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.

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 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.

Oocyte developmental competence declines in women aged 35 and older resulting in many women resorting to IVF. The present study determined whether adding Granulocyte-macrophage colony-stimulating factor (GM-CSF) during in vitro oocyte maturation (IVM) could improve oocyte developmental competence in a mouse model of advanced maternal age. Oocytes from 12–14 month C57BL6 J × CBA mice were treated with 10 ng/ml of GM-CSF during IVM, and embryo development, mitochondrial activity, spindle formation and chromosomal alignment were examined. The addition of GM-CSF tended to increase fertilisation rates (76.19 vs. 82.03%; P = 0.07) but did not affect cumulus expansion compared with control. The addition of GM-CSF also increased blastocysts rates (51.10 vs. 61.52%; P < 0.01) and the number of good quality blastocysts (33.31 vs. 44.13%; P < 0.05) present at 96 h of culture as well as inner cell mass (12.64 vs. 15.62 ; P < 0.01) and total cell number (42.98 vs. 48.78 ; P < 0.05). GM-CSF treatment also increased mitochondrial membrane potential two to three fold in the outer (2.86 vs. 0.97; P < 0.001), intermediate (3.25 vs. 0.89; P < 0.001) and peri nuclear areas (3.62 vs. 1.08; P < 0.001). GM-CSF treatment did not influence spindle formation or chromosomal alignment. Together our results indicate that the addition of GM-CSF during IVM may improve oocyte quality in women of advanced maternal age.

The developmental activation of the corpus luteum (CL) structurally and functionally is critical for the temporally regulated establishment, maintenance, and termination of pregnancy in rats. In this study, we have investigated the possible involvement of autophagy in the regulation of the CL during pregnancy in rats. The expression ratio of microtubule-associated protein light chain 3 (LC3)-II/-I, a widely used indicator of autophagic activity, in the CL remained relatively stable until day 15 of pregnancy. Subsequently, it progressively increased until day 21, and then declined until day 3 postpartum. This fluctuation was closely associated with the tissue weight of the CL rather than progesterone (P4) production activity. Light and electron microscopy revealed the presence of immunoreactive LC3 aggregates and irregularly shaped autolysosome-like microstructures in the cytoplasm of luteal cells during late pregnancy. Notably, a bolus intrabursal injection of the autophagy inhibitor bafilomycin A1 on day 15 of pregnancy resulted in a significant reduction in luteal cell size and disrupted the normal alteration of circulating P4 levels. Consequently, treatment with this inhibitor increased the likelihood of the varied timing (both advanced and delayed) of delivery and led to reduced body weight in neonates when compared with the vehicle-treated control group. Our findings suggest that autophagy in the rat CL contributes to luteal tissue growth, influences P4 production, and thereby fine-tunes the regulation of gestation length in rats.

Mammalian reproduction is more inefficient than expected and embryo/conceptus implantation into the maternal endometrium is considered to be a rate-limiting process. Although extensive physiological and structural diversity exists among mammalian species, the basic molecular mechanisms underlying successful implantation are conserved. The extensive use of genetically engineered mouse models has provided considerable information on uterine receptivity for embryo implantation. The molecular mechanisms and cellular processes identified thus far require further validation in other mammalian species. In this review, representative ovarian steroid hormone-induced signaling pathways controlling uterine adaptation are presented based on the results of rodent studies. Selected examples of functional conservation in mammals, such as humans and cattle, are briefly described. To date, molecular therapeutic trials for fertility improvement have not been conducted. Considerable efforts are required to provide further understanding of these molecular mechanisms. Such understanding will contribute to the development of reliable clinical diagnostics and therapeutics for implantation failure, leading to reproductive success in a wide variety of mammals in the future.

Pregnancy is intricately regulated by the interactions between various bioactive substances secreted by the conceptus, uterus, and corpus luteum (CL). Interferon-τ, synthesized and secreted by the conceptus, plays a central role in the interaction mechanism of maternal recognition in cows. Chemokines, chemotaxis mediators that are primarily secreted by immune cells, regulate various reproductive responses in various species. Although there are scattered reports on the potential roles of chemokines in the bovine CL and the uterus during the estrous cycle, there is little information on chemokines in these organs during pregnancy. Therefore, in this review, we discuss the possible physiological roles of chemokines in the CL and uterus of pregnant cows, focusing on our recent findings on chemokines and changes in their receptor expression in the CL and endometrium of cows at some stages of pregnancy.

The induction of the germ cell lineage from pluripotent stem cells (in vitro gametogenesis) will help understand the mechanisms underlying germ cell differentiation and provide an alternative source of gametes for reproduction. This technology is especially important for cattle, which are among the most important livestock species for milk and meat production. Here, we developed a new method for robust induction of primordial germ cell-like cells (PGCLCs) from newly established bovine embryonic stem (bES) cells. First, we refined the pluripotent culture conditions for pre-implantation embryos and ES cells. Inhibition of RHO increased the number of epiblast cells in the pre-implantation embryos and dramatically improved the efficiency of ES cell establishment. We then determined suitable culture conditions for PGCLC differentiation using bES cells harboring BLIMP1-tdTomato and TFAP2C-mNeonGreen (BTTN) reporter constructs. After a 24-h culture with bone morphogenetic protein 4 (BMP4), followed by three-dimensional culture with BMP4 and a chemical agonist and WNT signaling chemical antagonist, bES cells became positive for the reporters. A set of primordial germ cells (PGC) marker genes, including PRDM1/BLIMP1, TFAP2C, SOX17, and NANOS3, were expressed in BTTN-positive cells. These bovine PGCLCs (bPGCLCs) were isolated as KIT/CD117-positive and CD44-negative cell populations. We anticipate that this method for the efficient establishment of bES cells and induction of PGCLCs will be useful for stem cell-based reproductive technologies in cattle.

Incorporation of bovine serum-derived albumin formulation (AlbuMAX) into a basic culture medium, MEMα, enables the completion of in vitro spermatogenesis through testicular tissue culture in mice. However, this medium was not effective in other animals. Therefore, we sought an alternative approach for in vitro spermatogenesis using a synthetic medium without AlbuMAX and aimed to identify its essential components. In addition to factors known to be important for spermatogenesis, such as retinoic acid and reproductive hormones, we found that antioxidants (vitamin E, vitamin C, and glutathione) and lysophospholipids are vital for in vitro spermatogenesis. Moreover, based on our experience with microfluidic devices (MFD), we developed an alternative approach, the PDMS-ceiling method (PC method), which involves simply covering the tissue with a flat chip made of PDMS, a silicone resin material used in MFD. The PC method, while straightforward, integrates the advantages of MFD, enabling improved and uniform oxygen and nutrient supply via tissue flattening. Furthermore, our studies underscored the significance of lowering the oxygen concentration to 10–15%. Using an integrated cultivation method based on these findings, we successfully achieved in vitro spermatogenesis in rats, which has been a long-standing challenge. Further improvements in culture conditions would pave the way for spermatogenesis completion in diverse animal species.

The adenohypophysis is comprised of the anterior and intermediate lobes (AL and IL, respectively). Cluster of differentiation 9 (CD9)- and sex-determining region Y-box 2 (SOX2)-positive cells are stem/progenitor hormone-producing cells in the AL. They are located in the marginal cell layer (MCL) facing Rathke’s cleft between the AL and IL (primary niche) and the parenchyma of the AL (secondary niche). We previously showed that, in rats, CD9/SOX2-positive cells in the IL side of the MCL (IL-side MCL) migrate to the AL side (AL-side MCL) and differentiate into prolactin-producing cells (PRL cells) in the AL parenchyma during pregnancy, lactation, and diethylstilbestrol treatment, all of which increase PRL cell turnover. This study examined the changes in CD9/SOX2-positive stem/progenitor cell niches and their proportions by manipulating the turnover of growth hormone (GH)- and thyroid-stimulating hormone (TSH)-producing cells (GH and TSH cells, respectively), which are Pit1 lineage cells, as well as PRL cells. After induction, the isolated CD9/SOX2-positive cells from the IL-side MCL formed spheres and differentiated into GH and TSH cells. We also observed an increased GH cell proportion upon treatment with GH-releasing hormone and recovery from continuous stress and an increased TSH cell proportion upon propylthiouracil treatment, concomitant with alterations in the proportion of CD9/SOX2-positive cells in the primary and secondary niches. These findings suggest that CD9/SOX2-positive cells have the potential to supply GH and TSH when an increase in GH and TSH cell populations is required in the adult pituitary gland.

Hypothalamic kisspeptin neurons are master regulators of mammalian reproduction via direct stimulation of gonadotropin-releasing hormone and consequent gonadotropin release. Here, we generated novel Kiss1 (kisspeptin gene)-Cre rats and investigated the developmental changes and sex differences in visualized Kiss1 neurons of Kiss1-Cre-activated tdTomato reporter rats. First, we validated Kiss1-Cre rats by generating Kiss1-expressing cell-specific Kiss1 knockout (Kiss1-KpKO) rats, which were obtained by crossing the current Kiss1-Cre rats with Kiss1-floxed rats. The resulting male Kiss1-KpKO rats lacked Kiss1 expression in the brain and exhibited hypogonadotropic hypogonadism, similar to the hypogonadal phenotype of global Kiss1 KO rats. Histological analysis of Kiss1 neurons in Kiss1-Cre-activated tdTomato reporter rats revealed that tdTomato signals in the anteroventral periventricular nucleus (AVPV) and arcuate nucleus (ARC) were not affected by estrogen, and that tdTomato signals in the ARC, AVPV, and medial amygdala (MeA) were sexually dimorphic. Notably, neonatal AVPV tdTomato signals were detected only in males, but a larger number of tdTomato-expressing cells were detected in the AVPV and ARC, and a smaller number of cells in the MeA was detected in females than in males at postpuberty. These findings suggest that Kiss1-visualized rats can be used to examine the effect of estrogen feedback mechanisms on Kiss1 expression in the AVPV and ARC. Moreover, the Kiss1-Cre and Kiss1-visualized rats could be valuable tools for further detailed analyses of sexual differentiation in the brain and the physiological role of kisspeptin neurons across the brain in rats.

In this study, we evaluated the effects of pre-maturational culture (pre-IVM) on the developmental competence of bovine oocytes derived from an 8-day in vitro growth (IVG) culture system. IVG oocytes were subjected to 5 h pre-IVM before in vitro maturation, followed by in vitro fertilization (IVF). The proportion of oocytes that progressed to the germinal vesicle breakdown stage was similar between groups with and without pre-IVM. Although metaphase II oocytes and cleavage rates after IVF were similar regardless of pre-IVM culture, the blastocyst rate was significantly higher in the group with pre-IVM (22.5%) than without pre-IVM (11.0%, P < 0.05). In conclusion, pre-IVM culture improved the developmental competence of bovine oocytes derived from an 8-day IVG system.

In mouse fetal gonads, germ cell development is accompanied by changes in cell cycle mode in response to external signals and intrinsic mechanisms of cells. During fetal development, male germ cells undergo G0/G1 arrest, while female germ cells exit the mitotic cell cycle and enter meiosis. In fetal testes, NANOS2 and CYP26B1 force germ cells to stay in G0/G1 arrest phase, preventing them from entering the meiotic cell cycle. In the fetal ovary, external signals, such as RA, BMP, and WNT, promote the competency of female germ cells to enter the meiotic cell cycle. MEIOSIN and STRA8 ensure the establishment of the meiotic cell cycle by activating meiotic genes, such that meiotic entry coincides with the S phase. This review discusses germ cell development from the viewpoint of cell cycle regulation and highlights the mechanism of the entry of germ cells into meiosis.

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.

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.

NANOS3 is an evolutionarily conserved gene expressed in primordial germ cells that is important for germ cell development. Germ cell deletion by NANOS3 knockout has been reported in several mammalian species, but its function in pigs is unclear. In the present study, we investigated the germline effects of NANOS3 knockout in pigs using CRISPR/Cas9. Embryo transfer of CRISPR/Cas9-modified embryos produced ten offspring, of which one showed wild-type NANOS3 alleles, eight had two mutant NANOS3 alleles, and the other exhibited mosaicism (four mutant alleles). Histological analysis revealed no germ cells in the testes or ovaries of any of the nine mutant pigs. These results demonstrated that NANOS3 is crucial for porcine germ cell production.

Conventional culture systems for bovine embryos are unable to support sustained embryonic development until the developmentally mature blastocyst stage. Although we have previously developed an on-gel culture system that enables bovine blastocysts to complete cell segregation events at day (D) 10 following in vitro culture, the development of D10 blastocysts to term has yet to be achieved. In this study, we attained full-term development of D10 mature blastocysts produced using an on-gel culture system. Two calves derived from on-gel-cultured embryos were vaginally born, showing normal birth and placental weights and no obvious morphological abnormalities. Moreover, we detected no abnormalities in blood metabolic profile analyses. Our findings indicate that on-gel culturing can be used to facilitate the development of developmentally mature blastocysts to term, and produce healthy viable calves. This culture system could make a valuable contribution to cattle production and would enable a range of analyses for characterizing bovine-specific pre-implantation development.

In ruminants, uterine glands play key roles in the establishment of pregnancy by secreting various factors into the uterine lumen. Although a three-dimensional (3D) culture system has been used for investigating cellular functions in vitro, the detailed functions of uterine gland have not been fully elucidated. In this study, we examined the benefits of 3D culture system to examine the innate functions of bovine uterine glands. Isolated bovine uterine glands were cultured on Matrigel (2D) or in Matrigel (3D), respectively, and the mRNA levels of secreted proteins (SERPINA14, MEP1B, APOA1, ARSA, CTGF, and SPP1) were measured in isolated and cultured uterine glands. The protein expression of estrogen receptor β (ERβ) and progesterone receptor (PR) and the establishment of apico-basal polarity were examined. In isolated uterine glands, the mRNA levels of secreted proteins changed during the estrous cycle. Although uterine glands cultured in both 2D and 3D expressed ERβ and PR, progesterone did not affect SERPINA14 mRNA expression. The expression of APOA1 mRNA in 2D cultured uterine glands did not respond to estrogen and progesterone. Additionally, the mRNA levels of secreted proteins in the 3D culture system were significantly higher than those in the 2D culture system, which might be attributed to the different cellular morphology between them. The locations of ZO-1 and β-catenin in 2D cultured uterine glands were disordered compared with 3D cultured uterine glands. These results showed that the hormonal responsiveness of secreted factor expression and cellular morphology were different between 2D and 3D cultured bovine uterine glands.

Reproductive function is suppressed during lactation owing to the suckling-induced suppression of the kisspeptin gene (Kiss1) expression in the arcuate nucleus (ARC) and subsequent suppression of luteinizing hormone (LH) release. Our previous study revealed that somatostatin (SST) neurons mediate suckling-induced suppression of LH release via SST receptor 2 (SSTR2) in ovariectomized lactating rats during early lactation. This study examined whether central SST-SSTR2 signaling mediates the inhibition of ARC Kiss1 expression and LH release in lactating rats during late lactation and whether the inhibition of glutamatergic neurons, stimulators of LH release, is involved in the suppression of LH release mediated by central SST-SSTR2 signaling in lactating rats. A central injection of the SSTR2 antagonist CYN154806 (CYN) significantly increased ARC Kiss1 expression in lactating rats on day 16 of lactation. Dual in situ hybridization revealed that few ARC Kiss1-positive cells co-expressed Sstr2, and some of the ARC Slc17a6 (a glutamatergic neuronal marker)-positive cells co-expressed Sstr2. Furthermore, almost all ARC Kiss1-positive cells co-expressed Grin1, a subunit of N-methyl-D-aspartate (NMDA) receptors. The numbers of Slc17a6/Sstr2 double-labeled and Slc17a6 single-labeled cells were significantly lower in lactating dams than in non-lactating rats whose pups had been removed after parturition. A central injection of an NMDA antagonist reversed the CYN-induced increase in LH release in lactating rats. Overall, these results suggest that central SST-SSTR2 signaling, at least partly, mediates the suppression of ARC Kiss1 expression and LH release by inhibiting ARC glutamatergic interneurons in lactating rats.

Epithelial-mesenchymal transition (EMT), which is common in cancer metastasis, is also observed during developmental processes such as embryo implantation into the maternal endometrium in humans and rodents. However, this process has not been well characterized in the non-invasive type of implantation that occurs in ruminants. To understand whether EMT occurs in ruminant ungulates, ovine conceptuses (embryo plus extraembryonic membranes) from days 15 (P15: pre-attachment), 17 (P17: during attachment), and 21 (P21: post-attachment, day 0 = day of estrus) were evaluated. RNA-seq analysis revealed that the expression of EMT-related transcripts increased on P21. Real-time PCR and western blotting analyses indicated that levels of transcripts and proteins indicative of mesenchyme-related molecules increased on P21, but a minor expression of epithelium-related molecules remained. Immunohistochemical analysis revealed that E-cadherin (CDH1) was localized in the elongated trophectoderm on P15 and P17. On P21, CDH1 was localized to the trophectoderm and on the conceptus cells undergoing differentiation. Vimentin (VIM) was localized in the uterine stroma on P15 and P17, and its expression was observed at the edge of elongating trophoblast on P21. Further, it was found that some bi-nucleated trophoblast cells were present on P17; however, numerous bi- and multi-nucleated trophoblast cells on the uterine epithelium or next to the uterine stroma were found on P21. A minor expression of pregnancy-associated glycoprotein (PAG) transcripts was found on P15 and P17, but a definitive expression of PAGs, transcripts, and proteins was found on P21. Although further investigation is required, these observations indicate that bi-nucleated trophoblast cell formation begins on the day conceptus implantation to the maternal endometrium is initiated, followed by EMT in trophoblast cells. These results suggest that these sequential events are required if pregnancy is to be established in ruminants.