Biology of Reproduction最新文献

Phoenixin (Pnx) exerts its physiological function primarily through its putative receptor GPR173 in mammals. However, the signaling pathways and the function of Pnx in the regulation of reproduction in teleosts remain poorly understood. Accordingly, this study presents an investigation of the signaling pathways and their functions in the regulation of reproduction in the spotted scat (Scatophagus argus). After transfection of Gpr173a into HEK-293 T cells, the results of the dual luciferase reporter assay indicated that Pnx-14 did not alter CRE-luc or SRE-luc activity. In addition, Pnx-20 could significantly increase SRE-luc activity. After transfection with Gpr173b, Pnx-14 could significantly increase CRE-luc activity while simultaneously inhibiting SRE-luc activity. Pnx-20 could significantly enhance SRE-luc activity. In vitro hypothalamic experiments, the expression of genes regulated by Pnx-14 and Pnx-20 can be suppressed in the presence of the cAMP/PKA inhibitor H89. Pnx-20 can also significantly increase the phosphorylation level of ERK1/2. Intracerebroventricular (ICV) injection of Pnx-20 significantly elevated estradiol levels in females. Conversely, ICV administration of Pnx-14 and Pnx-20 significantly reduced 11-ketotestosterone levels in males. Furthermore, ICV injection of Pnx-14 and Pnx-20 was shown to regulate the expression of reproduction-related genes in the hypothalamus, pituitary, and gonads of both females and males. These findings suggest that Pnx-14 may activate the cAMP/PKA or ERK pathway by Gpr173b. Similarly, Pnx-20 may activate the ERK pathway by Gpr173a and Gpr173b, thereby influencing the reproductive axis in the spotted scat.

This study investigated the modulation of cellular stress in the male reproductive system of Daurian ground squirrels (Spermophilus dauricus) across distinct phases of hibernation under extreme environmental conditions. Morphological and volumetric changes in the testes and epididymides were assessed through paraffin-embedded sections and hematoxylin-eosin (H&E) staining, while oxidative stress (OS) and endoplasmic reticulum stress (ERS) markers were quantified using western blotting, colorimetric assays, and immunofluorescence histochemistry. Relative protein expression within key signaling pathways was also evaluated. Compared to the summer active stage, (1) reproductive hormone concentrations and testicular and epididymal visceral mass increased during hibernation and post-hibernation, indicating sustained reproductive activity. (2) Malondialdehyde (MDA) and hydrogen peroxide (H2O2) levels remained stable in the testes and epididymides during hibernation, suggesting effective suppression of oxidative stress during this stage. (3) Superoxide dismutase (SOD) activity and total antioxidant capacity (T-AOC) increased in the testes during pre-hibernation, potentially supporting germ cell differentiation. (4) Protein expression of unfolded protein response (UPR) markers, including CHOP, p-PKR, GRP78, p-IRE1, and p-PERK, was elevated during hibernation in both the testes and epididymides, implicating UPR activation in the preservation of reproductive tissue integrity and subsequent recovery. Despite stage-specific variations, the testes and epididymides of Daurian ground squirrels maintained a well-regulated cellular stress response throughout hibernation, preventing structural degeneration of the reproductive system. Furthermore, hibernation markedly reduced the abundance of common internal reference proteins such as β-actin and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), underscoring the need to use total protein normalization for accurate quantification in hibernation-related western blot analyses.

Ovarian stimulation medications are critical in assisted reproductive technology (ART), with growing demand for long-acting follicle-stimulating hormone (FSH). Although luteinizing hormone (LH) plays a pivotal role in oocyte maturation, recombinant long-acting LH remains underexplored. Here we develop a novel recombinant protein with an extended in vivo half-life and dual FSH and LH bioactivity to improve ovarian stimulation efficacy. This protein successfully induced ovarian stimulation in both mice and cynomolgus monkeys, confirming robust reproductive hormonal activity. The findings indicate its potential as an ovarian stimulation agent in ART, although further optimization of stimulation protocols is required.

Cervical insufficiency (CI) affects 0.1-2% of pregnancies and represents a significant cause of second-trimester pregnancy loss and preterm birth, yet its pathophysiology remains incompletely understood. This study investigated whether aquaporin-3 (AQP3) facilitates hydrogen peroxide (H₂O₂) transport into cervical macrophages, driving their polarization toward a pro-inflammatory phenotype and subsequent cervical matrix degradation. Cervical tissues from women with CI demonstrated 1.45-fold higher AQP3 expression compared to gestational age-matched controls, with increased colocalization with macrophages. Tissue H₂O₂ levels were elevated 2.43-fold in CI, accompanied by increased oxidative damage markers and reduced collagen content. In vitro studies using THP-1 macrophages revealed that AQP3 knockdown prevented intracellular H₂O₂ accumulation despite pro-inflammatory stimulation, blocking M1 polarization and NF-κB activation. Co-culture experiments demonstrated that AQP3-dependent M1 macrophages increased matrix metalloproteinase (MMP)-9 activity 3.8-fold and reduced fibroblast collagen content by 59%. Both H₂O₂ scavenging with PEG-catalase and NF-κB inhibition with Bay 11-7082 prevented macrophage-mediated matrix degradation. These findings suggest that AQP3 may serve as an important mediator linking oxidative stress to inflammatory cervical remodeling through facilitation of H₂O₂ influx, NF-κB activation, and M1 macrophage polarization. Targeting AQP3 or its downstream signaling may represent a potential therapeutic approach that requires preclinical validation to prevent CI-associated pregnancy complications.

The efferent ductules 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 efferent ductules, 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.

The metabolic activity of stallion spermatozoa was assessed using flow cytometry to detect NAD(P)H and FAD fluorescence without labels. Sperm were incubated with different energy sources-glucose, lactate, and pyruvate-individually or combined, and measurements of NAD(P)H, FAD, NAD(P)H/FAD ratio, and Optical Redox Ratio (ORR) were taken. Additionally, a metabolic assay based on resazurin reduction and flow cytometry detection was developed. Changes in NAD(P)H and FAD fluorescence, the NAD(P)H/FAD ratio, and ORR were observed. The NAD(P)H/FAD ratio increased significantly, especially when glucose and pyruvate (P<0.0001), glucose and lactate (P<0.0001), or all three substrates (P<0.0001) were present together. The mitochondrial activity index (MAI) and kinematic efficiency (KE) were also analyzed. Both indices showed positive correlations with the NADH/FAD ratio (0.6 and 0.7; P<0.00001) and negative correlations with the ORR (-0.6 and -0.7; P<0.000001). Furthermore, flow cytometry was used to evaluate the spermatozoa's ability to metabolize different substrates via resazurin reduction. Results indicated that stallion sperm preferentially oxidized glucose, lactate, and oxoglutarate. In summary, we developed a straightforward flow cytometry-based redox and metabolic assay. This, combined with the resazurin reduction test, revealed a preference for glucose and lactate oxidation. This suggests specialized metabolic compartmentalization, with glycolysis occurring in the flagella and oxidative phosphorylation in mitochondria. Additionally, this assay could be useful for clinical sperm assessment, as metabolic changes often reflect physiological alterations in sperm function.

Ovulation failure accounts for over 25% of infertility cases in reproductive-age women in the U.S., 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.

Hormonal contraceptives modulate the hypothalamic-pituitary-ovarian (HPO) axis; however, underlying mechanisms and differences between contraceptives are underexplored. The Women's Health Injectable Contraception and HIV trial randomised 521 women to intramuscular depot medroxyprogesterone acetate (DMPA-IM) or norethisterone enanthate (NET-EN) and showed similar decreased estradiol levels, but more amenorrhea for DMPA-IM users. This secondary study excluded for misreporting contraceptive use for 128 participants (DMPA-IM n = 65; NET-EN n = 63). Peripheral blood serum collected at initiation and one week after the 24-week injection (25W), at peak progestin levels, was analysed for gonadal steroids, progestins and peptide hormones. While no changes were detected in peripheral gonadotropin-releasing hormone levels, DMPA-IM decreased luteinising hormone (LH) less than NET-EN. DMPA-IM increased, while NET-EN decreased follicle-stimulating hormone (FSH). Both contraceptives substantially decreased gonadal steroid levels, more so in NET-EN users for testosterone and estradiol. Post-menopausal-like hypoestrogenic effects were greater than previously reported, consistent with the substantial reduction in LH levels. Whether reduced LH levels are due to direct pituitary, hypothalamic, or supra-hypothalamic effects by progestins, is unclear. MPA, unlike NET, increased fsh expression in LβT2 cells, likely via the glucocorticoid receptor, consistent with direct effects on the pituitary by MPA in women. Amenorrhea associated in a time-varying manner with MPA and HPO hormone levels and LH/FSH, for DMPA-IM but not NET-EN users. HPO hormone profiles differ between DMPA-IM and NET-EN users and compared to pre- and post-menopausal women. Mechanisms affecting amenorrhea likely differ between contraceptives, with lower 25W LH/FSH being consistent with more amenorrhea for DMPA-IM.