To improve the vitrification efficiency of bovine germinal vesicle (GV) oocytes, the use of hydroxyapatite (HA) nanoparticles as a novel cryopreservation additive represents a promising approach. This study aimed to investigate the effects of HA nanoparticles and permeable cryoprotective agents (CPAs) on the ultrastructure, developmental competence, and gene expression of bovine GV oocytes following vitrification. Oocytes were vitrified in vitrification solutions containing HA nanoparticles of different sizes (20, 40, or 60 nm) and concentrations (0.01%, 0.05%, or 0.1%) to determine the optimal conditions based on survival rate, mitochondrial membrane potential (MMP) level, and developmental competence. Subsequently, the synergistic effects of HA nanoparticles and permeable CPAs (VS: 20% EG + 20% DMSO; VS1: 17.5% EG + 17.5% DMSO) were further evaluated. The optimal treatment (40 nm 0.05% HA nanoparticles) significantly increased MMP level, and improved developmental competence compared with the vitrified control group (p < 0.05). Among the vitrified groups, vitrified oocytes in the VS1-HA group (combining HA nanoparticles with reduced concentrations of permeable CPAs) exhibited the highest MMP level (1.89), maturation rate (50.39%), cleavage rate (27.07%), and blastocyst rate (10.53%) (p < 0.05). Ultrastructural analysis further revealed that the VS1-HA group maintained more intact zona pellucida structures and showed reduced mitochondrial swelling compared with the vitrified control group. Moreover, the expression levels of genes associated with zona pellucida formation (ZP3), mitochondrial fusion (MFN1), and chromatin remodeling (NPM2) were significantly upregulated in the VS1-HA group relative to the vitrified control group. Overall, these findings indicate that the combination of HA nanoparticles with lower concentrations of permeable CPAs enhances MMP level, alleviates vitrification-induced ultrastructural damage, and upregulates the expression of key developmental genes, thereby improving the developmental competence of vitrified bovine GV oocytes.

Embryo implantation is a highly selective and hormonally regulated event, critically dependent on endometrial receptivity and the molecular microenvironment of uterine fluid (UF). UF is actively secreted by endometrial glands and epithelial cells and comprises proteins, metabolites, hormones, and extracellular vesicles (EVs) carrying miRNAs, lncRNAs, and regulatory proteins. These components coordinate embryo development, trophoblast invasion, angiogenesis, and maternal–fetal immune tolerance. Dysregulation of UF composition is increasingly implicated in reproductive disorders, including recurrent implantation failure, unexplained infertility, endometriosis, adenomyosis, and polycystic ovary syndrome, highlighting its potential as a noninvasive biomarker of endometrial function. Despite advances, challenges remain in standardizing UF sampling, addressing temporal and inter-individual variability, and elucidating upstream regulatory networks. Integrative multi-omics analyses, functional organoid–embryo models, and longitudinal clinical studies are essential to translate UF insights into predictive diagnostics and therapeutic strategies, ultimately improving implantation success and reproductive outcomes.

Background: Decidualization requires stromal reprogramming to establish pregnancy, yet the upstream mechanisms governing cytoskeletal remodeling remain obscure. While primary cilia direct this transformation, how stromal cells integrate post-translational modifications to regulate the mechanics of ciliogenesis remains unknown. This study aims to investigate the role of neddylation, a ubiquitin-like modification, in coordinating the structural and transcriptional plasticity required for mouse decidualization. Methods: We utilized the NAE inhibitor MLN4924 in an in vivo mouse model of artificial decidualization. To decipher the underlying molecular mechanism, we combined pharmacological blockade with genetic silencing of Uba3 and Cul3 in mouse uterine stromal cells. We employed RNA-sequencing to map global transcriptomic landscapes, complemented by real-time PCR, immunofluorescence, and Western blotting to assess ciliary dynamics and cytoskeletal architecture. Additionally, the ciliogenesis inhibitor Ciliobrevin A (CBA) was used to study the functional link between primary cilia and decidualization. Finally, we performed rescue experiments with small-molecule inhibitors of RhoA (Rhosin) and ROCK (Y27632), alongside the microtubule stabilizer Tubastatin A (TubA), to dissect the specific mechanotransduction pathways driving the observed phenotypes during mouse decidualization. Results: We identified neddylation as a key regulator of stromal remodeling during decidualization. Pharmacological inhibition and genetic silencing significantly suppressed primary cilia formation and impaired the decidual response. Transcriptomic profiling revealed that neddylation inhibition aberrantly reactivates hormone-suppressed actomyosin contractile genes, particularly Myl9 . Mechanistically, we demonstrate that neddylation selectively activates the E3 ligase Cullin 3 (Cul3) to target RhoA for degradation. Disruption of this axis leads to RhoA accumulation and excessive actomyosin contractility, creating a rigid cytoskeletal environment that restrains ciliogenesis. Remarkably, relieving cytoskeletal tension (via Rhosin or Y27632) or stabilizing microtubules (via TubA) functionally rescued the decidualization defects, overcoming the upstream transcriptional dysregulation. Conclusions: These findings establish the Cul3-RhoA axis as a critical mechanical checkpoint linking protein modification to the plasticity required for pregnancy success. By maintaining a low-tension state permissive for ciliogenesis, neddylation ensures the execution of the decidual program. Furthermore, our data suggest potential reproductive risks and highlight the neddylation-ciliary axis as a novel theoretical target for disorders of impaired decidualization.