Evaluation of the NOX5 protein expression and oxidative stress in sperm from asthenozoospermic men compared to normozoospermic men.

PURPOSE: NADPH oxidase 5 (NOX5), the main isoform of NOX in spermatozoa, has been recognized as the main active generators of reactive oxygen species (ROS), including superoxide anion (O 2 -. ) and hydrogen peroxide (H2O2). ROS have been shown to play important roles in many physiological and pathological conditions in spermatozoa. The present study aims to investigate the alterations of NOX5 protein expression and oxidative stress (OS) status in asthenozoospermic men compared to normozoospermic men. METHODS: Semen samples were collected from 25 asthenozoospermic men and 28 normozoospermic men. In this study, NOX5 protein expression was evaluated by Western blotting. An OS status was evaluated by measuring of ROS (O 2 -. and H2O2), DNA damage and plasma membrane integrity in spermatozoa. RESULTS: The protein expression of NOX5 (p < 0.0001) was remarkably higher in asthenozoospermic men in comparison to normozoospermic men. In addition, the percentages of intracellular O 2 -. (p < 0.0001), H2O2 (p < 0.0001) in viable spermatozoa, apoptotic sperm cells with altered plasma membrane (p < 0.001) and DNA damage (p = 0.001) were significantly increased in asthenozoospermic men compared to normozoospermic men. CONCLUSIONS: The present study provides evidence that the overexpression of NOX5 protein may induce excessive ROS production and oxidative stress damages to DNA and plasma membrane integrity in asthenozoospermic men.

Immobilising effect of Ruta graveolens L. on human spermatozoa: coumarin compounds are involved.

This study was designed to find out Ruta graveolens L. functional components, which have immobilisation effect on human spermatozoa for contraceptive use. A five-step fractionation method was used to derive different components from rue aqueous extract by using hexane, chloroform, ethanol, acetone and ultrapure water. Gas chromatography-mass spectrophotometery (GC-MS) of all fractions and the aqueous extract were performed to determine the chemical components. The immobilisation assay and membrane integrity test were also performed with four different coumarins, which were found in GC-MS in a concentration of 10 µm. Hexane, chloroform, acetone and ethanol fractions could significantly decrease motility of sperms within the first and the second hours. Hexane fraction had also significant immediate effect. The aqueous fraction had no effect on sperm motility. Meanwhile, GC-MS revealed that aqueous extract and effective fractions had similar coumarin compounds. We performed the immobilisation assay on four different coumarins, which were found in GC-MS in a concentration of 10 µm. Reduction of sperm motility was only significant for xanthotoxin. In the sperm viability and membrane integrity tests, hexane and ethanolic fractions could impair sperm vitality significantly, in contrast to coumarins. These results indicated that a part of immobilising effect of rue could be due to its coumarins. The possible mechanism could be blocking of spermatozoa potassium channels.

Regeneration of spermatogenesis in a mouse model of azoospermia by follicle-stimulating hormone and oestradiol.

Busulfan is a chemotherapeutic drug that induces sterility, azoospermia and testicular atrophy. To induce degeneration of spermatogenesis, we used different amounts of busulfan. Adult male C57Bl/6 mice were treated with 15, 30 and 45 mg kg(-1) of busulfan. After 5 weeks, animals had daily injections of 7.5 IU human follicle-stimulating hormone (hFSH) and 12.5 µg kg(-1) oestradiol benzoate (EB), separately or simultaneously. After this time, the animals were killed and blood samples were taken through cardiac puncture. Testes were used for histopathology experiments, DNA flow cytometry and RNA extraction for expression of c-kit and cyclin B1 genes. EB unlike FSH has induced stimulatory effects on spermatogenesis, increased the level of serum testosterone 2-fold and caused a 2-fold increase in the number of haploid cells. The result showed that hFSH with EB multiplied EB stimulatory effects on spermatogenesis up to four times. Expression of c-kit and cyclin B1 genes increased in EB and hFSH+EB groups. These findings suggest that EB regulates spermatogonial stem cells via hFSH. hFSH with EB had synergistic effect on regeneration of spermatogenesis.

The micronutrient supplements, zinc sulphate and folic acid, did not ameliorate sperm functional parameters in oligoasthenoteratozoospermic men.

We investigated the effects of folic acid and zinc sulphate supplementation on the improvement of sperm function in subfertile oligoasthenoteratozoospermic (OAT) men. Eighty-three OAT men participated in a 16-week intervention randomised, double-blind clinical trial with daily treatment of folic acid (5 mg day(-1) ) and zinc sulphate (220 mg day(-1) ), or placebo. Before and after treatment, semen and blood samples were obtained for determining sperm concentration, motility, and morphology, sperm viability, sperm mitochondrial function, sperm chromatin status using toluidine blue, aniline blue, acridine orange and chromomycin A3 staining; and semen and blood folate, zinc, B12 , total antioxidant capacity (TAC) and malondialdehyde (MDA) concentrations. Sperm concentration (×10(6)  ml(-1) ) increased in subfertile men receiving the combined treatment of folic acid and zinc sulphate and also in the group receiving only folic acid treatment; however, it was not statistically significant (P = 0.056 and P = 0.05, respectively). Sperm chromatin integrity (%) increased significantly in subfertile men receiving only zinc sulphate treatment (P = 0.048). However, this improvement in sperm quality was not significant after adjusting placebo effect. This study showed that zinc sulphate and folic acid supplementation did not ameliorate sperm quality in infertile men with severely compromised sperm parameters, OAT. Male infertility is a multifactorial disorder, and also nutritional factors play an important role in results of administration of supplementation on sperm parameters. However, these results should be confirmed by multiple studies in larger populations of OAT men.

Regulation of luteinizing hormone receptor in hippocampal neurons following different long-lasting treatments of castrated adult rats.

The aim of this study was to investigate the effects of different Luteinizing hormone (LH) and steroid hormones levels on LH receptor (LHR) expression in the hippocampal cells. Rats (24 males and 24 females) were assigned to four groups: one control and three experimental [gonadectomy (GDX), gonadectomy + gonadotropin releasing hormone analogue (GDX+GnRHa) and GDX+GnRHa+estradiol (E2) or testosterone (T)] independently for each gender. All experimental rats were gonadectomized; then GnRHa was administrated to GDX+GnRHa group, and GnRHa plus steroid hormone to GDX+GnRHa+E2 or T group in both genders for four-month. LHR mRNA expression and its protein level in hippocampal cells were measured using QRT-PCR and Western blotting. Quantification of mRNA revealed a decrease in LHR transcripts level in GDX+GnRHa group of females. A significant change was observed between GDX groups and GDX+GnRHa+E2 or T versus GDX+GnRHa group in females. High levels of LH decreased significantly the immature isoform of LHR in GDX group compared to control group in both genders, but low LH concentrations in GDX+GnRHa group induced immature LHR isoform production only in females. Therefore increased LH concentration induces production of incomplete LHR transcripts in hippocampal cells and decreases immature LHR at the protein level. This implies that LH decreases the efficiency of translation through either producing non-functional LHR molecules or preventing their translation.

Relationship between sperm chromatin status and ICSI outcome in men with obstructive azoospermia and unexplained infertile normozoospermia.

This study was done to evaluate the effect of sperm source on chromatin integrity and ICSI outcomes. One hundred and thirteen samples containing epididymal aspirates of 57 obstructive azoospermic men and 56-ejaculated semen of normozoospermic men were included in this study. Sperm chromatin status was evaluated by Chromomycin A3 (CMA3), Aniline Blue (AB) and Toluidine Blue (TB). Fertilization rate and embryo quality were recorded. In epididymal group the percentage of sperms stained with AB, CMA3 and TB were significantly higher compared to ejaculate group while fertilization rate (60.6% vs. 74.04%) was significantly lower. However, embryo quality was not significantly different between two groups. In addition, abnormal sperm chromatin condensation and DNA fragmentation were not correlated with fertilization rate and embryo quality. Our results highlight the role of epididymis in sperm maturation and confirm that ICSI using ejaculated sperm is the gold standard for treatment of infertile men.