nucleotide sequences are highly conserved among species. Transmembrane topology prediction showed that both NMBR and GRPR possess seven transmembrane domains (Figure S1 ). We then examined NMB and receptor expression and localization in goat ovaries at 3 and 9 months of age. Compared with 3-month-old goats, 9-month-old ovaries exhibited significantly higher NMB and GRPR mRNA and protein levels, whereas NMBR expression was markedly reduced (Fig. 1 A-B). Meanwhile, IHC results showed that NMB and its receptors are expressed during goat ovarian follicular development, and in antral follicles, NMB and its receptors are mainly localized in GCs, with no positive signals observed in the negative control group (Fig. 1 C). Moreover, we analyzed the expression of NMB and its receptors in healthy follicles of different diameters. The results showed that the mRNA and protein relative expression levels of NMB and GRPR significantly increased with the enlargement of follicle diameter, whereas the gene and protein expression levels of NMBR significantly decreased with increasing follicle diameter (Fig. 1 D-E). Additionally, both NMB and its receptors were expressed in GCs, and IF experiments demonstrated that NMB and NMBR were primarily localized in the cytoplasm of GCs, while GRPR was expressed in both the nucleus and cytoplasm (Figure S2). These results collectively indicate that NMB plays a role in ovarian follicle development in goats. 3.2 NMB Promotes Goat GC Proliferation via NMB To investigate the effect of NMB on the proliferation of goat GCs, we treated goat GCs with different concentrations of NMB (10⁻¹⁰, 10⁻⁹, 10⁻⁸, 10⁻⁷, and 10⁻⁶ M) for varying durations (3, 6, 12, 24, and 48 h). We found that treatment with 10⁻⁷ M NMB for 24 h significantly enhanced the proliferative capacity of goat GCs (Figure S3 A-F). Additionally, to determine the receptor through which NMB promotes goat GC proliferation, we examined the effects of NMBR and GRPR antagonists on the proliferation of goat GCs. The results showed that antagonizing GRPR had no significant effect on the protein expression level of PCNA in goat GCs (Figure S3H). However, antagonizing NMBR inhibited the proliferation rate of goat GCs (Fig. 2 A) and blocked the proliferative effect of NMB, leading to significant decreases in both mRNA and protein expression levels of PCNA (Fig. 2 B-C). These results indicate that NMB primarily enhances the proliferative capacity of goat GCs by binding to NMBR. To investigate the underlying reasons for changes in goat GC proliferation rates after NMB treatment, we analyzed the number of cells in G1, S, and G2 phases using flow cytometry. Compared with the control group, NMB significantly increased the number of cells in S phase, while co-treatment with NMBR antagonist and NMB significantly reduced the number of S-phase cells (Fig. 2 D-E). Additionally, the protein expression levels of cyclin (CCNE1) and cyclin-dependent kinases (CDK2, CDK6, and CDK1) were detected (Fig. 2 F). NMB markedly upregulated CCNE1, CDK2, CDK6, and CDK1 protein expression, and these increases were reversed by NMBR antagonism. Together, these data indicate that NMB regulates GC cell-cycle progression via NMBR. 3.3NMB Regulates Ca²⁺ in Goat GCs via Activating the NMBR-PLCβ1-IP3R Pathway To investigate whether NMB regulates goat GC proliferation by affecting Ca²⁺ homeostasis, we first measured intracellular Ca²⁺ levels. The results showed that NMB treatment significantly increased Ca²⁺ concentration in goat GCs compared to the control group, whereas antagonizing NMBR markedly reduced Ca²⁺ levels (Fig. 3 A). We subsequently examined the expression changes of key proteins in the endoplasmic reticulum calcium pathway and found that NMB treatment significantly upregulated PLCβ1 and IP3R protein expression, whereas NMBR antagonism reversed these effects (Fig. 3 B). Meanwhile, the ER Ca²⁺ fluorescence intensity was significantly decreased in the NMB-treated group but restored upon NMBR antagonism (Fig. 3 C). ER-Tracker staining revealed enhanced fluorescence intensity and uniform ER morphology in the NMB group, indicating ER activation, while reduced fluorescence in the antagonist-treated group suggested ER dysfunction (Fig. 3 D). These data demonstrate that NMB regulates intracellular Ca²⁺ homeostasis via the NMBR–PLCβ1–IP3R axis, thereby modulating GC cell-cycle progression and proliferation. 3.4 NMB enhances the proliferation ability of goat GCs by regulating the IRE1α pathway of UPR er through NMBR To further elucidate the mechanism by which NMB affects goat GC proliferation through the ER, we examined the expression of proteins associated with the three key regulatory pathways of the unfolded protein response (UPR) in the ER. The results showed that compared with the control group, NMB treatment significantly decreased the expression of phospho-IRE1α and XBP1S, while co-treatment with NMBR antagonist and NMB significantly upregulated the expression levels of phospho-IRE1α and XBP1S (Fig. 4 A-B). However, the related proteins of the other two pathways (ATF6 and PERK) showed no significant changes in expression compared to the control group after NMB treatment (Figure S4). Additionally, interference with XBP1S inhibited goat GC proliferation and hindered the transition of the cell cycle from G1 to S phase, but NMB treatment rescued the effects caused by XBP1S deficiency (Fig. 4 C-F). These results demonstrate that the binding of NMB to NMBR modulates Ca²⁺ homeostasis, thereby regulating the IRE1α pathway of the UPR er , which ultimately influences the cell cycle and proliferation of goat GCs. 3.5 NMB regulates mitochondrial morphology and function through NMBR To investigate the effect of NMB on mitochondrial morphology, we visualized mitochondria using Mito Tracker under confocal microscopy. The results demonstrated that NMB treatment induced mitochondrial fusion in goat GCs, characterized by an increased proportion of elongated mitochondria, whereas antagonizing NMBR triggered mitochondrial fission (Fig. 5 A-B). TEM confirmed that NMB-treated cells exhibited elongated mitochondria with increased length and diameter, while NMBR antagonism caused structural damage, including disorganized cristae and pale matrix (Fig. 5 C). Quantitative morphometric analysis showed that NMB significantly increased mitochondrial area, perimeter, aspect ratio, and form factor (Fig. 5 D-G). Additionally, we examined the expression of key mitochondrial fusion and fission proteins. NMB treatment significantly upregulated the expression of mitochondrial fusion proteins MFN1, MFN2, and OPA1, while downregulating the fission protein DNM1L. However, co-treatment with the NMBR antagonist reversed the effects of NMB on the expression levels of mitochondrial fusion and fission proteins (Fig. 5 H). Additionally, to investigate the effect of NMB on mitochondrial function, we measured the activities of MMP, ATP production, MRC complexes (CI-CV), and cellular reactive ROS in goat GCs treated with NMB. The results revealed that NMB significantly increased MMP, ATP production, and the activities of MRC complexes CI-CV, while decreasing cellular ROS generation. However, co-treatment with the NMBR antagonist reversed the effects induced by NMB (Fig. 6 A-H). These findings indicate that NMB binding to NMBR regulates mitochondrial function through mitochondrial dynamics, thereby modulating the proliferative capacity of goat GCs. 3.6 NMB-NMBR regulates Ca²⁺ transport in goat GCs via MAMs To elucidate how NMB influences Ca²⁺ translocation and thereby regulates goat GC proliferation, we measured Ca²⁺ levels in the ER, cytosol, and mitochondria of GCs following NMB treatment. NMB significantly reduced ER Ca²⁺ levels (Fig. 3 C) while markedly elevating cytosolic and mitochondrial Ca²⁺ (Fig. 3 A; Fig. 7 A). Because MAMs mediate inter-organelle Ca²⁺ flux, we examined ultrastructural MAMs by TEM. NMB increased ER-mitochondrial contact sites, as indicated by a higher ratio of MAMs associated ER length to mitochondrial perimeter; NMBR antagonism reversed this effect (Fig. 7 B). Complementary confocal co-staining with ER and mitochondrial probes revealed increased yellow overlap in merged images, denoting MAMs. Fluorescence colocalization analysis showed that NMB treatment significantly enhanced MAMs coupling in goat GCs, whereas co-treatment with the NMBR antagonist and NMB markedly reduced MAMs coupling (Fig. 7 C). IRE1α physically interacts with IP3R to regulate calcium flux between mitochondria and the ER. Our experiments showed that NMB treatment significantly upregulated the protein expression levels of IRE1α and IP3R in goat GCs compared to the control group (Fig. 7 E). Furthermore, Co-IP assays confirmed the interaction between IRE1α and IP3R (Fig. 7 D). These results demonstrate that the binding of NMB to NMBR modulates Ca²⁺ transport between mitochondria and the ER by regulating the MAMs structure via the IRE1α-IP3R-VDAC1 pathway. 4. Discussion As the fundamental functional units of the ovary, follicular development is central to oogenesis and directly determines female reproductive capacity. GCs, the most abundant somatic cells within follicles, participate in folliculogenesis, oocyte maturation, and atresia regulation throughout the entire follicular lifespan. GC proliferation and estrogen synthesis constitute critical factors supporting follicular growth and development. This study demonstrates that NMB promotes goat GC proliferation through binding to NMBR, providing a theoretical basis for further investigating NMB's mechanism in the female reproductive system while offering insights into potential mechanisms governing caprine follicular growth and development. NMB can affect cell proliferation and thereby participate in animal reproductive functions [ 26 ]. Through cloning and sequencing, we found that the coding nucleotide sequences of goat NMB and its receptors (NMBR/GRPR) exhibit high homology with ruminants such as cattle and sheep. Both NMBR and GRPR possess seven transmembrane domains and are typical G protein-coupled receptors [ 27 ], consistent with previous reports [ 23 ]. Studies have shown that NMB regulates the release of LH and prolactin [ 10 , 11 ], and NMB and its receptors are expressed in the hypothalamus, pituitary, and ovaries [ 23 ], indicating that NMB influences follicular development and function via the hypothalamic-pituitary-ovarian axis. Our results confirmed that NMB and its receptors are highly expressed in ovarian follicles, with predominant localization in granulosa cells of antral follicles. IF revealed that both NMB and its receptors are expressed in granulosa cells. Accumulating evidence suggests that NMB/NMBR contributes to regulating proliferation across diverse cell types [ 28 , 29 ]. Our findings provide compelling evidence that NMB exerts an enhancing effect on the proliferation of goat GCs, as reflected by the increased G1/S cell population and elevated expression of PCNA, CCNE1, and CDK2. Furthermore, we observed that antagonizing NMBR impedes the effects of NMB on goat GC proliferation and cell cycle progression, aligning with existing research findings [ 28 , 30 ]. These results demonstrate that NMB primarily promotes goat GC proliferation through binding to NMBR, thereby playing a critical role in follicular growth. Upon binding to its receptor, NMB activates multiple intracellular signaling pathways, leading to cell-specific responses to exert biological functions [ 27 ]. Our results demonstrate that NMB regulates calcium homeostasis in goat GCs by activating the NMBR-PLCβ1-IP3R pathway. Moreover, studies have shown that ER Ca²⁺ homeostasis modulates cell proliferation [ 31 ] and affects cell cycle progression [ 32 ]. Our findings support this notion, as NMB treatment significantly alters ER Ca²⁺ levels, thereby promoting cell proliferation. In mouse GCs, disruption of ER Ca²⁺ homeostasis triggers endoplasmic reticulum stress (ERS) activation and impairs follicular development [ 33 ]. ERS activation induces cell cycle arrest (e.g., CHOP-mediated suppression of Cyclin D1 expression) and apoptosis (e.g., JNK-mediated activation of Bax) through the PERK/eIF2α/CHOP and IRE1α/JNK/XBP1s pathways [ 34 ]. Our study reveals that antagonizing NMBR disrupts ER Ca²⁺ dynamics, leading to ERS activation, characterized by increased phosphorylation of IRE1α and elevated XBP1s splicing, which ultimately induces cell cycle arrest in goat GCs. In diabetic models, reducing ERS mitigates β-cell apoptosis and delays disease progression [ 35 ]. Therefore, we propose that NMB/NMBR signaling modulates the ERS threshold via Ca²⁺ homeostasis, thereby determining granulosa cell fate (proliferation/apoptosis) and influencing follicular development. Mitochondrial morphology and function are critical for maintaining normal cell proliferation. Our results revealed that the binding of NMB to NMBR maintains mitochondrial dynamics by inducing mitochondrial fusion and promoting the expression of fusion-associated proteins. Mitochondrial dynamics are central to their quality control, which relies on the coordinated regulation of fission and fusion to adjust mitochondrial morphology and thereby modulate their function [ 36 ]. We found that NMB treatment upregulated the expression of MFN1, MFN2, and OPA1 while downregulating DNM1L and FIS1, leading to enlarged mitochondrial morphology and a greater tendency toward fusion. Fusion is considered beneficial for mitochondrial function, as it allows metabolites and proteins to distribute throughout the mitochondrial network, maximizing bioenergetic capacity [ 37 , 38 ]. Studies have shown that downregulation of OPA1 or dysfunction of MFN1 and MFN2 significantly reduces mitochondrial respiratory capacity when fueled by substrates for electron transport chain complexes I, II, and IV [ 39 , 40 ]. Our results corroborate this finding, demonstrating that NMB enhances mitochondrial fusion, increases MMP, boosts respiratory chain enzyme activity, and elevates ATP production. This indicates a close association between mitochondrial morphological changes and functional outcomes. Research has found that elevated MMP and low ROS levels activate pro-proliferative pathways, thereby promoting the expression of cell cycle-related proteins [ 41 , 42 ]. Our data support this hypothesis, showing that NMB sustains high MMP to ensure sufficient ATP supply and maintain low ROS levels, which in turn promotes the expression of CCNE1 and CDK2 proteins. Therefore, we propose that NMB enhances mitochondrial function by regulating the balance of mitochondrial dynamics (fusion/fission), thereby coordinating the expression of cell cycle proteins and proliferation processes. This mechanism likely underlies the maintenance of goat GCs homeostasis and the promotion of follicular development. MAMs serve as critical platforms for material exchange between mitochondria and the ER [ 43 ], with ER-mediated Ca²⁺ release and recycling being closely linked to MAMs structure and function [ 44 ]. Our study demonstrated that NMB treatment facilitates Ca²⁺ transfer from the ER to mitochondria, leading to elevated mitochondrial Ca²⁺ levels. By stimulating electron transport chain (ETC) complexes, mitochondrial Ca²⁺ enhances the tricarboxylic acid cycle and OXPHOS, thereby boosting ATP synthase activity and ATP generation, ultimately facilitating cell proliferation [ 45 ]. Our findings validate this cascade: NMB-induced elevation of mitochondrial Ca²⁺ levels enhance respiratory chain complex activity and ATP synthase function, resulting in increased ATP generation and augmented proliferative capacity of goat GCs. Studies indicate that MAMs integrity is essential for Ca²⁺ transfer and intracellular homeostasis [ 46 ], with enhanced mitochondrial-ER coupling promoting mitochondrial respiration and bioenergetics, thereby improving cellular adaptability to stress [ 47 ]. Consistent with this, NMB treatment strengthens MAMs coupling efficiency, optimizing mitochondrial function to promote goat GC proliferation. The IP3R–VDAC1 complex primarily regulates MAMs associated Ca²⁺ transport [ 48 ], while the ER stress sensor protein IRE1α physically interacts with IP3R within MAMs, influencing its subcellular localization, controlling mitochondrial Ca²⁺ uptake, and playing a pivotal role in cell survival [ 49 ]. Our results revealed that NMB treatment upregulates IRE1α and IP3R expression, and CO-IP confirmed protein-protein interaction between IRE1α and IP3R. Therefore, we propose that NMB modulates the structure of MAMs via the IRE1α-IP3R-VDAC1 complex, thereby regulating inter-organellar Ca²⁺ flux between mitochondria and the ER and influencing goat GC proliferation. This study has several limitations. Firstly, the study primarily utilized NMBR antagonists for receptor antagonism experiments. Subsequent work should employ CRISPR-Cas9 technology to knock down the NMBR gene, thereby achieving receptor-specific silencing. Secondly, the conclusions drawn are mainly based on in vitro experiments. Future research needs to enhance clinical relevance through in vivo models and cross-species validation. Conclusion In summary, our results indicate that NMB exerts its effects by binding to NMBR, regulating intracellular Ca²⁺ homeostasis, alleviating ER stress, triggering mitochondrial metabolic reprogramming, and promoting cell proliferation. These findings expand the functional understanding of NMB in reproductive biology, provide novel evidence for calcium signaling-mediated organelle crosstalk in proliferation regulation, and suggest potential targets for optimizing reproductive efficiency and treating ovarian dysfunction. Abbreviations NMB Neuromedin B NMBR GRPR Neuromedin B receptor Gastrin releasing peptide receptor GCs Granulosa cells PCNA Proliferating cell nuclear antigen CCNE1 Cyclin E1 CDK1 CDK2 CDK6 Cyclin dependent kinase 1 Cyclin dependent kinase 2 Cyclin dependent kinase 6 ER Endoplasmic reticulum Ca 2+ Calcium ion PLCβ1 Phospholipase C-beta1 IP3R/ITPR1 Inositol 1,4,5-trisphosphate receptor type 1 SERCA1/ATP2A1 ATPase sarcoplasmic/endoplasmic reticulum Ca 2+ transporting 1 UPR er Endoplasmic reticulum unfolded protein response ATF4 Activating transcription factor 4 ATF6 Activating transcription factor 6 ERp57/PDIA3 Protein disulfide isomerase family A member 3 IRE1 α Inositol-requiring enzyme 1α XBP1S X-box binding protein 1 BIP/HSPA5 Heat shock protein family A (Hsp70) member 5 PERK/EIF2AK3 Eukaryotic translation initiation factor 2 alpha kinase 3 MAMs Mitochondria-associated ER membranes VDAC1 Voltage-Dependent Anion Channel 1 ATP Adenosine triphosphate eIF2α Eukaryotic translation initiation factor 2α MFN1 Mitofusion 1 MFN2 Mitofusion 2 OPA1 Optic Atrophy 1 GAPDH Glyceraldehyde-3-phosphate dehydrogenase Declarations Ethics approval and consent to participate All experiments were conducted in accordance with the approved Guidelines for Animal Experiments of Nanjing Agricultural University and received approval from the Animal Care and Use Committee of Nanjing Agricultural University (Approval ID: SYXK2022-0031). All antibodies and other chemicals of reagent grade were purchased from biotechnology companies. Consent for publication Not applicable. Availability of data and materials Data sharing is not applicable to this article as no datasets were generated or analysed during the current study. Funding This study was supported by the National Key R&D program of China (2021YFD1200902), the National Natural Science Foundation of China (No. 32472993), the Fundamental Research Funds for the Central Universities (No. KYTZ2023003). Authors' contributions Rongxin Xia: Writing – original draft, Writing – review & editing, Methodology, Investigation, Formal analysis, Data curation. Qi Zhang: Writing – review & editing, Methodology, Investigation, Data curation. Junhui Shao: Writing – review & editing, Formal analysis, Data curation. Yuan Wang: Methodology, Investigation, Data curation. Xinyi Lv: Methodology, Investigation, Data curation. Rui Chen: Methodology, Investigation, Data curation. Zhen Lu: Writing – review & editing, Supervision, Methodology. Yanli Zhang: Project administration, Supervision, Funding acquisition. Feng Wang: Project administration Supervision, Funding acquisition. Guomin Zhang：Writing – review & editing, Writing – original draft, Supervision, Project administration, Funding acquisition, Conceptualization. Acknowledgements The authors also thank all the members of Feng Wang’s laboratory who contributed to sample collection. References Mcgee EA, Hsueh AJ. Initial and cyclic recruitment of ovarian follicles. Endocr Rev. 2000;21(2):200–14. Filatov M, Khramova Y, Parshina E, Bagaeva T, Semenova M. Influence of gonadotropins on ovarian follicle growth and development in vivo and in vitro. Zygote. 2017;25(3):235–43. Irusta G, Parborell F, Peluffo M, Manna PR, Gonzalez-Calvar SI, Calandra R, et al. Steroidogenic acute regulatory protein in ovarian follicles of gonadotropin-stimulated rats is regulated by a gonadotropin-releasing hormone agonist. 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Supplementary Files supplementalinformation.docx Cite Share Download PDF Status: Published Journal Publication published 19 Nov, 2025 Read the published version in Journal of Ovarian Research → Version 1 posted Editorial decision: Revision requested 21 Jul, 2025 Reviews received at journal 08 Jul, 2025 Reviews received at journal 07 Jul, 2025 Reviewers agreed at journal 30 Jun, 2025 Reviewers agreed at journal 29 Jun, 2025 Reviewers invited by journal 27 Jun, 2025 Editor assigned by journal 08 Jun, 2025 Submission checks completed at journal 05 Jun, 2025 First submitted to journal 03 Jun, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6811713","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":478660545,"identity":"71322055-b3a9-434b-b6e4-ee3abd1f6793","order_by":0,"name":"Rongxin Xia","email":"","orcid":"","institution":"Nanjing Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Rongxin","middleName":"","lastName":"Xia","suffix":""},{"id":478660546,"identity":"51852f66-f93c-4839-8325-9b6fe8c41377","order_by":1,"name":"Qi Zhang","email":"","orcid":"","institution":"Nanjing Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Qi","middleName":"","lastName":"Zhang","suffix":""},{"id":478660547,"identity":"d3523fc1-aa5e-496d-8d64-6c9f47a11858","order_by":2,"name":"Junhui Shao","email":"","orcid":"","institution":"Nanjing Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Junhui","middleName":"","lastName":"Shao","suffix":""},{"id":478660548,"identity":"57863e65-c1a2-4311-8f2f-76140be10eb6","order_by":3,"name":"Yuan Wang","email":"","orcid":"","institution":"Nanjing Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yuan","middleName":"","lastName":"Wang","suffix":""},{"id":478660549,"identity":"3d0c251d-63e3-4d18-9a7b-37ffd18c8870","order_by":4,"name":"Xinyi Lv","email":"","orcid":"","institution":"Nanjing Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Xinyi","middleName":"","lastName":"Lv","suffix":""},{"id":478660550,"identity":"fdfaf608-bff9-4f85-be68-0bef63671e94","order_by":5,"name":"Rui Chen","email":"","orcid":"","institution":"Nanjing Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Rui","middleName":"","lastName":"Chen","suffix":""},{"id":478660551,"identity":"d7a3494f-2c6a-4a43-a526-8dc9880cb68e","order_by":6,"name":"Zhen Lu","email":"","orcid":"","institution":"Mashan County Centre for Animal Disease Control and prevention，Nanning City","correspondingAuthor":false,"prefix":"","firstName":"Zhen","middleName":"","lastName":"Lu","suffix":""},{"id":478660552,"identity":"1d0a5d8c-ed97-4028-be3f-b3db68242809","order_by":7,"name":"Yanli Zhang","email":"","orcid":"","institution":"Nanjing Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yanli","middleName":"","lastName":"Zhang","suffix":""},{"id":478660553,"identity":"07c6b739-b58b-41d5-99f6-ad27cc5bbcf9","order_by":8,"name":"Feng Wang","email":"","orcid":"","institution":"Nanjing Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Feng","middleName":"","lastName":"Wang","suffix":""},{"id":478660555,"identity":"36a29835-5f43-4e13-bde2-eb0b4871f273","order_by":9,"name":"Guomin Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+0lEQVRIiWNgGAWjYDACCRiDmYHxAQMPmGlAtBZmAxK1MDCwwdj4tcjPbn728Muvw3K67bzHqgtk7BIb2Ju3STDU3MGphXHOMXNj2b7DxmaH+dJuz+BJTmzgOVYmwXDsGU4tzBIJZtKSPYcTtx3mMbvNw8Oc2CCRYybB2HAYpxY2ifRvcC3FPDz1iQ3yb/Br4QGaKfnhB0QLMw/PYaAtPPi1SEjklEkzNqQD/cJjLM3Dc9y4jSet2CLhGG4t8jPSt0n++GMtZ3b+jOFn3p5q2X72wxtvfKjBrQUcBLxtUBZjD9B3IEYCXg1AhT/+wJg/CCgdBaNgFIyCEQkASetOHT/oJZkAAAAASUVORK5CYII=","orcid":"","institution":"Nanjing Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Guomin","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2025-06-03 13:23:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6811713/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6811713/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13048-025-01844-7","type":"published","date":"2025-11-19T15:57:51+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":85787478,"identity":"0257cb57-b952-4106-abac-fce97979ac5f","added_by":"auto","created_at":"2025-07-01 16:38:00","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":177441,"visible":true,"origin":"","legend":"\u003cp\u003eExpression and localization of NMB and its receptors in ovarian follicles​​. (A) qRT-PCR analysis of mRNA expression levels of NMB and its receptors in ovaries of 3-month-old and 9-month-old goats. (B) Western blot detection of protein expression levels of NMB and its receptors in ovaries of 3-month-old and 9-month-old goats. (C) Localization of NMB and its receptor proteins in goat ovaries. Tissue sections were stained with specific antibodies and counterstained with hematoxylin. Positive signals appear brown, and counterstained backgrounds are blue. ​​a-a2: Stained with NMB polyclonal antibody; ​​b-b2​​: Stained with NMBR polyclonal antibody; ​​c-c2​​: Stained with GRPR polyclonal antibody; ​​d-d2: Negative control (no significant immunoreactivity observed when primary antibody was replaced with normal rabbit serum). ​​EN​​: Oocyte nest; ​​PR​​: Primary follicle; ​​SC​​: Secondary follicle. Scale bar: 100 μm. (D) qRT-PCR analysis of mRNA expression of NMB and its receptors in follicles of different diameters. (E) Western blot analysis of protein expression of NMB and its receptors in follicles of different diameters. Data are expressed as fold changes and presented as mean ± S.E.M. from at least three independent experiments. Different letters indicate significant differences between groups (\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6811713/v1/90ba1f065b7384b875d9b6b7.png"},{"id":85787477,"identity":"5a899505-2949-4516-aa65-17ebae10f156","added_by":"auto","created_at":"2025-07-01 16:38:00","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":102617,"visible":true,"origin":"","legend":"\u003cp\u003eNMBR regulates proliferation and cell cycle in goat GCs​​. (A) EDU assay analyzing the proliferative capacity of goat GCs under NMB treatment with or without NMBR antagonism (PD168368). (B) qRT-PCR and (C) Western blot analysis of PCNA expression levels in goat GCs under NMB treatment with or without NMBR antagonism. (D-E) Flow cytometry analysis of cell cycle distribution in goat GCs under NMB treatment with or without NMBR antagonism. (F) Western blot analysis of CCNE1 and CDK2/6/1 expression levels in goat GCs under NMB treatment with or without NMBR antagonism. Data are expressed as fold changes and presented as mean ±S.E.M. from at least three independent experiments. Different letters indicate significant differences between groups (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6811713/v1/174771cb055565c3019a2d59.png"},{"id":85788739,"identity":"ef697806-1709-4880-bc6d-039288d1eba3","added_by":"auto","created_at":"2025-07-01 16:54:00","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":66465,"visible":true,"origin":"","legend":"\u003cp\u003eNMB maintains Ca²⁺ homeostasis in goat GCs via the NMBR-PLCβ1-IP3R Axis​​. (A) Quantitative analysis of intracellular Ca²⁺ concentration in goat GCs under NMB treatment with or without NMBR antagonism. (B) Western blot analysis of PLCβ1, IP3R, and SERCA1 expression levels in goat GCs under NMB treatment with or without NMBR antagonism. (C) Histogram statistics of relative ER Ca²⁺ fluorescence intensity in goat GCs under NMB treatment with or without NMBR antagonism. (D) Confocal microscopy images of ER Ca²⁺ dynamics in goat GCs under NMB treatment with or without NMBR antagonism. ER was stained with ER-Tracker (red), and nuclei were labeled with Hoechst (blue). Data are expressed as fold changes and presented as mean ± S.E.M. from at least three independent experiments. Different letters indicate significant differences between groups (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6811713/v1/1b3104cfa3c2e499050ba14d.png"},{"id":85788194,"identity":"aff95de0-dc38-4250-8dd6-777ecdb90728","added_by":"auto","created_at":"2025-07-01 16:46:00","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":73828,"visible":true,"origin":"","legend":"\u003cp\u003eNMB enhances the proliferative capacity of goat GCs by regulating the UPR\u003csup\u003eer\u003c/sup\u003e IRE1α pathway. (A) Western blot analysis of phospho-IRE1α, XBP1S, and Bip expression levels in goat GCs under NMB treatment with or without NMBR antagonism. (B) Statistical analysis of changes in the p-IRE1α/IRE1α protein expression ratio in goat GCs under NMB treatment with or without NMBR antagonism. (C) Western blot analysis of the effects of different interference sequences (siXBP1S-1/2/3/4) on XBP1S expression levels in goat GCs. (D) Western blot analysis of PCNA expression levels in goat GCs with or without NMB treatment under siXBP1S conditions. (E) Flow cytometry analysis of cell cycle distribution in goat GCs with or without NMB treatment under siXBP1S conditions. (F) Western blot analysis of CCNE1 and CDK2/6/1 expression levels in goat GCs with or without NMB treatment under siXBP1S conditions. Data are expressed as fold changes and presented as mean ± S.E.M. from at least three independent experiments. Different letters indicate significant differences between groups (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6811713/v1/5f0d6c88b885939abbfec0f1.png"},{"id":85787482,"identity":"9271520a-8502-4dc1-a21f-af5ea37cb7fa","added_by":"auto","created_at":"2025-07-01 16:38:00","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":206178,"visible":true,"origin":"","legend":"\u003cp\u003eNMB modulates mitochondrial morphology and structure in goat GCs. (A) Confocal microscopy images of mitochondria in goat GCs under NMB treatment with or without NMBR antagonism. Mitochondria were stained with Mito-tracker (green), and nuclei (blue) were labeled with Hoechst. Scale bar: 20 μm. (B) Statistical analysis of fused and fragmented mitochondria by Mitochondria Analyzer. (C) Representative TEM images of mitochondria in goat GCs under NMB treatment with or without NMBR antagonism. Arrows indicate mitochondria. (D-G) Software-based quantification of mitochondrial morphology parameters from TEM observations: (D) aspect ratio, (E) perimeter, (F) surface area, and (G) shape factor. Mitochondrial shape factor was calculated as perimeter\u003csup\u003e 2\u003c/sup\u003e /4π⋅surface area, reflecting structural complexity and branching. (H) Western blot analysis of mitochondrial dynamics-related genes in goat GCs under NMB treatment with or without NMBR antagonism. Data represent mean ± S.E.M. from at least three independent experiments. Different letters indicate significant differences between groups (\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6811713/v1/e429cd32e450094869d49ddf.png"},{"id":85788196,"identity":"c21444d2-1e04-4605-b758-2bfb05b97afa","added_by":"auto","created_at":"2025-07-01 16:46:00","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":95583,"visible":true,"origin":"","legend":"\u003cp\u003eNMB affects mitochondrial function in goat GCs​​. (A) Representative images (left) and statistical quantification (right) of mitochondrial membrane potential in goat GCs under NMB treatment with or without NMBR antagonism, stained with JC-1. (B) Representative images (left) and statistical quantification (right) of ROS production in goat GCs under NMB treatment with or without NMBR antagonism, stained with DCFH-DA. DCFH-DA: green; Hoechst: blue. (C) Intracellular ATP levels in goat GCs under NMB treatment with or without NMBR antagonism. (D-H) Activities of MRC complexes I-V in goat GCs under NMB treatment with or without NMBR antagonism. Data represent mean ± S.E.M. from at least three independent experiments. Different letters indicate significant differences between groups (\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-6811713/v1/73894e809c024b4d366fc2f4.png"},{"id":85787492,"identity":"207898b3-a2f2-4436-bec0-a40b155af4fc","added_by":"auto","created_at":"2025-07-01 16:38:00","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":193974,"visible":true,"origin":"","legend":"\u003cp\u003eNMB modulates calcium transport via MAMs in goat GCs​​. (A) Histogram statistics of relative mitochondrial Ca²⁺ fluorescence intensity in goat GCs under NMB treatment with or without NMBR antagonism. (B) Representative transmission electron microscopy images (left) and statistical quantification (right) of MAMs in goat GCs under NMB treatment with or without NMBR antagonism. Green lines mark mitochondrial components, and red lines mark ER components within MAMs. ER-mitochondria contacts with a distance \u0026lt;50 nm was defined as MAMs. (C) Confocal microscopy images (left) showing Mito-ER colocalization in goat GCs stained with Mito-Tracker (green) and ER-Tracker (red) under NMB treatment with or without NMBR antagonism. Quantification of Pearson’s correlation coefficient for ER-mitochondria colocalization (right). Scale bar: 20 μm. (D) CO-IP the interaction between IP3R and IRE1α proteins. (E) Western blot analysis of IRE1α and VDAC1 expression levels in goat GCs under NMB treatment with or without NMBR antagonism. Data represent mean ±S.E.M. from at least three independent experiments. Different letters indicate significant differences between groups (\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-6811713/v1/88361c4625fd0ca978e0bd6a.png"},{"id":96650285,"identity":"8144d8dc-283b-44db-9ca0-1db9a62bab16","added_by":"auto","created_at":"2025-11-24 16:10:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2323253,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6811713/v1/9d11936b-f37e-4688-923e-7113062d297f.pdf"},{"id":85787488,"identity":"68a4f620-46d6-4176-adeb-064f039e6d1c","added_by":"auto","created_at":"2025-07-01 16:38:00","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":2311776,"visible":true,"origin":"","legend":"","description":"","filename":"supplementalinformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-6811713/v1/a746c110fc4a0cc9c26291d8.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Neuromedin B Drives Goat Granulosa Cell Proliferation via NMBR-Mediated Calcium Homeostasis","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe ovary, as the central organ of the female reproductive system, determines the reproductive potential of females through the developmental status of its functional unit-the ovarian follicle [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Follicular development is coordinately regulated by endocrine signals (e.g., gonadotropins) and local ovarian factors (e.g., growth factors and cytokines) [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Granulosa cells (GCs), the core functional cells within follicles, critically govern key reproductive events including follicular growth, ovulation efficiency, luteal function, and steroidogenesis through their proliferation and differentiation states [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Therefore, elucidating the regulatory mechanisms of GC proliferation is pivotal for understanding ovarian physiology.\u003c/p\u003e \u003cp\u003eNeuromedin B (NMB), a member of the mammalian bombesin like peptide family, is a decapeptide composed of 10 amino acids [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. NMB is widely distributed in the central nervous system and peripheral tissues (e.g., gastrointestinal tract and gonads) [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], mediating diverse biological functions by binding to specific receptors and activating downstream signaling pathways [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Notably, NMB and its receptor exhibit high expression in the reproductive systems of model animals such as humans and mice [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], suggesting their potential regulatory roles in reproductive function. Moreover, studies show that NMB enhances the excitability of gonadotropin-releasing hormone (GnRH) neurons in female mice [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], promoting hypothalamic GnRH secretion to regulate pituitary luteinizing hormone (LH) release [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. In addition, NMB directly stimulates pituitary prolactin secretion [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], indicating its involvement in follicular development via the hypothalamic\u0026ndash;pituitary\u0026ndash;ovarian axis. Together, these findings indicate that NMB-mediated regulation of reproductive function involves multiple pathways, genes, and factors, yet the precise mechanisms remain to be elucidated.\u003c/p\u003e \u003cp\u003eMitochondria and the endoplasmic reticulum (ER) are core organelles regulating cellular biological functions. Approximately 5\u0026ndash;20% of the mitochondrial outer membrane forms highly dynamic physical contacts with the ER, known as mitochondria-associated ER membranes (MAMs) [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. MAMs formation is not merely membrane contact between organelles but a complex, highly regulated process. MAMs mediate the transfer of calcium ion (Ca\u0026sup2;⁺), phospholipids, and metabolites between the two organelles [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], thereby regulating lipid metabolism [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], mitochondrial dynamics [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], ER stress responses [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], calcium homeostasis [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], and cell fate [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Notably, MAMs formation is essential for optimal cholesterol transfer from the ER to mitochondria and serves as a central hub for initiating mitochondrial steroidogenesis [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], suggesting a critical regulatory role of MAMs in reproductive regulation. Meanwhile, a high-fat diet can increase the levels of MAMs related proteins in mouse germ cells, elevate mitochondrial calcium levels, and promote apoptosis [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Exposure to endocrine-disrupting chemicals induces MAMs alterations that disrupt ovarian function in mice [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. These observations underscore the potential importance of MAMs in follicular development.\u003c/p\u003e \u003cp\u003eUsing goat GCs as a model, this study combined NMB treatment and Neuromedin B receptor (NMBR) antagonism experiments to reveal that NMB regulates ER and intracellular Ca\u0026sup2;⁺ homeostasis through binding to NMBR, thereby influencing goat GCs proliferation and cell cycle progression. We further investigated how NMB-NMBR interaction modulates the structural coupling efficiency of MAMs and its impact on mitochondrial function and dynamics. The results confirmed that NMB promotes goat GC proliferation via a \"Ca\u0026sup2;⁺ homeostasis-MAMs-mitochondrial metabolism\" cascading signaling axis. This study systematically elucidates a novel mechanism of NMB/NMBR in ovarian function regulation, providing a theoretical breakthrough for reproductive biology research.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cp\u003e\u003cstrong\u003e2.1 Goat Ovaries and Follicles Collection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGoat ovary tissues were collected from the slaughterhouse and stored in a thermos containing sterilized normal saline (30-35 ℃, including 100 IU/L penicillin and 50 mg/L streptomycin) and transported to the laboratory within 1 h after collection. After washing five times with normal saline, the connective tissue on the ovary and the attached fallopian tube were removed. Part of the collected ovarian samples were fixed with Bouin's fixative for immunohistochemical detection; One part was immediately frozen in liquid nitrogen for subsequent extraction of total RNA and protein. According to previous methods [22]and others, healthy follicles were isolated from the ovary and divided into three diameters (≤\u0026nbsp;2 mm, 2-5 mm and\u0026nbsp;≥\u0026nbsp;5 mm, with at least 80, 60 and 40 follicles in each diameter group). The isolated follicles were rapidly cryopreserved in liquid nitrogen for subsequent analysis of gene and protein expression.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2 Cell Culture and Treatments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGCs were isolated from healthy follicles (2-7 mm in diameter) using a 24-gauge needle connected to a 5 mL syringe according to established laboratory protocols after ovarian collection from the slaughterhouse\u0026nbsp;[22]. The GCs were cultured in DMEM/F12 (1:1) medium containing 10% fetal bovine serum (FBS), 100 IU/mL penicillin, and 100 IU/mL streptomycin, and incubated at 37\u0026nbsp;°C in a 5% CO\u003csub\u003e2\u003c/sub\u003e environment.\u003c/p\u003e\n\u003cp\u003eGoat NMB (RP10507, purity \u0026gt; 95% by HPLC analysis) was synthesized by GenScript Biotech. GCs were cultured in 6-well plates until reaching 50% - 60% confluence, then treated with either different NMB concentrations (10\u003csup\u003e-10\u003c/sup\u003e, 10\u003csup\u003e-9\u003c/sup\u003e, 10\u003csup\u003e-8\u003c/sup\u003e, 10\u003csup\u003e-7\u003c/sup\u003e, 10\u003csup\u003e-6\u003c/sup\u003e M) for 48 h or 10\u003csup\u003e-7\u003c/sup\u003e M NMB for varying durations (3, 6, 12, 24, 48 h), followed by an additional 24 h treatment with 10\u003csup\u003e-7\u003c/sup\u003e M NMB.\u003c/p\u003e\n\u003cp\u003eThe NMBR antagonist PD168368 (MCE, HY-116216) was dissolved in dimethyl sulfoxide (DMSO) and stored at a stock concentration of 50 mM. For treatment, 1 μL of the stock solution or DMSO (vehicle control) was added per milliliter of medium, yielding final concentrations of 50 μM and 0.1% DMSO, respectively.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3 RNA extraction, cDNA synthesis and gene clone\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe total RNA was extracted from goat ovaries using TRIzol reagent (Vazyme, R401-01). RNA concentration was determined using the ND1000 spectrophotometer (Thermo Scientifific). RNA was reverse-transcribed into cDNA via the Primer Script RT reagent kit with gDNA eraser (TaKaRa Biotechnology). The cDNA samples were stored at -20 ℃ until use.\u003c/p\u003e\n\u003cp\u003eCloning method conducted according to the former research with minor modifications\u0026nbsp;[23]. Nucleotide sequence of PCR primers were listed in Table S1. Positive clones were randomly chosen and commercially sequenced through General Biological Technology (Beijing qingke biotechnology Ltd, china). The obtained nucleotide sequences of NMB and its two receptors were uploaded to NCBI. The similarity of amplified sequences and NCBI reference sequences was performed using BLAST (http://www.ncbi.nlm.nih.gov/blast). The phylogenetic tree analysis and homology assessments were performed using the neighbor joining and dislocation comparison method in MEGA-X software. Amino acid sequences were predicted with the Expasy Proteomics Server (http:// www.expasy.ch/tools/dna.html) and then aligned with other species using GenomeNet (http:// www.genome.jp/tools/clustalw). The accession numbers of NMB and its receptors for five species were listed in Table S2. The prediction of transmembrane domains was performed using TMHMM (http://www. cbs.dtu.dk/services/TMHMM-2.0).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4 Quantitative real-time polymerase chain reaction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted from the samples using TRIzol reagent (Vazyme, R401-01) according to the manufacturer’s instructions, and 1 μg of total RNA from each sample was reverse-transcribed into first-strand cDNA using a reverse transcription kit (Vazyme, R323). Real-time PCR (qRT-PCR) with SYBR Green was conducted on the ABI 7500 Real-Time PCR system (Applied Biosystems) using 1 μL cDNA in a total reaction volume of 20 μL, following the manufacturer’s protocol. Amplification specificity was confirmed by melting curve analysis, and the relative mRNA expression levels of target genes were quantified using the 2\u003csup\u003e−ΔΔCt\u003c/sup\u003e method, normalized against glyceraldehyde-3-phosphate dehydrogenase (\u003cem\u003eGAPDH\u003c/em\u003e) mRNA expression levels. All experiments were independently performed in triplicate, and the primer sequences for target gene amplification are provided in Table S3.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5 Western Blot Assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProteins were extracted from the samples using RIPA lysis buffer (Thermo Scientific, 89901), and the total protein concentration was determined with a BCA protein assay kit (Beyotime, P0010). Equal amounts of protein (20 μg) were separated by 4%-20% polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, ISEQ00010), and blocked with 5% skimmed milk. After overnight incubation with primary antibodies at 4\u0026nbsp;°C, the membranes were treated with secondary antibodies for 1 h at room temperature. Protein bands were visualized using the Western Bright ECL kit (Biosharp, No. BL520B-2) on an Image Quant LAS 400 system (Fiji film), and target protein quantification was performed with Image J software (Wayne Rasband), normalized to the internal control (GAPDH). Detailed information on antibodies used in this study is provided in Table S4.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.6 Immunohistochemistry analysis (IHC)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGoat ovaries were fixed, processed, and paraffin-embedded for sectioning as previously described\u0026nbsp;[24].\u0026nbsp;Following dewaxing, antigen retrieval was performed on the sections according to the protocol of the M\u0026amp;R HRP/DAB Detection IHC Kit (Vazyme, HC301-01). After blocking endogenous peroxidase activity, the sections were incubated with primary antibodies at 4\u0026nbsp;°C overnight, followed by incubation with secondary antibodies at room temperature for 2 h. Subsequently, DAB chromogenic staining and counterstaining were carried out, and the sections were dehydrated and mounted. Negative controls were run by substituting 5% rabbit serum for the primary antibody. Brown-colored positive signals were visualized, and images were captured using a Nikon microscope (T300). Antibody details are listed in Table S4.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.7 Immunofluorescence analysis (IF)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter treatment of goat GCs, the cells were fixed in 4% paraformaldehyde (PFA) (Beyotime, No. P0099), permeabilized with 0.5% Triton X-100, blocked with 5% bovine serum albumin (BSA), and then incubated with primary antibodies overnight at 4 °C. The next day, cells were incubated with secondary antibodies for 1 h, followed by nuclear staining with Hoechst 33258 (Yeasen, 40730ES03) for 10 min. Finally, coverslips were mounted onto glass slides, and images were captured using a confocal laser scanning microscope (Zeiss, LSM900). Positive signals appeared green, and negative controls were prepared by replacing the primary antibody with 1% rabbit serum.\u0026nbsp;Details of antibodies for this study are provided in Table S4.\u003c/p\u003e\n\u003cp\u003eLive-cell fluorescence staining of goat GCs was performed using either a mitochondrial green fluorescent probe (Yeasen, 40742ES50) diluted to 500 nM working solution and incubated at 37\u0026nbsp;°C in the dark for 30 min, or an ER red fluorescent probe (Yeasen, 40764ES20) diluted to 1 µM working solution and incubated at 37\u0026nbsp;°C in the dark for 20 min, followed by nuclear staining with Hoechst 33258 for 10 min. For MAMs structure analysis, cells were first incubated with the ER red fluorescent probe working solution at 37\u0026nbsp;°C in the dark for 20 min, the solution was then removed, followed by incubation with the mitochondrial green fluorescent probe working solution at 37\u0026nbsp;°C in the dark for 30 min, and finally nuclear staining with Hoechst for 10 min. Images were observed and captured using a laser confocal microscope (Zeiss, LSM900), and quantitative assessment was performed with Image J software (Wayne Rasband).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.8 Cell proliferation assay\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCell proliferation was assessed using the BeyoClick™\u0026nbsp;EdU-594 Cell Proliferation Detection Kit (Beyotime, C0078S). Briefly, cells were seeded into 24-well plates pre-placed with sterile cell slides and treated for the indicated durations. Subsequently, cells were incubated with 50 nM EdU working solution for 2 h, fixed with 4% PFA, washed with PBS containing 3% BSA, and permeabilized with PBS containing 0.3% Triton X-100. The Click reaction solution was then applied to the cells and incubated in the dark for 30 min. After washing, nuclei were counterstained with 5 μg/mL Hoechst. Finally, cells were mounted with anti-fade medium, sealed with coverslips, and imaged using a confocal laser scanning microscope (Zeiss, LSM900). Quantification of results was performed using ImageJ software (Wayne Rasband).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.9 Cell cycle distribution analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCell cycle analysis was performed via flow cytometry as follows: harvested cells were fixed overnight in ice-cold 70% ethanol, washed once with ice-cold DPBS, and stained with propidium iodide (PI) staining solution (Beyotime, C1052) prepared according to the manufacturer’s instructions, followed by incubation at 37\u0026nbsp;°C in the dark for 30 min. Red fluorescence emitted by PI-DNA complexes was detected at an excitation wavelength of 488 nm using a flow cytometer (Becton, Dickinson and Company).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.10 Intracellular Ca\u003csup\u003e2+\u003c/sup\u003e content determination\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIntracellular Ca\u003csup\u003e2+\u003c/sup\u003e levels were measured using a Calcium Colorimetric Assay Kit (Beyotime, S1063S). Briefly, after cell lysis, the supernatant was incubated with the working solution at room temperature in the dark for 10 min, while a standard curve was prepared in parallel using the provided standards. Absorbance was measured with a multimode microplate reader (Thermo Scientific), and Ca\u003csup\u003e2+\u003c/sup\u003e concentrations in each sample were calculated based on the standard curve.\u003c/p\u003e\n\u003cp\u003eIntracellular ER Ca²⁺ levels were measured using a commercial kit (BestBio, BB-481159). Briefly, after standard treatments, the culture medium was removed, and cells were washed three times with HBSS. Subsequently, cells were incubated with BBcellProbe® C93 staining working solution at 37 °C for 45 min, followed by three additional HBSS washes. Cells were then covered with HBSS and incubated again at 37 °C for 30 min. Mitochondrial Ca²⁺ levels were assessed using another kit (Beyotime, S1062S). In brief, after treatments, the medium was discarded, cells were washed three times with HBSS, and Rhod-2 staining solution was added. Cells were incubated at 37 °C for 20 min, after which the supernatant was removed, and cells were washed three times. Fluorescence images were captured using a laser scanning confocal microscope (Zeiss, LSM900), and the average fluorescence intensity of each image was quantified with Image J software (Wayne Rasband).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.11 Small interfering RNA transfection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003esiNC and siXBP1S-1/2/3/4 were purchased from Genepharma and transfected into GCs using Lipofectamine™ 3000 (Invitrogen, L3000150). The sequence information of the siRNAs is listed in Table S5.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.12 Transmission Electron Microscopy Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGCs were fixed with 2.5% glutaraldehyde (in 0.1 M PBS, pH 7.4) at 4°C for 4 h, followed by dehydration through a graded ethanol series. Samples were embedded in resin, sectioned into ultrathin slices, and dual-stained with uranyl acetate-lead citrate. Mitochondrial morphology, ultrastructural changes, and MAMs integrity were observed using TEM. Image J quantified mitochondrial aspect ratio and MAMs contact points. The fraction of mitochondria in contact with ER within a 50 nm range was calculated and normalized to the mitochondria perimeter, as previously described [25].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.13 Co-immunoprecipitation (CO-IP)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImmunoprecipitation was performed using an immunoprecipitation kit (Proteintech, PK10008). Cultured GCs were lysed with lysis buffer according to the manufacturer’s protocol, followed by incubation with specific antibodies under rotation at 4 °C overnight. Subsequently, pre-resuspended Protein A/G beads slurry was added to precipitate the immune complexes, which were further incubated under rotation at 4 °C for 4 h. After washing, the complexes were eluted, and the eluate was mixed with alkali neutralization buffer and 5×\u0026nbsp;sample buffer, heated in boiling water for 5 min, and analyzed by western blotting for the input lysates.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.14 Mitochondrial Membrane Potential Assay\u003c/strong\u003e\u003cstrong\u003e（\u003c/strong\u003e\u003cstrong\u003eJC-1\u003c/strong\u003e\u003cstrong\u003e）\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAccording to the manufacturer’s protocol (Solarbio, M8650), treated GCs were incubated with JC-1 staining working solution and thoroughly mixed. The cells were then maintained in a cell culture incubator at 37 °C for 20 min. After incubation, the liquid was aspirated, and the cells were washed twice with JC-1 staining buffer (1×). Images were observed and captured using a laser confocal microscope (Zeiss, LSM900).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.15 Reactive Oxygen Species Assay\u003c/strong\u003e\u003cstrong\u003e（\u003c/strong\u003e\u003cstrong\u003eROS\u003c/strong\u003e\u003cstrong\u003e）\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAccording to the manufacturer’s protocol (Beyotime, S0033S), treated GCs were incubated with DCFH-DA diluted to a final concentration of 10 µM and thoroughly mixed. The cells were then maintained in a cell culture incubator at 37 °C for 20 min. After incubation, the liquid was aspirated, and the cells were washed twice with DPBS. Images were observed and captured using a laser confocal microscope (Zeiss, LSM900).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.16 Mitochondrial complex reductase activity Assay (MRC)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAccording to the manufacturer’s protocols for mitochondrial respiratory chain complexes I, II, III, IV, and V (Solarbio, BC0515, BC3235, BC3245, BC0945, BC1445), the complexes were extracted from collected cells, incubated with their respective working solutions, and the absorbance was measured using a multifunctional microplate reader (Thermo Scientific). The activity levels of the complexes in the samples were subsequently calculated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.17 Statistical analysis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experiments were repeated at least three times. Statistical analyses were analyzed with SPSS 24.0 (SPSS Inc., IL, USA). Shapiro-Wilk test was used to determine the normality of data. F test (two groups) and Brown-Forsythe test (three or more groups) were used to test the homogeneity of variance. Continuous variables with normal distribution were presents as mean ± SEM. Data with normal distribution were compared using Student’s t-test for two groups, and one-way analysis of variance (ANOVA) with Turkey’s or Dunnett’s post-hoc analyses were employed for comparisons involving three groups or more. A value of \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 are considered as statistically significant.\u003c/p\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Dynamic Expression of NMB and Its Receptors in Ovarian Follicle Development of Goats\u003c/h2\u003e \u003cp\u003eTo explore the roles of NMB and its receptors in ovarian follicular development, we first cloned and performed structural analyses of NMB and its receptors, revealing that goat NMB and receptor nucleotide sequences are highly conserved among species. Transmembrane topology prediction showed that both NMBR and GRPR possess seven transmembrane domains (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). We then examined NMB and receptor expression and localization in goat ovaries at 3 and 9 months of age. Compared with 3-month-old goats, 9-month-old ovaries exhibited significantly higher NMB and GRPR mRNA and protein levels, whereas NMBR expression was markedly reduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-B). Meanwhile, IHC results showed that NMB and its receptors are expressed during goat ovarian follicular development, and in antral follicles, NMB and its receptors are mainly localized in GCs, with no positive signals observed in the negative control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMoreover, we analyzed the expression of NMB and its receptors in healthy follicles of different diameters. The results showed that the mRNA and protein relative expression levels of NMB and GRPR significantly increased with the enlargement of follicle diameter, whereas the gene and protein expression levels of NMBR significantly decreased with increasing follicle diameter (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD-E). Additionally, both NMB and its receptors were expressed in GCs, and IF experiments demonstrated that NMB and NMBR were primarily localized in the cytoplasm of GCs, while GRPR was expressed in both the nucleus and cytoplasm (Figure S2). These results collectively indicate that NMB plays a role in ovarian follicle development in goats.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.2 NMB Promotes Goat GC Proliferation via NMB\u003c/h2\u003e \u003cp\u003eTo investigate the effect of NMB on the proliferation of goat GCs, we treated goat GCs with different concentrations of NMB (10⁻\u0026sup1;⁰, 10⁻⁹, 10⁻⁸, 10⁻⁷, and 10⁻⁶ M) for varying durations (3, 6, 12, 24, and 48 h). We found that treatment with 10⁻⁷ M NMB for 24 h significantly enhanced the proliferative capacity of goat GCs (Figure S3 A-F). Additionally, to determine the receptor through which NMB promotes goat GC proliferation, we examined the effects of NMBR and GRPR antagonists on the proliferation of goat GCs. The results showed that antagonizing GRPR had no significant effect on the protein expression level of PCNA in goat GCs (Figure S3H). However, antagonizing NMBR inhibited the proliferation rate of goat GCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) and blocked the proliferative effect of NMB, leading to significant decreases in both mRNA and protein expression levels of PCNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-C). These results indicate that NMB primarily enhances the proliferative capacity of goat GCs by binding to NMBR.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo investigate the underlying reasons for changes in goat GC proliferation rates after NMB treatment, we analyzed the number of cells in G1, S, and G2 phases using flow cytometry. Compared with the control group, NMB significantly increased the number of cells in S phase, while co-treatment with NMBR antagonist and NMB significantly reduced the number of S-phase cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD-E). Additionally, the protein expression levels of cyclin (CCNE1) and cyclin-dependent kinases (CDK2, CDK6, and CDK1) were detected (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). NMB markedly upregulated CCNE1, CDK2, CDK6, and CDK1 protein expression, and these increases were reversed by NMBR antagonism. Together, these data indicate that NMB regulates GC cell-cycle progression via NMBR.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.3NMB Regulates Ca\u0026sup2;⁺ in Goat GCs via Activating the NMBR-PLCβ1-IP3R Pathway\u003c/h2\u003e \u003cp\u003eTo investigate whether NMB regulates goat GC proliferation by affecting Ca\u0026sup2;⁺ homeostasis, we first measured intracellular Ca\u0026sup2;⁺ levels. The results showed that NMB treatment significantly increased Ca\u0026sup2;⁺ concentration in goat GCs compared to the control group, whereas antagonizing NMBR markedly reduced Ca\u0026sup2;⁺ levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). We subsequently examined the expression changes of key proteins in the endoplasmic reticulum calcium pathway and found that NMB treatment significantly upregulated PLCβ1 and IP3R protein expression, whereas NMBR antagonism reversed these effects (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Meanwhile, the ER Ca\u0026sup2;⁺ fluorescence intensity was significantly decreased in the NMB-treated group but restored upon NMBR antagonism (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). ER-Tracker staining revealed enhanced fluorescence intensity and uniform ER morphology in the NMB group, indicating ER activation, while reduced fluorescence in the antagonist-treated group suggested ER dysfunction (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). These data demonstrate that NMB regulates intracellular Ca\u0026sup2;⁺ homeostasis via the NMBR\u0026ndash;PLCβ1\u0026ndash;IP3R axis, thereby modulating GC cell-cycle progression and proliferation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e3.4 NMB enhances the proliferation ability of goat GCs by regulating the IRE1α pathway of UPR\u003c/b\u003e \u003csup\u003e \u003cb\u003eer\u003c/b\u003e \u003c/sup\u003e \u003cb\u003ethrough NMBR\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo further elucidate the mechanism by which NMB affects goat GC proliferation through the ER, we examined the expression of proteins associated with the three key regulatory pathways of the unfolded protein response (UPR) in the ER. The results showed that compared with the control group, NMB treatment significantly decreased the expression of phospho-IRE1α and XBP1S, while co-treatment with NMBR antagonist and NMB significantly upregulated the expression levels of phospho-IRE1α and XBP1S (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-B). However, the related proteins of the other two pathways (ATF6 and PERK) showed no significant changes in expression compared to the control group after NMB treatment (Figure S4). Additionally, interference with XBP1S inhibited goat GC proliferation and hindered the transition of the cell cycle from G1 to S phase, but NMB treatment rescued the effects caused by XBP1S deficiency (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC-F). These results demonstrate that the binding of NMB to NMBR modulates Ca\u0026sup2;⁺ homeostasis, thereby regulating the IRE1α pathway of the UPR\u003csup\u003eer\u003c/sup\u003e, which ultimately influences the cell cycle and proliferation of goat GCs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.5 NMB regulates mitochondrial morphology and function through NMBR\u003c/h2\u003e \u003cp\u003eTo investigate the effect of NMB on mitochondrial morphology, we visualized mitochondria using Mito Tracker under confocal microscopy. The results demonstrated that NMB treatment induced mitochondrial fusion in goat GCs, characterized by an increased proportion of elongated mitochondria, whereas antagonizing NMBR triggered mitochondrial fission (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-B). TEM confirmed that NMB-treated cells exhibited elongated mitochondria with increased length and diameter, while NMBR antagonism caused structural damage, including disorganized cristae and pale matrix (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Quantitative morphometric analysis showed that NMB significantly increased mitochondrial area, perimeter, aspect ratio, and form factor (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD-G). Additionally, we examined the expression of key mitochondrial fusion and fission proteins. NMB treatment significantly upregulated the expression of mitochondrial fusion proteins MFN1, MFN2, and OPA1, while downregulating the fission protein DNM1L. However, co-treatment with the NMBR antagonist reversed the effects of NMB on the expression levels of mitochondrial fusion and fission proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAdditionally, to investigate the effect of NMB on mitochondrial function, we measured the activities of MMP, ATP production, MRC complexes (CI-CV), and cellular reactive ROS in goat GCs treated with NMB. The results revealed that NMB significantly increased MMP, ATP production, and the activities of MRC complexes CI-CV, while decreasing cellular ROS generation. However, co-treatment with the NMBR antagonist reversed the effects induced by NMB (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-H). These findings indicate that NMB binding to NMBR regulates mitochondrial function through mitochondrial dynamics, thereby modulating the proliferative capacity of goat GCs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e3.6 NMB-NMBR regulates Ca\u0026sup2;⁺ transport in goat GCs via MAMs\u003c/h2\u003e \u003cp\u003eTo elucidate how NMB influences Ca\u0026sup2;⁺ translocation and thereby regulates goat GC proliferation, we measured Ca\u0026sup2;⁺ levels in the ER, cytosol, and mitochondria of GCs following NMB treatment. NMB significantly reduced ER Ca\u0026sup2;⁺ levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC) while markedly elevating cytosolic and mitochondrial Ca\u0026sup2;⁺ (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA; Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Because MAMs mediate inter-organelle Ca\u0026sup2;⁺ flux, we examined ultrastructural MAMs by TEM. NMB increased ER-mitochondrial contact sites, as indicated by a higher ratio of MAMs associated ER length to mitochondrial perimeter; NMBR antagonism reversed this effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). Complementary confocal co-staining with ER and mitochondrial probes revealed increased yellow overlap in merged images, denoting MAMs. Fluorescence colocalization analysis showed that NMB treatment significantly enhanced MAMs coupling in goat GCs, whereas co-treatment with the NMBR antagonist and NMB markedly reduced MAMs coupling (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIRE1α physically interacts with IP3R to regulate calcium flux between mitochondria and the ER. Our experiments showed that NMB treatment significantly upregulated the protein expression levels of IRE1α and IP3R in goat GCs compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). Furthermore, Co-IP assays confirmed the interaction between IRE1α and IP3R (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). These results demonstrate that the binding of NMB to NMBR modulates Ca\u0026sup2;⁺ transport between mitochondria and the ER by regulating the MAMs structure via the IRE1α-IP3R-VDAC1 pathway.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eAs the fundamental functional units of the ovary, follicular development is central to oogenesis and directly determines female reproductive capacity. GCs, the most abundant somatic cells within follicles, participate in folliculogenesis, oocyte maturation, and atresia regulation throughout the entire follicular lifespan. GC proliferation and estrogen synthesis constitute critical factors supporting follicular growth and development. This study demonstrates that NMB promotes goat GC proliferation through binding to NMBR, providing a theoretical basis for further investigating NMB's mechanism in the female reproductive system while offering insights into potential mechanisms governing caprine follicular growth and development.\u003c/p\u003e \u003cp\u003eNMB can affect cell proliferation and thereby participate in animal reproductive functions [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Through cloning and sequencing, we found that the coding nucleotide sequences of goat NMB and its receptors (NMBR/GRPR) exhibit high homology with ruminants such as cattle and sheep. Both NMBR and GRPR possess seven transmembrane domains and are typical G protein-coupled receptors [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], consistent with previous reports [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Studies have shown that NMB regulates the release of LH and prolactin [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], and NMB and its receptors are expressed in the hypothalamus, pituitary, and ovaries [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], indicating that NMB influences follicular development and function via the hypothalamic-pituitary-ovarian axis. Our results confirmed that NMB and its receptors are highly expressed in ovarian follicles, with predominant localization in granulosa cells of antral follicles. IF revealed that both NMB and its receptors are expressed in granulosa cells. Accumulating evidence suggests that NMB/NMBR contributes to regulating proliferation across diverse cell types [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Our findings provide compelling evidence that NMB exerts an enhancing effect on the proliferation of goat GCs, as reflected by the increased G1/S cell population and elevated expression of PCNA, CCNE1, and CDK2. Furthermore, we observed that antagonizing NMBR impedes the effects of NMB on goat GC proliferation and cell cycle progression, aligning with existing research findings [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. These results demonstrate that NMB primarily promotes goat GC proliferation through binding to NMBR, thereby playing a critical role in follicular growth.\u003c/p\u003e \u003cp\u003eUpon binding to its receptor, NMB activates multiple intracellular signaling pathways, leading to cell-specific responses to exert biological functions [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Our results demonstrate that NMB regulates calcium homeostasis in goat GCs by activating the NMBR-PLCβ1-IP3R pathway. Moreover, studies have shown that ER Ca²⁺ homeostasis modulates cell proliferation [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] and affects cell cycle progression [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Our findings support this notion, as NMB treatment significantly alters ER Ca²⁺ levels, thereby promoting cell proliferation. In mouse GCs, disruption of ER Ca²⁺ homeostasis triggers endoplasmic reticulum stress (ERS) activation and impairs follicular development [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. ERS activation induces cell cycle arrest (e.g., CHOP-mediated suppression of Cyclin D1 expression) and apoptosis (e.g., JNK-mediated activation of Bax) through the PERK/eIF2α/CHOP and IRE1α/JNK/XBP1s pathways [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Our study reveals that antagonizing NMBR disrupts ER Ca²⁺ dynamics, leading to ERS activation, characterized by increased phosphorylation of IRE1α and elevated XBP1s splicing, which ultimately induces cell cycle arrest in goat GCs. In diabetic models, reducing ERS mitigates β-cell apoptosis and delays disease progression [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Therefore, we propose that NMB/NMBR signaling modulates the ERS threshold via Ca²⁺ homeostasis, thereby determining granulosa cell fate (proliferation/apoptosis) and influencing follicular development.\u003c/p\u003e \u003cp\u003eMitochondrial morphology and function are critical for maintaining normal cell proliferation. Our results revealed that the binding of NMB to NMBR maintains mitochondrial dynamics by inducing mitochondrial fusion and promoting the expression of fusion-associated proteins. Mitochondrial dynamics are central to their quality control, which relies on the coordinated regulation of fission and fusion to adjust mitochondrial morphology and thereby modulate their function [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. We found that NMB treatment upregulated the expression of MFN1, MFN2, and OPA1 while downregulating DNM1L and FIS1, leading to enlarged mitochondrial morphology and a greater tendency toward fusion. Fusion is considered beneficial for mitochondrial function, as it allows metabolites and proteins to distribute throughout the mitochondrial network, maximizing bioenergetic capacity [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Studies have shown that downregulation of OPA1 or dysfunction of MFN1 and MFN2 significantly reduces mitochondrial respiratory capacity when fueled by substrates for electron transport chain complexes I, II, and IV [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Our results corroborate this finding, demonstrating that NMB enhances mitochondrial fusion, increases MMP, boosts respiratory chain enzyme activity, and elevates ATP production. This indicates a close association between mitochondrial morphological changes and functional outcomes. Research has found that elevated MMP and low ROS levels activate pro-proliferative pathways, thereby promoting the expression of cell cycle-related proteins [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Our data support this hypothesis, showing that NMB sustains high MMP to ensure sufficient ATP supply and maintain low ROS levels, which in turn promotes the expression of CCNE1 and CDK2 proteins. Therefore, we propose that NMB enhances mitochondrial function by regulating the balance of mitochondrial dynamics (fusion/fission), thereby coordinating the expression of cell cycle proteins and proliferation processes. This mechanism likely underlies the maintenance of goat GCs homeostasis and the promotion of follicular development.\u003c/p\u003e \u003cp\u003eMAMs serve as critical platforms for material exchange between mitochondria and the ER [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], with ER-mediated Ca²⁺ release and recycling being closely linked to MAMs structure and function [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Our study demonstrated that NMB treatment facilitates Ca²⁺ transfer from the ER to mitochondria, leading to elevated mitochondrial Ca²⁺ levels. By stimulating electron transport chain (ETC) complexes, mitochondrial Ca²⁺ enhances the tricarboxylic acid cycle and OXPHOS, thereby boosting ATP synthase activity and ATP generation, ultimately facilitating cell proliferation [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Our findings validate this cascade: NMB-induced elevation of mitochondrial Ca²⁺ levels enhance respiratory chain complex activity and ATP synthase function, resulting in increased ATP generation and augmented proliferative capacity of goat GCs. Studies indicate that MAMs integrity is essential for Ca²⁺ transfer and intracellular homeostasis [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e], with enhanced mitochondrial-ER coupling promoting mitochondrial respiration and bioenergetics, thereby improving cellular adaptability to stress [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Consistent with this, NMB treatment strengthens MAMs coupling efficiency, optimizing mitochondrial function to promote goat GC proliferation. The IP3R–VDAC1 complex primarily regulates MAMs associated Ca²⁺ transport [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e], while the ER stress sensor protein IRE1α physically interacts with IP3R within MAMs, influencing its subcellular localization, controlling mitochondrial Ca²⁺ uptake, and playing a pivotal role in cell survival [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Our results revealed that NMB treatment upregulates IRE1α and IP3R expression, and CO-IP confirmed protein-protein interaction between IRE1α and IP3R. Therefore, we propose that NMB modulates the structure of MAMs via the IRE1α-IP3R-VDAC1 complex, thereby regulating inter-organellar Ca²⁺ flux between mitochondria and the ER and influencing goat GC proliferation.\u003c/p\u003e \u003cp\u003eThis study has several limitations. Firstly, the study primarily utilized NMBR antagonists for receptor antagonism experiments. Subsequent work should employ CRISPR-Cas9 technology to knock down the NMBR gene, thereby achieving receptor-specific silencing. Secondly, the conclusions drawn are mainly based on in vitro experiments. Future research needs to enhance clinical relevance through in vivo models and cross-species validation.\u003c/p\u003e "},{"header":"Conclusion","content":"\u003cp\u003eIn summary, our results indicate that NMB exerts its effects by binding to NMBR, regulating intracellular Ca²⁺ homeostasis, alleviating ER stress, triggering mitochondrial metabolic reprogramming, and promoting cell proliferation. These findings expand the functional understanding of NMB in reproductive biology, provide novel evidence for calcium signaling-mediated organelle crosstalk in proliferation regulation, and suggest potential targets for optimizing reproductive efficiency and treating ovarian dysfunction.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"586\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 25.7679%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNMB\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74.2321%;\"\u003e\n \u003cp\u003eNeuromedin B\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 25.7679%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNMBR\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eGRPR\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74.2321%;\"\u003e\n \u003cp\u003eNeuromedin B receptor\u003c/p\u003e\n \u003cp\u003eGastrin releasing peptide receptor\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 25.7679%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGCs\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74.2321%;\"\u003e\n \u003cp\u003eGranulosa cells\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 25.7679%;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePCNA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74.2321%;\"\u003e\n \u003cp\u003eProliferating cell nuclear antigen\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 25.7679%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCCNE1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74.2321%;\"\u003e\n \u003cp\u003eCyclin E1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 25.7679%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCDK1\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eCDK2\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eCDK6\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74.2321%;\"\u003e\n \u003cp\u003eCyclin dependent kinase 1\u003c/p\u003e\n \u003cp\u003eCyclin dependent kinase 2\u003c/p\u003e\n \u003cp\u003eCyclin dependent kinase 6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 25.7679%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eER\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74.2321%;\"\u003e\n \u003cp\u003eEndoplasmic reticulum\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 25.7679%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCa\u003csup\u003e2+\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74.2321%;\"\u003e\n \u003cp\u003eCalcium ion\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 25.7679%;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePLC\u0026beta;1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74.2321%;\"\u003e\n \u003cp\u003ePhospholipase C-beta1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 25.7679%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eIP3R/ITPR1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74.2321%;\"\u003e\n \u003cp\u003eInositol 1,4,5-trisphosphate receptor type 1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 25.7679%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSERCA1/ATP2A1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74.2321%;\"\u003e\n \u003cp\u003eATPase sarcoplasmic/endoplasmic reticulum Ca\u003csup\u003e2+\u003c/sup\u003e transporting 1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 25.7679%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eUPR\u003csup\u003eer\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74.2321%;\"\u003e\n \u003cp\u003eEndoplasmic reticulum unfolded protein response\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 25.7679%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eATF4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74.2321%;\"\u003e\n \u003cp\u003eActivating transcription factor 4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 25.7679%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eATF6\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74.2321%;\"\u003e\n \u003cp\u003eActivating transcription factor 6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 25.7679%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eERp57/PDIA3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74.2321%;\"\u003e\n \u003cp\u003eProtein disulfide isomerase family A member 3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 25.7679%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eIRE1\u003c/strong\u003e\u003cstrong\u003e\u0026alpha;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74.2321%;\"\u003e\n \u003cp\u003eInositol-requiring enzyme 1\u0026alpha;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 25.7679%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eXBP1S\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74.2321%;\"\u003e\n \u003cp\u003eX-box binding protein 1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 25.7679%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBIP/HSPA5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74.2321%;\"\u003e\n \u003cp\u003eHeat shock protein family A (Hsp70) member 5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 25.7679%;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePERK/EIF2AK3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74.2321%;\"\u003e\n \u003cp\u003eEukaryotic translation initiation factor 2 alpha kinase 3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 25.7679%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMAMs\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74.2321%;\"\u003e\n \u003cp\u003eMitochondria-associated ER membranes\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 25.7679%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eVDAC1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74.2321%;\"\u003e\n \u003cp\u003eVoltage-Dependent Anion Channel 1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 25.7679%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eATP\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74.2321%;\"\u003e\n \u003cp\u003eAdenosine triphosphate\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 25.7679%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eeIF2\u0026alpha;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74.2321%;\"\u003e\n \u003cp\u003eEukaryotic translation initiation factor 2\u0026alpha;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 25.7679%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMFN1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74.2321%;\"\u003e\n \u003cp\u003eMitofusion 1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 25.7679%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMFN2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74.2321%;\"\u003e\n \u003cp\u003eMitofusion 2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 25.7679%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eOPA1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74.2321%;\"\u003e\n \u003cp\u003eOptic Atrophy 1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 25.7679%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGAPDH\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 74.2321%;\"\u003e\n \u003cp\u003eGlyceraldehyde-3-phosphate dehydrogenase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experiments were conducted in accordance with the approved Guidelines for Animal Experiments of Nanjing Agricultural University and received approval from the Animal Care and Use Committee of Nanjing Agricultural University (Approval ID: SYXK2022-0031). All antibodies and other chemicals of reagent grade were purchased from biotechnology companies.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData sharing is not applicable to this article as no datasets were generated or analysed during the current study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the National Key R\u0026amp;D program of China (2021YFD1200902), the National Natural Science Foundation of China (No. 32472993), the Fundamental Research Funds for the Central Universities (No. KYTZ2023003).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRongxin Xia: Writing\u0026nbsp;–\u0026nbsp;original draft, Writing\u0026nbsp;–\u0026nbsp;review \u0026amp; editing, Methodology, Investigation, Formal analysis, Data curation. Qi Zhang: Writing\u0026nbsp;–\u0026nbsp;review \u0026amp; editing, Methodology, Investigation, Data curation. Junhui Shao: Writing\u0026nbsp;–\u0026nbsp;review \u0026amp; editing, Formal analysis, Data curation. Yuan Wang: Methodology, Investigation, Data curation. Xinyi Lv: Methodology, Investigation, Data curation. Rui Chen: Methodology, Investigation, Data curation. Zhen Lu: Writing\u0026nbsp;–\u0026nbsp;review \u0026amp; editing, Supervision, Methodology. Yanli Zhang: Project administration, Supervision, Funding acquisition. Feng Wang: Project administration Supervision, Funding acquisition. Guomin Zhang：Writing\u0026nbsp;–\u0026nbsp;review \u0026amp; editing, Writing\u0026nbsp;–\u0026nbsp;original draft, Supervision, Project administration, Funding acquisition, Conceptualization.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors also thank all the members of Feng Wang’s laboratory who contributed to sample collection.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMcgee EA, Hsueh AJ. Initial and cyclic recruitment of ovarian follicles. Endocr Rev. 2000;21(2):200\u0026ndash;14.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFilatov M, Khramova Y, Parshina E, Bagaeva T, Semenova M. Influence of gonadotropins on ovarian follicle growth and development in vivo and in vitro. 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Nat Cell Biol. 2019;21(6):755\u0026ndash;67.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-ovarian-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jovr","sideBox":"Learn more about [Journal of Ovarian Research](http://ovarianresearch.biomedcentral.com)","snPcode":"13048","submissionUrl":"https://submission.nature.com/new-submission/13048/3","title":"Journal of Ovarian Research","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Neuromedin B, granulosa cells, cell proliferation, endoplasmic reticulum, mitochondria, goat","lastPublishedDoi":"10.21203/rs.3.rs-6811713/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6811713/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eNeuromedin B (NMB) has been implicated in the regulation of female reproductive functions, yet its precise role and underlying mechanisms in ovarian follicular development remain undefined. Granulosa cells (GCs), the principal functional cells within ovarian follicles, directly govern follicular growth and maturation through their proliferation and differentiation. In this study, we explored the regulatory effects and molecular mechanisms of NMB and its receptor (NMBR) on goat GC proliferation.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eWe documented dynamic expression patterns of NMB and NMBR throughout ovarian and follicular development. Exogenous NMB treatment markedly enhanced GC proliferation, as evidenced by an increased fraction of S-phase cells and upregulation of CCNE1 and CDK1/2/6. Mechanistically, NMB bound to NMBR to activate phospholipase C β1 (PLCβ1), triggering endoplasmic reticulum (ER) Ca\u0026sup2;⁺ release and significantly raising cytosolic Ca\u0026sup2;⁺ levels while alleviating ER stress. Further analyses revealed that NMB strengthened mitochondria-associated ER membranes (MAMs) formation via the IRE1α\u0026ndash;IP3R\u0026ndash;VDAC1 axis, facilitating Ca\u0026sup2;⁺ transfer into mitochondria. This led to enhanced mitochondrial function, including increased mitochondrial membrane potential, elevated respiratory chain complex activities, augmented ATP production, and promotion of mitochondrial network fusion. Importantly, these effects were abolished by an NMBR antagonist.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThe molecular mechanism by which NMB-mediated activation of NMBR enhances mitochondrial metabolism through modulation of intracellular calcium homeostasis, thereby promoting proliferation of goat GCs. These findings shed new light on the regulation of follicular development and may inform strategies to improve reproductive efficiency in goats.\u003c/p\u003e","manuscriptTitle":"Neuromedin B Drives Goat Granulosa Cell Proliferation via NMBR-Mediated Calcium Homeostasis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-01 16:37:55","doi":"10.21203/rs.3.rs-6811713/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-07-21T10:07:37+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-08T07:42:13+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-07T20:40:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"31843491149465859126151148440341556344","date":"2025-06-30T08:12:02+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"80012050306185452030397667414640803072","date":"2025-06-29T18:25:35+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-27T15:26:34+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-08T12:26:02+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-05T10:21:13+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Ovarian Research","date":"2025-06-03T13:08:03+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-ovarian-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jovr","sideBox":"Learn more about [Journal of Ovarian Research](http://ovarianresearch.biomedcentral.com)","snPcode":"13048","submissionUrl":"https://submission.nature.com/new-submission/13048/3","title":"Journal of Ovarian Research","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a5c781cf-5050-4475-bbaa-55f8384704cb","owner":[],"postedDate":"July 1st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-11-24T16:05:53+00:00","versionOfRecord":{"articleIdentity":"rs-6811713","link":"https://doi.org/10.1186/s13048-025-01844-7","journal":{"identity":"journal-of-ovarian-research","isVorOnly":false,"title":"Journal of Ovarian Research"},"publishedOn":"2025-11-19 15:57:51","publishedOnDateReadable":"November 19th, 2025"},"versionCreatedAt":"2025-07-01 16:37:55","video":"","vorDoi":"10.1186/s13048-025-01844-7","vorDoiUrl":"https://doi.org/10.1186/s13048-025-01844-7","workflowStages":[]},"version":"v1","identity":"rs-6811713","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6811713","identity":"rs-6811713","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}