Molecular Reproduction and Development最新文献

The circadian clock is not functional during preimplantation development because lack of transcription prevents the feedback loop required for circadian cyclicity. Moreover, transcript abundance for the core clock genes (CLOCK, ARNTL, PER1, PER2, CRY1, and CRY2) declines in the embryo after embryonic genome activation. Nonetheless, transcripts for each of the clock genes are present in the embryo. The potential for translation of these transcripts in the bovine embryo was evaluated by assessing whether circadian clock genes are associated with polysomes and whether these transcripts are preferentially retained as development proceeds to the blastocyst stage. Transcript abundance declined at the eight-cell stage for BMAL1 and CRY1 and at the morula stage for PER1, PER2, and CRY2. Before embryonic genome activation at the eight-cell stage, a large fraction of transcripts for each of the genes was associated with polysomes. At specific later stages of development, there was less transcript associated with polysomes than with other fractions. This was true for PER1 at the morula and blastocyst stage, PER2 at the morula stage, and CRY1 at the eight-cell stage. The percent of transcripts associated with polysomes was also calculated. This value was lower after the two-cell stage for CLOCK and PER1. Based on the decrease in transcript abundance and proportional association with polysomes after the two-cell stage, it was concluded that capacity for translation of circadian clock genes declines in the preimplantation embryo as development proceeds. Thus, de novo synthesized proteins involved in the circadian clock mechanism are unlikely to play an important function in the preimplantation embryo following embryonic genome activation.

Circular RNAs are associated with important pathophysiological characteristics of gestational diabetes mellitus (GDM). This study preliminarily explored circular polyribonucleotide nucleotidyltransferase 1 (circ-PNPT1) expression in placental tissues and serum of GDM patients and its role in regulating the miR-144-3p/UBE2G1 axis to affect endothelial dysfunction in GDM. Twenty GDM patients and 20 pregnant women with normal glucose tolerance were enrolled. Human umbilical vein endothelial cells (HUVECs) were cultured with high glucose (HG) to establish an in vitro GDM model, then treated with short hairpin-circ-PNPT1 and a lentiviral empty plasmid. Circ-PNPT1 levels, HUVEC viability, endothelial function, and oxidative damage were assessed using RT-qPCR tube formation, CCK-8, flow cytometry, and Transwell assays, respectively. Normally cultured and HG-cultured HUVEC samples underwent small RNA sequencing to analyze differentially expressed miRNAs. The possible miRNAs and mRNAs downstream of circ-PNPT1 were screened using bioinformatics and verified using RT-qPCR, dual luciferase reporter assay, and Ago2-RIP. GDM patients exhibited highly expressed circ-PNPT1 and UBE2G1, and weakly expressed miR-144-3p in placental tissues and serum. In vitro, HG-treated HUVECs displayed highly expressed circ-PNPT1 and cellular dysfunction, as evidenced by reduced cell survival, enhanced apoptosis, decreased cell migration and angiogenesis, an elevated MDA level, and downregulated SOD and GSH-Px levels. Circ-PNPT1 knockdown alleviated HG-induced HUVEC dysfunction. circ-PNPT1 might target and bind miR-144-3p; miR-144-3p might target and bind UBE2G1. Additionally, circ-PNPT1 might act as a competing endogenous RNA for miR-144-3p to regulate UBE2G1 expression in HG-induced HUVECs. Taken together, circ-PNPT1 modulates HG-induced HUVEC dysfunction via the miR-144-3p/UBE2G1 axis.

Thirteen-lined ground squirrels (Ictidomys tridecemlineatus Mitchill 1821; 13-LGS) are useful diurnal rodent models of human cone-mediated vision due to their cone photoreceptor-dominant retinas. To develop the 13-LGS as a better model of inherited human visual disorders, we report a gene-editing protocol targeting the 13-LGS tyrosinase (Tyr) gene. CRISPR/Cas9 microinjection into donor embryos, followed by transfer to pseudo-pregnant recipients, yielded two Tyr-mutated founders. Mating these two to wild-type 13-LGS resulted in 22 offspring, of which five were genotyped with either a 17-bp deletion, 1-bp insertion, or 7-bp deletion Tyr mutation. These results demonstrated that this valuable mammalian model is amenable to germline gene editing by conventional methods.

Endometrial fibrosis in mares compromises fertility through aberrant extracellular matrix deposition and sustained myofibroblast activation. Conventional interventions fail to reverse these pathological alterations, necessitating innovative, mechanism-focused therapies. In this study, we pioneered the use of prostaglandin E2 (PGE2) preconditioning of equine endometrial-derived mesenchymal stem cells (ET-eMSCs) and their extracellular vesicles (EVs) to target fibrotic processes directly. ET-eMSCs were isolated from mare endometrial biopsies pretreated with PGE2 to enhance their anti-fibrotic secretome, and EVs were subsequently harvested. In vitro assays demonstrated that PGE2-preconditioned ET-eMSCs and their EVs inhibited α-smooth muscle actin expression, reduced collagen I deposition, and restored key endometrial markers of receptivity and proliferation. These findings establish the first evidence that PGE2-enhanced ET-eMSC-derived EVs can exert anti-fibrotic effects in an in vitro model of equine endometrial fibrosis. This study provides a robust translational framework for developing targeted regenerative therapies for fibrotic diseases across species, with potential applications in human reproductive medicine.

Phospholipase D2 (PLD2) modulates cytoskeletal dynamics and membrane trafficking processes by converting phosphatidylcholine (PC) into phosphatidic acid (PA) and choline within somatic cells. Nonetheless, the role in oocyte meiosis remains largely unknown. Here, we demonstrate that PLD2 is selectively targeted to the meiotic spindle in mouse oocytes. The knockdown of PLD2 via the specific morpholino oligonucleotides (MOs) or its inhibition with VU 0364739 led to a marked increase in α-tubulin acetylation and induced a meiotic arrest at metaphase I (MI), accompanied by misaligned chromosomes. These defects were effectively rescued by the ectopic expression of Pld2 complementary RNA (cRNA). Furthermore, our findings implicate the Pld2 MO-induced alterations in the AKT-GSK3-Tau signaling cascade in oocytes. The overexpression of a gain-of-function GSK3β mutant (GSK3β^S9A) and a Tau-phosphorylation-enhancing mutant (Tau^P301L) substantially reversed the increased microtubule acetylation and the reduced rate of the first polar body extrusion (PBE) in oocytes lacking PLD2 activity. Additionally, the co-immunoprecipitation revealed a direct physical interaction between PLD2, GSK3β, and Tau in mouse oocytes. Together, PLD2 finely regulates α-tubulin acetylation through the modulation of the AKT-GSK3β-Tau signaling axis, thereby preserving an optimal microtubule dynamic equilibrium and ensuring the fidelity of the spindle apparatus function during oocyte meiosis.

There is a small number of studies describing the uterine biology of Bos indicus cows. We hypothesized that there is a transcriptional signature in endometrial epithelial cells 4 days after estrus (D4) that predicts the ability of the cow to remain pregnant/artificial insemination (AI). Brahman cows were submitted to an estrous synchronization protocol and AI. On D4 cows were submitted to endometrial cytology. Pregnancy/AI was diagnosed on day 30 and endometrial cytology samples were submitted to RNA-seq (n = 32 nonpregnant and n = 32 pregnant). Based on RNA-seq we performed targeted analysis using pathways previously reported in the literature and untargeted analysis using Ingenuity Pathway Analysis. A total of 975 genes were significantly associated (p ≤ 0.1) with pregnancy/AI, 64.9% of them (620/975) showed a negative association. Targeted and untargeted analysis showed a downregulation of Th2 immune response and activation of cholesterol biosynthesis in pregnant cows. Prediction analysis resulted in greater accuracy for the targeted transcripts than the whole transcriptome (0.91 vs. 0.86) but reduced precision (0.64 vs. 0.74). In conclusion, the endometrial receptivity was predominantly marked by an overall reduction in the molecular response. Th2 response and cholesterol biosynthesis are promising pathways to understand uterine biology, and the use of a set of genes rather than a single gene appears to be the future for prediction of pregnancy in bovine.