Journal of Reproduction and Development最新文献

Bovine blastocysts cultured under mild hypothermia (MH) can be maintained with non-hatching viable embryos compared to normothermic controls (38.5°C). However, the mechanism by which mildly hypothermic culture delays embryonic growth has not yet been elucidated. This study evaluated the number of cells in embryos cultured under MH conditions and the expression of genes involved in embryonic differentiation. Bovine blastocysts cultured under MH conditions exhibited reduced cell numbers and interferon-tau mRNA expression. Both forkhead box O3 (FOXO3) mRNA expression and FOXO3 protein level in blastocysts cultured under MH conditions were higher than those in normothermic controls (P < 0.05). On the phosphorylated FOXO3 protein level, there was no significant difference between blastocysts cultured under MH and normothermic conditions. In contrast, no significant difference was observed in the ATP content of blastocysts between the MH and normothermic groups. In blastocysts cultured under MH conditions, cold-inducible RNA-binding protein (CIRP) and RNA-binding motif protein 3 (RBM3) mRNA expression increased, and heat shock protein 70 (HSP70) mRNA expression decreased compared to that in normothermic controls (P < 0.05). Considering that HSP70 is involved in preventing apoptosis, these results suggest that MH retards embryonic development via apoptosis induced by HSP70 downregulation during the culture period.

Ethanolamine plasmalogens (EPls) and choline plasmalogens (CPls), unique glycerophospholipids may play important roles in milk production and reproduction in postpartum dairy cows. While CPls are more abundant in bovine blood, EPls are predominant in the brain. Brain EPls are the only recognized ligands of G protein-coupled receptor 61 (GPR61), a receptor that co-localizes with GnRH receptors on gonadotrophs. We hypothesized that chemosynthetic CPls stimulate gonadotropin secretion from bovine gonadotrophs, similar to the reported effects of chemosynthetic EPls. Anterior pituitary cells from healthy, post-pubertal heifers, were cultured for 3.5 days and then treated with increasing concentrations (0, 0.7, 7, 70, or 700 pM) of EPl with vinyl-ether-bonded stearic acid and ester-bonded oleic acid (C18:0-C18:1EPl) as a positive control, or CPls with vinyl-ether-bonded stearic acid and ester-bonded oleic acid (C18:0-C18:1CPl), arachidonic acid (C18:0-C20:4CPl), or docosahexaenoic acid (C18:0-C22:6CPl). After 2 h, the medium samples were harvested for FSH and LH assays. C18:0-C18:1EPl (7-700 pM) stimulated basal FSH and LH secretion (P < 0.01). None of the tested CPl concentrations stimulated LH secretion. Only 700 pM of C18:0-C18:1CPl, but not lower concentrations, stimulated FSH secretion (P < 0.05), an effect that was inhibited by a SMAD pathway inhibitor. However, both C18:0-C18:1CPl and C18:0-C20:4CPl synergized with GnRH to stimulate FSH secretion. In silico molecular-docking simulations using the deep-learning algorithm ColabFold revealed that CPls bind to the three-dimensional structural model of GPR61. In conclusion, C18:0-C20:4CPl stimulated FSH secretion exclusively in the presence of GnRH, whereas C18:0-C18:1CPl weakly stimulated FSH secretion and showed potential interaction with the GnRH signaling pathways.

Cyclic cell proliferation and endometrial remodeling during the estrous cycle are important for maintaining normal endometrial function. However, the regulatory mechanisms underlying this cell proliferation have not yet been elucidated. In this study, the function of matrix metalloproteinase 3 (MMP3) on endometrial cell proliferation was analyzed. Gene expression in bovine endometrial stromal (BES) and epithelial (BEE) cells was analyzed using qPCR. The protein expression of MMP3 and heparin-binding EGF-like growth factor (HB-EGF) was analyzed using casein zymography and western blotting, respectively. Cell proliferation was analyzed using an automated cell counter. The results revealed that MMP3 was highly expressed at the follicular stage compared to that at the luteal and implantation stages. Estrogen (E2) increased the gene expression and release of MMP3 protein in BES in vitro, whereas progesterone (P4) and interferon alpha (IFNα) decreased mRNA and protein expression. E2 also increased the proliferation of BES, but the inhibitors of MMP3 and epidermal growth factor receptor (EGFR) inhibited the proliferation induced by E2. Furthermore, E2 increased the release of HB-EGF from BES, whereas the MMP3 inhibitor suppressed this release. The effect of E2 on BEE cell proliferation was not reported. However, the conditioned medium of BES treated with E2 increased BEE cell proliferation but was inhibited by an EGFR inhibitor. E2 induced MMP3 protein expression and promotes HB-EGF release from BES. These results suggest that MMP3 is involved in endometrial cell proliferation during the follicular stage.

Spermatogenesis is a complex process that is required for sperm production. Multiple RNA-binding proteins participate in regulating spermatogenesis. Y-box-binding protein 1 (YBX1) is involved in transcriptional regulation, mRNA stabilization, and translational repression. However, its specific role in spermatogenesis remains unclear. This study investigated the role of YBX1 in spermatogenesis using a Ybx1 conditional knockout (Ybx1 cKO) mouse model. By analyzing the phenotype of Ybx1 cKO mice, we investigated the role of YBX1 in spermatogenesis and male fertility. The morphology and weight of Ybx1 cKO mouse testes were similar to those of wild-type (WT) testes. Sperm count and motility were lower in Ybx1 cKO mice than in WT mice. Histological analysis showed reduced numbers of elongated spermatids in seminiferous tubules and spermatozoa in tubules of the epididymis in Ybx1 cKO mice. Although YBX1 was highly expressed in the cytoplasm of spermatocytes, meiosis progressed normally in Ybx1 cKO spermatocytes. Finally, the fertilization potential of spermatozoa from Ybx1 cKO epididymis was decreased. In conclusion, our results indicate that YBX1 participates in the regulation of spermatid development but is dispensable for meiosis.

In vitro fertilization (IVF) is crucial for livestock reproduction; however, pregnancy rates after embryo transfer vary depending on the developmental speeds of the embryos. Although quantitative PCR (qPCR) is used to predict developmental potential, its reliability depends on the selection of appropriate reference genes (RGs) for normalization. To determine suitable RGs in bovine blastocysts with different developing speed, we evaluated the stability of eight candidate RGs (18S, ACTB, GAPDH, HMBS, PPIA, TBP, HPRT1, and SDHA) in early-, mid-, and late-developing IVF blastocysts (E-BL, M-BL, and L-BL, respectively) using RefFinder. Despite morphological similarities, E-BL, M-BL, and L-BL exhibited different biological features, including significantly lower pregnancy rates in L-BL than in the other groups, and less abundant transcript levels of five candidate RGs in L-BL than in E-BL. RefFinder revealed that ACTB was the most stable RG, whereas TBP was the least stable. To emphasize the critical importance of selecting stable RGs, we analyzed the expression of key developmental markers including those of the inner cell mass (ICM; OCT4, SOX2) and trophectoderm (TE; CDX2, GATA3, IFNτ), using various RGs for normalization. For ICM markers, normalization with ACTB showed results consistent with pregnancy rates, whereas moderately stable (18S) and less stable (TBP) RGs yielded contradictory outcomes. Normalization with unstable RGs produced inconsistent TE marker expression patterns (CDX2, GATA3) and overestimated (IFNτ) results across groups, compared with the results of ACTB. These results demonstrate that selecting inappropriate RGs for qPCR normalization can lead to misinterpretation, highlighting the necessity of proper RG evaluation to ensure accurate results in bovine embryo research.

The Rec8 gene is specifically expressed in fetal and adult gonads. Although the importance of REC8 in gametogenesis is widely acknowledged, the mechanisms underlying its germ cell-specific expression remain unclear. In this study, we utilized the mouse Rec8 gene sequence to construct a 2577 bp sequence, which included intron 1 (180 bp), exon 1 (118 bp), and an upstream 2279 bp region. The dual-luciferase assay results showed significant differences in promoter activity between -650 bp and -385 bp and between -89 bp and -35 bp. This indicated that the core promoter region of the Rec8 gene may exist within these regions. Bisulfite sequencing PCR results showed that CpGs 10-19 were largely unmethylated in the testes but hypermethylated in other tissues. Interestingly, correlation analysis between CpG methylation status and Rec8 mRNA expression levels showed that methylation of CpGs 10 to 19 was negatively correlated with Rec8 mRNA expression levels (Pearson's r = -0.991, P = 0.009). Furthermore, RNA-Seq data and bioinformatic analyses suggested that the specific expression of Rec8 may be linked to the presence of TATA-like sequences within its core promoter region. Overall, these findings indicate that Rec8 expression is regulated by the low methylation of CpG sites and the presence of TATA-like sequences in its core promoter.

An association has been reported between a lower pH in the uterus and an increased rate of implantation. How low pH regulates endometrial function is unclear. This study investigated the effect of low pH on the expression of leukemia inhibitory factor (LIF), which is crucial for implantation, in a human endometrial carcinoma cell line, rat endometrial stromal cells, and porcine endometrial cells. LIF mRNA expression was quantified by real-time PCR and protein expression was assessed using western blot analysis. LIF mRNA and protein expression increased at low pH in human endometrial carcinoma cells. Increased LIF mRNA expression was also detected at low pH in rat endometrial stromal and porcine endometrial cells, suggesting that low intrauterine pH may create favorable conditions for implantation and endometrial receptivity across species. The increase in LIF mRNA expression in the three cell types was attenuated by the addition of amiloride, indicating that low pH promotes the expression of LIF via amiloride-sensitive molecules in the endometrium.

This study evaluated the viability of in vitro embryo production using ovum pick-up (OPU) and intracytoplasmic sperm injection (ICSI) as breeding techniques for pure and crossbred Hokkaido native ponies (n = 9). Oocytes were collected using transvaginal ultrasound-guided follicle aspiration. ICSI was performed on in vitro matured oocytes using frozen semen. Embryonic cultures were monitored using time-lapse cinematography. Blastocysts were cryopreserved and, after thawing, were transferred non-surgically into recipient mares. Over nine OPU sessions, the mean number of aspirated follicles was 23.9 (range, 13-49). The oocyte recovery and maturation rates were 35.3% (76/215) and 61.5% (40/65), respectively. The cleavage rate was 57.5% (23/40). Of cleaved embryos, 56.5% (13/23) were arrested at the 4-cell to 8-cell stage, and five developed into early-blastocyst. Three embryos were transferred, resulting in a successful pregnancy. In conclusion, OPU-ICSI is a viable assisted reproductive technology for enhancing the population of Japanese native horses.

A thin endometrium can lead to low clinical pregnancy rates, low live birth rates, high spontaneous abortion rates, and low birth weight. However, current methods of treating thin endometria do not achieve ideal results. This study explored the effect of Indian Hedgehog (IHH) on thin endometrium and its mechanism of action. A thin endometrial rat model was established by infusion of 95% ethanol. IHH was overexpressed in model rats using adeno-associated viruses. The endometrial thickness and number of glands and vessels were determined using H&E staining. Endometrial fibrosis was detected using Masson's trichrome staining. Immunohistochemistry was performed to detect α-SMA, MUC-1, and CK19. After modeling, the rats were mated, and the number of gestational sacs was counted for fertility assessment. Western blotting was used to detect the angiogenesis markers vWF, PCNA, and vim and Hedgehog signaling-related proteins SMO, GLI1, and GLI3. IHH overexpression reduced ethanol-induced edema and bruising, repaired the appearance of damaged tissue, increased endometrial thickness, promoted glandular and vascular regeneration, and alleviated endometrial fibrosis. IHH overexpression inhibited the expression of fibroblast marker α-SMA while promoting the expression of vWF, PCNA, vim, CK19, and MUC-1. It also increased the number of gestational sacs and promoted the expression of SMO, GLI1, and GLI3. In conclusion, IHH ameliorates ethanol-induced thin endometrium and improves fertility by activating the Hedgehog signaling pathway.

During the periovulatory period, local production of cortisol surges in the bovine cumulus-oocyte complex (COC), although its physiological significance is not well understood. As a potent anti-inflammatory agent, cortisol may protect the COC from inflammation caused by lipopolysaccharide (LPS), an endotoxin known to cause infertility in postpartum cows. This study examined the effect of cortisol, together with progesterone (P4), on LPS-challenged bovine oocyte maturation. COCs were aspirated from follicles 2-5 mm in diameter and subjected to in vitro maturation for 21 h with various combinations of LPS, cortisol, cortisone (a substrate for cortisol production), trilostane (a P4 synthesis inhibitor), and nomegestrol acetate (NA; a synthetic progestogen). LPS (0.001, 0.01, 0.1, 1 μg/ml) suppressed oocyte maturation in a dose-dependent manner, and this effect was reversed by concomitant treatment with cortisol (0.1 μM). COCs converted cortisone to cortisol, and the locally produced cortisol (approximately 0.01 μM) was capable of negating the suppressive effect of LPS (1 μg/ml) on oocyte maturation. Trilostane suppressed oocyte maturation by eliminating P4 production, indicating the crucial role of P4 in this process. LPS equally suppressed oocyte maturation, regardless of the presence or absence of P4 or the various doses of NA (0.001-1 μM). This suggests that P4 alone does not inhibit the action of LPS. However, in the absence of P4, cortisol could not suppress the LPS effect on oocyte maturation. Collectively, these findings suggest that the bovine COC can protect itself from the suppressive effects of LPS by producing cortisol, with P4 being essential for this function.