Journal of Reproduction and Development最新文献

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.

Male infertility contributes substantially to overall infertility, with temperature changes adversely affecting the sperm quality. During infertility treatments, the exposure of home-collected semen to extreme temperatures during transport deteriorates the semen parameters. This study investigates the effectiveness of thermal containers and insulation materials for preserving semen quality at low temperatures. Semen samples from 35 healthy male partners undergoing fertility treatments were analyzed. The samples were segregated in three groups and assessed: standard collection containers (Group A), thermal containers (Group B), and thermal containers with warming materials (Group C). Samples exposed to 4°C exhibited a notable decline in motility and forward motility over time, whereas groups B and C maintained these parameters better. Group C maintained the internal temperature at approximately 20°C for up to 90 min, reducing cold-induced deterioration. These findings demonstrate that cost-effective thermal retention methods can preserve semen quality during transport, and potentially improve the outcomes of intrauterine insemination and in vitro fertilization.

The enzymatic isolation of preantral follicles (PAFs) is considered the most efficient method for retrieving a large number of intact follicles, offering significant advantages in terms of yield and processing time. However, the low success rate of enzymatically isolated follicles in long-term culture raises concerns regarding their impact on oocyte quality and developmental potential. This study addresses a critical gap in understanding how enzymatic retrieval of PAFs affects the oocyte-granulosa cell connection and its relationship with high mortality and culture failure observed during in vitro growth (IVG). By systematically comparing crude collagenases (IA and IV) and purified collagenases (Liberase TM and DH) with a mechanical isolation protocol, we identified the optimal enzyme concentrations that maximize follicle yield while minimizing cellular damage. Our results reveal that the enzymatic retrieval of PAFs corresponds to the loss of transzonal projections (TZPs) post-isolation, as well as premature oocyte extrusion and follicle deformities during IVG. Our findings also highlight the differential apoptotic responses in oocytes and granulosa cells. Although these enzymes sustain follicle cell integrity, they compromise oocyte viability during isolation. Notably, crude collagenases impair oocyte growth during prolonged culture, whereas purified collagenases preserve the developmental potential of oocytes. This study also provides the first evidence that enzymatic isolation of PAFs adversely affects TZPs. Overall, our study highlights the importance of selecting an appropriate method, enzyme type, and concentration for preserving the integrity of oocytes, follicles, and their connections, thereby supporting successful in vitro culture. Additionally, our results suggest that mechanical protocols and high-purity enzymes are preferred for maintaining oocyte competence.

Climate change has caused heat stress (HS) to become an increasingly severe problem for high-producing dairy herds. Although cooling systems allow milk production to remain nearly constant throughout the year, fertility decreases during summer. Physiological counter-current heat transfer mechanisms maintaining brain/hypothalamic and reproductive functions in cattle are vulnerable to HS. In this study, I propose strategies to improve cooling systems, particularly in zones with the highest risk of increased body temperature, such as milking areas. In addition, heat transfer mechanisms to protect the brain-hypothalamus axis from hyperthermia must be considered when implementing measures to reduce HS-related problems.

In Japanese Black (JB) cattle, the number of transferable embryos produced after superovulation is crucial for the economic success of embryo production for both farmers and veterinarians. Anti-Müllerian hormone (AMH) has emerged as a key reproductive marker for predicting the number of embryos produced in vivo and oocytes retrieved through transvaginal pickup. This study investigated the relationship between AMH, inflammatory markers, including serum amyloid A (SAA) and albumin/globulin (A/G) ratio, and the number of embryos recovered and transferable after superovulation in JB cows. A total of 96 JB donor cows underwent artificial insemination after superovulation, and embryo recovery was performed 7 days later. Embryos recovered were classified based on the International Embryo Technology Society criteria, wherein "transferable embryos" included those with codes 1 or 2, while "total embryos" included transferable embryos as well as those with codes 3 and 4. Blood samples collected during embryo recovery were used to measure serum AMH, SAA, and A/G ratios. When grouped by AMH quartiles, the high-AMH and middle-high-AMH groups produced significantly more total embryos compared to the low-AMH group. The total number of embryos increased with higher AMH levels (r = 0.3336, P = 0.0009). Correlation analysis revealed associations between AMH, α₁-globulin and SAA. Additionally, a significant positive correlation was observed between total and transferable embryos (r = 0.6339, P < 0.0001) and between AMH and the yield ratio (r = 0.25583, P = 0.0119). These findings confirm that AMH concentration is a valuable reproductive marker for predicting the total and transferable embryos produced by JB donor cows.

The neurotransmitter, 5-hydroxytriptamine (5-HT), is well known. Furthermore, it enhances the acrosome reaction, hyperactivation, and in vitro fertilization (IVF) success in hamsters and mice. In the present study, we examined whether 5-HT enhances hyperactivation and increases IVF success in rats. When rat sperm was exposed to 5-HT, hyperactivation was significantly enhanced. Only the 5-HT₄ receptor agonists significantly enhanced hyperactivation. Additionally, both 5-HT and 5-HT₄ receptor agonists significantly increase the success of IVF. These results suggested that 5-HT increases IVF success by enhancing hyperactivation and effects of 5-HT are associated with the 5-HT₄ receptor. Therefore, in rats, 5-HT enhances capacitation and the 5-HT₄ receptor is the key molecule for capacitation.

Hypothalamic arcuate (ARC) kisspeptin neurons are considered the gonadotropin-releasing hormone pulse generator in rats. In virgin rats, the expression of the ARC kisspeptin gene (Kiss1) is repressed by proestrous levels of estradiol-17β (high E2) but not by diestrous levels of E2 (low E2). In lactating rats, ARC Kiss1 expression is repressed by low E2 during late lactation. This study aimed to investigate whether nuclear receptor corepressor 2 (NCOR2, encoded by Ncor2), an estrogen receptor α corepressor, is involved in the estrogen-induced repression of ARC Kiss1 expression in rats. Double in situ hybridization for Kiss1 and Ncor2 revealed that approximately 80% of ARC Kiss1-expressing cells co-expressed Ncor2 in ovariectomized (OVX) + low E2 virgin rats, while approximately 90% of ARC Kiss1-expressing cells co-expressed Ncor2 in OVX + low E2 lactating rats. To further examine the role of Ncor2, we studied the effects of Kiss1-dependent Ncor2 knockdown on ARC Kiss1 expression and luteinizing hormone (LH) pulses. An adeno-associated virus vector carrying Cre-activated short hairpin RNA (shRNA) for Ncor2 was administered to the ARC in two Kiss1-Cre rat models: OVX + high E2 Kiss1-Cre virgin rats and OVX + low E2 Kiss1-Cre lactating rats. Ncor2-shRNA treatment significantly increased the number of ARC Kiss1-expressing cells and the intensity of Kiss1 signals in OVX + high E2 virgin rats but failed to fully restore low E2-induced Kiss1 repression in lactating rats. The Ncor2-shRNA treatment failed to affect LH pulses in both models. These findings suggest that NCOR2 in ARC kisspeptin neurons mediates high E2-induced repression of ARC Kiss1 expression in virgin rats.

In vitro production of porcine-hatched blastocysts is important for various applications. However, the mechanobiology of blastocoel expansion and hatching remains poorly understood. Our study aimed to efficiently produce hatched blastocysts and investigate the hatching mechanics of late-stage porcine embryos using time-lapse assessment. In this study, fetal bovine serum (FBS), amino acids (AA), and insulin-transferrin-selenium (ITS) were added individually or in combination to culture day 5 parthenogenetic morulae and early blastocysts to investigate their developmental capacity. We found that FBS-AA-ITS supplementation significantly promoted blastocoel expansion (4.4-fold), accelerated early hatching (at day 6 vs. day 7.5) and improved hatching rates (39.59 ± 4.34% vs. 0 ± 0% on day 7, and 80.02 ± 2.93% vs. 2.78 ± 1.60% on day 8) compared with non-supplemented controls. It also increased the number of trophectoderm (2.5-fold) and inner cell mass (2.7-fold). Furthermore, FBS-AA-ITS elevated BCL2 expression and reduced BAX expression, potentially inhibiting apoptosis. In in vitro fertilized embryos, the combination also significantly improved the hatching rate (8.3-fold) and cell number (2.7-fold). Analysis of the effects of FBS on hatching dynamics revealed that FBS enhanced hatching by increasing the number of blastomeres and blastocoel diameter. Blastocysts supplemented with FBS had significantly smaller hatching sizes (320.09 ± 5.01 µm vs. 343.95 ± 5.67 µm) and faster rate of hatching progress (5.6-fold) compared with non-FBS groups. In conclusion, FBS-AA-ITS played a pivotal role in supporting the development of late-stage porcine embryos and significantly accelerated their progression through the embryonic developmental phase.

The efficiency of porcine in vitro fertilized (IVF) embryo production remains low. Polyspermy is considered a contributing factor to this result. In this study, we investigated the effects of mechanical vibrations during the co-culture of oocytes and spermatozoa on fertilization parameters and subsequent embryonic development. The rate of polyspermy decreased significantly in all vibration culture groups compared with the stationary culture (control) group (P < 0.05). Regarding subsequent embryonic development, the blastocyst formation rate was significantly improved in the middle-vibration culture group compared with the control group (P < 0.05). However, the high-vibration culture group had the lowest sperm penetration rate and did not show any improvement in monospermy rate and normal fertilization efficiency. In addition, their in vitro developmental status was the lowest. These results indicate that moderate mechanical vibrations during insemination effectively suppress polyspermy and improve porcine IVF embryo production efficiency.