Diabetes mellitus is a serious health issue that adversely affects male reproductive function and contributes to the rise in male infertility. This study examined the effectiveness of Casimiroa edulis leaf extract in improving reproductive impairment in diabetic male rats. Forty male rats were divided into control, Casimiroa edulis extract-treated, diabetic, and diabetic + Casimiroa edulis groups. Diabetic rats showed a notable reduction in sperm quality, a considerable rise in serum cholesterol levels, accompanied by a decrease in both androstenedione and testosterone levels. Histological examination of testicular tissue revealed degeneration of the seminiferous tubules, depletion of germinal cells, and lack of Leydig cells. They also exhibited a notable reduction in the activity of testicular enzymes, specifically 3β-hydroxysteroid dehydrogenase and 17β-hydroxysteroid dehydrogenase, along with a rise in the level of dehydroepiandrosterone. Casimiroa edulis treatment substantially reduced cholesterol levels, enhanced the activity of testicular enzymes, and restored testicle structure. Consequently, testosterone levels were elevated, and sperm quality was improved. To conclude, Casimiroa edulis may mitigate the negative effects of diabetes mellitus on the reproductive parameters of male rats and enhance fertility.

Monosodium glutamate (MSG) has been implicated in female reproductive toxicity through endocrine disruption, oxidative stress, inflammation, and apoptosis. This study investigated the protective role of Sorghum bicolor extract (SBE), a polyphenol-rich nutraceutical, against MSG-induced reproductive dysfunction in female Wistar rats, integrating in vivo outcomes with in silico docking analysis. Rats were exposed to MSG (4 g/kg) with or without SBE supplementation (100 mg/kg) orally for 28 days. Estrous cycle, sexual behaviors, hormonal profiles, oxidative and nitrosative stress markers, inflammatory mediators, and apoptotic regulators were assessed. Molecular docking evaluated the interactions of SBE phytochemicals with LHB and LHCGR. MSG disrupted the pituitary-gonadal axis, shortened estrous phases, suppressed reproductive behaviors, increased lipid peroxidation, activated NF-κB, and triggered mitochondrial apoptosis. SBE restored gonadotropins and steroid hormones, normalized estrous cycle, enhanced antioxidant defenses, suppressed inflammation, and prevented apoptosis. Docking analysis revealed strong ligand-protein interactions, supporting endocrine and cellular protection. Conclusively, SBE confers robust gonadoprotective effects against MSG toxicity through endocrine restoration, antioxidant, anti-inflammatory, and anti-apoptotic mechanisms validated in vivo and in silico.

Cervical cancer (CC) is one of the most common malignant tumors affecting the female reproductive system. Epithelial Cell Transforming Sequence 2 (ECT2), a guanine nucleotide exchange factor, is crucial in regulating cellular functions. This study focuses on elucidating the role of ECT2 in CC and the involved underlying mechanisms. Western blot verified protein expression in tissues and cells. Cell proliferation, migration, and invasion were assessed via clone formation, wound healing, and transwell assays. Glycolytic indicators (glucose uptake, lactate release, ATP levels, lactate dehydrogenase (LDH) activity, and pyruvate dehydrogenase (PDH) activity) were detected using specific kits. UbiBrowser was used to predict ubiquitin-specific protease 13 (USP13)-mediated ECT2 deubiquitination. Co-immunoprecipitation (Co-IP) was performed to validate USP13's deubiquitination on ECT2 and their interaction. In vivo validation employed a mouse xenograft model, with immunohistochemistry (IHC) assessing gene expression therein. This study demonstrated that ECT2 was upregulated in CC tissues and cells, and its downregulation effectively inhibited the proliferation, migration, invasion, and glycolysis of CC cells. USP13 exhibited high expression levels in CC and stabilized ECT2 expression through its deubiquitinating activity. Knockdown of USP13 significantly suppressed the malignant phenotypes of CC cells; however, this suppressive effect was markedly reversed upon ECT2 overexpression. In vivo experiments revealed that USP13 knockdown suppressed CC tumor growth by modulating ECT2 expression. Together, USP13 exacerbates the malignant progression of CC by inhibiting ECT2 ubiquitination, suggesting that targeting the USP13-ECT2 axis might be a potential therapeutic strategy for CC with notable clinical significance.

This study aimed to investigate the association between the SOD1 gene I>D polymorphism, a key component of the antioxidant defense system, and polycystic ovary syndrome (PCOS), as well as to evaluate the potential effects of this genetic variation on genotypic distribution and metabolic parameters. A total of 100 PCOS patients and 135 healthy controls were included, and genomic DNA extracted from peripheral blood was analyzed for the SOD1 I>D polymorphism using conventional PCR, while clinical and biochemical parameters such as body mass index (BMI), insulin levels, HOMA-IR, FSH, LH, and other hormone levels were assessed. The ID genotype was found to be less frequent in the PCOS group compared to controls, with borderline statistical significance (p = 0.050; OR = 0.57), and was significantly less common in PCOS patients with a positive family history (p = 0.013), suggesting a potential protective effect, while correlations were observed between the ID genotype and markers of insulin resistance, though no significant differences were detected in allele frequencies. These findings indicate that the SOD1 gene I>D polymorphism may influence both susceptibility and metabolic characteristics of PCOS, supporting the role of oxidative stress in its pathogenesis and emphasizing the need for further functional and large-scale studies to confirm these associations.

The overpopulation of free-roaming animals, particularly dogs and cats, poses a significant global public health concern, contributing to socioeconomic, ecological, political, and ethical issues. These animals reproduce freely, increasing the risk of zoonotic disease transmission, wildlife predation, and traffic accidents. Although surgical castration is the most widely used method for controlling the male animal population, it is costly, time-consuming, and requires specialized professionals and post-operative care. Therefore, there is an urgent need for practical, low-cost, large-scale, and welfare-friendly non-surgical sterilization methods. This systematic review aimed to evaluate the efficacy of intratesticular or intraepididymal injections of sclerosing agents for inducing permanent chemical sterilization. A total of 38 peer-reviewed articles were retrieved from Medline/PubMed, Web of Science, Embase, and Scopus. Among sixteen substances, calcium chloride was the most frequently studied, followed by zinc gluconate, hypertonic saline, chlorhexidine gluconate, mannitol, and glycerol. A single bilateral intratesticular and intraepididymal injection damaged testicular and epididymal tissues, thereby disrupting spermatogenesis, steroidogenesis, and sperm transport. Despite differences in the methodology and outcomes among studies, most tested agents showed promising results in disrupting reproductive function. None of the compounds fully met the two key criteria for their practical use, including the absence of side effects and consistent induction of irreversible azoospermia or infertility. Nevertheless, calcium chloride and hypertonic saline emerged as the most promising candidates, due to their robust mechanistic profiles, broad evidence base, and favorable cost-effectiveness. Therefore, researchers may prioritize these two agents in future studies to develop standardized, safe, and field-applicable protocols for large-scale population control.

Phloretin, a natural dihydrochalcone flavonoid, is recognized for its potent antioxidant, anti-apoptotic, and anti-inflammatory properties. This study investigated the effects of phloretin on the in vitro maturation (IVM) of sheep oocytes and subsequent early embryonic development. Treatment with 5 μmol/L phloretin significantly improved the rates of oocyte maturation, cleavage, and blastocyst formation compared to the control group. This treatment also significantly increased intracellular glutathione (GSH) levels and mitochondrial membrane potential (ΔΨm), while reducing reactive oxygen species (ROS) levels. Furthermore, the treatment up-regulated the expression of key antioxidant genes (CAT, SOD2, GPX3) and the follicular development factor gene BMP15. Similarly, supplementing the early embryo culture medium with 5 μmol/L phloretin elevated GSH levels and ΔΨm, and reduced ROS levels in morulae. It also up-regulated the expression of the antioxidant genes CAT, GPX3, and SOD2. Moreover, the treatment enhanced the expression of development-related genes (P53 and OCT4) and significantly decreased the pro-apoptotic Bax/Bcl-2 ratio. In conclusion, phloretin enhances the efficiency of sheep oocyte IVM and early embryonic development by bolstering antioxidant defenses, inhibiting apoptosis, and promoting development. These findings provide a foundation for optimizing in vitro embryo production (IVP) protocols in sheep.

Luteinization is a vital process in female reproduction, where granulosa cells differentiate into progesterone-secreting luteal cells. While luteinizing hormone (LH) and human chorionic gonadotropin (hCG) are established promoters of luteinization, the regulatory role of activin A remains incompletely understood. This study aimed to investigate whether activin A can prevent or reverse luteinization in granulosa cells from small antral and preovulatory follicles using an in vitro model. Granulosa cells were collected from women undergoing either IVF treatment or ovarian tissue cryopreservation. Cells were cultured for 48 h with FSH, hCG, or activin A. Hormone secretion (progesterone and estradiol) and content of steroidogenic markers (CYP19A1, STAR, 17βHSD1, 3βHSD2, FSHR, and LHCGR) were analyzed via ELISA, RT-qPCR, and Western blot. RESULTS: demonstrated that activin A suppressed luteinization markers and progesterone production while promoting estrogen synthesis and maintaining granulosa cell features. Conversely, hCG increased luteinization marker expression and progesterone secretion, consistent with its known role in luteal transformation. Notably, granulosa cells from small antral follicles showed a distinct, stage-specific response to activin A compared to those from preovulatory follicles, indicating a temporal regulation of luteinization competence. These findings support a regulatory role for activin A in maintaining granulosa cell identity and delaying luteinization. Understanding the differential responsiveness of granulosa cells across follicular development stages may inform new therapeutic approaches for luteal phase deficiencies and fertility preservation strategies.

We aimed to develop prediction models estimating (1) the probability of obtaining a target number of euploid embryos per individual and (2) the probability of euploidy at the embryo level using data from preimplantation genetic testing for aneuploidy (PGT-A). This retrospective observational study included 664 individuals who underwent PGT-A between March 2020 and October 2024, totaling 5539 biopsied embryos (23.2 % euploid). Among them, 153 had no euploid embryos and 511 had at least one. Four models were developed: Models 1-A, 1-B, and 1-C predicted the probability of obtaining ≥ 1, ≥ 2, and ≥ 3 euploid embryos, respectively. Model 2 predicted the probability of euploidy per embryo. Multivariate logistic regression was used for individual-level models (1-A to 1-C), and a generalized estimating equation was applied for Model 2. Predictors included maternal age at retrieval, number of biopsied embryos, and embryo morphology. Area under the receiver operating characteristic curve (AUC) was used to examine the discriminative ability of models. Internal validation was performed using 10-fold cross-validation. Decision curve analysis assessed clinical utility. Models 1-A, 1-B, and 1-C demonstrated good discrimination (AUCs: 0.85-0.87), while Model 2 showed fair discrimination (AUC: 0.77). Decision curve analysis supported the clinical utility of all models at wide range of thresholds probabilties. The individual-level prediction models demonstrated good discriminative ability, while the embryo-level model showed fair discriminative ability. All models demonstrated potential clinical utility in supporting embryo selection.

Polystyrene microplastics (PS-MPs) are extensively utilized in plastic goods worldwide. The ingestion of PS-MPs has resulted in a high rate of DNA fragmentation index (DFI), which can potentially result in infertility and recurrent spontaneous abortion. This study established and characterized a mouse model of polystyrene microplastic (PS-MP)-induced sperm DNA damage (DnaD), and concurrently analyzed the associated transcriptomic and proteomic profiles. Over a period of 60 days, male mice assigned to the PS group were given PS-MPs at a dose of 1 mg/kg/d while the control group was administered an equivalent volume of normal saline. Sperm DNA Fragmentation Index (DFI) was then assessed using the Sperm Chromatin Structure Assay (SCSA).The testis was examined using RNA-seq and data-independent acquisition (DIA) to detect the patterns of mRNA and protein expression. The PS group exhibited an significant increase in the sperm DFI. Compared with the control group, 874 differentially expressed genes (DEGs) and 164 differentially expressed proteins (DEPs) were identified in the PS group. These included Agt, Gstt1, Fetub, Akr1c12, Eln, Gaa, Ppic and Ltbp2. The PI3K/Akt and metabolic pathways exhibited significant enrichment of these genes. After a 60-day period of intragastric injection, our findings indicated that the administration of PS-MPs at a 1 mg/kg/d dosage can lead to DnaD in the sperm of male mice. The metabolic and PI3K/Akt signaling pathways could be associated with the reproductive toxicity of PS-MPs. SUMMARY SENTENCE: The intake of PS-MPs mainly reduces DFI in mice via the metabolic and PI3K/Akt signaling pathways.

To date, the Gardner Grading System remains the gold standard for the conventional evaluation of blastocysts. However, the use of Time-lapse technology in Assisted Reproductive Technologies (ART) cycles provides a dynamic, morphokinetic assessment that complements traditional morphological evaluation, as vitrification and subsequent warming process may induce morphological and biochemical variations in blastocysts. After warming, 203 blastocysts, obtained from homologous ART cycles, were cultured in EmbryoScope to evaluate their morphokinetic modifications using Time-Lapse Technology (TLT). (I) The degree of trophectoderm expansion at insertion into the incubator (t0 exp), (II) the first signal of trophectoderm re-expansion (t first exp), and (III) the degree of expansion and morphology at two hours (t2h exp, t2h grad) were recorded. Statistical analysis was performed by SPSS version 29.0 and relations between I, II, III parameters and beta-human Chorionic Gonadotropin (beta-hCG) outcome, heartbeat, and live birth were assessed. RESULTS: Showed that t first exp was significantly associated with beta-hCG levels, heartbeat, and live birth outcomes. Additionally, blastocysts achieving complete re-expansion within two hours and presented optimal morphology exhibited a higher likelihood of successful implantation. Early biomarkers were identified through morphokinetic parameters derived from Time-Lapse technology, and pregnancy outcome prediction was improved by the combination of morphological evaluation with post-warming kinetic assessments.

The aim of the present study was to investigate the activity of exogenous melatonin as a potential agent in reversing testicular structural damage induced by early weaning. For this purpose, Wistar rats were used and divided into four experimental groups: control; early weaning; early weaning treated with melatonin in a daily dose 10 mg/kg of body weight; and early weaning treated with a vehicle composed of ethanol and saline solution. Except for the control group, all animals were weaned on the 16th day after birth. Body weight was measured weekly, and euthanasia was performed on day 51. The testes were weighed and collected for histopathological, morphometric, immunohistochemical, and oxidative stress analyses, while serum was collected for biochemical and hormonal analyses. At the end of the experiment, the early weaning group exhibited an increase in body mass, as well as structural alterations, including a reduction in the diameter and height of the seminiferous tubule epithelium, along with atrophy, vacuolization, and degeneration of germ cells. This group also showed lower production of pachytene spermatocytes, Sertoli cells, and Leydig cells, increased oxidative stress, decreased testosterone concentration, an adverse lipid profile, and reduced PCNA labeling. In contrast, the group treated with melatonin demonstrated improvement in these parameters when compared to early weaning group. These findings suggest that melatonin exerts a restorative role in testicular tissue.

The heterotrimeric complex serine-threonine protein phosphatase 2 A (PP2A) is integral to the regulation of essential cellular processes. It is particularly crucial in spermatogenesis, where it is indispensable for meiosis, mitosis, sperm capacitation, and apoptosis. Previous research has concentrated on the knockdown of the catalytic subunit of PP2A, PPP2CA, in germ cells using Ddx4-Cre, resulting in male mouse sterility, disrupted meiotic recombination, and meiotic arrest of spermatocytes. To further elucidate the role of PP2A in spermatogenesis, we performed transcriptomic and proteomic sequencing analyses on the testes of knockout and control mice. A nine-quadrant map was developed to depict the differential expression of genes and proteins. Our analyses identified 1732 differentially expressed genes, which exhibited a strong positive correlation with the trends in differential protein expression. Gene Ontology (GO) enrichment analysis indicated a significant downregulation of genes involved in spermatogenesis, sperm cell development, and sperm cell differentiation. Furthermore, KEGG enrichment analysis revealed a notable enrichment of differentially expressed genes within the ubiquitin-mediated proteolysis pathway. In knockout mouse testicular tissue, testicular expression of the ubiquitin-related gene, UBE2K, was markedly downregulated, which was associated with the accumulation of histone H3, upregulation of the methyltransferase SETDB1, and increased levels of H3K9me3. Similarly, knockdown of Ppp2ca in GC2 cells resulted in decreased UBE2K expression, histone H3 accumulation, SETDB1 upregulation, and elevated H3K9me3 levels, consistent with mirroring the phenotype observed in the knockout mice. Notably, the ubiquitin-related gene UBE2K was identified as a significant outlier in the nine-quadrant map, and real-time quantitative PCR confirmed that UBE2K transcript levels were significantly reduced in knockout mice compared to wild-type controls. These findings suggest that PP2A may regulate histon.

Animal cloning remains inefficient, with live birth rates below 5 % in most mammalian species, slightly higher in bovines (20-25 %). Among various factors, nuclear donor cells play crucial roles, and their passage numbers may influence cloning efficiency. However, a definitive association between passage numbers and outcomes in cloning remains inconclusive due to insufficient data and inconsistent results. In the present meta-analytical study, we compared research using high (>6) and low (≤6) cell passage numbers across various types, breeds, and sexes of donor cells to assess their impact on embryo development and associated gene expressions in bovine clones. Our findings revealed that cell passaging influences developmental competence at the cleavage and blastocyst rates, with lower passage numbers yielding better results. Higher acetylation of H3K9 in low-passage cells is consistently associated with improved developmental competence through the blastocyst stage, although the difference was not statistically significant. Donor cells with higher histone acetylation may undergo reprogramming more easily and completely, thus improving cloning efficiency. Further analysis elucidated that the types and breeds of donor cells also affected the blastocyst outcome. Nevertheless, high heterogeneity and meta-bias were identified in the meta-analytical outcomes, particularly in cleavage, 2-cell, 8-cell, blastocyst, the total cell number (TCN), the ratio of inner cell mass and total cell number (ICM/TCN), NANOG, and birth rate, which may contribute to inconsistencies in embryo quality results and hinder comparisons between development-related gene expressions and the embryo transfer outcome.

Endometritis is the inflammation of the endometrial lining, frequently linked to pelvic pain. Endometritis is significantly associated with postpartum problems, including infertility. Programmed cell death (PCD) is a genetically regulated mechanism of autonomic and regulated cell death that preserves homeostasis and promotes development. PCD, which includes apoptosis, pyroptosis, autophagy, ferroptosis, and necroptosis, is essential in the pathogenesis of reproductive diseases, including endometritis. Despite the limited study on PCD in endometritis, it is essential to identify important molecules and comprehend their regulatory functions for effective disease prevention and management. This review delineates the different types of PCD and their interactions in endometritis. It also evaluates the progress in PCD research pertaining to endometritis. The objective is to create a basis for subsequent investigations into the function and interaction of PCD in endometritis. We anticipate that this study will facilitate the exploration of novel PCD targets for endometritis treatment, hence encouraging comprehensive research.

Annatto (Bixa orellana) carotenoids have antioxidant potential, but effects on placental biology and antioxidant parameters in ewes are unclear. We tested whether dietary annatto concentrate (ANC) in late gestation modulates immediate-postpartum blood oxidative markers and placental gene abundance. Thirty ewes were stratified by expected litter size (single/twin) and randomized within stratum to 0 %, 0.5 %, or 1.0 % ANC (dry-matter basis) from gestational day 100. ANC was a powdered concentrate, premixed into feed (1.5 % BW) and offered individually to ensure full intake. At lambing, we recorded maternal/neonatal descriptors (ewe weights, gestational length, time to placental expulsion, and lamb birth weight interpreted with litter size and sex), collected blood for FRAP, TBARS-MDA, H₂O₂, SOD, GPx, CAT, and -SH, and sampled placenta (cotyledon and intercotyledonary tissue) aseptically and froze it for RT-qPCR of VEGF-A, FGF-2, PAG1, GPX1, GSR, CAT, and SOD2. ANC produced marker-specific blood adjustments: lower H₂O₂ at 1.0 %, higher GPx in supplemented ewes, and lower -SH at 0.5 %, whereas FRAP, SOD, MDA, and CAT were unchanged. In placenta, cotyledonary FGF-2 showed higher relative abundance at 0.5 %, with no differences for VEGF-A, PAG1, GPX1, GSR, CAT, or SOD2 in either tissue. Ewe weights, gestational length, and time to placental expulsion were similar among groups; lamb birth weight showed no main effect of ANC. These results indicate that, in late gestation, ANC selectively increased the relative abundance of cotyledonary FGF-2 at 0.5 % and modulated peroxide handling in blood, supporting its use as a nutritional tool for antioxidant support near parturition.

This study examined pannexin 1 (PANX1) expression in ovarian granulosa cells of women with advanced maternal age and its role in cell proliferation and apoptosis, aiming to clarify mechanisms of age-related follicular dysplasia. Ninety women undergoing assisted reproductive technology were divided into reproductive-age (<35 years), advanced-age (35-41 years), and very advanced-age (≥42 years) groups. Clinical data and granulosa cell function were analyzed. PANX1 expression was detected in primary cells, while KGN cells were transfected with PANX1 plasmid or siRNA. Cell proliferation, apoptosis, and extracellular ATP levels were evaluated. With increasing age, oocyte yield, blastocyst formation, and pregnancy rates declined, granulosa cell proliferation decreased, apoptosis increased, and PANX1 expression was elevated. PANX1 overexpression inhibited proliferation and increased extracellular ATP, whereas knockdown enhanced proliferation without affecting apoptosis. PANX1 upregulation in aging granulosa cells mediates ATP efflux, depletes intracellular ATP, and suppresses proliferation, contributing to abnormal follicular development and reduced fertility.

Nicotine, a chemical component included in tobacco, has been demonstrated to influence various facets of spermatogenesis and sperm functionality. Moringa oleifera Lam. (Moringaceae) is used in traditional medicine to treat various ailments, including reproductive toxicity. The current study aims to investigate the ameliorative effect of Moringa oleifera leaves aqueous extract (MOE) on nicotine (NT)-induced reproductive toxicity in male rats. Forty rats were assigned into four groups (n = 10): control (distilled water), MOE (400 mg/kg), NT (1 mg/kg), and NT + MOE groups, and orally treated for 56 days. At the end of the experiment, body and organ weights, epididymal sperm analysis, oxidative stress, hormone levels, and histopathological abnormalities were assessed. Nicotine administration results in a significant increase in malondialdehyde (MDA) levels, accompanied by a decrease in glutathione (GSH), catalase (CAT), and superoxide dismutase (SOD). Consequently, NT decreases sperm quality and serum reproductive hormone levels, including testosterone, follicle-stimulating hormone (FSH), and luteinizing hormone (LH), and distorts testicular tissue architecture. The oral administration of MOE caused a significant decrease in MDA concentration and an increase in the levels of antioxidant molecules (GSH, CAT, SOD) and serum reproductive hormones levels. Additionally, it improved sperm characteristics, including sperm count, motility, viability, and morphology and partial improvement to the histological architecture of the testis. Our findings imply that 400 mg/kg of Moringa oleifera leaves may be utilized to enhance reproductive parameters against NT-induced reproductive toxicity in male rats, with the primary mechanism attributed to its antioxidant properties.

Reproduction is a complex and highly regulated biological process, underpinned by a range of hormones and unique biological fluids secreted by the reproductive system, including follicular fluid, oviductal fluid, uterine fluid, seminal fluid, amniotic fluid, etc. These fluids not only create an optimal environment by maintaining homeostasis for gamete development and fertilization but also ensure the successful adaptation of the developing embryo to its surrounding environments. Within this intricate network, Extracellular Vesicles (EVs), which are found in reproductive tracts associated biological fluids, have emerged as key players in regulating various reproductive processes. Unlike other micro and macromolecules found in these fluids, EVs are gaining significant attention due to their crucial roles in cellular communication across several biological barriers and their biological compatibility. Therefore, over the past few decades, EVs have emerged as a significant aspect in reproduction-related research, shedding light on underlying physiological mechanisms and harboring potential for clinical applications in fertility diagnosis, treatment, and beyond.

This study aimed to investigate the effects of gossypol on cell viability, oxidative stress, and gene expression in Primary Smooth Muscle of Myometrium from Ovis aries (PSMo) Cells. The cells were cultured and treated with varying concentrations of gossypol (1-2000 µg/mL), cytotoxicity was assessed using the MTT assay, oxidative stress were evaluated by fluorescence-based methods (DCFH-DA/ABAP), and the expression of structural, hormonal, apoptotic, and antioxidant genes was analyzed by RT-qPCR. The results showed that concentrations ≤ 5 µg/mL gossypol (G5) maintained cell viability, whereas higher doses induced a progressive loss of viability, indicating dose-dependent cytotoxicity. The production of reactive oxygen species was not significantly affected, suggesting that gossypol did not exhibit relevant antioxidant or pro-oxidant activity at the tested doses. Gene expression analysis revealed differential regulation of β-actin, myosin light chain kinase (MLCK), progesterone receptor (PR), estradiol receptor (ER), the pro-apoptotic gene BAX, and the anti-apoptotic gene BCL2. The transition observed between G7, G10, and G15 revealed a functional gradient: G7 reflected a cell survival profile; G10 marked the apoptotic inflection point; and G15 corresponded to programmed cell death. The antioxidant genes remained unchanged, indicating the maintenance of cellular oxidative homeostasis. In conclusion, gossypol induces dose-dependent cytotoxicity in uterine smooth muscle cells (PSMo), modulating structural, hormonal, and apoptotic genes without altering basal antioxidant activity. Low concentrations were well tolerated, while higher doses from 7 µg/mL activated pro-apoptotic pathways The results highlight the need for caution in reproductive contexts within the animal field and suggest potential use in oncological therapies.

The establishment of endometrial receptivity is required for successful embryo implantation during early pregnancy in many mammals. Extracellular vesicles (EVs) microRNAs (miRNAs) play an important role during embryo implantation. While their roles have been characterized in other species, the specific functions of trophoblast-derived EVs miRNAs in ovine endometrial receptivity remain undefined. In this study, we systematically investigated the effects of ovine placental trophoblast (OTR) cells-derived EVs on ovine endometrial epithelial cells (EECs) by cck-8 assay, EdU assay, cell migration assay, RT-qPCR and ultrastructural examination of apical plasma membranes. Subsequently, miRNA expression profiles of EV-treated EECs were identified and analyzed by miRNA-Seq. The results showed that OTR cells-derived EVs were taken up by EECs, enhancing the migration of EECs. EVs treatment reduced microvilli on the apical plasma membranes of EECs. The expression of genes involved in endometrial receptivity increased. OTR cells-derived EVs induced changes consistent with a receptive phenotype through coordinated cellular remodeling and gene expression changes. The miRNA-Seq results revealed 287 detectable miRNAs, including 34 with significant differential expression (20 upregulated and 14 downregulated) in EV-treated versus control EECs. The predicted target genes of these differentially expressed miRNAs were enriched in signaling pathways regulating embryo implantation and endometrial receptivity, such as MAPK, Toll-like receptor, adherens junction and focal adhesion. Our in vitro findings suggested that OTR cells-derived EVs may promote endometrial receptivity by facilitating the transformation of EECs, as indicated by receptivity-associated morphological and molecular changes. It provided novel insights for improving successful pregnancy rate in sheep.

Testosterone production by testicular Leydig cells (steroidogenesis) is vital to male fertility and overall male health. Information about how nutrition influences Leydig cell steroidogenesis is lacking. Branched chain amino acids (BCAAs - leucine, isoleucine, and valine) are essential amino acids and important regulators of protein synthesis and energy production. Circulating and tissue BCAA levels are tightly regulated by the enzyme branched chain a-keto acid dehydrogenase kinase (BCKDK), which inhibits their catabolism. This work explored how BCAAs, and especially leucine, modulate male fertility and testosterone production. In a mutant mouse model of Bckdk, breeding analysis showed reduced male fertility and circulating testosterone. Further, morphological evaluation demonstrated testicular and epididymal abnormalities consistent with abnormal testicular androgen signaling. Fertility was partially rescued by feeding a high protein diet while circulating testosterone was not. In wild type testes, Leydig cells were the primary cell type to express BCKDK. Leveraging a primary interstitial cell culture, cell survival and apoptosis analyses demonstrated Leydig cells are highly sensitive to leucine deprivation and this sensitivity is enhanced under steroidogenesis stimulating conditions. Lastly, using the same primary cell culture system, testosterone production was shown to be lost under leucine deprivation. In total, this work demonstrates Leydig cells are uniquely sensitive to BCAA status under steroidogenesis stimulation and that regulated BCAA catabolism may be important for optimal male fertility.