Journal of Reproduction and Development最新文献

Conservation of chicken germplasm is crucial in supporting commercial breeds for sustainable egg and meat production and preserving the genetic diversity of indigenous breeds for future breeding. Cryopreservation of chicken fertilized eggs or embryos is not feasible, owing to the large yolk-laden structure of the eggs. Primordial germ cells (PGCs), the embryonic precursors of gametes, are the best candidates for the cryobanking of chicken germplasm. Effective cryobanking of chicken PGCs requires an optimal cryopreservation protocol. Cryomedia containing dimethyl sulfoxide (DMSO) or DMSO combined with serum have been widely used for the cryopreservation of chicken PGCs. However, as cryoprotectants are yet to be optimized for chicken PGCs, the efficacy of cryomedia can be further improved. Here, we investigated the cryoprotective effects of propylene glycol (PG), an alternative to DMSO, on chicken PGCs. We found that the addition of non-permeable cryoprotectants, such as trehalose or chicken serum, to DMSO or PG cryomedia improved the recovery and survival rates of post-thawed PGCs. We further investigated the cryoprotective effects of trehalose and chicken serum and found that these additives have different cryoprotective actions. Based on these findings, we designed two different cryomedia: DTS, including 5% DMSO, 0.3 M trehalose, and 1% chicken serum, and PTS, including 7.5% PG, 0.1 M trehalose, and 5% chicken serum. Among the different PGC lines and freshly isolated PGCs, the cryomedia showed similar post-thaw recovery rates. Following transplantation, post-thawed male PGCs can colonize gonads and differentiate into functional sperm. We successfully revived the offspring of Kurokashiwa, a rare chicken breed in Japan, with cryopreserved PGCs. In conclusion, we developed two different cryomedia that achieved > 50% recovery of viable PGCs after thawing while maintaining germline competency.

Maternal RNA and proteins accumulate in mouse oocytes and regulate initial developmental stages. Sperm DNA combines with protamine, which is exchanged after fertilization with maternal histones, including H3.3; however, the effect of H3.3 on development post-fertilization remains unclear. Herein, we established an electroporation method to introduce H3.3 siRNA into germinal vesicle (GV)-stage oocytes without removing cumulus cells. Oocyte-attached cumulus cells need to be removed during the traditional microinjection method; however, we confirmed that artificially removing cumulus cells from oocytes reduced fertilization rates, and oocytes originally free of cumulus cells had reduced developmental competence. On introducing H3.3 siRNA at the GV stage, H3.3 was maintained in the maternal pronucleus and second polar body but not in the paternal pronucleus, resulting in embryonic lethality after fertilization. These findings indicate that H3.3 protein was not incorporated into the paternal pronucleus, as it was repeatedly translated and degraded over a relatively short period. Conversely, H3.3 protein incorporated into the maternal genome in the GV stage escaped degradation and remained in the maternal pronucleus after fertilization. This new method of electroporation into GV-stage oocytes without cumulus cell removal is not skill-intensive and is essential for the accurate analysis of maternal effect genes.

We investigated the effect of the temperature-humidity index (THI) on the conception rate (CR) in Holstein heifers and cows receiving in vitro-produced (IVP) Japanese Black cattle fresh embryos. IVP embryos were transferred to Holstein heifers (n = 1,407) and cows (n = 3,189) on 245 commercial farms. The monthly average ambient temperature (AT) and THI ranged from 4.7 to 29°C and 41 to 81, respectively; both were the highest in August. The monthly CR ranged from 16.3% to 46.7% in cows and 23.8% to 74.1% in heifers. The CR of heifers was unaffected by THI, AT, or the month of embryo transfer. However, these parameters affected the CR of cows. The CR at THI values of 61-65 and 71-75 was greater than that at THI > 75, whereas other THI values had no effect. The CR at temperatures > 25°C was lower (P = 0.008) than that at temperatures of 15-20°C and 20-25°C. Moreover, the CR was lowest (P = 0.003) in July. THI and parity (P = 0.057 and P = 0.001, respectively) and AT and parity (P = 0.019 and P = 0.001, respectively) showed significant effects on CR; however, there was no interaction between these two factors. In conclusion, AT > 25°C and THI > 75 adversely affect the CR outcome in cows but not in heifers.

RAD2lL and REC8, meiosis-specific paralogs of the canonical cohesin subunit RAD21, are essential for proper formation of axial/lateral elements of the synaptonemal complex, synapsis of homologous chromosomes, and crossover recombination in mammalian meiosis. However, how many meiotic cohesins are present in germ cells has not been investigated because of the lack of an appropriate method of analysis. In the present study, to examine the intracellular amount of meiotic cohesins, we generated two strains of knock-in (KI) mice that expressed a 3×FLAG-tag at the C-terminus of RAD21L or REC8 protein using the CRISPR/Cas9 genome editing system. Both KI mice were fertile. Western blot analyses and immunocytochemical studies revealed that expression levels and localization patterns of both RAD21L-3×FLAG and REC8-3×FLAG in KI mice were similar to those in wild-type mice. After confirming that tagging of endogenous RAD21L and REC8 with 3×FLAG did not affect their expression profiles, we evaluated the levels of RAD21L-3×FLAG and REC8-3×FLAG in the testes of 2-week-old mice in which only RAD21L and REC8 but little RAD21 are expressed in the meiocytes. By comparing the band intensities of testicular RAD21L-3×FLAG and REC8-3×FLAG with 3×FLAG-tagged recombinant proteins of known concentrations in western blot analysis, we found that there were approximately 413,000 RAD21L and 453,000 REC8 molecules per spermatocyte in the early stages of prophase I. These findings provide new insights into the role played by cohesins in the process of meiotic chromosome organization in mammalian germ cells.

Uterine peristalsis is essential for gamete transport and embryo implantation. It shares the characteristics of spontaneity, rhythmicity, and directivity with gastrointestinal peristalsis. Telocytes, the "interstitial Cajal-like cells" outside the digestive canal, are also located in the uterus and may act as pacemakers. To investigate the possible origin and regulatory mechanism of periodic uterine peristalsis in the human menstrual cycle, telocytes in the myometrium were studied to determine the effect of estradiol on T-type calcium channel regulation. In this study, biopsies of the human myometrium were obtained for cell culture, and double-labeling immunofluorescence screening was used to identify telocytes and T-type calcium channel expression. Intracellular calcium signal measurements and patch-clamp recordings were used to investigate the role of T-type calcium channels in regulating calcium currents with or without estradiol. Our study demonstrates that telocytes exist in the human uterus and express T-type calcium channels. The intracellular Ca²⁺ fluorescence intensity marked by Fluo-4AM was dramatically decreased by NNC 55-0396, a highly selective T-type calcium channel blocker, but enhanced by estradiol. T-type calcium current amplitude increased in telocytes incubated with estradiol in a dose-dependent manner compared to the control group. In conclusion, our study demonstrated that telocytes exist in the human myometrium, expressing T-type calcium channels and estradiol-enhanced T-type calcium currents, which may be a reasonable explanation for the origin of uterine peristalsis. The role of telocytes in the human uterus as pacemakers and message transfer stations in uterine peristalsis may be worth further investigation.

This in vivo study aimed to investigate local and systemic immune responses induced by sperm in cows after artificial insemination (AI). Initially, 12 multiparous Japanese Black cows were subjected to intrauterine AI (AI group, n = 6) or saline infusion (control group, n = 6). The uterine body and horn ipsilateral to the ovulatory follicle were mini-flushed with 2 ml of RPMI-1640 medium at different time points (0, 1, 6, 10, 24, 48 h, and 7 days after AI), centrifuged, and the sediments were examined under a light microscope. Vaginal smears were prepared at 0, 1, 6, and 10 h after AI to investigate the sperm backflow. Subsequently, another experiment was conducted by assigning cows to three groups: intrauterine AI (AI group, n = 5), heat-inactivated AI (Heat-AI group, n = 5), or saline infusion (control group, n = 5). Blood samples were collected, and polymorphonuclear neutrophils (PMNs) and peripheral blood mononuclear cells (PBMCs) were separated and analyzed for gene expression using real-time PCR. The results showed that most sperm were rapidly transported either forward into the uterine horn or backward into the vagina within 1 h after AI. The PMNs migrated into the uterine lumen 6 hours after AI. Only active sperm-induced proinflammatory responses in PMNs and PBMCs via upregulation of TNFa, IL8, IL1B, and PGES and downregulation of IL10 at 6 h after AI. These data provide evidence that sperm generate transient proinflammatory responses locally in the uterus and systemically in the peripheral immune cells, which may be prerequisites for uterine clearance, embryo receptivity, and implantation in cows.

Over the years, the utilization of in vitro fertilization (IVF) in bovine embryo production has increased globally to accelerate the selection of cows with high genetic values. The selection of embryos with high implantation potential is a critical factor in establishing pregnancy. Time-lapse monitoring (TLM) has emerged as a new technique that allows frequent and non-invasive imaging of developing embryos. TLM is considered to have several advantages over the conventional morphological evaluation of embryos, which has been widely used in bovine embryo production. Establishing a novel embryo selection algorithm specifically for bovine IVF embryos is a critical challenge, but information on the association between morphokinetic data obtained using TLM and the implantation potential of embryos is still limited. This review outlines the potential application of TLM technology to improve the fertility of bovine IVF embryos, focusing on the results of human and bovine TLM studies that can be applied to select bovine embryos with high implantation potential. First, the progress of the TLM technology in bovine embryo production is summarized. The association between kinetic and morphological parameters and the developmental and implantation potential of human and bovine embryos is outlined. Finally, the benefits of evaluating blastocyst collapse and re-expansion as indicators of bovine embryo viability and the possible application of TLM to detect chromosomal abnormalities and determine embryo sex will be discussed.

Dihydromyricetin (DHM), a dihydroflavonoid compound, exhibits a variety of biological activities, including antitumor activity. However, the effects of DHM on mammalian reproductive processes, especially during early embryonic development, remain unclear. In this study, we added DHM to porcine zygotic medium to explore the influence and underlying mechanisms of DHM on the developmental competence of parthenogenetically activated porcine embryos. Supplementation with 5 μM DHM during in vitro culture (IVC) significantly improved blastocyst formation rate and increased the total number of cells in porcine embryos. Further, DHM supplementation also improved glutathione levels and mitochondrial membrane potential; reduced natural reactive oxygen species levels in blastomeres and apoptosis rate; upregulated Nanog, Oct4, SOD1, SOD2, Sirt1, and Bcl2 expression; and downregulated Beclin1, ATG12, and Bax expression. Collectively, DHM supplementation regulated oxidative stress during IVC and could act as a potential antioxidant during in vitro porcine oocytes maturation.

In mice and humans, Nik-related protein kinase (Nrk) is an X-linked gene that encodes a serine/threonine kinase belonging to GCK group 4. Nrk knockout (Nrk KO) mice exhibit delayed delivery, possibly due to defective communication between the Nrk KO conceptus and its mother. However, the mechanism of delayed labor remains largely unknown. Here, we found that in pregnant mothers with the Nrk KO conceptus, the serum progesterone (P4) and placental lactogen (PL-2) concentrations in late pregnancy were higher than those in the wild type. Moreover, we demonstrated that Nrk is expressed in trophoblast giant cells (TGCs) and syncytiotrophoblast-2 (SynT-2) in the labyrinth layer of the mouse placenta. In the human placenta, NRK is also expressed in Syn-T in villi. Both human Syn-T and mouse TGCs of the labyrinth layer are present within fetal tissues that are in direct contact with the maternal blood. The labyrinth layer of the Nrk KO conceptus was gigantic, with enlarged cytoplasm and Golgi bodies in the TGCs. To investigate the function of Nrk in the labyrinth layer, a differentially expressed gene (DEG) analysis was performed. The DEG analysis revealed that labor-promoting factors, such as prostaglandins, were decreased, and pregnancy-maintaining factors, such as the prolactin family and P4 receptor, were increased. These findings suggest that the Nrk KO mice exhibit delayed delivery owing to high P4 concentrations caused by the hypersecretion of pregnancy-maintaining factors, such as PL-2, from the placenta.

The oviductal epithelium consists of ciliated and non-ciliated cells, and their numbers vary depending on the segment of the oviduct and stage of the estrous cycle. Compared with the ampulla, fewer cyclic changes in the number of the two types of cells occur in the isthmus. Recently, we have reported that the epithelium in the ampullary oviduct is composed of many types of cells during different translational/transcriptional states, and their numbers change during the estrous cycle. However, detailed information regarding the epithelial cell subtypes lining the isthmic oviductal epithelium has not yet been reported. In this study, we aimed to identify the epithelial subtypes in the isthmus of the oviduct using immunohistochemistry. Some similarities and differences were observed between the ampulla and isthmus. As observed in the ampulla, epithelial cells of the isthmus expressed either FOXJ1 (ciliogenesis marker) or PAX8 (non-ciliated cell marker). The estrous cycle affected the number of Ki67⁺ cells but not that of ciliated cells. A relatively high rate of Ki67⁺ cells (60%) was observed at 1-4 days after the ovulation. Interestingly, unlike the ampulla, Ki67⁺/FOXJ1⁺ cells (12.6 ± 1.1%) were discovered in the isthmus. Double staining for Ki67 with FOXJ1, PAX8, or Centrin-1 (a centriole marker) revealed that Centrin-1 was localized on the apical surface of some Ki67⁺/FOXJ1⁺ cells. In conclusion, some epithelial cell subtypes exist in the isthmus of the oviduct and isthmus-specific cell subtypes have been identified. These region-specific cells may provide functional and morphological differences between the ampulla and isthmus of the oviduct.