Reproduction最新文献

Accurate testicular tissue characterization is critical for diagnosing reproductive disorders and malignancies. We present a rapid, label-free 3D imaging technique that leverages endogenous autofluorescence and a fast, non-toxic optical clearing protocol to reveal the full 3D architecture of thick mouse testis samples, without sectioning. This approach clearly distinguishes structures like epithelial tubules, interstitial tissue, and key cell types (germ, Sertoli, Leydig) with high precision. Autofluorescence serves as a powerful structural counterstain, enhancing multiplexed analysis alongside immunofluorescent or transgenic markers. Compatible with high-resolution techniques like image-scanning confocal microscopy (AiryScan), our approach captures fine intracellular details while avoiding the artefacts of traditional histology. Accessible, affordable, and equally usable on standard confocal microscopes, this method democratizes advanced testis imaging and accelerates progress in reproductive research.

Uterine epithelial estrogen receptor α (ERα) deficiency in epiERα-/- (Esr1f/-Wnt7aCre/+) mice leads to dysregulated environment of uterine lumen, which is lined by uterine luminal epithelium (LE). We hypothesized that transcriptomes in the LE layer held molecular keys to understanding ERα in regulating preimplantation uterine environment. Day 0.5 post-coitum (D0.5) and D3.5 LE layer (isolated via enzymatic digestion), digested uterus (DU), and uterus (U) from Esr1f/- (control) and epiERα-/- mice were processed for mRNA-seq. There were minimal differentially expressed genes (DEGs: transcripts per million > 1, fold-change > 2, false discovery rate < 0.05) between DU and U. Between D0.5 and D3.5 Esr1f/- LE layer, the top upregulated and downregulated gene ontology biological process (GOBP) pathways were on cellular processes and innate immune responses, respectively. Compared to Esr1f/- LE layer, the most upregulated and downregulated pathways in D0.5 epiERα-/- LE layer were on innate immune responses and on biosynthesis and metabolism, respectively; while those in D3.5 epiERα-/- LE layer were on cell division and on signaling and metabolic processes, respectively. Na+ transmembrane transport was among shared upregulated pathways in D0.5 and D3.5 epiERα-/- LE layer. Between Esr1f/- and epiERα-/-, most DEGs in U were also DEGs in the LE layer, and limited DEGs in U only with higher expression than in the LE layer were related to immune responses, implying potential paracrine effects of uterine epithelial ERα. Selected DEGs were verified by realtime PCR and immunohistochemistry. This mRNA-seq dataset provides molecular keys to understanding temporal (e.g., innate immunity) and constituent (e.g., uterine fluid movement) functions, and potential paracrine effects of uterine epithelial ERα in regulating the preimplantation uterine environment.

Female reproductive aging is characterized by progressive deterioration of ovarian function, yet the molecular mechanisms driving these changes remain incompletely understood. Here, we used long-read direct RNA-sequencing to map transcript isoform changes in mouse ovaries across reproductive age. Comparing young and aged mice after controlled gonadotropin stimulation, we identified widespread alternative splicing changes, including shifts in exon usage, splice site selection, and transcript boundaries. Aged ovaries exhibited increased isoform diversity, favoring distal start and end sites, and a significant rise in exon skipping and intron retention events. Many of these age-biased splicing events altered open reading frames, introduced premature stop codons, or disrupted conserved protein domains. Notably, several mitochondrial genes involved in the respiratory chain were affected. We highlight Ndufs4, a mitochondrial Complex I subunit, as a case in which aging promotes the alternative splicing of a short isoform lacking the canonical Pfam domain. Structural modeling suggests this splice variant could impair Complex I function, resulting in increased ROS production. Our data suggest a mechanistic link between splicing and mitochondrial dysfunction in the aging ovary. These findings support the model of the splicing-energy-aging axis in ovarian physiology, wherein declining mitochondrial function and adaptive or maladaptive splicing changes are intertwined. Our study reveals that alternative splicing is not merely a byproduct of aging but a dynamic, transcriptome-wide regulatory layer that may influence ovarian longevity. These insights open new avenues for investigating post-transcriptional mechanisms in reproductive aging and underscore the need to consider isoform-level regulation in models of ovarian decline.

This study investigated the critical role of mitochondrial dysfunction in early-onset preeclampsia (EOPE), a major contributor to perinatal morbidity and mortality. We enrolled 12 patients diagnosed with EOPE and 8 healthy control women. Placental trophoblasts from these participants underwent comprehensive proteomic sequencing to identify differentially expressed proteins between the two groups. Key findings from proteomics were rigorously cross-verified using western blotting and immunofluorescence techniques. To further elucidate the functional consequences, we utilized the human placental trophoblast cell line HTR8/SVneo and BeWo, employing siRNA to reduce the expression of a target protein gene, subsequently observing its effects on mitochondrial function and overall trophoblast cell behavior. Our results revealed 280 differentially expressed proteins, with a notable downregulation of mitochondrial ribosomal proteins. Specifically, the expression of mitochondrial ribosomal proteins L13 (MRPL13) and MRPL9 was significantly decreased in the EOPE group, alongside a significant reduction in the mitochondrial unfolded protein response (UPRmt) related protein caseinolytic protease P (CLPP). In vivo experiments, we found that the UPRmt became more severe in HTR8/SVneo and BeWo with reducing MRPL13, leading to a significant inhibition of cell migration and an enhancement of autophagy. Specifically, under the tunicamycin (TM)-induced endoplasmic reticulum (ER) stress, MRPL13-knockdown also depleted HSP60 and CLPP, aggravated UPRmt and promoted mitochondrial dysfunction. In conclusion, our findings suggest that downregulation of MRPL13 may induce mitochondrial dysfunction via participating in the UPRmt, thereby negatively impacting the migration, proliferation, and invasion of trophoblast cells, and contributing to the pathogenesis of EOPE.

The well-studied role of the microbiome in gut physiology and pathology has led to revolutionary microbiome-targeted treatments, but the function of the reproductive tract microbiome is yet to be delineated. This underexplored avenue could shed light on historically neglected reproductive health symptoms such as heavy menstrual bleeding.

Acquisition of luteolytic capacity (ALC) is the developmental process by which the corpus luteum, which initially does not regress in response to prostaglandin (PG)F2A acquires this ability. Although the timing of ALC varies widely by species, it exists in all species in which it has been studied, including nonhuman primates, rodents, ruminants, and pigs. Because manipulation of the estrous cycle has been an essential part of reproductive management in livestock species, ALC has mostly been studied in ruminants and pigs, yet what information exists in nonhuman primate and murine models suggests that this is a mostly conserved process with only modest variation across species. Although there are substantial challenges to studying human luteal function, this luteal transition may also occur in humans. Critical regulators of acquisition of luteolytic capacity include both intracellular signaling mechanisms and cell-cell interaction networks. In luteal steroidogenic cells, signaling mechanisms downstream of the PGF2A receptor, including AMPK, and abundance and differential regulation of transcription factors appear to be critical regulators of ALC. Additionally, intraluteal prostaglandin synthesis and metabolism pathways are differentially regulated in CL of differing capacity to regress in response to PGF2A. Increasing luteal abundance of apoptotic regulators, or differential responsiveness of these to PGF2A, is also a crucial change that occurs during ALC. Finally, luteal vasculature, immune cell populations, and cytokine production are determinants of luteal PGF2A response. Understanding how the CL acquires the ability to regress will improve understanding of luteolysis and will lead to novel technologies to support luteal function and fertility.

Heat stress (HS) during gestation poses a major physiological challenge to the developing fetus, potentially inducing long-term adaptations that persist across generations. Understanding this process is crucial for reproductive biology, as climate change poses challenges to fertility. This study evaluated the effects of prenatal HS (41 °C, 65% RH) during early (FP), late (SP), or full-term pregnancy (TP) on postnatal development and reproductive function of F1 male mice. Additionally, the potential transmission of heat acclimation (HA) was evaluated by analyzing gene expression in the testes of F1 males and in F2 blastocysts derived from matings between F1 males and control females (maintained at 25 °C, 45% RH). Between the 3rd and 8th postnatal weeks, F1 males exposed to HS showed accelerated weight gain (P < 0.05). Genes related to glucose transport (GLUT1, GLUT3, GLUT8) and lipid metabolism (FASN, ACACB) were upregulated (P < 0.05) in both generations. HS-response genes (HSP60, HSPA1A, HSPA1B) and autophagy-related ATG8 were also upregulated in FP and TP groups. Despite these molecular changes, sperm parameters (concentration, motility, morphology) and fertilization potential remained unaffected. These findings suggest that in utero HS induces metabolic and stress-response adaptations, promoting reproductive HA that persists in the next generation. Understanding these mechanisms may offer insights into fertility resilience under thermal stress.

Quantifying ovarian follicles is a fundamental tool in reproductive biology, providing the only direct measure of the ovarian reserve. The characterisation of follicle dynamics and depletion across the reproductive lifespan, and in response of the compounding effects of pathological insults, is essential for understanding women's reproductive, endocrine, and systemic health. Manually tallying follicles across serial histological sections is the most widely used method for quantifying ovarian follicles. However, this approach is prone to protocol inconsistencies and interpretation bias. Consequently, the accuracy, reproducibility, and suitability of the data for cross-study comparisons or meta-analyses are often compromised. In this protocol, we outline key considerations for follicle counting and provide standardized approaches using the two principal methods: direct counting with paraffin-embedded tissue and stereological analysis with glycol methyl-acrylate sections. These guidelines are intended to ensure consistent, accurate, and objective follicle quantification, and to support researchers across disciplines in adopting ovarian health and function, measured by follicle number, as a routine outcome in preclinical animal studies.

Norandrogens are significant anabolic-androgenic steroids used initially for the treatment of debilitating conditions and later as performance-enhancing drugs. Regulatory challenges of their abuse in human, equine, and canine sports stem from the question of endogenous norandrogens. Evidence from mass spectrometry for 19-norandrostenedione (19-norA4) in the sulfate fraction of conjugated steroids in equine yolk-sac fluid suggested a need for reexamination. In vitro biosynthesis of 19-norandrogens was examined in highly purified preparations of porcine Leydig cells from mature pigs by incubation in -medium containing [3H]-androstenedione (A4) or nonradioactive A4. Steroids were recovered from media by solid-phase extraction, separately as unconjugated and conjugated fractions. High performance liquid chromatographic (HPLC) profiles were obtained from liquid-scintillaion counting of radioctivity. Several peaks in the conjugated fractions were investigated further by HPLC. Two peaks widely separated on HPLC as conjugated steroids yielded evidence of norandrostenedione (19-norA4) after solvolysis. Other peaks identified by HPLC retention times matching standards were 19-hydroxy-A4, A4 (substrate), and epiandrosterone. Incubations with non-radioactive A4 provided further evidence from radioimmunoassay of sulfated steroids as precursors of 19-norA4. With known high sulfotransferase activity in porcine Leydig cells, it is proposed that two 3-enol sulfates were formed as stable "precursors" of 19-norA4 by the distinctive action of the third aromatase isoform of the boar testes. Although their identities remain unknown, it was shown that 19-norA4 itself was not present as a 3-enol sulfate. These findings have implications regarding endogenous anabolic agents in normal growth and relevance to illegal use of anabolic androgens in sports and animal production.

Obesity-related spermatogenic dysfunction has attracted growing attention. Concurrently, gonadotropin-inhibitory hor-mone (GnIH) has been recognized as a pivotal node bridging metabolic dysregulation and testicular dysfunction. Therefore, this study investigates the mechanistic role of the GnIH/G protein-coupled receptor 147 (GPR147) signaling pathway in spermatogenic dysfunction and metabolic syndrome-associated male infertility. This study employed Gene Expression Omnibus (GEO) database analysis and molecular biology techniques to examine the effects of GnIH/GPR147 signaling ablation on testicular morphology, spermatogenic function, testosterone levels, and blood-testis barrier (BTB) integrity in mice under different dietary regimens. Transcriptomic approaches were employed to elucidate the molecular mechanisms underlying GnIH/GPR147 signaling ablation-induced male reproductive disorders. Results demonstrated a significant correlation between downregulated GnIH/GPR147 signaling and male reproductive disorders. Both GnIH deficiency and GPR147 ablation induced spermatogenic dysfunction and BTB impairment in mice under a normal diet. Under a high-fat and high-sugar diet background, these genetic manipulations only mildly exacerbated diet-induced spermatogenic abnormalities, including morphological sperm abnormalities and defective sperm motility. However, this effect was independent of changes in serum testosterone levels or BTB integrity. Instead, transcriptome analysis indicated that GPR147 knockout activates the phosphatidylinositol 3-kinase (PI3K) signaling pathway, thereby inducing downstream glycolytic dysfunction, cell cycle arrest, and apoptosis, which ultimately leads to impaired spermatogenesis and compromised sperm motility. In summary, deficiency of the GnIH/GPR147 system induces testicular metabolic dysfunction and apoptosis via PI3K hyperactivation, leading to spermatogenic dysfunction. This study highlights the essential role of the GnIH/GPR147 gene in reproductive function, providing novel insights into the pathological mechanisms and potential therapeutic strategies for male infertility.