A novel micropeptide 9790aa mediates melatonin regulation of granulosa cell proliferation, apoptosis, and estrogen secretion in Hu sheep through the MAPK1/3-Elk1 signaling pathway.

A novel micropeptide 9790aa mediates melatonin regulation of granulosa cell proliferation, apoptosis, and estrogen secretion in Hu sheep through the MAPK1/3-Elk1 signaling pathway. | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 9 October 2025 V1 Latest version Share on A novel micropeptide 9790aa mediates melatonin regulation of granulosa cell proliferation, apoptosis, and estrogen secretion in Hu sheep through the MAPK1/3-Elk1 signaling pathway. Authors : Jianyu Ma , Hua Yang , Caifang Ren , Zhibo Wang , Xinai Huang , Peiyong Chen , Shanglai Li , Yongjie Wan , Yanli Zhang , and Feng Wang 0000-0002-8887-8998 [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.176004948.80876527/v1 124 views 98 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Melatonin (MT), a hormone secreted by the pineal gland, plays a crucial role in regulating circadian rhythms and reproductive processes in mammals. Granulosa cells (GCs), essential for follicular development and hormone secretion, are significantly influenced by melatonin, with their dysregulation linked to reproductive disorders. This study explores the critical role of melatonin in regulating Hu sheep fertility and follicular development, unveiling a novel regulatory mechanism. We demonstrated that melatonin significantly influences GCs proliferation, reduces apoptosis, and increases estradiol (E2) synthesis. RNA-Seq and Ribo-Seq analyses of melatonin-treated GCs revealed a novel small open reading frame (sORF9790) on lncRNA1056 encoding a micropeptide, 9790aa. Melatonin regulates lncRNA1056 and 9790aa expression via its receptor MTNR1b. Mechanistically, 9790aa binds to MAPK1 and MAPK3 proteins, leading to the downregulation of the activity of the MAPK1/3-Elk1 signaling pathway, a critical regulator of GCs function. These findings establish a direct link between melatonin and reproductive outcomes in Hu sheep, mediated by this novel melatonin-micropeptide axis. This work highlights the therapeutic potential of targeting micropeptides to address reproductive disorders and optimize fertility management in livestock. 1.Introduction Granulosa cells (GCs), the most abundant cell type within the follicle, play a central role in follicular development and hormone secretion[1]. Enveloping the oocyte, GCs provide essential support and secrete various growth factors and hormones that drive both oocyte and follicle maturation[2]. Dysregulation in GCs function significantly impacts female reproductive health and may lead to numerous fertility and endocrine disorders, such as polycystic ovary syndrome (PCOS) [3] and premature ovarian insufficiency (POI)[4]. Thus, investigating the regulatory mechanisms governing GCs function holds critical implications not only for advancing female reproductive health but also for enhancing reproductive efficiency in livestock. Melatonin, an indoleamine hormone secreted by the pineal gland, follows a circadian rhythm regulated by the light-dark cycle, with low daytime and elevated nighttime levels[5]. Recent studies indicate that melatonin is involved in multiple reproductive processes in female mammals, such as estrus initiation and follicular development[6]. Melatonin can influence reproductive function both by acting on the hypothalamus to modulate GnRH secretion or directly on the ovary[7]. It has been reported that melatonin concentrations in follicular fluid increase with follicle diameter during follicular development, peaking just before ovulation[8]. Additionally, melatonin impacts GCs proliferation, apoptosis, and steroid hormone secretion, thereby regulating follicular growth and atresia[9]. However, most research has focused on the role of melatonin in scavenging excessive ROS within follicles, protecting GCs from oxidative stress and consequently affecting cell proliferation and apoptosis[10]. Whether melatonin influences GCs status and function through other mechanisms remains to be further explored. Micropeptides, defined as small proteins composed of fewer than 100 amino acids, are typically translated from non-coding regions of the genome or small open reading frames (sORFs), and are often overlooked in traditional protein annotations[11]. However, with advancements in sequencing technologies, an increasing number of sORFs and their encoded micropeptides have been found to play crucial roles in various physiological processes[12]. For example, Toddler, a micropeptide identified in zebrafish and encoded by the lncRNA LOC100506013, acts as an activator of the Apelin receptor (APJ), thereby promoting gastrulation[13]. Minion, recently discovered in skeletal muscle, forms a complex with the key muscle fusion protein Myomaker, thereby inducing myoblast fusion[14]. Another micropeptide, MOX1, is encoded by a lncRNA localized to the inner mitochondrial membrane, where it interacts with mitochondrial trifunctional protein (MTP) to enhance fatty acid β-oxidation[15]. However, whether melatonin regulates GCs function and status via micropeptides remains unreported. Here, we found that melatonin is significantly associated with Hu sheep fertility and follicular development. Melatonin influences the proliferation, apoptosis, and E2 synthesis of Hu sheep GCs. Through RNA-Seq and Ribo-Seq analyses on melatonin-treated GCs, we identified a novel sORF (sORF9790) located on lncRNA1056, which encodes a peptide, 9790aa. Melatonin, acting via its receptor MTNR1b, modulates the expression of lncRNA1056 and 9790aa. Furthermore, 9790aa binds to MAPK1 and MAPK3 proteins to regulate the MAPK1/3-Elk1 pathway activity, thereby controlling GC proliferation, apoptosis, and estrogen synthesis in sheep. 2.Materials and methods 2.1 Animals and Sample Collection This experiment was conducted at Xilai Ecological Agriculture Co., Ltd., Taizhou, Jiangsu Province, during the preliminary phase. Based on the reproductive pedigree records and genotyping (FecBB), 18 non-pregnant Hu sheep aged 2-3 years with similar body condition and reproductive physiological status were selected. These included three high-fecundity FecBB (HBB) ewes (producing three lambs per litter for three consecutive parities) and three low-fecundity FecBB (LBB) ewes (producing one lamb per litter for three consecutive parities). The experimental ewes were housed individually and fed separately. On day 1 at 16:00, intravaginal sponges were inserted, which were removed 11 days later, followed by an injection of 0.2 mg PG per ewe. Estrus detection was performed the next day using a teaser ram. Ewes that allowed mounting were considered in estrus, recorded as day 0 (D0). Blood samples were collected via the jugular vein daily at 10:00 to measure serum melatonin concentrations, continuing until the next natural estrus. Three ewes from each group with similar estrus onset were selected for slaughter. Samples of the pineal gland, hypothalamus, pituitary, ovary, and uterus were collected immediately post-slaughter, snap-frozen in liquid nitrogen, and stored at -80°C upon transfer to the laboratory. All animal experiments comply with the regulations for the use of experimental animals at Nanjing Agricultural University. 2.2 Cell culture Ovine GCs were carefully isolated from follicular fluid obtained from ovaries sourced from the Taicang abattoir in Jiangsu, China. Post-isolation, the cells were seeded onto 6-well culture plates. The culture medium utilized was a blend of DMEM/F12 glutaMAX (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), enriched with 10% fetal bovine serum (FBS) and a 1% antibiotic mixture of penicillin/streptomycin (P/S). Cultures were maintained at 37 °C in a 5% CO2 atmosphere, with the medium refreshed every 24 hours. 2.3 Cell treatment GCs were plated in 6-well plates and prepared for transfection. When reaching a confluency of 60-70%, cells were transfected using either RNA or DNA complexed with Lipofectamine 3000 (Thermo Fisher Scientific, USA). MT (Sigma, USA) was initially solubilized in DMSO, and then diluted to final concentrations of 1 nM, 10 nM and 100 nM in the culture medium. Both compounds were added to the culture 12 hours before cell harvest. Following transfection, cells were harvested at the 24-hour mark for RNA isolation and at the 48-hour mark for protein extraction. 2.4 CCK-8 assay GCs were plated in 96-well plates and subjected to diverse treatments and transfection methodologies. Following these procedures, each well was treated with the Cell Counting Kit-8 (CCK-8) reagent from Vazyme, China, and underwent an approximately 4-hour incubation at 37 °C with 5% CO 2 . Absorbance readings were measured at a wavelength of 450 nm using a spectrophotometer. 2.5 RNA Extraction and qRT-PCR Total RNA extraction utilized the Trizol method (Takara, Dalian, China), followed by cDNA synthesis using a reverse transcription kit (Takara, Dalian, China). Quantitative real-time PCR (qRT-PCR) was conducted using the SYBR Green Master mix (Vazyme, Nanjing, China) on the Step One Plus real-time PCR system (Life Technologies, USA), with primer sequences detailed in Table S1. 2.6 Co-IP The lysate was applied to the cell culture plate to ensure complete cell lysis at 4°C. Following this, the samples were centrifuged at 12,000 rpm for 10 minutes, and the supernatant was collected. A small portion of the lysate was reserved for additional experiments, while the remaining lysate was incubated with 10 μg of flag antibody overnight at 4°C. Pierce™ Protein A/G Agarose Beads (Thermo Fisher Scientific, Inc.) were thoroughly washed with lysis buffer before being added to the cell lysate for an overnight incubation at 4°C. The mixture was then centrifuged at 2,500 rpm for 3 minutes at 4°C. After removing the supernatant, the agarose beads were washed three times with 1 ml of lysis buffer. Finally, 100 μl of 2× SDS loading buffer was added, and the samples were heated at 95°C for 5 minutes before proceeding with WB and mass spectrometry (MS) analysis. The protein identification via MS was performed by Mabnus Biotech Co., Inc. in Wuhan, China. 2.7 Western blot Total protein was extracted with RIPA lysate (Thermo Fisher Scientific, USA) containing PMSF after a 12-hour treatment. This protein was then separated via SDS-PAGE and transferred onto a PVDF membrane (Millipore, USA). The membrane underwent overnight incubation with the primary antibody, followed by a 1-hour incubation with peroxidase-conjugated secondary antibody at room temperature. Antibody details are available in Table S2. Protein bands on the membrane were visualized using a chemiluminescence detection system (Fijifilm, Tokyo, Japan), and their intensities were quantified using Image J software (National Institutes of Health, Bethesda, MD, USA). 2.8 Edu assay Cell proliferation was evaluated using EDU staining following a previously published protocol[16]. Cells were seeded in a 12-well culture plate and cultured for 24 hours before treatment with and melatonin for the specified duration. Subsequently, the culture medium was replaced with complete medium containing 50 mmol/l EDU. After a 4-hour incubation, cells were washed thrice with DPBS and fixed with 4% paraformaldehyde (PFA) for 30 minutes at room temperature. Following this, cells were treated with 2 mg/ml glycine for 5 minutes and permeabilized with DPBS containing 0.5% Triton X-100 for approximately 20 minutes. After another round of DPBS washing, nuclei were stained with Hoechst 33342 for 5 minutes at room temperature. Finally, cells were observed and analyzed using a microscope. 2.9 Flow Cytometry Analysis of Apoptosis Cellular apoptosis rates were assessed using an Annexin V-FITC/PI Detection Kit (Nanjing, China) following the manufacturer’s guidelines rigorously. Post-treatment, GCs were harvested and washed with DPBS. Subsequently, these cells were resuspended in binding buffer and sequentially exposed to Annexin V-FITC and PI, followed by a 20-minute incubation in a light-protected environment. Flow cytometry was employed to measure apoptotic events within the cell population. 2.10 Elisa Assay After 48 hours of transfection and an additional 12-hour treatment with and melatonin, we harvested both the cell supernatant and the cells. Subsequently, we measured the concentration of E2 and melatonin using the Elisa assay, following the manufacturer’s guidelines meticulously. 2.11 Immunofluorescence Cells were seeded in glass-bottom dishes, fixed with ice-cold methanol for 20 minutes at room temperature, washed with PBS, and permeabilized with 0.25% Triton X-100 (Sigma-Aldrich, St. Louis, MO) for 10 minutes. Subsequently, cells were blocked with Immunol Staining Blocking Buffer (Beyotime, Shanghai, China) for 60 minutes at room temperature on a rocking platform and then rinsed with PBS. Primary antibodies were applied to the cells, which were then incubated overnight at 4°C. The next day, cells were washed three times with PBS and incubated with a 594-conjugated donkey anti-rabbit secondary antibody (1:200 dilution, Abcam, Boston, MA, USA) for 2 hours at room temperature in the dark. Finally, cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, Beyotime, Shanghai, China) for 10 minutes. Fluorescence was visualized using a Zeiss LSM 710 META confocal laser scanning microscope (Mannheim, Germany). 2.12 RNA-seq We employed RNA-seq and Ribo-seq techniques to explore the transcriptional dynamics in granulosa cells following MT treatment, alongside concurrent mRNA changes during translation. The NEB Next Ultra Directional RNA Library Prep Kit (NEB, MA, USA) facilitated cDNA library preparation, followed by sequencing on the Illumina HiSeq X. Ten platform at Gene Denovo (Guangzhou, China). Strand-specific transcriptomic sequencing was consistently applied. Post-sequencing, rigorous curation of low-quality reads yielded clean reads, subsequently aligned to the Ovis aries reference genome (Oar_v4.0) using TopHat2 software. This alignment procedure was succeeded by the identification of protein-coding genes. The fragments per kilobase of transcript per million mapped reads (FPKM) value, computed via Cuffdiff software (v2.1.1), served as a metric to quantify gene expression levels. 2.13 Ribo-seq To generate reading frames (RFs), we treated the lysate with RNase I and DNA I, followed by a 45-minute incubation at ambient conditions. Size-exclusion columns were prepared with polysome buffer before introducing the digested RFs for centrifugation. After elution, we added 10 μL of 10% SDS, selectively isolating RFs exceeding 17 nucleotides using the RNA Clean and Concentrator-25 protocol (Zymo Research; R1017). Following a previously established method, we depleted rRNA by using short antisense DNA probes (50-80 bases) targeting rRNA sequences in the RF mixture. Subsequent treatments with RNase H and DNase I ensured the digestion of rRNA and excess DNA probes. An additional purification step for RFs involved using magnetic beads. With the ribosomal footprints obtained, we constructed Ribo-seq libraries using the NEBNextR Multiple Small RNA Library Prep Set for Illumina. This process involved attaching adapters to RF ends, followed by reverse transcription and PCR amplification. PCR fragments, ranging from 140 to 160 base pairs, were concentrated to form a cDNA library, which was sequenced on the Illumina HiSeq X10 platform, courtesy of Gene Denovo Biotechnology. 2.14 GST pull down The target protein was expressed using the Escherichia coli expression system, and the specific protein sequence is outlined in Table S3. GST-fusion proteins and HIS-fusion proteins were obtained from Escherichia coli BL21 (expression strain) through lysis using muramidase and sonication. Following this, Glutathione-Sepharose beads were employed to isolate GST-fusion proteins overnight at 4°C. Subsequently, the mixture was incubated with HIS-tagged protein overnight, washed five times with PBST, and then treated with RIPA Buffer and Loading Buffer. After thorough mixing, the mixture was boiled and centrifuged at 12,000 rpm for 5 minutes to collect the supernatant, which was used for subsequent WB analysis. 2.15 Statistical analysis The statistical analysis of data in this study was performed using SPSS 20.0 software, including t-tests, one-way ANOVA, and the Kruskal-Wallis test. All experimental procedures were carried out in triplicate, and the results are presented as mean values with standard error of the mean (SEM) indicated. *, ** and *** indicate P < 0.05, P < 0.01 and P < 0.001, respectively. 3.1 Expression patterns of melatonin-related genes in Hu Sheep with different fertility rates After slaughter, we collected pineal glands from both groups and conducted RNA-Seq, identifying 847 differentially expressed genes (DEGs) (Fig. 1A), with significant differences in the tryptophan metabolism pathway, as shown in Figure. 1B. Genes associated with melatonin synthesis, including AANAT, HIOMT, and TPH1, were significantly higher in the high-fertility group (HBB) group compared to the low-fertility group (LBB) group. Simultaneously, we measured the serum melatonin (MT) concentration in Hu sheep with different fertility rates, and the results showed that the MT concentration in HBB was significantly higher than in the LBB. We further validated these findings using qRT-PCR (Fig. 1D) and western blot (Fig. 1E), confirming that both mRNA and protein expression levels were consistent with the RNA-Seq data. Next, we examined the expression of melatonin receptors, MTNR1a and MTNR1b, in reproductive organs (hypothalamus (Fig. S1), pituitary (Fig. S2), ovary (Fig. 1F and G), and uterus (Fig. S3)) of HBB and LBB groups. The results indicated that the expression levels of MTNR1a and MTNR1b in the ovaries of the HBB group were significantly higher than those in the LBB group, with no significant differences observed in the hypothalamus, pituitary, and uterus. We then isolated ovarian follicles of different sizes from Hu sheep, categorizing them into 5mm groups, and used the 3-5mm and >5mm follicles for subsequent experiments. Elisa results showed that the MT concentration in the follicular fluid of >5mm follicles was significantly higher than that in the 3-5mm follicles (Fig. 1H), with a similar trend observed for estradiol (E2) (Fig. 1I) concentration. Additionally, we assessed the expression of MT receptors in different-sized follicles and found that both MTNR1a and MTNR1b were higher in the >5mm group compared to the 3-5mm group (Fig. 1J and K). Moreover, the expression levels of E2secretion-related mRNAs (Fig. 1I) and proteins (Fig. 1M) were significantly higher in the >5mm follicles than in the 3-5mm follicles. These results indicate that melatonin is closely related to follicle development and may be an important factor influencing Hu sheep fertility. Fig 1. Melatonin-related gene expression in high and low prolificacy Hu Sheep. ( A): Volcano plot of the number of DEGs of high and low fecundity Hu sheep in the pineal gland; ( B): Heatmap of melatonin synthesis-related gene expression in the pineal gland; ( C): Elisa detection of melatonin concentration in the serum; ( D and E): qRT-PCR and WB were used to detect the mRNA and protein expression levels of key melatonin synthesis genes in the pineal gland, respectively; ( F) Melatonin receptor mRNA levels in the ovaries; ( G): Melatonin receptor protein expression levels in the ovaries of high and low prolificacy Hu sheep; ( H): Melatonin concentration in Hu sheep follicles of different sizes; ( I): Estrogen concentration in Hu sheep follicles of different sizes; ( J): Expression levels of melatonin receptor mRNA in follicles of different sizes; ( K): Expression levels of melatonin receptor protein in follicles; ( I and M): Expression levels of mRNA and protein related to estrogen synthesis in follicles of different sizes. HBB represents the high prolificacy Hu sheep group, LBB represents the low prolificacy Hu sheep group, 3-5 mm represents the group with follicle diameters of 3-5 mm, and >5 mm represents the group with follicle diameters greater than 5 mm. (n=3) 3.2 Melatonin Promotes E2 Secretion and Proliferation in Sheep Granulosa Cells in vitro To verify the impact of MT on follicle development, we isolated GCs from Hu sheep and treated them with various concentrations (1 nM, 10 nM and 100 nM) of MT for 12 hours. We found that MT at concentrations of 10 nM and 100 nM enhanced the viability of GCs (Fig. 2A). Furthermore, Edu assays demonstrated that MT significantly promoted the proliferation of GCs in vitro (Fig. 2C). Meanwhile, melatonin at concentrations of 10 nM and 100 nM also significantly promoted the increased expression of the proliferation-associated protein PCNA in GCs (Fig. 2B). Subsequently, we examined the effects of MT on GC apoptosis. Our results showed that MT effectively reduced the apoptosis rate (Fig. 2D) of GCs and decreased the expression level of the apoptosis-related protein BAX and p53 (Fig. 2E), while increasing the expression of Bcl-2 (Fig. 2E). Additionally, MT increased the concentration of E2 in the culture medium (Fig. 2F) and significantly upregulated the mRNA (Fig. 2G) and protein (Fig. 2H) expression levels of Star, CYP11A1, and CYP19. These data indicate that MT can promote GCs proliferation and E2 synthesis while inhibiting apoptosis. Fig 2. The effects of melatonin on apoptosis, proliferation, and E2 synthesis of Hu sheep GCs in vitro. (A) CC8 assay for cell viability of Hu sheep GCs treated with different concentrations of melatonin; (B) Effects of melatonin on the expression level of the proliferation-related protein PCNA in GCs; (C) Edu assay to evaluate the effect of melatonin treatment on GCs proliferation; (D) Effects of melatonin on GCs apoptosis; (E) Expression levels of apoptosis-related proteins in GCs after melatonin treatment; (F) Elisa assay for E2 concentration in the culture medium; (G and H) Expression levels of E2 synthesis-related mRNA and proteins in GCs exposed to melatonin. NC represents the group treated with 0.1% DMSO, while 1 nM, 10 nM, and 100 nM represent the GCs groups treated with the corresponding concentrations of melatonin. (n=3) 3.3 The effects of melatonin on the transcriptome and translatome of GCs. To investigate the effects of MT on GCs, we conducted RNA-Seq and Ribo-seq analyses on MT-treated GCs. Through RNA-Seq, we identified 2,186 DEGs (Fig. 3A). GO and KEGG enrichment analyses of these DEGs showed significant enrichment in pathways such as cGMP-PKG, cAMP (Fig. 3B), as well as in terms like reproduction and the ERK1/ERK2 cascade (Fig. S4). Notably, the mRNA expression levels of estrogen synthesis-related genes, including Star and CYP11A1 , were significantly upregulated in the MT-treated group (Fig. 3E). Subsequently, we extracted the ribosome mRNA, known as ribosome footprints (RFs). Aligning the 5’ ends of these RFs to the genome revealed that in both the NC and MT groups, RFs showed the highest abundance at the start codon, displaying a characteristic ”high-low-low” triplet periodicity, confirming the successful extraction of ribosome-associated mRNA (Fig. S5A). Next, we classified the RFs in both groups according to their alignment positions. As shown in Figure S5B, over 60% of RFs in both the NC and MT groups were located in the coding sequence (CDS) region of coding genes, about 30% in intron regions, and a relatively low proportion in UTR regions. In the NC group, RFs located in the 5’ UTR accounted for 1.71% and in the 3’ UTR for 2.81%, while in the MT group, RFs in the 5’ UTR accounted for 2.20% and in the 3’ UTR for 3.40%. Next, we analyzed the expression levels of RFs located in the CDS region detected by Ribo-Seq and found that melatonin led to differential expression of 1,253 genes within the ribosome, with 456 genes showing increased expression and 797 genes showing decreased expression (Fig. 3C). The top ten KEGG pathways included ribosome, apoptosis, cell cycle, and others (Fig. 3D). GO enrichment analysis showed significant differences in reproduction-related terms (Fig. S6). Notably, melatonin significantly upregulated the expression levels of steroid synthesis-related genes, such as Star , CYP11A1 , and STARD9 , within the GCs ribosome (Fig. 3F). The above results indicate that melatonin affects E2 secretion in GCs and also provide direction for future research. Fig 3. Effects of melatonin on the transcriptome and proteome of Hu Sheep GCs in vitro. ( A): Number of DEGs detected in the transcriptome by RNA-Seq in GCs treated with melatonin in vitro; (B) Top 20 KEGG pathway analysis of DEGs in the transcriptome; (C) DEGs detected in the proteome by Ribo-Seq; (D) Top 20 KEGG pathway analysis of DEGs in the proteome; (E) Heatmap of DEGs related to steroidogenesis in the transcriptome; (F) Heatmap of genes related to E2 synthesis in the proteome. (n=2) 3.4 Discovery of a novel peptide named 9790aa To further explore the sORFs and micropeptides involved in MT regulation of GCs, we first identified 6,478 lncRNAs via RNA-Seq (Fig. 4A). This included 445 novel lncRNAs identified through joint evaluation using Contrastive Predictive Coding2 (CPC2), Coding-Non-Coding Index (CNCI), and Flexible Extraction of Long Non-Coding RNAs (Feelnc) software (Fig. 4C). Among the 6,478 lncRNAs, there were 787 bidirectional lncRNAs, 91 sense lncRNAs, 3,873 long intergenic non-coding RNAs (lincRNAs), 1,212 antisense lncRNAs, and 103 intronic lncRNAs (Fig. 4A). Following MT treatment, 177 of these lncRNAs were differentially expressed (Fig. 4C). Next, by integrating RNA-Seq and Ribo-Seq results, we identified a total of 21,999 novel sORFs and 10 known sORFs in sheep (Fig. 4D). Alignment analysis revealed that 50.45% of these sORFs were located in the 3’ UTR, 10.23% in the 5’ UTR, 27.37% in the mRNA CDS region, and 11.95% in lncRNAs (Fig. 4E). Moreover, as shown in Figure 4F, melatonin collectively led to significant differential expression of 98 sORFs in GCs. Next, we predicted the coding potential of all sORFs. Briefly, ORFscore and RRS were calculated based on the abundance and positional distribution of sORFs, while Fickett score and Hexamer score were calculated based on sORF sequence features. By combining these four scores, we identified 91 sORFs with translational potential (Fig. 4G). Among the differentially expressed sORFs with coding potential, we noted sORF9790 (Fig. 4H). sORF9790 is located on lncRNA1056 (Fig. 4J), consisting of 138 bases and predicted to encode a 45-amino-acid peptide (Fig. 4K). Ribo-Seq analysis showed that the expression levels of both lncRNA1056 (Fig. 4O) and sORF9790 (Fig. 4I) were significantly reduced after MT treatment. Next, we validated the coding potential of sORF9790. By transfecting an overexpression vector containing 3xflag and GFP tags into sheep GCs, we observed normal expression of green fluorescence in the oe-sORF9790 group (Fig. 4L). WB using a 3xFlag antibody revealed that the fusion protein in the oe-sORF9790 group was noticeably larger than in the positive control group (Fig. 4M). These results confirm that sORF9790 can indeed encode a peptide, which we named 9790aa. Additionally, a specific antibody for 9790aa was custom-developed, and WB results showed that MT treatment significantly reduced the expression level of 9790aa (Fig. 4N). In conclusion, the above results indicate that sORF9790 can encode a peptide, named 9790aa, and that MT reduces the expression level of 9790aa in GCs. Fig 4. Identification and validation coding potential of sORF9790. (A) Types of lncRNAs identified in Hu sheep GCs by RNA-Seq; (B) Identification of newly discovered lncRNAs using CNCI, CPC2, and Feelnv; (C) Number of differentially expressed lncRNAs after melatonin treatment; (D) Statistical analysis of the number of sORFs identified by combined RNA-Seq and Ribo-Seq; (E) Classification of sORFs based on their genomic location; (F) Number of differentially expressed sORFs in GCs after melatonin treatment; (G) Number of sORFs with coding potential predicted by four software tools; (H) Coding potential score of sORF9790; (I) Effect of melatonin on the expression of sORF9790 in GCs detected by Ribo-Seq; (J) Full sequence of lncRNA1056 and sORF9790, with the red section representing the sORF9790 sequence; (K) Schematic diagram showing the location of sORF9790 on lncRNA1056; (L) Overexpression of sORF9790 using a fluorescent-tagged vector to identify its coding potential in GCs; (M) WB analysis to detect the size of the overexpressed sORF fluorescent-tagged fusion protein; (N) Effect of melatonin on the expression level of 9790aa; (O) Effect of melatonin on the expression level of lncRNA1056. NC represents the control group, and MT represents the GCs group treated with 10 nM melatonin. (n=3) 3.5 Effects of lncRNA1056 and sORF9790 on proliferation, apoptosis, and E2 secretion of GCs We further examined the role of lncRNA1056 and sORF9790 in regulating GCs function and viability. As illustrated in Fig. 5A, overexpression of lncRNA1056 (oe-lnc1056) and sORF9790 (oe-9790) reduced GC viability, whereas knockdown of lncRNA1056 (si-lnc1056) or sORF9790 (si-9790) markedly enhanced cell viability. Moreover, both oe-lnc1056 and oe-9790 significantly increased E2 secretion (Fig. 5B) and upregulated the expression of associated proteins (Fig. 5C), while their knockdown led to opposing effects. Additionally, silencing lncRNA1056 or sORF9790 promoted GC proliferation (Fig. 5F and G) and suppressed apoptosis (Fig. 5D and E), with overexpression yielding reverse outcomes. Collectively, these findings highlight the parallel influence of lncRNA1056 and sORF9790 on GC status and functionality. Fig 5. The effects of lncRNA1056 and 9790aa on proliferation, apoptosis, and E2 synthesis in GCs in vitro. (A and B): Effects of interfering with/overexpressing lncRNA1056 and 9790aa on GCs cell viability and E2 concentration in the culture medium; (C): WB analysis of the effects of knockdown/overexpression of lncRNA1056 and 9790aa on the expression levels of steroidogenesis-related proteins; (D and E): Effects of lncRNA1056 and 9790aa on GCs apoptosis rate and apoptosis-related expression levels; (F and G): Effects of lncRNA1056 and 9790aa on the key proliferation protein PCNA and GCs proliferation rate. si-NC, si-lnc1056, and si-9790aa represent the siRNA control group, the siRNA-mediated lncRNA1056 knockdown group, and the siRNA-mediated 9790aa knockdown group, respectively; oe-NC, oe-lnc1056, and oe-9790aa represent the overexpression control group, the lncRNA1056 overexpression group, and the 9790aa overexpression group, respectively. (n=3) 3.6 lncRNA1056 regulates GCs through encoding the 9790aa peptide rather than through a ceRNA mechanism. There are various mechanisms by which lncRNAs regulate cellular functions, such as encoding peptides or modulating miRNA pathways through a ceRNA mechanism[17]. To determine whether lncRNA1056 functions via the peptide 9790aa encoded by sORF9790, we performed a point mutation on the ATG start codon of sORF9790 (oe-lnc1056 mut) while overexpressing lncRNA1056 (Fig. 6A). We observed that in the oe-lnc1056 mut group, the expression level of 9790aa was significantly reduced compared to the oe-lnc1056 and oe-9790 groups, with no significant difference from the oe-NC group (Fig. 6B). Additionally, compared with oe-NC, oe-lnc1056 mut showed no significant differences in cell viability (Fig. 6C), E2 secretion (Fig. 6D and E), apoptosis (Fig. 6F and G), or proliferation (Fig. 6H and I). These findings indicate that lncRNA1056 regulates GCs via encoding 9790aa, rather than through a ceRNA mechanism. Fig 6. Verify whether lncRNA1056 affects GCs proliferation, apoptosis, and E2 synthesis through encoding 9790aa. (A): Schematic diagrams of the three overexpression plasmids, with red indicating the position of sORF9790, blue indicating the position of lncRNA1056, and yellow indicating the position of the point mutation; (B): The expression level of 9790aa. (C): CCK8 assay for cell viability; (D): E2 concentration in the culture medium; (E): Expression levels of key E2 synthesis proteins in GCs; (F and G): Apoptosis rate and expression levels of apoptosis-related proteins in GCs; (H): PCNA protein expression levels; (I): Cell proliferation rate. oe-lnc1056mut represents the overexpression group of lncRNA1056 with a point mutation at the initiation codon. 3.7 Melatonin influences GCs proliferation, apoptosis, and E2 secretion by suppressing 9790aa expression. Given that MT significantly reduced 9790aa expression, we hypothesized that MT may affect GCs biological functions by inhibiting 9790aa. To test this, we overexpressed 9790aa in the presence of MT. Results showed that 9790aa overexpression partially counteracted the regulatory effects of MT on GCs, including cell viability (Fig. 7A), E2 secretion (Fig. 7B and C), proliferation (Fig. 7D and G), and apoptosis (Fig. 7E and F). These findings indicate that MT indeed regulates GC proliferation, apoptosis, and estrogen secretion by suppressing 9790aa expression. Fig 7. Melatonin regulates GCs apoptosis, proliferation, and E2 synthesis through 9790aa. (A and B): GCs cell viability and E2 concentration; (C): Expression levels of Star, CYP11A1, and CYP19 proteins in GCs; (D and E): GCs cell proliferation rate and apoptosis rate; (F and G): Expression levels of proliferation and apoptosis-related proteins. (n=3) 3.8 9790-aa binds to MAPK1/3 proteins. To investigate the mechanism of 9790aa, we performed RNA-Seq on GCs overexpressing 9790aa. A total of 164 DEGs were identified (Fig. 8A). KEGG enrichment analysis revealed significant enrichment of DEGs in pathways such as MAPK and cAMP signaling (Fig. 8C), with several key genes in the MAPK pathway showing significantly decreased expression in the oe-9790aa group (Fig. 8B). Next, we introduced exogenous NC-3xFlag-GFP and 9790aa-3xFlag-GFP into ovine GCs for co-IP, followed by mass spectrometry (Fig. 8D), identifying peptides from MAPK1 and MAPK1/3 (Fig. 8E). To further confirm binding between 9790aa and MAPK1/3, we transfected GCs with 9790aa-3xFlag-GFP, MAPK1-HA, and MAPK3-HA constructs and conducted co-IP, which showed binding between MAPK1 (Fig. 8F) and MAPK3 with the 9790aa fusion protein (Fig. 8G). Software predictions indicated binding interactions between 9790aa and MAPK1 at Arg-359 and Ser-360, and with MAPK3 at Glu-378 and Ala-379 (Fig. S7A). To confirm this, we mutated each of these four amino acid sites individually and performed GST pull-down assays. The results showed significantly reduced binding of 9790aa to His-MAPK1 (mut) and His-MAPK3 (mut) compared to their wild-type counterparts, His-MAPK1 (wt) and His-MAPK3 (wt), indicating that 9790aa specifically binds to MAPK1 at Arg-359 and Ser-360, and to MAPK3 at Glu-378 and Ala-379 (Fig. S7B, C). Fig 8. 9790aa Validation of the interaction between 9790aa and MAPK1/3. (A): The number of differentially expressed genes (DEGs) produced by the overexpression of 9790aa in Hu sheep GCs. (B): The effects of overexpressing 9790aa on the expression levels of key genes in the MAPK signaling pathway; (C): KEGG enrichment analysis of MAPK-related signaling pathways; (D): WB detection of protein expression after co-IP. €: Mass spectrometry detection of peptide segments of MAPK1 and MAPK3; (F): Validation of the interaction between 9790aa and MAPK1; (G): Validation of the interaction between 9790aa and MAPK3. 3.9 9790aa regulates GCs proliferation, apoptosis, and E2 synthesis through the MAPK1/3-Elk1 pathway. MAPK1/3 plays a crucial role in regulating proliferation and apoptosis in GCs. Given that 9790aa binds to MAPK1/3 proteins, we further assessed the effects of 9790aa knockdown/overexpression on key proteins within the MAPK1/3 signaling pathway. As shown in Fig. 9A, knockdown or overexpression of 9790aa had no significant effect on MEK1/2 or p-MEK1/2. However, 9790aa knockdown markedly increased MAPK1/3, p-MAPK1/3, and Elk1 expression levels, whereas overexpression of 9790aa led to reduced expression of these proteins. To further confirm whether 9790aa regulates GC proliferation, apoptosis, and E2 secretion via the MAPK1/3-Elk1 pathway, we added SCH772984 (5 nM), a MAPK1/3 inhibitor, to cells with 9790aa knockdown. As shown in Fig. 9B, treatment with SCH772984 significantly decreased p-MAPK1/3 and Elk1 expression. Next, we examined the effects of MAPK1/3 inhibition on GCs with 9790aa knockdown. Results revealed that SCH772984 reduced the effects of 9790aa knockdown on cell viability (Fig. 9C), E2 synthesis (Fig. 9D and E), and GC proliferation (Fig. 9H and I). Additionally, MAPK1/3 inhibition increased apoptosis rates (Fig. 9F) and the ratio of BAX/Bcl-2 in GCs (Fig. 9G). These findings indicate that 9790aa modulates GC proliferation, apoptosis, and E2 synthesis by binding to MAPK1/3 proteins and inhibiting MAPK1/3 activity, thereby influencing the MAPK1/3-Elk1 signaling pathway. Fig 9. 9790aa affects GCs proliferation, apoptosis, and E2 secretion through the MAPK1/3-Elk1 signaling pathway. (A): Effects of knocking down and overexpressing 9790aa on the expression of key MAPK1/3 signaling pathway proteins in GCs; (B): Effects of knocking down 9790aa and inhibiting MAPK1/3 activity on the MAPK1/3-Elk1 signaling pathway; (C and D): Effects of interfering with 9790aa and adding SCH772984 on GCs cell viability and E2 concentration; (E): Expression levels of E2 synthesis-related proteins; (F and G): GCs apoptosis rate and expression levels of BAX and Bcl-2 proteins; (H and I): PCNA expression levels and GCs proliferation rate. MT + si-NC represents the GCs group treated with melatonin and interference with NC (negative control); MT + si-9790aa represents the GCs group treated with melatonin and interference with 9790aa; MT + si-9790aa+SCH772984 represents the GCs group treated with melatonin, interference with 9790aa, and SCH772984 (a MAPK1/3 inhibitor). (n=3) 3.10 9790aa is regulated by MTNR2 and is closely associated with follicular development. To investigate which receptor mediates the regulation of 9790aa expression by melatonin, we simultaneously treated GCs with melatonin and selectively knocked down MTNR1a or MTNR1b, followed by the assessment of 9790aa and MAPK1/3 pathway-associated protein levels. As shown in Fig. 10A, compared to the MT group, si-MTNR1a + MT had no significant effect on the expression of 9790aa, MAPK1/3, p-MAPK1/3, or Elk1. In contrast, si-MTNR1b + MT increased the expression of 9790aa while reducing the protein levels of MAPK1/3, p-MAPK1/3, and Elk1. Next, we examined the expression levels of lncRNA1056 and 9790aa across various tissues, including the heart, liver, spleen, lungs, kidneys, skeletal muscle, ovaries, and uterus, in sheep. We found that the expression levels of lncRNA1056 (Fig. 10B) and 9790aa (Fig. 10C) in the ovary and skeletal muscle were significantly higher than in other organs of sheep. Similarly, 9790aa exhibited significantly higher expression levels in the mice ovary than in other tissues (Fig. 10E). Meanwhile, through IF observation, we found that 9790aa was primarily expressed in the cytoplasm of GCs (Fig. 10D). We further analyzed the expression of lncRNA1056, 9790aa, and MAPK1/3 pathway-related proteins in the ovaries of Hu sheep with differing reproductive capacities and in ovarian follicles of varying sizes. As shown in Fig. 10E, in ovarian follicles larger than 5 mm in diameter, 9790aa were expressed at significantly lower levels compared to those in 3–5 mm follicles, while MAPK1/3, p-MAPK1/3, and Elk1 exhibited an inverse expression pattern. Moreover, the expression levels of 9790aa in the ovaries of the HBB group were significantly lower than those in the LBB group, whereas the levels of MAPK1/3, p-MAPK1/3, and Elk1 showed the opposite trend (Fig. 10G). These observations suggest that 9790aa plays an important role in the ovary and that 9790aa and the MAPK1/3 signaling pathway are closely associated with follicular development. Fig 10. The expression pattern of 9790aa and the MAPK1/3-Elk1 pathway during follicular development. (A): Expression levels of 9790aa and the MAPK1/3-Elk1 pathway in GCs after interference with melatonin receptors MTNR1a and MTNR1b; (B): Expression levels of lncRNA1056 in various tissues of sheep; (C and E): Expression levels of 9790aa in different organs of sheep and mice; (D): Localization of 9790aa in GCs; (F): Expression levels of 9790aa and the MAPK1/3-Elk1 signaling pathway in follicles of different sizes; (G): Expression levels of 9790aa and MAPK1/3 in the ovaries of high and low reproductive Hu sheep. (n=3) Discussion In addition to genetic factors, follicular development is regulated by various factors[19]. Studies have shown that melatonin is abundant in follicular fluid, where it scavenges ROS accumulated within follicles, protecting oocytes and granulosa cells (GCs) from oxidative damage[20]. Moreover, melatonin influences biological processes such as GC proliferation and apoptosis[21]. In recent years, increasing numbers of sORFs and their corresponding micropeptides have been found to play significant roles in biological activities, such as cancer progression[22] and embryonic development[12]. However, whether melatonin regulates GC function and state via sORFs and micropeptides, thereby impacting follicular development, remains unclear. In this study, we selected Hu sheep with identical genotypes (FecBB homozygotes) but varying reproductive capacities to examine the levels of melatonin secretion from the pineal gland and melatonin receptor expression in reproductive-related organs (hypothalamus, pituitary, ovary, and uterus). Subsequently, we isolated follicles of different sizes from Hu sheep and found that melatonin concentration and receptor expression levels were significantly higher in large follicles compared to small ones. In vitro experiments revealed that melatonin promotes GCs proliferation and E2 synthesis while inhibiting apoptosis. Through combined RNA-Seq and Ribo-Seq analysis, we discovered that melatonin reduces the expression of lncRNA1056 and sORF9790. Further validation demonstrated that lncRNA1056 regulates GCs proliferation, apoptosis, and E2 synthesis through the micropeptide 9790aa encoded by sORF9790. co-IP, RNA-Seq, and GST pull-down assays of GCs overexpressing 9790aa indicated that 9790aa impacts the MAPK signaling pathway and interacts with MAPK1 and MAPK3 proteins. Through additional in vitro and in vivo validation, we ultimately identified that melatonin, via its receptor MTRN1b, downregulates lncRNA1056 and 9790aa expression, activating the MAPK1/3-Elk1 signaling pathway, thereby regulating GCs proliferation, apoptosis, and E2 synthesis, ultimately affecting follicular development. Hu sheep, a renowned Chinese local breed, are characterized by year-round estrus and prolificacy[23]. While FecBB homozygous genotype is typically associated with multiple lambs, instances of single-lamb births occur in FecBB homozygous ewes under production conditions[24]. This suggests that factors beyond genetics may influence reproductive performance. To investigate the relationship between melatonin and reproductive traits in Hu sheep, we selected two groups of FecBB homozygous ewes with contrasting reproductive outcomes (triplets across three consecutive births vs. single lambs across three consecutive births). Elisa analysis revealed higher serum melatonin levels in HBB group. RNA-Seq showed that the expression of genes related to melatonin synthesis in the pineal gland was significantly upregulated in HBB group compared to LBB group, a finding further confirmed by qRT-PCR and western blot analyses. These results indicate elevated melatonin levels in high-prolificacy Hu sheep. In vivo, melatonin secreted by the pineal gland acts on the hypothalamus and pituitary, and directly on the gonads to regulate reproductive performance[25]. MTNR1a and MTNR1b are the primary melatonin receptors in mammals[26]. In this study, we examined the expression levels of MTNR1a and MTNR1b in the hypothalamus, pituitary, ovary, and uterus of two groups. Notably, differential expression of these melatonin receptors was observed exclusively in the ovary. This finding suggests that melatonin may directly act on the ovary to influence its function in female Hu sheep. To investigate this, we isolated Hu sheep follicles of varying sizes and grouped them based on follicle diameter. Our analysis revealed that both melatonin concentrations and melatonin receptor expression levels were significantly higher in large follicles compared to small follicles, indicating a close association between melatonin and follicular development. GCs, the predominant cell type within ovarian follicles, play a pivotal role in follicular development and oocyte maturation[27]. Here, we isolated GCs from Hu sheep to examine the impact of melatonin on GC proliferation, apoptosis, and hormone synthesis. Our findings reveal that melatonin enhances GC proliferation, suppresses apoptosis, and increases the synthesis of E2. Emerging evidence suggests that sORFs and their encoded micropeptides play critical roles in diverse biological processes[28]. However, no studies to date have reported sORFs associated with melatonin. Currently, the integration of Ribo-Seq and RNA-Seq represents a powerful approach for the identification of sORFs. In this study, we employed Ribo-Seq and RNA-Seq to uncover sORFs involved in melatonin-mediated regulation of GCs. Our analysis revealed that melatonin significantly upregulated key genes associated with E2 synthesis, such as Star and CYP11A1 . These findings align with a substantial body of research demonstrating the influence of melatonin on steroidogenesis in GCs, further corroborating this regulatory role in the current study. Through sequencing, we identified 22,009 sORFs, among which 91 were predicted to have coding potential. Among these, we focused on sORF9790, located within lncRNA1056. Sequencing data revealed that melatonin significantly downregulated the expression of both lncRNA1056 and sORF9790. Previous studies have shown that some lncRNAs exert biological functions by encoding peptides[29]. In this study, we first confirmed the coding capacity of lncRNA1056 and found that sORF9790 encodes a 45-amino-acid micropeptide, which we named 9790aa. Functional assays using knockdown and overexpression approaches demonstrated that both lncRNA1056 and 9790aa influence GC proliferation, apoptosis, and E2 synthesis. To determine whether the regulatory effects were mediated by lncRNA1056 itself or its encoded micropeptide 9790aa, we overexpressed a mutant form of lncRNA1056 with a point mutation in the start codon of sORF9790, abolishing its coding ability. Phenotypic analysis showed that the mutant lncRNA1056 lost its regulatory effects on GCs, indicating that the micropeptide 9790aa, rather than lncRNA1056, exerts the regulatory function in GCs. To further investigate the mechanisms by which 9790aa regulates GCs, we performed RNA-Seq and co-IP analyses in GCs overexpressing 9790aa. The results revealed that 9790aa interacts with MAPK1/3 proteins. MAPK1 (ERK2) and MAPK3 (ERK1) are critical members of the MAPK family, which play essential roles in GCs[30]. Their activation is dependent on the classical three-tier kinase cascade, known as the Raf-MEK-MAPK pathway, one of the most crucial signal transduction pathways in mammalian cells[31]. This pathway amplifies extracellular signals through a cascade mechanism to regulate various physiological processes, including cell proliferation[32], differentiation, and survival[33]. Typically, extracellular signals first activate Raf (MAP3K), which subsequently activates the MAP kinase kinases (MAP2Ks), namely MEK1 and MEK2. MEK1/2, dual-specificity kinases, phosphorylate both serine and threonine residues of downstream MAPK1 and MAPK3[34]. Activated MAPK1/3 translocate from the cytoplasm to the nucleus, where they activate a range of transcription factors and target proteins, such as Elk-1 and c-Fos, thereby modulating gene expression and regulating cellular proliferation and differentiation[35]. In this study, we observed that knockdown or overexpression of 9790aa selectively affected the expression of MAPK1/3, p-MAPK1/3, and Elk1, without altering MEK1/2 levels. This finding suggests that 9790aa may regulate GCs by modulating the activity of the MAPK1/3-Elk1 signaling pathway. To validate this hypothesis, we inhibited MAPK1/3 activity in GCs while simultaneously downregulating 9790aa. The inhibition of MAPK1/3 significantly alleviated the effects of 9790aa knockdown on GC proliferation, apoptosis, and E2 synthesis. These results indicate that 9790aa exerts its regulatory effects on GCs via the MAPK1/3-Elk1 signaling pathway. Given that melatonin reduces the expression of 9790aa, we hypothesized that melatonin may regulate GCs function via 9790aa. To test this, we treated GCs with melatonin while simultaneously overexpressing 9790aa. The results demonstrated that overexpression of 9790aa attenuated the effects of melatonin on GCs. Melatonin primarily exerts its biological effects through its receptors. To identify which receptor mediates the regulation of 9790aa by melatonin, we knocked down MTNR1a and MTNR1b in GCs and treated the cells with melatonin. Knockdown of MTNR1b, but not MTNR1a, significantly altered the protein levels of 9790aa, MAPK1/3, p-MAPK1/3, and Elk1. These findings suggest that melatonin regulates 9790aa expression and the MAPK1/3-Elk1 pathway via its receptor MTNR1b. To explore the in vivo role of 9790aa, we assessed its expression across various tissues in sheep and mice. The expression of 9790aa was significantly higher in the ovary than in other tissues, underscoring its critical role in ovarian function. Furthermore, we examined the expression of lncRNA1056, 9790aa, and key proteins in the MAPK1/3-Elk1 pathway in the ovaries of high- and low-fertility Hu sheep, as well as in ovarian follicles of different sizes. Our results revealed that the expression levels of lncRNA1056 and 9790aa decreased with enhanced ovarian development and reproductive capacity, while the expression of the MAPK1/3 pathway showed an opposite trend. These findings indicate that 9790aa suppresses the MAPK1/3-Elk1 signaling pathway and is closely associated with follicular development. Conclusion In this study, we identified a novel micropeptide, 9790aa, encoded by sORF9790 on lncRNA1056, as a key mediator of melatonin’s effects on GCs in Hu sheep. Melatonin, via its receptor MTNR1b, regulates the expression of lncRNA1056 and 9790aa, which in turn modulates GC proliferation, apoptosis, and estrogen secretion. Mechanistically, 9790aa binds to MAPK1 and MAPK3 proteins, suppressing the MAPK1/3-Elk1 signaling pathway, a crucial regulator of GCs function. These findings reveal a previously uncharacterized melatonin-micropeptide axis that connects melatonin signaling to ovarian follicular development. Importantly, we demonstrated that the suppression of 9790aa expression by melatonin enhances GCs function and promotes follicular development, contributing to improved fertility outcomes in Hu sheep. Funding statement This work was supported by National Natural Science Foundation of China (NSFC: 32472903) Declaration of competing interest No conflict of interest exits in the submission of this manuscript, and manuscript is approved by all authors for publication. Data Availability Statement The data that support the findings of this study are available from the corresponding author upon reasonable request. Conflicts of interest The authors have no conflicts of interest to disclose. References [1] Z. Zhu, M. He, T. Zhang, T. Zhao, S. Qin, M. Gao, W. Wang, W. Zheng, Z. Chen, L. Liu, M. Hao, B. Zhou, H. Zhang, J. Wang, F. Wang, G. Xia, C. Wang, Sci Bull (Beijing) 2024, 69 (8), 1122, https://doi.org/10.1016/j.scib.2024.01.015. [2] Q. Zhu, J. Du, Y. Li, X. Qin, R. He, H. Ma, X. Liang, Gene 2025, 933, 148979, https://doi.org/10.1016/j.gene.2024.148979. [3] N. Gleicher, S. Darmon, P. Patrizio, D. H. Barad, Biomedicines 2022, 10 (7), https://doi.org/10.3390/biomedicines10071505. [4] A. C. 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(A): Melatonin receptor mRNA expression levels in the pituitary of high and low prolificacy Hu sheep. (B): Melatonin receptor protein expression levels in the pituitary of high and low prolificacy Hu sheep. Fig. S3. Expression of melatonin receptors in the uterus of high and low prolificacy Hu sheep. (A): Melatonin receptor mRNA levels in the uterus of high and low prolificacy Hu sheep. (B): Melatonin receptor protein expression levels in the uterus of high and low prolificacy Hu sheep. Fig. S4. GO analysis of DEGs in RNA-Seq Figure S5. Analysis of Ribo-seq. (A): Distribution map of trinucleotide rhythmicity. (B):Distribution map of RFs compared to gene positions. Fig. S6. GO analysis of DEGs in Ribo-Seq Fig. S7. Validation of the binding sites between 9790aa and MAPK1/3. (A): Software prediction of the binding sites between 9790aa and MAPK1/3. (B): GST pull-down verification of the binding of 9790aa to the ARG-359 and SER-360 sites of MAPK1. (C): GST pull-down verification of the binding of 9790aa to the GLU-378 and ALA-379 sites of MAPK3. Information & Authors Information Version history V1 Version 1 09 October 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords 9790aa mapk1/3 sorf Authors Affiliations Jianyu Ma Nanjing Agricultural University Sanya Research Institute View all articles by this author Hua Yang Nanjing Agricultural University View all articles by this author Caifang Ren Jiangsu University View all articles by this author Zhibo Wang Nanjing Agricultural University View all articles by this author Xinai Huang Nanjing Agricultural University View all articles by this author Peiyong Chen Nanjing Agricultural University View all articles by this author Shanglai Li Nanjing Agricultural University View all articles by this author Yongjie Wan Nanjing Agricultural University View all articles by this author Yanli Zhang Nanjing Agricultural University View all articles by this author Feng Wang 0000-0002-8887-8998 [email protected] Nanjing Agricultural University Sanya Research Institute View all articles by this author Metrics & Citations Metrics Article Usage 124 views 98 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Jianyu Ma, Hua Yang, Caifang Ren, et al. A novel micropeptide 9790aa mediates melatonin regulation of granulosa cell proliferation, apoptosis, and estrogen secretion in Hu sheep through the MAPK1/3-Elk1 signaling pathway.. Authorea . 09 October 2025. DOI: https://doi.org/10.22541/au.176004948.80876527/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu . 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