Journal of Reproduction and Development最新文献

Immature zebrafish oocytes are sensitive to chilling, and their survival is markedly reduced by exposure to 0°C. In the present study, we investigated the involvement of cold-sensitive channels and lipid mediators in chilling injury in immature zebrafish oocytes. The oocytes were injected with inhibitors of a cold-sensitive channel (TRPA1), cytosolic phospholipase A₂α (cPLA₂α), cyclooxygenases (COXs), arachidonate 5-lipoxygenase (ALOX5), and lysophosphatidylcholine acyltransferase 2 (LPCAT2). The cells were then chilled at 0-12°C for 5-30 min, incubated at 25°C for 2 h, and stained with propidium iodide. Oocytes were damaged when exposed to temperatures below 12°C. When oocytes were chilled at 0°C for 15 min, the survival rate was very low (9%). However, when the oocytes were injected with a TRPA1-specific inhibitor, their survival markedly improved (70%). This strongly suggests that activation of the cold-sensitive TRPA1 channel triggers chilling injury in oocytes. When a cPLA₂α-specific inhibitor was injected, the survival of chilled oocytes markedly improved (60%). This strongly suggests that lipid mediators are involved in chilling injury in oocytes. When oocytes were injected with specific inhibitors of COXs, ALOX5, and LPCAT2, the survival of chilled oocytes significantly improved by 47%, 28%, and 43%, respectively. These results strongly suggest that eicosanoids and platelet-activating factor are involved in the chilling injury in oocytes. The results of this study may facilitate advancements in successful cryopreservation of fish oocytes.

L-tryptophan (Trp), an essential amino acid, is a precursor of 5-hydroxytryptamine (5-HT; also known as serotonin) that promotes mammalian sperm hyperactivation. Since mammalian sperm contain Trp hydroxylase (TPH), they may contribute to 5‑HT biosynthesis. Therefore, this study aimed to examine the effect of Trp on hamster sperm hyperactivation and determine whether sperm are involved in 5-HT biosynthesis. Trp significantly enhanced sperm hyperactivation via the 5-HT₄ receptor and its associated signals. In contrast, D-tryptophan did not affect sperm hyperactivation. Furthermore, hamster sperm contained the 5-HT biosynthesis enzymes TPH and aromatic L-amino acid decarboxylase (AADC). Additionally, hamster sperm secreted 5-HT. Trp-enhanced hyperactivation and 5-HT secretion were significantly inhibited by TPH and AADC inhibitors. Overall, our findings suggest that Trp enhanced sperm hyperactivation through the biosynthesis of 5-HT within the sperm.

Assisted reproductive technologies (ART) to generate developmentally competent oocytes necessitates light exposure due to the use of microscopes. Previous studies in several species have reported that the wavelength of light during the light exposure period is a critical factor in embryo development. However, the effects of different light wavelengths on embryo development in pigs remain unexplored. This study aimed to identify the optimal light conditions to enhance oocyte maturation, parthenogenetic activation of mature oocytes, and pre-implantation development of parthenogenetic embryos in pigs. Conducted irradiation experiments during in vitro maturation (IVM), parthenogenesis (PG), and in vitro culture (IVC) using visible (390-750 nm), blue (445-500 nm), green (500-575 nm), yellow (575-585 nm), and red (620-750 nm) light. Variation in light wavelengths during IVM, PG, or IVC did not significantly influence oocyte maturation of cumulus-oocyte complexes (COCs) derived from median antral follicles (MAFs), developmental competence of in vitro-culture COCs after PG, and the production of blastocysts. However, continuous irradiation with green light throughout the entire process (IVM, PG, and IVC) significantly enhanced in vitro blastocyst production, and the resulting blastocysts showed significantly elevated HSP70 expression and a numerical increase in PCNA expression. We recommend conducting all in vitro procedures (IVM, PG, and IVC) for producing blastocysts from MAFs in porcine models under green light. This study will contribute to yielding higher success rates of porcine ART and reduce artificial stress to oocytes and embryos caused by in vitro manipulation under strong light exposure.

Precise and early pregnancy detection is crucial for better breeding management and enhancing the overall production of ruminant livestock. Throughout the years, numerous methods have evolved for pregnancy detection in ruminants, each possessing specific advantages and limitations. This review thoroughly discusses both traditional and emergent diagnostic methods, emphasizing their principles, implementation, merits and challenges. Behavioral observation, rectal palpation and ultrasonography are the traditional approaches widely used because of their accessibility and direct detection of pregnancy conditions. Progesterone measurement, pregnancy-associated glycoprotein detection, and estrone sulfate examination are the hormonal assays that provide biochemical proof at specific phases of gestation. Recently, the analysis of interferon-stimulated gene expression and circulating microRNAs has shown promising roles in early pregnancy detection at the genetic and transcriptomic levels. The investigation of volatile organic compounds is a novel approach in pregnancy diagnosis, though it is non-invasive, and further confirmation is required for regular application. This review highlighted the importance of incorporating multiple examination strategies to enhance the accuracy and reliability of pregnancy detection in ruminants. Future research should center on the refinement and field application of advanced technologies to ensure their proper implementation in diverse ruminant production systems.

Golden hamsters (Mesocricetus auratus) have been extensively used in biomedical research. With the advent of genome-editing technology, it is now possible to generate gene-knockout hamsters, providing unique research models that cannot be achieved with mice or rats. Therefore, the development of cryopreservation techniques for hamster embryos is in high demand. In this study, we present a simplified vitrification protocol for hamster embryo preservation. In vivo-derived 8-cell or morula embryos (Day 3) were vitrified using Cryotop in modified HECM-3 medium containing ethylene glycol, DMSO, and sucrose. After warming, the embryos were transferred into the uteri of Day 3-pregnant females with a different coat color. The results showed that 21-26% of the transferred embryos developed to the term. The experiments were conducted in a conventional laboratory setting, avoiding direct light exposure. Given the reproducibility of our vitrification protocol, it has broad applicability in laboratories that use hamsters.

Luteal blood flow (LBF) is essential for progesterone (P₄) biosynthesis in the corpus luteum (CL) and affects bovine fertility. However, the mechanism by which LBF affects fertility remains unclear. This study was conducted to investigate the effects of LBF on endometrial P₄ concentrations and gene expression. Endometrial biopsies and blood samples were collected from 13 Japanese Black cows after ultrasound examination on Day 7 (Day 0 = day of estrus). Based on LBF, the cows were divided into low- (LV; n = 5), medium- (MV; n = 2), and high-vascularity (HV; n = 6) groups. Plasma and endometrial P₄ concentrations were measured using enzyme immunoassays. RNA sequencing was performed to compare the endometrial gene expression profiles from three cows in each of the LV and HV groups. Reverse transcription-quantitative PCR was performed for genes selected from the differentially expressed genes (DEGs), P₄ receptors (PGR, PGRMC1, and PGRMC2), and P₄-regulated genes (ANPEP, DGAT2, DKK1, and LTF). No differences were observed in plasma or endometrial P₄ concentrations between the HV and LV groups. CCN3 was identified as a DEG between the HV and LV groups and was upregulated in the HV group. Compared to those of the LV group, the HV group exhibited higher CCN3 and PGR mRNA expression levels and lower ANPEP, DGAT2, and DKK1 mRNA expression levels. In conclusion, LBF affects endometrial gene expression without changing plasma or endometrial P₄ concentrations on Day 7.

Climate change is causing heat stress (HS) in dairy cattle. This study aimed to compare the clinical efficacy of two GnRH synthetic analogs, dephereline and busereline, as ovulation inducers under HS conditions. The study population comprised 1,000 lactating dairy cows showing signs of spontaneous estrus which were assigned to the groups: DEPH (489 cows receiving 100 µg of dephereline) and BUS (511 cows receiving 10 µg of busereline) at the time of insemination. Cows were included only once in the study. Treatment with busereline increased the risk of multiple ovulations and twin pregnancies, with an odds ratio (OR) of 1.6, and twin pregnancies, with an OR of 2.8, when compared with dephereline. The likelihood of pregnancy in multiple-ovulating cows was significantly higher in the DEPH group than the BUS group. Collectively, our results comparing two ovulation inducers showed that dephereline treatment may improve the fertility of lactating dairy cows under HS conditions.

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.