Journal of Reproduction and Development最新文献

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.

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.

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 the future, human beings will surely expand into space. But given its unique risks, will humanity thrive in space environments? For example, when humans begin living and reproducing in space habitats or on other planets in the solar system, are there risks that future generations may suffer from adverse mutations induced by space radiation, or that embryos and fetuses will develop abnormally in gravitational environments that differ from that of Earth? Moreover, human expansion to other stellar systems requires that for each breed of animal, thousands of individuals must be transported to destination planets to prevent populations from experiencing inbreeding-related degeneration. In even more distant future, when humans have spread throughout the galaxy, all genetic resources on Earth, the planet where humans originated, must be permanently and safely stored- but is this even possible? Such issues with future space colonization may not be an urgent research priority, but research and technological development accompanying advancements in spaceflight will excite many people and contribute to technological improvements that can improve living standards in the present day (e.g., more effective treatments for infertility, etc.). This review will therefore focus primarily on issues related to mammalian reproduction in space environments.

To produce an embryo, a high conception rate must be complied along with four evaluation criteria based on the timing of early cleavage and proper embryo morphology (hereafter, these blastocysts will be referred to as "four-criteria-compliant blastocysts"). Therefore, it is necessary to construct a culture system for high efficiency production of embryos meeting these four criteria. Non-essential amino acids (NEAAs) are widely used for the culture of bovine embryos fertilized in vitro; however, the necessity and optimal concentration of individual NEAA must be verified to produce four-criteria-compliant blastocysts. DNA methylation is a critical event for blastocyst formation in bovines, and serine is a common NEAA that serves as a methyl donor and participates in DNA methylation. Serine is generally added at a concentration of 100 µM in bovine embryo culture medium. However, the rate of formation of four-criteria-compliant blastocysts was significantly improved when 1,000 µM of serine was added. Analysis of endogenous serine synthases gene expression in oocytes and embryos revealed that phosphoglycerate dehydrogenase, the rate-limiting enzyme in the serine synthesis pathway, is expressed at the morula stage and beyond. The addition of serine at 1,000 µM increased the amount of methyl donors; moreover, the addition of an inhibitor of serine-metabolizing enzymes decreased the number of methyl donors and markedly inhibited blastocyst formation. These results indicate that the addition of serine at an optimal concentration of 1,000 µM favors production of four-criteria-compliant blastocysts, and that methyl donor synthesis may be involved in this effect.

The objective of this study was to examine the relationship between the insulin-like peptide 3 (INSL3) receptor (RXFP2) expression levels on spermatozoa and INSL3 concentrations in the seminal plasma of fresh semen from beef bulls with different levels of sperm morphological normality. Ejaculates (n = 44) were collected from 21 yearling Japanese Black beef bulls and categorized into three groups based on the levels of sperm morphological normality: High (normal morphology ≥ 80%; n = 23), Mid (< 80% & ≥ 65%; n = 10) and Low (< 65%; n = 11). Immunofluorescence was used to determine the localization and expression levels of RXFP2 in spermatozoa. Sperm RXFP2 was detected in the principal and equatorial segments of the acrosomal region, postacrosomal region, and neck in all groups. The levels of RXFP2 in the acrosomal principal segment, postacrosomal region, and neck in the Low group were significantly lower than those in the High and Mid groups, and those in the equatorial segment tended to be lower than those in the High group. The total level of RXFP2 in the Low group was also significantly reduced compared with that in the other two groups. Seminal plasma INSL3 concentrations were significantly higher in the Low group than in the other two groups, whereas testosterone levels did not differ significantly between the groups. In conclusion, RXFP2 levels were reduced in the sperm head and neck in bovine semen, with a lower level of sperm morphological normality, suggesting possible associations between increased sperm deformity and INSL3 receptor reduction. Higher seminal INSL3 concentrations in abnormal semen are probably related to fewer INSL3 receptors in spermatozoa.

Due to the strong demand for embryo production from young and genotyped superior animals using ovum-pick up (OPU) combined with in vitro fertilization (IVF), the number of in vitro-produced embryos has exceeded that of in vivo-derived embryos globally since 2016. One of the merits of OPU-IVF is that the administration of follicle-stimulating hormone (FSH) is not essential, while FSH treatment prior to OPU promotes oocyte developmental competence. Thus, investigations are needed to optimize OPU-IVF protocols with and without FSH. In addition, OPU enables oocyte collection from antral follicles in living animals. However, there are numerous immature oocytes in follicles at earlier stages, which are potentially destined to degenerate in ovaries. The technology used to foster acquisition of maturational and developmental competences in these immature oocytes is called in vitro growth (IVG). IVG is expected to contribute to assisted reproductive technologies for livestock, humans, and endangered species. However, no offspring from preantral follicles has been reported using IVG in animals other than in mice. Furthermore, IVG can be used to investigate factors affecting the fertility and developmental competence of oocytes by reconstituting follicle growth at each stage in vitro, which cannot be evaluated in vivo. Here, the technological progress of the optimization of immature bovine oocyte utilization is reviewed alongside findings from a variety of other ruminants.

Calcium release from the endoplasmic reticulum via sperm-derived phospholipase C zeta is crucial for oocyte activation during fertilization. Chloroquine (CQ) inhibits the increase in cytoplasmic calcium. This study investigated the effects of CQ on fertilization and oocyte activation. Oocytes were collected from ICR mice, and in vitro fertilization and artificial oocyte activation with strontium ions (Sr²⁺) and ethanol were performed in the presence of 50 µM CQ. Pronuclear formation was assessed via Hoechst33242 nuclear staining, cortical granule release was evaluated using lens culinaris agglutinin-fluorescein isothiocyanate (LCA-FITC) staining, and cytosolic calcium levels were measured using fluorescence microscopy with Cal-520 AM. In the presence of CQ, no pronuclei were formed even 8 h after Sr²⁺-induced oocyte activation. Furthermore, cortical granule release in CQ-treated oocytes was significantly suppressed, although not completely inhibited, and no increase in cytosolic calcium was detected. CQ also inhibited pronuclei formation during ethanol-induced oocyte activation. In in vitro fertilization, although the fertilization rate was decreased in the CQ-treated group, in which CQ treatment was continuously applied during insemination, pronuclear formation and cortical granule release were observed. The decrease in the fertilization rate was likely attributable to reduced sperm motility and decreased penetration of the zona pellucida. The findings indicate that the oocyte activation pathways triggered by ethanol/Sr²⁺ and sperm are distinguishable by CQ, and that CQ can be used as a selective inhibitor of oocyte activation induced by Sr²⁺ or ethanol treatment.

Herein, we report a case of pregnancy of a female bottlenose dolphin (Tursiops truncatus) that was subjected to artificial insemination (AI) in water based on its estrous behavior using simple instruments. AI was performed on this female dolphin once or twice daily for 4 days at the detection of estrous behavior, such as floating horizontally and showing reduced responsiveness, likely indicating the appropriate timing for AI. The female was placed in supine a position in the water to position the genital slit above the water surface. A Nélaton catheter (Fr. 10, 40 cm length), with its tip modified, was inserted approximately 20 cm into the vagina through the genital slit, and 1-2 ml of fresh semen was injected. The AI procedure was performed within 1 min by two technicians. Thus, this AI method may be a new choice for artificial reproduction, as pregnancy success can be achieved with relatively less cost, less difficulty, and less invasive treatments of cetaceans.

CRISPR/Cas9-based multiplex genome editing via electroporation is relatively efficient; however, lipofection is versatile because of its ease of use and low cost. Here, we aimed to determine the efficiency of lipofection in CRISPR/Cas9-based multiplex genome editing using growth hormone receptor (GHR) and glycoprotein alpha-galactosyltransferase 1 (GGTA1)-targeting guide RNAs (gRNAs) in pig zygotes. Zona pellucida-free zygotes were collected 10 h after in vitro fertilization and incubated with Cas9, gRNAs, and Lipofectamine 2000 (LP2000) for 5 h. In Experiment 1, we evaluated the mutation efficiency of gRNAs targeting either GHR or GGTA1 in zygotes transfected using LP2000 and cultured in 4-well plates. In Experiment 2, we examined the effects of the culture method on the development, mutation rate, and mutation efficiency of zygotes with simultaneously double-edited GHR and GGTA1, cultured using 4-well (group culture) and 25-well plates (individual culture). In Experiment 3, we assessed the effect of additional GHR-targeted lipofection before and after simultaneous double gRNA-targeted lipofection on the mutation efficiency of edited embryos cultured in 25-well plates. No significant differences in mutation rates were observed between the zygotes edited with either gRNA. Moreover, the formation rate of blastocysts derived from GHR and GGTA1 double-edited zygotes was significantly increased in the 25-well plate culture compared to that in the 4-well plate culture. However, mutations were only observed in GGTA1 when zygotes were transfected with both gRNAs, irrespective of the culture method used. GHR mutations were detected only in blastocysts derived from zygotes subjected to GHR-targeted lipofection before simultaneous double gRNA-targeted lipofection. Overall, our results suggest that additional lipofection before simultaneous double gRNA-targeted lipofection induces additional mutations in the zygotes.