Junji Tsuchida

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.

Molecular Cloning and Characterization of Phosphatidylcholine Transfer Protein-Like Protein Gene Expressed in Murine Haploid Germ Cells

Abstract We have isolated a cDNA clone specifically expressed in spermiogenesis from a subtracted cDNA library of mouse testis. The cDNA consisted of 1392 nucleotides and had an open reading frame of 873 nucleotides encoding a protein of 291 amino acid residues. Computer-mediated homology search revealed that the nucleotide sequence was unique but the deduced amino acid sequence had similarity to mouse phosphatidylcholine transfer protein (PCTP). We named this newly isolated gene PCTP-like protein. Northern blot analysis revealed a 1.4-kilobase mRNA expressed in the testis, kidney, liver, and intestine with the highest level in the testis. Messenger RNA expression in the testis was detected first on Day 23 in postnatal development and then increased up to adulthood. The protein, having a molecular weight of approximately 40 000, was encoded by the mRNA and was detected at the tail of the elongated spermatids and sperm by immunohistochemical staining.

Cloning and characterization of the human Calmegin gene encoding putative testis-specific chaperone

The putative chaperone Calmegin is required for sperm fertility in mouse and the relevance of the gene to certain cases of human male infertility has been suggested. In the present paper, we have isolated and characterized the human homolog cDNA of the mouse germ cell-specific Calmegin. The entire coding region of the human cDNA showed 80% identity with the previously reported mouse Calmegin. The predicted amino acid sequence showed strong conservation of the two sets of internal repetitive sequences (Ca2+ binding motif), and the hydrophilic COOH terminus, which corresponds to the putative endoplasmic reticulum (ER) retention motif. Our finding will support diagnosis of male infertility. Northern blotting analysis of various human tissues showed that the transcript was 3 kb in length and was expressed exclusively in the testis. Using the fluorescence in situ hybridization (FISH) technique, human Calmegin gene was mapped to chromosome 4q28.3-q31.1.

Meichroacidin Containing the Membrane Occupation and Recognition Nexus Motif Is Essential for Spermatozoa Morphogenesis

Meichroacidin (MCA) is a highly hydrophilic protein that contains the membrane occupation and recognition nexus motif. MCA is expressed during the stages of spermatogenesis from pachytene spermatocytes to mature sperm development and is localized in the male meiotic metaphase chromosome and sperm flagellum. MCA sequences are highly conserved in Ciona intestinalis, Cyprinus carpio, and mammals. To investigate the physiological role of MCA, we generated MCA-disrupted mutant mice; homozygous MCA mutant males were infertile, but females were not. Sperm was rarely observed in the caput epididymidis of MCA mutant males. However, little to no difference was seen in testis mass between wild-type and mutant mice. During sperm morphogenesis, elongated spermatids had retarded flagellum formation and might increase phagocytosis by Sertoli cells. Immunohistochemical analysis revealed that MCA interacts with proteins located on the outer dense fibers of the flagellum. The testicular sperm of MCA mutant mice was capable of fertilizing eggs successfully via intracytoplasmic sperm injection and generated healthy progeny. Our results suggest that MCA is essential for sperm flagellum formation and the production of functional sperm.

Cloning and Characterization of a Complementary Deoxyribonucleic Acid Encoding Haploid-Specific Alanine-Rich Acidic Protein Located on Chromosome-X

Abstract We have isolated a cDNA clone encoding a germ cell-specific protein from an expression cDNA library prepared from the mouse testis using testis-specific polyclonal antibodies. Northern blot analysis showed a transcript of 1.1 kilobases exclusively expressed in haploid germ cells of the testis. Sequence analysis of the cDNA revealed one long open reading frame consisting of 238 deduced amino acids, rich in basic amino acids in the N-terminal one-third that also contained the nuclear localization signal, and rich in acidic amino acids, including two type of acidic alanine-rich repeats, in the rest of the deduced protein. The protein having a molecular weight of approximately 55 kDa and an isoelectric point of pH 4.3–4.7 was also exclusively detected in the testis by Western blot analysis. As the cDNA was located on chromosome-X, Halap-X (haploid-specific alanine-rich acidic protein located on chromosome-X) was proposed for the name of the protein encoded by the cDNA. Immunohistochemical observation revealed that the Halap-X protein was predominantly present in the nucleoplasm of round spermatids but gradually decreased as spermatids matured, followed by the subsequent appearance in the cytoplasm of elongating spermatids. Thus, the Halap-X protein was transferred from the nuclei to the cytoplasm during the spermatid maturation when the chromatin condensation and transformation of the nuclei occurred. The Halap-X may facilitate specific association of nuclear DNA with some basic chromosomal proteins and play important roles in the process of chromatin condensation.

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.

Mapping of six germ-cell-specific genes to mouse chromosomes.

compared with 11 days p.c. are: 7 days p.c. 80%, 11 days p.c. 100%, 15 days p.c. 89%, 17 days p.c. 29%. Expression of a 1.95kb signal was also detected in adult lung and testes but not heart, brain, spleen, skeletal muscle, or testes. Discussion: The molecular events involved in conversion of pleuripotent epithelial derivatives into various neural crest derivatives require complex cellular and environmental interactions modulated by lineage-specific transcription factors. One important event in the development of neural crest-derived cells is the transition of epithelial to mesenchymal characteristics during emigration from the neural tube. Slug, a zinc finger protein, is one gene believed to play an important role in this transition [2,3]. Slug is a neurogenic, transcription factor belonging to the Snail family in Drosophila melanogaster. Embryological studies in chick and frog demonstrated that Slug mRNA is expressed in the developing neural crest and in mesodermal cells emerging from the primitive streak [3-5]. Indirect functional analyses with antisense oligonucleotides to the Slug mRNA showed specific and transient developmental failures at the early embryonic stages. These failures resulted in defects in neural tube closure between the midbrain and cervical regions, block of the epithelial-mesenchymal transition in the neural crest, and in the emergence of mesoderm from the primitive streak. These anomalies suggest that SLUG is required for the genetic control of cell activity during early stages of neural tube and neural crest development. Consistent with a role for SLUGH function in mouse embryonic development, our Northern blot analyses demonstrated that Slugh is expressed at 7 days p.c., and the signal intensity decreases subsequent to 11 days p.c. Further experiments with in situ hybridization or immunohistochemistry will be necessary to determine the specific sites of Slug expression during mouse embryogenesis. Identification of known mutations caused by alterations of specific genes can provide essential clues for understanding the normal function of those genes in mammalian development. To determine whether Slugh is a candidate gene for a disease locus, we identified the chromosomal localization of Slugh in the mouse and human genomes. Segregation analysis of a Slugh RFLP in The Jackson Laboratory FI(C57BL/6J x Mus spretus) x Mus spretus (BSS) interspecific backcross panel [1] determined that the mouse Slugh gene is located on the proximal end of Chr 16 and cosegregated with four previously mapped loci: Tbxl (T/omb homologous domain containing gene 1), D16Bir4, D16Hun3, and Gplbb (Fig. 1). An interspecies somatic-cell hybrid (SCH) panel was used to determine the chromosomal localization of SLUGH in the human genome. The mouse Slugh cDNA hybridized to a human-specific 10 kb band in two SCH lanes: one SCH cell line containing only human Chr 8; the other SCH cell line containing three human chromosomes, 4, 8, and 20 (Fig. 2). This result indicated that SLUGH was located on human Chr 8. Only one other gene, a CCAAT/enhancer binding protein, C/EBP-delta (CRP3/CELF), has been localized to this portion of human Chr 8 and mouse Chr 16 ([6], mouse human homology map http://www3.ncbi.nlm.nih. gov/Homology/mousel6.html). Therefore, to confirm and further refine the localization of Slugh in the human genome, we identified a human SLUGH EST and determined its human map location with the Stanford G3 radiation hybrid (RH) mapping panel (http:// shgc.stanford.edu/RH/index.html). Comparison of RH mapping data with previously scored markers determined that SLUGH was closely linked to marker D8S2090 (LOD 15) on human Chr 8 at cM 66-69 (http://www/ncbi.nlm.nih.gov/cgi-bin/SCIENCE96/ loc?WI-8188, h t tp : / /www.ncbi .n lm.nih .gov/cgi -b in /Schuler / clust2html?Homo+sapiens+8760). These data are consistent with and further define a region of conserved linkage between mouse Chr 16 and human Chr 8ql 1. Survey of the biomedical literature did not indicate any genetically linked, mammalian disease loci that would suggest a defect in Slugh. Therefore, additional molecular and embryonic studies of SLUGH function are required to determine its role in mammalian development.

Sperm flagella protein components: Human meichroacidin constructed by the membrane occupation and recognition nexus motif

Background and AimsIn a previous study, the authors of the present study cloned mouse meichroacidin (MCA), which is expressed in stages of spermatogenesis from pachytene spermatocytes through round spermatid germ cells. MCA protein contains the membrane occupation and recognition nexus (MORN) motif and localizes to a male meiotic metaphase chromosome. Recently a MCA homolog of carp (Cyprinus carpio), MORN motif-containing sperm-specific axonemal protein (MSAP), was reportedly identified and localized in sperm flagella. Present knowledge of human spermiogenesis requires the identification of proteins in human sperm. The present study identified the human orthologue of MCA.MethodsColony hybridization using a human testis plasmid cDNA library was carried out to clone human MCA (h-MCA) cDNA. Northern blot, Western blot, and immunohistochemical analyses were carried out.Resultsh-MCA was found to be specifically expressed in the testes. The h-MCA amino acid sequence shared 79.8% identity with mouse MCA and contained MORN motifs. h-MCA localized in the sperm flagellum and basal body, as does MSAP in carp.ConclusionExpression and localization analyses showed that h-MCA is a component of the sperm flagellum and basal body and might play an important role in the development of the sperm flagellum in humans.