Biology of Reproduction最新文献

Organophosphate esters (OPEs), widely used as flame retardants and plasticizers, are frequently detected in indoor environments and human tissues, raising concerns about their potential endocrine-disrupting effects. In this study, we examined the effects of a household dust-based mixture of OPEs, along with two structural distinct sub-mixtures, on the phenotype, function, and lipidome on MA10 Leydig cells. Using high-content imaging, we identified increase in oxidative stress levels and accumulation of lipid droplets as common phenotypic effects across mixtures. Notably, the triaryl OPE sub-mixture exhibited greater potency, suggesting that specific structural features contribute to the toxicity of OPEs. While the OPE mixture did not impair basal steroid hormone production in MA-10 cells, changes were observed in stimulated progesterone levels and transcriptional regulation of key steroidogenic transcripts. When comparing lipidomic profiles across three steroidogenic cell lines (MA-10, H295R, and KGN), we found that glycerolipids, particularly triglycerides and diglycerides, consistently appeared to be the most affected lipid species, highlighting a common disruption in the composition of lipid droplet. However, cell line specific effects were also observed, especially in the regulation of cholesterol esters, likely reflecting differences in cholesterol sourcing and steroidogenic pathways. These findings emphasize the importance of evaluating environmentally relevant chemical mixtures and demonstrate that OPEs can disrupt steroidogenic function and lipid metabolism.

Cryopreservation methods for archiving and distributing mouse strains mostly focus on freezing embryos or sperm. Although protocols for the cryopreservation of wild-type mouse oocytes are available, these methods have not been widely adopted in large biomedical research facilities. This is partly due to a lack of validation of the available methods on a large scale using a range of genetically modified oocytes. Furthermore, some of the existing methods report a relatively low rate of fertilization, requiring either the zona pellucida to be physically breached or the inclusion of cumulus cells to enable efficient fertilization, interventions which might be incompatible with maintaining hygiene barriers. Existing methods also often use cryovials rather than straws or slimline vitrification devices, which are more practical for storage and handling. Here, we present a robust vitrification protocol for large-scale oocyte cryopreservation, achieving high viability and fertilization rates comparable to fresh oocytes. We have extensively tested the protocol for in vitro fertilization of many genetically altered strains, using both genetically altered and wild-type C57BL/6J oocytes and sperm. Providing an archive of cryopreserved oocytes harboring genetically modified alleles separately from archives of cryopreserved sperm allows multiple allele combinations to be generated during the rederivation process. This reduces subsequent breeding steps and the need to maintain live stocks of mice. Furthermore, cryopreserving oocytes for later use enables them to be obtained from females at the optimal age, thereby reducing the number of mice required and providing greater scheduling flexibility for subsequent genetic modification and rederivation work.

Genome editing is a rapidly advancing technology with transformative potential in livestock, offering opportunities that range from enhanced production traits to the generation of biomedical models for human disease and xenotransplantation. The CRISPR/Cas9 system, originally identified as a bacterial defense mechanism, has become the most widely used tool for precise genome editing. In this review, we first summarize the potential applications of CRISPR/Cas9 in livestock and highlight notable successes to date. We then address the ongoing challenges associated with delivering CRISPR/Cas9 into gametes and embryos, as current methods such as microinjection and electroporation often result in high mosaicism and cellular damage. We subsequently introduce extracellular vesicles (EVs) as a promising alternative delivery system. Secreted by virtually all cell types, EVs can efficiently transport bioactive molecules and are readily internalized by gametes and embryos. Although EV-mediated delivery of CRISPR/Cas9 has shown success in somatic cells, its use in reproductive cells remains largely unexplored. We review emerging strategies for loading EVs with CRISPR/Cas components and discuss the potential advantages of combining this approach with recently developed smaller Cas variants to overcome delivery barriers. Collectively, these innovations support the promise of EVs as a biologically compatible, efficient, and minimally invasive system for targeted genome editing in livestock reproduction.

Purpose: Polycystic ovary syndrome affects 5-10% of women of reproductive age, with insulin resistance playing a central role in its pathophysiology in up to 80% of cases. This review aims to elucidate the molecular mechanisms by which insulin resistance disrupts ovarian function, contributing to menstrual irregularities and hyperandrogenism. It also evaluates current and emerging therapeutic strategies, with an emphasis on individualized management.

Methods: A comprehensive review of recent literature was conducted, focusing on molecular studies, clinical trials, and meta-analyses related to insulin signaling pathways in polycystic ovary syndrome, as well as therapeutic interventions. Special attention was paid to ethnic variations, particularly in East Asian populations, and advances in genomic and metabolomic profiling.

Results: Polycystic ovary syndrome is characterized by selective insulin resistance, wherein metabolic insulin signaling is impaired, but steroidogenic and mitogenic pathways remain responsive, promoting hyperandrogenism and anovulation. East Asian women exhibit significant insulin resistance despite lower body mass index compared with Western populations. Insulin resistance in polycystic ovary syndrome also increases cardiometabolic risks and psychological burden. While lifestyle modification, insulin sensitizers, and hormonal therapy remain first-line treatments, novel approaches such as microbiome-targeted therapies and anti-inflammatory agents show promise.

Conclusion: Understanding the complex interplay between insulin resistance and ovarian dysfunction is crucial for effective polycystic ovary syndrome management. Integrating emerging molecular insights with digital health tools can facilitate personalized, multidisciplinary approaches that address both reproductive and metabolic aspects of polycystic ovary syndrome, ultimately improving patient outcomes.

Summary sentence: This study underscores the pivotal role of insulin resistance in ovarian dysfunction and advocates for integrated, technology-enhanced strategies to personalize PCOS management, thereby improving holistic patient outcomes.

Extracellular vesicles (EVs) are promising markers for biological processes, but their role in predicting superovulatory response remains largely underexplored. We investigated blood plasma-derived EV from ewes subjected to superovulation treatment in follicular or luteal phases. 20 Santa Inês ewes underwent an estrous cycle synchronization and superovulation protocol. Based on the number of corpora lutea (CL) and after transcervical embryo collection was accomplished, 10 ewes were allocated into high- (HR; ≥ 11 CL; n = 4) or no/low-response (N/LR; CL ≤ 4; n = 6) groups. The nanoparticle tracking analysis revealed similar EV size distributions and concentrations among groups, but higher concentrations during the follicular compared to the luteal phase. Transmission electron microscopy analysis confirmed the presence of microvesicles and exosomes, while western blotting and nano-flow cytometry revealed the expression of CD63, Syntenin-1, CD9, CD81, and Alix. Tandem mass spectrometry identified a total of 357 proteins, revealing differential protein expression between phases of the estrous cycle and the superovulation response. EV proteomes differed by phase, with 17 DAP: seven up-regulated proteins in the follicular phase and 10 proteins up-regulated in the luteal phase. Comparing ewes with HR versus N/LR responses revealed only a small number of DAP across both phases. In the luteal phase, a single DAP was detected in each group, whereas in the follicular phase, four significant DAP were observed exclusively in the HR group. In conclusion, physical and molecular differences in plasma EV across the follicular and luteal phases of superovulatory treatment highlight their potential as reproductive physiology and superovulation responsiveness biomarkers.

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.