Biology of Reproduction最新文献

The maintenance of mammalian spermatogenesis depends on the intricate molecular and cellular interactions between spermatogonial stem cells and their cognate niche in the seminiferous epithelium of the testis. To sustain the continuous production of sperm, spermatogonia proliferate and differentiate under the control of various niche factors, promoting either self-renewal or commitment to spermatogonial differentiation. Single-cell RNA sequencing analyses have identified different subpopulations of spermatogonia in primates based on the expression of specific marker genes (PIWIL4, GFRA1, NANOS3 and KIT). However, the spatial distribution of the different spermatogonial subpopulations and their relationship with the niche has not been described yet. Here, we investigate the topological localization of spermatogonia in primates. To this end, immunohistochemical stainings for PIWIL4, GFRA1, NANOS3 and KIT were performed on Bouin fixed samples of Macaca fascicularis and quantitatively analyzed. Strauss's linear selectivity index (Linear Index, Li) was employed to assess the regional distribution of spermatogonial subpopulations in the basal compartment of seminiferous tubules. Remarkably, PIWIL4+ spermatogonia showed a random distribution along the basal compartment across all the stages of the seminiferous epithelium cycle. In contrast, GFRA1+, NANOS3+ and KIT+ spermatogonia displayed stage-dependent localization patterns. The spatial organization of different spermatogonial subpopulations, appeared coordinated with the cycle of the seminiferous epithelium, suggesting a dynamic regulation of spermatogonial behavior throughout the process of sperm production. Our study contributes to the growing body of literature aimed at deciphering the complexities of SSC biology and the regulation of spermatogenesis in mammalian species, with implications for understanding male fertility.

Spermatozoan DNA is hyper-condensed by protamines, which are essential for sperm motility and function. Since defects in this process impact natural fertilization, little is known about how changes to protamine-mediated condensation impact the oocyte's ability to recognize and reprogram the paternal genetic material for the early stages of embryogenesis. Here, we performed intracytoplasmic sperm injection (ICSI) with sperm lacking Protamine 2 (Prm2 KO) and tracked preimplantation development in mice. We found that ICSI with Prm2 KO sperm leads to embryo fragmentation and arrest at the 2-cell stage. Surprisingly, injected Prm2 KO sperm DNA is rapidly depleted from the oocyte during the completion of maternal meiosis II, leading to a zygote with one morphologically abnormal pronucleus. Direct induction of DNA damage in wild type sperm did not recapitulate the pronuclear abnormalities found in Prm2 KO-derived zygotes. Co-injection with wild type sperm failed to rescue these defects, indicating that they were not caused by absence of a normally protaminated paternal genome. Finally, we find that testicular Prm2 KO sperm support progression to the blastocyst stage, suggesting a model where fertility-compromising factors are acquired during epididymal maturation. Our results demonstrate that Protamine 2 is necessary for correct maturation of epididymal sperm and essential for their ability to form of a functional zygote at fertilization.

Sex control technologies hold great promise for enhancing animal production efficiency. X chromosome-linked toll-like receptor 7/8 (TLR7/8) genes are specifically expressed in mammalian X sperm but not Y sperm. In vitro treatment with the TLR7/8 ligand R848 can separate X and Y sperm by selectively reducing X sperm motility via inhibition of energy production. Fertilization using such treated sperm leads to sex-skewed embryos or offspring. However, whether R848 can be used in vivo to control offspring sex remains unclear. This study tested this hypothesis. We found that the X/Y sperm ratio in the upper layer of caudal epididymal sperm collected at 24 and 72 hours after intraperitoneal R848 injection was 24.09%/75.91% and 63.16%/36.84%, respectively. Mating superovulated females with males at these time points resulted in male- and female-biased litters. Using the upper layer of epididymal sperm collected at 24 and 72 hours after R848 injection for in vitro fertilization, the embryo sex ratio also skew toward male and female, respectively. Our findings demonstrate that intraperitoneal injection of R848 into male mice can dynamically modulate the proportion of mature X and Y sperm in the cauda epididymis, allowing offspring sex control through timed mating. This approach provides a simple and potentially practical method for effective sex ratio intervention in mammals.

Exposure of the preimplantation bovine embryo to choline programs development to alter postnatal phenotype. To understand potential mechanisms, actions of choline on gene expression and DNA methylation of the bovine blastocyst were characterized. Embryos produced in vitro were cultured for 7 days with either 1.8 mM choline chloride or vehicle and RNA was extracted to assess gene expression. Using an adjusted p value of 0.05, the total number of differentially expressed genes (DEG) was 263 with 208 downregulated by choline. Analysis of gene ontologies of differentially expressed genes indicated choline causes reduced protein synthesis. DNA methylation was determined using whole genome enzymatic methyl sequencing. A total of 7983 differentially methylated regions (DMR) were identified, with 6174 hypermethylated (choline>vehicle) and 1809 hypomethylated. Thus, as expected given its role as a methyl donor, the major action of choline was to promote methylation. The correlation coefficient between DNA methylation percent in promoters and gene expression was -0.18 for both vehicle and choline groups. Comparison of a DNA methylation data set of blood cells from heifers derived from choline- or vehicle-treated embryos identified 26 overlapping DMR for the blastocyst and blood datasets. Twelve of these DMR exhibited a consistent trend of hypermethylation in both heifer blood cells and blastocysts, whereas two DMR consistently showed a trend toward hypomethylation. It was concluded that choline alters blastocyst gene expression in a manner consistent with reduced protein synthesis and causes small changes in DNA methylation. Moreover, a small number of DMR are retained into the postnatal period.

Endometriosis is a complex condition affecting 10% of reproductive-age women worldwide, yet its study has long been hindered by the shortcomings of traditional research models. This review aims to propose a novel theoretical framework and strategic roadmap to accelerate precision medicine in endometriosis. We systematically trace the evolution of research models in the field and critically assess the inherent constraints of conventional systems, including immortalized cell lines and primary cell cultures. While immortalized lines often fail to capture patient heterogeneity due to genetic drift, animal models cannot fully replicate the human immune microenvironment. Although primary cells preserve patient-specific traits and support personalized drug testing, their two-dimensional (2D) culture format limits their ability to mimic the three-dimensional (3D) architecture and dynamic cell-cell interactions of native tissues. Recent breakthroughs in organoid technology are reshaping endometriosis research by enabling 3D structural reconstruction, hormone responsiveness, and simulation of lesion progression. This review highlights the transformative potential of organoids in disease modeling, drug screening, and personalized therapy. We also discuss future opportunities for integrating organoids with emerging platforms-such as organ-on-a-chip systems, multi-omics analyses, and digital twin technology-to open new avenues for understanding disease mechanisms and advancing targeted treatments.

Sperm motility is essential for successful fertilization, with hyperactivated motility (HA) playing a critical role in sperm migration and penetration of the oocyte's protective layers. HA is regulated by intracellular Ca2+ influx through the sperm-specific CatSper channel. While CatSper activation is known to be influenced by various chemical factors, its sensitivity to physical stimuli such as temperature remain unclear. In this study, we investigated the impact of temperature on CatSper activity and HA in human sperm. The percentage of sperm exhibiting HA increased significantly in a temperature-dependent manner without affecting total motility or viability. Temperature-induced HA was dependent on CatSper-mediated Ca2+ influx, as pharmacological inhibition of CatSper abolished the response. Spectrofluorometric assays and patch-clamp experiments revealed that higher temperatures modestly but consistently enhanced CatSper activation as well as other capacitation-associated cAMP/PKA signaling pathways. These findings demonstrate that rather than acting as a dedicated thermosensor, CatSper displays physiologically relevant temperature-dependent modulation, suggesting a role for thermal cues in sperm capacitation and motility within the female reproductive tract.

Uterine adenomyosis is a widespread condition that impairs female fertility, with elevated serum levels of cancer antigen 125 (CA125) and prolactin (PRL) often observed in affected women. Mifepristone has been investigated as a potential therapeutic agent to improve pregnancy outcomes in uterine adenomyosis patients. This study aimed to examine the effects of mifepristone on serum CA125 and PRL levels in uterine adenomyosis mouse models. The ICR mice were treated with varying doses of mifepristone after uterine adenomyosis was induced. Immunohistochemistry was used to analyze estrogen receptor (ER) and CA125 expression in uterine tissues, while serum CA125 and PRL levels were measured by ELISA. The study found that mifepristone treatment significantly reduced serum CA125 and PRL levels, and the 20 mg/kg/day dose was most effective in improving pregnancy outcomes. These findings suggest that mifepristone can reduce key biomarkers associated with uterine adenomyosis and improve fertility outcomes, offering a promising treatment option for women suffering from this condition.

Background: Endometriosis-associated ovarian cancer (EAOC) is a distinct form of epithelial ovarian cancer that arises from the malignant transformation of benign endometriotic lesions. While epithelial-mesenchymal transition (EMT) is acknowledged as a crucial process in the progression of EAOC, the upstream regulatory mechanisms and key molecular drivers are not fully understood. This study focuses on the chemokine CXCL12, its biological function, molecular mechanisms, and clinical prognostic significance in the transition from endometriosis to EAOC.

Methods: Differentially expressed genes (DEGs) between benign endometriosis and EAOC tissues were identified using Gene Expression Omnibus (GEO) datasets. CXCL12 emerged as a candidate regulator. To further elucidate the functional role of CXCL12, we conducted in vitro studies by establishing cell models with either CXCL12 overexpression or knockdown. Additionally, we investigated the underlying mechanism of CXCL12's function, focusing on its interaction with the PI3K/Akt signaling pathway and its regulation of downstream EMT-associated proteins. A retrospective analysis of clinical data from 38 EAOC patients was performed to evaluate the association between CXCL12 expression levels and patient prognosis.

Results: CXCL12 expression was significantly elevated in EAOC tissues compared to benign endometriosis samples and was closely associated with EMT-related phenotypes. In vitro functional assays demonstrated that CXCL12 enhanced cellular migratory and invasive capacities. Mechanistically, CXCL12 was found to induce EMT by activating the PI3K/Akt signaling pathway. Clinical analysis further revealed that high CXCL12 expression was associated with reduced overall survival and increased recurrence risk in EAOC patients. Multivariate Cox regression analysis identified CXCL12 as an independent adverse prognostic factor in EAOC.

Conclusion: This study is the first to systematically define the critical role of CXCL12 in the malignant transformation of endometriosis to EAOC.Our findings demonstrate that CXCL12 promotes tumor cell invasion and metastasis through PI3K/Akt-mediated induction of EMT. These results provide novel insights into the pathogenesis of EAOC and highlight CXCL12 as a promising biomarker for early diagnosis and a potential therapeutic target, offering new avenues for precision management of EAOC.

Seminal plasma is commonly known to serve as a survival medium to transport sperm cells through the male and then female reproductive tract. In rodents, swine and humans, seminal plasma actively modulates the cellular and molecular maternal environment at the time of conception and early embryo development. Artificial insemination in cattle minimizes female exposure to seminal plasma and deposits semen directly into the uterus, bypassing the vagina as the natural site of insemination. Seminal plasma exposure at insemination increases heifer birth weight, but the mechanisms are unknown. We hypothesized that supplementation of seminal plasma at insemination would alter the endometrial transcriptome and program early embryo development. As such, cows were subjected to estrus synchronization and superovulation followed by timed artificial insemination using X-sorted semen. Immediately following insemination, cows received an intrauterine infusion of either saline (n = 12) or seminal plasma (n = 14). On day 7 after insemination, embryos were non-surgically recovered and the endometrium was sampled for RNA sequencing. The number, morphology and quality of recovered embryos were not affected by seminal plasma infusion. A total of 107 differentially expressed genes (FDR ≤ 0.1) were identified in the endometrium that were related to 1) cellular immune responses, 2) cellular growth, proliferation, and development, and 3) cell morphology and embryonic development. We conclude that seminal plasma exposure at insemination alters the endometrial environment seven days after insemination and does not adversely impact embryo development.

RTCB (RNA 2',3'-cyclic phosphate and 5'-OH ligase) is a unique 3'-5' RNA ligase with diverse physiological functions in metazoans. To further explore the role of RTCB in reproduction, we generated a Rtcb conditional knockout mouse model using the Ddx4-Cre system. The complete absence of viable Rtcb-/- offspring indicated embryonic lethality. Integrated analyses of histology, immunohistochemistry, quantitative PCR, and published single-cell RNA sequencing (scRNA-seq) data revealed that Rtcb is expressed throughout embryogenesis, with pronounced upregulation during gastrulation, and that Rtcb knockout resulted in gastrulation failure around embryonic day 6.5 (E6.5), which was accompanied by decreased proliferation and increased apoptosis of embryonic cells. Notably, NUSAP1 (nucleolar and spindle-associated protein 1) is vital for gastrulation as it participates in cell division. In NIH 3T3 cells, knockdown of Rtcb led to destabilization of Nusap1 mRNA, suggesting that NUSAP1 might function downstream of RTCB. Moreover, Rtcb is a target gene of YY1 (Yin-Yang-1), a transcription factor crucial for gastrulation. Expression profiling through scRNA-seq revealed spatiotemporal coordination among Yy1, Rtcb, and Nusap1 between E3.5 and E7.5. Collectively, our findings demonstrate that RTCB is essential for early mouse embryogenesis and propose the presence of a YY1-RTCB-NUSAP1 axis that maintains proper cell proliferation for successful gastrulation.