Yasuhide Yoshimura

A rapid and non‐invasive selection of transgenic embryos before implantation using green fluorescent protein (GFP)

Non‐invasive selection of transgenic mice was performed at the stage of preimplantation embryos. The morulae collected from wild female mated with hemizygous transgenic male expressing Aequorea victoria green fluorescent protein (GFP) under chicken β‐actin promoter could be classified as green or non‐green under a fluorescent microscope. All the green embryos were shown to carry the transgene by PCR analysis. Taking advantage of the detection of GFP expression can be done non‐invasively, the selected embryos were demonstrated to be able to developed to term with 100% of accuracy of the selection.

Induction of Pluripotent Stem Cells from Human Third Molar Mesenchymal Stromal Cells

The expression of four transcription factors (OCT3/4, SOX2, KLF4, and MYC) can reprogram mouse as well as human somatic cells to induced pluripotent stem (iPS) cells. We generated iPS cells from mesenchymal stromal cells (MSCs) derived from human third molars (wisdom teeth) by retroviral transduction of OCT3/4, SOX2, and KLF4 without MYC, which is considered as oncogene. Interestingly, some of the clonally expanded MSCs could be used for iPS cell generation with 30–100-fold higher efficiency when compared with that of other clonally expanded MSCs and human dermal fibroblasts. Global gene expression profiles demonstrated some up-regulated genes regarding DNA repair/histone conformational change in the efficient clones, suggesting that the processes of chromatin remodeling have important roles in the cascade of iPS cells generation. The generated iPS cells resembled human embryonic stem (ES) cells in many aspects, including morphology, ES marker expression, global gene expression, epigenetic states, and the ability to differentiate into the three germ layers in vitro and in vivo. Because human third molars are discarded as clinical waste, our data indicate that clonally expanded MSCs derived from human third molars are a valuable cell source for the generation of iPS cells.

Functional competence of T cells in the absence of glycosylphosphatidylinositol-anchored proteins caused by T cell-specific disruption of the Pig-a gene

T lymphocytes express various glycosylphosphatidylinositol (GPI)‐anchored surface proteins, such as Thy‐1 and Ly‐6A. However, functional contribution of GPI‐anchored proteins in T cell activation is as yet poorly understood. Here we report the generation of mutant mice deficient in the expression of GPI‐anchored molecules exclusively in their T cells. We established mice carrying three identically oriented lox‐P sites within the Pig‐a gene, which encodes a component essential for the initial step of GPI anchor biosynthesis. These mice were crossed with mice carrying the Cre recombinase gene driven by the T cell‐specific p56lck proximal promoter. Offspring carrying both the lox‐P ‐containing Pig‐a gene and the Cre transgene exhibited almost complete loss of the surface expression of GPI‐anchored molecules on peripheral T cells. Interestingly, those T cells deficient in GPI‐anchored mole cules were capable of responding to T cell receptor stimulation in vitro and in vivo. These results indicate that T cells lacking the expression of GPI‐anchored molecules are functionally competent in exerting TCR‐mediated immune responses.

Isolation and characterization of cDNA clones specifically expressed in testicular germ cells.

We have cloned cDNAs involved in germ cell‐specific expression. For this, a subtracted cDNA library was generated by subtracting cDNAs derived from supporting cells of mutant testis from wild‐type testis cDNAs. Detailed analyses of mRNA expression revealed that the genes corresponding to the cloned cDNAs were exclusively expressed in testes and were developmentally controlled.

Cell cycle-dependent expression of the mouseRad51 gene in proliferating cells

The mouseRad51 gene is a mammalian homologue of theEscherichia coli recA and yeastRAD51 genes, both of which are involved in homologous recombination and DNA repair in mitosis and meiosis. The expression of mouseRad51 mRNA was examined in synchronized mouse m5S cells. TheRad51 transcript was observed from late G1 phase through to M phase. During the period of late G1-S-G2, the RAD51 proteins were observed exclusively in nuclei. Activation by mitogens of T cell and B cell proliferation in spleen induced the expression ofRad51 mRNA. By immunohistochemical analyses, the mouse RAD51 protein was detected in proliferating cells: spermatogonia in testis, immature T cells in thymus, germinal center cells of the secondary lymphatic nodules of spleen and intestine, follicle cells in ovary and epithelial cells in uterus and intestine. It was also expressed in spermatocytes during early and mid-prophase of meiosis and in resting oocytes before maturation. Thus, mouseRad51 expression is closely related to the state of cell proliferation and is presumably involved in DNA repair coupled with DNA replication, as well as in meiotic DNA recombination in spermatocytes.

Identification and characterization of a haploid germ cell-specific nuclear protein kinase (Haspin) in spermatid nuclei and its effects on somatic cells.

We have cloned the entire coding region of a mouse germ cell-specific cDNA encoding a unique protein kinase whose catalytic domain contains only three consensus subdomains (I–III) instead of the normal 12. The protein possesses intrinsic Ser/Thr kinase activity and is exclusively expressed in haploid germ cells, localizing only in their nuclei, and was thus named Haspin (forhaploid germ cell-specific nuclearprotein kinase). Western blot analysis showed that specific antibodies recognized a protein ofM r 83,000 in the testis. Ectopically expressed Haspin was detected exclusively in the nuclei of cultured somatic cells. Even in the absence of kinase activity, however, Haspin caused cell cycle arrest at G1, resulting in growth arrest of the transfected somatic cells. In a DNA binding experiment, approximately one-half of wild-type Haspin was able to bind to a DNA-cellulose column, whereas the other half was not. In contrast, all of the deletion mutant Haspin that lacked autophosphorylation bound to the DNA column. Thus, the DNA-binding activity of Haspin may, in some way, be associated with its kinase activity. These observations suggest that Haspin has some critical roles in cell cycle cessation and differentiation of haploid germ cells.

Isolation and Characterization of a Haploid Germ Cell-Specific Novel Complementary Deoxyribonucleic Acid; Testis-Specific Homologue of Succinyl CoA:3-Oxo Acid CoA Transferase

Abstract We have isolated a cDNA clone encoding a mouse haploid germ cell-specific protein from a subtracted cDNA library. Sequence analysis of the cDNA revealed high homology with pig and human heart succinyl CoA:3-oxo acid CoA transferase (EC 2.8.3.5), which is a key enzyme for energy metabolism of ketone bodies. The deduced protein consists of 520 amino acid residues, including glutamate 344, known to be the catalytic residue in the active site of pig heart CoA transferase and the expected mitochondrial targeting sequence enriched with Arg, Leu, and Ser in the N-terminal region. Thus, we termed this gene scot-t (testis-specific succinyl CoA:3-oxo acid CoA transferase). Northern blot analysis, in situ hybridization, and Western blot analysis demonstrated a unique expression pattern of the mRNA with rapid translation exclusively in late spermatids. The scot-t protein was detected first in elongated spermatids at step 8 or 9 as faint signals and gradually accumulated during spermiogenesis. It was also detected in the midpiece of spermatozoa by immunohistochemistry. The results suggest that the scot-t protein plays important roles in the energy metabolism of spermatozoa.

Genomic analysis of male germ cell-specific actin capping protein α

The Gsg3 gene which expresses specifically in haploid germ cells is a mouse testicular homolog of somatic cell type actin capping protein alpha (ACP alpha). We have obtained a mouse Gsg3 genomic clone using cDNA as a probe. Sequencing data showed that the Gsg3 gene was not interrupted by introns. The transcription initiation site of the gene was preceded not by a TATA box or GC rich promoter motifs, but by two consensus cAMP-response element (CRE) motifs at the putative position. Southern blotting analysis showed that Gsg3 is a single copy gene in the mouse, and conserved in mammals. Phylogenetic analysis showed that Gsg3 is a novel ACP alpha specific for haploid germ cells.

Nested genomic structure of haploid germ cell specific haspin gene

The haspin gene specifically expressed in haploid germ cells encodes a unique Ser/Thr protein kinase. We have cloned a mouse haspin genomic clone using cDNA as a probe. Sequencing data showed that the haspin gene was not interrupted by introns and was bordered by appropriate direct repeat. The transcription start site of the gene was not preceded by a TATA box. The whole transcription unit was located at an intron of integrin alphaM290 gene, and transcription direction of these two genes was different. Southern blotting analysis under stringent condition showed that haspin was a single gene. Phylogenetic analysis suggested that the diversion of haspin gene from other kinase family might be very ancient: the early stage of plant-fungus-animal split.

Studies on the Mechanism of Sperm Production

Producing functional sperm in mammals requires two steps that take place in two different organs: the production of sperm in the testis and the maturation of sperm in the epididymis. The germ cell differentiation that produces sperm in males involves numerous morphological and physiological changes that are timed precisely. These complex processes, referred to as spermatogenesis, comprise: the proliferation and differentiation of spermatogonia, meiotic prophase of spermatocytes, and substantial morphological changes from postmeiotic haploid spermatids to sperm.