Proteomic analysis of testis biopsies in men treated with transient scrotal hyperthermia reveals the potential targets for contraceptive development.

Mild testicular heating safely and reversibly suppresses spermatogenesis. In this study, we attempted to clarify the underlying molecular mechanism(s) involved in heat-induced spermatogenesis suppression in human testis. We conducted global proteomic analyses of human testicular biopsies before, and at 2 and 9 wk after heat treatment. Thirty-one and Twenty-six known proteins were identified with significant differential expression at 2 and 9 wk after heat treatment, respectively. These were used to characterize the cellular and molecular events in the testes when seminiferous epithelia became damaged (2 wk) and recovered (9 wk). At 2 wk post-treatment, the changed expression of a series of proteins could promote apoptosis or suppress proliferation and cell survival. At 9 wk post-treatment, the changed expression of proteins mainly promoted cell proliferation, differentiation and survival, but resisted cell apoptosis. Among those heat-regulated proteins, HNRNPH1 was selected for the further functional study. We found that HNRNPH1 was an anti-apoptosis protein that could regulate the expression of other heat-induced proteins. In conclusion, heat-induced reversible suppression of spermatogenesis occurred by modulating the expression of proteins related to proliferation, differentiation, apoptosis and cell survival pathways. These differentially expressed proteins were found to be key molecular targets affecting spermatogenesis after heat treatment.

[A study on population-based prenatal screening and diagnosis of Down's syndrome in Jiangsu province].

OBJECTIVE: To screen and diagnose Down's syndrome during mid-term pregnancy to reduce the number of babies with Down's syndrome. METHODS: With the multi-level of stratified cluster sampling, twenty thousand and eight hundred and three women at 15-20 weeks gestation were screened by maternal serum AFP and beta-hCG using the time resolved fluoroimmunoassay (TRFIA). Then the screened high-risk women were diagnosed by amniocentesis, cell culture and chromosome analyses. The born children were diagnosed by follow-up and peripheral blood chromosome analyses. RESULTS: Six fetuses were diagnosed by serum screening and amniotic fluid chromosome analyses, and 3 born children were diagnosed by follow-up and peripheral blood chromosome analyses. Nine cases of Down's syndrome were detected in total, with the positive prenatal screen rate being 67% (6/9). CONCLUSION: The prenatal screening and diagnosis can reduce the birth of Down's syndrome patients and improve the population quality. However, the diagnosis accuracy still needs to be improved to further reduce the false negative rate and prevent misdiagnosis.

[Long-term toxicity of Shen Yan Ling tablet and its effect on male reproductive function in rats].

OBJECTIVE: Shen Yan Ling Tablet is an innovative compound of traditional Chinese medicine, scientifically prepared with Tripterygium wilfordii, Radix Astragali, and others, with precise efficacy on renal diseases and reduced adverse effects of Tripterygium wilfordii. Based on the Guiding Principles for New Drug Toxicity Research Before Clinical Application, we investigated the long-term toxicity of Shen Yan Ling Tablet and its effect on the reproductive function in rats. METHODS: According to the clinical therapeutic dose and the results of the acute toxicity test of Shen Yan Ling Tablet, we equally divided 80 rats (males and females half-and-half) into a low-dose (1.25 g/kg body wt), a medium-dose (2.50 g/kg body wt), a high-dose (5.00 g/kg body wt) and a control group. After a 3-month medication, we conducted standardized long-term toxicity tests and observed the effects of Shen Yan Ling on the serum sexual hormones and epididymal sperm count. RESULTS: After 3 months of treatment with Shen Yan Ling, no death occurred, the general status remained unchanged, and the parameters of blood cytology and biochemistry fluctuated within the normal range, without any significant changes (P > 0.05). Some blood parameters, RBC, WBC, HGB, AST and TBIL, showed statistic changes (P < 0.05), but with no clinical significance. There were no significant differences in the mass coefficients of the main organs between the medication and control groups. The high-dose group exhibited slight hepatic and pulmonary pathological changes and significantly reduced sperm counts in the epididymis, but no significant changes in serum sexual hormones (P < 0.05). CONCLUSION: Three-month medication of Shen Yan Ling at 1.25 - 5.00 g/kg produced no significant accumulated toxicity on rats, but it had a negative effect on their reproductive function at a higher dose of > or = 5.00 g/kg.

Proteomic analysis of testis biopsies in men treated with injectable testosterone undecanoate alone or in combination with oral levonorgestrel as potential male contraceptive.

Treatment with injectable testosterone undecanoate (TU) alone or in combination with oral levonorgestrel (LNG) resulted in marked decreases in sperm concentrations. In this study, we used proteomic analyses to examine the cellular/molecular events occurring in the human testis after TU or TU + LNG treatment. We conducted a global proteomic analysis of the human testicular biopsies before and at 2 weeks after TU alone or TU + LNG treatment. Proteins showing significant changes in expression were identified and analyzed. As a result, 17 and 46 protein spots were found with significant differential expression after the treatment with TU alone and TU + LNG, respectively. TU treatment changed the expression of heterogeneous nuclear ribonucleoprotein K (hnRNP K), proteasome inhibitor PI31 subunit (PSMF1), and superoxide dismutase [Mn] mitochondrial precursor (SOD2). These proteins inhibit "assembly", induce cell death, and promote compensatory "cell survival" in the testis. After TU + LNG treatment, "proliferation/cell survival" and "apoptosis/death" were the predominant responses in the testis. TU + LNG treatment inhibited the expression of Prolyl 4-hydroxylase beta subunit (P4HB) and Annexin A2 (Annexin II). These proteins are involved in apoptosis and cell proliferation, respectively. TU + LNG treatment also enhanced the expression of SOD2 and Parvalbumin alpha (Pvalb). These two proteins may protect testicular cells against apoptosis/death and promote cell survival. In conclusion, TU and TU + LNG treatments suppress spermatogenesis through different pathways by changing the expression of different proteins. hnRNP K, PSMF1, SOD2, P4HB, Annexin II, and Pvalb, are key proteins that may be early molecular targets responsible for spermatogenesis suppression induced by hormone treatment.

[Effect of large-dosage of 5alpha-reductase inhibitors on the spermatogenesis of male rats].

OBJECTIVE: To identify the role of 5alpha-reductase in the spermatogenesis of male rats by studying the effect of two 5alpha-reductase inhibitors, Epristeride and Finasteride, on the spermatogenesis in male Sprague-Dawley (SD) rats. METHODS: Changes in the weight of the testis, serum testosterone and dihydrotestosterone levels, epididymal sperm count, and reproductive function were observed and analyzed after the two 5alpha-reductase inhibitors were administered to male SD rats orally. RESULTS: The experiment showed that in comparison with control animals, both the two 5alpha-reductase inhibitors: 1. suppressed the development of the prostate and reduced the weight of the testis in the experimental groups (P < 0.05); 2. decreased the serum level of dihydrotestosterone and enhanced testosterone; 3. inhibited epididymal sperm count and productive function. CONCLUSION: High dosages of the 5alpha-reductase inhibitor, Epristeride, can suppress the development of the prostate and reduce the weight of the testis, decrease dihydrotestosterone, and inhibit spermatogenesis and productive function in male rats.

[Health investigation of 3 551 middle-aged and old males in Jiangsu Province].

OBJECTIVE: To understand the state of health of the middle-aged and old males in Jiangsu Province. METHODS: A total number of 3 551 middle-aged and old males aged 46 approximately 69 years were randomly selected from 13 counties of Jiangsu Province. All of them received physical examinations, including laboratory tests of liver and kidney function, sugar and cholesterols, radioimmunoassay of testosterone and free testosterone levels in the serum, and B-ultrasonic examination of the prostate volume and remaining urine. They also underwent inquiries according to the International Index of Erectile Function (IIEF-5) and the questionnaire for PADAM (partial androgen deficiency of the aging male). RESULTS: The prostate volume differed significantly (P < 0.05), while the testosterone level showed no significant difference in different age groups (P > 0.05). The level of the free testosterone in the serum descended with the increase of age. The incidence of ED and PADAM was also correlated with the increase of age. CONCLUSION: In the old and middle-aged population of males, with the increase of age, sexual function decreases, the prostate volume enlarges, and the incidence of ED and PADAM obviously increases (P < 0.001).

[Effect of selective 5alpha-reductase inhibitor or/and testosterone undecanoate on the reproductive function of male rats].

OBJECTIVE: To investigate whether 5alpha-reductase inhibitor and dihydrotestosterone (DHT) play a role in spermatogenesis in male rats. METHODS: Thirty-two male rats were divided into 4 groups (Groups C, T, F and FT). Group C received plant oil injection and oral starch perfusion, Group T testosterone undecanoate (TU, 20 mg/kg) injection and oral starch perfusion, Group F plant oil injection and oral Finasteride perfusion, and Group FT TU (20 mg/kg) injection and oral Finasteride perfusion. Data on serum T and DHT, sperm count, sperm mobility and reproductive function were collected and analysed. RESULTS: (1) 5alpha-reductase inhibitor, Finasteride and TU reduced the weight of the testis and epididymis in the experiment groups compared with the negative control (Group C), but TU increased the weight of the prostate while Finasteride decreased it compared with the positive control (Group T). TU combined with Finasteride could counteract the effect of the weight increase of the prostate, but not that of the testis. (2) Finasteride, or Finasteride combined with TU, reduced the DHT but increased the testosterone level in comparison with the control group. (3) Both Finasteride and TU could inhibit epididymal sperm count and reproductive function compared with the control, but the effect was less significant in Group FT than in Group F. CONCLUSION: High dosages of 5alpha-reductase inhibitor, Finasteride, can suppress male reproductive function, but the inhibiting effect could be counteracted by administration of 5alpha-reductase inhibitor along with TU.

Quantitative (stereological) study of incomplete spermatogenic suppression induced by testosterone undecanoate injection in rats.

AIM: To evaluate the key lesions in spermatogenesis suppressed partially by testosterone undecanoate (TU) treatment. METHODS: Adult male SD rats were treated with vehicle or TU (19 mg/kg) injection (i.m.) every 15 days for 130 days. The numbers of all types of cells (nuclei) in the seminiferous tubules and the interstitial tissue were estimated using a contemporary stereological tool, the optical disector. RESULTS: In response to TU treatment, the numbers of non-type B spermatogonia, type B spermatogonia and late elongated spermatids per testis were reduced to 51 %, 66 % and 14 % of the controls, respectively. The conversion ratios from type B spermatogonia to early spermatocytes and pachytene spermatocytes were not significantly affected and the ratios to the later germ cell types fell to 51 % - 65 % of the controls. Less than 1.0 % of immature round spermatids were seen sloughing into the tubule lumen, 4.0 % of elongated spermatids retained in the seminiferous epithelium, and about half of the elongated spermatid nuclei appreciably malformed. Leydig cells were atrophied but their number and the peritubular myoid cell number per testis were unchanged. CONCLUSION: Double inhibition of spermatogenesis (i.e. inhibition at spermiation and spermatogonial conversion to type B spermatogonia), a scenario seen in the monkey and human following gonadotrophin withdrawal, was not sufficiently effective for a complete spermatogenic suppression in the rat after TU treatment, probably due to ineffective inhibition of the Leydig cell population and therefore the intra-testicular testosterone levels.

[Effect of injection of testosterone undecanoate and depot medroxyprogesterone acetate on the suppression of spermatogenesis].

OBJECTIVE: To observe depot medroxyprogesterone acetate (DMPA) and testosterone undecanoate (TU) injected at 8-week intervals for the suppression of spermatogenesis in healthy Chinese men. METHODS: After screening, 30 healthy volunteers were enrolled and randomly assigned to 3 dosage-groups (n = 10/group): 1000 mg TU (Group A), 1000 mg TU plus 150 mg DMPA (Group B), 1000 mg TU plus 300 mg DMPA (Group C). All dosages were given as intramuscular injections at 8-week intervals. The study consisted of an 8-week control (baseline) period, a 24-week treatment period and a 24-week recovery period. RESULTS: Consistent azoospermia or severe oligozoospermia was achieved and maintained in all the volunteers during the treatment period, except 2 in the mere TU group who experienced a "rebound" in sperm concentrations. An 8-week regimen of TU plus DMPA at both tested combination dosages effectively suppressed spermatogenesis to azoospermia. All volunteers tolerated the injections; no serious adverse effects were reported. CONCLUSION: The lower combined dosage is recommended for further testing in an expanded clinical trial or contraceptive efficacy study.

Male hormonal contraception: effects of injections of testosterone undecanoate and depot medroxyprogesterone acetate at eight-week intervals in chinese men.

Surveys indicate that one form of acceptable male hormonal contraception would consist of injections given at 2- to 3-month intervals. This report describes a study of depot medroxyprogesterone acetate (DMPA) and testosterone undecanoate (TU) injected at 8-wk intervals for suppression of spermatogenesis in healthy Chinese men. After screening, 30 healthy volunteers were enrolled and randomly assigned to one of three dose groups (n = 10/group): 1000 mg TU (group A); 1000 mg TU plus 150 mg DMPA (group B); 1000 mg TU plus 300 mg DMPA (group C). All doses were given as im injections at 8-wk intervals. The study consisted of an 8-wk control (baseline) period, a 24-wk treatment period, and a 24-wk recovery period. Consistent azoospermia or severe oligozoospermia was achieved and maintained in all volunteers during the treatment period, except for two men in the TU-alone group who experienced a rebound in sperm concentrations. An 8-wk regimen of TU plus DMPA at both tested combination doses effectively suppressed spermatogenesis to azoospermia in Chinese men. All volunteers tolerated the injections; no serious adverse effects were reported. The lower-dose combination is recommended for further testing in an expanded clinical trial or contraceptive efficacy study.

[Studies and progress of male hormonal contraception].

It has become more and more urgent to develop a safe, effective, recoverable and acceptable contraceptive for males. Decades of studies have made much progress on male hormonal contraception, one of the promising contraceptive methods. The principle is based on the suppression of pituitary gonadotropin and intratesticular testosterone, then the suppression of spermatogenesis, and the supplement of androgen to maintain the male characteristics and sexual function. There are many male hormonal contraceptive methods being studied include androgen, androgen combined with progesterone, GnRH antagonists combined with androgen, as well as immunological methods. To develop a safe and convenient androgen preparation with longer action and fewer side effects is also one of the key items of present research in this field.

[Effect of androgen on erythropoietin in patients with hypogonadism].

OBJECTIVES: To observe the change of erythropoietin (EPO) in patients of hypogonadism who received androgen replacement treatment and explore the mechanism of androgen-induced increase of red blood cells and haemoglobin. METHODS: Eight patients with Klinefelter's syndrome, divided into two groups, received TU intramuscular injections of 500 mg or 1000 mg dose, respectively. After three months, seven patients received the second injection of crossover dose. Testosterone levels in serum were measured with RIA before and after the injections treatment. RBC count, impacted volume of blood cells and haemoglobin concentration were measured before treatment and 4, 8 weeks after treatment. At the same interval, EPO levels were measured with ELISA method. RESULTS: Development of the secondary sex characters was improved in all patients after the TU injection. Serum testosterone levels raised significantly and reached the peak one week after the injections. Effective level of testosterone lasted for over 6 weeks. RBC count, impacted volume of blood cells and haemoglobin increased at different degrees after TU injections, but these changes were not significant in statistic(P < 0.05). The increased levels remained for 8 weeks. EPO levels were elevated significantly (P < 0.01 or 0.05) after the TU injection(Pbat > 0.05). The second injection could still make the EPO level go up. CONCLUSIONS: Androgen replacement treatment can increase the EPO levels in patients of hypogonadism, which is one of the mechanism of RBC production increase.

[Effect of MPA and MPA + TU on the rat spermatogenesis and sexual hormones].

OBJECTIVES: To investigate the effect of administration of MPA with/without TU on serum sexual hormones and spermatogenesis of male rats. METHODS: Twenty rats had been classified into four groups. Each group received injection of saline(group A) or MPA(37.5 or 75 mg/kg) (group B or group C, respectively) or MPA (75 mg/kg) + TU (25 mg/kg) (group D) every month during three months. Data from serum sexual hormones (FSH, LH, T), sperm counting and motility had been collected and analysed. RESULTS: Spermatogenesis of rats undergoing administration of MPA with or without TU had been suppressed. Serum FSH and LH of group B, C, D declined, and so did serum T of group D. Testis of rats of group D atrophied and sperm counting of group D decreased remarkably compared with group B and C. But there was no statistics difference of the sexual hormone level among group B, C and D. CONCLUSIONS: Administration of MPA alone could suppress the levels of FSH and LH and block the spermatogenesis of male rats. MPA combined with TU could offer stronger suppression on spermatogenesis. Mechanism of the suppression on spermatogenesis of MPA + TU is not only limited in the feed-back of gonadotropin, but there maybe exist a direct suppression on testis.

[Study on the male contraceptive based on epididymis].

More and more study on the epididymal function and sperm maturation has shown that epididymis will be one of the best target organs of male contraception, although at present there is not a male contraceptive medicine based on epididymis for clinical practice. The promoting research aspects in epididymal contraception in animal included affecting directly epididymis (such as Sulpasalazine), interfering energy metabolism and sperm mobility (such as Chlorinated Glycerol), altering the internal environment of epididymis (such as copper particles and TW19). The epididymal specific proteins could bring out some new target antigens for immunological contraception, to produce contraceptive vaccine. Some special genes, which expressed distinctively in epididymis such as SC342, bin1, have been cloned and studied on their function. These works would be helpful not only for clinical diagnosis and treatment of epididymitis and male infertility, but also for male contraceptive research and progress.