Ken Sawai

Impeding Xist Expression from the Active X Chromosome Improves Mouse Somatic Cell Nuclear Transfer

Cloning Futures Cloning mammals by somatic cell nuclear transfer is a technique with many potential applications in regenerative medicine, agriculture, and pharmaceutics; however, it is inefficient because of the incidence of aberrant genomic reprogramming. Inoue et al. (p. 496, published online 16 September) found that the gene product of Xist, which normally inactivates one of the two X chromosomes in females, was unexpectedly expressed ectopically from active X chromosomes in cloned mice. When Xist was deleted from the mice, gene expression returned to normal and the efficiency of somatic cell nuclear transfer increased about ninefold, offering promise for future nuclear transfer technology. Efficiency of mouse nuclear transfer was improved by correcting aberrant gene expression on the active X chromosome. Cloning mammals by means of somatic cell nuclear transfer (SCNT) is highly inefficient because of erroneous reprogramming of the donor genome. Reprogramming errors appear to arise randomly, but the nature of nonrandom, SCNT-specific errors remains elusive. We found that Xist, a noncoding RNA that inactivates one of the two X chromosomes in females, was ectopically expressed from the active X (Xa) chromosome in cloned mouse embryos of both sexes. Deletion of Xist on Xa showed normal global gene expression and resulted in about an eight- to ninefold increase in cloning efficiency. We also identified an Xist-independent mechanism that specifically down-regulated a subset of X-linked genes through somatic-type repressive histone blocks. Thus, we have identified nonrandom reprogramming errors in mouse cloning that can be altered to improve the efficiency of SCNT methods.

Isolation of embryonic stem-like cells from equine blastocysts and their differentiation in vitro1

Embryonic stem (ES) cells are pluripotent cells with the potential capacity to generate any type of cell. We describe here the isolation of pluripotent ES‐like cells from equine blastocysts that have been frozen and thawed. Our two lines of ES‐like cells (E‐1 and E‐2) appear to maintain a normal diploid karyotype indefinitely in culture in vitro and to express markers that are characteristic of ES cells from mice, namely, alkaline phosphatase, stage‐specific embryonic antigen‐1, STAT‐3 and Oct 4. After culture of equine ES‐like cells in vitro for more than 17 passages, some ES‐like cells differentiated to neural precursor cells in the presence of basic fibroblast growth factor (bFGF), epidermal growth factor and platelet‐derived growth factor. We also developed a protocol that resulted in the differentiation of ES‐like cells in vitro to hematopoietic and endothelial cell lineages in response to bFGF, stem cell factor and oncostatin M. Our observations set the stage for future developments that may allow the use of equine ES‐like cells for the treatment of neurological and hematopoietic disorders.

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.

Characteristics of stimulation of gonadotropin secretion by kisspeptin-10 in female goats.

The aims of the present study were to clarify the effect of kisspeptin-10 (Kp10) on the secretion of luteinizing hormone (LH), follicle stimulating hormone (FSH), growth hormone (GH) and prolactin (PRL) in goats, and compare the characteristics of any response with those of the response to gonadotropin-releasing hormone (GnRH). The experiments were performed using four female goats (4-5 years old) in the luteal phase of estrous cycle. A single intravenous (i.v.) injection of 1, 5 and 10 microg/kg b.w. (0.77, 3.85 and 7.69 nmol/kg b.w.) of Kp10 stimulated the release of LH. Maximum values were observed 20-30 min after the injection. On the other hand, Kp10 did not alter plasma GH and PRL concentrations significantly. Three consecutive i.v. injections of Kp10 (5 microg/kg b.w.) or GnRH (5 microg/kg b.w.: 4.23 nmol/kg b.w.) at 2-h intervals increased both plasma LH and FSH levels after each injection (P<0.05); however, the responses to Kp10 were different from a similar level of GnRH. The rate of decrease in LH and FSH levels following the peak was attenuated in Kp10-treated compared to GnRH-treated animals. These results show that Kp10 can stimulate the release of LH and FSH but not GH and PRL in female goats and suggest that the LH- and FSH-releasing effect of the i.v. injection of Kp10 is less potent than that of GnRH.

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.

Effects of Exogenous Tumour Necrosis Factor-α on the Secretory Function of the Bovine Reproductive Tract Depend on Tumour Necrosis Factor-α Concentrations

The aim of study was to correlate tumour necrosis factor-alpha (TNF) infused doses used with the TNF concentrations achieved and with the secretory function of both the ovary and the uterus in cows. We evaluated the concentrations of progesterone (P4), prostaglandin (PG)F(2alpha), PGE(2) nitric oxide (NO) and TNF in the jugular vein and vena cava caudalis as parameters of exogenous TNF action on the female reproductive tract. Aortae abdominalis of cows (n = 18) were infused with saline or two doses of TNF (luteolytic--1 microg or luteotrophic--10 microg). In the peripheral blood, 1 microg TNF concentrations achieved within the range of 30-45 pg/ml, and 10 microg TNF provoked a sharp increase in achieved concentrations at a range of 250-450 pg/mL). The TNF concentrations achieved in vena cava caudalis were five to six times higher than that in peripheral blood (p < 0.001). One microgram TNF increased PGF(2alpha) and NO (p < 0.001) and decreased P4 (p < 0.05). The higher TNF dose stimulated P4 and PGE(2) (p < 0.01). TNF infusion at luteolytic dose achieved its concentrations at the physiological range previously observed in cows. Luteotrophic TNF dose achieved the concentrations in vena cava caudalis that are much higher than physiological level and were previously noted in pathological circumstances (i.e. mastitis, metritis).

Derivation, Maintenance, and Induction of the Differentiation In Vitro of Equine Embryonic Stem Cells

We describe here the isolation and maintenance of pluripotent embryonic stem (ES) cells from equine blastocysts that have been frozen and thawed. Equine ES cells appear to maintain a normal diploid karyotype in culture. These cells express markers that are characteristic of mouse ES cells, namely, alkaline phosphatase, stage-specific-embryonic antigen 1, STAT3, and Oct4. We also describe protocols for the induction of differentiation in vitro to neural precursor cells in the presence of basic fibroblast growth factor (bFGF), epidermal growth factor, and platelet-derived growth factor and to hematopoietic and endothelial cell lineages in the presence of bFGF, stem cell factor, and oncostatin M. Equine ES cells provide a powerful tool for gene targeting and the generation of transgenic clonal offspring.

Kisspeptin‐10 stimulates the release of luteinizing hormone and testosterone in pre‐ and post‐pubertal male goats

The aims of the present study were to clarify the effect of kisspeptin-10 (Kp10) on the secretion of luteinizing hormone (LH) and testosterone (T) in pre-pubertal and post-pubertal male ruminants. Four male goats (Shiba goats) were given an intravenous (i.v.) injection of Kp10 (5 µg/kg body weight (b.w.)), gonadotoropin-releasing hormone (GnRH, 1 µg/kg b.w.), or 2 mL of saline as a control at the ages of 3 (pre-pubertal) and 6 (post-pubertal) months. A single i.v. injection of Kp10 significantly stimulated the release of LH and T in both groups. The area under the response curve (AUC) of LH for a 60-min period after the i.v. injection of Kp10 was significantly greater in the pre-pubertal goats (P < 0.05). The AUC of T for a 120 min period post-injection did not differ between the two age groups. A single i.v. injection of GnRH also significantly stimulated the release of LH and T in both groups (P < 0.05). The secretory pattern of LH and T in response to GnRH resembled that in response to Kp10. These results show that the LH-releasing response to Kp10 is greater in pre-pubertal than post-pubertal male goats. They also show that Kp10, as well as GnRH, is able to stimulate the release of T in male goats.

The role of sexual steroid hormones in the direct stimulation by Kisspeptin-10 of the secretion of luteinizing hormone, follicle-stimulating hormone and prolactin from bovine anterior pituitary cells

The aims of the present study were to clarify the effect of Kisspeptin-10 (Kp10) on the secretion of luteinizing hormone (LH), follicle-stimulating hormone (FSH) and prolactin (PRL) from bovine anterior pituitary (AP) cells and evaluate the ability of sex steroids to enhance the sensitivity of gonadotropic and lactotropic cells to Kp10. AP cells prepared from 7-week-old male calves were incubated for 12h with estradiol (E(2); 10(-8)M), progesterone (P(4); 10(-8)M), testosterone (T; 10(-8)M), or vehicle only (control), and then for 2h with Kp10 (10(-6)M). The amounts of LH, FSH and PRL released into the culture medium after the 2-h incubation period were examined. Kp10 significantly stimulated the secretion of LH from the AP cells treated with E(2) and T (P<0.05), but not from the P(4)-treated cells. In contrast, Kp10 had no effect on the secretion of FSH regardless of the steroid treatment. Kp10 significantly stimulated the secretion of PRL (P<0.05), the sexual steroid hormones having no effect. The LH- or FSH-releasing response to gonadotropin-releasing hormone (GnRH; 10(-8)M) and PRL-releasing response to thyrotropin-releasing hormone (TRH; 10(-8)M) were significantly greater than those to Kp10 (P<0.05). The present results suggest that E(2) and T, but not P(4), enhance the sensitivity of gonadotropic cells to the secretion of LH in response to Kp10. However, Kp10 had no stimulatory effect on the secretion of FSH regardless of the effect of sex steroids. Kp10 directly stimulates the secretion of PRL from the pituitary cells, and sex steroids do not enhance the sensitivity of lactotropic cells to Kp10. Furthermore, the LH- and FSH-releasing effect and the PRL-releasing effect of Kp10 are less potent than that of GnRH and TRH, respectively.

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.