Mammalian sperm must undergo capacitation, a process marked by substantial phenotypic heterogeneity among sperm. How heterogeneity relates to fertilizing potential remains unresolved, in part because sperm subpopulations are typically analyzed independently despite being intrinsically interdependent. We combine flow cytometry with statistical modeling to quantify how sperm subpopulation compositions respond to a range of capacitation signal intensity in vitro. Using cauda epididymal mouse sperm, we systematically varied extracellular bicarbonate and free calcium-key regulators of capacitation-and classified millions of individual cells into four irreversible physiological states. We show that bicarbonate and calcium interact nonlinearly to redistribute sperm across these subpopulations, revealing responses that would be otherwise obscured in measurements lacking single-cell resolution. Elevated intracellular calcium was associated with increased cell death, while the highest proportions of live, acrosome-reacted sperm occurred under relatively low extracellular calcium conditions. We then applied a hierarchical Dirichlet-multinomial regression model that accounts for multinomial sampling noise and between-male variability, yielding posterior probability surfaces that describe how sperm subpopulations reallocate across functional states as signaling conditions change. We then tested the model predictions using IVF and found that the proportion of live, acrosome reacted sperm was not predictive of early embryonic developmental success. Together, these results suggest that capacitation, though tightly regulated at the single cell scale, is a stochastic process shaped by structured phenotypic heterogeneity at the cell population scale. This framework provides a quantitative foundation for linking sperm subpopulation composition to measures of fertility competence.
Abstract Sepsis, a serious life-threatening systemic inflammatory disease, has not received enough attention for the testicular dysfunction it causes. The testis is not only a reproductive organ, but also an important endocrine gland that secretes testosterone and other sex hormones. This study aimed to systematically investigate the persistent damage caused by sepsis to testicular function and to explore the underlying mechanisms, with a focus on macrophages. Clinical observations were performed in sepsis patients, and animal experiments were conducted using the cecal ligation and puncture model. Macrophage depletion experiments were further performed to confirm the mechanistic role of macrophages. Clinical observations showed significant low testosterone levels in patients during both acute and recovery phases. In mice, testicular tissue damage and spermatogenic dysfunction occurred at 24 hours post-cecal ligation and puncture and persisted through day 14. Mechanistic studies revealed that sepsis triggers oxidative stress injury by inducing testicular macrophages proliferation and promoting excessive release of pro-inflammatory factors and reactive oxygen species. Macrophage depletion largely abolished sepsis-induced testicular injury. In conclusion, sepsis causes persistent testicular injury through macrophage-associated inflammatory and oxidative stress responses. These findings elucidate the pathogenesis of sepsis-induced testicular injury and identify potential therapeutic targets for improving reproductive-endocrine function and overall prognosis in sepsis patients.
Abstract In vitro maturation (IVM) of oocytes plays a crucial role in assisted reproductive technologies in livestock production, enabling efficient large-scale embryos production and supporting genetics improvement programs. However, the efficiency of this technology remains limited as only a small proportion of cumulus-oocytes complexes (COCs) matured in vitro reach the blastocyst stage, which exhibit lower quality than those produced in vivo. Recent efforts have focused on delivering biological materials to cumulus cells during IVM to improve oocyte quality. Herein, we hypothesized that extracellular vesicles (EVs) isolated from ovarian follicular fluid (FF) can be engineered to deliver synthetic microRNAs (miRNAs) to cumulus cells during IVM. Here, we demonstrate for the first time that follicular fluid-derived EVs are enriched with synthetic miRNAs and successfully delivered to COCs, resulting in altered gene expression in cumulus cells, and transfer of miRNAs to the oocyte cytoplasm. Specifically, EVs loaded with bta-miR-20a-5p were effectively taken up by cumulus cells during IVM, enhanced PI3K/AKT signaling with significant increase in downstream genes, such EIF4b and GSK3, which are involved in cell cycle progression, DNA repair and protein synthesis. Additionally, bta-miR-20a-5p-loaded-EVs supplementation to COCs, during IVM, appeared to reduce cleavage and blastocyst development rates. Collectively, our findings demonstrated that engineered EVs can deliver a synthetic miRNA to COCs during IVM, representing highlighting their potential to modulate oocyte cytoplasm content and embryo development.
The bovine embryo expresses interleukin-6 (IL6) as well as its receptor system, which includes the ligand-binding IL6 receptor subunit (IL6R) and the common IL6 family signal-transducing component (IL6ST). Our laboratory recently determined that IL6ST activity facilitates normal in vitro embryo development and proper inner cell mass (ICM) size and morphology. We therefore hypothesized that embryo-derived IL6, acting through the IL6R-IL6ST receptor complex, drives these developmental outcomes. To test this, two single guide RNAs (sgRNAs) targeting exon 2 of IL6R were designed and electroporated with Cas9 protein into bovine zygotes. Editing efficiency was assessed in 16-cell embryos through targeted IL6R genotyping. Disruption of IL6R did not alter cleavage or development to the 16-cell stage but reduced blastocyst formation on days 7 and 8. The IL6R-edited blastocysts that were generated had reduced ICM cell numbers, no changes in trophectoderm (TE) cell number, and a reduced ICM:TE ratio. In summary, loss of IL6R signaling compromises blastocyst formation and alters cellular composition by reducing ICM cell numbers, thus supporting an embryotrophic role for embryo-derived IL6 acting through the IL6R/IL6ST complex. However, the absence of earlier developmental defects, which occurred in our prior IL6ST-targeting studies, suggests that additional embryo-derived IL6-family cytokines likely also contribute to pre-blastulation development in cattle.
Calcium and vitamin D are essential for cellular function and fetal and placental development, yet the mechanisms regulating their availability within utero-placental tissues remain poorly understood. This study characterized calcium and 25-hydroxyvitamin D [25(OH)D] abundance and the spatiotemporal expression of calcium- and vitamin D-regulatory molecules in bovine utero-placental tissues and evaluated the influence of fetal sex. Pregnant bovine uteri (n=95) were collected between gestational days (GD) 46 and 136 at 7-day intervals (n=3-10/group). Calcium concentrations varied with gestational day in allantoic and amniotic fluids and placentomes (P ≤ 0.05), declining with advancing gestation in amniotic fluid and placentomes (P≤0.001), but were unchanged in endometria. Concentrations also differed between fetal fluid and utero-placental tissue types (P≤0.0001). Tissue 25(OH)D abundance was not affected by gestational day, tissue type, or fetal sex. Gestational day affected the expression of VDR, S100G, STC1, STC2, and S100A8 mRNAs in endometria and VDR, S100G, STC1, and S100A9 mRNAs in placentomes (P≤0.05). Expression of STC1 mRNA was greater in endometria associated with female than male fetuses (P≤0.05). VDR protein localized to the uterine epithelia and stratum spongiosum stroma, as well as the chorionic epithelium and syncytium within placentomes. S100B and S100G proteins localized to the uterine epithelia, chorionic epithelium and syncytium, and trophoblast binucleate and multinucleated giant cells. These findings demonstrate that calcium- and vitamin D-regulatory molecules are differentially expressed and localized throughout bovine utero-placental tissues across gestation, providing a foundation for understanding local regulation of calcium availability during pregnancy.
