Selenium (Se) and vitamin E (Vit-E), as integral parts of antioxidant systems, play important roles for sperm and embryos in vitro. In this study, the effects of Se and Vit-E on the maturation, in vitro fertilization and culture to blastocysts of porcine oocytes and accumulation of ammonia in the culture medium during different development stages were investigated. The maturation was performed in modified tissue culture medium (mTCM)-199 supplemented with 10% (v/v) porcine follicular fluid, the fertilization medium was modified Tyrode’s albumin lactate pyruvate (mTALP), and the embryo culture medium was modified North Carolina State University (mNCSU)-23. Se in the form of sodium selenite (SS) and seleon-L-methionine (SeMet) and Vit-E at different concentrations were also used. The incorporation and oxidation of ¹⁴C(U)-glucose were assessed with a liquid scintillation counter. In this study, SeMet and SeMet+Vit-E increased oocyte maturation, fertilization and incorporation and oxidation of ¹⁴C(U)-glucose significantly (P<0.05) compared with the control and other treatments. In addition, embryo development, specifically in terms of the numbers of morulae and blastocysts, significantly increased (P<0.05) with SeMet and SeMet+Vit-E. In contrast, the accumulation of ammonia was reduced with SeMet and SeMet+Vit-E compared with other treatments. These findings indicate that SeMet and SeMet+Vit-E may play important roles in reducing the accumulation of ammonia and subsequently in increasing the rate of maturation of porcine oocytes and fertilization, as well as development of the blastocyst and utilization of glucose in in vitro maturation, fertilization and culture to blastocysts of porcine oocytes.

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Androgens and androgen receptor play a critical role in spermatogenesis and fertility in mammals, and estrogens and their receptors contribute to regulation of testicular function through initiation and maintenance of spermatogenesis and germ cell division and survival. However, results from different species are still far from establishing a clear understanding of these receptors in the different cell types from the testis. We analyzed the expression of androgen receptor, estrogen receptors α and β and aromatase protein by immunohistochemistry and real-time PCR, in relation to proliferation followed by the expression of proliferation cell nuclear antigen (PCNA) and germinal identity by VASA protein, in fetal, perinatal, prepubertal and adult testes of Lagostomus maximus, a rodent with sustained germ cell proliferation and an increasing number of OCT-4-expressing gonocytes in the developing ovary. AR expression was restricted to Leydig cells and peritubular cells before sexual maturity, at which point it also became expressed in Sertoli cells. ERα and ERβ were expressed in seminiferous tubules and the interstitium, respectively, in both fetal and prepubertal testes. In adult testes, both ERα and ERβ co-localized in Leydig and peritubular cells. The aromatase enzyme, which converts androgenic precursors into estrogens, was detectable in all developmental stages analyzed and was restricted to Leydig cells. PCNA remained high until sexual maturity. ERα nuclear detection in germ cells and AR in Leydig cells in PCNA-positive cells suggest the possibility of a stimulatory effect of estrogens on spermatogonia proliferation. This effect might explain the increase found in VASA-expressing cells in the adult testis.

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Mitochondrial bioenergetics in mammalian oocytes has not been sufficiently characterized. In this study, the function of oxidative phosphorylation (OXPHOS), a major pathway in mitochondria, was investigated in individual bovine oocytes by monitoring oxygen consumption using modified scanning electrochemical microscopy (SECM). At the germinal vesicle (GV) stage, 65% of basal respiration was used for mitochondrial respiration, which was inhibited by complex IV inhibitor. Around 63% of mitochondrial respiration was coupled to ATP synthesis, as determined by sensitivity to an ATP synthase inhibitor, and the remaining 37% was attributed to proton leak. In contrast, 50% and 43% of mitochondrial respiration were used for ATP synthesis in in vivo- and in vitro-derived metaphase II (MII)-stage oocytes, respectively. ATP-linked respiration, in both in vivo- and in vitro-derived MII-stage oocytes, was significantly lower than in GV-stage oocytes, suggesting that OXPHOS in bovine oocytes is more active at the GV stage compared with the MII stage. Interestingly, basal respiration in in vitro-derived MII oocytes was significantly higher than for in vivo-derived oocytes, reflecting an increase in proton leak. Next, we assessed respiration in MII oocytes cultured for 8 h. The aged oocytes had a significantly reduced maximum respiratory capacity, which was stimulated by a mitochondrial uncoupler, and reduced ATP-linked respiration compared with non-aged oocytes. However, the aging-related phenomenon could be prevented by caffeine treatment. We conclude that OXPHOS in bovine oocytes varies in the transition from GV to MII stage, in vitro maturation and the aging process. This approach will be particularly useful for analyzing mitochondrial bioenergetics in individual mammalian oocytes.

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Parthenote-derived human embryonic stem cells (phESCs) have many advantages over conventionally derived human embryonic stem cells (hESCs), but a more thorough investigation of these cells is needed before they can be implemented in cell therapies. In this work, we used a Cytogenetics Whole-Genome Array to study the copy number variation (CNV) status in phESCs and hESCs. We also investigated X chromosome inactivation (XCI) and expression levels of marker genes in these cells. More CNVs were found in phESCs than in hESCs in the present study, and gene expression appeared to be associated with the gain or loss of CNVs. In addition, a variable XCI status and different expression pattern of paternally expressed imprinted gene were also found in phESCs. In conclusion, although phESCs had a similar pluripotent profile to conventionally derived hESCs, these cells differed in imprinted gene expression, XCI status and number of CNVs. Our work highlights the need for a deeper investigation to elucidate the genetic and epigenetic characteristics of these cells.

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Increased vascular permeability and angiogenesis are hallmarks of the implantation process in the uterus. Angiomotin (Amot), which is a vascular angiogenesis-related protein, belongs to the motin family. There are two other members of the motin family, angiomotin-like 1 and 2 (Amotl1 and 2), which are also thought to be involved with angiogenesis. In the present study, the distribution of motin mRNAs in the mouse uterus during the peri-implantation period was investigated by in situ hybridization. Amot and Amotl1 were expressed in the stromal cells on days 3 and 4; expressions of Amotl2 during the same period were low. During the postimplantation period, Amot and Amotl1 were expressed in secondary decidual cells, while Amotl2 expression fell to an undetectable level. We also examined hormonal regulation of motin expression by steroid hormone treatment in ovariectomized mice. We found that expression of Amot was induced by P₄ in stromal cells. Additionally, Amotl1 expression was upregulated by both P₄ and estrogen (E₂) in stromal cells, whereas E₂ increased this gene expression for only a limited time; after 12 h, expression dissipated. In contrast, P₄ regulated the expression of Amotl2 in stromal cells, while E₂ regulated its expression in luminal epithelium cells. Our results demonstrated that Amot, Amotl1, and Amotl2 were differentially expressed in uterine cells during the peri-implantation period, and that their expressions were differentially regulated by P₄ and E₂.

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During mammalian sex determination of XY fetuses, SRY induces SOX9 in Sertoli cells, resulting in formation of testes with seminiferous tubules, interstitial Leydig cells and peritubular myoid cells. Meanwhile XX fetuses without SRY develop ovaries. In cattle, most XX heifers born with a male twin, so-called freemartins, develop nonfunctioning ovaries and genitalia with an intersex phenotype. Interestingly, freemartins sometimes develop highly masculinized gonads with seminiferous tubule-like structures despite the absence of SRY. However, in these cases, the degree of masculinization in each gonadal somatic cell type is unclear. Here, we report a rare case of a freemartin Japanese black calf with almost complete XX sexreversal. Gross anatomical analysis of this calf revealed the presence of a pair of small testis-like gonads with rudimentary epididymides, in addition to highly masculinized genitalia including a pampiniform plexus, scrotum and vesicular gland. Histological and immunohistochemical analyses of these masculinized gonads revealed well-defined seminiferous tubule-like structures throughout the whole gonadal parenchyma. In epithelia of these tubules, SOX9-positive supporting cells (i.e., Sertoli cells) were found to be arranged regularly along the bases of tubules, and they were also positive for GDNF, one of the major factors for spermatogenesis. 3β-HSD-positive cells (i.e., Leydig cells) and SMA-positive peritubular myoid cells were also identified around tubules. Therefore, for the first time, we found the transdifferentiation of ovarian somatic cells into all testicular somatic cell types in the XX freemartin gonads. These data strongly support the idea of a high sexual plasticity in the ovarian somatic cells of mammalian gonads.

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We examined whether the CL is a site for lysophosphatidic acid (LPA) synthesis and/or a target for LPA action in the bovine reproductive tract. LPA concentrations in the CL tissue increased towards the end of the cycle and were stable during early pregnancy. No changes in the expression of LPA receptors (LPARs) occurred during the estrous cycle. The expressions of LPAR2 and LPAR4 on days 17–19 of pregnancy were higher than those on the respective days of the estrous cycle and higher than those on days 8–10 of pregnancy. LPA stimulated P4 synthesis via 3βHSD stimulation but did not modulate the interferon–tau (IFNτ) influence on P4 synthesis in steroidogenic cells. Moreover, we found LPA-dependent stimulation of IFNτ action on 2,5’-oligoadenylate synthase (OAS1) and ubiquitin-like IFN-stimulated gene 15-kDa protein (ISG15) expression. The present study demonstrated that the CL might be a site of LPA synthesis and target of LPA action in the bovine reproductive tract. We postulate that during the estrous cycle and early pregnancy, LPA exerts autocrine and paracrine effects on the CL mainly via LPAR2 and LPAR4. The stimulatory effect of LPA on P4 synthesis via 3βHSD stimulation and LPA-dependent stimulation of IFNτ action on OAS1 and ISG15 expression suggest that LPA is an additional auxiliary luteosupportive factor in steroidogenic cells.

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The use of transgenic farm animals as “bioreactors” to address the growing demand for biopharmaceuticals, both in terms of increased quantity and greater number, represents a key development in the advancement of medical science. However, the potential for detrimental side-effects as a result of uncontrolled constitutive expression of foreign genes in transgenic animals is a well-recognized limitation of such systems. Previously, using a tetracycline-inducible expression system, we demonstrated the induction of expression of a transgene encoding green fluorescent protein (GFP) in transgenic chickens by feeding with doxycycline, a tetracycline derivative; expression of GFP reverted to pre-induction levels when the inducer was removed from the diet. As a proof of principle study, however, quantitative assessment of expression was not possible, as only one G0 and one G1 transgenic chicken was obtained. In the current study, a sufficient number of G2 and G3 transgenic chickens were obtained, and quantification analysis demonstrated up to a 20-fold induction of expression by doxycycline. In addition, stable transmission of the transgene without any apparent genetic modifications was observed through several generations. The use of an inducible expression system that can be regulated by dietary supplementation could help mitigate the physiological disruption that can occur in transgenic animals as a result of uncontrolled constitutive expression of a transgene. Importantly, these results also support the use of the retroviral system for generating transgenic animals with minimal risk in terms of biosafety.

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Endothelin (ET)-2 plays a crucial role in ovarian ovulation in mammals. The present study was designed to test the hypothesis that hypoxia-inducible factor (HIF)-1α-mediated transcriptional activation contributes to the increased expression of ET-2 gene in response to hCG in rat ovarian granulosa cells (GCs) during gonadotropin-induced superovulation. By real-time RT-PCR analysis, ET-2 mRNA expression was found to significantly increase in cultured ovarian GCs after treatment with hCG, or even N-carbobenzoxyl-L-leucinyl-L-leucinyl-L-norvalinal (MG-132), while this increased ET-2 mRNA expression could also be blocked by ferrous ammonium sulfate (FAS) under human chorionic gonadotropin (hCG) treatment. Further analysis also found that these changes of ET-2 mRNA were consistent with HIF-1α expression or HIF-1 activity, and HIF-1α inhibitor echinomycin inhibited ovulation in rats. Taken together, these results indicate that ET-2 is transcriptionally activated by hCG through HIF-1α-mediated mechanism in GCs. This HIF-1α-induced transcriptional activation may be one of the important mechanisms mediating the increase of ET-2 expression in GCs during the gonadotropin-induced mammalian ovulatory process in vivo.

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The aims of the present study were to investigate the profiles of ovarian steroids and luteinizing hormone (LH) and the appearance of estrous signs in relation to luteolysis and ovulation in lactating and non-lactating cows and to examine the influence of lactation on those observations. Five lactating (daily milk yield of 28.4 ± 3.2 kg; mean ± SD) and five non-lactating cycling Holstein cows were examined. Their ovaries were monitored by ultrasonography daily during one estrous cycle. Blood samples were collected daily and then at 3-h intervals after luteolysis until ovulation. Estrous signs in terms of behavior, the vulva and the vagina were checked at 8-h intervals after luteolysis until ovulation. Profiles of progesterone, estradiol-17β and LH did not differ between the groups. There were no differences in the interval from luteolysis to ovulation (4.6 ± 0.5 and 4.2 ± 0.8 days) and the interval from the estradiol-17β peak to ovulation (34.2 ± 4.5 and 30.6 ± 3.9 h) between lactating and non-lactating cows. The interval from the peak of the LH surge to ovulation was 27 h in all cows examined. Appearance of estrous signs did not differ between the groups. The vaginal estrous signs were observed conspicuously in all cows examined, but the behavioral signs were not observed in 20.0% of the cows. The duration of behavioral signs (41.3 ± 23.6 h) was shorter (P<0.05) than that of the vagina (68.9 ± 25.4 h). These results imply that lactation might not interfere with the hormonal profiles from luteolysis to ovulation.

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Mouse embryonic stem (ES) cells consist of heterogeneous populations with differing abilities to proliferate and differentiate. We previously demonstrated that the expression level of platelet endothelial cell adhesion molecule 1 (PECAM1)/CD31 was positively correlated with the undifferentiated state of mouse ES cells. In order to screen for a novel gene(s) involved in ES cell pluripotency, we performed an oligo microarray analysis and identified that B-box and SPRY domain containing protein (BSPRY) was expressed at high levels in PECAM1-positive cells. Two splice isoforms of BSPRY, BSPRY-1 and BSPRY-2, were expressed in undifferentiated ES cells and in blastocysts. Knockdown of BSPRY-1/2 in ES cells significantly reduced the number of undifferentiated colonies and caused increased expression of primitive ectoderm marker gene Fgf5. The overexpression of BSPRY-2 reciprocally increased the number of undifferentiated ES cells in the presence of LIF. Similarly, injection of BSPRY-1/2 siRNAs into 2-cell embryos caused developmental retardation and degeneration of embryos, and a significant decrease in the number of cells, especially in the inner cell mass (ICM), was observed at the blastocyst stage. Furthermore, microinjection of a BSPRY-1 expression vector into pronuclear stage embryos resulted in an increase in the hatching blastocysts rate after 120 h of culture. These results suggest that BSPRY-1 and BSPRY-2 are associated with both ES cell pluripotency and early embryonic development.

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Human genetic studies have suggested that kisspeptin and neurokinin B (NKB) play pivotal roles in the control of gonadotropin-releasing hormone (GnRH) secretion. However, the role of NKB in this context is less clear compared with that of kisspeptin. In the present study, we investigated the ratio of colocalization of kisspeptin and NKB in neurons in the arcuate nucleus (ARC), the effects of intracerebroventricular infusion of NKB on luteinizing hormone (LH) secretion and whether the treatment activates ARC kisspeptin/NKB neurons in seasonally anestrous ewes. Double-labeling immunohistochemistry revealed that the majority of kisspeptin neurons coexpressed NKB in the ARC. Infusion of NKB for 2 h into the lateral ventricle elicited a discharge of LH, which resulted in significant increases in LH concentrations between 20 and 50 min after the start of infusion compared with a saline-infused control. Animals were sacrificed immediately after the end of infusion, and Fos expression in ARC kisspeptin neurons was immunohistochemically examined. The NKB treatment activated kisspeptin neurons throughout the ARC, and approximately 70% of kisspeptin neurons expressed Fos immunoreactivity at the caudal portion of the nucleus. The present study demonstrated that a central infusion of NKB elicited a discharge of LH, which was associated with the activation of a large population of ARC kisspeptin/NKB neurons in seasonally anestrous ewes. The results suggest that NKB plays a stimulatory role in the control of pulsatile GnRH secretion and that the population of ARC kisspeptin/NKB neurons is one of sites of the NKB action in sheep.

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Although the establishment of putative porcine embryonic stem cells (ESCs) has been reported, such cell lines quickly lose their self-renewal ability, as they easily differentiate or become extinct after only a limited number of passages in culture. ESC-like cells exhibiting self-renewal rather than pluripotency are considered to be a valuable resource in applications such as drug screening and toxicology testing in humans, livestock and veterinary medicine. Here, we report the generation of unique cell lines established from the inner cell mass (ICM) of porcine embryos by using inhibitors of glycogen synthase kinase 3β and mitogen-activated protein kinase kinase 1. These ICM-derived cell lines were initially cultured and passaged in conventional ES medium for human ESCs and showed porcine ESC-like morphology with alkaline phosphatase (AP) activity. After transfer to culture in ES medium containing inhibitors, the morphology of the colonies was dramatically changed, i.e., they were closely packed smooth-edged colonies with close cell-cell boundaries and showed the expression of undifferentiated markers including OCT4 (POU5F1) and NANOG. Notably, the self-renewal capacity and morphology of the cells were LIF-dependent, consistent with the expression of LIF receptors and phosphorylation of signal transducer and activator of transcription 3. To date, our established cell lines have been cultured continuously for over 100 passages without any overt morphological changes. Thus, the established cell lines reported here provide a new ESC-like cell culture system for use not only in the fields of veterinary medicine and livestock but also human medical research, since porcine physiology closely resembles that of humans.

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To improve the development of singly cultured bovine embryos, we developed a co-culture method with trophoblastic vesicles. The growth of trophoblastic cells was markedly increased in vitamin-supplemented medium 199 compared with medium 199. Upon co-culture of a single embryo with trophoblastic vesicles in vitamin-supplemented medium 199, embryo development to the blastocyst stage was significantly higher than in embryos co-cultured with trophoblastic vesicles in RPMI 1640 or with cumulus cells in medium 199 (control). In the absence of the vitamin cocktail, co-culture with trophoblastic vesicles in medium 199 did not improve embryo development compared with that of the control. The vitamin cocktail was effective in embryo development when co-cultured with trophoblastic vesicles, but not with cumulus cells. Embryo development was not improved in the absence of co-cultured trophoblastic vesicles, even in the presence of vitamin cocktail. In conclusion, the co-culture system with trophoblastic vesicles in vitamin-supplemented medium 199 efficiently enhances the development of singly cultured embryos.

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