Biology of Reproduction最新文献

Circadian disruption affects various physiological processes, including male fertility. However, the effects and mechanism of circadian disruption on sperm DNA fragmentation (SDF) remain largely unexplored. In this study, we investigated this relationship using a mouse model of circadian disruption to assess in vivo effects, human sperm samples to evaluate clinical associations, and GC2 spermatocyte cells for mechanistic insights. A model of circadian disruption was established using C57BL/6 J male mice subjected to a weekly 12-h inversion of the light-dark cycle for 8 weeks, which resulted in significant sperm DNA damage in mice. Analysis of differentially expressed genes from RNA sequencing of testes from circadian-disrupted mice revealed that the homologous recombination (HR) repair pathway was significantly affected. Potential correlations between CREB1 expression and SDF were explored in human sperm (n = 40). CREB1 mRNA expression detected using droplet digital PCR was lower in patients with high sperm DNA damage than in the control group. In GC2 germ cells, Creb1 knockdown reduced cell proliferation and increased sensitivity to oxidative stress; moreover, it increased double-strand breaks and decreased protein levels of the DNA repair genes BRCA1, MRE11, and RAD51. Luciferase reporter and ChIP assay confirmed the transcriptional regulation of CREB1 on BRCA1 expression. Overall, circadian disruption suppressed CREB1 expression, which contributed to deficient HR repair and ultimately led to SDF. CREB1 expression was associated with sperm DNA damage in human. Our findings may have implications for understanding male health under circadian disruption and could provide novel avenues for therapeutic strategies to address SDF.

Australia's most iconic animal, the koala (Phascolarctos cinereus), faces significant population decline and while conservation has focused on assisted breeding technology and reproductive pathologies, koala reproductive biology remains poorly understood. In particular, the koala (P. cinereus) prostate has a vital role in the production of seminal plasma and facilitating successful reproduction. Furthermore, prostatitis regularly occurs as a consequence of chlamydiosis, a substantial factor in the ongoing decline of koala populations. Despite this, little is known about the koala prostate's molecular physiology. This study presents the first proteomic profile of the koala prostate, offering insights into its histological segmentation and broader functional significance. Prostatic tissue was collected from six mature male koalas, with samples taken from the anterior and posterior segments. Proteins were digested using filter-aided sample preparation and analysed via liquid chromatography-tandem mass spectrometry with Zeno-SWATH acquisition. Peptide spectra were processed using DIA-NN and evaluated in RStudio to identify differentially expressed proteins and compare the koala prostate proteome with those of other species. Functional annotation and pathway analysis revealed that whole prostate activity was primarily centered on protein translation and muscle function. Segment-specific proteomic profiles demonstrated slight proteomic differentiation, with secretory proteins contributing to segment-specific functions. Cross-species comparisons showed strong homology between the koala and human prostate proteomes. This proteome provides a foundation for future investigations into prostate-related pathologies in koalas. Furthermore, understanding the koala prostate at a molecular level helps advance wildlife conservation through a better comprehension of its role in male fertility and offers broader evolutionary insight into marsupial reproduction.

Theca cells are a critical steroidogenic cell type of the ovarian follicle and corpus luteum. The ovulatory luteinizing hormone (LH) surge (or human chorionic gonadotropin (hCG)) stimulates theca cell relocation from the stroma surrounding the dominant follicle to full integration into the developing corpus luteum. Luteinizing hormone/human chorionic gonadotropin also stimulates granulosa cells to produce local mediators of ovulation, including the peptide neurotensin (NTS). To determine if hCG-stimulated NTS regulates theca cell relocation within the ovulatory follicle, vehicle or an NTS receptor antagonist was injected into a macaque dominant follicle, and ovaries were removed 48 h after hCG administration. Additional ovaries with dominant follicles were collected without administration of hCG (pre-hCG). Theca cells were sparse in the ovarian stroma surrounding pre-hCG follicles, while theca cells were abundant in the stroma and granulosa cell layer of recently-ovulated, hCG-treated follicles. Intrafollicular injection of a general NTS receptor antagonist or antagonist selective for a specific NTS receptor (NTSR1 or SORT1) reduced theca migration into the granulosa cell layer after hCG. In vitro, NTS stimulated macaque theca cell migration in conventional and 3-dimensional migration assays, and NTS receptor antagonists blocked NTS-stimulated migration. Neurotensin-stimulated theca cell migration in vitro was influenced by ovarian extracellular matrix components, with laminin reducing theca cell migration. NTS also increased theca cell number in vivo and stimulated theca cell proliferation in vitro. In summary, hCG-stimulated NTS acts directly at theca cells via NTSR1 and SORT1 to stimulate theca cell migration during ovulation and transformation of the ovulatory follicle into the corpus luteum.

The global decline in human fertility has become an increasing public health concern, marked by notable regional disparities and a growing reliance on assisted reproductive technologies (ART). Among the environmental contributors to reproductive dysfunction, phthalates, ubiquitous endocrine-disrupting chemicals, have been implicated in adverse reproductive outcomes. This study aimed to evaluate the effects of a biologically relevant phthalate mixture on preimplantation embryo development using an in vitro mouse model. Dosing proportions were based on phthalate concentrations measured in urine samples from pregnant participants in the Illinois Kids Development Study (I-KIDS), with the following composition: 35% diethyl phthalate (DEP), 21% di(2-ethylhexyl) phthalate (DEHP), 15% dibutyl phthalate (DBP), 15% diisononyl phthalate (DiNP), 8% diisobutyl phthalate (DiBP), and 5% benzyl butyl phthalate (BBzP). Embryos were exposed to this mixture at concentrations of 0.001, 0.01, 0.1, and 1 μg/mL, alongside control groups (culture medium only and 0.075% DMSO vehicle control), from the zygote to the hatched blastocyst stage. Exposure resulted in a significant reduction in developmental progression, with increased cytoplasmic fragmentation observed during the 2- to 8-cell transition in embryos treated with 0.1 and 1 μg/mL. Concentrations of 0.01, 0.1, and 1 μg/mL caused a marked decrease in E-cadherin expression at the 8-cell stage, and a significant increase in micronucleus formation was observed at the blastocyst stage after exposure to 0.1 and 1 μg/mL. These findings suggest that phthalate exposure disrupts critical processes in early embryogenesis, including cell adhesion, and nuclear integrity, potentially compromising embryo viability.

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.