Search Results (89)

The activation mechanism of the reproductive axis in Kazakh ewes during the non-breeding season was explored by supplementation with glycerol complex (7% glycerol + tyrosine + vitamin B9). The experiment divided 50 ewes into five groups (n = 10). After 90 days of intervention, it was found that significant changes in serum DL-carnitine, N-methyl-lysine and other differential metabolites were observed in the GLY-Tyr-B9 group (p < 0.05, “p < 0.05” means significant difference, “p < 0.01” means “highly significant difference”). The bile acid metabolic pathway was specifically activated (p < 0.01). The group had a 50% estrus rate, ovaries contained 3–5 immature follicles, and HE staining showed intact granulosa cell structure. Serum E2/P4 fluctuated cyclically (p < 0.01), FSH/LH pulse frequency increased (p < 0.01), peak Glu/INS appeared on day 60 (p < 0.05), and LEP was negatively correlated with body fat percentage (p < 0.01). Molecular mechanisms revealed: upregulation of hypothalamic kiss-1/GPR54 expression (p < 0.01) drove GnRH pulses; ovarian CYP11A1/LHR/VEGF synergistically promoted follicular development (p < 0.05); the HSL of subcutaneous fat was significantly increased (p < 0.05), suggesting involvement of lipolytic supply. Glycerol activates the reproductive axis through a dual pathway—L-carnitine-mediated elevation of mitochondrial β-oxidation efficacy synergizes with kisspeptin/GPR54 signalling enhancement to re-establish HPO axis rhythms. This study reveals the central role of metabolic reprogramming in regulating seasonal reproduction in ruminants. Full article

Heat stress (HS) is a major environmental factor negatively impacting the reproductive performance of livestock. This study investigates the molecular mechanisms of heat stress on the hypothalamic–pituitary–ovarian (HPO) axis in Hu sheep. A heat-stressed animal model was established, and high-throughput RNA sequencing (RNA-seq) was employed to analyze gene expression in the hypothalamus, pituitary, and ovarian tissues of both control and heat-stressed groups. The results revealed significant changes in estrus behavior, hormone secretion, and reproductive health in heat-stressed sheep, with a shortened estrus duration, prolonged estrous cycles, and decreased levels of FSH, LH, E₂, and P4. A total of 520, 649, and 482 differentially expressed genes (DEGs) were identified in the hypothalamus, pituitary, and ovary, respectively. The DEGs were enriched in pathways related to hormone secretion, neurotransmission, cell proliferation, and immune response, with significant involvement of the p53 and cAMP signaling pathways. Tissue-specific responses to heat stress were observed, with distinct regulatory roles in each organ, including GPCR activity and cytokine signaling in the hypothalamus, calcium-regulated exocytosis in the pituitary, and cilium assembly and ATP binding in the ovary. Key genes such as SYN3, RPH3A, and IGFBP2 were identified as central to the coordinated regulation of the HPO axis. These findings provide new insights into the molecular basis of heat stress-induced impairments in reproductive function—manifested by altered estrous behavior, reduced hormone secretion (FSH, LH, E₂, and P4), and disrupted gene expression in the hypothalamic–pituitary–ovarian (HPO) axis—and offer potential targets for improving heat tolerance and reproductive regulation in sheep. Full article

Reproductive efficiency in pigs is regulated by hormonal pathways that control follicular development at Day 15 of the estrous cycle, during corpus luteum regression. Miniature pigs are extensively employed as human-relevant models in biomedical research, yet their reproductive characteristics during mid-luteal regression remain inadequately characterized, limiting assessments of their translational reliability. Differences in follicular morphology, hormonal signaling, and vascular development may underlie their lower fertility compared to conventional pigs. In this study, follicular development after corpus luteum formation was compared between conventional pigs and minipigs using histological staining, immunofluorescence, hormonal assays, and transcriptomic profiling. The expression of VEGF, mTOR, LH, FSH, PAPP-A, and apoptosis markers was evaluated across the granulosa and thecal regions. Differential gene expression was analyzed using microarray data followed by GO categorization. Minipigs exhibited smaller follicles, reduced vascularization, and lower VEGF and MMP activity compared to conventional pigs. Expression of LH and PAPP-A was higher in conventional pigs, while minipigs showed relatively elevated E2 and FSH levels. Transcriptomic data revealed greater upregulation of cell-survival- and angiogenesis-related genes in conventional pigs, including genes involved in IGF pathways. Apoptosis and poor extracellular matrix remodeling were more pronounced in minipigs. Minipigs demonstrated impaired follicular remodeling and weaker hormonal signaling after corpus luteum formation, which likely contributed to their reduced reproductive efficiency. Understanding these species differences can guide breeding strategies and fertility management in biomedical and agricultural settings. 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

Nile tilapia, as an ideal model for studying sex differentiation, is a popular farmed fish worldwide with a stable XX/XY sex-determination system. In tilapia, ovarian differentiation is triggered by estradiol-17β (E2) production in undifferentiated gonads. In a previous study, we suggested that follicle-stimulating hormone (FSH) signaling might be involved in ovarian differentiation in Nile tilapia. In this study, we further investigated the role of FSH signaling in ovarian differentiation via aromatase expression, which converts testosterone to E2. Masculinization of XX fry by aromatase inhibitor or 17α-methyltestosterone leads to suppression of fshr expression. Feminization of XY fry by E2 treatment increased fshr expression from 15 days after hatching, when E2 treatment was terminated. XX tilapia developed ovaries harboring aromatase expression if fsh and fshr were double knockdowns by morpholino-oligo injections. Finally, the transcriptional activity in the upstream region of the aromatase gene (cyp19a1a) was further increased by FSH stimulation when HEK293T cells were co-transfected with foxl2 and ad4bp/sf1. Collectively, this study suggests that the role of FSH signaling is not critical in tilapia ovarian differentiation; however, FSH signaling may have a compensatory role in ovarian differentiation by increasing cyp19a1a transcription in cooperation with foxl2 and ad4bp/sf1 in Nile tilapia. Full article

Glioblastoma (GBM) is a fatal brain cancer known for its rapid and aggressive growth, with some studies indicating that females may have better survival outcomes compared to males. While sex differences in GBM have been observed, the underlying biological mechanisms remain poorly understood. Feature selection can lead to the identification of discriminative key biomarkers by reducing dimensionality from high-dimensional medical datasets to improve machine learning model performance, explainability, and interpretability. Feature selection can uncover unique sex-specific biomarkers, determinants, and molecular profiles in patients with GBM. We analyzed high-dimensional proteomic and metabolomic profiles from serum biospecimens obtained from 109 patients with pathology-proven glioblastoma (GBM) on NIH IRB-approved protocols with full clinical annotation (local dataset). Serum proteomic analysis was performed using Somalogic aptamer-based technology (measuring 7289 proteins) and serum metabolome analysis using the University of Florida’s SECIM (Southeast Center for Integrated Metabolomics) platform (measuring 6015 metabolites). Machine learning-based feature selection was employed to identify proteins and metabolites associated with male and female labels in high-dimensional datasets. Results were compared to publicly available proteomic and metabolomic datasets (CPTAC and TCGA) using the same methodology and TCGA data previously structured for glioma grading. Employing a machine learning-based and hybrid feature selection approach, utilizing both LASSO and mRMR, in conjunction with a rank-based weighting method (i.e., GLIO-Select), we linked proteomic and metabolomic data to clinical data for the purposes of feature reduction to identify molecular biomarkers associated with biological sex in patients with GBM and used a separate TCGA set to explore possible linkages between biological sex and mutations associated with tumor grading. Serum proteomic and metabolomic data identified several hundred features that were associated with the male/female class label in the GBM datasets. Using the local serum-based dataset of 109 patients, 17 features (100% ACC) and 16 features (92% ACC) were identified for the proteomic and metabolomic datasets, respectively. Using the CPTAC tissue-based dataset (8828 proteomic and 59 metabolomic features), 5 features (99% ACC) and 13 features (80% ACC) were identified for the proteomic and metabolomic datasets, respectively. The proteomic data serum or tissue (CPTAC) achieved the highest accuracy rates (100% and 99%, respectively), followed by serum metabolome and tissue metabolome. The local serum data yielded several clinically known features (PSA, PZP, HCG, and FSH) which were distinct from CPTAC tissue data (RPS4Y1 and DDX3Y), both providing methodological validation, with PZP and defensins (DEFA3 and DEFB4A) representing shared proteomic features between serum and tissue. Metabolomic features shared between serum and tissue were homocysteine and pantothenic acid. Several signals emerged that are known to be associated with glioma or GBM but not previously known to be associated with biological sex, requiring further research, as well as several novel signals that were previously not linked to either biological sex or glioma. EGFR, FAT4, and BCOR were the three features associated with 64% ACC using the TCGA glioma grading set. GLIO-Select shows remarkable results in reducing feature dimensionality when different types of datasets (e.g., serum and tissue-based) were used for our analyses. The proposed approach successfully reduced relevant features to less than twenty biomarkers for each GBM dataset. Serum biospecimens appear to be highly effective for identifying biologically relevant sex differences in GBM. These findings suggest that serum-based noninvasive biospecimen-based analyses may provide more accurate and clinically detailed insights into sex as a biological variable (SABV) as compared to other biospecimens, with several signals linking sex differences and glioma pathology via immune response, amino acid metabolism, and cancer hallmark signals requiring further research. Our results underscore the importance of biospecimen choice and feature selection in enhancing the interpretation of omics data for understanding sex-based differences in GBM. This discovery holds significant potential for enhancing personalized treatment plans and patient outcomes. Full article

Granulosa cell (GC) differentiation, stimulated by FSH and LH, drives oocyte maturation and follicle development. FSH promotes GC proliferation, and LH triggers ovulation. In clinical practice, hCG is used to mimic LH. Despite various controlled ovarian stimulation (COS) protocols employing exogenous gonadotropins and GnRH analogs to prevent premature ovulation, their effectiveness and safety remain debated. To identify markers predicting a positive treatment response, the secretome of gonadotropin-stimulated GC using the human granulosa-like tumor cell line (KGN) via proteomics was analyzed. Additionally, a novel 2D-FFT quantitative method was employed to assess cytoskeleton fiber aggregation and polymerization, which are critical processes for GC differentiation. Furthermore, the activation of key kinases, focal adhesion kinase (FAK), and Rho-associated coiled-coil-containing protein kinase 1 (ROCK-1), which are implicated in cytoskeleton dynamics and hormone signaling, was evaluated. The proteomic analysis revealed significant modulation of proteins involved in extracellular matrix organization, steroidogenesis, and cytoskeleton remodeling. Notably, the combined FSH/hCG treatment led to a dynamic upregulation of the semaphorin pathway, specifically semaphorin 7A. Finally, a significant reorganization of the cytoskeleton network and signaling was detected. These findings enhance our understanding of folliculogenesis and suggest potential novel molecular markers for predicting patient responses to gonadotropin stimulation. Full article

This study aimed to investigate the effect of Artemisia absinthium extract on testicular dysfunction in rats and explain the involvement of the androgen receptor signaling pathway as a biomarker in maintaining fertility during sodium fluoride (NaF) treatment. Thirty-two male rats were divided equally into four groups and received treatment for 60 days. The control group (I) received normal saline; group II received Artemisia extract at 100 mg/kg b.w.; group III received NaF at 12 mg/kg b.w. orally; and group IV received NaF (12 mg/kg b.w.) and Artemisia extract (100 mg/kg b.w.). The testis weights and the lipid peroxidation, luteinizing hormone (LH), follicle-stimulating hormone (FSH), and testosterone levels were estimated. The genital organs were prepared and immunoreacted with a receptor. Histomorphometric analyses were performed to obtain the diameter of the seminiferous tubules and the height of the germinal epithelia in the testes. The results showed that exposure to NaF caused a significant increase in testis weight and malondialdehyde (MDA) and a decrease in serum LH, FSH, and testosterone concentrations compared to the control group, while extract administration induced an increase in the levels of these hormones in group IV. Testicular histological and histomorphometric changes were observed in group III: degenerative seminiferous tubules with vascular congestion, disorganization of the germinal layer, and decreased seminiferous tubule diameter and germinal epithelium height. The expression of androgen receptors in the testes of the NaF-treated rats was significantly reduced. In contrast, these testicular histological changes were ameliorated in rats treated with the extract. The results allow us to conclude that the administration of Artemisia absinthium confers positive effects on male reproductive function by inhibiting fluoride, maybe via ameliorative testicular function. Full article

Ovarian endometriosis (OEM) is a chronic inflammatory condition that impairs ovarian function. While its effects on ovarian reserve are well established, the long-term consequences of OEM on ovarian dysfunction, premature ovarian failure (POF), and systemic health, particularly in the context of accelerated aging, remain insufficiently studied. In this study, we employed an OEM mouse model and bulk RNA sequencing to investigate the underlying mechanisms. Our results show that OEM accelerates primordial follicle depletion and upregulates mTOR signaling pathway gene expression, along with mechanical stress and paracrine signaling via the Hippo and Myc pathways. OEM also induces irregular estrus and ovarian fibrosis in aging mice, decreases serum AMH levels, and increases FSH levels. Systemically, elevated serum IgG levels contribute to osteoporosis and cognitive decline, both linked to ovarian dysfunction and POF in OEM. These findings elucidate the mechanisms driving premature ovarian reserve depletion in OEM and highlight its broader systemic effects. This study emphasizes the importance of monitoring ovarian health and ectopic tissue to safeguard ovarian reserves and mitigate long-term risks such as osteoporosis and cognitive decline. Full article

Maturation of follicles is the primary condition for the initiation of puberty, and excessive apoptosis of granulosa cells (GCs) will hinder the normal development of follicles in pigs. Signal Transducer and Activator of Transcription 4 (STAT4) plays an important role in cell proliferation and apoptosis. However, the mechanism of DNA methylation regulating STAT4 transcription and affecting follicle development in pigs remains unclear. To resolve this problem, we constructed a STAT4 overexpression vector and interference fragment to explore the effects of STAT4 on GC function and investigate the effects of changes in methylation status of the STAT4 promoter region on cell function and kisspeptin-1 (KISS1) expression, as well as the STAT4 effects on the development of the follicles of pigs and mice in vitro. We found that the expression of STAT4 decreased, while DNA methylation of the STAT4 promoter region increased with the growth of the follicles. After overexpression of STAT4, the apoptosis of GCs was increased but the proliferation, cell cycle and estrogen secretion of GCs were inhibited. When GCs were treated with DNA methyltransferase inhibitor (5-Aza-CdR), the methylation of the STAT4 promoter region decreased, resulting in a significant increase in the expression of STAT4. Consequently, the expression of KISS1 was inhibited. At the same time, the expressions of genes related to cell proliferation, cell cycle and estrogen secretion signaling pathways decreased, while the expressions of genes related to the apoptosis signaling pathway increased. After infection with the STAT4 lentiviral vector (LV-STAT4) in follicles of mice, the expression of STAT4 in ovaries of mice significantly increased, and the expression of KISS1 was significantly decreased. The capillaries on the surface of follicles were constricted, the age of puberty onset in mice was delayed while the levels of GnRH, LH, FSH and E2 in serum were decreased. In conclusion, we found that reduced methylation status of the STAT4 promoter region promoted the transcription of STAT4 and then inhibited the expression of KISS1, as well as promoted the apoptosis of GCs and ultimately inhibited the normal development of follicles in mammals. Full article

Background: This study aims to investigate the regulation of small mothers against decapentaplegic 2 and 3 (Smad2/3) protein phosphorylation and nuclear exclusion in follicular granulosa cells (GCs) by chicken follicle-stimulating hormone (FSH) through the phosphatidylinositol 3-kinase (PI3K) signaling pathway, as well as the effect of Smad2/3 proteins on forkhead box O 3 and 4 (FoxO3/4). This lays the foundation for exploring the regulatory functions of signaling pathways closely related to follicular growth and development, as well as the molecular mechanisms of subcellular localization and nuclear exclusion of various effector factors (including transcription factors). Methods: In this study, we used granulosa cells from 6–8 mm prehierachical follicles of chickens and performed immunofluorescence, quantitative real-time PCR (RT-qPCR), and Western blotting analysis to detect the phosphorylation and nuclear exclusion of Smad2/3 induced by FSH, as well as the regulatory effect of Smad2/3 on FOXO3/4 proteins. Results: The results showed that 10 ng/mL FSH and 50 μg/mL PI3K activator significantly reduced the phosphorylation level of Smad2/3 (p < 0.05), while no nuclear exclusion was observed. On the other hand, 16 nM/mL PI3K inhibitor and 50 μg/mL alkaline phosphatase significantly increased the phosphorylation level of Smad2/3 (p < 0.05). Overexpression of Smad2/3 increased the phosphorylation level of FOXO3/4 (p < 0.05); Smad2/3 interference resulted in a decrease in FOXO3/4 phosphorylation levels (p < 0.05). Conclusions: FSH can inhibit Smad2/3 phosphorylation and retain it in the nucleus through the PI3K signaling pathway. Smad2/3 and FOXO3/4 act as downstream effectors of the PI3K signaling pathway, and Smad2/3 can promote the phosphorylation of FOXO3/4. Full article

Equine chorionic gonadotropin (eCG) hormone, comprising highly glycosylated α- and β-subunits, elicits responses similar to follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in non-equid species. This study aimed to establish a mass production of recombinant eCG (rec-eCG) using CHO DG44 cells. Single-chain rec-eCG β/α was expressed in CHO DG44 cells. FSH- and LH-like activities were evaluated in CHO-K1 and HEK 293 cells expressing the equine LH/CG receptor (eLH/CGR), rat LH/CGR (rLH/CGR), and rFSHR. pERK1/2 activation and β-arrestin 2 recruitment were assessed in PathHunter CHO-K1 cells. The expression from one, among nine isolates, peaked at 364–470 IU/mL on days 9 and 11. The molecular weight of rec-eCG β/α ranged from 40 to 47 kDa, with two distinct bands. PNGase F treatment reduced the molecular weight by 8–10 kDa, indicating N-glycosylation. Rec-eCG β/α demonstrated dose-responsive cAMP activity in cells expressing eLH/CGR, with enhanced potency in rLH/CGR and rFSHR. Phospho-ERK1/2 activation peaked at 5 min before declining rapidly. β-arrestin 2 recruitment was receptor-mediated in cells expressing hFSHR and hLH/CGR. This study provides insights into the mechanisms underlying eCG’s FSH- and LH-like activities. Stable CHO DG44 cells can produce large quantities of rec-eCG. eCG activates pERK1/2 signaling via the PKA/cAMP pathway and facilitates β-arrestin 2 recruitment. Full article

How can cellular electrophysiology measurements and mathematical modeling of ionic channels help to identify pivotal targets in disease-related cell signaling? The purpose of this review is to highlight the advantages and disadvantages of using both of these complementary techniques to determine molecular targets that may be structurally or functionally altered in a specific disease. In addition, both electrophysiology measurements and mathematical modeling may improve coordinated drug development, accelerate the prediction of new drugs, and facilitate repositioning of pharmacological agents. This review focuses on the data obtained from electrophysiology and mathematical model approaches, including intracellular recording, cellular patch clamp measurements, and the Hodgkin and Huxley equation, as key precision methodologies. To this end, seminiferous tubules, the Sertoli cell line (TM4), and/or primary cultures of Sertoli cells were used to explore the role of follicle-stimulating hormone (FSH), thyroid hormones, retinol, testosterone, and 1,25(OH)₂ vitamin D₃ in the coordinated activation or inhibition of ionic channels essential for male fertility. Based on the discussed data, Sertoli cells precisely regulate their biological activity by coordinating channel activity according to the hormonal environment and the nutritional requirements required for germ cell development. Full article

Radiotherapy is a critical treatment for cancer but poses significant risks to ovarian tissue, particularly in young females, leading to premature ovarian failure (POF). This study examines the therapeutic potential of etoricoxib nanostructured lipid carriers (ETO-NLC) in mitigating radiation-induced ovarian damage in female Wistar rats. Twenty-four female rats were randomly assigned to four groups: a control group receiving normal saline, a group exposed to a single dose of whole-body gamma radiation (6 Gy), a group treated with etoricoxib (10 mg/kg) post-radiation, and a group treated with ETO-NLC for 14 days following radiation. Histopathological evaluations and oxidative stress biomarker assessments were conducted, including ELISAs for reactive oxygen species (ROS), pro-inflammatory cytokines (IL-1β, TNF-α), and signaling molecules (PI3K, AKT, P38MAPK, AMH). Serum levels of estrogen, FSH, and LH were measured, and gene expression analysis for TGF-β and Nrf2 was performed using qRT-PCR. The findings indicate that ETO-NLC has the potential to ameliorate the harmful effects of ovarian damage induced by γ-radiation. These therapeutic effects were achieved through the modulation of oxidative stress, inflammation, augmentation of antioxidant defenses (including Nrf2 activation), support for cell survival pathways (via PI3K/Akt signaling), regulation of MAPK, mitigation of fibrosis (TGF-β), and preservation of ovarian reserve (as evidenced by AMH, FSH/LH, and estrogen levels). ETO-NLC shows promise as an effective strategy for attenuating radiation-induced ovarian damage, highlighting the need for further research to enhance therapeutic interventions aimed at preserving ovarian function during cancer treatment. Full article

This article presents a narrative review that explores the potential link between kisspeptin—a key regulator of the hypothalamic-pituitary-gonadal axis—and the pathogenesis of endometriosis. Kisspeptin plays a significant role in regulating reproductive functions by modulating the release of gonadotropin-releasing hormone (GnRH), which in turn stimulates the secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Recent studies suggest that kisspeptin may also impact peripheral reproductive tissues and influence inflammatory processes involved in the development of endometriosis. Altered kisspeptin signaling has been associated with the abnormal hormonal environment observed in endometriosis, which affects menstrual cycles and ovarian function. Research indicates that women with endometriosis exhibit altered levels of kisspeptin and its receptor, KISS1R, in both eutopic and ectopic endometrial tissues, suggesting a role in disease progression, particularly in tissue invasion and lesion formation. Kisspeptin’s role in regulating matrix metalloproteinases (MMPs), enzymes essential for tissue remodeling, further supports its potential contribution to the pathophysiology of endometriosis. Moreover, kisspeptin-based therapeutic strategies are currently under investigation, with the aim of providing targeted treatments that reduce the side effects commonly associated with existing therapies. Despite promising findings, further research is needed to fully understand the mechanisms by which kisspeptin influences endometriosis. Full article