Journal of Reproduction and Development最新文献

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.

This study aimed to clarify the association between the percentage of follicle number by size over antral follicle count (AFC) and subsequent reproductive performance. A total of 306 Japanese Black cattle underwent timed artificial insemination (TAI) 41-62 days postpartum; the AFC and numbers of small, medium, and large follicles were recorded 10 days before TAI. The cross-sectional and blood flow areas of the dominant follicle (DF) on the day of TAI and the corpus luteum (CL) six days after TAI were recorded. The total number of follicles ≥ 2 mm was defined as the AFC, and the percentages of follicle number by each size defined as small (S-AFC%; 2-2.9 mm), medium (M-AFC%; 3-8.4 mm), and large (L-AFC%; ≥ 8.5 mm) follicles. The AFC and S-, M-, and L-AFC% were further grouped into low, medium, and high tertiles, and the subsequent reproductive performance compared among the groups. Plasma anti-Müllerian hormone (AMH) levels were quantified on the day of AFC measurement. No differences were observed in reproductive performance between the AFC and L-AFC% groups. The high-S-AFC% group showed a 20.6% lower conception rate, 0.58 more AI numbers, and 21.9 longer days open than those of the low-S-AFC% group (P < 0.05). The low-M-AFC% group showed an 18.0% lower conception rate after TAI and 0.54 more AI numbers than those of the high-M-AFC% group (P < 0.05). DF and CL parameters did not differ among the AFC, S-, M-, and L-AFC% groups. Plasma AMH levels in the low-AFC group were the lowest in the tertile. In conclusion, the percentage of follicles by size could be used to estimate subsequent reproductive performance.

The cytoplasmic poly(A)-binding protein (PABPC) plays a central role in the life of poly(A) mRNAs, including their stability, translation, and decay. In addition to the nearly ubiquitous PABPC1, two testis-specific PABPCs, PABPC2 and PABPC6, are present in rodents, while one specific PABPC, PABPC3, is found in primate testes. These three PABPC proteins are each encoded by intronless genes that may have diverged independently due to the retroposition of prototypical Pabpc1 or PABPC1. PABPC2 and PABPC6 are distinguished from PABPC1 in that they barely associate with translationally active polysomal mRNAs and are enriched in male germ cell-specific nuage, termed chromatoid bodies. Despite these unique characteristics, spermatogenesis and male fertility were not compromised in mutant mice lacking either PABPC2 or PABPC6, suggesting functional redundancy between the two proteins. Here, we produced double-mutant mice lacking both PABPC2 and PABPC6 and found that the simultaneous absence of these two proteins failed to affect testicular protein synthesis, spermatogenesis, or male fertility in vivo. These results suggest that the functions of PABPC2 and PABPC6 are redundant with those of other co-existing PABPC proteins, including PABPC1. We propose that testis-specific PABPC proteins emerged because of transcriptional promiscuity in the testis.

During mouse preimplantation development, zygotic genome activation (ZGA), which synthesizes new transcripts in the embryo, occurs during the 1-cell to 2-cell stage. Embryos at the 1- and 2-cell stages are totipotent, and as embryonic development progresses, their differentiation potential decreases, and the embryos become pluripotent. However, the roles of genes expressed during ZGA in mouse embryonic differentiation remain incompletely understood. Here, we show that periodic tryptophan protein 1 (Pwp1), a WD-repeat protein, is expressed from the ZGA and controls embryonic differentiation at later stages. Developmental potential was reduced when siRNAs or antisense oligonucleotides targeting Pwp1 were introduced into 1-cell stage mouse embryos. Further, Pwp1 knockdown resulted in irregular localization of YAP1 at the morula stage, upregulation of the inner cell mass marker Nanog, and downregulation of the trophectoderm marker Cdx2 at the blastocyst stage. Transcriptome analysis showed that Pwp1 knockdown upregulated ZGA gene expression at the morula stage. Because Pwp1 contributes to H4K20me3 histone modification, these results suggest that Pwp1 is required for mouse preimplantation development to control differentiation-associated genes via H4K20me3 modification. Elucidating the role of Pwp1 in embryonic differentiation is expected to contribute toward the advancement of assisted reproductive technologies.