Masanori Kanzaki

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.

Y-chromosome microdeletion and phenotype in cytogenetically normal men with idiopathic azoospermia

OBJECTIVE To determine the prevalence of microdeletions of the long arm of chromosome Y within the AZFa, AZFb, and AZFc subregions in patients with idiopathic azoospermia, and then correlate the microdeletions with clinical phenotypes to determine the most important subregion for screening. DESIGN Controlled clinical study. SETTING Male infertility clinic, Kobe University Hospital. PATIENT(S) Among 89 consecutive azoospermic patients, those whose infertility was related to known hereditary, endocrine, or obstructive causes or a cytogenetic abnormality were excluded; 54 remaining patients were studied using a polymerase chain reaction (PCR). Of these patients, 33 had Sertoli cell only syndrome, 10 had maturation arrest, and 11 had hypospermatogenesis. INTERVENTION(S) Blood and semen samples and testicular biopsies were obtained from all of the participants. MAIN OUTCOME MEASURE(S) We performed semen analysis, polymerase chain amplification of 28 DNA loci on the long arm of the Y chromosome involving the DAZ (deleted in azoospermia), and measured the plasma FSH, LH, testosterone, prolactin, and estradiol levels. RESULT(S) Microdeletions were detected in 14 of the 54 patients (nine with Sertoli cell only, three with maturation arrest, and two with hypospermatogenesis). Most microdeletions involved AZFb or AZFc. Patients with hypospermatogenesis or maturation arrest showed deletion only in AZFc. The DAZ gene was deleted in four patients with Sertoli cell only and one patient with maturation arrest. The RBM gene was deleted in two patients with Sertoli cell only who had particularly large deletions, but in no patients with arrest or hypospermatogenesis. CONCLUSION(S) Cytogenetically azoospermic patients should be examined for microdeletions before undertaking assisted reproduction. AZFc may be the most important subregion to screen. In addition, intact AZFa and AZFb subregions may be important for the presence of germ cells.

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.

Effect of erythropoietin on Leydig cell is associated with the activation of Stat5 pathway

In order to elucidate whether erythropoietin (EPO) influences male reproduction, EPO activation via erythropoietin receptor (EPOR) of Stat5 was studied in Leydig cell cultures. RT-PCR analysis of mRNA that had been prepared from primary rat Leydig cell culture and the mouse Leydig tumor cell line (I-10 cells) each demonstrated amplification of the appropriate fragment for the respective genes. Both I-10 cells and primary rat Leydig cell cultures were treated with 1U/ml recombinant human erythropoietin (rHuEPO) for various time periods upto 60 min, and cytoplasmic proteins, total cellular proteins, and nuclear extracts were isolated. Expression of Stat5b protein and phosphorylated Stat5b protein were investigated by Western blotting using anti-Stat5b pAb and anti-phospho-Stat5a/b pAb. Stat5b protein exists at the cytoplasmic level irrespective of exposure to rHuEPO, while phosphorylated Stat5b protein increases upon rHuEPO stimulation. DNA binding of nuclear protein to synthetic double-stranded oligonucleotides containing a DNA response element derived from PRL-inducible element (PIE) was examined using a gel mobility shift assay. The oligonucleotide representing the PIE of the rat beta-casein promoter was demonstrated to bind to the nuclear extract of rHuEPO-treated cells, time dependently forming a single mobility shift complex X. These data suggest that EPO binds to a receptor expressed on the surface of Leydig cells. This EPOR regulates expression of specific gene, through a Jak-Stat pathway to influence male reproductive function.

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.

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.

Metenkephalin in Seminal Plasma of Infertile Men

Background: Enkephalin is one of the opioids, which is expressed widely in reproductive organs. However, the function of enkephalin in male reproduction is not completely understood. The effect of metenkephalin on sperm motility remains especially controversial. In this study we examined the level of metenkephalin in seminal plasma from men with normal sperm production and patients with asthenospermia, oligospermia, and azoospermia to investigate the role of metenkephalin in seminal plasma on sperm function. We also investigated the effect of metenkephalin on sperm motility in vitro.

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.