Masanori Ohta

Expression of the Ha-ras Suppressor Family Member 5 Gene in the Maturing Rat Testis

We analyzed the gene expression of Ha-ras suppressor family member 5 (Hrasls5), which is considered to modulate the Ha-ras signaling cascade, from maturing rat testis. Expression was detected primarily in the spermatocytes in the maturing rat testis. The Hrasls5 gene product might function as a tumor suppressor as well as in spermatogenesis, as deduced from its amino acid sequence.

Expression of Rat Sperm Flagellum-Movement Associated Protein Genes under 2,3,7,8-Tetrachlorodibenzo-p-dioxin Treatment

We analyzed the gene expression of sperm flagellum-movement associated proteins, namely, adenylate kinase domain containing protein (RGD1303144) and outer dense fiber protein 1. These gene expressions were up-regulated in a maturing rat testis, and RGD1303144 gene was expressed dominantly in spermatocytes. Although 2,3,7,8-tetrachlorodibenzo-p-dioxin administration reduced sperm motility, these gene expressions were not affected.

Expression of the Centrosomal Colon Cancer Autoantigen Gene during Spermatogenesis in the Maturing Rat Testis

We analyzed the gene and protein expression of serologically defined colon cancer antigen 8. Gene expression was upregulated in the maturing rat testis, and was localized to the spermatocytes. Protein was detected in the spermatids and at the sites of mRNA expression. Specific expression of colon cancer antigen 8 was observed in the maturing rat testis.

Expression of anti-Müllerian hormone and its type II receptor in germ cells of maturing rat testis

This work aimed to clarify the expression and roles of anti-Müllerian hormone (AMH) and its type 2 receptor (AMHR2) in seminiferous tubules of maturing rat testes. By quantitative RT-PCR, we determined the relative expressions of Amh, Amhr2, Scp1, Rsbn1, Ngfr, and Rhox5 in rat testes aged 5-49 days (d), and in germ cells and Sertoli cells isolated from 21d testes. Smad 1,5 and 8 expressions were also determined in 21d testes and isolated germ cells. Moreover, we performed in situ hybridization (ISH) of Amh and Amhr2 in 21d testes, and immunohistochemical staining (IHCS) in 10, 15 and 21d testes using antibodies of AMH and AMHR2. In 21d testes, expression of the spermatocyte specific gene, Scp1, increased but that of the round spermatid specific gene, Rsbn1, was faint. By ISH and IHCS, expressions of AMH and AMHR2 were strongly observed in spermatocytes of 21d testes, but not in spermatogonia. In 21d testes, expressions of immature Sertoli cell specific gene, Ngfr, and mature Sertoli cell specific gene, Rhox5, were observed. IHCS confirmed the presence of AMH and AMHR2 in Sertoli cells. Smad 1, 5 and 8 were highly expressed in 21d testes and isolated germ cells. These results indicate that not only immature Sertoli cells but also spermatocytes express AMH and AMHR2 in maturing testes. In this study, we first clarified that spermatocytes coexpressed AMH and AMHR2 in rats. We speculated that AMH produced by spermatocytes and Sertoli cells binds AMHR2 of spermatocytes and acts through SMADs.

Expression of a Novel Sphingosine 1-Phosphate Receptor Motif Protein Gene in Maturing Rat Testes

We screened a novel sphingosine 1-phosphate receptor type 5 motif-containing gene, LOC290876, from maturing rat testes by differential display. Gene expression was testis-specific, increased at week 7, and continued for 15 weeks. PCR analysis clarified two gene transcript isoforms, which were expressed at the same level in all samples detected in Northern blot. The deduced amino acid sequences of the two isoforms revealed differences in carboxyl terminal sequences. Gene and protein expression in the testes was dominant in the spermatocytes, and protein expression was localized to the nucleus. Taken together, these findings suggest that the LOC290876-encoded gene product is not involved in sphingosine signaling, but has distinct roles in the nucleus during the processes of spermatocyte maturation and meiosis producing spermatids.

Rat Stem-Cell Leukemia Gene Expression Increased during Testis Maturation

We found that stem-cell leukemia (SCL), also known as T cell acute-lymphocytic leukemia (Tal-1) gene expression, was upregulated in the maturing rat testis. Strong expression of Tal-1 was detected in the normal maturing rat testis by Northern blotting. Western blotting revealed the protein size to be about 34 kDa. Protein expression was wide-spread in spermatocytes, spermtids and spermatogonia in accordance with the seminiferous epithelium cycle, as determined by an analysis of immunohistochemistry. Gene expression of Tal-1 regulatory gene, NKX3.1, was negatively correlated with Tal-1 expression. Human Tal-1 expression in the maturing testis as well as in bone marrow was observed, which suggests that the gene product is a novel cancer-testis antigen candidate. Taken together, TAL-1 may be involved in cell division, morphological changes, and the development of spermatogenic cells in the normal rat testis.

Plasma Active and Inactive Renin Concentrations in Children

Plasma active and inactive renin concentrations (PARC and PIRC) were measured by immunoradiometric assay. Age-related changes in PARC, PIRC and the ratio of PARC/PIRC were studied in 78 normal children, age 1 month to 15 years. The effects of upright position for 15 min were also investigated in 7 postmenarcheal girls. PARC and PIRC in infants were significantly higher than in older children and their ratio of PARC/PIRC was significantly lower than in prepubertal children. During puberty, PARC, PIRC and their ratio were higher in premenarcheal girls than in postmenarcheal girls. In the upright position, PARC, PIRC and the ratio were increased significantly. These finding suggest that: (1) the production of inactive renin is increased but the activation of renin may be lowered in infants; (2) the activation of renin is affected by the menstrual cycle, and (3) the production and activation of renin are increased during short term standing.

Role of Counterregulatory Hormones for Glucose Metabolism in Children and Adolescents with Type 1 Diabetes

To elucidate the mechanism of insulin resistance due to insulin counterregulatory hormones (ICRHs) and evaluate ICRH secretion kinetics, ICRH concentrations were measured and correlated with blood glucose levels in 28 type 1 diabetic patients. Blood glucose was measured before bedtime. Early morning urine samples were collected the next morning before insulin injection and breakfast. Fasting blood glucose, cortisol, glucagon and HbA1c levels were measured. Growth hormone (GH), adrenaline, cortisol and C-peptide levels in morning urine samples were measured; SD scores were calculated for urine GH. The laboratory values (mean ± SD) were as follows; HbA1c of 8.1% ± 1.4%; pre-bedtime glucose of 203 ± 105 mg/dl; fasting blood glucose of 145 ± 87 mg/dl; serum cortisol of 21.6 ± 5.5 µg/dl; plasma glucagon of 98 ± 41 pg/ml; urinary GH, 27.2 ± 13.0 ng/gCr; urinary cortisol of 238 ± 197 ng/gCr; and urinary Adrenaline of 22.9 ± 21.0 ng/gCr. The mean urinary GH SD score was increased (+1.01 ± 0.70; p=0.000); the mean plasma glucagon lebel (98 ± 41 pg/ml) was not. Fasting blood glucose was positively correlated with plasma glucagon (R=0.378, p=0.0471) and negatively correlated with urinary cortisol (R=–0.476, p=0.010). Urinary adrenaline correlated positively with urinary GH (R=0.470, p=0.013) and urinary cortisol (R=0.522, p=0.004). In type 1 diabetes, GH, glucagon and cortisol hypersecretion may contribute to insulin resistance, but the mechanism remains unclear.

Effect of Cyproterone Acetate on Active and Inactive Renin Secretion in Patients with Precocious Puberty and Genetic Short Stature

To evaluate the effect of cyproterone acetate (CA) on the renin-angiotensin-aldosterone axis, we measured the plasma active, inactive and total renin concentrations (PARC, PIRC and PTRC) during and after CA treatment in patients with precocious puberty and genetic short stature. CA was administered at a daily dose of 150-170 mg/m2 in all subjects. PARC and PTRC were measured by immunoradiometric assays. During CA treatment, PARC, PIRC, PTRC and the PARC/PTRC ratio were significantly decreased. The plasma renin activity, measured by enzymatic assay, and the plasma aldosterone concentration were also decreased. After CA discontinuation, all of these were increased immediately along normal ranges. PARC closely correlated with plasma renin activity. These results suggest that CA produces mineralocorticoid action and suppresses the production and activation of renin.

Regulation of Rat Tetratricopeptide Repeat Domain 29 Gene Expression by Follicle-Stimulating Hormone

We screened the gene that encodes tetratricopeptide repeat domain 29 (Ttc29) in the maturing rat testis. Gene expression was determined by Northern blotting of 7-week-old rat testes, and a strong signal was detected close to the 18S rRNA band in addition to two weak high-molecular-weight signals. In situ hybridization revealed that Ttc29 was expressed primarily in the spermatocytes. We evaluated the effect of gonadotropin on Ttc29 expression using hypophysectomized rats. The pituitary was removed from 3-week-old rats, gonadotropin was injected at 5 weeks, and Ttc29 expression was determined at 7 weeks. Although testicular development and hyperplasia of interstitial cells were observed following chorionic gonadotropin treatment after hypophysectomy, Ttc29 expression was upregulated by treatment with follicle-stimulating hormone. Ttc29 encodes axonemal dynein, a component of sperm flagella. Taken together, these data indicate that axonemal dynein expression starts in the spermatocytes and is regulated by follicle-stimulating hormone.