Yoshihiro Okuda

INHIBITION OF STEROIDOGENESIS IN LEYDIG CELLS BY EXOGENOUS NITRIC OXIDE OCCURS INDEPENDENTLY OF STEROIDOGENIC ACUTE REGULATORY PROTEIN (StAR) MRNA

Nitric oxide (NO) plays multiple roles in the reproductive system. The authors studied the effect of NO on LH-stimulated steroidogenesis in primary cultures of rat Leydig cells, particularly seeking a link between inhibition of steroidogenesis and changes in expression of steroidogenic acute regulatory protein (StAR). Sodium nitroprusside (SNP), an NO generator, did not alter basal testosterone, but dosedependently reduced testosterone production in the Leydig cells stimulated by LH (100 ng/mL) at 3 h after addition of SNP. Induction of StAR mRNA transcripts could be detected as early as 1 h after the addition of LH, but no effect was detected of SNP on LH induction of StAR mRNA. StAR, then, is not affected in the inhibition by NO of LH-stimulated steroidogenesis in Leydig cells.

Stem cell factor in human seminal plasma as a marker for spermatogenesis

OBJECTIVES To measure the level of stem cell factor (SCF) in human seminal plasma to determine whether SCF may be useful in evaluating the ability to produce sperm and search the role of SCF in the testes. METHODS We measured the level of SCF in seminal plasma obtained from 108 males, including idiopathic azoospermia due to germ cell aplasia (n = 10), oligospermia (n = 50), asthenospermia (n = 31), and normospermia (n = 1 7). The expression of SCF messenger ribonucleic acid in the human testis was evaluated by reverse transcriptase polymerase chain reaction (RT-PCR). The correlation between its level and clinical findings was also evaluated. RESULTS RT-PCR showed a larger form that encoded the soluble protein and a smaller form that encoded the membrane-associated form of SCF in the human testis. The similar ratio of the larger form to the smaller one was observed both in the testis of normal and oligospermic men. The level of SCF is significantly correlated with the sperm count (r = 0.214; P < 0.05). CONCLUSIONS The level of SCF in seminal plasma appeared to predict the ability to produce sperm. Thus, this factor may play an important role in spermatogenesis.

Nitric oxide production of rat Leydig and Sertoli cells is stimulated by round spermatid factor(s).

In this study, we provide evidence of cell-to-cell interaction between rat germ cells and Leydig or Sertoli cells in relation to nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) messenger RNA (mRNA) expression. As a result of being cultured in a round spermatid-conditioned medium (RSd-CM), NO production in both Leydig and Sertoli cells increased in proportion to the length of the culture period. iNOS mRNA expression in both types of cells also increased in a dose-dependent manner as a result of being cultured with RSd-CM. This increase was detected as early as 3 h and was maintained up to 24 h. In contrast, neither NO production nor iNOS mRNA increased in either type of cell following culture in a pachytene spermatocyte-conditioned medium (PS-CM). Our findings suggest that RSd may control NO production of Leydig and Sertoli cells. This cell-to-cell interaction may be an important mechanism of regulation of testicular function.

Testosterone Dependent Regulation of the Enzymes involved in DNA Synthesis in the Rat Ventral Prostate

The effects of testosterone on the activities of DNA polymerase alpha, beta, and gamma as well as topoisomerase I (all enzymes involved in DNA synthesis) were examined in the rat ventral prostate. The activities of these four enzymes decreased gradually after castration in comparison with normal controls, and continued to decrease until the 14th day. Enzyme activities were almost restored to normal within 48 to 72 hr. of the commencement of daily testosterone injections (0.3 mg./0.2 ml.), and increased successively until the 14th day. The wet weight, protein content, and DNA content of the rat ventral prostate decreased after castration, while following testosterone therapy all these parameters increased to equal or exceed the control levels. These results suggested that the activities of DNA polymerase alpha, beta, and gamma and topoisomerase I are at least partially regulated by testosterone, and that these enzymes play an important role in the regulation of prostatic cellular proliferation.

EXPRESSION AND REGULATION OF GP130 MESSENGER RIBONUCLEIC ACID IN CULTURED IMMATURE RAT SEROLI CELLS

The expression of glycoprotein 130 (gp130) was studied in rat primary Sertoli cells by Northern blot analysis. Gp130 mRNA of 9.0 and 7.5 kb were detected in a variety of rat tissues including the testis. Gp130 mRNAs were detected in isolated, immature Sertoli cells. The levels of gp130 were significantly stimulated by the addition of interleukin-1-ß (IL-1-beta) or interleukin-6 (IL-6) but not by the addition of follicle-stimulating hormone (FSH). IL-6 activates intracellular signaling by binding with a receptor consisting of an 80-kDa ligand-binding protein, IL-6 receptor (IL-6R), and a second 130-kDa protein, gp130. As previously reported, expression of IL-6R mRNA in rat Sertoli cells was stimulated not only by IL-1-beta and IL-6 but also by FSH. In contrast, gp130 mRNA expression was not stimulated by FSH in our analysis. These data suggest that gp130 expression may be regulated by more than 1 mechanism and that production of gp130 and IL-6R, the 2 components of the IL-6 receptor system, may be regulated, at least in part, by a different pathway.

Inhibition Of Apoptosis In Cultured Immature Rat Leydig Cells By Human Chorionic Gonadotropin Associated With Bcl-2 Mrna Expression

Bcl-2 is the first identified negative regulator of apoptotic cell death. When the level of Bcl-2 mRNA in rat whole testes was examined in the present study, it gradually decreased in rats from 2.5 to 9 weeks old. We also examined the effect of human chorionic gonadotropin (hCG) on apoptosis and the level of Bcl-2 mRNA expression in immature Leydig cells in vitro. When the cells were cultured with serum free media (SFM), Bcl-2 mRNA levels gradually decreased. On the other hand, the level of Bcl-2 mRNA in cells treated with 50 ng/ml of hCG decreased at 6 h, but increased after 12 h. At 24 h, the level of Bcl-2 mRNA in the treated cells was almost the same as that of control cells (time = 0). At 12 h after the addition of various concentrations (from 0.1-1000 ng/ml) of hCG, Bcl-2 mRNA levels increased in a dose-dependent manner. An analysis of DNA fragmentation showed that treatment with hCG prevents the apoptosis of immature Leydig cells. Our findings suggest that Bcl-2 mRNA may be related to the programmed cell death of immature rat Leydig cells in vitro, which are inhibited by hCG.

SERTOLI CELLS INHIBITED APOPTOSIS OF PACHYTENE SPERMATOCYTES AND ROUND SPERMATIDS

Apoptosis in the testis represents an important physiological mechanism that regulates the number of germ cells in the seminiferous epithelium. This apoptosis is believed to be regulated by many factors, including growth factors and cytokines, which appear to suppress apoptosis of the germ cells. In this study, we examined the roles of Sertoli cells on the regulation of pachytene spermatocyte (PS) and round spermatid (RSd) apoptosis with either a co-culture trans-well system or a direct contact system. Apoptosis was detected by low molecular weight DNA fragmentation assay, in situ end labeling, and an LDH assay. In addition, the level of Bcl-2, Bax, and ICE mRNAs in PS and RSd by Northern blot analysis. When PS and RSd were cultured with Sertoli cells in either a trans-well system or direct contact system, the extent of apoptotic DNA fragmentation and LDH level were both significantly lower than those control values. TUNEL staining also revealed the inhibition of apoptosis of PS and RSd when they were cultured with Sertoli cells compared with controls (p < 0.05). Moreover, the extent of apoptotic DNA fragmentation and LDH level were significantly lower in the direct contact system than in the trans-well system. TUNEL staining also demonstrated a more decrease in apoptosis of PS and RSd in the direct contact system compared with the trans-well system (p < 0.05). PS and RSd cultured with Sertoli cells exhibited an increase in Bcl-2 mRNA, whereas those cultured with serum-free medium did not show any change. The levels of Bax and ICE mRNAs decreased in PS and RSd cultured with Sertoli cells in comparison with control values. These results suggest that Sertoli cells can prevent apoptosis of germ cells, and that the effect of Sertoli cells on germ cells is mediated by cell to cell interaction or, remote effects of inhibitory factors on apoptosis.

A GIANT RENAL CYST IN A YOUNG MAN WITH INCREASED CARBOHYDRATE ANTIGEN 19–9 IN SERUM AND CYSTIC FLUID

Simple renal cysts are uncommon in patients younger than 30 years and most are benign. However, a few cases have been reported of carcinoma in the wall of renal cysts. Carbohydrate antigen 19-9 is well known as a serum marker for various cancers of digestive system. We describe a rare case of a giant renal cyst in a young man with increased carbohydrate antigen 19-9 in the serum and cystic fluid.

MULLERIAN DUCT CYST WITH 46, XYq‐

We report a case of a mullerian duct cyst associated with a 46, XYq‐ chromosome anomaly. Mullerian duct cyst is a consequence of the abnormal regression of paramesonephric derivative. In the past many different manifestations have been associated with the 46, XYq‐, anomaly. We discuss the possibility that a mullerian duct cyst may be one clinical manifestation of 46, XYq‐.