Showing papers in "Journal of Andrology in 2002"

Oxidative stress and male infertility: from research bench to clinical practice.

Abstract: Extensive research in our center at the Cleveland Clinic indicates that the seminal oxidative stress test has diagnostic and prognostic capabilities beyond those of conventional tests of sperm quality or functions. An oxidative stress test can accurately discriminate between fertile and infertile men and identify patients with a clinical diagnosis of male-factor infertility who are likely to initiate a pregnancy if they are followed over a period of time. In addition, the test can help select subgroups of patients with infertility in which oxidative stress is a significant factor, and who may benefit from antioxidant supplementation. Incorporation of such a test into routine andrology laboratory practice may be of particular importance to the future management of male infertility. In recent years, the generation of reactive oxygen species (ROS) in the male reproductive tract has become a real concern because of their potential toxic effects at high levels on sperm quality and function. ROS are highly reactive oxidizing agents belonging to the class of free radicals (Aitken, 1994). A free radical is defined as ‘‘any atom or molecule that possesses one or more unpaired electrons’’ (Warren et al, 1987). Recent reports have indicated that high levels of ROS are detected in semen samples of 25% to 40% of infertile men (de Lamirande et al, 1995; Padron et al, 1997). However, a strong body of evidence suggests that small amounts of ROS are necessary for spermatozoa to acquire fertilizing capabilities (Aitken, 1999). Spermatozoa, like all cells living in aerobic conditions, constantly face the oxygen (O2) paradox: O2 is required to support life, but its metabolites such as ROS can modify cell functions, endanger cell survival, or both (de Lamirande and Gagnon, 1995). Hence, ROS must be continuously inactivated to keep only a small

Real-time fine morphology of motile human sperm cells is associated with IVF-ICSI outcome.

Abstract: The aim of the present prospective study was to determine whether subtle sperm morphological characteristics affect the outcome of intracytoplasmic sperm injection (ICSI), and if so, to identify those that are relevant. For this purpose, we developed a new method, the motile sperm organelle morphology examination (MSOME). The examination is performed in real time using an inverted light microscope equipped with high-power Nomarski optics enhanced by digital imaging to achieve a magnification up to 6300x. MSOME was applied to the leftover sperm fraction selected for microinjection in 100 random couples referred for ICSI treatment at 3 major in vitro fertilization centers. We found that the morphological normalcy of the entire sperm cell, according to MSOME criteria, was positively associated with ICSI fertilization rate (area under the receiver operating characteristics [ROC] curve, 88%) but not with pregnancy outcome. The morphological normalcy of the sperm nucleus, defined by MSOME, was significantly and positively associated with both fertilization rate and pregnancy outcome (areas under the ROC curve, 72% and 74%, respectively). These findings indicate that ICSI-associated pregnancy rate may be affected by subtle morphological malformations of the sperm nucleus, which may remain undetected by the embryologist during the routine selection procedure.

Localization of androgen and estrogen receptors in adult male mouse reproductive tract.

Abstract: There is considerable variation, both within and between species, in reports of nuclear steroid receptor localizations in the male reproductive tract. In this study, androgen receptor (AR) and estrogen receptors ERalpha and ERbeta were visualized by immunohistochemistry in adult male mice reproductive tracts, including testes, efferent ductules; initial segment, caput, corpus, and cauda epididymides; and vas deferens. Antibody specificity was demonstrated by Western blot and antibody competition. In testis, AR was expressed in Leydig cells, Sertoli cells, and most peritubular cells, but not in germ cells; Sertoli cells showed more intense staining in stages VI-VII; ERalpha was present in Leydig and some peritubular cells; ERbeta was in Leydig, some peritubular, all Sertoli and germ cells except in spermatids and meiotic spermatocytes. In efferent ductules, AR was strongly expressed in ciliated and nonciliated epithelial cells and in stromal cells; ERalpha was strongly expressed in ciliated and nonciliated epithelial cells; stromal cells were negative; and ERbeta was strongly expressed in ciliated and nonciliated epithelial cells and also in stromal cells. In epididymis, AR was strongly expressed in all epithelial cells (not in intraepithelial lymphocytes); ERalpha was strongly expressed in apical, narrow, and some basal cells of the initial segment, and in caput, principal cells of the caput, clear cells of the distal caput through cauda; stromal cells were negative in the initial segment, but more stromal cells were stained from caput to cauda; ERbeta was strongly expressed in most of epithelial cells of the epididymis, but stromal cells were inconsistently stained. In vas deferens, AR was weakly expressed or absent in principal cells but moderately stained in basal cells, smooth muscle cells of stroma were stained intensely, ERalpha was absent in epithelial cells but present in a subepithelial smooth muscle layer, and ERbeta was strongly expressed in all epithelial cells and most stromal cells. This study demonstrates that the reproductive tracts of male mice differ considerably from those of rats in expression of ARs and ERs and that caution is needed when extrapolating nuclear steroid receptor data across mammalian species.

CAG Repeat Expansion in the Androgen Receptor Gene Is Not Associated With Male Infertility in Indian Populations

Abstract: CAG repeat expansion in exon 1 of the androgen re- ceptor (AR) gene has been reported to be associated with male infertility in some but not all populations Until now, studies have not been carried out to examine this among Indian populations For the first time, we have analyzed the CAG repeat motif in the AR gene in 280 men with azoospermia and in 201 men with normal fertility The mean number of CAG repeats in the AR gene of men with azoospermia was 217 6 018, with a high incidence of repeat num- ber 22 Among fertile-control men, the mean number of CAG repeats was 224 6 019, with a predominance of repeat number 23 The highest number of CAG repeats (32) was found with low frequency in both fertile and azoospermic groups Comparison of fertile men and those with azoospermia on the basis of CAG repeats revealed that the number of CAG repeats in both groups were similar, as revealed with a paired t test (t 5 004; P 5 967) Expansion of the CAG repeat in the AR gene is therefore not associated with male infertility in Indian populations This suggests that what is true for one population may not be true for other populations

Development of a cryopreservation protocol for type A spermatogonia

Abstract: The aim of this study was to develop a cryopreservation protocol for type A spermatogonia. Testes from 5- to 7-month-old calves were collected, and type A spermatogonia were isolated using two-step enzymatic digestion and Percoll separation. Cells were resuspended in minimum essential medium (MEM) supplemented with 1% bovine serum albumin (BSA) in a final concentration of 6 x 10(6) per mL, and the effects of different cryoprotectants and freezing protocols were tested. Cells frozen/thawed in medium containing 10% fetal calf serum (FCS) and 1.4 M glycerol or dimethyl sulfoxide (DMSO) had a significantly (P <.05) higher percentage of living cells compared to medium with only FCS, whereas DMSO gave a significantly better cell survival rate than glycerol did. An increase in the concentration of FCS in the DMSO-based medium to 20% had no effect on survival after freezing and thawing. Furthermore, inclusion of 0.07, 0.14, or 0.21 M sucrose in DMSO-based medium resulted in a significant improvement of cell survival, cell proliferation in culture, and colonization efficiency in recipient testes. A controlled slow-freezing rate (1 degrees C/min) resulted in significantly (P <.05) more viable cells than fast (5 degrees C/min) freezing. However, noncontrolled-rate freezing, with a comparably low cooling rate, gave even better results than the controlled-rate slow freezing. Cryopreservation in MEM-based medium containing 10% FCS, 10% DMSO, and 0.07 M sucrose using a non-controlled-rate freezing protocol appeared to be a simple and effective way to preserve type A spermatogonia, with a high yield of almost 70% living cells after thawing. Frozen/thawed spermatogonia survived in culture and retained the ability to proliferate as determined by colorimetric and bromodeoxyuridine incorporation assays. To test whether the stem cells among the A spermatogonia retained their ability to colonize the testis of a recipient mouse, bovine spermatogonia were transplanted. This resulted in colonization 2-3 months after transplantation. In conclusion, for the first time, a method specific for cryopreservation of type A spermatogonia, including spermatogonial stem cells was developed, which allows long-term preservation of these cells without apparent harmful effects to their function.

Hormonal regulation of spermatogenesis in primates and man: Insights for development of the male hormonal contraceptive

Abstract: A better understanding of the hormonal regulation of spermatogenesis may provide new avenues for management of human infertility, and development of safe and reversible contraceptives. Although much basic work must be conducted in lower mammals, primarily rodents, concepts must, at some stage, be tested for their relevance to humans. In this review, we will draw together existing data in humans and monkeys in formulating an overview of the hormonal regulation of spermatogenesis, focusing particularly on the pursuit of the male hormonal contraceptive. Reference to data from lower mammals will be made when these highlight interesting and relevant differences or similarities with primates. The dual functions of the adult testis; namely, the production of spermatozoa (fertility) and the secretion of testosterone (virility), are both dependent on stimulation by the pituitary gonadotropins, follicle-stimulating hormone (FSH) and luteinizing hormone (LH), which are secreted in response to hypothalamic gonadotropin-releasing hormone (GnRH). Testosterone is essential for promoting spermatogenesis and is secreted by the adult Leydig cell under LH stimulation, and acts via androgen receptors (ARs) on Sertoli cells, Leydig cells, and peritubular cells. That testosterone exerts its effects on somatic cells rather than germ cells was highlighted by recent germ cell transplantation studies (Johnston et al, 2001). FSH acts via

Treatment of male hypogonadism with testosterone undecanoate injected at extended intervals of 12 weeks: a phase II study.

Abstract: This paper reports the result of an open-label, non-randomized clinical trial investigating the efficacy and safety of an injectable preparation of testosterone undecanoate (TU) dissolved in castor oil and given over a 3.2-year period. In a previous study we demonstrated that injections of TU every 6 weeks resulted in satisfactory substitution but a tendency toward testosterone accumulation. Here we investigate prolonged TU treatment at extended injection intervals in 7 hypogonadal men. Injections were given at gradually increasing intervals between the fifth and 10th injection, and from then on every 12 weeks. Steady state kinetics were obtained after the 13th injection. Well-being, sexual activity, clinical chemistry, prostate volume, and prostate-specific antigen (PSA) and serum hormone levels were monitored. Patients were clinically well-adjusted throughout the study. Before the next injection, testosterone, dihydrotestosterone, and estradiol levels were mostly within the normal range and showed a tendency to decrease with increasing injection intervals. Body weight, hemoglobin, serum lipids, PSA, and prostate volume did not change significantly during the 3.2 years of treatment. PSA levels were always within the normal limit. Maximal testosterone levels during steady state kinetics were measured after 1 week with 32.0 +/- 11.7 nmol/L (mean +/- SD). Before the last injection, mean testosterone concentrations were 12.6 +/- 3.7 nmol/L. Compared with conventional testosterone enanthate or cypionate treatment requiring injection intervals of 2-3 weeks and resulting in supraphysiological serum testosterone levels, injections of TU at intervals of up to 3 months offer an excellent alternative for substitution therapy of male hypogonadism.

Expression and regulation of aquaporins 1, 8, and 9 in the testis, efferent ducts, and epididymis of adult rats and during postnatal development.

Abstract: Aquaporins (AQPs) are membrane protein channels that allow the rapid passage of water through an epithelium containing tight junctions In the present study, light microscope immunocytochemistry was utilized to localize several members of the AQP family in the testis, efferent ducts, and epididymis of normal adult animals during postnatal development and after various experimental procedures on adult animals In the testis of normal adult animals, AQP-8 was expressed exclusively in Sertoli cells, while AQP-9 outlined Leydig cells In the efferent ducts, AQP-1 was expressed on the microvilli, basolateral plasma membranes, and apical endosomes of the nonciliated cells and cilia of ciliated cells, while AQP-9 was present only on the microvilli of nonciliated cells In the epididymis, AQP-9 was localized to the microvilli of the principal cells of all regions, with the most intense reaction being noted in the initial segment and cauda regions The clear cells of the cauda region expressed only AQP-9 AQP-1 was not expressed in the testis or the epididymal epithelium, but it was expressed over the endothelial cells of the vascular channels of the efferent ducts and epididymis After efferent duct ligation or orchidectomy, there was no change in the expression of AQP-1 or -9 over the microvilli or cilia of epithelial cells in the case of the efferent ducts, suggesting that testicular factors do not regulate their expression in this region In contrast, AQP-9 expression in the principal cells of the initial segment, but not of other regions, and also in the clear cells of the cauda region was dramatically reduced after both treatments As the expression was not restored to control levels by testosterone replacement, the data suggest that a luminal factor(s) derived from the testis regulates AQP-9 expression in the principal cells of the initial segment and in the clear cells of the cauda region Postnatal studies revealed that the expression of AQP-1 and -9 in the different cell types of the efferent ducts and epididymis occurred between days 7 and 29, eliminating sperm and high androgen levels as possible regulating factors Taken together, these data suggest cell specificity with respect to the expression of AQP-8 and -9 in the testis In the efferent ducts and epididymis, specificity exists in cell, region, and tissue distribution with respect to the expression of AQP-1 and -9, and their expression does not appear to be regulated by androgens

Effect of leukocytospermia on sperm DNA integrity: a negative effect in abnormal semen samples.

Abstract: Controversy exists over levels of DNA integrity in the sperm of fertile and infertile men. In addition, the effect of leukocytospermia on sperm DNA in these 2 groups is unclear. We decided to address these questions by collecting semen samples from men known or presumed to be fertile and men from infertile couples. Samples were analyzed and assessed for sperm concentration, motility, and morphology. Samples failing to meet World Health Organization (WHO) standards in one or more of these parameters were judged abnormal. Samples were then arbitrarily assigned normalized scores in each of the above parameters, and scores were summed to give a normalized value for overall sperm quality. DNA abnormality was determined by an in situ DNA denaturation test with acridine orange and expressed as a percentage of cells with abnormal DNA integrity (ADI). Assessment of 187 samples revealed a moderate inverse correlation between ADI and sperm quality (r =.58), although a large degree of ADI dispersion was observed in abnormal semen samples. The average ADI for normal and abnormal semen samples was 18% +/- 2.8% and 36% +/- 5.8%, respectively, with the threshold of 95% probability set at 30%. When sorted for leukocytospermia, the difference in ADI between normal and abnormal semen groups without leukocytospermia was much smaller (17% +/- 2.2% and 22% +/- 4.6%; P =.023). Leukocytospermia had no significant effect on ADI in the normal semen group (P = .46); however, ADI was more than double the ADI in the abnormal semen group (18% +/- 2.4% and 50% +/- 11%; P < .001). The results of our analysis show that at least 3 factors affect net DNA integrity in leukocytospermic samples that fail to meet WHO standards: 1) primary DNA damage, which is moderately inverse to sperm quality, in particular to sperm concentration; 2) effect of leukocytes increasing primary or provoking potential DNA damage in a cascade-like manner, particularly in sperm with poor morphology and motility; and 3) a decreasing proportion of cells with damaged DNA in semen with the worst quality.

Preclinical Evaluation of Sodium Cellulose Sulfate (Ushercell) as a Contraceptive Antimicrobial Agent

Abstract: The spread of sexually transmitted infections (STIs) and limited methods for control of pregnancies presents high risks to the reproductive health of women. Methods con- trolled by women and directed toward disease prevention and contraception are needed. We report on preclinical studies of the biological properties of sodium cellulose sulfate (Ushercell) cur- rently being developed for use as a topical contraceptive anti- microbial agent. Ushercell was evaluated with tests designed to identify its contraceptive and antimicrobial properties. Ushercell inhibits hyaluronidase (reversible; IC50 5 1.7 mg/mL), impairs sperm penetration of cervical mucus (approximately 70% inhibi- tion at 1 mg/mL), and acts as a stimulus for acrosomal loss (IC50 5 52 ng/mL). It prevents conception in rabbits when added to spermatozoa (approximately 95% inhibition at 1 mg/mL) or when vaginally applied (complete contraception by 45 mg) before in- semination. However, up to 50 mg/mL, Ushercell does not irre- versibly immobilize spermatozoa, suggesting that Ushercell is not cytotoxic. Ushercell has a broad spectrum of antimicrobial activity in vitro. Inhibited microbes include human immunodeficiency vi- ruses (different laboratory strains and clinical isolates; IC50 val- ues range from 3 to 78 mg/mL), herpes viruses, HSV-1 (IC50 5 59 ng/mL) and HSV-2 (IC50 5 24 ng/mL), Neisseria gonorrhoeae (IC50 5 2 mg/mL), and Chlamydia trachomatis (IC50 5 78 mg/mL). In contrast, Ushercell does not inhibit growth of beneficial vaginal bacteria, Lactobacillus gasseri, at 5 mg/mL. These results sug- gest that the antimicrobial effects of Ushercell are selective, and not likely mediated by nonspecific cytotoxic mechanisms. These data provide the basis for further clinical development of Usher- cell as a vaginal agent to prevent unplanned pregnancy and STIs.

Bicarbonate Stimulation of Boar Sperm Motility via a Protein Kinase A—Dependent Pathway: Between-Cell and Between-Ejaculate Differences Are Not Due to Deficiencies in Protein Kinase A Activation

Abstract: Because poorly motile sperm samples can often be stimulated by treatments that increase intracellular levels of cyclic adenosine monophosphate (cAMP), it has been supposed that such samples are unable to maintain an adequate supply of the cyclic nucleotide with which to activate protein kinase A (PKA). To investigate this hypothesis, we incubated boar sperm samples with bicarbonate (a stimulator of adenylyl cyclase) and compared its effect with that of 5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole 3‘,5’-cyclic monophosphothioate (cBIMPS, a highly permeable and stable cAMP analog). Videomicroscopy assessment of motility was followed by computer analysis of the sperm tracks to produce motility descriptor values for many individual cells in each sample, whence “cluster” analysis of these data identified groups of spermatozoa that differed in motility characteristics. Bicarbonate stimulation of motility was characterized by an increase in the linearity (LIN) and progressive velocity of part of the sperm population only. The size of this “fast linear” subpopulation varied considerably between ejaculates. However, treatment with cBIMPS did not induce significantly more “fast linear” sperm than treatment with bicarbonate. In further experiments investigating the role of protein kinases in motility control, bicarbonate stimulation was greatly inhibited by H89 (a specific inhibitor of PKA), whereas GF109203X and lavendustin A (inhibitors of protein kinase C [PKC] and protein tyrosine kinase [PTK], respectively) had essentially no effect. While inclusion of the protein phosphatase inhibitor calyculin stimulated motility, it failed to increase the overall percentage of “fast linear sperm” induced by bicarbonate. We conclude that intersperm and interejaculate differences in boar sperm motility are not due to inadequacy in cAMP supply or to ineffective PKA activity.

Activation of protein kinase A during human sperm capacitation and acrosome reaction.

Abstract: Spermatozoa undergo a variety of changes during their life that are prerequisites to their maturation and ability to fertilize eggs. Mammalian sperm capacitation and acrosome reaction are regulated by signal transduction systems involving cyclic adenosine monophosphate (cAMP) as a second messenger. This second messenger acts through the activation of protein kinase A (PKA) and indirectly regulates protein tyrosine phosphorylation. cAMP levels are controlled by a balance of phosphodiesterases (PDEs) and adenylyl cyclase (AC) enzymatic activities, which are responsible for its degradation and production, respectively. The aim of this study was to evaluate the possible relationship between the intracellular levels of cAMP and PDE and PKA activities during human sperm capacitation induced by fetal cord serum ultrafiltrate (FCSu) and acrosome reaction induced by calcium ionophore A23187. We report that PKA activity was higher in capacitating than in noncapacitating spermatozoa and that intracellular levels of cAMP decreased but that PDE activity remained constant during capacitation. The acrosome reaction induced by A23187 was associated with increases in cAMP and PKA activity but not in PDE activity. These results strongly suggest that net cAMP concentration is under the control of AC, since PDE activity is constant during sperm capacitation and the acrosome reaction. Moreover, the results suggest that low levels of cAMP are sufficient for capacitation and PKA activation and/or that the cAMP concentration measured in whole spermatozoa does not reflect the effective intracellular cAMP levels present in specific compartments of these cells.

Detection of Lipid Peroxidation in Equine Spermatozoa Based Upon the Lipophilic Fluorescent Dye C11‐BODIPY581/591

Abstract: The lipophilic fluorescent probe, 4,4-difluoro-5-(4-phenyl-1 ,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid (C11-BODIPY581/591) was used to evaluate changes in lipid peroxidation in equine spermatozoa during both short-term exposure to ferrous sulfate and sodium ascorbate in the presence of cumene hydroperoxide as well as during storage of spermatozoa at 5 degrees C for 48 hours. Peroxidation of C11-BODIPY581/591 was accompanied by a shift in fluorescence from red to green, and the relative amount of nonoxidized probe was determined as the ratio of red:(red + green) fluorescence as detected by either fluorescence microplate reader or by flow cytometry. The addition of Fe2SO4 (0 to 0.5 mM), low concentrations of sodium ascorbate, and the addition of cumene hydroperoxide increased peroxidation of C11-BODIPY581/591. The addition of high concentrations (10 or 20 mM) of sodium ascorbate or alpha-tocopherol reduced peroxidation of C11-BODIPY581/591 during short-term incubations. During storage at 5 degrees C in a skim milk-based extender, equine spermatozoa demonstrated a progressive decline in motility and a small but significant increase in lipid peroxidation based upon ratiometric analysis of C11-BODIPY581/591. The addition of Fe2SO4 increased lipid peroxidation in cooled spermatozoa in a dose-dependent fashion and decreased sperm motility. The addition of alpha-tocopherol, however, did not reduce lipid peroxidation during cooled semen storage. These data demonstrate that the lipophilic fluorescent probe C11-BODIPY581/591 is a useful measurement of lipid peroxidation in equine spermatozoa and that there is an increase in lipid peroxidation during cooled storage of equine spermatozoa that is increased in the presence of ferrous promoters.

P53 and Fas are sequential mechanisms of testicular germ cell apoptosis

Abstract: Testicular germ cell apoptosis in the cryptorchid testis is induced by abdominal heat stress. p53-dependent apoptosis appears responsible for the initial phase of germ cell loss in experimental cryptorchidism based on a 3-day delay of apoptosis in p53-/- mice. However, the mechanisms underlying the subsequent p53-independent apoptosis have not been identified. Although studies have suggested that Fas plays a role in testicular germ cell apoptosis, no direct evidence has been shown. To test the hypothesis that Fas is involved in testicular germ cell apoptosis and is responsible for the p53-independent phase of apoptosis in the cryptorchid testis, p53-/-, lpr/lpr (a spontaneous mutation in the Fas gene, which causes autoimmune disease) double-mutant mice were generated and unilateral cryptorchidism was induced in these mice. It was found that testicular weight reduction and germ cell apoptosis were delayed by an additional 3 days, and the Fas production increased in the time frame of p53-independent apoptosis in the experimental cryptorchid testis of wild-type mice. These results suggest that Fas is involved in testicular germ cell apoptosis, and that Fas-dependent apoptosis is responsible for the p53-independent phase of germ cell apoptosis in the cryptorchid testis. The cascade of testicular germ cell apoptosis in response to heat stress implies the existence of sequential quality control mechanisms in spermatogenesis.

Noncholinergic penile erection in mice lacking the gene for endothelial nitric oxide synthase.

Abstract: With the current understanding that nitric oxide (NO) mediates penile erection, the endothelial isoform of NO synthase (eNOS) has been implicated in this function. We undertook this study applying transgenic mice with targeted deletion of the eNOS gene (eNOS-/- mice) as an experimental approach to evaluate the importance of eNOS in cholinergically stimulated erectile function in vivo. Combined pharmacostimulation with intracavernosal carbachol (3 ng) administration and submaximal cavernous nerve (CN) electrical stimulation (16 Hz, 5 millisecond, 1 V) simultaneous with intracavernosal pressure (ICP) monitoring, and both biochemical assay of NO synthase activity and Western blot analysis of eNOS protein content in penile tissue, were performed on eNOS-/- mice and wild-type controls. Combined intracavernosal carbachol administration and submaximal CN electrical stimulation raised the recorded ICP, elicited by CN electrical stimulation alone in wild-type mice (from 35.7 +/- 2.7 to 48.1 +/- 5.5 mm Hg, P < .05) but not in eNOS-/ - mice (from 54.9 +/- 6.3 to 51.0 +/- 9.5 mm Hg, not significant [NS]). Pretreatment with the nonselective nitric oxide synthase inhibitor nitro-L-arginine methyl ester (L-NAME; 100 mg intracavernosally) blocked electrically stimulated ICP responses in eNOS-/- mice to baseline levels (37.8 +/- 4.4 vs 12.7 +/- 4.0 mm Hg, P < .05). In penes of eNOS-/- mice, approximately 60% NO synthase activity of wild-type penis levels was retained (NS), and eNOS protein was absent. We concluded that eNOS-/- mice preserve erectile function on the basis of a noncholinergic but NO-dependent mechanism and that eNOS physiologically mediates penile erection under cholinergic stimulation.

Peptide and nonpeptide reactive oxygen scavengers provide partial rescue of the testis after torsion.

Abstract: Ischemia-reperfusion (IR) of the testis results in germ cell-specific apoptosis, followed by a reduction in testis weight and daily sperm production (DSP). This has been associated with an increase in the adhesion of neutrophils to testicular subtunical venules and an increase in reactive oxygen species (ROS). The present study investigated: 1) the effects of a direct, non-IR-related ROS insult to the testis and 2) the effects of catalase, superoxide dismutase (SOD), and a novel nonpeptide mimic of SOD, M40403, on neutrophil recruitment, ROS production, testis weight, and DSP following IR of the rat testis. Results revealed that the infusion of H2O2 increased testicular lipid peroxidation 1 hour after administration and increased germ cell apoptosis within 24 hours of administration. Four hours after the repair of torsion plus vehicle infusion, there was a significant increase in myeloperoxidase (MPO) activity, an indicator of neutrophil accumulation, and thiobarbituric acid reactive substances (TBARS), a measure of ROS production, compared to equivalent data in sham-treated testes. Animals sacrificed 30 days after the torsion plus vehicle infusion revealed a significant decrease in testis weight and DSP compared to the same parameters in sham-operated animals. The treatment of animals with catalase plus SOD or M40403 showed a significant decrease in MPO activity and TBARS 4 hours after IR of the testis. Animals treated with SOD, SOD plus catalase, and M40403 provided a partial rescue of DSP 30 days after IR of the testis. These results demonstrate that oxidative stress can directly cause germ cell apoptosis, even outside the IR model, and confirm the importance of oxidative stress in testicular IR injury. Also, following testicular IR, there is a recruitment of neutrophils and an increase in ROS production in the testis. The administration of ROS scavengers significantly reduced the IR-induced responses. Interestingly, the administration of all ROS scavengers also blocked neutrophil recruitment to the testis. The mechanism by which ROS modulates neutrophil adhesion to venules is presently under investigation, as are additional therapeutic regimens to block oxidative stress.

Host defense proteins of the male reproductive tract.

Abstract: Innate immunity, the rapidly responsive and phylogenetically ancient system of host defense, is generating increasing interest in part due to the growing appreciation for its remarkable broad spectrum effectiveness. We dwell in a sea of micropredators that are well equipped to colonize and invade our tissues. Yet we coexist with these organisms day after day without chronic inflammation, tissue damage, or even awareness of their presence. Coexistence occurs largely because the innate immune system, which includes antimicrobial proteins, complement, and phagocytes, prevents invasion by destroying or evicting the microbes. Only those maintaining commensal status are allowed to colonize. In addition, evidence is emerging to indicate that antimicrobial proteins stimulate the adaptive immune response. When the innate immune system is overwhelmed and tissue invasion does occur, the T lymphocytes and antibody-producing B lymphocytes of the adaptive system are recruited to eliminate the infection. The success of plants, insects, and other lower animals that lack adaptive immunity affirms the effectiveness of the innate immune system. Today, more than 700 antimicrobial proteins are known in the plant and animal kingdoms. The Antimicrobial Sequences Database lists 752 eukaryotic entries as of February 12, 2002 (Tossi et al, 2002); this is a dramatic increase since the original discoveries in plants 30 years ago (Fernandez-de-Caleya et al, 1972), and in animals 20 years ago (Hultmark et al, 1980; Steiner et al, 1981; Selsted et al, 1983). Through improved understanding of innate antimicrobial proteins, ways to mimic or enhance their activities

Mild testicular hyperthermia induces profound transitional spermatogenic suppression through increased germ cell apoptosis in adult Cynomolgus monkeys (Macaca fascicularis)

Abstract: We have previously demonstrated that mild testicular hyperthermia induces stage-specific and germ cell-specific apoptosis in rat and mouse testes. The objectives of this pilot study were to examine whether mild testicular hyperthermia induces azoospermia and oligozoospermia in nonhuman primates, and to determine whether spermatogenesis suppression was due to acceleration of germ cell apoptosis. Three adult Cynomolgus monkeys (Macaca fascicularis) were used in this study. The scrota containing the testes were immersed in a water bath at 43 degrees C for 30 minutes once daily for 6 consecutive days. Semen and blood samples were collected at 2 and 1 weeks before, and 2, 4, 6, 8, 10, and 12 weeks after the first heat treatment. Testicular biopsies were performed before and at 3 and 7 days, and 12 weeks after the first heat exposure. Apoptosis in testicular biopsy was assessed by TUNEL assay, by electron microscopy, and by detection of cleaved Poly(ADP-ribose)polymerase with Western blotting. A transient decrease in serum testosterone levels was observed in 2 monkeys 2 weeks after heat treatment. Serum inhibin B levels declined in all 3 monkeys 2 weeks after testicular hyperthermia and remained at relatively low levels throughout the study in 2 of 3 monkeys. Two of 3 monkeys exhibited azoospermia by 6 or 8 weeks after the first heat treatment; the remaining monkey had marked oligozoospermia (8 x 10(6)/ejaculate, 10.89% of pretreatment levels) 6 weeks after the first heat treatment. Increased germ cell apoptosis in testicular biopsy samples was found at 3 and 7 days after the first heat exposure. Using immunohistochemistry, we observed that the immunoactivity of proapoptotic Bax protein accumulated in heat-induced apoptotic germ cells. Full recovery of spermatogenesis was noted 12 weeks after the first heat treatment. We conclude that, similar to rodents, mild testicular hyperthermia results in azoospermia and oligozoospermia in monkeys through increased germ cell apoptosis with minimal effect on the hormonal milieu.

Detection of DNA Damage in Response to Cooling Injury in Equine Spermatozoa Using Single-Cell Gel Electrophoresis

Abstract: Single-cell gel electrophoresis (SCGE), or comet as- say, has the ability to detect damage at the single cell level and has not been reported for equine sperm. The ability to detect nuclear damage at the single cell level could aid in the advancement of protocols for optimal semen preservation. The goals of these ex- periments were to adapt this assay for use with equine sperm and to utilize the assay for determining the integrity of equine sperm DNA following treatments with storage at various decreased temperatures (20C and 5C). Results from experiments in which sperm were frozen (20C) in the absence of cryoprotectants revealed that sig- nificantly more cells with fragmented tails of DNA, or comets, oc- curred among those exposed to 1, 3, and 5 freeze-thaw cycles (65% 6%, 76% 11%, 92% 6%, respectively) compared with fresh, untreated sperm (19% 16%, P.05). In addition, DNA damage was different (P.05) between the three freeze-thaw treatments. Sensitivity of SCGE on equine sperm was further tested with known ratios of frozen-thawed and fresh cells. The amount of detectable DNA damage was positively correlated with the percentage of cryo- damaged cells in each treatment (r 2 0.92, P.05). Potential damage as a result of cooled storage was also investigated and results revealed that sperm stored for 48 hours (at 5C) had a higher percentage of comets than that of fresh sperm (63% 13.9% and 28% 15.6%, respectively, P.05). The percentage of viable sperm also decreased linearly over time and was inversely corre- lated with percent of comets (r 2 0.805, P.001). Detection of sublethal and/or uncompensable fertility factors in semen, such as DNA fragmentation, could be useful for detecting male differences in semen for cooling or cryopreservation potential and could provide a tool for monitoring and preserving fertility for individual stallions.

Cholesterol transport, peripheral benzodiazepine receptor, and steroidogenesis in aging Leydig cells. Commentary. Author's reply

Abstract: The cellular mechanisms responsible for age-related decline in the ability of Leydig cells to produce testosterone are not yet fully understood. The decline in testosterone production could result from a reduction in the Leydig cell enzymatic activities mediating testosterone synthesis, the amount of substrate available for these enzymes, or both. In the present study, we examined the effect of age on a critical early step in the steroidogenic pathway, the transport of cholesterol into mitochondria. Leydig cells were isolated from the testes of young and old Brown Norway rats and incubated with human chorionic gonadotropin (hCG) and the side-chain cleavage cytochrome P450scc inhibitor aminoglutethimide (AMG). Mitochondria were isolated from these cells in the presence of AMG. Upon removal of AMG, the mitochondria from old cells produced 80% less steroid than those from young cells, only a fraction of which could be accounted for by a decrease in P450scc activity. These results suggest that the accumulation of hormonally recruited cholesterol into mitochondria is defective in old Leydig cells. With this in mind, we turned our attention to peripheral benzodiazepine receptor (PBR), a mitochondrial cholesterol-binding protein known to be involved in mediating cholesterol transport. PBR messenger RNA (mRNA) and protein expression were decreased in old cells. Moreover, both the dissociation constant (Kd) and the number of binding sites (Bmax) of the PBR were decreased in the old cells by 50% and 30%, respectively. Taken together, these results suggest that alterations in cholesterol transport and in PBR may play critical roles in age-related decreases in testosterone production in Brown Norway rat Leydig cells.

Melatonin administration alters semen quality in healthy men.

Abstract: The role of melatonin in the regulation of reproduction in humans is unknown. We conducted a 6-month, double-blind, crossover study of a daily treatment dose of 3 mg melatonin or placebo given orally at 1700 hours in 8 healthy men. Semen quality (concentration, motility, and morphology), serum and seminal plasma 17-beta-estradiol (E(2)), testosterone, melatonin, and serum gonadotropin levels were determined every 3 months throughout the study. In 6 men, there was no change in semen quality or in serum and seminal plasma hormone levels during the study period. In 2 men, during the melatonin treatment period, sperm concentration decreased to 3 x 10(6)/mL and 12 x 10(6)/mL, and motility declined to 32% and 30%. These coincided with a decline in seminal plasma and serum E(2) levels and with an increase in testosterone:E(2) ratios. Six months after the cessation of melatonin, sperm concentration and motility were normal in 1 man but remained abnormal in the other one with a still elevated testosterone:E(2) ratio. Serum gonadotropin levels were unchanged during the study in all 8 men. Our preliminary observations suggest that long-term melatonin administration is associated with decreased semen quality in a number of healthy men, probably through the inhibition of aromatase at the testicular level.

Cholesterol Transport, Peripheral Benzodiazepine Receptor, and Steroidogenesis in Aging Leydig Cells

Abstract: The cellular mechanisms responsible for age-related decline in the ability of Leydig cells to produce testosterone are not yet fully understood. The decline in testosterone production could result from a reduction in the Leydig cell enzymatic activities mediating testosterone synthesis, the amount of substrate available for these enzymes, or both. In the present study, we examined the effect of age on a critical early step in the steroidogenic pathway, the transport of cholesterol into mitochondria. Leydig cells were isolated from the testes of young and old Brown Norway rats and incubated with human chorionic gonadotropin (hCG) and the side-chain cleavage cytochrome P450scc inhibitor aminoglutethimide (AMG). Mitochondria were isolated from these cells in the presence of AMG. Upon removal of AMG, the mitochondria from old cells produced 80% less steroid than those from young cells, only a fraction of which could be accounted for by a decrease in P450scc activity. These results suggest that the accumulation of hormonally recruited cholesterol into mitochondria is defective in old Leydig cells. With this in mind, we turned our attention to peripheral benzodiazepine receptor (PBR), a mitochondrial cholesterol-binding protein known to be involved in mediating cholesterol transport. PBR messenger RNA (mRNA) and protein expression were decreased in old cells. Moreover, both the dissociation constant (Kd) and the number of binding sites (Bmax) of the PBR were decreased in the old cells by 50% and 30%, respectively. Taken together, these results suggest that alterations in cholesterol transport and in PBR may play critical roles in age-related decreases in testosterone production in Brown Norway rat Leydig cells.

Vitamin D and Prostate Cancer

Abstract: The original hypothesis of Schwartz and Hulka (1990) proposing that vitamin D deficiency may be a risk factor for prostate cancer has triggered many studies. Epidemiological studies have supported this hypothesis with findings that sunlight exposure is inversely proportional to prostate cancer mortality and that prostate cancer risk is greater in men with lower levels of vitamin D (Hanchette and Schwartz, 1992; Corder et al, 1993; Ahonen et al, 2000). Prostate cancer cells express receptors for 1,25(OH)2D3 and some cell lines are growth inhibited when treated with 1,25(OH)2D3 (reviewed in Blutt and Weigel, 1999). The mechanism of action of these growth inhibitory effects of 1,25(OH)2D3 in LNCaP cells involves G1 accumulation, induction of quiescence, and an increase in apoptosis of the cancer cells (Blutt et al, 1997, 2000a; Zhuang and Burnstein, 1998). In vivo, 1,25(OH)2D3 and its analogs slow tumor growth and hinder metastasis of prostate tumors in rodent models (Schwartz et al, 1995; Getzenberg et al, 1997; Lokeshwar et al, 1999; Blutt et al, 2000b), and 1,25(OH)2D3 may have clinically relevant effects (Gross et al, 1998). More work is required to elucidate the mechanism of 1,25(OH)2D3 action in prostate cancer cells and to identify optimal 1,25(OH)2D3 analogs in a search for compounds with a better separation of growth inhibitory effects from hypercalcemic effects.

Localization of the sperm protein SP22 and inhibition of fertility in vivo and in vitro.

Abstract: We previously established that levels of the sperm membrane protein, SP22, are highly correlated with the fertility of sperm from the cauda epididymidis of rats exposed to both epididymal and testicular toxicants, and that a testis-specific SP22 transcript is expressed in postmeiotic germ cells. In this study, polyclonal and monoclonal antibodies were generated to study the expression of SP22 in the testis and epididymis, and to determine whether SP22 plays a coincidental or causal role in fertility. Polyclonal antiserum was raised in sheep against full-length recombinant rat SP22 (rSP22). Hybridoma clones were generated from mice immunized with rSP22 and boosted with native SP22; positive clones were used for ascites production. Immunoblots indicated that affinity-purified anti-rSP22 immunoglobulin (Ig) and ascites Ig recognized denatured and native SP22, respectively. Linear epitope mapping of the 189-amino acid SP22 sequence revealed 3 distinct peptide sequences recognized by anti-rSP22 Ig, and 1 sequence recognized by ascites Ig. Cytoplasm of round spermatids and heads of elongating/elongated spermatids immunostained with both anti-rSP22 and ascites antibodies. Isolated rete testis sperm revealed discrete staining over the cytoplasmic droplet, whereas staining was apparent over the equatorial segment of the head by the time sperm reached the caput epididymidis. Clear cells were, interestingly, immunostained along the length of the epididymis. Ascites Ig and anti-SP22 Ig each recognized the equatorial segment of sperm heads from rat, hamster, bull, rabbit, and human. Ascites Ig and affinity-purified anti-rSP22 Ig each significantly inhibited the fertility of cauda epididymal sperm from the rat in vivo, as well as the fertilization rates of cauda epididymal sperm in vitro. Moreover, affinity-purified anti-rSP22 significantly inhibited in vitro fertilization of both zona-intact and zona-free hamster oocytes, suggesting that SP22 may play a role in both the zona penetration and membrane fusion steps of fertilization.

Puberty is delayed in male growth hormone receptor gene-disrupted mice.

Abstract: The role of insulin-like growth factor-I (IGF-I) in the initiation of puberty and testicular function is poorly understood Growth hormone (GH) receptor (R) gene-disrupted mice or GHR gene "knockouts" (GHR-KO) are GH resistant and IGF-I deficient To assess whether the age of sexual maturation is affected by the absence of IGF-I, various parameters of sexual development including testicular and accessory reproductive organ weights, balanopreputial separation, germ cell development, and intratesticular testosterone levels were determined in normal and GHR-KO mice between the ages of 25 and 60 days In addition, at 36 days of age, the testosterone response to luteinizing hormone (LH) treatment was assessed in these mice The results indicate that the balanopreputial separation was delayed 5 days, and a significant increase in the weights of the seminal vesicles (SV) occurred later in GHR-KO mice than in normal animals (between 30 and 35 days and between 35 and 40 days, respectively) Also, the weights of testes and epididymii were significantly reduced in GHR-KO mice The intratesticular testosterone levels and the testosterone response to LH treatment were attenuated in GHR gene-disrupted mice Furthermore, elongated spermatids appeared later in the testes of GHR-KO mice than in the testes of normal mice These results suggest that the absence of IGF-I secretion delays the normal course of sexual maturation in male GHR-KO mice, indicating that IGF-I plays an important role in the initiation of puberty in male mice