Biology of Reproduction最新文献

The peri-implantation period of pregnancy in pigs is characterized by rapid morphological transitions of the conceptus necessitating a precisely regulated uterine environment to support elongation, survival, and implantation. Uterine histotroph, composed of nutrients and signaling molecules secreted by or transported by endometrial epithelia, plays a central role in mediating these events. However, dynamic changes in the metabolic composition of uterine luminal fluid (ULF) during early pregnancy are incompletely defined. In this study, we performed stage-resolved, untargeted metabolomic profiling of ULF collected from cyclic and pregnant gilts on Days 10, 12, 14, and 16 of the estrous cycle and pregnancy (n = 2-6/group). A total of 206 metabolites were identified, with amino acids, fatty acids, and carbohydrates being the dominant classes. Principal component and supervised learning analyses revealed progressive divergence in ULF composition between pregnant and cyclic gilts with the most distinct profiles observed by Day 16. Notably, pregnancy induced substantial increases in amino acids associated with mechanistic target of rapamycin (mTOR) signaling and trophectoderm proliferation, including arginine, glutamine, proline, lysine, and phenylalanine. Kyoto Encyclopedia of Genes and Genomes enrichment analyses identified gestational age-dependent activation of pathways involved in amino acid biosynthesis, nucleotide metabolism, and phospholipid turnover. Metabolites such as phosphorylcholine, succinic acid, and asymmetric dimethylarginine increased markedly in pregnancy, suggesting coordinated regulation of membrane remodeling, energy production, and nitric oxide signaling. Targeted quantification of 19 amino acids revealed both linear and quadratic trends across time and pregnancy status, with distinct differences in glycine and serine trajectories between pregnant and cyclic ULF. Collectively, these findings describe the evolving biochemical landscape of the uterine lumen during early pregnancy and highlight key metabolic pathways that likely support conceptus development and uterine receptivity to implantation.

Maternal excess adiposity during pregnancy is linked to placental malperfusion and inflammatory injury. Obesity-associated placental malperfusion may induce fetoplacental hypoxia, contributing to adverse health outcomes within and beyond the perinatal period. However, direct comparisons of tissue oxygen saturation at the uteroplacental interface in pregnancies complicated by excess adiposity are lacking. Using a mouse model of preconception high-fat, high-sucrose (HFHS) diet-induced excess adiposity, we found that both placental junctional and labyrinth zones at E17.5 were hypoxic compared to chow-fed controls (CON). HFHS placentas had a greater burden of histopathological lesions, including tissue calcification and fibrinoid deposition within the labyrinth zone. Calcified placental tissue coincided with the destruction of vasculosyncytial membranes and macrophage-dense foci, alongside altered expression of immunomodulatory and chemotactic cytokines, which differed in magnitude with fetal sex. While fetal growth was not impaired, fetuses from HFHS pregnancies exhibited higher levels of circulating IL-6, prolactin, CXCL1, and CCL2. Collectively, these data confirm that maternal diet-induced excess adiposity leads to a reduction in placental oxygen saturation, even in the absence of marked growth restriction or fetal demise. While this hypoxic state was not linked to gross morphological abnormalities, it was associated with markers indicative of local malperfusion and inflammation, and an altered fetal inflammatory and endocrine milieu in late gestation. Together, these findings demonstrate that a state of placental hypoxia may contribute to the increased risk of adverse perinatal outcomes and long-term disease programming in pregnancies affected by maternal obesity.

Deep three-dimensional imaging of oocytes shows several difficulties. Their large size and spherical shape cause depth-dependent artefactual shadow in the middle, resulting from refractive index mismatches induced by turbid organelles and lipid droplets. These mismatches lead to optical aberrations, increasing the laser spot size at the confocal pinhole plan and causing significant attenuation of fluorescence intensity, making it difficult to clearly image fine structures such as the transzonal projections (TZPs) connecting cumulus cells and the oocyte. To overcome these challenges, various methods of sample preparation and confocal imagery settings were compared. To clearly show the depth limitation, a clearing protocol was used to image entire fixed embryos. As expected, limiting diffraction, namely, by removing lipid droplets and harmonizing extra- and intracellular media, resulted in more uniform staining and distribution, compared to uncleared specimens. The density of the cumulus cloud and fixation protocols were shown to have a profound impact on image quality. Gentle partial stripping and low fixation reduced noise in imagery, while permeabilization with Triton enhanced antibody penetration, resulting in efficient protein labeling with the zona pellucida-enclosed TZPs. Control samples were employed to exemplify unspecific and specific signals to determine optimal confocal settings. Careful consideration of confocal parameters was shown to be crucial for well-adjusted imagery. Moreover, the choice of mounting medium and slide assembly impacts the shape and resolution of the specimen. These findings provide valuable insights into challenges associated with cumulus-oocyte complex imaging, offering solutions for optimizing sample preparation and image quality.

Endometriosis markedly compromises female fertility, and although endometrial dysfunction likely plays a role in this pathology, its precise mechanistic contributions remain poorly understood. This study aims to investigate the decidualization and angiogenic capacity of eutopic endometrial in endometriosis patients. The study enrolled 30 participants, including 15 infertile patients with endometriosis (EMS group) and 15 patients with benign gynecological conditions who underwent laparoscopic treatment (negative control group, NC group). Immunohistochemistry, F-actin staining, qRT-PCR, Western blot, ELISA, and tube formation assays were used to analyze decidual endometrial stromal cell (ESC) morphology, measure protein expression associated with decidualization and angiogenesis. Secretory phase endometrium from endometriosis patients EMS group showed significantly reduced expression of decidual markers (PRL, IGFBP1, HOXA10, BMP2) and angiogenic-related proteins (ANG2, VEGFA, VEGFR1) versus NC group. Primary ESCs isolated from proliferative phase endometrium tissue of endometriosis patients showed impaired decidualization under hormonal induction, with attenuated morphological transformation, downregulated decidual-related proteins (P < 0.05). Angiogenic dysfunction was evidenced by decreased VEGFA secretion (P = 0.026), reduced angiogenic-related proteins (P < 0.05), and impaired HUVEC tube formation in cocultures (P < 0.05). Overall, endometriosis-associated infertility involves intrinsic defects in decidualization and angiogenesis. This may provide new perspectives for improving the reproductive outcomes of patients with endometriosis.

Introduction: Despite the increasing body of evidence that autophagy implicate in intrahepatic cholestasis of pregnancy (ICP), we are still far from a mechanistic understanding of the autophagy in ICP pathogenesis.

Methods: In this study, we performed untargeted lipidomics, scRNA-seq, and RNA-seq data to systematically demonstrate the lipid alterations, cells and gene expression closely related to autophagy in placenta. These results were also verified by tissue, cell, and animal experiments.

Results: We confirmed that autophagy played a pivotal role in ICP, autophagy-related lipids including PE (38:2e) and PE (54:5) had a good diagnostic value. Autophagy genes were mainly concentrated in villous cytotrophoblast (VCT), extravillous trophoblast (EVT), and macrophage. VCT was found increased while EVT decreased in ICP. In the subcluster analysis of VCT, VCT-EVT, the precursor cells of EVT, was significantly reduced. In the subcluster analysis of EVT, EVT2 cells with epithelial migration and regulatory functions were significantly reduced. Furthermore, autophagy gene TNFSF10 was decreased in ICP, while supplementation of soluble TNFSF10 could restore the expression of TNFSF10 in ICP cell model, which alleviated the autophagic damage and improved the invasion and migration ability of trophoblast cells.

Conclusion: Our study established the causal linkage between autophagy and ICP, providing a potential therapeutic avenue to improve trophoblast function accounting for ICP by targeting autophagy gene TNFSF10.

The oviductal fluid (OF) provides essential nutritional, mechanical, and physical support during the first four days of embryonic development. Among its physical properties, viscosity has been largely overlooked in embryo in vitro culture (IVC) media due to limited available data. In this study, we measured the viscosity of bovine OF from ex vivo samples and mimicked it during in vitro embryo culture. OF viscosity remained consistent across estrous cycle stages and between oviductal antimeres, and was over three times higher than that of standard IVC media (3.4 ± 1 vs. 0.9 ± 0 mPa·s, P < 0.05). To replicate this property, in vitro bovine embryos were cultured in media supplemented with sodium alginate (0%, 0.25%, or 0.75% w/v), corresponding to low (control), physiological (3.3 ± 0.1 mPa·s), and supraphysiological (6.8 ± 0.02 mPa·s) viscosity levels. While cleavage and blastocyst rates were unaffected, embryos cultured at physiological viscosity (0.25%) showed improved quality indicators: increased total cell number, lower apoptosis, reduced reactive oxygen species, and decreased global DNA methylation compared to the high-viscosity (0.75%) group. Notably, embryos in the 0.25% group also showed lower apoptosis and inner cell mass methylation than controls. These findings suggest that standard IVC media fail to replicate key rheological features of the oviductal environment and that alginate is a safe, non-Newtonian viscosity modulator. Mimicking OF viscosity during IVC improves embryo quality and epigenetic outcomes and may represent a valuable refinement in assisted reproductive technologies.

Purpose: Developing protocols for ovarian cortex preservation paves the way for collection of valuable early germ cells from all female individuals, regardless of age and reproductive status. This study aimed to further optimize the microwave-assisted dehydration protocol by characterizing ovarian tissue stress response to drying in the domestic cat model.

Methods and findings: Ovaries from prepubertal cats were dissected using different techniques, exposed to different treatments, and analyzed for ovarian cortex integrity and function after dehydration and rehydration protocols. Using RNA sequencing, we highlighted functions and pathways most affected by dehydration and selected marker genes for quantitative PCR to test different protocol conditions. Switching to a biopsy puncher over our previously used dissection technique introduced the first major advancement in the protocol, improving follicular integrity and transcriptional activity after 10 min of drying compared to our previous reports. This change also improved the expression of genes related to signaling, membrane transport, and transcription regulation after 10 min of drying (loss of 87.3% of water), while almost all genes were significantly downregulated after 15 min of drying (loss of 89.7% of water). Using lucifer yellow as a proxy for trehalose, we indirectly estimated trehalose uptake into follicles after membrane permeabilization with digitonin. Although increased digitonin incubation resulted in potentially higher uptake of trehalose, the associated membrane damage led to compromised follicular integrity and decreased transcriptional activity after dehydration.

Conclusions: These results expand our knowledge of the ovarian tissue stress response to the dehydration challenge and bring us closer to optimizing the tissue preservation protocol at ambient temperatures.

Summary sentence: This study advances ambient-temperature preservation of ovarian cortex by characterizing tissue stress response to the microwave-assisted dehydration protocol and exploring ways to reduce stress-induced damage in the domestic cat model.

Previous studies have shown that the metabolite of gonadotropin-releasing hormone (GnRH), GnRH-(1-5), promotes migration and invasion in endometrial cancer cell lines through a non-canonical mechanism from its parental peptide. These studies showed that GnRH-(1-5) transactivates the epidermal growth factor receptor/extracellular signal-regulated kinases (EGFR/ERK) signaling pathway through an orphan G-protein-coupled receptor, GPR101, to stimulate matrix metalloproteinase-9 (MMP-9)-mediated EGF release to augment cellular migration and invasion. However, inhibition of the EGFR/ERK signaling pathway showed an incomplete ablation of the effects of GnRH-(1-5) in these studies to suggest that alternative signaling pathways are also involved. Given the incomplete inhibition of GnRH-(1-5) effects by EGFR/ERK pathway blockade, the present study sought to investigate the potential role of transforming growth factor beta (TGF-beta) in complementing the previously observed EGF effects on cellular function. As our previous studies were conducted in Phosphatase and Tensin homolog (PTEN)-negative cell lines, we sought to elucidate the involvement of the TGF-beta signaling pathway and the role of PTEN status in mediating the cellular responses to GnRH-(1-5). The present results show that cellular migration responses to GnRH-(1-5) involve both TGF-beta and EGF signaling pathways and are differentially regulated based on PTEN status. In addition to these cell line studies, we performed differential gene expression analysis of PTEN-positive and PTEN-negative cell lines and tumors using The Cancer Genome Atlas database. Identifying markers associated with PTEN status will allow for a more precise and rapid investigation of GnRH-(1-5) signaling mechanisms in endometrial cancer pathophysiology.

For many years, activin A, encoded by Inhba, has been thought to be present in both mouse and human oocytes and preimplantation embryos. However, its deficiency does not impede the proper embryonic development of the embryo until birth. It has been suggested that the lack of a phenotype in zygotic knockout embryos may be masked by the presence of maternal protein deposited in the oocyte during oogenesis or provided from the reproductive tract. Therefore, to explore whether maternally supplied activin A is required for embryo development, we carried out a conditional Inhba knockout in oocytes using Zp3-Cre/LoxP strategy. By examining Inhba maternal and maternal/zygotic knockout embryos, individually recorded using time-lapse imaging, immunostained, and genotyped, we revealed that the maternal pool of activin A affects the dynamics of mouse preimplantation development. These alterations are accompanied by impaired mitochondrial activity in oocytes. Surprisingly, using the droplet digital polymerase chain reaction approach, we provided evidence that the Inhba mRNA of zygotic origin is undetectable in mouse embryos.

In ungulates such as pigs and sheep, blastocyst elongation is essential for implantation, driven by coordinated trophoblast proliferation, cytoskeletal remodeling, and nutrient signaling. L-arginine (Arg), a conditionally essential amino acid enriched in uterine histotroph during early pregnancy, plays a key role in conceptus development; however, its genomic effects on the trophectoderm are poorly defined. Therefore, we investigated Arg-responsive transcriptomic changes in porcine (pTr2) and ovine (oTr1) trophectoderm cells using RNA-seq and integrative pathway analyses. Arg significantly stimulated proliferation in both cell types in a non-linear, dose-dependent manner, with maximal effects at 0.2 mM, consistent with physiological levels. Transcriptomic profiling identified 2723 and 5369 differentially expressed genes in pTr2 and oTr1 cells, respectively, encompassing genes involved in cell proliferation, metabolism, cytoskeletal remodeling, and implantation. Canonical pathway and upstream regulator analyses revealed both conserved and species-specific responses to Arg, including shared activation of actin cytoskeleton and adrenomedullin signaling and suppression of p53 signaling. Species-specific enrichment of cholesterol biosynthesis (pigs) and interferon signaling (sheep) further highlighted divergent strategies for pregnancy recognition. Comparative analyses identified 615 commonly regulated differentially expressed genes and highlighted O-linked N-acetylglucosamine transferase as a conserved upstream regulator, implicating nutrient-responsive O-GlcNAcylation in trophectoderm function. Arg promoted gene programs supporting mitosis, oxidative phosphorylation, and extracellular matrix remodeling while repressing pathways related to apoptosis and pluripotency. These results advance understanding of how Arg activates conserved transcriptional networks to support trophectoderm proliferation and differentiation, while also fine-tuning species-specific pathways aligned with pregnancy recognition and implantation of conceptuses in sheep and pigs.