Search Results (28)

Normal proliferation of ovarian granulosa cells is essential for follicular development. The results of this study showed that ADAMTS1 was primarily localized in the cytoplasm of granulosa cells in sheep ovarian follicles, as revealed by immunohistochemistry and immunofluorescence staining. Knockdown and overexpression experiments of ADAMTS1 in granulosa cells demonstrated that the number of EdU-positive cells significantly decreased in the knockdown group (p < 0.05), while the expression levels of Bax (p < 0.05), Bax/Bcl2 (p < 0.01), and caspase3 (p < 0.05) were significantly upregulated, indicating that knockdown of ADAMTS1 markedly inhibited granulosa cell proliferation. In contrast, overexpression of ADAMTS1 significantly promoted cell proliferation. Transcriptome sequencing revealed that PSAT1 and SLC6A9 were significantly downregulated in the knockdown group and significantly upregulated in the overexpression group, which was confirmed by Quantitative Polymerase Chain Reaction (Q-PCR) (p < 0.05). KEGG enrichment analysis showed that PSAT1 was significantly enriched in the glycine, serine and threonine metabolism and vitamin B6 metabolism pathways. Molecular docking analysis indicated a stable binding interface between ADAMTS1 and PSAT1. Based on these findings, we speculate that ADAMTS1 may regulate amino acid metabolism in ovarian granulosa cells by modulating the expression of SLC6A9, which in turn affects PSAT1 in the glycine, serine, and threonine metabolism and vitamin B6 metabolism pathways, thereby influencing granulosa cell proliferation. Full article

Seasonal reproduction in sheep reduces reproductive efficiency. Melatonin (MT) plays a crucial role in reproductive processes. The purpose of this study was to assess the effects of a 5-day MT implant pretreatment on estrus synchronization and reproductive performance in sheep during seasonal anestrus. A total of 40 multiparous Mongolian sheep were selected and randomly divided into two groups. In the MT group (n = 20), the ewes received an MT implant for 5 days, and then, they were given a progesterone (P4)-containing vaginal sponge for 14 days with equine chorionic gonadotropin (eCG) administered (330 I.U. per ewe; I.M.) at sponge removal. Control (CON) ewes (n = 20) were similarly treated but did not receive MT implants. The results demonstrated that MT implantation significantly improved serum levels of total antioxidant capacity (T-AOC), superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), and glutathione peroxidase (GSH-Px), increased post-ovulatory luteal diameter and serum P4 levels, and reduced ovarian apoptosis. Compared with the CON group, the MT group showed significantly higher pregnancy (68.23% vs. 50.59%) and lambing rates (63.53% vs. 47.06%; number of lambed ewes/number of total ewes) following cervical-timed artificial insemination. Ovarian transcriptome analysis revealed 522 differentially expressed genes (DEGs) in the MT group compared with the CON group, including 355 upregulated and 167 downregulated DEGs. In addition, MT significantly enhanced proliferation and inhibited apoptosis in cultured granulosa cells (GCs) and luteal cells (LCs) in vitro. Moreover, it enhanced the antioxidant capacity of GCs and LCs probably by activating the NRF2 signaling pathway as well as stimulating steroid hormone synthesis. In conclusion, MT implantation 5 days before applying the conventional P4-eCG protocol enhances ovine reproductive outcomes during seasonal anestrus. MT implantation has a beneficial role on the growth and function of ovarian cells. These findings offer novel evidence supporting the functional role of MT in mammalian reproduction, and would be informative for optimizing estrus synchronization in sheep. Full article

Climate change poses an increasing threat to livestock reproduction, with heat stress (HS) known to significantly impair ovarian function. This study aimed to elucidate the impact of HS on ovarian function and circRNA expression profiles in Hu sheep. Twelve ewes were randomly assigned to a control (Con, n = 6) or HS group (n = 6) and exposed to different temperatures for 68 days. Compared with the Con group, HS significantly increased the respiratory rate (108.33 ± 3.72 vs. 63.58 ± 2.42 breaths/min), pulse rate (121.17 ± 3.98 vs. 78.08 ± 3.31 beats/min), and rectal temperature (40.17 ± 0.14 °C vs. 39.02 ± 0.21 °C; p < 0.05). Concurrently, serum antioxidant levels were markedly decreased, including total antioxidant capacity (T-AOC), total superoxide dismutase (T-SOD), and glutathione peroxidase (GSH-Px) (p < 0.05). Histological analysis revealed a significant reduction in the numbers of primordial, primary, secondary, and mature follicles, alongside an increase in antral follicles (p < 0.05). TUNEL staining demonstrated enhanced granulosa cell apoptosis (p < 0.05), accompanied by the upregulation of pro-apoptotic genes Bax and Caspase-3 and downregulation of the anti-apoptotic gene Bcl-2, as confirmed by qPCR (p < 0.05). CircRNA sequencing identified 152 differentially expressed circRNAs (120 upregulated, 32 downregulated), and enrichment analyses indicated their involvement in apoptosis, mitophagy, and the FoxO signaling pathway. Collectively, these findings demonstrate that HS impairs ovarian physiology and antioxidant defense, induces follicular damage and cell apoptosis, and alters circRNA expression profiles, providing new insights into the molecular mechanisms underlying HS-induced reproductive dysfunction in Hu sheep. Full article

The transforming growth factor-beta (TGF-β) superfamily plays a crucial role in regulating female reproductive traits, particularly litter size, in small ruminants, such as sheep and goats. This review comprehensively examines the molecular mechanisms through which TGF-β superfamily members—including bone morphogenetic proteins (BMPs), growth differentiation factor 9 (GDF9), inhibin (INHA and INHB), and associated signaling genes—influence ovarian follicular development, ovulation rate, and ultimately, litter size. We synthesize recent findings on polymorphisms in key genes, such as BMPR1B, BMP15, GDF9, inhibins and SMADs family genes, across diverse sheep and goat breeds worldwide. The manuscript highlights how specific mutations in these genes create an intricate signaling network that modulates granulosa cell proliferation, follicular sensitivity to FSH, and the prevention of dominant follicle selection. These molecular interactions result in increased ovulation rates and larger litter sizes in prolific breeds. The gene dosage effects observed in heterozygous versus homozygous mutation carriers further illuminate the complex nature of these reproductive regulations. This improved the understanding of the genetic basis for prolificacy provides valuable insights for marker-assisted selection strategies aimed at enhancing reproductive efficiency in small ruminant breeding programs, with significant implications for improving livestock productivity and economic outcomes. Full article

Background/Objectives: Puberty is a critical stage in sheep development when reproductive capability is established, but the hormonal mechanisms underlying this transition remain incompletely understood. This study aimed to investigate the dynamic changes in estradiol (E2) levels and the expression patterns of estrogen receptors (ERα and ERβ) during puberty in Duolang sheep, a breed characterized by early sexual maturity and high reproductive efficiency. Methods: A total of 18 female Duolang sheep were assigned to three developmental stages (n = 6 per group): prepuberty (145 days), puberty (within 0 h of first estrus), and postpuberty (+3 days). Serum E2 concentrations and the mRNA and protein levels of ERα and ERβ were assessed in the hypothalamus, pituitary, and ovary. Additionally, primary ovarian granulosa cells (GCs) were isolated and stimulated in vitro with increasing concentrations of E2 (0–1000 ng/mL) to evaluate the dose-dependent expression of ERα, ERβ, and gonadotropin-releasing hormone (GnRH). Results: E2 levels peaked at the onset of puberty and declined thereafter. ERα expression in the hypothalamus and pituitary decreased during puberty but rebounded postpuberty, indicating a role in negative feedback regulation. In contrast, ovarian ERα expression reached its highest level during puberty, while ERβ expression in the ovary gradually increased from prepuberty to postpuberty. In GCs, ERα exhibited a biphasic expression pattern, peaking at 250 ng/mL E2 and decreasing at higher concentrations. ERβ and GnRH expression levels increased in a dose-dependent manner. Conclusions: These findings suggest that ERα primarily mediates E2 feedback within the hypothalamus–pituitary axis, whereas ERβ is associated with ovarian development and may regulate GnRH expression during the pubertal transition. The study provides new insights into the hormonal regulation of puberty in Duolang sheep and offers potential biomarkers for improving reproductive efficiency through targeted breeding strategies. Full article

Toll-like receptors (TLRs) play crucial roles in innate immunity, but their function in reproduction remains poorly understood. This study investigated the expression patterns and localization of TLR1-9 in the reproductive system of Hu sheep and their potential association with prolificacy. All TLRs were expressed in the oviduct, uterus, and ovary, with TLR6 showing significantly higher expression in the oviduct, while TLR3, TLR6, and TLR7 were predominantly expressed in the ovary. Following this initial screening, we focused on TLR2, TLR6, and TLR7 for detailed analysis. Immunohistochemical analysis revealed that TLR2, TLR6, and TLR7 were localized in the luminal epithelium and circular muscle of the oviduct, the luminal and superficial glandular epithelium of the uterus, and in ovarian follicles at all developmental stages. A comparative analysis between high-prolificacy (HP) and low-prolificacy (LP) Hu sheep demonstrated significantly lower TLR2 expression in the reproductive organs of HP sheep, while TLR6 expression was higher and TLR7 expression was lower in HP ovaries compared to LP ovaries. Notably, TLR7 was observed around apoptotic bodies of granulosa cells, suggesting a potential role in follicular development through the regulation of granulosa cell apoptosis. These findings establish a novel link between innate immunity and reproductive function in sheep, suggesting that TLRs, particularly TLR2, TLR6, and TLR7, may serve dual roles as immune sentinels and reproductive regulators influencing ovine fertility. Full article

Background: The diameter of mature follicles in donkeys is several times larger than in cattle and sheep, but the key genes responsible for maintaining follicular development and preventing apoptosis remain unclear. Methods: This study observed the process of donkey follicular development using ultrasound and analyzed the changes in common reproductive hormones in serum. Granulosa cells (GCs) were collected from large (mature follicles, diameter ≥ 37 mm) and small (atretic follicles, diameter 10–25 mm) follicles for sequencing to screen differentially expressed genes (DEGs) and signaling pathways influencing the development of mature follicles. The roles of selected genes were further validated in in vitro cultured GCs. Results: Donkey follicles exhibited rapid growth 5–7 days before ovulation, reaching maturity at a diameter of 37 mm. The maximum diameter of ovulatory follicles was approximately 40.7 mm, while non-ovulatory follicles began to undergo atresia when reaching about 25 mm. Serum reproductive hormone levels aligned with follicular developmental status. RNA sequencing identified 3291 DEGs between large and small follicles, with KEGG analysis highlighting enrichment in the PI3K-Akt signaling pathway, focal adhesion, amoebiasis, and cancer pathways. Lentiviral overexpression and interference assays targeting the DEGs EMCN and SYT12 revealed that EMCN positively regulates FOXO3, a key gene in the PI3K-Akt pathway. Conclusions: The EMCN gene in mature donkey follicles regulates FOXO3 in the PI3K-Akt signaling pathway, potentially inhibiting apoptosis in follicular granulosa cells and sustaining follicular development until ovulation. This study provides insights into the mechanisms underlying follicular development in donkeys. Full article

Anti-Müllerian hormone (AMH), produced only by granulosa cells, is a biomarker for ovarian reserve in sheep. This study compared AMH levels in two Slovenian breeds: Istrska pramenka (IP), a seasonal breeder, and Jezersko–Solčava (JS), which reproduces year-round. Blood serum samples from 78 sheep were analyzed during the estrus period using an ovine AMH ELISA. JS sheep were grouped by age (1–3, 4–6, ≥ 7 years), while the majority of IP sheep were ≥7 years. AMH levels differed significantly between breeds (p < 0.001), with JS sheep showing higher concentrations. No age-related differences were found within the JS breed (p = 0.752), but JS sheep ≥ 7 years had higher AMH than IP sheep of the same age (p < 0.001). AMH levels were also higher in ewes lambing two lambs (p < 0.001) and were positively correlated with litter size, particularly in the JS sheep ≥ 7 years (p < 0.001). These findings suggest AMH is a useful fertility marker in sheep, although breed-specific reference values are essential for an accurate interpretation. The study highlights the influence of breed and age on AMH levels and its potential role in predicting reproductive performance. Full article

MiRNAs regulate follicle development and atresia, steroid production, granulosa cell (GC) proliferation, and apoptosis. However, the target genes and the functioning of novel miRNAs remain unexplored. We reveal the targeting relationship between novel-m0297-5p and WNT5A and the specific regulatory mechanism of GC proliferation in small-tailed Han sheep using whole transcriptomic sequencing. We performed whole transcriptomic sequencing on small-tailed Han sheep ovarian GCs supplemented with 10 ng/mL of transforming growth factor-β1 (TGF-β1) during the early stages. This led to identifying the differential expression of novel-m0297-5p and Wnt family member 5A (WNT5A) and predicting their targeting relationship. Based on this, we hypothesized that TGF-β1 could mediate novel-m0297-5p targeting WNT5A to participate in the proliferation process of GCs in small-tailed sheep. We confirmed the relationship between TGF-β1 and both novel-m0297-5p and WNT5A. The mimicry of novel-m0297-5p inhibited GC activity and proliferation. However, the inhibition of novel-m0297-5p yielded the opposite effect. We validated the binding site for novel m0297-5p within the 3′UTR of WNT5A using dual-luciferase reporter gene. TGF-β1 alleviated the impact induced by the mimicry of novel-m0297-5p on cell viability. Inhibitor co-transfection for both novel-m0297-5p and si-WNT5A suppressed the granulocyte proliferation induced by novel-m0297-5p inhibition. These findings suggest that TGF-β1 can mediate the inhibitory effect of novel-m0297-5p targeting WNT5A on GC proliferation and activity in small-tailed Han sheep. This study provides an experimental basis for research on the biological function of GCs and their impact on follicle development. Full article

The glucose metabolism homeostasis in the follicular fluid microenvironment plays an important role in follicular maturation and ovulation, and excessively high or low glucose concentrations have adverse effects on the differentiation of follicular granulosa cells (GCs). However, a limited number of microRNAs (miRNA) have been reported to be involved in glucose-stimulated GCs differentiation. In this study, we characterized the miRNA expression profiles of sheep ovarian GCs cultured in high-glucose and optimal glucose concentrations and focused on a differentially expressed miRNA: miR-17-5p, which may be involved in regulating high-glucose-induced GC apoptosis by targeting KPNA2. We found that overexpression of miR-17-5p significantly promoted GCs proliferation and inhibited cell apoptosis, while downregulated the mRNA and protein expression of apoptosis-related makers (Bax, Caspase-3, Caspase-9, and Bcl-2). In contrast to the classical mechanism of miRNA silencing target gene expression, miR-17-5p overexpression significantly upregulated the expression of target gene KPNA2. A dual luciferase reporter gene assay verified the targeted binding relationship between miR-17-5p and KPNA2 promoter. Meanwhile, overexpression of KPNA2 further promoted the downregulation of apoptosis-related genes driven by miR-17-5p mimics. Knockdown of KPNA2 blocked the inhibitory effect of miR-17-5p mimics on the expression of apoptosis-related genes. Our results demonstrated that miR-17-5p activated the KPNA2 promoter region and upregulated KPNA2 expression, thereby inhibiting GCs apoptosis under high glucose. Full article

Oxidative stress is a significant factor in the death of granulosa cells (GCs), leading to follicular atresia and consequently limiting the number of dominant follicles that can mature and ovulate within each follicular wave. Follicular fluid contains a diverse array of metabolites that play crucial roles in regulating GCs’ proliferation and oocyte maturation, which are essential for follicle development and female fertility. However, the mechanisms behind metabolite heterogeneity and its effects on GCs’ function remain poorly understood. Here, we identified elevated nicotinamide levels in the follicular fluid of high-prolificacy sheep, correlated with oxidative stress in GCs, by an integrated analysis. In vitro experiments demonstrated that supplementation with β-nicotinamide mononucleotide (NMN) significantly increased the levels of nicotinamide adenine dinucleotide (NAD+) and adenosine triphosphate (ATP) in GCs. NMN treatment effectively reduced Lipopolysaccharide (LPS)-induced apoptosis and mitigated mitochondrial dysfunction, while also decreasing the production of reactive oxygen species (ROS), thereby enhancing the activity of the antioxidant defense system. Importantly, NMN treatment improved the impairments in steroid hormone levels induced by LPS. Mechanistically, the protective effects of NMN against GCs function were mediated via the AMPK/mTOR pathway. Collectively, our findings elucidate the metabolic characteristics associated with sheep prolificacy and demonstrate that NMN effectively protects GCs from LPS-induced dysfunction and enhances ovarian responsiveness via the AMPK/mTOR pathway. These findings also position NMN as a potential novel metabolic biomarker in enhancing ovarian function. Full article

Inhibin β-A (INHBA), a TGF-β superfamily member, is crucial for developing follicles. Although miRNAs are essential for post-transcriptional gene regulation, it is not yet known how they affect the expression of INHBA during follicle development. Using bioinformatics analyses, miR-134-3p was found, in this investigation, to be a crucial microRNA that targets INHBA in sheep GCs. Furthermore, when the follicular diameter expanded, there was a discernible decline in miR-134-3p expression. The miR-134-3p overexpression markedly reduced the proliferation of GCs, whereas its knockdown augmented it. Moreover, cell cycle progression was enhanced by miR-134-3p overexpression. Furthermore, miR-134-3p overexpression heightened GC apoptosis, while its knockdown reduced it. Importantly, miR-134-3p overexpression blocked the PI3K/AKT/mTOR axis, whereas its knockdown stimulated it. Overall, the outcomes of transfections with INHBA and miR-134-3p showed that, in sheep GCs, miR-134-3p targets INHBA to control cell proliferation and apoptosis. In summary, these results add to our understanding of the molecular mechanisms involving important miRNAs in ewe fecundity by indicating that miR-134-3p influences cell proliferation, cell apoptosis, and the TGF-β/PI3K/AKT/mTOR axis, which, in turn, influences the follicular development of sheep GCs. Full article

Prolactin (PRL) plays a key role in the growth and ovulation of animal follicles, but its impact on follicular recruitment in ewes remains uncertain. In this study, a total of sixteen healthy ewes (Hu sheep, aged 2–3 years, with continuous reproduction and housed separately), matched for parity and weight (52.98 ± 0.96 kg), were randomly assigned to two groups: a control group (C) and a treatment group (T, PRL inhibition). Ovaries were collected in vivo after anesthesia during the estrus stage, and tissue morphology was observed using hematoxylin–eosin (HE) staining. By using RNA sequencing on the ovaries of C and T groups and conducting bioinformatics analysis, the essential genes and pathways involved in the regulation of PRL inhibition were pinpointed. Subcellular localization of key genes in ovarian tissue was determined using a fluorescence in situ hybridization (FISH) assay and immunohistochemistry. The function of key genes was validated using knockout and overexpression techniques. During the estrus phase, we noted a marked rise in the count of large follicles within ovarian tissue following the inhibition of prolactin. In total, 328 differentially expressed genes (DEGs) were detected, with 162 upregulated and 166 downregulated. The results indicated that inhibiting PRL primarily influences follicle recruitment by acting on the target gene PIKfyve. Following the inhibition of PRL during the estrus phase, there was an increase in the expression of PIKfyve. PIKfyve was primarily localized in the ovarian granulosa cells (GCs) and cumulus cells (CCs) in the ovarian tissue of ewes. The overexpression of PIKfyve decreased cell apoptosis and enhanced steroid hormone release, whereas knockout of PIKfyve had the reverse effect. In conclusion, PRL inhibition promoted follicle recruitment in ewes by upregulating PIKfyve during the estrus stage. Full article

Atresia is a process in ovarian follicles that is regulated by hormone-induced apoptosis. During atresia, granulosa cell (GC) apoptosis is a key mechanism orchestrated through diverse signaling pathways. Cocaine- and amphetamine-regulated transcript (CART) signaling within ovarian GCs has been demonstrated to play a key role in the regulation of follicular atresia in cattle, pigs, and sheep. The present work aimed to investigate the potential local regulatory role of CART in GC apoptosis-induced follicular atresia in buffalo, focusing on the modulation of the AKT/GSK3β/β-catenin signaling pathways, which are the intracellular signaling pathways involved in cell viability. Our findings revealed increased expression of CARTPT and BAX and decreased levels of AKT, β-catenin, and CYP19A1 genes in atretic follicles compared to healthy follicles. Subsequently, CART treatment in the presence of FSH inhibited the FSH-induced increase in GC viability by reducing estradiol production and increasing apoptosis. This change was accompanied by an increase in the gene expression levels of both CARTPT and BAX. At the protein level, treatment with CART in the presence of FSH negatively affected the activity of AKT, β-catenin, and LEF1, while the activity of GSK3β was enhanced. In conclusion, our study shows how CART negatively influences buffalo GC viability, underlying the modulation of the AKT/GSK3β/β-catenin pathway and promoting apoptosis—a key factor in follicular atresia. Full article

Neurotrophin receptor B (NTRK2), also named TRKB, belongs to the neurotrophic factor family. Previous studies have shown that NTRK2 is associated with high fertility in mammals. However, the molecular mechanism and regulatory pathway of this neurotrophic factor remain unclear. In this study, NTRK2 overexpression and NTRK2-siRNA were constructed to detect the effects of NTRK2 on the proliferation and hormone secretion of the ovarian granulosa cells (GCs) of sheep. We successfully isolated follicular phase granulosa cells in vitro from the ovaries of sheep in simultaneous estrus, and the immunofluorescence results confirmed that NTRK2 was expressed in the collected cells. Subsequently, the effect of NTRK2 on the proliferation of sheep granulosa cells was examined via cell transfection experiments. The results showed that the expression of CDK4 and CyclinD2 was significantly increased after NTRK2 overexpression, while the opposite trend was observed after the inhibition of NTRK2 expression (p < 0.05). The EdU and CCK-8 assays showed that the proliferation rate of sheep GCs was significantly increased after NTRK2 overexpression, while the opposite trend was observed after the inhibition of NTRK2 expression (p < 0.05). Moreover, NTRK2 significantly increased the expression of steroidogenesis-related genes, including steroidogenic acute regulatory protein (STAR) and hydroxy-δ-5-steroid dehydrogenase (HSD3B1), and cytochrome P450 family 19 subfamily A member 1 (CYP19A1). The ELISA results showed that the secretion levels of E₂ and P₄ significantly increased after NTRK2 overexpression, while the opposite trend was observed after the inhibition of NTRK2 expression (p < 0.05). Previous studies had confirmed that NTRK2 gene belongs to the PI3K-AKT signaling pathway and participates in the signaling of this pathway. This was demonstrated by protein–protein interaction analysis and NTRK2 belongs to the PI3K-AKT pathway. The modification of PI3K and AKT, markers of the PI3K-AKT pathway, via phosphorylation was increased after NTRK2 overexpression in the sheep GCs, while the opposite trend was observed after the inhibition of NTRK2 expression (p < 0.05). Overall, these results suggest that the NTRK2 gene regulates the proliferation of GCs and the secretion of steroid hormones in sheep, and that it influences the phosphorylation level of the PI3K/AKT signaling pathway. These findings provided a theoretical basis and new perspectives for exploring the regulation of NTRK2 gene in the development of ovine follicles. Full article