In mammalian embryos, the first visible differentiation event is the segregation of the inner cell mass (ICM) and trophectoderm (TE) during the transition from the morula to the blastocyst stage. The ICM, which is attached to the inside of the TE, develop into the fetus and extraembryonic tissues, while the TE, which is a single layer surrounding the fluid-filled cavity called the blastocoel, will provide extraembryonic structures such as the placenta. ICM/TE differentiation is regulated by the interaction between various transcriptional factors. However, little information is available on the segregation of the ICM and TE lineages in preimplantation embryos of domestic animals, such as cattle and pigs. This review focuses on the roles of cell differentiation factors that regulate the ICM/TE segregation of preimplantation bovine and porcine embryos. Understanding the mechanism of cell differentiation in early embryos is necessary to improve the in vitro production systems for bovine and porcine embryos.

This review discusses the production and sale of fertile oocytes for in vitro fertilization technology, calf production through transplantation and delivery, and the current circulation of calves produced by in vitro production (IVP) embryos.

Genetically modified animals, especially rodents, are widely used in biomedical research. However, non-rodent models are required for efficient translational medicine and preclinical studies. Owing to the similarity in the physiological traits of pigs and humans, genetically modified pigs may be a valuable resource for biomedical research. Somatic cell nuclear transfer (SCNT) using genetically modified somatic cells has been the primary method for the generation of genetically modified pigs. However, site-specific gene modification in porcine cells is inefficient and requires laborious and time-consuming processes. Recent improvements in gene-editing systems, such as zinc finger nucleases, transcription activator-like effector nucleases, and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (CRISPR/Cas) system, represent major advances. The efficient introduction of site-specific modifications into cells via gene editors dramatically reduces the effort and time required to generate genetically modified pigs. Furthermore, gene editors enable direct gene modification during embryogenesis, bypassing the SCNT procedure. The application of gene editors has progressively expanded, and a range of strategies is now available for porcine gene engineering. This review provides an overview of approaches for the generation of genetically modified pigs using gene editors, and highlights the current trends, as well as the limitations, of gene editing in pigs.

In female reproduction, the oocyte number is limited after birth. To achieve a continuous ovulatory cycle, oocytes are stored in primordial follicles. Therefore, the regulation of primordial follicle dormancy and activation is important for reproductive sustainability, and its collapse leads to premature ovarian insufficiency. In this review, we summarize primordial follicle development and the molecular mechanisms underlying primordial follicle maintenance and activation in mice. We also overview the mechanisms discovered through in vitro culture of functional oocytes, including the establishment of primordial follicle induction by environmental factors, which revealed the importance of hypoxia and compression by the extra cellular matrix (ECM) for primordial follicle maintenance in vivo.

Mouse trophoblast stem cells (TSCs) can differentiate into trophoblast cells, which constitute the placenta. Under conventional culture conditions, in a medium supplemented with 20% fetal bovine serum (FBS), fibroblast growth factor 4 (FGF4), and heparin and in the presence of mouse embryonic fibroblast cells (MEFs) as feeder cells, TSCs maintain their undifferentiated, proliferative status. MEFs can be replaced by a 70% MEF-conditioned medium (MEF-CM) or by TGF-ß/activin A. To find out if KnockOut^TM Serum Replacement (KSR) can replace FBS for TSC maintenance, we cultured mouse TSCs in KSR-based, FBS-free medium and investigated their proliferation capacity, stemness, and differentiation potential. The results indicated that fibronectin, vitronectin, or laminin coating was necessary for adhesion of TSCs under KSR-based conditions but not for their survival or proliferation. While the presence of FGF4, heparin, and activin A was not sufficient to support the proliferation of TSCs, the addition of a pan-retinoic acid receptor inverse agonist and a ROCK-inhibitor yielded a proliferation rate comparable to that obtained under the conventional FBS-based conditions. TSCs cultured under the KSR-based conditions had a gene expression and DNA methylation profile characteristic of TSCs and exhibited a differentiation potential. Moreover, under KSR-based conditions, we could obtain a suspension culture of TSCs using extracellular matrix (ECM) coating-free dishes. Thus, we have established here, KSR-based culture conditions for the maintenance of TSCs, which should be useful for future studies.

The underlying functional and molecular changes in canine primary uterine inertia (PUI) are still not clarified. Leptin (Lep) and obesity negatively affect uterine contractility in women, partly mediated by the RhoA/Rho associated kinase pathway, affecting myometrial calcium sensitization. We hypothesized that increased uterine Lep/Lep receptor (LepR) or decreased RhoA/Rho associated kinase expression contributes to PUI in dogs, independent of obesity. Dogs presented for dystocia were grouped into PUI (n = 11) or obstructive dystocia (OD, still showing strong labor contractions; n = 7). Interplacental full-thickness uterine biopsies were collected during Cesarean section for relative gene expression (RGE) of RhoA, its effector kinases (ROCK1, ROCK2), Lep and LepR by qPCR. Protein and/or mRNA expression and localization was evaluated by immunohistochemistry and in situ hybridization. RGE was compared between groups by one-way ANOVA using body weight as covariate with statistical significance at P < 0.05. Uterine ROCK1 and ROCK2 gene expression was significantly higher in PUI than OD, while RhoA and Lep did not differ. LepR RGE was below the detection limit in five PUI and all OD dogs. Litter size had no influence. Lep, LepR, RhoA, ROCK1, ROCK2 protein and/or mRNA were localized in the myometrium and endometrium. Uterine protein expression appeared similar between groups. LepR mRNA signals appeared stronger in PUI than OD. In conclusion, lasting, strong labor contractions in OD likely resulted in downregulation of uterine ROCK1 and ROCK2, contrasting the higher expression in PUI dogs with insufficient contractions. The Lep-LepR system may affect uterine contractility in non-obese PUI dogs in a paracrine-autocrine manner.

Functional relationship between nuclear receptor subfamily 4 group A member 3 (Nr4a3) and annexin A5 (Anxa5), which are two gonadotropin-releasing hormone (GnRH)-inducible genes, has been established while evaluating pituitary gonadotropes in relation to follicle-stimulating hormone beta (Fshb) expression. However, the physiological variations that arise due to the differential expression of these genes in the pituitary gland during rat estrous cycle remain unknown. This study aimed to evaluate the Nr4a3 and Anxa5 mRNA expression during the estrous cycle in rats in comparison with the expression of the gonadotropin subunit genes, luteinizing hormone beta (Lhb) and Fshb. Nr4a3 mRNA expression showed a single peak at 1400 h of proestrus during the 4-d estrous cycle. Anxa5 mRNA level was elevated along with increased Fshb mRNA expression after the decline of Nr4a3 mRNA until 2300 h. Lhb mRNA expression levels were not significantly changed during the estrous cycle. Notably, addition of a GnRH antagonist at 1100 h completely eradicated luteinizing hormone secretion at 1400 h and 1700 h of proestrus, and significantly reduced the Nr4a3 mRNA expression level at both the time points. These results suggest that GnRH is, at least partly, responsible for the increase in pituitary Nr4a3, and that the interaction between NR4A3 and ANXA5 is required to regulate Fshb expression during the preovulatory gonadotropin surge.

Upon mammalian fertilization, zygotic genome activation (ZGA) and activation of transposable elements (TEs) occur in early embryos to establish totipotency and support embryogenesis. However, the molecular mechanisms controlling the expression of these genes in mammals remain poorly understood. The 2-cell-like population of mouse embryonic stem cells (mESCs) mimics cleavage-stage embryos with transient Dux activation. In this study, we demonstrated that deficiency of the transcription factor OTX2 stimulates the expression of ZGA genes in mESCs. Further analysis revealed that OTX2 is incorporated at the Dux locus with corepressors for transcriptional inhibition. We also found that OTX2 associates with TEs and silences the subtypes of TEs. Therefore, OTX2 protein plays an important role in ZGA and TE expression in mESCs to orchestrate the transcriptional network.

Conditional knockout technology is a powerful tool for investigating the spatiotemporal functions of target genes. However, generation of conditional knockout mice involves complicated breeding programs and considerable time. A recent study has shown that artificially designed microRNAs (amiRNAs), inserted into an intron of the constitutively expressed gene, induce knockdown of the targeted gene in mice, thus creating a simpler method to analyze the functions of target genes in oocytes. Here, to establish an oocyte-specific knockdown system, amiRNA sequences against enhanced green fluorescent protein (EGFP) were knocked into the intronic sites of the Zp3 gene. Knock-in mice were then bred with EGFP transgenic mice. Our results showed that Zp3-derived amiRNA successfully reduced EGFP fluorescence in the oocytes in a size-dependent manner. Importantly, knockdown of EGFP did not occur in somatic cells. Thus, we present our knockdown system as a tool for screening gene functions in mouse oocytes.

Saving space for sperm cryopreservation would aid mouse genetics research. We previously developed the ST (sperm freezing in ShorT STraw to reduce STorage space) method for cryopreserving mouse sperm in a smaller storage space than conventional methods. However, our ST method has two drawbacks: difficulties during freeze-thaw procedures and the potential risk of sperm loss during storage. Here, we refine ST, terming the new method improved ST (iST). In iST, the straw has an air-permeable filter and the straw container (2-ml cryotube) is endowed with air vents. As in our ST method, iST frozen-thawed sperm showed good performance upon in vitro fertilization. Moreover, up to nine straws can be stored in one cryotube, occupying less storage space than conventional methods. This method provides an easy and space-saving cryopreservation method for mouse sperm, and thus will be valuable for mouse genetics researchers.