Hiroki Hirayama

Generation of cloned calves and transgenic chimeric embryos from bovine embryonic stem-like cells.

Bovine embryonic stem-like cells (ES-like cells) appear to maintain a normal diploid karyotype indefinitely during culture in vitro and to express marker proteins that are characteristic of ES cells from mice, namely, alkaline phosphatase (AP), stage-specific embryonic antigen-1 (SSEA-1), STAT-3, and Oct 4. After proliferation of undifferentiated ES-like cells in vitro, some bovine ES-like cells differentiated to neural precursor cells, which were cultured in the presence of basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and platelet-derived growth factor (PDGF). In addition, calves were successfully cloned using ES-like cells and the frequency of term pregnancies for blastocysts derived from ES-like cells was higher than those of early pregnancies and maintained pregnancies after nuclear transplantation (NT) with bovine somatic cells. Successful cloning from bovine ES-like cells should allow the introduction into cattle of specific genetic characteristics of biomedical and/or agricultural importance.

Aberrant Expression Patterns of Genes Involved in Segregation of Inner Cell Mass and Trophectoderm Lineages in Bovine Embryos Derived from Somatic Cell Nuclear Transfer

High rates of embryonic, fetal, or placental abnormalities have consistently been observed in bovine cloning. Segregation of inner cell mass (ICM) and trophectoderm (TE) lineages in early embryos is an important process for fetal and placental formation. In mouse embryos, differentiation of ICM and TE is regulated by various transcription factors, such as OCT-4, CDX2, and TEAD4, but molecular mechanisms that regulate differentiation in bovine embryos remain unknown. To clarify gene transcripts involved in segregation of ICM and TE lineages in bovine embryos, we examined the relative abundances of OCT-4, CDX2, TEAD4, GATA3, NANOG, and FGF4 transcripts in blastocyst embryos derived from in vitro fertilization (IVF). Furthermore, transcript levels of such genes in bovine embryos derived from somatic cell nuclear transfer (NT-SC) and in vivo (Vivo) were also compared. OCT-4, NANOG, and FGF4 transcript levels in IVF embryos were significantly higher in ICM than in TE. In contrast, the CDX2 transcript level was lower in ICM than in TE. In NT-SC embryos at the blastocyst stage, transcript levels of all genes except CDX2 were lower than that in Vivo embryos. In the elongated stage, expression levels of the six genes did not differ between NT-SC and Vivo embryos. We observed aberrant expression patterns of various genes involved in segregation of ICM and TE lineages in bovine NT-SC embryos. These results raise the possibility that abnormalities in the cloned fetus and placenta are related to the aberrant expression of genes involved in segregation and differentiation process in the early developmental stage.

Changes in the expression patterns of the genes involved in the segregation and function of inner cell mass and trophectoderm lineages during porcine preimplantation development.

Abstract In mouse embryos, segregation of the inner cell mass (ICM) and trophectoderm (TE) lineages is regulated by genes, such as OCT-4, CDX2 and TEAD4. However, the molecular mechanisms that regulate the segregation of the ICM and TE lineages in porcine embryos remain unknown. To obtain insights regarding the segregation of the ICM and TE lineages in porcine embryos, we examined the mRNA expression patterns of candidate genes, OCT-4, CDX2, TEAD4, GATA3, NANOG, FGF4, FGFR1-IIIc and FGFR2-IIIc, in blastocyst and elongated stage embryos. In blastocyst embryos, the expression levels of OCT-4, FGF4 and FGFR1-IIIc were significantly higher in the ICM than in the TE, while the CDX2, TEAD4 and GATA3 levels did not differ between the ICM and TE. The expression ratio of CDX2 to OCT-4 (CDX2/OCT-4) also did not differ between the ICM and TE at the blastocyst stage. In elongated embryos, OCT-4, NANOG, FGF4 and FGFR1-IIIc were abundantly expressed in the embryo disc (ED; ICM lineage), but their expression levels were very low in the TE. In contrast, the CDX2, TEAD4 and GATA3 levels were significantly higher in the TE than in the ED. In addition, the CDX2/OCT-4 ratio was markedly higher in the TE than in the ED. We demonstrated that differences in the expression levels of OCT-4, CDX2, TEAD4, GATA3, NANOG, FGF4, FGFR1-IIIc and FGFR2-IIIc genes between ICM and TE lineages cells become more clear during development from porcine blastocyst to elongated embryos, which indicates the possibility that in porcine embryos, functions of ICM and TE lineage cells depend on these gene expressions proceed as transition from blastocyst to elongated stage.

Changes in the DNA Methylation Status of Bovine Embryos from the Blastocyst to Elongated Stage Derived from Somatic Cell Nuclear Transfer

The epigenetic reprogramming of the donor cell nucleus is an important factor in the development of embryos and production of normal offspring derived by somatic cell nuclear transfer (NT-SC). During early development, a dramatic reduction in methylation levels occurs in mouse. In early embryos, this process makes it possible to erase gamete-specific methylation patterns and induce de novo methylation at defined developmental time-points. To clarify changes in DNA methylation in bovine NT-SC embryos, we examined satellite I sequences in bovine embryos derived in vivo (Vivo) and by NT-SC at the blastocyst (BC) and elongated (EL) stages. Because the EL stage embryo consists of the embryo disc (ED) and trophectoderm (TE), the methylation status of each part was analyzed with respect to the progress of differentiation. DNA methylation levels in Vivo embryos were increased during the elongation stage. In contrast, DNA methylation levels in NT-SC embryos remained unchanged in the ED and significantly decreased in the TE. Real-time PCR analysis showed that Dnmt-1 expression in BC embryos derived by NT-SC was significantly lower than that in Vivo embryos; thus, differences in the DNA methylation status may reflect transcript levels of Dnmt-1. Our results suggest that the aberrant methylation level of bovine NT-SC embryos in the satellite I region is corrected as a result of demethylation and retention of methylation as the embryo develops and differentiates.

Excess estrogen sulfoconjugation as the possible cause for a poor sign of parturition in pregnant cows carrying somatic cell clone fetuses

We conducted this study to elucidate a factor causing a poor sign of parturition and prolonged gestation, which is frequently observed in cows carrying somatic clone fetuses. Pre-partum rises in concentrations of plasma estrone and estradiol-17beta in the recipient cows pregnant with clones were subtle. By contrast, the plasma concentration of estrone sulfate in clone pregnancies increased gradually from pre-initiation of parturition induction whereas control cows that received in vivo-derived embryos showed a significant increase at parturition. Therefore, in clone pregnancies, the ratio of estrone/estrone sulfate was low during the pre-partum period compared with control. Messenger RNA expression of estrogen sulfotransferase (SULT1E1) in the placenta at parturition was significantly higher in clone pregnancies than control pregnancies and was localized in binucleate cells (BNC). SULT1E1 mRNA abundance was negatively and positively correlated with concentrations of maternal estrone and estrone sulfate at parturition respectively. Messenger RNA expressions of estrogen sulfatase (STS) and aromatase (CYP19) were similar between clone and control pregnancies and were localized in BNC and caruncular epithelial cells. STS and CYP19 mRNA abundances showed positive correlations with maternal estradiol-17beta concentration. The population of BNC in the placenta did not differ between clone and control pregnancies. Plasma cortisol concentration of vaginally delivered newborn clone calves was comparable with those of control, although cesarean section delivered clone calves showed a low concentration. These results suggest that excess estrogen sulfoconjugation is the reason for the perturbed low ratio of active to inactive estrogens and the resulting hormonal imbalance contributes to the lack of overt signs of readiness for parturition in cows pregnant with clones.

Regulation of bovine oviductal NO synthesis by follicular steroids and prostaglandins

Nitric oxide (NO) is a regulator of sperm motility, oocyte/embryo survival, and waves of contraction/relaxation in mammalian oviducts. As follicles control oviductal functions by two routes at least, (1) a systemic way via blood vessels before ovulation, (2) a direct way by entering of follicular fluid through fimbria at ovulation, we hypothesized that NO synthesis in the bovine oviduct is regulated by follicular steroids and prostaglandins (PGs). Quantification of mRNA expressions in the ampullary tissues showed that inducible NO synthase (NOS2) mRNA expression was highest on the day of ovulation (day 0). By contrast, NOS2 mRNA expression in the isthmus was highest on days 5-6 and lowest on days 19-21. Endothelial NOS (NOS3) mRNA expressions in either the ampulla or the isthmus did not change during the estrous cycle. PGE2 and PGF2α increased NOS2 mRNA expressions in cultured ampullary oviductal epithelial cells after 1-h incubation. These increases were suppressed by an antagonist of E-prostanoid receptor type 2, one of the PGE2 receptor. Estradiol-17β decreased the expression of NOS2 mRNA expression in cultured isthmic epithelial cells 24h after treatment. This effect was suppressed by an antagonist of estrogen receptorα(ESR1). Expression of ESR1 was highest on days 19-21 in the isthmic tissues. The overall findings indicate region-specific difference of NO synthesis in the oviduct. PGs flowed from ruptured follicle may up-regulate NO synthesis in the oviductal epithelium, whereas circulating E2 seems to inhibit NO synthesis via ESR1 in the isthmus at the follicular stage.

Embryo sexing and sex chromosomal chimerism analysis by loop-mediated isothermal amplification in cattle and water buffaloes.

Abstract In domestic animals of the family Bovidae, sex preselection of offspring has been demanded for convenience of milk/beef production and animal breeding. Development of the nonsurgical embryo transfer technique and sexing methods of preimplantation embryos made it possible. Sexing based on detection of Y chromosome-specific DNA sequences is considered the most reliable method to date. PCR enables amplification of a target sequence from a small number of blastomeres. However, it requires technical skill and is time consuming. Furthermore, PCR has the risk of false positives because of DNA contamination during handling of the PCR products in duplicate PCR procedures and/or electrophoresis. Therefore, for embryo sexing to become widely used in the cattle embryo transfer industry, a simple, rapid and precise sexing method needs to be developed. Loop-mediated isothermal amplification (LAMP) is a novel DNA amplification method, and the reaction is carried out under isothermal conditions (range, 60 to 65 C) using DNA polymerase with strand displacement activity. When the target DNA is amplified by LAMP, a white precipitate derived from magnesium pyrophosphate (a by-product of the LAMP reaction) is observed. It is noteworthy that LAMP does not need special reagents or electrophoresis to detect the amplified DNA. This review describes the development and application of an embryo sexing method using LAMP in cattle and water buffaloes.

Enhancement of maternal recognition of pregnancy with parthenogenetic embryos in bovine embryo transfer

This study was performed to elucidate the changes in IFNT messenger RNA (mRNA) levels in in vivo-fertilized and parthenogenetic bovine embryos and their interferon-τ (IFNT) secretion amounts during the elongation phase. We assessed the induction capability of maternal recognition of pregnancy by parthenogenetic embryos and attempted cotransfer of in vivo-fertilized and parthenogenetic embryos. The expression level of IFNT mRNA in in vivo-fertilized embryos peaked on Day 18 after estrus, and the highest amount of uterine IFNT was observed on Day 20. Transfer of 10 parthenogenetic embryos produced a detectable amount of uterine IFNT. Transfer of one or three parthenogenetic embryos inhibited luteolysis. An increase in ISG15 mRNA levels in peripheral granulocytes was induced by the transfer of three parthenogenetic embryos. Cotransfer of three parthenogenetic embryos significantly improved the pregnancy rate on Day 40 in code 3 in vivo-fertilized embryos compared with single transfer without parthenogenetic embryos (65% vs. 35%). However, the pregnancy rate on Day 90 (35%) in cotransfer of code 3 in vivo-fertilized embryos did not differ from that upon single transfer (29%), because the cotransfer group had a higher incidence of pregnancy loss than with single transfer (47% vs. 17%) after Day 40. Cotransfer did not affect the pregnancy rate of code 2 in vivo-fertilized embryos. The incidence of pregnancy loss was higher in cotransfer of code 2 in vivo-fertilized embryos than in single transfer (30% vs. 7%). In conclusion, parthenogenetic embryos in the elongation phase secreted IFNT, enabling induction of maternal recognition of pregnancy. The present study revealed that enhancement of the maternal recognition of pregnancy using parthenogenetic embryos promoted the viability of poor-quality embryos until Day 40 of gestation. However, the incidence of pregnancy loss increased after Day 40 in the cotransfer of parthenogenetic embryos. A technique for promoting the full-term survival of poor-quality embryos is needed.

Long-term changes in plasma anti-Müllerian hormone concentration and the relationship with superovulatory response in Japanese Black cattle

The concentration of circulating anti-Müllerian hormone (AMH) in cattle is a useful endocrine marker for ovarian response to superovulation. Although the AMH concentration undergoes little variation throughout the estrous cycle, its long-term changes remain incompletely understood. Here, we investigated the relationship between superovulation response and plasma AMH concentration in Japanese Black cattle and the long-term changes in plasma AMH concentration of embryo donor cows and heifers. The median, 25th percentile, and 75th percentile of AMH concentrations in 222 mature animals were 0.265, 0.118, and 0.488 ng/ml, respectively. The numbers of ova/embryos, fertilized embryos, and transferable embryos in a total of 295 superovulations were significantly different among the H (AMH ≥ 0.488 ng/ml), M (AMH 0.487–0.119 ng/ml), and L (AMH ≤ 0.118 ng/ml) groups. AMH concentrations during repeated superovulation in ten donor cows were significantly decreased after the third treatment. In heifers, the highest AMH concentration was observed in individuals during 2–13 months of age, with considerable individual variability. AMH concentrations of heifers at 10 or 11 months correlated with the number of ova/embryos during superovulation at 13–18 months (r = 0.641, P < 0.05). These results suggest that the 25th and 75th percentile values of AMH concentration would give a useful rough estimate of ovarian response; however, repeated superovulation may reduce the predictive accuracy of single measurements of AMH concentration. It would be possible to evaluate AMH concentration in heifers after approximately 11 months of age.

Production of calves by the transfer of cryopreserved bovine elongating conceptuses and possible application for preimplantation genomic selection

Preimplantation genomic selection based on single nucleotide polymorphism (SNP) genotypes is expected to accelerate genetic improvement in cattle. However, genome-wide genotyping at the early embryonic stage has several limitations, such as the technical difficulty of embryonic biopsy and low accuracy of genotyping resulting from a limited number of biopsied cells. After hatching from the zona pellucida, the morphology of the bovine embryo changes from spherical to filamentous, in a process known as elongation. The bovine nonsurgical elongating conceptus transfer technique was recently developed and applied for sexing without requiring specialized skills for biopsy. In order to develop a bovine preimplantation genomic selection system combined with the elongating conceptus transfer technique, we examined the accuracy of genotyping by SNP chip analysis using the DNA from elongating conceptuses (Experiment 1) and optimal cryopreservation methods for elongating conceptuses (Experiment 2). In Experiment 1, the call rates of SNP chip analysis following whole genome amplification in biopsied cells from two elongating conceptuses were 95.14% and 99.32%, which were sufficient for estimating genomic breeding value. In Experiment 2, the rates of dead cells in elongating conceptuses cryopreserved by slow freezing were comparable to those in fresh elongating conceptuses. In addition, we obtained healthy calves by the transfer of elongating conceptuses cryopreserved by slow freezing. Our findings indicate that the elongating conceptus transfer technology enables preimplantation genomic selection in cattle based on SNP chip analysis. Further studies on the optimization of cryopreservation methods for elongating conceptuses are required for practical application of the selection system.