Search Results (68)

Follicular development is recognized as a highly complex biological process regulated by multiple factors. Thyroid hormone (TH) is considered one of the key regulators of female reproduction, and its dysregulation can significantly impair follicular development. Epigallocatechin gallate (EGCG), the main active component of green tea, possesses strong antioxidant properties. Numerous studies have demonstrated that EGCG positively influences reproductive function in both humans and animals. However, whether EGCG directly affects follicular development under conditions of TH dysregulation remains poorly understood. The primary objective of this study was to investigate the impact of hyperthyroidism on ovarian development, examine whether EGCG could mitigate the adverse effects of TH dysregulation, and elucidate the underlying molecular mechanisms. In the T₄-induced hyperthyroidism rat model, ovarian tissues were serially sectioned for Hematoxylin-Eosin (HE) and Masson’s trichrome staining to assess morphological changes, and follicle numbers were quantified at each developmental stage. Granulosa cell (GC) viability, proliferation, and apoptosis induced by T₃ were evaluated using CCK8, EdU, and TUNEL assays, respectively. Antioxidant enzyme activity was measured, and the expression levels of related proteins were analyzed via Western blotting. Results showed that hyperthyroidism altered ovarian structure, significantly increasing the number of atretic follicles. Levels of antioxidant enzymes, including Superoxide Dismutase (SOD), Glutathione Peroxidase (GSH-PX), and Catalase (CAT), were markedly decreased, whereas the lipid peroxidation product malondialdehyde (MDA) was significantly elevated. Furthermore, all ERS-related proteins, phosphorylated Eukaryotic Initiation Factor 2 Alpha (p-eIF2α), Activating Transcription Factor 4 (ATF4), C/EBP homologous protein (CHOP), and Caspase-3, were upregulated, accompanied by decreased glucose-regulated protein 78 (GRP78) expression. Treatment with EGCG alleviated these detrimental effects of hyperthyroidism. At the cellular level, high concentrations of T₃ reduced GC viability and proliferation while increasing apoptosis. Reactive oxygen species levels were elevated, and GRP78 expression was decreased. Notably, all T₃-induced effects were reversed by EGCG treatment. In summary, this study demonstrates that hyperthyroidism induces oxidative stress in GCs, which triggers endoplasmic reticulum stress via the eIF2α/ATF4 pathway and leads to apoptosis. EGCG mitigates apoptosis by enhancing antioxidant capacity, thereby preserving ovarian function. These findings establish EGCG as a protective agent for maintaining ovarian health and fertility. Full article

Background/Objectives: Hair loss, driven by disrupted hair cycles, age-related hormonal imbalances, and oxidative stress, poses significant psychological challenges, necessitating the development of safe and effective therapies. This research investigates the trichogenic potential and underlying mechanisms of a standardized Ageratum conyzoides extract (ACE) using human follicle dermal papilla cells (HFDPCs) and C57BL/6 mice as models. Methods: HFDPCs were treated with ACE to assess its effects on 5α-reductase activity, estrogen receptor (ERα/ERβ) signaling, and activation of Wnt/β-catenin and MAPK pathways. Reactive oxygen species (ROS) levels and antioxidant enzyme expression were also evaluated. In vivo, C57BL/6 mice were administered ACE orally, and hair regrowth, follicle number and depth, and histological changes were measured. Results: In HFDPCs, ACE inhibited 5α-reductase activity, modulated ERα and ERβ signaling, and activated Wnt/β-catenin and MAPK pathways. ACE treatment at 100 μg/mL significantly increased β-catenin, p-GSK3β, and vascular endothelial growth factor (VEGF) expression (p < 0.01) and decreased Dickkopf-related protein-1 (DKK-)1 expression (p < 0.05). It also upregulated VEGF and other hair-growth-related factors and exhibited substantial antioxidant properties by reducing reactive oxygen species (ROS) and elevating the expression of antioxidant enzymes, notably SOD2 at 100 μg/mL. In C57BL/6 mice, oral administration of ACE significantly increased hair regrowth, with the 50 mg/kg group showing the most prominent effects, including increased hair follicle number and depth compared to the negative control group (p < 0.05). These effects were observed to be dose-dependent and comparable to those of minoxidil. Histological analysis confirmed enhanced anagen-phase follicle development. Conclusions: These findings highlight ACE’s multifaceted biological activity in promoting hair growth through hormonal modulation, pathway activation, and antioxidant protection, positioning it as a promising natural supplement for hair growth and health, although further clinical studies are required to confirm its efficacy in humans. Full article

Premature ovarian insufficiency (POI) is an important factor in female infertility and is often associated with oxidative stress. Alginate oligosaccharides (AOSs), derived from the degradation of alginate, have been demonstrated to have protective effects against various oxidative stress-related diseases. However, the impact of AOSs on POI has not been previously explored. The current study explored the effects of AOSs on ovarian dysfunction in a mouse model of POI induced by D-galactose (D-gal). Female C57BL/6 mice were randomly divided into five groups: the control (CON), POI model (D-gal), and low-, medium-, and high-dose AOS groups (AOS-L, 100 mg/kg/day; AOS-M, 150 mg/kg/day; AOS-H, 200 mg/kg/day). For 42 consecutive days, mice in the D-gal, AOS-L, AOS-M, and AOS-H groups received daily intraperitoneal injections of D-gal (200 mg/kg/day), whereas those in the CON group received equivalent volumes of sterile saline. Following D-gal injection, AOSs were administered via gavage at the specified doses; mice in the CON and D-gal groups received sterile saline instead. AOS treatment markedly improved estrous cycle irregularities, normalized serum hormone levels, reduced granulosa cell apoptosis, and increased follicle counts in POI mice. Moreover, AOSs significantly reduced ovarian oxidative stress and senescence in POI mice, as indicated by lower levels of malondialdehyde (MDA), higher activities of catalase (CAT) and superoxide dismutase (SOD), and decreased protein expression of 4-hydroxynonenal (4-HNE), nitrotyrosine (NTY), 8-hydroxydeoxyguanosine (8-OHdG), and p16 in ovarian tissue. Analysis of the gut microbiota through 16S rRNA gene sequencing and short-chain fatty acid (SCFA) analysis revealed significant differences in gut microbiota composition and SCFA levels (acetic acid and total SCFAs) between control and D-gal-induced POI mice. These differences were largely alleviated by AOS treatment. AOSs changed the gut microbiota by increasing the abundance of Ligilactobacillus and decreasing the abundance of Clostridiales, Clostridiaceae, Marinifilaceae, and Clostridium_T. Additionally, AOSs mitigated the decline in acetic acid and total SCFA levels observed in POI mice. Notably, the total SCFA level was significantly correlated with the abundance of Ligilactobacillus, Marinifilaceae, and Clostridium_T. In conclusion, AOS intervention effectively mitigates ovarian oxidative stress, restores gut microbiota homeostasis, and regulates the microbiota–SCFA axis, collectively improving D-gal-induced POI. Therefore, AOSs represent a promising therapeutic strategy for POI management. Full article

(1) Background: As society progresses, increasing numbers of individuals are experiencing hair loss, which can be attributed to factors such as unhealthy diets, insufficient sleep, stress, and hormonal imbalances. Currently available pharmacological treatments for hair loss often cause undesirable side effects, highlighting the urgent need to explore safer and more effective agents to promote hair restoration. This study investigated the role of recombinant human type XVII collagen derived from the α1 chain (rhCOL17A1) in facilitating hair growth and restoration. (2) Methods: We analyzed the impact of rhCOL17A1 on the mRNA expression of several growth factors, as well as Bcl-2 and Bax, at the cellular level. Moreover, the effects of rhCOL17A1 on the expression of key proteins in the Wnt/β-catenin and Sonic Hedgehog (SHH)/GLI signaling pathways were examined by Western blotting (WB). At the organismal level, we established a model in C57BL/6 mice through chronic subcutaneous administration of 5% testosterone propionate. We subsequently assessed the effect of rhCOL17A1 on hair regrowth via histological analysis using hematoxylin and eosin (H&E) staining and immunofluorescence staining. (3) Results: rhCOL17A1 contributes to the resistance of hair follicle dermal papilla cells (HFDPCs) to apoptosis. rhCOL17A1 activates the Wnt/β-catenin and SHH/GLI signaling pathways, and increases the expression of type XVII collagen (COLXVII), thereby creating a favorable environment for hair growth. Furthermore, rhCOL17A1 exerts a significant growth-promoting effect at the animal level. (4) Conclusions: rhCOL17 promotes hair growth by activating the Wnt/β-catenin and SHH/GLI signaling pathways and upregulating COLXVII expression. Full article

Stem cell-derived secretome and exosomes present a promising cell-free strategy for tissue repair and wound healing. This study aimed to isolate and characterize, for the first time, exosomes derived from rat hair follicle stem cells (rHFSCs) and to evaluate their wound-healing potential alongside rHFSC secretome. Exosomes were isolated via ultracentrifugation and characterized using Reverse Transcriptase Polymerase Chain Reaction (RT-PCR), biomarker profiling and protein quantification. Scanning electron microscopy (SEM) with energy-dispersive X-ray spectroscopy (EDS) confirmed their spherical morphology, diameter and elemental composition. Protein quantification showed higher protein content in the secretome than in exosomes. RT-PCR and biomarker profiling highlighted the therapeutic relevance of the exosomal cargo compared to parent rHFSCs. Functional analysis of 30 wound-healing biomolecules validated their pro-regenerative potential. Cytocompatibility was confirmed via the PrestoBlue™ viability assay, while scratch assays demonstrated significant wound closure in the treated groups, both with and without mitomycin C. These findings highlight the potential of rHFSC-derived exosomes and secretome as innovative, cell-free therapeutic agents for cutaneous regeneration. This study advances our understanding of their role in wound healing and underscores their broader applicability in regenerative medicine. Full article

Follicle stimulating hormone (FSH) stands as one of the most prevalently used reproductive hormones in the field of animal-assisted reproduction. Conventionally, pituitary FSH is sourced from the heterologous pituitary glands of pigs and sheep procured from slaughterhouses, and it typically exists in the form of crude FSH. The specific challenges inherent in FSH-based assisted reproduction drugs has significantly spurred the interest in exploring novel alternatives, aiming to reduce the reliance on these traditional sources in relevant production processes. In this study, the α- and β-FSH genes were retrieved from pituitary cDNA libraries. These genes were selected to construct a recombinant protein—the novel cow recombinant FSH (CrFSH)—through the application of the homologous recombination method. Notably, the β-subunit was extended by a carboxy-terminal peptide (CTP). After successfully integrating the two genes into Chinese hamster ovary (CHO) cells, the recombinant protein (approximately 33 kDa) in the culture supernatant was detected using Western blotting (WB). The results of the GCs proliferation experiment indicated that both 1.2 µg/mL pFSH and 20–20,000 ng/mL CrFSH could significantly promote the proliferation of GCs in vitro. Remarkably, on the 4th day after treatment, 20 ng/mL of CrFSH had a higher GCs proliferation rate than 1.2 μg/mL of pFSH (p < 0.001). Additionally, cyclic adenosine monophosphate (cAMP) induction assay in GCs unequivocally confirmed that CrFSH possesses superior activity compared to pFSH. These findings underscore that this recombinant protein holds great potential as a promising candidate for FSH production in assisted reproduction approaches for dairy herds. Full article

O-glycosylation is a common post-translational modification on extracellular and secreted proteins driving biochemical and biophysical interactions at the cell surface. Glycosylation affects drug immunogenicity, efficacy, and clearance, making it a critical attribute of biotherapeutics. Unlike N-linked glycans, O-linked glycans are difficult to characterize because there is no consensus sequence for glycosylation sites on the polypeptide and a universal enzyme to release O-glycans from proteins. To overcome these hurdles, O-glycan analysis and localization require an appropriate and well-validated approach, particularly for recombinant human follicle stimulating hormone-C-terminal peptide (rhFSH-CTP). FSH-CTP consists of a native FSH α/β subunit fused with the C-terminal fragment of a human chorionic gonadotropin (hCG) β subunit, which is heavily O-glycosylated. However, few FSH-CTP O-glycosylation identification methods exist. Thus, we developed a characterization method for the O-linked glycan profile and glycosylation sites of rhFSH-CTP. By means of O-glycan profiling, we identified predominantly core 1-based structures with good reproducibility. For site-specific localization, the O-glycopeptidase OpeRATOR, used with sialidase, helped identify O-glycosylated peptides. Electron transfer/higher-energy collision dissociation (EThcD), combined with OpeRATOR, identified all six glycosylation sites. This approach improves quality control for rhFSH-CTP biosimilars and other CTP-fusion proteins, contributing to the development of standardized O-glycan identification methods. Full article

Spermatogenesis is a complex and highly regulated process involving the proliferation, differentiation, and apoptosis of germ cells. This process is controlled by various hormonal, genetic, and environmental factors, including temperature. In hormonal regulation, follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone (T) are essential for correct spermatogenesis development from the early stages and spermatogonia proliferation to germ cell maturation. Other hormones, like inhibin and activin, finely participate tuning the process of spermatogenesis. Genetic regulation involves various transcription factors, such as SOX9, SRY, and DMRT1, which are crucial for the development and maintenance of the testis and germ cells. MicroRNAs (miRNAs) play a significant role by regulating gene expression post-transcriptionally. Epigenetic modifications, including DNA methylation, histone modifications, and chromatin remodelling, are also vital. Temperature regulation is another critical aspect, with the testicular temperature maintained around 2–4 °C below body temperature, essential for efficient spermatogenesis. Heat shock proteins (HSPs) protect germ cells from heat-induced damage by acting as molecular chaperones, ensuring proper protein folding and preventing the aggregation of misfolded proteins during thermal stress. Elevated testicular temperature can impair spermatogenesis, increasing germ cell apoptosis and inducing oxidative stress, DNA damage, and the disruption of the blood–testis barrier, leading to germ cell death and impaired differentiation. The cellular mechanisms of germ cell proliferation, differentiation, and death include the mitotic divisions of spermatogonia to maintain the germ cell pool and produce spermatocytes. Spermatocytes undergo meiosis to produce haploid spermatids, which then differentiate into mature spermatozoa. Apoptosis, or programmed cell death, ensures the removal of defective germ cells and regulates the germ cell population. Hormonal imbalance, genetic defects, and environmental stress can trigger apoptosis during spermatogenesis. Understanding these mechanisms is crucial for addressing male infertility and developing therapeutic interventions. Advances in molecular biology and genetics continue to uncover the intricate details of how spermatogenesis is regulated at multiple levels, providing new insights and potential targets for treatment. Full article

Equine follicle-stimulating hormone receptor (eFSHR) contains four extracellular N-linked glycosylation sites, which play important roles in agonist-induced signal transduction. Glycosylation regulates G protein-coupled receptor mechanisms by influencing folding, ligand binding, signaling, trafficking, and internalization. Here, we examined whether the glycosylated sites in eFSHR are necessary for cyclic adenosine monophosphate (cAMP) signal transduction and the phosphate extracellular signal-regulated kinase 1/2 (pERK1/2) response. We constructed mutants (N191Q, N199Q, N268Q, and N293Q) of the four N-linked glycosylation sites in eFSHR using site-directed mutagenesis. In wild-type (wt) eFSHR, the cAMP response gradually increased dose-dependently, displaying a strong response at the EC₅₀ and Rmax. Two mutants (N191Q and N199Q) considerably decreased the cAMP response. Both EC₅₀ values were approximately 0.46- and 0.44-fold compared to that of the eFSHR-wt, whereas Rmax levels were 0.29- and 0.45-fold compared to eFSHR-wt because of high-ligand treatment. Specifically, the EC₅₀ and Rmax values in the N268Q mutant were increased 1.23- and 1.46-fold, respectively, by eFSHR-wt. pERK1/2 activity in eFSHR-wt cells was rapid, peaked within 5 min, consistently sustained until 15 min, and then sharply decreased. pERK1/2 activity in the N191Q mutant showed a pattern similar to that of the wild type, despite impaired cAMP responsiveness. The N199Q mutant showed low pERK1/2 activity at 5 and 15 min. Interestingly, pERK1/2 activity in the N268Q and N298Q mutants was similar to that of eFSHR-wt at 5 min, but neither mutant showed any signaling at 15 min, despite displaying high cAMP responsiveness. Overall, eFSHR N-linked glycosylation sites can signal to pERK1/2 via PKA and the other signals, dependent on G protein coupling and β-arrestin-dependent recruitment. Our results provide strong evidence for a new paradigm in which cAMP signaling is not activated, yet pERK1/2 cascade remains strongly induced. Full article

Follicle development in dairy goats is lower after induced estrus during the non-breeding season, reducing conception rates and challenging year-round milk supply. This study investigated follicle development during the breeding and non-breeding seasons and explored molecular mechanisms for variations in the proportions of follicles of different sizes using ovarian RNA-seq and in vitro experiments. Induced estrus during the non-breeding season used a simulated breeding season short photoperiod and male effect methods, while the male effect method was used during the breeding season. This study identified an increase in follicle size during the breeding season and performed RNA-seq on ovaries to explore the underlying causes. The RNA-seq analysis elucidated pathways associated with cellular and hormonal metabolism and identified adenylyl cyclase 5 (ADCY5) as a key differentially expressed gene. In vitro experiments demonstrated that interfering with ADCY5 in ovarian granulosa cells (GCs) reduced steroid synthesis. Conversely, the overexpression of ADCY5 increased steroid synthesis. ADCY5 affects the biological function of GCs and consequently influences follicle development through the cAMP-response element binding protein (CREB) and p38 mitogen-activated protein kinase phosphorylation (MAPK) pathways. Overall, our findings demonstrate that follicle development in dairy goats differs between the breeding and non-breeding seasons and that the differential expression levels of the ADCY5 gene contribute to this discrepancy. Full article

The study objectives were to investigate the heat resistance using peripheral blood mononuclear cells (PBMCs) and hair follicles in beef and dairy calves based on heat shock protein (HSP) 70 genetic polymorphisms. The hair follicle samples from sixty calves (6 months old; 30 Korean native beef calves and 30 Holstein dairy calves) were collected for DNA extraction. The HSP70 single nucleotide polymorphism (SNP) was genotyped using a 5′-exonuclease activity (TaqMan) assay. In Study 1, PBMCs were isolated from 20 calves categorized by their HSP70 genotypes during a thermoneutral period: 10 Korean native beef calves (B-CC and B-C/-type) and 10 Holstein dairy calves (D-CC, D-C/-type). The PBMCs were then exposed to heat stress at 37 °C (control, CON) and 42 °C (heat stress, HS) for 3 h. Following this, the cells were returned to the 37 °C incubator at 0, 1, 3, 6, and 12 h for further recovery analysis. In Study 2, hair follicles were collected from 20 calves (six times every 30 days; threshold, mild, and moderate stress levels) and HSP70 gene expression was measured. Data were analyzed via two-way analysis of variance (ANOVA) and Tukey’s honestly significant difference (HSD) test. The cell proliferation in the D-C/-group was significantly higher (p < 0.05) than in the D-CC and B-C/-groups at 0 and 1 h after HS for 3 h. The mRNA gene expression of HSP70 was greater (p < 0.01) in all HS groups compared to the CON groups after heat exposure. The expression of the HSP70 gene in the D-C/-group was significantly higher (p < 0.05) compared to the B-CC and B-C/-groups immediately (0 h) following 3 h of HS. The expression in the D-CC group also higher (p < 0.05) than in the B-C/-group. The gene expression of HSP70 in hair follicles increased more at the moderate HS level than that at the threshold level. In addition, overexpression of HSP70 was noted (p < 0.05) in the D-CC and D-C/-groups compared to the B-CC and B-C/-groups. In conclusion, our results indicate that breeds and HSP70 genetic polymorphisms exhibit a distinctive pattern of immune cell proliferation and HSP70 expression profiles. Additionally, the HSP70 gene expression in hair follicles may serve as an indicator of heat resistance across different breeds, making it a potential novel barometer for HS. Full article

Background/Objectives: Cutibacterium acnes (C. acnes), a bacterium residing in hair follicles, triggers acne by inducing monocyte-mediated inflammatory cytokine production. Gedunin, a limonoid derived from Azadirachta indica (commonly known as neem), is renowned for its antifungal, antimalarial, anticancer, anti-inflammatory, and neuroprotective effects. However, its role in mitigating C. acnes-induced skin inflammation remains unexplored. This study investigates the anti-inflammatory effects of gedunin on C. acnes-induced skin inflammation and elucidates the underlying mechanisms. Methods: The anti-inflammatory activity of gedunin was assessed using RAW 264.7 mouse macrophage cells and mouse bone-marrow-derived macrophages (BMDMs). Key inflammatory mediators, including interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and interleukin-6 (IL-6), were evaluated. Mechanistic studies focused on the nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways, along with the NOD-like receptor pyrin domain-containing 3 (NLRP3) inflammasome. An in vivo acne model was employed to examine gedunin’s therapeutic efficacy. Results: Gedunin significantly reduced the expression of IL-1β, TNF-α, iNOS, COX-2, and IL-6 in RAW 264.7 cells. It inhibited NF-κB activation without affecting the MAPK pathways, including JNK/SAPK, ERK, and p38 MAPK. Gedunin also suppressed the activation of the NLRP3 inflammasome in BMDMs. In the mouse acne model, gedunin effectively alleviated C. acnes-induced inflammation, primarily by targeting NF-κB signaling. Conclusions: Gedunin demonstrates potential as a therapeutic agent for acne treatment by targeting key inflammatory pathways, particularly NF-κB signaling. This study highlights gedunin’s promise as an alternative approach to managing C. acnes-induced skin inflammation. Full article

Ovarian tissue cryopreservation has been gradually applied. It is essential to elucidate the differences between cryopreserved and fresh ovarian tissue and to refine cryopreservation protocols for improved outcomes. To explore the transcriptomic differences between fresh ovarian tissue and tissue cryopreserved with an elevated thawing rate. Ovarian tissue samples were collected and cryopreserved (frozen and thawed) following RNA sequencing and histological evaluation. Three groups were formed: fresh tissue (Group 1), frozen tissue after quick thawing at 100 °C (Group 2), and frozen tissue after slow thawing at 37 °C (Group 3). KEGG analysis showed that in comparison with Group 1, DEGs in Group 2 were mainly enriched in the cortisol synthesis and ovarian steroidogenesis pathways, and DEGs in the cells of Group 3 were mainly enriched in the ovarian steroidogenesis pathway. GO analysis showed that compared to cells of Group 2, DEGs in Group 3 were primarily enriched in the SRP-dependent co-translational protein targeting pathway and co-translational protein targeting to the membrane. The results were formulated with a minimal difference in the histological evaluation of cells after quick and slow thawed tissue. Cryopreservation of ovarian tissue by the described method does not decrease follicle production but downregulates the ovarian steroidogenesis pathway, reducing estrogen and progesterone secretion. The quick thawing of ovarian tissue increases the proliferation and apoptosis pathways of cells. Full article

Androgenetic alopecia (AGA) is characterized by microinflammation and abnormal immune responses, particularly in the upper segment of hair follicles (HFs). However, the precise patterns of immune dysregulation remain unclear, partly due to limitations in current analysis techniques to preserve tissue architecture. The infundibulum, a major part of the upper segment of HFs, is associated with significant clusters of immune cells. In this study, we investigated immune cells around the infundibulum, referred to as peri-infundibular immune infiltration (PII). We employed spatial transcriptome profiling, a high-throughput analysis technology, to investigate the immunological disruptions within the PII region. Our comprehensive analysis included an evaluation of overall immune infiltrates, gene set enrichment analysis (GSEA), cellular deconvolution, differential expression analysis, over-representation analysis, protein-protein interaction (PPI) networks, and upstream regulator analysis to identify cell types and molecular dysregulation in immune cells. Our results demonstrated significant differences in immune signatures between the PII of AGA patients (PII-A) and the PII of control donors (PII-C). Specifically, PII-A exhibited an enrichment of CD4⁺ helper T cells, distinct immune response patterns, and a bias toward a T helper (Th) 2 response. Immunohistochemistry revealed disruptions in T cell subpopulations, with more CD4⁺ T cells displaying an elevated Th2 response and a reduced Th1-cytotoxic response compared to PII-C. These findings reveal the unique immune landscapes of PII-A and PII-C, suggesting potential for the development of innovative treatment approaches. Full article

Litter size is a significant economic trait during animal reproduction. This current study attempted to decipher whether MTHFR promotes the apoptosis of granulosa cells (GCs) and inhibits their proliferation by investigating the effects of the MTHFR gene using flow cytometry and a Cell Counting Kit-8 (CCK-8) assay. MTHFR is linked with ovarian follicle development in the reproductive performance of 104 female New Zealand rabbits. We observed that MTHFR could regulate the mRNA of follicular development-related genes (TIMP1, CITED1, FSHR, GHR, HSD17B1, and STAR) with a qRT-PCR, and we observed the protein expression of CITED1 and GHR using a western blot (WB) analysis. The dual luciferase activity assays helped identify the core promoter region of the MTHFR gene, and the polymorphism of the MTHFR promoter region was studied using Sanger sequencing. The results indicated four single nucleotide polymorphisms (SNPs) within the core promoter region, among which the g.-680C>A locus was significantly associated with both the total and alive litter sizes. Additionally, the CC genotype was associated with the largest total and alive litter sizes, compared to the CA and AA genotypes (p < 0.05). In conclusion, this study investigated the effects of MTHFR on ovarian granulosa cells and its association with selected reproductive parameters in rabbits. The results provide a theoretical foundation for the use of MTHFR as a molecular marker in rabbits. Full article