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Interindividual differences in gonadotropin suppression achieved by short- and long-acting intramuscular testosterone (T) preparations were studied to detect factors hindering complete suppression of gonadotropins as the prerequisites for effective male contraception. Forty healthy men received a single injection of T propionate; 4 weeks later they received 2 injections of 1000 mg of T undecanoate (TU) given 6 weeks apart. Following TU, declines of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels were consistent in 17.5% and almost absent in 25% of men. Men showing the most rapid and consistent declines in LH and FSH levels received a slightly higher dose per body weight of TU (13.1 +/- 0.6 vs 11.3 +/- 0.6 mg/kg; P = NS) and reached higher maximal concentrations of total T (40 +/- 4.8 vs 18.4 +/- 2.4 nmol/L; P < .001) and free T as well as estradiol. Men with high fat mass (mean +/- SEM, 10.3 +/- 1.5 vs 23.2 +/- 6.4 kg) had a delayed increase in T levels and an impaired relative decline in LH (12 +/- 2% vs 53 +/- 10%) and FSH (17 +/- 6%. vs 70 +/- 25%) levels within the first 2 weeks after the first TU injection. We conclude that overweight reduces the chance of rapid and profound gonadotropin suppression during treatment with TU. Body weight needs to be considered to avoid failure of hormonal male contraception.

Isolation and culture of spermatogonial stem cells (SSCs) has become an approach to study the milieu and the factors controlling their expansion and differentiation. Traditional conventional cell culture does not mimic the complex situation in the seminiferous epithelium providing a basal, intraepithelial, and adluminal compartment to the developing male germ cells. SSCs are located in specific stem cell niches whose features and functional parameters are thus far poorly understood. It was the aim of this study to isolate SSCs and to explore their expansion and differentiation potential in a novel three-dimensional Soft-Agar-Culture-System (SACS). This system provides three-dimensional structural support and multiple options for manipulations through the addition of factors, cells, or other changes. The system has revolutionized research on blood stem cells by providing a tool for clonal analysis of expanding and differentiating blood cell lineages. In our studies, SSCs are enriched using Gfralpha-1 as a specific surface marker and magnetic-activated cell sorting as a separation approach. At termination of the culture, we determined the type and number of germ cells obtained after the first 24 hours of culture. We also determined cell types and numbers in expanding cell clones of differentiating germ cells during the subsequent 15 days of culture. We analyzed a supportive effect of somatic cell lineages added to the solid part of the culture system. We conclude that our enrichment and culture approach is highly useful for exploration of SSC expansion and have found indications that the system supports differentiation up to the level of postmeiotic germ cells.

A significantly greater percentage of motile than immotile spermatozoa bore droplets at the osmolality of semen and cervical mucus. The percentage of spermatozoa with droplets was not significantly correlated with the osmolality of semen or the extent of cell swelling in response to quinine in hypotonic medium. Droplets appeared slightly more frequently (ie, were more obvious) in the presence of quinine, which blocks regulatory volume decrease, indicating that they are the major site of volume expansion. There was no selection for or against droplet-bearing spermatozoa migrating through viscous surrogate mucus at high or low osmolality. Sperm swelling in response to quinine at mucus osmolality was significantly greater in fathers than in patients whose partners had no fertility problem. Therefore, cytoplasmic droplets are not deleterious to sperm motility and may be related to physiological volume regulation, which may be predictive of some forms of human male infertility.

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.

Although cryopreservation of sperm is performed routinely in adults, only a small amount of information is available on its feasibility in adolescent patients with malignancies. Of 936 patients who were candidates for sperm cryopreservation, 851 (111 adolescents and 740 adults) were eligible for this retrospective analysis after excluding patients with relapses of the original or secondary cancers, known bitesticular lesions, or an unknown diagnosis. In general, patients were seen before initiation of treatment for malignancies. However, unilateral ablation of the testis was performed in 61% of patients with testicular cancer before cryopreservation of samples. Patients were grouped according to primary diagnosis and age. Measurements included testicular volume, semen analysis, and serum hormones (luteinizing hormone [LH], follicle-stimulating hormone [FSH], and testosterone). The youngest patient with an ejaculate containing sperm was 13.5 years old. No significant differences in any investigated parameter could be detected for any diagnosis among the 111 adolescents (age, <20 years). In contrast, adult patients with testicular cancer showed higher FSH values and lower sperm concentrations than adult patients with lymphomas, leukemias, and bone cancers. Patients younger than 16 years had lower ejaculate volumes than men older than 25 years, and testosterone levels were higher in patients aged 20-29 years than in the youngest patient group. Cryopreservation of sperm can be performed in adolescent patients with overall success rates (defined as the observation of at least a single motile sperm after the thawing procedure) similar to those observed in adults and should be recommended even to oncological patients younger than 15 years, provided that these patients can produce a semen sample.

Currently available testosterone (T) preparations differ substantially in their pharmacokinetic profile that might influence their androgenic properties in terms of suppression of the gonadal axis, effects on anabolic parameters, lipid metabolism, and erythropoiesis. The present work was undertaken to determine the physiological effects of three T preparations with different serum kinetics. Twenty adult male cynomolgus monkeys (Macaca fascicularis) were randomly assigned to receive treatment for 28 weeks with either T enanthate (TE) every 4 weeks, T buciclate (TB) every 7 weeks, or T undecanoate (TU) every 10 weeks or remaining untreated (controls). Each injection delivered 20 mg pure T per kilogram body weight. Pharmacokinetic profiles demonstrated higher peak levels of T for TE-treated animals; serum half-lives were longer for TU or TB. Estradiol levels (area under the curve) were significantly higher in TB vs TU or TE. All T regimens suppressed serum luteinizing hormone bioactivity and testicular volumes declined (all P <.001 vs controls). Sperm counts were markedly lowered in all animals but least in TE (P <.01 vs TB or TU). During recovery phase, return to normal for all three parameters occurred significantly earlier in TE-treated animals, followed by those given TU, compared with TB (all P <.001 between groups). Body weight increased significantly during T exposure. This effect was stronger and more sustained in TB vs TU or TE (both P <.001). Serum creatinine and hemoglobin increased with high significance in all T-treated animals (all P <.001 vs controls). The lowering impact of T on serum lipids was markedly stronger in the longer-acting T preparations in comparison with TE, as were effects on purine metabolism (all P <.001). The pattern of exposure and degree of aromatization rather than overall exposure to T determine its effects in the preclinical primate model. Both fluctuations of androgen concentrations and the conversion rate to estradiol influence gonadal suppression as well as metabolism. These results have to be considered in men receiving treatment for hypogonadism or regimens for hormonal contraception.

A multicenter, open-label, randomized efficacy and safety study was performed with combined human chorionic gonadotropin (hCG) and recombinant follicle-stimulating hormone (recFSH) (Puregon(R)) treatment to induce spermatogenesis in hypogonadotropic hypogonadal male patients. Patients were pretreated for 16 weeks with hCG to normalize testosterone levels. A total of 30 of 49 (61%) subjects had normalized testosterone levels but were still azoospermic after the hCG-alone phase. These patients were randomized into 2 treatment schemes with recFSH (2 x 225 IU recFSH per week [group A] or 3 x 150 IU recFSH per week [group B]), in combination with hCG for a period of 48 weeks. Total testosterone increased during the hCG-alone period from 1.08 and 1.22 ng/mL to 6.26 and 4.52 ng/mL for groups A and B, respectively. Combined gonadotropin treatment was effective in inducing spermatogenesis (sperm count >/=1 x 10(6)/mL) in 14 of 30 subjects (47%) and this was achieved after a median duration of treatment of approximately 5.5 months. Treatment time necessary for first sperm cells to appear in the ejaculate was related to the initial testicular volume. Subjects with a history of maldescended testes (11 of 30 subjects, 37%) showed a lower mean response to treatment as indicated by the relatively lower number of subjects reaching levels of at least 1 x 10(6) sperm cells per milliliter. Combined testicular volume increased during combined gonadotropin treatment from 11.4 to 24.0 mL. Although subjects with a history of maldescended testes had a lower starting testicular volume, subjects with and without a history of maldescended testes showed approximately the same relative increase in testicular volume. Total testosterone levels showed only a minor further increase during the combined gonadotropin treatment period. In conclusion, a weekly dose of 450 IU (3 x 150 IU or 2 x 225 IU) recFSH, in addition to hCG, was able to induce spermatogenesis in many hypogonadotropic azoospermic men who failed to respond to treatment with hCG alone.

Klinefelter syndrome, with an incidence of 1:600 male newborns, is the most frequent form of male hypogonadism. However, despite its relatively high frequency, the syndrome is often overlooked. To prevent such oversights, the clinical features should be better characterized, and simple screening tests should be used more frequently. In a cohort of 309 patients suspected of having Klinefelter syndrome, we evaluated the clinical symptoms as well as the diagnostic value of the Barr body test for screening procedures. On the basis of chromosome analysis, 85 patients (group I) were diagnosed as having Klinefelter syndrome, and 224 patients had a 46,XY karyotype (group II). Barr body analysis revealed a specificity of 95% and a sensitivity of 82% for the diagnosis of Klinefelter syndrome. General features (eg, reason for admission, age, age of the parents, body weight, and frequency of maldescended testes) were not different between the groups, except that group I had a higher proportion of patients with a lower educational background. Compared to group II, patients with Klinefelter syndrome were taller (P <.001); had smaller testis volumes (P <.0001), higher follicle-stimulating hormone (FSH) and luteinizing hormone (LH) values; and carried a tendency for less androgenic phenotype and secondary hair distribution. Testosterone, estradiol, sex hormone-binding globulin (SHBG), and prostate-specific antigen (PSA) serum levels as well as prostate volume were not significantly different between the groups. In patients who provided an ejaculate, azoospermia was found in 54% of the patients in group II and in 93% of the patients with Klinefelter syndrome. Although not exclusively characteristic for Klinefelter syndrome, the combination of low testicular volume and azoospermia, together with elevated gonadotropins, is highly indicative for a Klinefelter syndrome and should stimulate further clinical investigations. Barr body analysis provides a quick and reliable screening test, which, however, must be confirmed by karyotyping.

Transgenic male mice carrying inactive mutations of the receptor tyrosine kinase c-ros lack the caput epididymidis initial segment and are infertile because sperm volume regulation is compromised. Complementary DNA arrays were used to detect differences in gene expression in the caput epididymidis of heterozygous fertile and homozygous infertile males. The glutamate transporter excitatory amino acid carrier 1 (EAAC1) was expressed in all epididymal regions with high expression in the initial segment and cauda epididymidis. Homozygous knockout mice did not express EAAC1 messenger RNA (mRNA) in the caput but they did express the gene in the corpus and cauda. Immunohistochemical staining for EAAC1 confirmed regional mRNA expression and demonstrated an adluminal location on stereocilia/microvilli of principal cells. The glutamate transporter-associated protein (GTRAP) 3-18 was detected in all epididymal regions independent of genotype, but a highly abundant novel transcript of 4.2 kilobases was found only in the initial segment of heterozygous c-ros mice. High-performance liquid chromatography measurement of glutamate revealed a significantly higher content in the proximal caput of infertile mice than fertile mice, and tissue glutamate content decreased distally in both genotypes. Because glutamate is used as an osmolyte in somatic cells, the lack of EAAC1 reported here may disturb normal osmolyte balance in the proximal epididymal lumen and compromise sperm maturation, in particular the development of sperm volume regulatory mechanisms.

Hormonal contraception for men requires administration of testosterone and gestagens. The effects of a long-acting testosterone ester and 2 different progestins on hemostatic activation parameters were studied in relation to cardiovascular risk. In phase 1, 7 healthy men aged 28-38 years received a single intramuscular injection of 200 mg norethisterone-enanthate (NET-EN) on Day 0. Plasma samples were obtained on Days 0, 14, 41, and 84. In phase 2, 3 groups of 14 healthy men aged 18-45 years received four injections (every 6 weeks) of 1000 mg testosterone undecanoate (TU), plus daily oral placebo or daily oral levonorgestrel (LNG, 250 microg); or four injections (every 6 weeks) of NET-EN. Treatment lasted 24 weeks. Plasma samples were obtained at weeks 0, 16, 24, and 52. All samples were assayed for levels of coagulation factors VIIc, VIIa, XIIc, and XIIa; prothrombin fragment F1+2 (F1+2); antithrombin; plasmin-alpha(2)-antiplasmin-complex (PAP); and fibrinogen. NET-EN alone led to a depletion of sexual hormones and a marked shift in hemostatic parameters with increasing levels of FXIIc, fibrinogen, antithrombin, and F1+2, whereas FVIIc and FVIIa levels decreased. PAP levels increased significantly. Opposite effects were seen in the TU/placebo group, with a significant down-regulation of fibrinolysis and the hemostatic turnover rate. Testosterone effects were attenuated by additional administration of gestagens. The effect of hormonal male contraception using long-acting testosterone esters with or without gestagens was significantly measurable within the hemostatic system. Down-regulation of the hemostatic system with testosterone alone may indicate an antithrombotic effect, whereas clinical consequences of an additional gestagen compound cannot be derived.

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

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 (2.3% [32/1399] of infertile patients, 2.3% [5/219] of other patients without infertility, and 1.5% [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.

Nonhormonal contraceptives that act by blocking energy metabolism within sperm have the advantage over spermatogenic inhibitors by their fast onset of infertility and their almost immediate restoration of fertility after withdrawal of the contraceptive agent. This study was done to test new chlorinated compounds for their contraceptive potency on rodent and human sperm in vitro. Cells were incubated in a medium containing glucose as the sole energy source with 1-chloro-3-hydroxypropanone (CHOP) and 1,6-dichloro-1,6-dideoxy-D-fructose (DCDF), chlorinated analogues of glycolytic substrates, as well as racemic (R,S)-alpha-chlorohydrin (ACH). After incubation, enzymatic activity and kinematic parameters were estimated. A dose-dependent inhibition of the glycolytic enzyme, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), of rat and mouse distal cauda epididymidal and human ejaculated sperm by ACH, CHOP, and DCDF was demonstrated. Triosephosphate isomerase (TPI) was inhibited by ACH, but not by CHOP and DCDF, irrespective of species. All compounds inhibited sperm motility and kinematic parameters with increasing concentration. The results confirm that inhibition of glycolytic enzymes of sperm, including those of human, can be effectively brought about by a variety of chloro-compounds that can be converted to (S)-3-chlorolactaldehyde, the stereospecific chloro-derivative of the enzyme's natural substrate, (R)-glyceraldehyde 3-phosphate, and could be developed into contraceptive agents for men.

During spermiogenesis, histones are replaced by transition proteins, which in turn are replaced by protamines. The TNP1 gene-encoding TP1 (transition protein 1) protein contains a cAMP-responsive element (CRE) that serves as binding site for the CRE modulator (CREM). To gain further insight into the complex regulation of nucleoprotein exchanges in haploid spermatids and its potential role for spermatogenic impairment, we studied the gene expression of testis-specific histone H1t, CREM, and TNP1 in testicular biopsies from men with normal spermatogenesis (n = 20) and with round spermatid maturation arrest (n = 16). During normal spermatogenesis, H1t messenger RNA (mRNA) was present in 86.2%+/-8.7% of pachytene spermatocytes (stages III-V), whereas H1t protein was synthesized in 83.5%+/-13.0% of pachytene spermatocytes (stages III-V) and persisted in 95.2%+/-3.1% of spermatids (steps 1-5). CREM mRNA was detectable in 74.2%+/-9.4% of pachytene spermatocytes (stages IV-V) and in 78.7%+/-10.0% of spermatids (steps 1-3). CREM protein was synthesized in 81.2%+/-14.2% of spermatids (steps 1-3). TNP1 mRNA was present in 80.0%+/-13.5% of spermatids (steps 2-4), whereas TP1 protein was synthesized in 89.7%+/-5.3% of spermatids (steps 3-4). In men with round spermatid maturation arrest, spermatids only develop to step 3 of differentiation. These spermatids were devoid of both CREM and TP1 but did contain H1t. These results indicate that TP1 is likely to be an important parameter in the histone-to-protamine exchange and in the initiation of spermatid elongation. CREM is involved in the regulation of TNP1 gene expression and consequently plays a vital role in the correct differentiation step from round spermatids to mature spermatozoa.

Factors controlling the appearance in the epididymis of basal cells and their expression of macrophage antigens were examined by ligating the efferent ducts to prevent the entry of spermatozoa in adult and juvenile mice. Fixation and antigen retrieval techniques were developed to preserve tissue morphology and expression of two macrophage antigens in paraffin-embedded epididymal tissue. A combination of periodate-lysine-phosphate fixation, low-temperature embedding and enzyme predigestion of sections permitted immunohistochemical detection of the mature macrophage antigen Mac-1, whereas the panmacrophage marker F4/80 required fixation in neutral-buffered formalin. Epididymal basal cells were immunostained for F4/80 and quantified with avidin-biotin-peroxidase. In the adult mouse, the total number of basal cells per millimeter length of tubule cross section perimeter and the percentage expressing the F4/80-antigen were significantly higher in the initial segment and caput region than in all other epididymal regions. In the initial segment, immunostained basal cells surrounded the tubule in a network, and some extended towards the lumen. Ligation of the efferent ducts to prevent inflow of testicular secretions significantly reduced the number of basal cells per cross section in the initial segment of the adult and juvenile; the percentage of basal cells expressing macrophage antigens in the initial segment and the caput epididymidis was also reduced. Since basal cells still appeared in the ligated postpubertal epididymis, it is concluded that testicular exocrine secretions entering the epididymal lumen around puberty are not the major influence on basal cell appearance in the murine epididymis, but they may modulate their expression of macrophage antigens.

To evaluate the effects of testosterone (T), dihydrotestosterone (DHT), and estradiol (E2) on the regulation of prostate growth and tissue composition, the following study was conducted in a nonhuman primate model. Fifteen adult, long-term castrated cynomolgus monkeys were randomly assigned to receive implants filled with T (0.19 +/- 0.01 g), DHT alone (0.21 +/- 0.01 g), or (99%) DHT + (1%) E2 (0.21 +/- 0.01 g). Prior to and at 4-week intervals during the treatment phase of 252 days, prostate volumes (PV), body weight, ejaculate weight, hormone levels (of T, DHT, and E2), and red blood cell count were measured. Five adult, intact monkeys served as controls for prostate volume and histology. At the end of the study, histological analysis of an ultrasound-guided prostate biopsy was performed. T levels increased significantly in the T group compared with baseline (P < 0.01) and with the DHT and DHT + E2 groups (P < 0.05). Both groups receiving DHT showed higher DHT levels than did animals in the T group (P < 0.001). E2 levels in all groups increased over time (P < 0.05), although significant differences (P < 0.01) could only be detected between the DHT + E2 and the DHT group. Prostate volume in all groups increased (at baseline: T = 1.03 +/- 0.12 ml, DHT = 1.08 +/- 0.15 ml, DHT + E2 = 1.13 +/- 0.09; at day 252: T = 5.83 +/- 1.00, DHT = 4.72 +/- 0.9, DHT + E2 = 5.05 +/- 0.62) over time (P < 0.001), whereas no differences could be detected between the groups. Prostate biopsy could be performed successfully in 15 out of 20 monkeys. Prostate tissue evaluation between the treatment groups and the evaluated intact monkeys revealed no differences in the status of secretory epithelia, nuclear chromatin, excretory vacuoles, interstitial stroma, smooth muscles, and total functional status, whereas the prostate of a long-term castrated monkey showed severe atrophy. Thus, both androgens fully restored prostate volume and ejaculatory function. Highly supraphysiological DHT serum levels are not associated with abnormal volumetric or histological changes of the prostate. Comparing the DHT group with the DHT + E2 group, an additional stimulatory effect of normal or slightly elevated estrogens on the prostate cannot be found in the presence of highly supraphysiological DHT levels.

Several studies have reported the occurrence and significance of programmed cell death (apoptosis) of testicular germ cells in mammals. In those studies, apoptotic germ cells were identified by morphological criteria or by in situ end labeling (TUNEL) and were enumerated from histological sections by semi-quantitative and time-consuming techniques. In the present study, we have established a flow cytometric technique for quantification of TUNEL-positive cells in the mouse testis. Groups of five adult mice each received 0, 650, or 1300 mg/kg (IP) of methoxyacetic acid (MAA), and testes were collected 24 hours later. MAA is known to induce germ cell apoptosis in rodent testes. MAA induced a significant (P < 0.01) dose-dependent decline in the percentage of pachytene spermatocytes (4C cells). DNA strand breaks generated by the activation of endogenous endonuclease in the apoptotic germ cells were detected by the in situ labeling of the 3'-OH termini with biotinylated dUTP in the presence of terminal deoxynucleotidyl transferase (TUNEL technique). Histologically, TUNEL-positive germ cells were observed in control testes, and the number of these cells was visibly increased following MAA exposure. As determined by flow cytometry, four cell populations contained TUNEL-positive cells: 1C cells (round spermatids), 2C cells (mainly spermatogonia), S-ph cells (spermatogonial cells and preleptotene spermatocytes synthesizing DNA [the S-phase]), and 4C cells (primary spermatocytes). Analysis of the percentages of TUNEL-positive cells within each population yielded values of 1.57+/-0.23% for 1C cells, 1.65+/-0.27% for 2C cells, 6.26+/-1.03% for S-ph cells, and 3.24+/-0.39% for 4C cells. Hence, a substantial proportion of proliferating cells are undergoing apoptosis during normal spermatogenesis. The overall incidence of apoptotic cells among all testicular cells was around 2%. At 650 mg per kilogram of body weight, MAA induced a fourfold to eightfold increase (P < 0.001) in the percentage of TUNEL-positive cells, compared with saline-treated controls, and, overall, 17% of testicular cells were apoptotic. This effect of MAA was most pronounced for S-ph and 4C cells, with 25-30% of cells being affected in each of those populations. At 1300 mg per kilogram of body weight, MAA had no further effect. These quantitative data demonstrate that 1) in the normal testis, it is mainly proliferating cells that undergo apoptosis, and 2) MAA induces primary spermatocyte loss by germ cell apoptosis.

This study analyzed the hormonal requirements necessary for the development of primate testicular peritubular cells. Alpha-smooth muscle actin, as a specific differentiation marker for peritubular cells, was immunohistochemically detected in the testes of immature rhesus and adult rhesus and cynomolgus monkeys. Positive staining was localized in the wall of blood vessels and in peritubular myoid cells of adult animals. In the testis of vehicle-treated immature monkeys no positive staining could be detected in peritubular cells. Following follicle-stimulating hormone (FSH) or testosterone treatment, immunostaining for alpha-smooth muscle actin appeared in peritubular cells of immature animals. In comparison to FSH, testosterone was more effective in inducing the differentiation of peritubular cells. The most intense label for alpha-smooth muscle actin was, however, obtained after combined treatment with FSH and testosterone. A computer-assisted image analysis system was used to evaluate semiquantitative data for the alpha-smooth muscle actin positive area; this confirmed a significantly higher expression of alpha-smooth muscle actin in peritubular cells of immature monkeys after treatment with testosterone plus FSH in comparison to controls. These data present evidence that the differentiation of primate peritubular cells during pubertal sexual development is a hormone-dependent process, which may be predominantly regulated by androgens. FSH exerted additive effects on the androgen-induced differentiation of peritubular cells, suggesting that paracrine communication between peritubular cells and Sertoli cells existed. Maintenance of peritubular cell differentiation after withdrawal of hormonal stimuli in adult monkeys may be a possible explanation for different hormonal requirements during reinitiation of spermatogenesis after testicular involution.

Most computer-aided sperm analysis (CASA) systems for human spermatozoa use positive phase-contrast optics. In this study the performance of one of these (HTM) and two other systems using negative phase optics (IVOS and SM) were assessed by scrutinizing the video-playback of the analyzed images for errors in the recognition of motile and immotile spermatozoa and in the tracking of motile cells. Whereas both the HTM and IVOS provided accurate measurements of motile sperm concentrations, errors in total sperm concentrations and motility percentages were greater in the IVOS than the HTM. However, IVOS appeared to be more accurate in tracking motile spermatozoa. Individual sperm track data suggested that velocity measurements may be biased by the elimination of incomplete tracks from the analysis. With the SM system, discrimination of immotile spermatozoa from non-sperm particles in semen was poor despite an additional algorithm for tail detection. The use of negative phase optics in CASA may be superior to that of positive phase in the analysis of motile spermatozoa but inferior in the recognition of immotile spermatozoa.

Earlier reports indicated that sperm from 25% of patients from infertile couples, but not from normal or fertile donors, show deviations from the theoretical Poisson distribution of the number of sperm penetrating zona-free hamster ova. Using semen samples from 15 grandfathers (aged 60 to 84 years) and 24 young fathers (aged 25 to 36 years), this study analyzed whether age also has an effect on the distribution. It was found that the overall fit to the Poisson distribution of the samples from grandfathers was very poor; in contrast, the samples from young fathers fit well. The observed deviations from the Poisson distribution among grandfathers may be a consequence of their long periods of sexual abstinence. Decrease in sexual activity produces age-different populations of sperm that probably differ in penetrating ability. Samples from older fathers also show a worse fit to the Poisson distribution than do those from younger fathers. These results suggest that the duration of sperm storage in the genital tract after maturation has an effect on sperm function.

Alpha-glucosidase, glycerophosphocholine, and L-carnitine were measured in sperm-free seminal plasma to determine whether these markers reflected the epididymal function of men attending an infertility clinic. The putative markers correlated well with each other (r = 0.66 to 0.70) and in 92% of 283 cases were accurate in categorizing semen as containing normal or subnormal amounts of markers. Glucosidase was considered the best index of epididymal function and was used for a further 306 samples. The ejaculate content of epididymal markers was correlated with testicular volume and serum testosterone below values of 30 ml and 30 nmol/l, respectively. Markers were also correlated with the concentration and motility of spermatozoa in semen. Seventy-one of 425 patients (17%) displayed subnormal epididymal secretions, mainly in association with hypogonadism (Klinefelter syndrome, Kallman syndrome, idiopathic hypogonadotropic hypogonadism) but also in cases of obstructed ducts, maldescended testicles, and local irradiation following hemicastration. Because azoospermic patients had reduced epididymal markers with both high and low FSH levels and a large proportion of men with reduced glucosidase and normal FSH suffered from testicular failure, it is suggested that other indices of testicular function are required for correct interpretation of reduced epididymal markers. Thirteen patients (3%) had low markers for which no cause was apparent; these may be cases of infertility due to isolated epididymal dysfunction.

The present investigation evaluates the long-term effects of GnRH agonist treatment on testicular histology, sperm production and the subsequent recovery of these parameters. Four adult rhesus monkeys (M. mulatta) were treated with the GnRH agonist nafarelin (D-Nal(2)6-GnRH), released from i.m.--injected poly-D,L-lactic-co-glycolide microspheres for 20 months. Monthly injection of the GnRH agonist preparation uniformly suppressed serum levels of bioactive LH and testosterone. The size of the testis was reduced to about 30% of pretreatment. Sperm counts were suppressed to azoospermia for a total period of 53 and 77 weeks, respectively, in two monkeys and the other two animals were extremely oligozoospermic. Evaluation of testicular biopsy material after 6, 12 and 20 months of treatment revealed decreased seminiferous tubule diameter, spermatogenic disruption at the level of spermatogonia or spermatocytes, accumulation of lipid droplets and secondary lysosomes in the Sertoli cell cytoplasm, and increased thickness of the tubular wall compared with pretreatment histology. Electron microscopic examination revealed that the increased wall thickness was due to an enlargement of the inner collagen layer. No evidence of fibrosis or calcification could be obtained. Leydig cells were atrophic. Serum hormones, testis size and sperm counts returned to pretreatment values within 5 to 8, 13 to 16, and 18 weeks, respectively, after termination of treatment. Testicular histology, assessed 8 months after cessation of treatment, was indistinguishable from pretreatment. It is concluded that GnRH agonist-containing microspheres are a feasible modality for sustained administration of GnRH agonists and GnRH agonist-induced suppression of pituitary and testicular function is reversible following withdrawal of treatment. Thus, GnRH agonists may have a potential for regulation of male fertility and, presumably, also for treatment of precocious puberty.

Sperm penetration rates in the heterologous ovum penetration test were correlated with results of routine semen analysis in 30 fertile and 50 infertile men. There was no difference in penetration rates when comparing infertile men with normal and abnormal seminal parameters, nor was any difference seen between fertile (15-83%) and infertile men (8-83%). Of the 22 infertile men with normal seminal characteristics, seven had partners with no discernible reproductive dysfunction. The penetration rates of these men (38-81%) did not differ from those of fertile men. Stepwise regression analysis of seminal characteristics, with penetration as the dependent variable, indicated that sperm count and morphology are the most important seminal parameters for fertility assessment. Discrimination analysis revealed that sperm numbers and morphologic variables provide significant information for discriminating between fertile men and infertile men with normal or abnormal seminal characteristics. Sperm penetration and motility were not indicated as important factors. The present data suggest that, in unselected male patients seeking reproductive evaluation, the sperm penetration assay did not yield any additional information on the cause of infertility.

To evaluate the influence of indomethacin and oxaprozin on reproductive function in healthy young men, 34 volunteers with normal semen parameters were recruited. In a randomized double-blind design, 12 men were treated with placebo, 12 received 600 mg/day of oxaprozin and 10 took indomethacin 25 mg t.i.d. This treatment phase lasted for 14 days after which a follow-up period extended for another 10 weeks. Sperm counts, percentage of motile and normally formed sperm cells, sperm velocity, linearity, lateral head displacement and beat frequency were evaluated by computerized image analysis once before treatment and at weekly intervals during the rest of the study. Prostaglandin levels in seminal plasma were significantly reduced after 2 weeks of treatment and remained suppressed for at least 2 additional weeks. In spite of this long lasting impairment of physiologic prostaglandin concentrations, no changes in any of the measured parameters were detectable when compared with the placebo group. Basal levels of testosterone, estradiol, LH, FSH, TSH and prolactin were unchanged. The response of hypophyseal hormones to a combined GnRH/TRH test before, during and after the treatment also was not affected. Overall, no negative influence of indomethacin or oxaprozin treatment on male reproductive function could be found in healthy volunteers. Since the active treatment phase was only 14 days, one can only speculate about long term effects of the tested drugs on reproductive parameters in men.