Noboru Tatsumi

Formation of Reactive Oxygen Species by Spermatozoa From Asthenospermic Patients: Response to Treatment With Pentoxifylline

PURPOSE We determined the incidence of reactive oxygen species formation by spermatozoa from asthenospermic patients, and the relationship between reactive oxygen species formation and sperm motion parameters. We also assessed the efficacy of in vitro and in vivo pentoxifylline treatment of asthenospermic patients whose spermatozoa generated high reactive oxygen species levels. MATERIALS AND METHODS Reactive oxygen species formation by spermatozoa from asthenospermic patients and fertile volunteers was measured by chemoluminescence. Reactive oxygen species formation by the sperm preparations was investigated without stimulation (steady state), or after stimulation with N-formyl-methionyl-leucyl-phenylalanine (f-MLP) or phorbol-12-myristate-13-acetate. Spermatozoa from 15 asthenospermic patients whose spermatozoa produced high levels of reactive oxygen species at steady state were treated in vitro with pentoxifylline to determine its effect on reactive oxygen species generation and sperm motion parameters. These same 15 patients and 18 with asthenospermia whose spermatozoa did not produce reactive oxygen species at steady state were treated with pentoxifylline at 2 different dosages (300 and 1,200 mg. daily) to determine its effect on reactive oxygen species generation, sperm motion parameters and sperm fertilizing ability in vivo. RESULTS When reactive oxygen species formation was detected in the steady state that was not stimulated by f-MLP, the source of reactive oxygen species could be attributed to the spermatozoa themselves. Spermatozoa from 15 of 71 asthenospermic patients generated reactive oxygen species at steady state. Pentoxifylline decreased reactive oxygen species generation by spermatozoa in these patients, and preserved the decrease of curvilinear velocity and beat cross frequency for 6 hours in vitro. For these patients orally administered pentoxifylline failed to decrease reactive oxygen species generation by spermatozoa, and had no effect on sperm motility, sperm motion parameters and sperm fertilizing ability at low dosage (300 mg. daily). However, it increased motility and beat cross frequency at high dosage (1,200 mg. daily) but it had no effect on sperm fertilizing ability. CONCLUSIONS Stimulation of sperm preparations with f-MLP can identify the source of reactive oxygen species generated at steady state. Among asthenospermic patients there were some whose spermatozoa produced detectable steady state levels of reactive oxygen species. In this group pentoxifylline appeared to be effective for decreasing reactive oxygen species formation and preserving sperm motion parameters in vitro. Orally administered pentoxifylline had no effect at low dosage but it increased sperm motility and some sperm motion parameters without altering sperm fertilizing ability at high dosage.

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.

Assisted reproduction technology for patients with congenital bilateral absence of vas deferens.

PURPOSE We investigate the frequency of cystic fibrosis transmembrane conductance regulator gene mutations in Japanese patients with congenital bilateral absence of the vas deferens, and assess treatment outcomes of assisted reproduction interventions. MATERIALS AND METHODS In 10 Japanese patients with bilateral congenital absence of the vas deferens genetic analysis was performed for known frequent mutations of the cystic fibrosis transmembrane conductance regulator gene using polymerase chain reaction amplification followed by dot-blot hybridization with the allele-specific oligonucleotide probes and direct sequencing. Intracytoplasmic sperm injection using spermatozoa retrieved from the testes was performed in 7 of the couples. RESULTS No known mutations of the gene were detected in the patients. However, analysis of the polythymidine tract polymorphism in intron 8 revealed 30% allele frequency of 5T. Pregnancy was achieved in 7 cycles of intracytoplasmic sperm injection using spermatozoa retrieved from the testes. CONCLUSION The 5T variant in intron 8 polythymidine tract was identified with high allelic frequency in Japanese patients with congenital bilateral absence of the vas deferens, suggesting that the disease in Japan is also partially caused by this particular mutation of the cystic fibrosis transmembrane conductance regulator gene. Modern assisted reproduction technology offers an important option for patients with congenital bilateral absence of the vas deferens.

Levels of Interferon α and γ in Seminal Plasma of Normozoospermic, Oligozoospermic, and Azoospermic Men

The functions of the interferons in the testis are unclear. We investigated the role of interferon alpha and gamma in the seminal plasma on spermatogenesis. The levels of interferon alpha and gamma were determined in the seminal plasma of 101 males including normozoospermic (n = 31), oligozoospermic (n = 57) and azoospermic men (n = 13). The correlation between such levels and clinical parameter (seminogram and serum hormone levels) was evaluated. The seminal plasma level of interferon alpha (92.0 + 110.3 ng/mL) in the normozoospermic men was significantly lower than that of oligozoospermic men (162.4 +/- 130.3 ng/mL) (p < .05). The three groups (normozoospermic, oligozoospermic, and azoospermic group) did not differ as to the level of interferon gamma. No significant correlations were observed between the levels of interferon alpha or gamma in seminal plasma and the serum levels of follicle stimulating hormone (FSH), luteinizing hormone (LH), testosterone, prolactin, or estradiol. Theses results suggest that the level of interferon alpha in the seminal plasma may be related to sperm production.

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.

TREATMENT OF PATIENTS WITH RETROGRADE EJACULATION IN THE ERA OF MODERN ASSISTED REPRODUCTION TECHNOLOGY

PURPOSE We determined a rational strategy for treatment of patients with retrograde ejaculation in the era of modern assisted reproduction technology. MATERIALS AND METHODS In 7 consecutive patients medical treatment or retrieval of spermatozoa from the bladder was performed at a male infertility clinic. RESULTS Antegrade ejaculation was restored in 3 patients, and spermatozoa were retrieved from the bladder and used for assisted reproduction in 3. Spermatozoa with good oolemma penetrating ability were collected by seminal vesicle massage. CONCLUSIONS Modern assisted reproduction technology is a powerful treatment option for retrograde ejaculation when combined with a technique to retrieve spermatozoa of good quality from the bladder.

Hyperprolactinaemia among infertile patients and its effect on sperm functions

Summary. The effects of hyperprolactinaemia on sperm function were investigated in 264 men with oligozoo‐, asthenozoo‐, or teratozoospermia and who were attending a male infertility clinic. None of the patients exhibited galactorrhea or complained of impotence. There was no correlation between abnormal values in spermiogram and hyperprolactinaemia. After multiple measurements of serum prolactin concentration, 15 cases (5.7%) were diagnosed as hyperprolactinaemic (≧ 10 ng ml−1). Six of these patients were taking cimetidine and six were taking anti‐anxiety drugs. Serum prolactin returned to the normal level after discontinuation of these drugs; thus these 12 cases were considered as drug‐induced hyperprolactinaemia. The other three patients were diagnosed as having pituitary microadenomas and received bromocriptine treatment; the serum prolactin levels normalized within 1 month. No changes in sperm concentration, motility or morphology were found after normalization of serum prolactin levels. Sperm fertilizing ability was monitored by the hamster test for 10 months in the three patients with pituitary microadenoma, and no improvement was observed. Results suggest that hyperprolactinaemia, which does not cause symptoms, has little effect on the impairment of sperm functions. Measurement of serum prolactin in infertile men could be justified, however, for early detection of pituitary adenomas.

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.