To determine the physiological role of the c-kit receptor, which is highly expressed in Leydig cells, the regenerative differentiation of Leydig cells was studied following transient degeneration induced by ethane dimethyl sulphonate (EDS) in c-kit-deficient mutant rats (Ws/Ws). EDS caused the destruction of Leydig cells; their functional recovery was evaluated by the weight change of the target organs of androgens, which occurred at the same rate in Ws/Ws and wild-type rats. These results indicate that the tyrosine kinase activity of the c-kit receptor does not play an essential role in the regenerative differentiation of Leydig cells.

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.

We have isolated a monoclonal antibody (mAb) TRA 55, which recognizes mouse testicular germ cells from mid-pachytene spermatocytes to the early stages of haploid spermatids during differentiation. Immunohistochemical analysis produced strong positive staining of the nuclei and faint staining in the cytoplasm of germ cells. At meiotic division, when the nuclear membrane disappeared, a specific positive signal could be observed on metaphase chromosomes. When germ cells produced haploid spermatids, antigenicity became suddenly weak and soon disappeared. TRA 55 did not react with testicular somatic cells, such as Sertoli cells or Leydig cells. Western blot analysis of the whole testis showed four positive bands with molecular weights of 43, 46, 49 and 55 kDa. Three bands of 43, 49 and 55 kDa, and a single band of 46 kDa were recovered in cytoplasmic and nuclear fractions of testicular germ cells, respectively. Chronological changes in the Western blot pattern indicated that these antigens became detectable in the testis at the age of 10 days. Furthermore, all antigens were resistant to periodate treatment, suggesting that the epitope was in an amino acid rather than a sugar moiety. These antigen molecules may play important roles in the differentiation of germ cells at the later stages of meiotic prophase and meiotic division in the mouse testis.

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.

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.

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 (for haploid germ cell-Specific nuclear protein kinase). Western blot analysis showed that specific antibodies recognized a protein of M-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 G(1), 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.

It has been suggested that a plasma protein, selenoprotein P, functions as an antioxidant and that its mRNA is expressed ubiquitously, including in the testis. To determine its physiological function, we have investigated the expression of selenoprotein-P mRNA in the rat testis. Northern blot analysis showed that selenoprotein P was exclusively expressed in the Leydig cell fraction. In situ hybridization experiments further supported this observation. Testes of rats administered ethylene dimethane sulfonate (EDS) were also examined by Northern blot analysis. Selective degeneration of Leydig cells by EDS treatment resulted in disappearance of selenoprotein-P mRNA from the testis. Furthermore, upon recovery, in association with regenerative differentiation of Leydig cells, reappearance of the selenoprotein-P mRNA was observed. These results indicated that selenoprotein-P mRNA was predominantly expressed in the interstitial Leydig cells.

We have molecularly cloned a cDNA encoding a new Rel-related protein in Xenopus laevis. The product is most homologous to mammalian p100-NFkappaB2. Furthermore, the putative protein kinase A-phosphorylation site (RRPS), which is found in most of the Rel family proteins and is replaced by KRKR in mammalian p100, is also replaced by KRKK in our clone, indicating that our cDNA most likely encodes the Xenopus p100 (Xp100). Like mammalian p52, a processed product of p100, Xp52 alone binds to the kappaB site but does not activate transcription, while the XRelB/Xp52 heterodimer activates transcription, which is inhibited by the carboxyl-terminal half of Xp100 (XIkappaBdelta). Xp100 transcripts are present at all stages of oocyte maturation and in all adult tissues examined. Xp100 transcripts decrease at the gastrula stage and resume their expression at the neurula stage, which is different from other Xenopus rel family. Xp100 is highly expressed in somitogenic mesoderm at the neurula stage, while in the gastrula and tailbud stages, Xp100 transcripts are not localized to restricted regions. These results suggest that Xp100 could be involved in the late-stage development of Xenopus laevis, especially in the maturation of somites.

A monoclonal antibody (mAb TRA 104) raised against mouse testicular germ cells was able to recognize the nuclei of testicular germ cells at all the stages of differentiation from embryonic gonocytes to spermatids and did not react with any somatic cells. The antigen recognized by mAb TRA 104 was exclusively present in testicular extracts. The molecular weights and isoelectric point (pI) of the antigens determined by Western blotting analysis were 60-110 kDa and 7.2, respectively. This antigen(s) is referred to as a germ cell-specific nuclear antigen(s) (GENA) since GENA was first detected specifically in the genital ridge at around 12 days of gestation by Western blotting analysis. In the testis, the expression increased gradually until adulthood whereas it was lost in the ovary by postpartum day 5. Thus, GENA is a molecule(s) exclusively present in the nuclei of germ cells and may be a useful marker with which to study the mechanism of germ cell development and differentiation at the molecular level.

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. Sequence analysis of the cDNA revealed that the deduced amino acid sequence consisted of 284 residues, including a nominal repeat structure in the N-terminal region. Northern blot analysis revealed the presence of a transcript of 1.3 kb exclusively expressed in the testis and ovary, but at relatively low levels in the ovary. In contrast, no other tissues and organs expressed significant levels of the transcript. Expression of the mRNA in the testis was first detected on day 14 in postnatal development. Western blot analysis showed the presence of the protein with a molecular weight of approximately 40 kDa and an isoelectric point of 4.9. The protein was exclusively found in the testis and ovary, but in a far lesser amount in the ovary as was the case with the transcript. Immunohistochemical examination revealed that the protein was predominantly present in the cytoplasm in pachytene spermatocytes through to round spermatids. However, during the disappearance of the nuclear envelope at both the first and second meiotic divisions, the protein was localized around the metaphase chromosomes and spindles. Because of this, the name meichroacidin which stands formale meiotic metaphasechromosome-associatedacidic proteinis proposed for this antigen. The highly regulated stage-specific expression of meichroacidin and its specific association with the metaphase chromosomes and spindles suggest that the protein plays important roles in male meiosis.

We have been trying to clone testicular germ cell-specific genes. To accomplish this aim, we have combined three approaches. First, we obtained monoclonal antibodies recognizing germ cells at specific steps of differentiation. Meanwhile, we raised rabbit antisera against mouse resticular germ cells capable of reacting with all types of mouse germ cells. Using monoclonal and polyclonal and bodies we isolated cDNA clones encoding specific antigenic molecules expressed in various steps of germ cell differentiation. However, our third approach was employed when the amount of antigens was insufficient or the antigenicity was low. We made a cDNA library from wild type testes and mutant mice (W/W-gamma). In the latter, germ cells do not differentiate in the testes. Thus, we were able to subtract a cDNA library of such mutant testes from that of wild-type, and isolate many cDNA clones specifically expressed in testicular germ cells. Among the cloned genes, ''calmegin'' was chosen to be characterized further by producing a calmegin-knockout mouse line.

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 3kb 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.

Antisera were raised by immunizing rabbits with mouse testicular germ cells, and absorbed in vivo by injection into castrated male whole mice to obtain a specific antiserum which reacted with mouse germ cells. The expression of mouse testis-specific antigenic macromolecules was then studied immunochemically with the antiserum. Approximately 20 antigenic macromolecules with molecular weights ranging from 26 to 110 kD were detected in the normal adult testis. At least 12 of these were differentiation-specific antigens appearing during development of germ cells, while others were expressed in testicular germ and/or somatic cells or detected only in mature spermatozoa. This technique for raising testis-specific antisera could be useful for isolation of cDNA clones encoding their antigens, as well as for investigation of the physiological roles of these molecules in germ cell differentiation at the molecular level.

We have identified a novel cDNA clone, named AZ1, obtained from a cDNA library of mRNA prepared from C3H10T1/2 cells that had been transiently exposed to 5-azacytidine, a potent demethylating reagent. The amount of transcript increased with 5-azacytidine treatment of C3H10T1/2 cells and the transcript was highly expressed in mouse testis. As the mutant mouse jsd/jsd, which has a defect in germ cell maturation, barely expressed the transcript, the message was expected to be expressed specifically in spermatocytes. The mRNA was detected at significant levels in the testes from mice aged 16 days after birth, suggesting that its expression started at the pachytene spermatocyte stage. The elucidated nucleotide sequence contained a 2841-nucleotide open reading frame, and the expected amino acid sequence had a molecular mass of 107 254 Da. Specific antibodies raised against the fusion protein including glutathione S-transferase revealed an approximately 130-kDa band of a translation product in testis and in cultured cells transfected with AZ1 cDNA in the expression vector on Western-blot analysis. The protein was localized to the pre-acrosome region of round and elongated spermatids. However, it was not detected at a more advanced stage of spermatids, i.e. just before their release from Sertoli cells. This protein may play an important role in spermatogenesis.

(1983). HB St. Luke's [α95 (G2) Pro → Arg] in Japan. Hemoglobin: Vol. 7, No. 5, pp. 471-472.