Biology of Reproduction最新文献

Porcine conceptus elongation during the peri-implantation period is essential for establishment of pregnancy and involves rapid morphological and biochemical remodeling. However, metabolomic alterations within developing porcine conceptuses during this developmental interval are poorly understood. Therefore, the objective of this study was to characterize metabolomic remodeling in porcine conceptuses between gestational Days (GD) 10 and 16 using liquid chromatography-mass spectrometry (LC-MS)-based metabolomics and targeted amino acid analyses. A total of 230 metabolites were identified in conceptuses collected on GD10, 12, 13, 14, 15, and 16. Principal component analyses revealed progressive temporal separation of metabolomic profiles during conceptus elongation and implantation. Conceptus elongation was associated with progressive remodeling of metabolites related to nucleotide biosynthesis, phospholipid metabolism, amino acid metabolism, redox regulation, and energy metabolism. Random forest analyses identified cytidine diphosphate-ethanolamine (CDP-ethanolamine) and nucleotide-associated metabolites as key discriminators of progression in conceptus development. Targeted amino acid analyses of conceptuses demonstrated progressive increases in glutamine, glutamate, histidine, valine, arginine, and cystine, whereas tryptophan, glycine, and serine decreased during later stages of development. These coordinated changes support increased requirements for nucleotide synthesis, membrane biogenesis, redox homeostasis, and nutrient metabolism during rapid elongation and subsequent extraembryonic growth and differentiation. Collectively, these findings define metabolic transitions associated with porcine conceptus elongation and post-elongation development and provide candidate metabolic processes for future mechanistic investigation.

The standard protocols used in clinics to assess quality of embryos are still unable to reliably assess the embryo's ability to implant and develop full term. On the other hand, being able to predict the implantation potential of a given embryo appears to be crucial, as implantation abnormalities are one of the major causes of reproductive loss in mammals. Notwithstanding the differences in the implantation mechanism between different species, the functional trophectoderm is prerequisite in this process. In the present study we investigated whether the velocity of cytoplasmic movement in mural and polar TE cells of mouse blastocysts can be used to assess the embryo implantation potential. The cytoplasmic velocity was measured using time-lapse imaging in E3.5 and E4.5 mouse embryos and analyzed using Particle Image Velocimetry. We found that it decreases during late preimplantation embryo development and is higher in polar than in mural trophectoderm cells of mouse blastocysts. Our data indicate that cytoplasmic movement velocity was only minimally affected by alterations in the keratin cytoskeleton, despite its established role in determining the biomechanical properties of trophectoderm cells. Furthermore, cytoplasmic movement velocity was not affected by either maternal or postovulatory aging, both of which are known to impair the developmental potential of embryos. However, using an outgrowth assay, we showed that the velocity of cytoplasmic movement in polar, but not mural, trophectodermal cells in E4.5 blastocysts reflects the embryo's ability to implant in vitro. Therefore, analysis of cytoplasmic speed may support evaluation of the embryo quality.

Primordial germ cells (PGCs) are the embryonic precursors of gametes, essential for transmitting genetic and epigenetic information across generations. However, PGC specification occurs within a narrow developmental window and involves only a small number of cells, making it difficult to study in vivo. In vitro models using pluripotent stem cells have enabled the generation of primordial germ cell-like cells, but these systems often rely on exogenous signaling and exhibit variability in efficiency and epigenetic fidelity. In this review, we synthesize current understanding of PGC specification in mouse and human systems, emphasizing the integration of signaling pathways, transcriptional networks, epigenetic reprogramming, and metabolic regulation. Canonical regulators, including PRDM1, PRDM14, TFAP2C, and SOX17, function within a broader, interconnected network that establishes for PGC competence. Understanding these interactions will be crucial for advancing in vitro gametogenesis and improving mammalian reproduction.

Phosphate is critical for pregnancy maintenance, placental, and fetal development. Although the mechanisms regulating postnatal phosphate homeostasis are well described, those regulating phosphate availability during gestation in cattle remain poorly understood. Tissue non-specific alkaline phosphatase (TNSALP, encoded by ALPL), is a postnatal regulator of phosphate availability, yet its role in the regulation of utero-placental phosphate availability in ruminants remains poorly understood. This study characterized phosphate abundance and TNSALP activity in bovine utero-placental tissues and fetal fluids, and the expression of ALPL mRNA, and TNSALP protein and activity localization in bovine utero-placental tissues during early-mid gestation. Fetal fluids and utero-placental tissues were collected (n=3-10 per group; range: 48-133 days). Phosphate concentrations and TNSALP activity were quantified spectrophotometrically. Expression of ALPL mRNA was quantified using qPCR. TNSALP protein and activity localization were determined using histological staining. Phosphate concentrations increased in allantoic fluid and decreased in amniotic fluid as gestation progressed (P ≤ 0.0001). Phosphate abundance did not change in utero-placental tissues throughout gestation. Gestational day affected TNSALP activity in allantoic fluids, amniotic fluids, and endometria (P ≤ 0.001), but not placentomes. Gestational day affected expression of ALPL mRNA in endometria and placentomes (P ≤ 0.01). TNSALP protein and enzymatic activity localized to the endometrial luminal and glandular epithelia, endometrial and placental vasculature, and the caruncular-cotyledonary epithelial interface. Collectively, these findings establish a spatiotemporal profile of phosphate abundance and TNSALP activity in bovine utero-placental tissues and fetal fluids and demonstrate TNSALP is localized to regions associated with maternal-fetal nutrient exchange.

THOR (testis-associated highly conserved oncogenic long non-coding RNA) is a highly conserved and testis-enriched lncRNA across vertebrates that plays diverse roles in various cancers. However, its physiological function and regulatory mechanism in testes remain largely unknown. Here, we investigated the genomic location and expression pattern of THOR in the model organism zebrafish, and generated a homozygous THOR knockout model using CRISPR-Cas9 technology. Loss of THOR in zebrafish impaired spermatogenesis, leading to oligospermia (38.7% reduction in sperm count), reduced sperm motility, sperm ultrastructural defects, and decreased fertilization rates. RNA-seq analysis of WT and THOR knockout testes revealed dysregulation of cell cycle-related genes, including cdkn1d, foxo1a, tsc1a, tsc2, atrx, and rad21b. RNA pulldown assays in zebrafish testes identified 486 potential THOR-interacting proteins primarily involved in ribosome biogenesis, RNA splicing, chromatin architecture, and meiotic progression. Notably, the core synaptonemal complex components Sycp1, Sycp2, and Sycp3 were all captured as THOR-binding partners. We further demonstrated that THOR directly interacts with Sycp3 and positively regulates its protein levels. Immunostaining assays on chromosome spreads revealed a significantly higher frequency of discontinuous Sycp3 signals in THOR-/- testes, suggesting the presence of meiosis defects caused by Sycp3 downregulation. Our findings expand the understanding of lncRNA-mediated control of spermatogenesis and male infertility by providing the first evidence that lncRNA THOR interacts with the synaptonemal complex to regulate meiosis progression.

Early embryonic development occurs in a low oxygen environment, and mitochondrial morphology and function are distinct in embryonic cells and pluripotent stem cells (PSC), which rely less on oxidative phosphorylation than differentiated cells. Oxidative phosphorylation increases with differentiation to trophoblast (TB) and this process is reversed by reprogramming of adult cells to pluripotency, but the influence of oxygen conditions on this process has not been characterized. When PSC were differentiated to trophoblast by treatment with BAP (BMP4, A83-01 and PD173074), cellular ATP concentrations increased equally in 5% and 20% oxygen conditions. Although oxygen conditions in culture altered transcripts encoding mitochondrial proteins, particularly by suppressing COX4l2 at 20% oxygen, there were no consistent differences in mitochondrial morphology in either cytotrophoblast (CTB) or syncytiotrophoblast (STB) cells with changing oxygen. These results suggest that TB adapt to maintain mitochondrial function at high and low oxygen during differentiation. In a previous study, iPSCs were derived from both control and early onset preeclampsia (EOPE) pregnancies and differentiated with BAP; high oxygen conditions impaired TB invasion only in cells from EOPE pregnancies. Here, the increase in ATP concentration with TB differentiation was less robust in EOPE cells, and cytochrome C and ATPase subunit transcripts differed between EOPE and control cells at high oxygen. However, investigation of mitochondrial morphology revealed no excess damage in EOPE-derived lines, and no difference in mitochondrial respiration was detected. Collectively, these data provide limited support for the hypothesis that intrinsic differences in mitochondria underlie poor TB invasion in EOPE.

Testicular tissue cryopreservation (TTC) is currently the only option to preserve fertility in pre-pubertal individuals with testes. However, reimplantation of tissue collected during TTC has yet to result in a live human birth, despite recent progress in rhesus macaque models. Additionally, cell or tissue implantations are not options for individuals with metastatic disease, making it critical to develop alternative fertility restoration strategies using TTC specimens. Our goal was to generate primary cell-derived testicular assembloids with native-similar internal cell organization, enclosed seminiferous tubule-like structures (TLS), and surviving germ cells. Organoids derived from primary testicular cells harvested from 5-day old mice developed native-similar organization and enclosed TLSs when seeded at ~9,300 cells/microwell. We assessed the fusion and internal organization of merged organoids ("assembloids") in multiple open channel designs with differing physical constraints and access to media and oxygen. These conditions impacted assembloid ellipticity, cellular organization, TLS formation, TLS diameter, and germ cell survival. Open channels with 400- and 600-micron widths that were submerged within culture media most consistently produced assembloids with native-similar architecture. These data support a robust framework for the generation of murine testicular assembloids with germ cell-containing tubule-like structures and interstitial compartments. Optimization of this in vitro platform is an important step towards expanding fertility restoration options for prepubertal patients at increased risk for infertility.

Phthalates and per- and polyfluoroalkyl substances (PFAS) are widespread environmental contaminants that are consistently detected in follicular fluid, serum, and reproductive tissues. This review synthesizes current experimental and epidemiological evidence on the molecular and cellular mechanisms by which phthalates and PFAS impair ovarian function and female fertility. Human studies primarily identify exposure-outcome associations, whereas animal, ex vivo, and in vitro models provide most of the causal and mechanistic evidence. Phthalates and PFAS disrupt interconnected pathways involved in folliculogenesis, steroidogenesis, mitochondrial homeostasis, inflammatory signaling, and cell survival. These chemicals dysregulate pathways governing primordial follicle activation and ovarian reserve maintenance, and impair estradiol and progesterone synthesis. A central mechanistic theme by which phthalates and PFAS impair ovarian function and fertility involves mitochondrial dysfunction, which promotes oxidative stress and contributes to apoptosis. However, evidence for phthalate- and PFAS-induced ovarian necroptosis, pyroptosis, immune-cell infiltration, and inflammation-driven fibrosis remains limited and is often based on a small number of studies conducted at doses above typical human exposures. Although mixture studies are relatively scarce, available data indicate that mixtures can perturb mitochondrial activity, steroid secretion, follicle dynamics, inflammatory signaling, and Hippo-pathway endpoints. Overall, altered folliculogenesis and steroidogenesis, mitochondrial dysfunction, oxidative stress, and apoptosis emerge as the best-supported mechanisms linking phthalate and PFAS exposure to ovarian toxicity. This review also highlights the need for exposure-relevant studies, quantitative pathology, and stronger integration of experimental mechanisms with human biomonitoring data.

In vitro embryo production (IVP) has achieved remarkable gains in efficiency; however, these advances are still based on oversimplified models. By optimizing blastocyst yield rather than biological quality, the field has systematically overlooked the critical reality that embryos reaching the blastocyst stage in vitro frequently exhibit profound molecular and functional deviations from their in vivo counterparts, with measurable consequences for implantation success, placental function, and long-term offspring health. Current IVP systems continue to impose non-physiological conditions during the most epigenetically vulnerable windows of preimplantation development. Converging evidence from omics technologies demonstrates that in vitro environments do not merely stress the embryo; they probably rewire its regulatory architecture. Disruptions in metabolic flux alter the epigenome, and redirect gene expression networks, thereby reducing developmental fidelity and generating molecular signatures consistent with the Developmental Origins of Health and Disease (DOHaD) concept. A central limitation is the prevalence of descriptive omics studies that describe molecular differences without establishing causal mechanisms. Advancing embryo quality requires a shift toward functional perturbation approaches, multi-omics integration, and mechanistically based experimental design. In this review, we propose a conceptual model for next-generation IVP systems built on adaptive culture conditions responsive to embryo-derived metabolic signals, restoration of epigenetic integrity, and re-establishment of bidirectional embryo-environment communication through oviductal signals, extracellular vesicles, and bioengineered interfaces. Success metrics must be redefined from blastocyst formation to functionally validated outcomes. The future of IVP lies not in producing more embryos, but in producing embryos whose molecular architecture reflects the regulatory precision of the beginning of life.