Showing papers in "Journal of Andrology in 2001"

Altered protamine 2 expression is uncommon in donors of known fertility, but common among men with poor fertilizing capacity, and may reflect other abnormalities of spermiogenesis.

Abstract: During the spermatid elongation stage of spermiogenesis approximately 85% of sperm nuclear histones are replaced by protamines. Protamines increase the packing ratio of sperm chromatin, presumably facilitating sperm motility and function. In this study we evaluated the incidence of abnormal protamine expression in 75 patients undergoing in vitro fertilization (IVF) and 50 donors of known fertility by isolation of sperm nuclear proteins, quantitative gel electrophoresis, and Western blot analysis. In addition, we evaluated the relationship between abnormal protamine expression and semen quality, sperm penetration ability, chromatin stability, and IVF outcome. Seventeen percent (13/75) of IVF patients had no measurable protamine 2 (P2) versus 0% (0/50) of donors of known fertility (P < .005). Sperm penetration rates were decreased in 12 of 13 patients without P2, and mean penetration rates (4.6 +/- 1.2 vs 32.8 +/- 2.9, P < .005), normal morphology (22.4 +/- 3.6 vs 48.7 +/- 4.2, P < .05), and progressive motility (22.3 +/- 2.5 vs 35.4 +/- 2.1, P < .05) were all significantly decreased compared with patients with measurable P2. The mean sperm concentration was not significantly different. The presence of protamine precursor bands was also associated with a diminished penetration capacity (18.4 +/- 2.8 vs 36.7 +/- 3.0, P < .05). Sperm chromatin decondensation following exposure to heparin sulfate was significantly increased in patients without a measurable P2 band. Twelve patients with no measurable P2 underwent intracytoplasmic sperm injection (ICSI), with 6 patients (6/12, 50%) becoming pregnant. ICSI fertilization and subsequent embryo cleavage were not different in patients without P2 compared with other patients undergoing ICSI. These data indicate that abnormal sperm protamine levels are a common defect in infertility patients, but not in donors of known fertility. It appears that abnormal protamine levels may reflect defects of late spermiogenesis, including sperm penetration capacity.

Relationship between seminal white blood cell counts and oxidative stress in men treated at an infertility clinic.

Abstract: In semen, granulocytes are major producers of reactive oxygen species (ROS), which can damage sperm. The diagnosis of leukocytospermia is usually based on the World Health Organization (WHO) definition of 1 x 10(6) white blood cells per milliliter, but controversy remains over the minimum leukocyte level that impairs fertility. The goals of this study were to clarity the relationship between leukocyte count and oxidative stress and to establish the minimum leukocyte count associated with oxidative stress. To do so, we compared oxidative stress in semen samples with different leukocyte counts (by the Endtz test) after a simple wash-and-resuspend procedure and determined the correlation between leukocyte counts and oxidative stress (expressed as ROS-TAC score, a composite score calculated from ROS levels and total antioxidant capacity (TAC), both measured with chemiluminescence assays). ROS-TAC decreases as oxidative stress rises. We compared specimens from 271 men attending an infertility clinic and 28 healthy controls. About 9% of patients had WHO-defined leukocytospermia and an additional 16% had some leukocytes. Samples with no seminal leukocytes had significantly lower ROS levels and significantly higher ROS-TAC scores than samples with any seminal leukocytes, even very low levels. Oxidative stress was correlated with rising white blood cell (WBC) count (r = .39; P < .001). Receiver operating characteristics curves showed that ROS-TAC score would be fairly accurate at distinguishing between patients with any leukocytes and those with no leukocytes (area under the curve, 75%). In conclusion, oxidative stress occurs even in patients with very low seminal WBC counts (between 0 and 1 x 10(6)/mL) and rises with an increase in WBC count. Therefore, we are unable to determine a safe minimum WBC count; the presence of any WBCs is associated with oxidative stress and may therefore impair fertility. Complete removal of WBCs from semen samples used for assisted reproduction may help reduce oxidative stress.

The Role of Glucose in Supporting Motility and Capacitation in Human Spermatozoa

Abstract: Glucose has been reported to be beneficial to human sperm for optimal capacitation and fertilization, although it is unclear whether glucose is required for providing extra metabolic energy through glycolysis, or for generating some other metabolic product. In this study, the effects of sugars on human sperm capacitation, motility, and energy production were investigated. The glucose concentration that supported the greatest number of acrosome reactions was 5.56 mmol L(-1). Compared with incubations with no added sugar, this concentration of glucose, fructose, mannose, or galactose appeared to slightly increase the number of acrosome reactions occurring after 18 hours of capacitation, or following induction by 2 micromol A23187 + 3.6 mmol pentoxifylline L 1, but only glucose had a statistically significant effect. Glucose supported increased penetration of zona-free hamster oocytes, but its advantage was not statistically significant. The addition of 5.56 mmol glucose or fructose L(-1) to sugar-free medium immediately increased the adenosine triphosphate (ATP) concentration and motility of sperm. These parameters were then stable for 3 hours, but declined markedly after 18 hours. In the absence of a glycolysable sugar, motility began to decline in the first hour and only 2% or 3% of sperm remained motile after 18 hours. Glucose or fructose was required to support hyperactivated motility. 2-Deoxyglucose was detrimental to the ATP concentration and motility of sperm, and supported fewer spontaneous or progesterone-stimulated acrosome reactions than were observed in the absence of a sugar. We conclude that glycolytic ATP production is required for vigorous motility and hyperactivation in human sperm. Other products of glucose metabolism are not essential to support capacitation, but they may have a small, enhancing effect.

Osmotic tolerance of equine spermatozoa and the effects of soluble cryoprotectants on equine sperm motility, viability, and mitochondrial membrane potential.

Abstract: Osmotic stress attributed to differences in the relative permeability of cryoprotectants, such as glycerol and water, appears to be an important factor in cryodamage. The objective of this study was to characterize the osmotic tolerance of equine spermatozoa, and to evaluate the effects of addition and removal of cryoprotectants from equine spermatozoa on their motility, and membrane and acrosomal integrity, as well as their mitochondrial membrane potential. Equine spermatozoa had a limited osmotic tolerance to anisosmotic conditions. Although the addition of increasing concentrations of glycerol decreased the motility and viability of equine spermatozoa, the rapid removal of glycerol by dilution in isosmotic media resulted in an even greater decline in motility and viability compared with spermatozoa maintained under anisosmotic conditions. Likewise, the addition and rapid removal of 1.0 M glycerol, ethylene glycol, dimethylsulfoxide, or propylene glycol resulted in a significant decline in sperm motility and viability. Among these cryoprotectants, ethylene glycol had the least detrimental effect on either viability or motility of spermatozoa following the rapid addition and removal of these cryoprotectants. These data demonstrate that equine spermatozoa have a limited osmotic tolerance compared with published reports for mouse or human spermatozoa, and appear to be more similar to boar spermatozoa in their osmotic tolerance. Of the 4 cryoprotectants evaluated in equine spermatozoa, the addition and removal of glycerol resulted in a more marked osmotic stress as indicated by alterations in motility, viability, and acrosomal integrity. These data suggest that alternative cryoprotectants should be considered for cryopreservation of equine spermatozoa in order to reduce osmotic stress associated with the addition of these agents during semen freezing.

Morphologically distinct sperm subpopulations defined by Fourier shape descriptors in fresh ejaculates correlate with variation in boar semen quality following cryopreservation.

Abstract: This study investigated two hypotheses: 1) that consistent between-boar variation in frozen semen quality exists and is genetically determined, and 2) that morphologically distinct subpopulations of spermatozoa exist within fresh boar ejaculates and that the incidence of these subpopulations is correlated with semen quality following cryopreservation. Five ejaculates were collected from each of 15 boars (5 boars from each of 3 breeds). An objective sperm morphology analyzer used Fourier shape descriptors to describe variation in the morphology of 300 spermatozoa per ejaculate before freezing. Semen was diluted into a commercial freezing buffer (700 mOsm/kg, 3% glycerol) and 5 straws (0.5 mL) per ejaculate were cryopreserved (to -5 degrees C at 6 degrees C/min, then -5 degrees C to -80 degrees C at 40 degrees C/min). Semen was assessed for percentage of motile cells and motility characteristics (with computer-aided sperm analysis), plasma membrane integrity (SYBR-14 positive), and acrosome integrity (fluorescein-labeled peanut agglutinin positive). Consistent between-boar variability was detected for post-thaw sperm motility (P < .01), membrane integrity (P < .01), acrosome integrity (P < .01), curvilinear velocity (P < .01), straight-line velocity (P < .05), beat cross-frequency (P < .05), and amplitude of lateral head displacement (P < .01). Three morphologically distinct subpopulations of spermatozoa, defined by Fourier descriptors, were detected. The proportion of these subpopulations within the fresh ejaculate correlated with semen quality assessments made following cryopreservation. These findings support the hypothesis that consistent interindividual variation in sperm freezability is genetically determined and may relate to processes that occur during spermatogenesis. Subsequent characterization of these genetic differences between "good" and "poor" freezers may ultimately identify biophysical components of the spermatozoa that are essential for successful cryopreservation.

Quality control of reactive oxygen species measurement by luminol-dependent chemiluminescence assay.

Abstract: A total of 28 donor semen samples were used to evaluate the characteristics of laboratory variability in measuring reactive oxygen species (ROS). The objectives of this study were to assess the interassay (same sample observed on different days by the same observers) variability; interdonor, intraobserver (replications of the same sample on the same day) variability; and interobserver (multiple observers on the same day with the same sample) variability of the luminol-dependent chemiluminescence assay and to establish an optimal semen age and sperm concentration. Semen samples were collected from 6 healthy donors for 108 measures of ROS. ROS levels were measured by the assay using luminol as the probe. An additional assessment measured the effect of time (age of the sample) on ROS production in 12 donor samples at 60, 120, 180, and 240 minutes after the specimen was produced. Last, to evaluate the effect of sperm concentration on ROS production, ROS levels were measured in 10 donor sample aliquots with sperm concentrations ranging from 1 to 120 x 10(6)/mL. In the controls, the mean ROS level was 0.218 x 10(6) counted photons per minute; the interassay variability standard deviation (SD) was 0.077. The interobserver SD was 0.002 for an interobserver reliability of 97.5% (coefficient of variation [CV] = 0.9%). The intraobserver (between replication) SD was 0.001 for an intraobserver reliability of 98.7% (CV = 0.5%). The interassay SD was 0.005 for an interassay reliability of 93.8% (CV = 2.0%). There was no statistically significant interobserver, intraobserver, or interassay variation (P > .80). ROS levels decreased significantly with time; a dramatic decline in ROS production was seen in the specimens that were more than 60 minutes old (P < .001). ROS values decreased by 31% at 120 minutes and 62% at 180 minutes compared with the 60-minute-old specimens. A linear relationship was seen between the ROS levels and sperm concentration in 8 of the 10 samples analyzed (R2 = .99). Our results demonstrate that the luminol-dependent chemiluminescence assay for ROS measurement is both accurate and reliable when the sperm concentration is greater than 1 x 10(5)/mL and the samples are analyzed within the first hour after specimen collection.

Objectively measured sperm motility and sperm head morphometry in boars (Sus scrofa): relation to fertility and seminal plasma growth factors.

Abstract: This study was conducted to investigate the relationships between results of computer-assisted semen analysis (spermatozoal motility and sperm head morphometry) and fertility of boars. In addition, concentrations of insulin-like growth factor (IGF)-I and IGF-II in seminal plasma were determined. The nonreturn rate (NRR) and the number of live-born piglets were compatible with the requirements of artificial insemination for all boars included in this study. Semen samples of 12 boars (Pietrain; 3 ejaculates each) were evaluated for spermatozoal motility and sperm head dimensions using computer-assisted methods. Native semen samples were centrifuged, and seminal plasma was frozen at -20 degrees C until assayed for IGF-I and IGF-II by specific radioimmunoassays. Spermatozoa of boars with a higher NRR (>86%) had a significantly slower average velocity of motile spermatozoa when compared with that of boars with an NRR below 86%. High-fertility boars (NRR > 86%) had significantly smaller sperm heads than did boars with an NRR below 86%, and their sperm heads were less elongated. Substantial concentrations of IGF-I (8.4-22.2 ng/mL) and IGF-II (12.1-19.8 ng/mL) could be measured in porcine seminal plasma; however, there was no correlation between IGF levels and semen parameters or individual fertility.

Relationship of Bull Fertility to Sperm Nuclear Shape

Abstract: The relationship between sperm nuclear shape and bull fertility was determined. Two groups of bulls, 3 per group, were selected. Bulls differed in fertility based on lifetime nonreturn rates. Digital images of propidium iodide-stained sperm from each bull were collected and shape-evaluated by Fourier harmonic amplitudes 0 to 5. A discriminant function (P < .05) was constructed based on harmonic amplitudes and the 2 fertility groups. When individual sperm were classified as being of high or lower fertility, the percentage of each bull's sperm placed in the high-fertility group had a linear relationship (r = .89, P < .05) with fertility. To construct a plot of mean sperm shapes, a novel technique to automatically orient and identify the anterior tip of the sperm head was developed. The mean nuclear shape of high-fertility sperm was more elongated and tapered than those of lower fertility. A discriminant function (P < .05) was also constructed that separated the 6 bulls into 2 groups based only on the harmonic amplitudes or sperm nuclear shape. The bulls were correctly classified into the 2 fertility groups. A comparison of sperm chromatin structure analysis (SCSA) and harmonic amplitudes found that overall size variance, anterior roundness, and posterior taperedness of sperm nuclei were related to chromatin stability (P < .05). Some of the differences observed in sperm nuclear shape between the high- and lower-fertility bulls may be explained by varying levels of chromatin stability. However, sperm nuclear shape appears to contain additional information from chromatin stability alone. In this particular study, with 6 bulls, all with good chromatin quality, sperm nuclear shape was a better predictor of bull fertility.

Oxidative stress in normospermic men undergoing infertility evaluation.

Abstract: The purpose of this study was to determine whether normospermic infertile men have high seminal oxidative stress, using 3 measures of oxidative stress: reactive oxygen species (ROS), total antioxidant capacity (TAC), and a composite ROS-TAC score. Forty-three normospermic men without leukocytospermia and 19 healthy donors who came to our infertility clinic were included. Patients were categorized into 3 groups: group I, varicocele and no female factor (n = 16); group II, positive female factor (n = 16); and group III, idiopathic infertility (n = 11). In addition, 52 treated male factor patients and 19 donors were included as reference groups. We measured seminal ROS, TAC, and the ROS-TAC score in the patient groups and the controls. Normospermic infertile patients as a group had higher ROS levels (mean log [ROS + 1] 1.76 +/- 0.13) compared with controls (1.39 +/- 0.16; P = .03). Patients in the idiopathic subgroup had significantly higher ROS levels (2.29 +/- 0.25; P = .004) than controls. Normospermic infertile patients as a group not only had reduced TAC levels (970.18 +/- 73.95 Trolox equivalents), but each subgroup also had significantly lower TAC than controls (1650.93 +/- 95.87; P < .003). The ROS-TAC scores in all normospermic infertile patients as a group (35.7 +/- 1.8) as well as in each subgroup was significantly reduced compared with the ROS-TAC levels in the controls (50.0 +/- 2.1; P < .005). We conclude that oxidative stress is associated with male factor infertility. The presence of oxidative stress in infertile normospermic men may explain previously unexplained cases of infertility otherwise attributed to female factors.

Suppression of androgen action and the induction of gross abnormalities of the reproductive tract in male rats treated neonatally with diethylstilbestrol.

Abstract: This study evaluated whether androgen action is altered in rats treated neonatally with diethylstilbestrol (DES) at a dose that induced reproductive tract abnormalities. Rats were treated on alternate days 2-12 with 10 microg DES and studied on Day 18. DES-induced abnormalities included a 70% reduction in testis weight, distension and overgrowth of the rete, distension and reduction in epithelial height of the efferent ducts, underdevelopment of the epididymal duct epithelium, reduction in epithelial height in the vas deferens, and convolution of the extra-epididymal vas. In DES-treated rats, androgen receptor (AR) immunoexpression was virtually absent from all affected tissues and the testis, whereas AR expression in controls was intense in epithelial and stromal cells. The DES-induced change in AR immunoexpression was confirmed by Western analysis for the testis. In rats treated neonatally with 1 microg DES, reproductive abnormalities were absent or minor, except for a 38% reduction in testis weight; loss of AR immunoexpression also did not occur in these rats. Treatment-induced changes in AR expression were paralleled by changes in Leydig cell volume per testis (91% reduction in the 10-microg DES group; no change in the 1-microg DES group). To test whether suppression of androgen production or action alone could induce comparable reproductive abnormalities to 10 microg DES, rats were treated neonatally with either a potent gonadotropin-releasing hormone antagonist (GnRHa) or with flutamide (50 mg/kg/day). These treatments reduced testis weight (68% for GnRHa, 40% for flutamide), and generally retarded development of the reproductive tract but failed to induce the abnormalities induced by 10 microg DES. GnRHa and flutamide caused no detectable change in AR immunoexpression in target tissues, with the exception of minor changes in the testes of flutamide-treated males. GnRHa treatment caused a reduction (83%) in Leydig cell volume comparable to that caused by 10 microg DES. Immunoexpression of estrogen receptor alpha (ER alpha) in the efferent ducts and of ER beta in all tissues studied were unaffected by any of the above treatments. Neonatal coadministration of testosterone esters (TE; 200 microg) with 10 microg DES prevented most of the morphological abnormalities induced by 10 microg DES treatment alone, though testis weight was still subnormal (46% reduction in DES + TE vs 72% in DES alone and 49% with TE alone) and some lumenal distension was still evident in the efferent ducts. Coadministration of TE with DES prevented DES-induced loss of AR immunoexpression (confirmed for testis by Western blot analysis). It is concluded that 1) reproductive tract abnormalities induced in the neonatal male rat by a high (10 microg) dose of DES are associated with reduced AR expression and Leydig cell volume; 2) these changes are largely absent with a lower dose of DES (1 microg); 3) treatments that interfere with androgen production (GnRHa) or action (flutamide) alone failed to induce reproductive tract abnormalities or alter AR expression as did 10 microg DES; 4) a grossly altered androgen:estrogen balance (low androgen + high estrogen) may underlie the reproductive tract abnormalities, other than reduced testis weight, induced by high doses of DES.

Atrazine effects on testosterone levels and androgen-dependent reproductive organs in peripubertal male rats.

Abstract: Previous studies have reported that atrazine, a widely used herbicide that selectively inhibits photosynthesis in broadleaf and grassy weeds, has adverse effects on reproductive function in the male, suggesting a direct effect of atrazine on the hypothalamicpituitary-testicular axis. As yet, however, no studies have critically examined the doses of atrazine that elicit such effects, and few have focused on the mechanism by which atrazine acts. Herein we report a dose-response study of the effects of atrazine ingestion on reproductive function in male Sprague-Dawley rats during a critical developmental period, the peripubertal period. Atrazine was administered by gavage to rats from day 22 to day 47 of age, at doses of 1-200 mg/kg body weight per day. Atrazine administration of up to 50 mg/kg per day had no effect on any of the measured variables. Serum testosterone concentration was reduced by atrazine at doses of 100 and 200 mg/kg per day, as were seminal vesicle and ventral prostate weights. Intratesticular testosterone concentration was reduced in parallel with serum testosterone, suggesting that the reductions in serum testosterone resulted from reduced testosterone production by Leydig cells or from changes in testosterone metabolism within the testis, or both. Serum luteinizing hormone (LH) concentration was reduced despite the reduced serum testosterone, suggesting an effect on the hypothalamus, the pituitary gland, or both. At the termination of the study, the average body weight of rats receiving atrazine at 100 mg/kg per day was found to be reduced by approximately 9%. This suggested the possibility that the effects of atrazine on the reproductive tract may not be direct, but rather, the noted deficits of the male reproductive tract resulted from reduced food intake by the treated rats. We tested this by feeding control (vehicle-gavaged) rats amounts of food equivalent to that consumed by the atrazine-fed rats, and then assessing reproductive tract endpoints. Even mild food restriction resulted in reductions in serum testosterone concentration, in the weights of androgen-dependent organs, and in serum LH concentration; the same deficits that were seen in atrazine-gavaged rats. Indeed, the effects of atrazine on the male reproductive tract seen in rats receiving atrazine at greater than 50 mg/kg per day could not be distinguished from the effects of reduced food consumption. These results suggest that caution must be exercised before concluding that atrazine (or any potentially toxic chemical) has direct, detrimental effects.

Changes in motion characteristics, plasma membrane integrity, and acrosome morphology during cryopreservation of buffalo spermatozoa

Abstract: Motion characteristics, plasma membrane integrity, and acrosome morphology of buffalo spermatozoa after different stages of cryopreservation (ie, dilution, cooling to 4 degrees C, equilibration at 4 degrees C, and freezing and thawing) were examined. Semen ejaculates from 4 buffalo bulls were pooled (n = 5), diluted in tris-citric acid extender, cooled to 4 degrees C over 2 hours, equilibrated at 4 degrees C for 4 hours, dispensed into 0.5-mL straws, and frozen in a programmable cell freezer before plunging into liquid nitrogen. Frozen semen was thawed at 37 degrees C for 15 seconds. After completion of each stage, sperm motion characteristics, plasma membrane integrity, and acrosomal morphology were determined using computer-assisted semen analysis, hypo-osmotic swelling assay, and phase-contrast microscopy, respectively. Data were presented as mean +/- standard error of the mean. Visual and computerized motility did not differ due to dilution, cooling, or equilibration (77.3% +/- 2.3% and 90.5% +/- 1.2%, respectively), but was reduced (P < .05) after freezing and thawing (53.0% +/- 4.6% and 48.6% +/- 6.5%, respectively). Linear motility of spermatozoa was lower (P < .05) after dilution or equilibration (56.2% +/- 2.4%) than after cooling or freezing and thawing (79.6% +/- 1.4%). Sperm curvilinear velocity was reduced (P < .05) from 112.4 +/- 5.3 microm/sec after dilution to 96.0 +/- 5.8 microm/s after cooling, and from 87.6 +/- 4.1 microm/s after equilibration to 69.4 +/- 2.0 microm/s after freezing and thawing. Sperm lateral head displacement differed (P < .05) after each stage (ie, dilution, 3.9 +/- 0.2 microm; cooling, 2.3 +/- 0.2 microm; equilibration, 3.1 +/- 0.3 microm; and freezing and thawing, 1.7 +/- 0.2 microm). Spermatozoa with intact plasma membranes were 80.2% +/- 3.9% after dilution, reduced (P < .05) to 60.4% +/- 5.6% after equilibration, and then to 32.6% +/- 3.8% after freezing and thawing. The percentage of spermatozoa with normal acrosomes remained higher after dilution, cooling, or equilibration (73.2% +/- 2.4%) than after freezing and thawing (61.8% +/- 2.4%; P < .05). In conclusion, the maximal damage to the motility apparatus, plasma membrane, and acrosomal cap of buffalo spermatozoa occurs during freezing and thawing followed by equilibration.

Effects of dietary selenium on sperm motility in healthy men

Abstract: A deficiency of dietary selenium leads to immotile, deformed sperm and infertility in rats, whereas supplementation of the diet with selenium compounds has been associated with both beneficial and deleterious effects on sperm function, depending on the chemical form of selenium. We conducted a randomized, controlled, and blinded intervention study on the effects of selenium in food on semen quality. Eleven healthy men were fed a controlled diet of foods naturally high or low in selenium for 120 days while confined in a metabolic research unit. Dietary selenium was 47 microg/d for the first 21 days, then either 13 microg/d or 297 microg/d for 99 days, resulting in significant changes in selenium concentrations in blood and semen. Seminal plasma selenium concentration increased 50% with high selenium and decreased 40% with low selenium. The fraction of motile sperm in the high-selenium group decreased by 32% by week 13 and ended 18% lower than baseline. Selenium concentrations changed in seminal plasma but not in sperm, and serum androgen concentrations were unchanged in both groups, indicating this effect was neither androgen dependent nor caused by a change in the selenium supply to the testes. Serum triiodothyronine decreased and thyroid-stimulating hormone increased in the high-selenium group, suggesting that altered thyroid hormone metabolism may have affected sperm motility. Although this decrease in sperm motility does not necessarily predict decreased fertility, the increasing frequency of selenium supplementation in the healthy population suggests the need for larger studies to more fully assess this potential side effect.

Efficacy and safety of sildenafil citrate for treatment of erectile dysfunction in a population with associated organic risk factors.

Abstract: The objective of this study was to determine the efficacy and safety of sildenafil in patients with erectile dysfunction (ED) and associated organic risk factors in a multispecialty clinic. Patients (n = 521) were diagnosed with ED based on self-assessment. Associated risk factors were managed by medication or life-style modifications, or both, before treatment with sildenafil for ED. Patients received a 50-mg dose of sildenafil that could be adjusted to 100 mg or 25 mg based on tolerability and efficacy. Patients recorded the number of successful intercourse encounters for 6 to 8 weeks, and the number of adverse events. Overall, there was an 82% successful intercourse rate with sildenafil treatment. The predominant associated risk factors for ED were hypertension (39%), hypogonadism (37%), and multiple medications (34%). Common adverse events due to sildenafil treatment were mild to moderate in nature and resulted in <2% patient discontinuation. Clinicians should be particularly careful to evaluate patients presenting with ED because the condition can be accompanied by a wide spectrum of risk factors requiring monitoring and treatment. However, with adequate treatment and control of these risk factors, the use of sildenafil in a representative population of men with ED in a multispecialty clinic can achieve a higher efficacy rate than previous studies have indicated.

Testosterone Supplementation in Older Men: A Rational Idea Whose Time Has Not Yet Come

Abstract: Forty years after the introduction of estrogens, the debate over the risks and benefits of estrogen replacement therapy in postmenopausal women is still not entirely settled. In contrast, the issues of testosterone replacement in older men are just beginning to be addressed. There is agreement that total, free, and bioavailable testosterone (ie, not bound by sex hormone-binding globulin) levels decline progressively with advancing age (Harman and Tsitouras, 1980, 1982; Murono et al, 1982; Nieschlag et al, 1982; Zumoff et al, 1982; Bremner and Prinz, 1983; Davidson et al, 1983; Nankin and Calkins, 1986; Tenover et al, 1987; Gray et al, 1991; Vermeulen, 1991; Simon et al, 1992; Rudman and Shetty, 1994; Morley et al, 1997; Zmuda et al, 1997; Harmon et al, in press), and that many of the physiological changes that occur with advancing age such as loss of bone and muscle mass; increased fat mass; impairment of physical, sexual, and cognitive functions; loss of body hair; and decreased hemoglobin levels, are similar to those associated with androgen deficiency in young men. However, the beneficial effects of testosterone replacement on health-related outcomes in older men with low testosterone levels have yet to be demonstrated, and the risks of long-term testosterone administration, particularly the risks of prostate cancer and heart disease, remain unknown. Therefore, it is premature to make a general recommendation about testosterone supplementation in older men. Despite the paucity of efficacy and safety data, the sales of testosterone and other androgenic products have witnessed explosive growth recently

Characterization of the fertility of male aromatase knockout mice

Abstract: Previous studies employing the male aromatase knockout (ArKO) mouse have indicated that local expression of estrogens appears to be important for the progression of spermatogenesis. In the absence of estrogen biosynthesis round spermatids are observed to undergo apoptosis and thus fail to differentiate into mature, elongated spermatids. This lesion appears to arise between the ages of 18 weeks and 1 year. To ultimately determine if the disruption to spermatogenesis arises earlier than 18 weeks, we performed an intensive study to examine the fertility of younger male ArKO mice. This involved an analysis of their mating capacity together with an extensive stereological analysis, determination of the in vitro potential of mature sperm, and sexual behavior. ArKO and wild-type (w/t) males at 7 weeks of age were placed with w/t females for 7 weeks. At age 14 weeks, the males were killed and the testes removed. ArKO mice were observed to sire significantly fewer litters than the w/t mice; 5 out of the 10 sired no litters at all. Stereological analysis performed on the removed testes found a significant decrease in round spermatid numbers between w/t and ArKO mice at this age; however, there were no differences in all other germ cells and Sertoli cell numbers. When mature spermatozoa were analyzed, sperm from 15-week-old ArKO mice had a significant reduction in motility. This was further reduced by 1 year of age with a decrease in concentration. A preliminary examination of sexual behavior found that ArKO mice did not attempt to mount the females, in contrast to the w/t mice, which mounted consistently during the time period. In conclusion, we observed that ArKO mice have reduced fertility at age 14 weeks. This may be due in part to a disruption in spermatogenesis because the phenotype does appear to arise earlier than 18 weeks, possibly leading to abnormalities in the mature spermatozoa. Or, in part, this may be attributable to an impairment in the development of copulatory behavior, which is consistent with the available evidence that points to a crucial role for estrogens in the neural development and initiation of male sexual behavior.

Comparative study of cytochemical tests for sperm chromatin integrity.

Abstract: Tests were carried out on sperm from 40 fertile and infertile men to evaluate 2 DNA in situ denaturation methods using acridine orange (AO; the modified Rigler-Roschlau method and the Tejada method), alongside routine aniline blue (AB) and toluidine blue (TB) tests in our modification, and in order to estimate and compare the practical value of different in situ cytochemical tests for sperm chromatin structure. In addition, the methods were applied to rat and boar spermiogenesis models. The sperm heads with abnormal versus normal chromatin structure were specified as orange-red versus green by the AO method, blue versus uncolored by the AB method, and purple-violet versus light blue by the TB method. A good correlation for the proportion of sperm heads with abnormal chromatin structure was found among all the methods (r = .63-.70; P < .01), which characterized all 4 techniques as sensitive enough to estimate in situ sperm DNA integrity. In our study, the average value of abnormal cells was 17% +/- 3.8% and 30.2% +/- 6.8% for the fertile and infertile groups of men, respectively, setting a threshold of 95% probability at 23% as judged by the Rigler-Roschlau method. This compared with 23.9% +/- 7.5% and 52.1% +/- 20.8% (P < or = .05) for the fertile and infertile groups, respectively, setting a threshold at 31%, as judged by the Tejada method. The technical advantages and disadvantages of each method are briefly reported. Key words: Fertility, DNA normality, sperm maturation.

Semenogelin, the main protein of semen coagulum, inhibits human sperm capacitation by interfering with the superoxide anion generated during this process

Abstract: Semenogelin (Sg), the major protein of the human semen coagulum, is present at high concentrations in seminal vesicle secretions It is degraded by the prostate-specific antigen (PSA) to generate peptides of various biological activities that were found on and inside spermatozoa Our aim was to determine the effect of Sg on capacitation, which is the series of transformations that spermatozoa must undergo to become fertile At concentrations of 01 to 10 mg/mL (600- to 20-fold lower than those of semen), Sg did not affect sperm motility (%) but completely prevented capacitation induced by fetal cord serum ultrafiltrate; a partial inhibition of capacitation was noted with 003 mg Sg/mL There was also a dose-dependent decrease in the tyrosine phosphorylation of fibrous sheath proteins and in the O2--related chemiluminescence Ribonuclease (RNase), which has as high an isoelectric point (pI = 97) as Sg (pI = 95), also prevented sperm capacitation and O2--related chemiluminescence but to a lower extent, suggesting that one mechanism of Sg action on spermatozoa could be related to its positive charge at physiological pH Sg at 1, but not 03 or 01 mg/mL, scavenged the O2- generated by the mix of xanthine + xanthine oxidase and modified the kinetics of the reaction; RNase did not have such effects Therefore, Sg is a potential scavenger for O2- but probably also affects the sperm oxidase Spermatozoa rapidly processed Sg; a high proportion of Sg was degraded after 15 minutes of incubation The resulting polypeptide patterns were reminiscent of those obtained with PSA as a proteolytic enzyme These data suggest that Sg, its degradation products, or both may be natural regulators of sperm capacitation and could prevent this process from occurring prematurely One mechanism by which Sg acts could involve an interference with the O2- that is normally generated during this process

Cryopreservation-Thawing of fractionated human spermatozoa is associated with membrane phosphatidylserine externalization and not DNA fragmentation.

Abstract: The objective of these studies was to evaluate the effect of cryopreservation-thawing of human spermatozoa on DNA fragmentation and membrane integrity. This was a prospective, controlled cohort study, performed at a university-based infertility center. Ejaculates were examined from 5 donors and 16 men undergoing infertility evaluation. Purified sperm populations were prepared by gradient centrifugation, cryopreserved using a manual method and TEST-yolk buffer and glycerol (TYB-G), followed by quick-thaw. Annexin V binding was used for assessing membrane translocation of phosphatidylserine, and terminal deoxynucleotidyl transferase-me-diated dUTP nick end labeling (TUNEL) was utilized for the evaluation of DNA fragmentation. The results were as follows: the percentage of live cells with intact membranes (annexin V−, live) was significantly reduced after cryopreservation-thawing. On the other hand, the percentages of live cells with phosphatidylserine translocation (annexin V+, live) and of necrotic (dead) cells increased significantly after thawing. TUNEL revealed percentages of cells with DNA fragmentation in the prefreeze and postthaw samples that were not significantly different. In a further attempt to examine differences in response to various cryoprotection protocols, experiments were carried out using no cryoprotection, glycerol alone, or TYB-G. Samples frozen with TYB-G demonstrated significantly higher percentages of live cells without phosphatidylserine translocation than the other conditions. We concluded that cryopreservation-thawing of human sperm from patients and donors was associated with membrane change, as revealed by membrane translocation of phosphatidylserine, while having no major impact on DNA fragmentation.

Epidermal Growth Factor Modulates the Expression of Vascular Endothelial Growth Factor in the Human Prostate

Abstract: The growth and dissemination of tumors in the body has been associated with angiogenesis. Vascular endothelial growth factor (VEGF) is an angiogenic factor that stimulates endothelial cell growth and enhances vascular permeability. VEGF exerts its action by binding to specific cell surface receptors. Three receptors, VEGFR-1 (flt-1), VEGFR-2 (flk-1), and VEGFR-3 (flt-4) have been identified. Very little information on the coordinated expression of VEGF and its receptors in normal prostate, benign prostatic hyperplasia (BPH), and prostate carcinoma is available. Therefore, we examined the immunohistochemical localization of VEGF and its receptors in tissues derived from normal human prostate, BPH, and prostatic carcinoma. Immunostaining for VEGF was absent in the normal prostate. Epithelium lining the glands of prostate derived from patients with BPH exhibited strong immunostaining. The intensity of staining was relatively less in prostate carcinoma. It is interesting that VEGFR-1 and VEGFR-3 were strongly expressed in both stromal and epithelial tissues in normal prostate, BPH, and carcinoma. In comparison, VEGFR-2 was not localized to normal prostate and its expression in the stroma of BPH and epithelium of carcinoma was very weak. Because progression of prostate cancer is accompanied by altered expression of epidermal growth factor (EGF) and its receptor (EGFR) in malignant cells, we investigated the effect of EGF on VEGF gene expression by Northern blot analysis in 2 human prostate cancer cell lines that express EGFR. EGF greatly enhanced the expression of VEGF messenger RNA in DU145 and PC3 cell lines in a dose-dependent manner. The EGF induction of VEGF gene expression suggests a mechanism by which angiogenesis could be accelerated in BPH and prostate carcinoma.

A redox-regulated tyrosine phosphorylation cascade in rat spermatozoa.

Abstract: Rat spermatozoa from both the caput and cauda epididymidis were shown to generate superoxide anion (O 2 ) both spontaneously and following stimulation with NAD(P)H. Caput spermatozoa gave a significantly greater O 2 response to NADPH stimulation than caudal cells, whereas in both cell types the responses to exogenous NADPH and NADH were approximately equivalent. Analysis of H 2 O 2 production revealed that this oxidant was generated only by caudal epididymal cells and only in these cells did the stimulation of reactive oxygen species (ROS) production with NADPH lead to an increase in tyrosine phosphorylation. Stimulation of ROS production with NADPH increased intracellular cyclic adenosine monophosphate (cAMP) levels in both caput and caudal epididymal cells, but only in caudal cells did cAMP stimulate tyrosine phosphorylation, in keeping with the NADPH results. On the basis of these findings we propose that tyrosine phosphorylation in rat spermatozoa is driven by ROS acting via 2 different but complementary mechanisms; O 2 stimulates tyrosine kinase activity indirectly through the elevation of intracellular cAMP while H 2 O 2 acts directly on the kinase/phosphatase system, stimulating the former and inhibiting the latter. Zinc was examined as a potential regulator of this signal transduction cascade and was shown to suppress tyrosine phosphorylation in caput cells but to promote this activity in caudal spermatozoa, possibly through an inhibitory effect on tyrosine phosphatase activity. These results reveal the maturation of a redox-regulated, cAMP-mediated, signal transduction cascade during epididymal transit in the rat that is sensitive to zinc and plays a key role in the control of tyrosine phosphorylation events associated with capacitation.

Molecular genetic analysis of two human sperm fibrous sheath proteins, AKAP4 and AKAP3, in men with dysplasia of the fibrous sheath.

Abstract: Dysplasia of the fibrous sheath (DFS) is characterized by male infertility, asthenozoospermia, and morphologically abnormal flagella that possess a severely malformed fibrous sheath In many cases, DFS is familial, suggesting a genetic component Human AKAP4 and AKAP3 are structural proteins of the fibrous sheath that also function to anchor protein kinase A to this structure via the regulatory subunit of the kinase We hypothesized that defects in either AKAP4 or AKAP3 might cause DFS No quantitative or qualitative differences between patients with DFS and normal controls were detected when sperm proteins were analyzed by either silver staining or immunoblot analysis using antibodies raised against AKAP4 and AKAP3 Additionally, AKAP4 and AKAP3 from DFS sperm retained the ability to bind the regulatory subunit of protein kinase A Localization at the light and electron microscopic levels showed that AKAP3 and AKAP4 localized correctly to the FS of the amorphous flagellum in DFS sperm Partial sequence analysis of the AKAP4 and AKAP3 genes in patients with DFS did not identify any significant alterations in potential AKAP4/AKAP3 binding regions, suggesting that the two proteins interact normally in DFS sperm Our results did not find evidence to support the hypothesis that mutations in either gene are responsible for DFS in humans

Structure and control of a cell-cell adhesion complex associated with spermiation in rat seminiferous epithelium.

Abstract: Spermiation, the release of late spermatids from the Sertoli cell, is disrupted by a number of toxicants. Control of the spermiation process, and the proteins that interact to adhere mature spermatids to Sertoli cells, is poorly understood. In these studies we used immunohistochemistry, coimmunoprecipitation/Western blotting, and mass spectrometry to refine an earlier model of sperm adhesion proposed by our laboratory. We have identified specific proteins linked together as part of a multiprotein complex, as well as several additional proteins (cortactin, ERK1/2, and 14-3-3 zeta) that may be functioning in both structural and signal transduction roles. The current and prior data suggest that protein phosphorylation is central to the control of spermiation. We also present and characterize an in vitro tubule culture system that allowed functional testing of the spermiation model by pharmacologic manipulation, and yielded data consistent with the importance of protein phosphorylation in spermiation.

Assessment of the androgen environment within the human testis: minimally invasive method to obtain intratesticular fluid.

Abstract: Previous studies of the rat have shown that testosterone concentrations within the interstitial and seminiferous tubularfluids of the testes are significantly higher than normal serum levels, and further, that although intratesticular testosterone concentration can be substantially reduced without an effect on spermatogenesis, the concentration that is minimally required to maintain spermatogenesis is also substantially higher than serum levels. The purpose of the present study was to adapt a minimally invasive technique to sample human intratesticular fluid to enable parallel observations in man. To this end, aspiration methods were first developed for the rat testis and then adapted to the human. The testosterone concentration in fluid obtained by unilateral aspiration of rat testes was approximately 50 ng/mL, similar to the known concentration in seminiferous tubular fluid. These aspiration methods were then adapted to obtain intratesticular fluid from human testes. Studies of 12 fertile human subjects demonstrated that percutaneous testicular aspiration could be performed safely and successfully using a 19-gauge needle. Nine additional human subjects had bilateral testicular aspiration and simultaneous measurement of peripheral blood testosterone levels. Testicular aspirations yielded 8 to 117 microL of fluid from each testicle. The mean concentration of testosterone in aspirates obtained from the 21 patients was 609 +/- 50 ng/mL. Dihydrotestosterone and 3alpha-androstanediol concentrations were quite low, below the limits of detection of our assay. The SHBG/ABP concentration in the aspirates was 8.5 +/- 1.1 nM. These results define testosterone as the major androgenic steroid in the human testis, as in the rat testis, and indicate that the testosterone concentration within the human testis is approximately 200-fold greater than that of SHBG/ABP, and more than 100-fold greater than the concentration of testosterone found in normal human serum.

Sonographic testicular microlithiasis as an indicator of premalignant conditions in normal and infertile men.

Abstract: Sonographic detection of multiple, small hyperechogenic lesions in the testis (testicular microlithiasis; TM) can indicate germ cell tumors However, it has not been well established whether this finding signifies a risk factor for development of testicular neoplasm in all cases or whether it indicates premalignant changes only in those men with additional risk factors for germ cell cancer, such as infertility, a history of testicular maldescent, or the presence of an atrophic testis In a retrospective analysis of 1701 consecutively performed scrotal sonographies of patients with (n = 1399) and without (n = 219) infertility or with contralateral testicular tumors (n = 83), the prevalence of TM was compared with that in 198 healthy men who volunteered for different clinical trials TM was equally frequent in all groups (23% [32/1399] of infertile patients, 23% [5/219] of other patients without infertility, and 15% [3/198] of healthy men) Results of testicular biopsies were available for a subgroup of infertile men Carcinoma in situ (CIS) was present only in cases with TM (2/11) In addition, sonographic follow-up examinations were performed in another 14 men with TM Testicular tumors had developed in 2 patients, one whom was infertile and one in the control group None of these patients had a history of testicular maldescent but all testes affected either by CIS or tumors were reduced in volume We conclude that diagnosis of TM, especially if it is present in an atrophic testis, demands a diagnostic biopsy or at least sonographic follow-up examinations

Immunomagnetic isolation and long-term culture of mouse type a spermatogonia

Abstract: In the mammalian testis, type A spermatogonia proliferate and differentiate into sperm under the tight control of both endocrine and paracrine factors. In order to study the complex process of spermatogenesis at the molecular level, an in vitro system must be devised in which type A spermatogonia can be cultured for a prolonged period of time. Therefore, cocultures including type A spermatogonia and Sertoli cells, which act as nurse cells to the developing germ cells, are desirable. We have developed a method for the specific isolation of type A spermatogonia using magnetic beads and antibodies that recognize the c-kit receptor or the homophilic adhesion molecule, Ep-CAM. Purified spermatogonia could survive for a period of 25 days when cocultivated on Sertoli cell monolayers. Moreover, we recently established Sertoli cell lines that produce growth factors that are essential for the maintenance of spermatogonia in a proliferative state. Some of these Sertoli cell lines are able to reorganize into tubular structures when cultivated on a layer of Matrigel as extracellular matrix. We show here that type A spermatogonia associate specifically with the Sertoli cell tubules, and are able to replicate their DNA in this environment. Thus, these in vitro culture systems could be used for the long-term culture of primary, nonimmortalized type A spermatogonia.

Impaired seminal antioxidant capacity in human semen with hyperviscosity or oligoasthenozoospermia.

Abstract: Antioxidant capacity of seminal plasma was evaluated in 120 semen samples subdivided into asthenozoospermic and oligoasthenozoospermic specimens with normal consistency and into asthenozoospermic and oligoasthenozoospermic specimens with hyperviscosity. Semen samples (n = 25) from normozoospermic donors were used as a control group. Scavenger antioxidant capacity of reactive oxygen species was evaluated by superoxide dismutase and catalase activity measurements, whereas the chain-breaking antioxidant efficiency was detected by total antioxidant status assessment. In semen with normal viscosity, unaltered enzymatic and nonenzymatic antioxidant capacity was revealed in the asthenozoospermic specimens, whereas low superoxide dismutase activity was detected in oligoasthenozoospermic samples. On the contrary, impairment of both the scavenger and chain-breaking antioxidative systems was revealed in asthenozoospermic and oligoasthenozoospermic hyperviscous ejaculates, regardless of sperm count. Catalase activity and total antioxidant status values were also reduced in the 2 subgroups of hyperviscous ejaculates compared with their respective matched controls, whereas similar superoxide dismutase activities were detected in oligoasthenozoospermic samples with normal and high consistencies. These results suggest that asthenozoospermia could be related to an antioxidant deficiency only in combined ejaculate pathologies, and that a severe impairment of the low and high molecular weight seminal antioxidative capacities could be associated with semen hyperviscosity.

Sex‐Sorting Mammalian Sperm: Concept to Application in Animals

Abstract: Sperm sexing can be used to produce sexed offspring with 85%-95% accuracy (Amann, 1999; Johnson and Seidel, 1999; Seidel et al 1999a). On September 1, 2000, the sale of sexed bovine sperm commented in the United Kingdom. It will be interesting to see to what degree sexed sperm penetrate the semen market. This verified sexed product sets the stage for commercialization around the world in major animal producing countries. This commercialization of sexed sperm occurred nearly 20 years after technology for accurately determining the proportion of X and Y sperm in semen was first developed at Lawrence Livermore National Laboratory. It came about due to advances in both the hardware and the software componenets of computer science, biophysic, cell biology and applied reproductive physiology plus efforts of innovative scientist. Many individuals have contributed in making semen sexing in animals a commercial reality since the research team of Bart Gledhill, Dan Pinkel, Duane Garner, Susan Lake, and Larry Johnson began following up on the first flow cytometric studies on human sperm by Friedrich Otto, Wolfgang Gohde, and Marvin Meistrich. There was also major input from personnel at USDA Beltsville Agricultural Research Center as well as scientists at Cambridge University, Atlantic Breeders Cooperative, Colorado State University and XY Inc. These include Chuck Allen, Rupert Amann, David Cran, Patrick Doyle, Mike Evans, Lisa Herickhoff, Mervyn Jacobson, Kehuan Lu, Chris Polge, Wim Rens, John Schenk, George Seidel, Glenn Welch, and many others.