Biology of Reproduction最新文献

Sox proteins constitute a family of transcription factors now thought to play essential roles in cell fate decisions and tissue regeneration. SOX30 is a testis-specific transcription regulator for activating the postmeiotic haploid gene program, and knockout of Sox30 results in defects in meiotic exit with an arrest at the early stage of round spermatids. Here, we aim to investigate the impact of human SOX30 mutagenesis on this transcriptional regulatory axis and its contribution to male infertility. Genetic screen of a cohort of 620 patients with non-obstructive azoospermia identified six heterozygous sequence variations in SOX30. Its coding region contains five missense mutations and one stop-gained mutation (Arg478*), which is predicted to lead to the production of a C-terminal truncated protein. In vitro functional evaluation shows that this C-terminal truncated SOX30 protein exhibits a dramatic loss of its protein association with the histone deacetylase HDAC3, and the missense mutation in the HMG domain reduces its DNA-binding ability. We chose to model the human P353S mutation, by generating Sox30P382S knock-in mice with a point mutation in the HMG domain of the mouse Sox30 gene. Sox30P382S mutants are fertile, but this P382S point mutation causes defects in the late stages of spermatocytes that reduce mature sperm. These results suggest SOX30 mutations may contribute to the risk of non-obstructive azoospermia in humans.

Ovulation failure accounts for >25% of infertility cases in reproductive-age women in the United States, with obese women three times more likely to experience anovulatory infertility. The mechanisms by which obesity impairs ovulation remain unclear. The preovulatory luteinizing hormone (LH) surge induces changes in ovarian vasculature crucial for ovulation. We hypothesized that obesity disrupts ovarian vascular function, contributing to impaired ovulation. Using Doppler ultrasonography, we assessed ovarian hemodynamics in adult normal-weight mice and two obese groups: high-fat diet (HFD) and Agouti viable Yellow (AvY) mice. Both obese groups had reduced ovulation rates following superovulation. In normal-weight mice, flow velocity in the ovarian vessels increases within the first hour following ovulation induction by human chorionic gonadotropin, whereas this increase was absent in obese mice. This change in velocity correlated with ovulation rates, suggesting its importance for ovulation success. Ovarian hemodynamics were disturbed in naturally cycling obese mice compared to normal-weight controls during diestrus. Immediately before ovulation induction, ovarian vasculature analysis through lectin labeling revealed an increased abundance of capillaries in HFD ovaries compared to AvY; transcriptomic analysis of granulosa cells and ovarian stroma identified differentially expressed genes involved in vascular development, extracellular matrix organization, and inflammation, all crucial for vascular function and ovulation. This study characterizes for the first time in vivo ovarian hemodynamics in normal-weight adult mice, and demonstrates disrupted ovarian hemodynamics in obese mice during the preovulatory and luteal phases. These findings pinpoint that improving ovarian vascular function could be a therapeutic target for enhancing ovulation in obese women.

The efferent ductules (ED) function as essential conduits for spermatozoa transport from the rete testis to the epididymis. The nonciliated and ciliated cells within the efferent ductal epithelium are responsible for fluid reabsorption and stirring the luminal fluid to prevent sperm agglutination, respectively. Dysfunction in either cell type can result in obstructive azoospermia. To systematically investigate the molecular mechanisms underlying efferent ductal development and function, we successively developed two novel knock-in mouse models via CRISPR/Cas9-mediated insertion of Cre-P2A or CreERT2-P2A cassettes into the Adgrg2 locus, enabling Adgrg2 promoter-driven co-expression of endogenous Adgrg2 and Cre recombinase. Cre-active tissues were examined in Cre-positive males crossed to Rosa26LacZ or Rosa26tdTomato reporter mice. Adgrg2-Cre mice exhibited embryonic Cre activity, as evidenced by tdTomato fluorescence in embryonic ED, proximal epididymis, and precursor cells, while postnatal males showed widespread genetic recombination across multiple tissues. In contrast, in postnatal Adgrg2-CreERT2 males under tamoxifen administration, Cre activity was prominently present in nonciliated cells within the efferent ductule epithelium and proximal epididymis, with minimal activity in other tissues. These models provide precise tools for cell type- and stage-specific genetic manipulation, facilitating studies on efferent ductal development, fluid homeostasis, and obstructive male infertility. The Adgrg2-CreERT2 line, in particular, offers a unique platform for nonciliated cell-specific genetic studies. This study opens new avenues for understanding the genetic and molecular basis of male reproductive tract function and associated pathologies.

Metabolism provides sperm with the energy needed to swim to and fertilize the oocyte. While mammalian sperm become motile during ejaculation and undergo maturation in the female genital tract, their energy demand increases. Investigations into the metabolism of sperm and the capacitation-induced increase in energy demand have been stymied by a lack of appropriate methodologies. Here, we present a detailed methodology to perform stable isotope labeling mass spectrometry in isolated mouse sperm, allowing to follow the fate of exogenous energy substrates through their metabolic pathways. As an example, mouse sperm are exposed to ubiquitously and positionally labelled 13C-glucose and the rate of accumulation of 13C in different metabolites is detected and analyzed. Using this assay in the presence of different exogenous energy substrates, with sperm from different species, genetically modified mouse lines, and/or pharmacological activators and/or inhibitors can provide important insight into the contribution of different metabolic enzymes and pathways to sperm energy homeostasis.

Anti-Müllerian hormone is a promising fertoprotective agent, demonstrating particularly strong efficacy against doxorubicin-induced ovarian toxicity. However, the impact of chemotherapy on the uterus, and the potential benefits of anti-Müllerian hormone in this context, remain poorly understood. In this study, we characterized doxorubicin-induced uterine damage and assessed the fertoprotective effect of anti-Müllerian hormone co-treatment in mice. Acutely, doxorubicin treatment caused the accumulation of DNA damage in multiple uterine cell-types, including the myometrium, as evidenced by both increased γ-H2AX staining and upregulation of Cdkn1a, Trp53, and other downstream Trp53 pathway targets, both at the mRNA and protein levels. Anti-Müllerian hormone co-treatment counteracted these effects by reducing γ-H2AX-positive DNA damage lesion accumulation and by suppressing Trp53 and its downstream pathway. Furthermore, anti-Müllerian hormone co-treatment significantly reduced the incidence of doxorubicin-induced labor dystocia, a complication of parturition, in pregnancies following chemotherapy treatment. These findings suggest that, in addition to ovarian protection, anti-Müllerian hormone may have benefits in preserving myometrial integrity and long-term uterine function following chemotherapy, further supporting its therapeutic potential for fertility preservation in cancer patients receiving chemotherapy.

As a member of the G protein-coupled receptor (GPCR) family, the follicle-stimulating hormone receptor (FSHR) plays a central role in the regulation of female reproduction. By specifically binding to follicle-stimulating hormone (FSH), FSHR regulates the proliferation and differentiation of granulosa cells, follicular development and estradiol (E2) synthesis. In this article, we summarized 37 clinically relevant mutations of the FSHR gene. These mutations are classified according to their functional impacts as follows: (1) Inactivating mutations are primarily located in the extracellular domain (ECD; e.g., p.Ala189Val) and the transmembrane domain (TMD; e.g., p.Asp224Val). These mutations cause receptor expression defects or signaling impairment, clinically manifesting as resistant ovary syndrome (ROS), premature ovarian failure (POF), or related disorders. These conditions are typically characterized by a preserved primordial follicle reserve but an arrested antral follicle development; (2) Activating mutations are concentrated in the TMD (e.g., p.Asp567Gly) and intracellular loops (ICLs; e.g., p.Val514Ala). They exhibit relaxed ligand specificity and result in ligand-independent constitutive activation. This leads to pregnancy-associated spontaneous ovarian hyperstimulation syndrome (sOHSS), characterized by enlarged ovaries containing multiple follicles and supraphysiologically elevated E2 levels. The review highlights the clinical utility of in vitro maturation (IVM) technology in assisted reproduction for patients with FSHR mutations. Clinical evidence demonstrates that mature oocytes are successfully obtained by circumventing FSH stimulation. Through a "molecular pathology → mutation classification → clinical phenotypes → therapeutic strategies" framework, this review establishes a theoretical foundation for precise classification and individualized management of FSHR mutation-related reproductive disorders.

Our previous research highlighted the importance of hormones, free fatty acids, lysophospholipids, and antioxidants in supporting in vitro spermatogenesis in mice through the development of a chemically defined medium (CDM). While it was possible to induce round spermatids through the culture of testicular tissue in medium containing these factors, challenges remained with the low efficiency of spermatogenesis and the differentiation into elongating spermatids. This study aimed to further improve the in vitro spermatogenesis system by exploring optimal oxygen concentrations and identifying additional factors necessary for spermiogenesis. In addition to the conventional oxygen concentration of 20%, three hypoxic environments (15%, 10%, and 7%) were tested, and an oxygen concentration of 10% was found to be optimal for the maintenance and differentiation of germ cells in vitro. To address the limited tissue growth observed under low oxygen conditions, we further supplemented the culture medium with glucose and insulin, which led to a significant increase in tissue size. However, this enhancement in growth did not translate into improved spermatogenic differentiation. Following this, we explored factors involved in the induction of elongating spermatids. The addition of glycosphingolipids to the culture medium modestly promoted the formation of elongating spermatids, suggesting a potential role of glycosphingolipids in haploid cell differentiation. This study offers new insights into the environmental conditions and factors that influence spermatogenesis in mice.

Early and accurate assessment of bull fertility is critical for the success of artificial insemination (AI) programs in cattle production. However, current selection tools, including genomic predictions and standard semen evaluations, offer limited reliability in forecasting field fertility outcomes. To address this limitation, we explored the sperm metabolome as a potential source of novel fertility-associated biomarkers. Using high-throughput untargeted metabolomics, we profiled frozen-thawed sperm from Holstein-Friesian bulls with high (n = 12) and low (n = 12) adjusted fertility scores, each with a minimum of 500 AI service records (range from 519 to 99,953 per bull). Raw peak intensities for 615 metabolites were normalized to the total protein concentration of each sample, and, following data filtration, 547 metabolites were retained for downstream analyses. Unpaired t-tests combined with fold-change thresholding identified 18 differentially abundant metabolites between high fertility and low fertility groups (P < 0.1, absolute fold change >1.5), with significant enrichment in pathways relating to lipid and energy metabolism. Further interrogation of these differentially abundant metabolites in the literature revealed possible metabolic differences associated with calcium channel inhibition and reactive oxygen species production in the low fertility bulls. Machine learning-based biomarker discovery further identified a subset of five metabolites-3-phosphoglycerate, phenylalanine, ceramide, citrate, and citrulline-capable of distinguishing fertility status with high predictive accuracy (AUROC = 0.877; P = 0.02). Overall, these data support sperm metabolomics as a promising omics-based approach to enhance bull fertility evaluation and improve selection strategies in AI programs.

Maternal obesity (MO) predisposes male offspring to impaired fertility and premature aging of reproductive function. We hypothesized that postnatal high-fat diet (HF) from early childhood to adulthood (second hit) exacerbates MO programmed outcomes in male offspring reproductive capacity. Female Wistar rats ate chow (5%-fat (C)) or HF diet (25%-fat (MO)) from weaning through pregnancy and lactation. Male offspring were weaned on chow (C/C and MO/C) or HF diet (C/HF and MO/HF) and euthanized at postnatal-day 110. Scrotal temperature and epididymal fat were higher in C/HF and MO/HF. Testicles: malondialdehyde (MDA) concentrations were increased in MO/HF, reactive oxygen species (ROS) rose in MO/C and MO/HF, superoxide dismutase (SOD) activity increased in C/HF and MO/HF, and glutathione peroxidase (GPx) activity was higher in C/HF, MO/C, and MO/HF. Testicular mRNA expression of Nrf2, SOD-1, GPx-4, and Bax/Bcl2 ratio was lower in MO/HF. Sperm: MDA concentrations increased in MO/HF, ROS concentrations were elevated in MO/C and exacerbated in C/HF and MO/HF, SOD activity decreased in MO/HF. Sperm motility decreased, while sperm abnormalities increased in C/HF, MO/C, and MO/HF. DNA fragmentation: yellow-stained sperms (low-fragmentation) were increased in MO/C and exacerbated in C/HF and MO/HF; C/HF, MO/C, and MO/HF showed more orange-stained sperms (medium-fragmentation), but MO/HF had the highest increase. MO/HF exhibited an increase in red-stained sperms (high-fragmentation). MO/HF had a decreased fertility rate. MO combined with postnatal environmental factors, has a negative impact on offspring health and leads to greater reproductive alterations in comparison with unhealthy postnatal dietary habits or the programming caused by MO.

To determine the effects of a GnRH antagonist on ovarian function in cattle, we tested the hypotheses that cetrorelix will (1) cause regression of the extant dominant follicle by altering gonadotrophin secretion and (2) induce a new follicular wave emergence at a consistent time after treatment. In Experiment 1, heifers were given 1.5 mg cetrorelix im on Days 1-2 (Day 0 = wave emergence), Days 3-4, and Days 6-7, or normal saline (Control, n = 8 per group). The dominant follicle was smaller and regressed earlier in groups treated with cetrorelix on Days 1-2 or Days 3-4 than in Controls (P = 0.01). Plasma LH concentrations were lower (P = 0.04) for 3 days after treatment, and the pre-wave surge in FSH occurred earlier in groups treated with cetrorelix on Days 1-2 or Days 3-4 than Controls (P = 0.01). Corpus luteum diameter was smaller (P = 0.01) in the cetrorelix groups, but luteal vascularity, life-span, or progesterone production was not affected. In Experiment 2, heifers were treated with a single 3 mg dose of cetrorelix on Day 1, 3, or 6, or normal saline (Control, n = 8 per group), given prostaglandin F2a and inseminated. Wave emergence occurred synchronously 3.6 ± 0.3 days after cetrorelix treatment, ovulations occurred 12.4 ± 0.3 days later, and 13/23 heifers became pregnant. In Experiment 3, cetrorelix given at random days of the cycle induced a new wave 3.4 ± 0.3 days later and wave emergence was more synchronous than in saline-treated cows (Barlett test P = 0.04). Results supported hypotheses and provide rationale for the development of new cetrorelix-based protocols for breeding management in cattle.