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.

Comparison of intracytoplasmic sperm injection outcomes between spermatozoa retrieved from testicular biopsy and from ejaculation in cryptozoospermic men.

The infrequent presence of spermatozoa in cryptozoospermic men ejaculate is a limiting factor in the treatment of them. Sometimes, this consideration impels us to apply meticulous microscopic search in ejaculate or testicular sperm extraction (TESE) method. The aim of this study was to assess putative effectiveness of sperm origin, ejaculated or testicular, in cryptozoospermia treatment. In this context, were evaluated intracytoplasmic sperm injection (ICSI) outcomes in two parameters including fertilisation rate (2PN) and embryo quality, independently. We compared the outcome in two groups: patients who underwent ejaculate/ICSI and ones who underwent TESE/ICSI process. Nineteen ICSI cycles performed with testicular spermatozoa and the rest of cycles (n = 208) carried out with ejaculated spermatozoa. Result analysis showed similar fertilisation rate between testicular and ejaculated spermatozoa (respectively, 60% versus 68%, P ≥ 0.05). Also, on the other hand, embryo quality did not show significant differences between two groups, except grade A with low significance. With regard to almost equal performance of both methods in results and being invasive of TESE as surgical sperm retrieval method, the use of ejaculated sperm more than testicular sperm should be recommended in patients with cryptozoospermia whenever possible.

Evaluation of semen quality in patients with malignancies referred for sperm banking before cancer treatment.

Different cancer treatments such as chemotherapy and radiotherapy can lead to azoospermia and even sterility for an unknown period. Whether the type of cancer could affect semen quality or not is under debate. In this study, we have reviewed semen parameters of men with cancer who deposited their sperm samples at the Avicenna Research Institute tissue bank before undergoing cytotoxic treatment. This descriptive retrospective study examined 73 cases referred to sperm bank, because of malignancy, prior to initiation of cancer treatments including chemotherapy and radiotherapy. The data recorded were age, marital status, reproductive history, semen analysis reports and cancer history of the patients. Semen samples were analysed according to recommendations of the World Health Organization (1999) before freezing. Results of the analysis showed that 71.2% (52) of patients had oligozoospermia, 93.2% (68) teratozoospermia and 86.3% (63) asthenozoospermia. Different groups of cancer patients did not show any differences in oligozoospermia, teratozoospermia and asthenozoospermia. Impaired spermatogenesis even prior to cancer treatment indicates the importance of fertility preservation. As the majority of patients had suitable specimens for freezing and assisted reproduction, sperm banking is recommended to be performed promptly and before any treatment, especially surgery.

Coenzyme Q10 improves seminal oxidative defense but does not affect on semen parameters in idiopathic oligoasthenoteratozoospermia: a randomized double-blind, placebo controlled trial.

BACKGROUND: Several lines of evidence show the implication of oxidative stress in the etiology of male infertility. Recently, the role of coenzyme Q10 (CoQ10) in the prevention and treatment of disease has been intensively probed. However, definitive efficacy studies in oligoasthenoteratozoospermia (OAT) have not been completed yet. AIM: To evaluate the effect of CoQ10 supplementation on semen parameters in idiopathic OAT (iOAT). MATERIAL/SUBJECTS AND METHODS: A double-blind placebo controlled clinical trial was carried out. A total of 47 infertile men with iOAT were randomly assigned to receive 200 mg CoQ10 daily or placebo during a 12- week period. Semen parameters were determined using microscopic evaluation according to World Health Organization guidelines. Lipid peroxidation was assessed by measuring the concentration of plasma malondialdehyde. We evaluated the total antioxidant capacity of seminal plasma. To compare variables between and within the 2 groups we used independent t-test and Paired t-test. RESULTS: The trial showed non-significant changes in semen parameters of CoQ10 group. However, concentrations of thiobarbituric acid-reactive substances were significantly (p<0.05) reduced in serum of treated groups compared with the control. Furthermore, total antioxidant capacity of seminal plasma significantly increased in the CoQ10 group (p<0.05). CONCLUSION: Our results provide further evidence suggesting that CoQ10 supplementation is associated with alleviating oxidative stress, although it does not show any significant effects on sperm concentration, motility and morphology. It may be suggested that CoQ10 could be taken as an adjunct therapy in cases of OAT. Further studies are needed to draw a final conclusion.

Indoleamine 2,3-dioxygenase is expressed in the endometrium of cycling mice throughout the oestrous cycle.

Indoleamine 2,3-dioxygenase (IDO), an enzyme responsible for tryptophan catabolism, is thought to be required to prevent the rejection of the allogenic fetus by maternal T cells and to protect against intra- and extra-cellular pathogens. Consequently, we studied the expression of IDO in the endometrium of female Balb/c mice during the oestrous cycle. At each phase, the endometrium was peeled away and the relative expression of IDO mRNA was detected by semi-quantitative RT-PCR. The presence of IDO protein was confirmed in each phase by Western blotting and immunohistochemistry. Our results showed that IDO is expressed in the endometrium of cycling mice during all the phases of oestrous cycle. The expression of IDO was highest at the oestrus and lowest at the dioestrus. By means of Western blotting and immunohistochemistry, we obtained evidence that IDO protein is synthesised in the endometrium of cycling mice throughout the oestrous cycle. In accordance with RT-PCR results, IDO protein was predominant at the oestrus phase. IDO protein was mainly localised in the glandular and luminal epithelial cells. Our results support the concept of IDO providing a mechanism of innate immunity to protect from ascending infections of the female reproductive tract. In addition, considering the fact that mating only occurs during the oestrus phase, the high expression of IDO in this phase is likely to be a mechanism that induces immunological tolerance of the fetus.

Increased sperm ubiquitination correlates with abnormal chromatin integrity.

Ubiquitin, a 8.5 kDa peptide that marks other proteins for proteasomal degradation, tags defective spermatozoa during epididymal passage and is proposed as a biomarker for sperm quality. The present study was designed to evaluate the relationships between sperm ubiquitination, sperm chromatin integrity and semen parameters. Semen samples from 63 couples were collected and analysed according to World Health Organization criteria. Each sample was evaluated for sperm ubiquitination by the direct immunofluorescence method, using anti-ubiquitin antibodies. Chromatin integrity of the same samples was analysed using acridine orange (AO) and toluidine blue (TB) tests. A positive correlation was found between ubiquitinated spermatozoa and the percentage of spermatozoa with abnormal chromatin (AO: r = 0.58, P < 0.001 and TB: r = 0.48, P < 0.001). Negative correlations were obtained between sperm ubiquitination and: sperm count (r = -0.2, P = 0.048), sperm morphology (r = -0.36, P = 0.003), rapidly progressive motility (r = -0.25, P = 0.044) and slow progressive motility (r = -0.28, P = 0.022). Sperm ubiquitination was positively correlated with the percentage of immotile spermatozoa. These results show that among semen parameters, chromatin abnormality is more closely associated with sperm ubiquitination and further validate sperm ubiquitination as a suitable marker for sperm quality.

Prolonged survival of human spermatozoa when co-incubated with epididymal cell cultures.

Human epididymal tissue was recovered from 11 patients undergoing orchidectomy without anti-androgen treatment. Everted epithelial fragments from the caput and corpus epididymis of six patients were successfully cultured in a modified RPMI 1640 medium supplemented with HEPES and androgens for up to 110 days (mean 56 +/- 28) in 5% CO(2) in air at 37 degrees C. Epithelial cells from human oviduct and non-reproductive tract cells (breast epithelial cells, fibroblasts) were also cultured for comparison. The proportion of epididymal epithelial cells in primary cultures assessed by immunofluorescent localization using a cytokeratin monoclonal antibody was shown to be >70% for the first 6-8 weeks of culture. Light and electron microscopy indicated that epithelial cells maintained polarity and some normal morphology during the culture period. Washed epididymal or ejaculated spermatozoa prepared by a 'swim-up' procedure were co-incubated (i) directly with epididymal cells in culture wells, (ii) in 12 mm Millicell inserts within culture wells, thereby preventing contact of spermatozoa with culture cells; and (iii) in culture medium alone. A significant proportion of spermatozoa in direct contact with culture cells or in Millicell inserts were viable after 6 days of co-incubation (30-45%) and exhibited progressive motility, while all spermatozoa in medium alone were non-motile by 3 days. Using computer-assisted sperm analysis it was shown that the progressive motility of viable spermatozoa decreased gradually for the first 5 days in culture and then remained constant (approximately 30 microm/s, average path velocity). After 12 days of co-incubation, 15 +/- 4% of spermatozoa in direct contact with epithelial cells remained motile; in one experiment, a few spermatozoa (<1%) were motile at 17 days. Light and electron microscope observations indicated that prolonged sperm survival was associated with close apposition of spermatozoa (by equatorial segment) to the apical membrane of epithelial cells. Oviductal epithelial cells were also beneficial for sperm survival, but other cell types had no effect.

Fertilizing capacity of rat spermatozoa is correlated with decline in straight-line velocity measured by continuous computer-aided sperm analysis: epididymal rat spermatozoa from the proximal cauda have a greater fertilizing capacity in vitro than those from the distal cauda or vas deferens.

Rat spermatozoa recovered from different regions of the excurrent ducts of 10 adult males (proximal cauda epididymidis [PC], distal cauda epididymidis [DC], and vas deferens [VD]) were assessed by in vitro fertilization (LVF) using limited sperm numbers, and by continuous evaluation of motility parameters during 5 hours of incubation in vitro with automated computer-aided sperm analysis (CASA). Spermatozoa from the PC region fertilized (68 +/- 6%) a significantly greater (P < or = 0.005) number of oocytes than those from the DC (44 + 5%) or VD (47 +/- 7%). For pooled samples from all three regions, the mean fertilization rate (51 +/- 14%) was less tan for spermatozoa from the PC (P < 0.05) but was not significantly different from spermatozoa from the DC or VD. For each time point and sample, 1,592 +/- 428 sperm tracks were analyzed. CASA was verified by comparison with manual still-frame analysis of video recordings, by repeated analysis of the same or different samples of spermatozoa, and by examination of computer tracks. The coefficients of variation for various motion parameters suggested that the CASA obtained a high degree of precision. There were no significant differences in motility parameters for spermatozoa recovered from equivalent regions of the left or right tract or in motility parameters for spermatozoa from different regions of the tract immediately after recovery. However, during incubation in vitro, spermatozoa from the DC or VD regions exhibited a marked decline in straight-line velocity (VSL) compared with spermatozoa from the PC region. The reduction in VSL (combined values from right and left tract) for DC or VD spermatozoa compared with PC spermatozoa was significant at 2.5 hours of incubation (P < or = 0.05) and highly significant (P < or = 0.005) by the end of the incubation period. Differences in average path velocity (VAP) were also apparent after 4 hours (p < or = 0.05), but no significant differences were observed for measurements of curvilinear velocity (VCL) or lateral bead displacement (ALH). Overall, the decline in VSL over 5 hours was highly correlated (P < or = 0.001) with the outcome of fertilization in vitro. In contrast, initial VSL and changes in VCL of spermatozoa were not correlated with fertilization rate. These results indicate that the in vitro fertilizing capacity of rat spermatozoa is correlated with 1) the decline in straight-line velocity (VSL) as measured by repeated CASA during incubation in vitro and 2) with the site of recovery of mature rat spermatozoa from the distal excurrent duct. It is suggested that the deterioration of the quality of rat spermatozoa in the distal epididymidis and vas deferens during storage may occur sooner than previously realized, and therefore care must be taken when recovering samples for fertility assessment. In keeping with findings in other species, immediate "snapshot" analysis of rat motility was a poor predictor of sperm fertility. In contrast, continuous CASA provided significant information for determining sperm fertilizing capacity and will be a useful technique for reproductive toxicology.

In vitro maturation of mammalian spermatozoa.

During epididymal transit, mammalian spermatozoa undergo maturation and acquire full fertilizing capacity. The contribution of factors from the epididymal epithelium appears to be essential for this process. Although complete in vitro maturation of epididymal spermatozoa has not been achieved, stages of maturation can be induced under various conditions. The most successful results have been obtained by incubating epididymal spermatozoa with primary cultures of epididymal epithelium. These co-incubation methods promote sperm motility and the capacity of spermatozoa to bind to and fertilize oocytes, and extend the viability of spermatozoa in vitro. Specific androgen-dependent secretory proteins from epididymal principal cells that may be involved in this maturation process have been identified using pulse-labelling techniques.