Abstract: The objective of this study was to examine the relationship between DNA integrity and protamines in human sperm. One hundred forty-nine male infertility patients were included in an Institutional Review Board-approved study. Sperm were evaluated for DNA fragmentation using the DNA Integrity Assay, a test equivalent to the sperm chromatin structure assay (SCSA). Additionally, nuclear proteins were extracted and the protamine-1/protamine-2 ratio (P1/P2), protamine-1 (P1), protamine-2 (P2), and total protamine concentrations were evaluated. We identified 37 patients with abnormally low P1/P2 ratios, 99 patients with normal P1/P2 ratios, and 13 patients with abnormally high P1/P2 ratios. DNA fragmentation was significantly elevated in patients with low P1/P2 ratios (37.1 +/- 6.02) vs those with normal and high P1/P2 ratios (26.7 +/- 1.9 and 23.8 +/- 3.2, respectively; P < .05) and was inversely correlated with the P1/P2 ratio (R(s) -0.18, P < .05), P1 concentration (R(s) -0.29, P < .001), P2 concentration (R(s) - 0.24, P < .005), and total protamine concentration (R(s) -0.28, P < .001). Furthermore, chi2 analysis revealed a significant increase in the incidence of marked DNA fragmentation in patients with diminished levels of either P1 or P2. The present study is the first to report that human sperm protamine content is significantly related to DNA fragmentation. In particular, sperm P1 and P2 concentrations inversely correlate with DNA fragmentation, indicating a protective role of the protamines against sperm DNA damage. In light of recent studies highlighting the negative effect of sperm DNA damage on ART outcomes, these findings indicate a possible clinical significance for human sperm protamine levels.

Abstract: We recently described a complex population of spermatozoal coding RNAs that are delivered to the oocyte on fertilization. These are derived throughout spermatogenesis, representing a record of past events. Recently, evidence has been provided that micro-RNAs are present in testes, suggesting that they might also be carried in ejaculate spermatozoa. To directly test this hypothesis, a unique microarray system capable of directly identifying antisense RNAs and predicted transcripts was utilized. RNA isolated from the ejaculate spermatozoa of 6 normal fertile men was directly hybridized to sense oligonucleotide arrays containing 10 000 elements. This revealed 68 shared RNAs, some of which are similar to those previously defined as micro-RNAs, whereas others were the antisense of previously in silico-predicted transcripts. The results and implications of this study are described in this communication.

Abstract: Testosterone (T) administration to men increases lean body mass and decreases fat mass. Adiponectin is produced by adipocytes and is thought to influence insulin sensitivity. In this study, we sought to determine whether experimental alterations in serum T change adiponectin levels in normal men. We measured adiponectin levels in 28 healthy men ages 18-35 years before and during treatment with a potent gonadotropin-releasing-hormone (GnRH) antagonist, acyline. Decreased T levels led to increased serum adiponectin within 7 days; maximal adiponectin levels were reached on day 21 (baseline 8.6 +/- 0.9 compared with 12.2 +/- 1.0 microg/mL on day 21, P <.05) and persisted through day 30, despite no significant changes in body mass index (BMI) and an increase in leptin. The addition of T to acyline, maintaining serum T levels within the normal range, prevented the increase in adiponectin following acyline alone. In a second study, 25 men aged 55-85 years were treated with 3 weeks of high-dose T (testosterone enanthate [TE], 600 mg/wk intramuscularly). With high serum T levels, adiponectin levels decreased significantly by day 21 of treatment (baseline 14.3 +/- 1.9 compared with 10.8 +/- 1.5 microg/mL, P <.05 vs baseline and placebo), BMI slightly increased, and leptin levels were decreased. We conclude that adiponectin levels increase within days of experimental T deficiency in normal men, and the increase in adiponectin is prevented by T replacement. Furthermore, supraphysiologic T administration results in decreased adiponectin levels. Our data support the hypothesis that T, its metabolites, or both directly suppress adipocyte production of adiponectin.

Abstract: Androgens are deemed to be critical for the development, growth, and maintenance of penile tissue as well as for erectile function Androgens are also reported to inhibit differentiation of stroma progenitor cells into adipocytes and promote differentiation into smooth muscle The objective of this study was to investigate whether androgen deprivation results in accumulation of adipocytes in the corpus cavernosum Mature, New Zealand white male rabbits were subjected to sham surgery (control) or orchiectomy Two weeks after surgery, erectile function was assessed by monitoring changes in intracavernosal blood pressure (ICP) in response to pelvic nerve stimulation All ICP measurements were normalized to the mean systemic arterial blood pressure In parallel studies, penile cross sections from control and orchiectomized rabbits were fixed and stained with either Masson's trichrome or hematoxylin and eosin to assess smooth muscle and connective tissue content Alternatively, tissue sections were stained with Toluidine blue to assess accumulation of fat-containing cells Orchiectomy resulted in loss of erectile function and penile atrophy, associated with reduced trabecular smooth muscle and increased connective tissue content Most strikingly, tissue from orchiectomized animals exhibited accumulation of fat-containing cells (adipocytes) in the subtunical region of the corpus cavernosum We hypothesize that androgen deprivation promotes differentiation of progenitor stroma cells into an adipogenic lineage producing fat-containing cells, thus altering erectile function

Abstract: A statistical approach using sequentially principal component analysis (PCA), clustering, and discriminant analyses was developed to identify sperm morphometric subpopulations in well-defined portions of the fresh boar ejaculate. Semen was obtained as 2 portions (the first 10 mL of the sperm-rich fraction and the rest of the ejaculate, respectively) and frozen using a conventional protocol. Before freezing, an aliquot was used for computer-assisted sperm morphometry analysis (ASMA). Postthaw quality was evaluated using computer-assisted sperm analysis (CASA), and an annexin-V/PI assay evaluated sperm membranes. The PCA revealed that 3 variables represented more than 78% of the cumulative variance in sperm subpopulations. The clustering and discriminant analyses, based on 5780 individual spermatozoa, revealed the existence of 4 sperm subpopulations. The relative percentage of these subpopulations varied between boar and ejaculate portions. Linear regression models based on measured morphometric characteristics could account for up to 36% of the percentage of intact sperm membranes postthaw. The ASMA protocol used in our study was useful to detect subtle morphometric differences between spermatozoa, and the combination of this analysis with a multivariate statistical procedure gave new information on the biological characteristics of boar ejaculates that is not given by conventional sperm analysis.

Abstract: The present study evaluates the effect that various steps of a conventional cycle of cryopreservation have on the patterns of movement exhibited by boar spermatozoa. Sperm-rich ejaculate fractions collected from 24 mature fertile boars (1 ejaculate per boar) were cryopreserved following a standard freeze-thaw procedure with 0.5-mL plastic straws. Overall sperm motility and the individual kinematic parameters of motile spermatozoa (assessed by the computer-aided sperm analysis system Sperm Class Analyzer [SCA]) were recorded in 5 steps of the cryopreservation procedure. These steps were as follows: 1) at the time that the fresh semen was extended, 2) at 17 degrees C, after sperm concentration by centrifugation and re-extension of the pellet with lactose-egg yolk extender; 3) at 5 degrees C, after added freezing extender; 4) at the time that thawed semen was held in a water bath at 37 degrees C for 30 minutes; and 5) at the time that thawed semen was held in a water bath at 37 degrees C for 150 minutes. Data from individual motile spermatozoa, defined by 7 kinematic parameters (curvilinear velocity [VCL], straight-line velocity [VSL], average path velocity [VAP], linearity [LIN], straightness [STR], mean amplitude of lateral head displacement [ALH], and beat cross frequency [BCF]), were analyzed using a pattern analysis technique (PATN) to identify and quantify populations and subpopulations of motile sperm within the semen samples. After the first cluster analysis, 3 motile sperm populations (P) were identified (P1: progressive and/or vigorous cells [90.4%], P2: poorly progressive cells [8.3%], and P3: nonprogressive cells [1.3%]). These populations remained constant (P greater than .05) throughout the 5-step cryopreservation procedure. A second PATN was carried out within the P1 sperm population, which identified 3 sperm subpopulations (sP) (eg, sP1: cells with progressive and vigorous movement [58.7%], sP2: progressive cells only [24.6%], and sP3: vigorous cells only, hyperactive-like [16.7%]). Although the relative frequency of these 3 subpopulations varied among ejaculates (boars), there was no interaction with any cryopreservation step we examined. Whereas sP1 remained constant (P greater than .05), sP2 and sP3 varied significantly (P less than .05) through the cryopreservation procedure, with the increase in sP3 after centrifugation at 17 degrees C and during cooling at 5 degrees C considered particularly relevant. In conclusion, the present study confirms the heterogeneity of sperm movement patterns in boar semen, patterns that vary through the cryopreservation procedure, especially after removal of the seminal plasma by centrifugation and subsequent extension at 17 degrees C and after the slow cooling at 5 degrees C, when obvious increases in hyperactivated movement appeared. The vast majority of spermatozoa, those exhibiting progressive and vigorous movement, remained constant during the cryopreservation procedure, although the proportion differed among boars.

Abstract: We have already shown that seminal plasma proteins in the ram can repair cold-shock sperm membrane damage and that the addition of seminal plasma proteins before cold-shock treatment prevents sperm membrane injury. In this study, we prove that 2 protein bands of approximately 14 (P14) and 20 (P20) kd isolated from seminal plasma are responsible for this protective effect. In vitro capacitation (CA) and acrosome reaction (AR) modified the content of both proteins on the sperm surface. P20 release began at the beginning of CA, and the induction of the AR accounted for an additional release of both proteins; not more than 35% of these proteins remained on acrosome-reacted sperm. Cytochemical analysis detected that there are several binding sites for P14 and P20 on the sperm surface and that membrane alterations induced by CA and the AR accounted for the loss and redistribution of both proteins to the equatorial and postequatorial regions. The P14-sequenced fragment showed a high homology with several seminal plasma proteins of different species and contained the FN 2 domain like bovine PDC-109. However, the sequence of the P20 fragment was not homologous with any reported protein. By immunochemical analysis, we obtained evidence that P14 is phosphorylated in serine and threonine residues and that P20 is a glycosylated protein. These results suggest that both proteins are involved in sperm CA and gamete interaction, first by stabilizing the sperm membrane and then by participating in CA in the female reproductive tract. The protective effect of P14 and P20 could be related to their decapacitating role.

Abstract: Exposure to cisplatin results in impaired spermatogenesis, azoospermia, and, sometimes, permanent infertility in male patients. The mechanism(s) by which cisplatin induces damage to testicular cells is poorly understood. We previously reported that acute exposure to cisplatin results in elevated germ cell apoptotic rates and that this indicates long-term damage to the seminiferous epithelium. Here, we present data that implicate an injury to Sertoli cells as a possible mechanism to explain an elevated rate of germ cell apoptosis and consequent infertility. Normal adult C57/Bl/6J mice were exposed to 1, 2, or 4 rounds of 1, 2.5, or 5 mg/kg cisplatin in a regimen designed to resemble clinical chemotherapeutic exposure (1 injection daily for 5 days with a recovery phase of 16 days between cycles). A dose-dependent reduction in testicular weight due to germ cell loss was observed. While exposure to 1 mg/kg caused only temporary germ cell depletion, higher doses (2.5 and 5 mg/kg) revealed widespread testicular atrophy as evidenced by gaps in the epithelium due to cytoplasmic vacuolization and loss of differentiating germ cells. Although the acute loss of germ cells by apoptosis can result in temporary infertility, the testis has the ability to repopulate itself with mature cells, provided the stem germ cell population remains unharmed. Here, we demonstrate that a sustained disruption of spermatogenesis occurs despite the continued presence of stem spermatogonia in the seminiferous epithelium. These results suggest that cisplatin-induced germ cell loss may occur, in part, as a result of Sertoli cell injury-dependent alterations in germ cell microenvironment.

Abstract: Chronic pelvic pain syndrome is a common and se- rious health problem affecting the quality of life in men. Limited stud- ies exist on the relation of this condition to premature ejaculation. We evaluated prevalence rates of premature ejaculation in Turkish male patients with chronic pelvic pain syndrome and compared them with healthy control subjects. Sixty-six men with chronic pelvic pain syndrome were included in the study (group 1). A questionnaire con- sisting of 2 parts—demographic data and a Turkish version of the National Institutes of Health Chronic Prostatitis Symptom index— was administered to all patients. Premature ejaculation was defined as intravaginal ejaculation latency of less than 2 minutes with the same partner for at least 6 months. All patients were evaluated with physical examinations and routine laboratory tests. If erectile dys- function was noted from the medical history, penile Doppler ultra- sonography also was performed. The results were compared with the results of 30 healthy men without urinary symptoms (group 2). The x2 test was used for statistical analyses. Of 66 patients with chronic pelvic pain syndrome, 51 had premature ejaculation (77.3%), and in 10 (15.2%) patients, premature ejaculation and erectile dys- function were found together. Penile Doppler ultrasonography showed no vascular pathology in patients with erectile dysfunction. The rate of premature ejaculation was higher in patients in the study group than it was in patients in the control group, and this difference was statistically significant (P , .05). Both chronic pelvic pain syn- drome and premature ejaculation are common disorders, but their ethiopathogeneses are not well understood. In Turkish men with chronic pelvic pain syndrome, the incidence of psychogenic sexual problems was higher than in the normal population.

Abstract: Mammalian members of the cysteine-rich secretory protein (CRISP) family are expressed predominantly in the male reproductive tract and are implicated in the process of reproduction from spermiogenesis, posttesticular sperm maturation, and capacitation to oocyte-sperm fusion, and possibly also penetration of the zona pellucida. Rodents express only 2 CRISPs (CRISP-1 and CRISP-2) in their male reproductive system, whereas humans and horses express an additional third member named CRISP-3. We have previously demonstrated that this protein is present in human seminal plasma as well as in other exocrine secretions, in blood plasma, and in neutrophilic granulocytes. To characterize the protein in seminal plasma and localize the production of CRISP-3 in the human male reproductive tract, we performed immunoblotting and enzyme-linked immunosorbent assay measurements of seminal plasma and immunohistochemistry and in situ hybridization of tissue specimens. We were able to show that human CRISP-3 is a quantitatively minor seminal plasma protein not associated with prostasomes. Furthermore, CRISP-3 expression was found in the secretory epithelium throughout the male genital tract, with particularly high expression in the cauda epididymis and ampulla vas deferens. Examination of seminal plasma from vasectomized males indicates that organs downstream of the epididymis are probably the major sources of seminal plasma CRISP-3.

Abstract: Capillary loaded chambers are frequently used for semen analysis. Poiseuille flow of specimen into these chambers causes migration of suspended particles or cells in a direction transverse to the flow, which results in their preferential accumulation in the Segre-Silberberg (SS) planes. This SS effect depends on the transverse velocity gradient in the laminar flow. For semen analysis in thin capillary-loaded slides, the SS effect can lead to erroneous estimation of sample sperm-cell concentration. To better understand chamber flow dynamics and SS effect significance, we assessed flow uniformity, inflow cell velocity, and results of concentration measurements under different flow conditions for latex bead and porcine and human sperm suspensions. Overall, a concentration peak was present at the meniscus, which continued through chamber loading. High-velocity SS preferred planes, which channeled particles toward the meniscus, were located at the fractional positions of beta = .27 and beta = .73, where beta is the distance from wall to plane normalized to the chamber depth. In computer-automated semen analysis, a standard 20-microm x 18-mm x 6-mm chamber is commonly used, and these studies supported our previously published fluid-flow theory for this type of chamber. Conversely, the SS effect does not appear to have time to develop in the 100-microm-depth hemacytometer, which is deeper than the standard slide, has lower transverse velocity gradient, and consequently does not exhibit concentration variation due to the SS effect. These findings provide further support that hemacytometry, when performed properly, remains the gold standard. Applicability of our findings to routine semen analyses was then tested in 2 studies performed with independent boar studs. These studies compared diluted boar semen concentrations estimated by standard hemacytometry and in capillary-loaded 20-microm slides, using a computer-automated semen-analysis system designed to compensate for the SS effect. Good numerical agreement for sperm concentration with a high degree of correlation (r(2) = .936) was found between the 2 techniques. These findings reaffirm the need to critically assess new technologies for accuracy, repeatability, and precision.

Abstract: In this study, we evaluated the effects of glutathione (L- g-glutamyl-L-cysteinylglycine; GSH) supplementation of the thawing extender on semen parameters to compensate for the decrease in GSH content observed during sperm freezing. To fully address these questions, we used a set of functional sperm tests. These included tests of motility and motion parameters, changes in sulfhydryl group content in membrane proteins, capacitation status, measures of intra- cellular reactive oxygen species generation, sperm chromatin conden- sation, and in vitro penetration of immature oocytes. The main findings emerging from this study were that addition of GSH to the thawing media resulted in a lower number of capacitated viable spermatozoa, a decrease in the number of spermatozoa with changes in the sulf- hydryl groups in membrane proteins, a reduction of the reactive oxy- gen species generation, a lower chromatin condensation, and a higher penetration ability of oocytes in vitro and a higher proportion of de- condensated sperm heads. GSH appears to play an important role in sperm antioxidant defense strategy. Addition of GSH to the thawing extender could be of significant benefit in improving the function and fertilizing capacity of frozen boar spermatozoa.

Abstract: The human follicle-stimulating hormone (FSH) receptor (FSHR) gene possesses single nucleotide polymorphisms (SNP) in exon 10, which influence serum FSH levels in women, but not in men. In the present study we extend our previous investigation and for the first time analyze a novel, common SNP at position -29 of the FSHR core promoter in men. The SNP in codon 680 was analyzed in 438 men with nonobstructive azoospermia and in 304 controls. The SNP in codon 307 and at position -29 was analyzed in 345 men with nonobstructive azoospermia and 186 controls. SNPs were determined by allelic discrimination. No significant difference in the frequency of the polymorphism at position 680 and serum FSH levels was found. At position -29 (A/G) the A(-29) allele was less frequent than the G(-29) allele both in controls (25% vs 75%) and in patients (30% vs 70%) (P not significant). Together the three SNPs form four discrete haplotypes (A-Thr-Asn, G-Thr-Asn, A-Ala-Ser, and G-Ala-Ser) occurring in 10 combinations. A statistically significant difference in the allelic distribution between controls and azoospermic men was found (P < .05 by chi2 test). The A-Ala-Ser allele was more frequent in patients (9.1%) than in controls (5.4%), whereas the G-Thr-Asn allele was less frequent in patients (33.1%) than in controls (40.6%) (P < .01 by Fisher's exact test). No significant correlation between serum FSH levels and FSHR allele was found. We conclude that the FSHR haplotype does not associate with different serum FSH levels but it is differently distributed in normal and azoospermic men. The A-Ala-Ser and the G-Thr-Asn allele might represent genetic factors contributing to phenotypic expression of severe spermatogenetic impairment.

Abstract: Physiological aging is a significant risk factor in the on-set of male erectile dysfunction (ED) and an imbalance in factors that modulate cavernosal smooth-muscle tone may play a role in these altered penile hemodynamic mechanisms. To evaluate the association between aging and male erectile function, we monitored neurogenic erectile response and its correlation to systemic arterial pressure changes in old (21-23 months of age) vs young (6-9 months of age) Brown-Norway (BN) rats. We tested the hypothesis that age-associated ED is due to unregulated vasoconstrictive tone, contributed in part by an increased Rho-kinase activity, and that antagonism of Rho-kinase activity attenuates the age-related decline in male erectile function. We also examined the hypothesis that a combination of Rho-kinase antagonism and phosphodiesterase-5 (PDE-5) inhibition has a synergistic effect in improving the erectile response in these aging animals. Erectile function in old BN rats was evaluated before and after intracavernosal injection of a specific inhibitor of Rho-kinase (Y-27632) alone or in combination with zaprinast, a PDE-5 inhibitor. Erectile capabilities of the young and old BN rat groups were significantly different in corpus cavernosum pressure response after electrical-field stimulation of the major pelvic ganglion. Y-27632 administration attenuated the aging-related changes in male erectile function seen in BN rats. Rho-kinase antagonism and PDE-5 inhibition had a synergistic effect in improving erectile function in old rats. Our data indicate that aging leads to impairment in the neurogenic erectile response in BN rats involving a possible derangement in penile hemodynamic mechanisms of the erectile tissue. Rho-kinase inhibition may be of value in treating age-related ED.

Abstract: Accurate determination of sperm concentration in fluid suspension is a critical component in a semen analysis. Inaccurate estimations can lead to misinterpretation of the spermiogram and, in the case of livestock production, can lead to faulty insemination doses, which can adversely affect stud power, fertility, fecundity, and cost effectiveness of breeding programs. Capillary-loaded slides, like the hemacytometer, have been the standard for calibration of other concentration estimation modalities such as photometry, Coulter counter, flow cytometry, and computer-automated semen analysis (CASA). Single-use capillary-loaded slides, much smaller than the hemacytometer, are frequently used by many of the current CASA systems. As the use of CASA increases, more field reports are suggesting differences between CASA results and hemacytometry. In this article, we establish that these differences are, in large part, due to the Segre-Silberberg effect, which occurs during Poiseuille flow in high-gradient fluid flow in thin capillary-loaded slides. We develop the theory of this phenomenon and derive the scaling and significance of the effect. Finally, we graphically provide a means for predicting the necessary compensation factor when using capillary-loaded slides to determine sperm concentration.

Abstract: A newly developed flow cytometric method for determination of sperm concentration and viability was tested in an insemination trial with cryopreserved bull sperm to establish the relationship between sperm viability and nonreturn rates. Semen for experimental inseminations was produced from 157 young sires (114 Holstein and 43 Jersey), each contributing 4 experimental semen collections. Straws containing approximately 15 × 106 motile sperm before freezing were used in 118 680 experimental inseminations performed by 254 artificial insemination technicians in 6352 Danish herds. Statistical analysis based on 44 946 experimental first inseminations showed that the major part (95.4%) of variation in the 56-day nonreturn rate (NRR56) was residual. Only 0.38% of the total variation in NRR56 was due to bulls and differences between ejaculate within bull. However, bulls were preselected, and a relatively high insemination dose was used. Correlations between sperm viability as assessed by flow cytometry and NRR56 was slightly lower than observed for microscopic assessment of sperm motility. However, flow cytometry makes it possible to achieve an objective and precise determination of sperm viability. It was therefore possible to calculate the effect on NRR56 provided selection of semen is based on the flow cytometric method. Three freezing extenders were used in this experiment, but a significant difference in NRR56 was not observed. Flow cytometric results for 1 extender (Biociphos Plus) indicated poorer sperm survival during postthaw incubation compared with Triladyl extender with whole and with clarified egg yolk.

Abstract: The Sertoli cell ectoplasmic specialization (ES) is a specialized domain of the calcium-dependent Sertoli cell-spermatid junctional complex. Not only is it associated with the mechanical adhesion of the cells, but it also plays a role in the morphogenesis and differentiation of the developing germ cells. Abnormal or absent Sertoli ESs have been associated with step-8 spermatid sloughing and subsequent oligospermia. With a micropipette pressure trans- ducing system (MPTS) to measure the force needed to detach germ cells from Sertoli cells, this study examined, for the first time, the strength of the junction between Sertoli cells and spermatids and between Sertoli cells and spermatocytes. The mean force needed

Abstract: Chromosome abnormalities in embryos are a major cause of implantation and development failures. Some couples with normal karyotypes have repeated implantation failures after intracytoplasmic sperm injection (ICSI). In order to value patients at risk for genetic ICSI failures and the validity of sperm aneuploidy analysis, we have studied cytogenetic abnormalities in sperm from ICSI patients. Twenty-nine patients with normal karyotypes were included. Ten patients had at least 4 ICSI treatments without pregnancy (group A). Nine patients had a pregnancy after 1 to 3 ICSI treatments (group B). Ten fertile men with normal semen parameters were studied as controls (group C). Fluorescent in situ hybridization (FISH) was used for sperm nucleus cytogenetic analysis using chromosomes 8, 9, 13, 18, 21, X, and Y specific probes. Aneuploidy for each chromosome and diploidy rates were significantly higher in group A than in group B and in group B than in group C (P < .05). Considering each patient in groups A and B, aneuploidy rate for each chromosome was too variable to be considered as a significant test. We proposed analysis of the total sperm aneuploidy. Chromosomal sperm nuclei profile could be used as a predictive biological test before ICSI in order to improve genetic counseling for oligoasthenoteratozoospermia patients.

Abstract: Surgical induction of cryptorchidism in experimental animals causes testicular germ cell apoptosis and infertility. The mechanisms of germ cell apoptosis have been associated with oxidative stress or testicular exposure to elevated temperature. Nitric oxide (NO) has been associated with apoptosis in a number of cell types. The objective of this study was to investigate whether overexpression of endothelial NO synthase (eNOS) could accelerate apoptosis of germ cells in the testes of transgenic mice. There are 3 NOS isoforms, and we restricted the analysis to eNOS at this time. For the colocalization of eNOS, staining in degenerating germ cells that were apoptotic cells suggested that eNOS may be related to germ cell apoptosis. eNOS overexpression in the testes of eNOS transgenic (eNOS-Tg) mice was examined using Western blot analysis. Unilateral cryptorchidism was surgically induced in both eNOS-Tg and wild-type (WT) adult mice. The testes were evaluated 1, 3, 5, 7, and 14 days after the operation by weighing the testes and examining histopathologic features and cell apoptosis using in situ microscopic analysis of DNA fragmentation. Immunoblotting for eNOS protein demonstrated increases in eNOS protein expression in testes, as well as the lung and aorta. In eNOS-Tg mice, weight reduction of cryptorchid testis was significantly increased on days 3, 5, and 7 (P = .02, .02, and .04, respectively). The numbers of spermatocytes and spermatids of eNOS-Tg cryptorchid testis significantly decreased compared with those of WT cryptorchid testis from day 3 (spermatocytes: P = .04; spermatids: P = .02). Moreover, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling demonstrated that eNOS-Tg mice significantly accelerate germ cell apoptotic changes induced by experimental cryptorchidism compared with WT mice from day 3 (P = .03). We have provided evidence that eNOS plays a functional role in mouse spermatogenesis in cryptorchidism-induced apoptosis.

Abstract: In a previous study assessing tadalafil for the treatment of erectile dysfunction (ED), tadalafil 20 mg was shown to improve erectile function for up to 36 hours vs placebo. This study sought to demonstrate the effectiveness of both 10- and 20-mg tadalafil vs placebo at 2 prespecified assigned times of 24 and 36 hours postdosing. This double-blind, placebo-controlled, parallel-group study randomized 483 men with ED into 6 groups according to a combination of treatment (placebo, tadalafil 10 or 20 mg) and assigned time (24 or 36 hours) for intercourse attempts. Patients were stratified by baseline ED severity based on Erectile Function Domain scores. The study had 4 phases: a 4-week run-in (no ED medication taken); a 2- to 4-week equilibration (dosing as needed); a 4- to 6-week assessment; and a 6-month open-label extension. During the assessment phase, men took a total of 4 doses of study medication, each dose separated by more than or equal to 7 days. Efficacy was measured as the mean per-patient percentage of successful intercourse attempts (Sexual Encounter Profile Diary Question 3: SEP3) during the assessment phase. Men taking either 10- or 20-mg tadalafil had a significant increase in SEP3 from baseline scores vs placebo at both 24 hours (P = .038 and <.001 for 10 and 20 mg, respectively) and 36 hours (P < .001 for both doses) postdose. The mean per-patient percentages of successful intercourse attempts for the 24-hour time point were 41.8%, 55.8%, and 67.3% for placebo and tadalafil 10 and 20 mg, respectively; for the 36-hour time point, the mean per-patient percentages were 32.8%, 56.2%, and 61.9% for placebo and tadalafil 10 and 20 mg, respectively. The most common treatment-emergent adverse events were headache, back pain, dyspepsia, and nasopharyngitis. Both 10- and 20-mg tadalafil improved erectile function for up to 36 hours postdosing in men with ED of varied severity.

Abstract: It is known that abnormal androgen dynamics in the tissues is a cause of androgen-dependent disorders. Investigation of tissue androgen levels could provide a clue to the elucidation of disorders. However, it is difficult to measure a trace amount of androgen in the tissues. We established a highly sensitive simultaneous quantification method of testosterone and dihydrotestosterone (DHT), which play the most important roles in the body among androgenic steroids in trace amounts, and investigated time course changes in testosterone and DHT levels in male accessory sex organs, serum, and seminal fluid after castration in rat models. In addition, changes in the testosterone/DHT ratio of male accessory sex organs and seminal fluid were observed. The simultaneous testosterone and DHT measurement method established by us was validated. Intra-assay variation and interassay precision and accuracy were all within +/-20%, and the quantification limits of testosterone and DHT were both 15.6 pg/g. With the use of this method, the testosterone and DHT levels in the prostate, seminal vesicles, and serum immediately after castration were similar to those previously reported. The testosterone and DHT levels were 350 pg/g and 605 pg/g, respectively; which showed dominance of DHT in seminal fluid, although it was not as marked as that in the male accessory sex organs. Androgens decreased with time after castration in the accessory sex organs, serum, and seminal fluid. In the prostate and seminal vesicles, testosterone and DHT decreased to about 50% and about 2% of the normal levels, respectively, 72 hours after castration. The serum levels were under the quantification limits 6 hours after castration and thereafter. In seminal fluid, the testosterone and DHT levels decreased to 49% and 35% of normal levels, respectively, 72 hours after castration. The testosterone/DHT ratio in the male accessory sex organs was lower in the prostate (0.06) than in the seminal vesicles (0.13) immediately after castration. In the seminal fluid, changes in the ratio were small compared with those in the accessory sex organs and serum. These results showed that our method was capable of measuring testosterone and DHT in very small amounts of samples such as prostate biopsy specimens, and it might provide a clue to the elucidation of the pathology of androgen-dependent disorders.

Abstract: The cryoprotective effects of 11 different extenders, TTE, DM, mDM, LG-DM, G-DM, TCG, TEST, TSM, Test-M, Test- H, and LM, on sperm cryopreservation of cynomolgus monkey (Macaca fascicularis) have been compared with glycerol as cryo- protectant. Sperm motility, plasma membrane, and acrosomal in- tegrity were examined to evaluate frozen-thawed sperm function. The results showed that TTE, DM, mDM, LG-DM, G-DM, and TCG exhibited the best and similar protective efficiencies for cynomol- gus monkey sperm cryopreservation in terms of sperm motility and plasma membrane integrity (P. .05). The acrosomal integrity for spermatozoa cryopreserved in TCG was statistically lower than that of TTE, DM, mDM, LG-DM, and G-DM (P , .05) but was significantly higher than that of TEST, TSM, Test-M, Test-H, and LM (P , .05). The postthaw sperm motility for 5 other extenders (TEST, TSM, Test-M, Test-H, and LM) did not exceed 30%, and the 3 sperm parameters evaluated for them were significantly lower than that of TTE, DM, mDM, LG-DM, G-DM, and TCG (P , .05). On the basis of these findings, 5 commonly used permeating cryo- protectants, glycerol, ethylene glycol, dimethyl sulfoxide, acet- amide and propylene glycol have further been tested for their ef- fectiveness on sperm cryopreservation in extenders of TTE, DM, mDM, LG-DM, G-DM, and TCG. The results showed that the sperm cryoprotective efficiencies of glycerol and ethylene glycol were similar and best among 5 permeating cryoprotectant treat- ments (P. .05). Dimethyl sulfoxide or acetamide resulted in av- erage cryoprotection for cynomolgus monkey spermatozoa: poorer than glycerol or ethylene glycol but better than that of propylene glycol (P , .05). In addition, the action of permeating cryoprotec- tant appeared to be independent of extenders. The results in the present study demonstrate that 1) TTE, DM, mDM, LG-DM, G-DM, and TCG are excellent extenders and suitable for cynomolgus monkey sperm cryopreservation; 2) the mechanism of action of permeating cryoprotectants are not affected by extender compo- sition; 3) ethylene glycol has a similar cryoprotective efficacy to glycerol that makes it a successful cryoprotectant for sperm cryo- preservation in cynomolgus monkeys.

Abstract: Little is known about how human spermatogenesis is regulated, so it is not surprising that there have been few breakthroughs in the treatment of male infertility resulting from abnormalities of spermatogenesis. Testosterone is the predominant intratesticular steroid in both the rat and man. Previous studies have shown that the testosterone concentration within the rat testis that is required for the quantitative maintenance of spermatogenesis is far higher than the total testosterone concentration in rat blood, indicating that much of the testosterone within the testis might be biologically inactive. In contrast to the rat, little is known about the androgen requirements for human spermatogenesis, in part because, until recently, a minimally invasive method suitable for obtaining intratesticular fluids from the human testis has not been available. Percutaneous aspiration now makes it feasible to do so. A major objective of the present study was to assay the bioactive androgen concentration within the testes of normal, fertile men. Percutaneous aspiration was used to obtain intratesticular fluid from such men, and we adapted a highly sensitive recombinant protein mammalian cell-based bioassay to measure androgen bioactivity. Total intratesticular testosterone concentration, which we define as immunoreactive testosterone as measured by radioimmunoassay, was well in excess of that in serum (1236 ± 86 nM vs 11.7 ± 0.7 nM). The concentration of bioactive androgens within the normal human testis was found to be about two thirds that of the total testosterone concentration. Interestingly, the concentration of the major, known binding proteins for testosterone within the testis, serum hormone-binding globulin (SHBG)/ABP (52.4 ± 9.7 nM), was insufficient to account for the difference between total testosterone and bioactive androgens. This indicates that, in addition to its binding to SHBG/ABP, androgens may also be bound by unknown molecules, and that this contributes to reducing androgen bioactivity. These observations could have relevance for understanding the relationship between spermatogenesis and intratesticular androgens in normal men and in men diagnosed with infertility.

Abstract: Treatment of men of reproductive age with radiation or alkylating agents often produces prolonged azoospermia. We previously demonstrated that suppression of testosterone (T) with gonadotropin-releasing hormone (GnRH) analogs restored spermatogenesis following atrophy induced by radiation or chemotherapy in rats. This study tested whether GnRH antagonist therapy could reverse radiation-induced testicular injury in primates with a similar protocol. Adult male stump-tailed macaques were given either 6.7 Gy radiation to the testis alone, 6.7 Gy radiation combined with GnRH-antagonist treatment starting on the day of exposure, or daily injections of the GnRH antagonist Cetrorelix for 3 months alone and were monitored for 18 months. Cetrorelix alone produced a 20-40-week fully reversible suppression of serum T, but although spermatogenic recovery was incomplete, 40%-90% of tubules contained differentiating germ cells. Following radiation alone, testis volumes were reduced to approximately 28% and sperm counts to less than 1% of pretreatment values. A biopsy at 18 months after radiation showed that only 3.0% of seminiferous tubule cross sections had germ cells. In irradiated animals that received GnRH antagonist, testis volumes were reduced to 18% of pretreatment volume, and at 18 months, only 1.9% of seminiferous tubule cross sections contained germ cells. Inhibin B values were reduced to 10% and 3% of pretreatment levels in the radiation-only and the radiation plus GnRH antagonist groups, respectively. Species differences exist in the testicular response to radiation, GnRH antagonist therapy, or both, so that rescue protocols that were successful in rodents might not work in primates.

Abstract: The frequencies of aneuploid and diploid spermatozoa were determined in 3 patients presenting a complete asthenozoospermia due to a primary and specific flagellar anomaly: patients 1 and 2 presented a “stump tail syndrome,” more than 50% of spermatozoa with a short flagella, patient 3 had a Kartagener syndrome including situs inversus, sinusitis, and bronchiectassis. No pregnancy was obtained after 3 intracytoplasmic sperm injection (ICSI) attempts in patients 1 and 2. A 3-color fluorescence in situ hybridization analysis was performed on their spermatozoa using centromeric probes for chromosomes X, Y, and 18 and compared with those of 8 fertile males. The frequency of disomic 18 and hyperhaploid XY spermatozoa was not significantly increased in the 3 patients when compared with controls. However, the 3 patients showed elevated frequencies of XX, YY, and diploid spermatozoa. These data add to growing evidence that systematic sperm anomalies of flagella increase the rate of spermatozoa aneuploidy and may also reduce the chances of pregnancy after intracytoplasmic sperm injection.

Abstract: Our aim was to study retrospectively the destiny of the deep dorsal vein of the penis in the event of its stripping surgery or its simple ligation in patients diagnosed with venoocclusive dysfunction 17 years ago. From June 1986 to May 1987, a total of 31 men were seen for erectile dysfunction due to venous leakage resulting from priapism, aging, or congenital or idiopathic factors. Of these, 23 men underwent venous stripping of the deep dorsal vein and are referred to as the stripping group. The remaining 8 patients received a simple ligation of the deep dorsal vein and are classified as the ligation group. A total of 21 patients (16 of the 23 and 5 out of the 8) were available for follow-up by using the abridged 5-item version of the International Index of Erectile Function (IIEF-5) scoring system and cavernosograms. In the ligation group, the imaging demonstrates some compensatory veins that are commensurate with impotence postoperatively. In the stripping group, however, the follow-up cavernosograms disclosed no venous recurrence, but residual ones that were not crucial to the rigidity. The IIEF-5 scoring in the ligation group changed from a preoperative mean IIEF-5 score of 10.0 +/- 4.5 to 9.8 +/- 3.6 postoperatively. In the stripping group, however, the mean preoperative IIEF-5 score of 9.8 +/- 4.1 increased to a mean postoperative IIEF-5 score of 18.9 +/- 2.1. Although there was no significant difference between the 2 groups' preoperative IIEF-5 score, there was a statistically significant difference between treatments (P <.001). The penile venous vasculature bears no evidence of regeneration even as long as 17 years after their removal. This finding is in contrast to what is commonly believed, that erectile dysfunction will recur about 2 years after ligation of the deep dorsal vein. We therefore believe that the clinical recurrence may not be due to venous regeneration, and penile venous surgery, if properly performed, may be durable, although larger studies will be required.

Abstract: The development of a safe, well-tolerated, effective, and reversible male hormonal contraceptive would be a major clin- ical advance for couples planning their family size and for control of population growth. High-dosage parenteral testosterone (T) esters alone or in combination with a progestogen (eg, depot medroxypro- gesterone) have been shown to confer effective and reversible male contraception in clinical trials, but these regimens are associated with weight gain and suppression of serum high-density lipoprotein cholesterol (HDL) levels. We have previously demonstrated that in- tramuscular T enanthate 100 mg weekly plus oral levonorgestrel (LNG) 125, 250, or 500 mg daily suppresses spermatogenesis to levels associated with effective contraception, but there is a LNG- dosage-dependent effect of weight gain and HDL suppression. We hypothesized that intramuscular T enanthate 100 mg weekly plus a very low dosage of oral LNG would effectively suppress spermato- genesis in normal men without inducing weight gain or HDL sup- pression. We conducted a randomized trial comparing 6 months of intramuscular T enanthate (100 mg weekly) plus 31.25 mg of oral LNG daily (T1LNG 31; n 5 20) or 62.5 mg of oral LNG daily (T1LNG 62; n 5 21). The 2 regimens were equally effective in suppressing spermatogenesis to azoospermia, fewer than 1 million sperm/mL and fewer than 3 million sperm/mL (T1LNG 31 (60%, 85%, and 90%) vs T1LNG 62 (62%, 91%, and 95%) for azoospermia, fewer than 1 million and fewer than 3 million, respectively; P 5 NS). The T1LNG 31 group did not gain weight (0.25 6 1.08 kg; P 5 NS compared with baseline), but the T1LNG 62 group gained 2.5 6 0.77 kg (P , .05 compared with baseline). Serum HDL cholesterol levels declined significantly in both groups (percentage decline month 6 of treatment vs baseline: 12.0% 6 2.6% and 15.1% 6 3.0%; P , .05 for T1LNG 31 and 62 respectively). Serum low-den- sity lipoprotein cholesterol levels also declined in both groups (per- centage decline month 6 of treatment vs baseline: 6.9 6 3.9 and 6.0% 6 4.1%; P , .05 for T1LNG 31 and P 5 NS for T1LNG 62). There were no clinically significant adverse events or significant changes in hematology or chemistry profiles in either group during the study. We conclude that 1) intramuscular T plus oral LNG has a very potent synergistic effect in suppressing spermatogenesis at LNG dosages equal to or lower than dosages used in common fe- male oral contraceptive regimens and 2) large, long-term contracep- tive efficacy trials should be conducted with a variety of androgen- progestogen combinations including long-acting T formulations such as depot T pellets or intramuscular T undecanoate plus depot LNG or very low dosage oral LNG.

Abstract: To elucidate the anatomic distal ligament of the human glans penis and associated clinical implications, we compared the structures of the glans penis and corpora cavernosa in dogs, rats, and humans. From May 2001 to March 2003, gross dissection, microscopic examinations, and stains for elastic fibers and collagen subtypes were made in the penises of 11 adult human male cadavers, 7 dogs, and 5 rats. A distal ligament in the human glans penis replaces the os penis that is present in dogs or rats, also termed the baculum, but retains collagen types I and III as common structural and interlocking components, respectively. The intercavernosal septum is complete, and intracavernosal pillars (ICPs) are abundant in dogs, absent in rats, and moderately developed in humans. A tunica with numerous elastic fibers exists to fulfill the requirements of erectile function in humans but not in dogs or rats, since it is essential for establishing tissue strength to serve as a buttress. We may conclude that in dogs and rats, the strong os penis is designed for ready intromission and is associated with a pair of well-developed nonelastic corpora to serve as a buttress for the os penis. These structures are necessary for the rigorous coitus observed in dogs. The less compliant corpus cavernosum is suitable for the flipping action observed in a mating male rat. These specific anatomic designs may provide explanations for the individual requirements for the specific physiologic functions that differ from species to species. Although there is no os in the human glans, a strong equivalent distal ligament is arranged centrally and acts as a supporting trunk for the glans penis. Without this important structure, the glans could be too weak to bear the buckling pressure generated during coitus and too limber to serve as a patent passage for ejaculation, and it could be too difficult to transmit the intracavernosal pressure surge along the entire penis during ejaculation. Given the common histologic nature of the distal ligament, which is associated with the tunica albuginea and serves a similar function as the os penis observed in the dog and the rat, one may ask whether the healing process of a tunica may take as long as that required in a bony structure. Further research is required to answer this question.