The effects of humanin and its analogues on male germ cell apoptosis induced by chemotherapeutic drugs.

Human (HN) prevents stress-induced apoptosis in many cells/tissues. In this study we showed that HN ameliorated chemotherapy [cyclophosphamide (CP) and Doxorubicin (DOX)]-induced male germ cell apoptosis both ex vivo in seminiferous tubule cultures and in vivo in the testis. HN acts by several putative mechanisms via binding to: an IL-12 like trimeric membrane receptor; BAX; or insulin-like growth factor binding protein-3 (IGFBP-3, a proapoptotic factor). To understand the mechanisms of HN on male germ cell apoptosis, we studied five HN analogues including: HNG (HN-S14G, a potent agonist), HNG-F6A (no binding to IGFBP-3), HN-S7A (no self-dimerization), HN-C8P (no binding to BAX), and HN-L12A (a HN antagonist) on CP-induced male germ cell apoptosis in mice. CP-induced germ cell apoptosis was inhibited by HN, HNG, HNG-F6A, HN-S7A, and HN-C8P (less effective); but not by HN-L12A. HN-L12A, but not HN-S7A or HN-C8P, blocked the protective effect of HN against CP-induced male germ cell apoptosis. HN, HN-S7A, and HN-C8P restored CP-suppressed STAT3 phosphorylation. These results suggest that HN: (1) decreases DOX (ex vivo) and CP (in vivo) induced male germ cell apoptosis; (2) action is mediated by the membrane receptor/STAT3 with minor contribution by BAX-binding pathway; (3) self-dimerization or binding to IGFBP-3 may not be involved in HN's effect in testis. HN is an important molecule in the regulation of germ cell homeostasis after injury and agonistic analogues may be developed for treating male infertility or protection against chemotherapy side effects.

Integrity of the blood-testis barrier in healthy men after suppression of spermatogenesis with testosterone and levonorgestrel.

STUDY QUESTION: Do exogenous male hormonal contraceptives that suppress intratesticular testosterone and spermatogenesis interfere with the blood-testis barrier integrity in men? SUMMARY ANSWER: When spermatogenesis was suppressed by testosterone alone or combined with levonorgestrel (LNG) treatment in men, the structural appearance of Sertoli cell tight junctions remained intact in the human testis. WHAT IS ALREADY KNOWN: Testosterone promotes the integrity of the blood-testis barrier. Intratesticular androgen deprivation induced by exogenous testosterone plus a progestin to suppress spermatogenesis in a contraceptive regimen may disturb the structural and functional integrity of the blood-testis barrier. STUDY DESIGN, SIZE AND DURATION: Testicular biopsies were obtained from a sub-study of a randomized clinical trial of 36 healthy Chinese men who were treated for 18 weeks and followed for at least a 12-week recovery period. PARTICIPANTS/MATERIAL, SETTING, METHODS: Healthy Chinese male volunteers (27-48 years) were randomized to two treatment groups (n = 18/group) for 18 weeks: (1) testosterone undecanoate (TU) 1000 mg i.m. injection followed by a 500 mg injection every 6 weeks and (2) TU + LNG 250 µg orally daily. Blood samples were obtained from all participants before and during treatment and at the end of the recovery phase. Open testicular biopsies for this study were obtained from four men before treatment and from four men in each of the TU and TU + LNG groups at 2 and 9 weeks of treatment. The presence of antisperm antibodies was checked in the archived serum samples of the subjects at baseline, during treatment and at the end of the recovery period. Stored testicular biopsy samples from cynomolgus monkeys treated with either sub-cutaneous testosterone or placebo for 12 weeks were used for additional protein expression studies. MAIN RESULTS AND ROLE OF THE CHANCE: Expression of blood-testis barrier associated proteins quantified by immunohistochemistry (claudin 3, claudin 11, junctional adhesion molecule-A, zonula occludens-1) remained unchanged despite a significant decrease in the numbers of pachytene spermatocytes and round spermatids in the seminiferous tubules at 9 weeks in the TU + LNG group. This was confirmed by immunoblots showing a lack of quantitative change in these tight junction proteins in monkeys after testosterone treatment. There were no increases in serum antisperm antibodies in the volunteers during the study. LIMITATIONS/REASONS FOR CAUTION: The duration of the study was short and the long-term effects of male hormonal contraceptive treatments on the integrity of the blood-testis barrier remain to be determined. WIDER IMPLICATIONS OF THE FINDINGS: This study supports the safety of male hormonal contraceptive treatment and does not corroborate the previous findings of disturbed immunological integrity of the blood-testis barrier from animal studies such as androgen receptor knockout mice and exogenous hormonal treatment in rats. STUDY FUNDING/COMPETING INTEREST: The study was supported by grants from the Contraceptive Research and Development Program and the Mellon Foundation (MFG-02-64, MFG-03-67), Endocrine, Metabolism and Nutrition Training Grant (T32 DK007571), the Clinical and Translational Science Institute at Los Angeles Biomedical and Harbor-UCLA Medical Center (UL1RR033176 and UL1TR000124) and the Los Angeles Biomedical Research Institute Summer High School Student Program.

Interaction of insulin-like growth factor-binding protein-3 and BAX in mitochondria promotes male germ cell apoptosis.

Germ cell apoptosis is crucial for spermatogenesis and can be triggered by various stimuli, including intratesticular hormone deprivation. This study proposes a role for insulin-like growth factor binding protein-3 (IGFBP-3) in male germ cell apoptosis. Groups of adult Sprague-Dawley male rats received one of the following treatments for 5 days: (i) daily intratesticular (IT) injections with saline (control); (ii) a single subcutaneous injection of the gonadotropin-releasing hormone antagonist (GnRH-A), acyline, on day 1 and a daily IT injection of saline; (iii) daily IT injection of IGFBP-3; and (iv) a GnRH-A injection on day 1 and a daily IT injection of IGFBP-3. Germ cell apoptosis increased significantly after IGFBP-3 or GnRH-A treatment which was further enhanced by the combined treatment. After co-immunoprecipitation with BAX antibody, IGFBP-3 association with BAX was demonstrated in total and mitochondrial fractions but not in the cytosol of testis extracts. BAX-associated IGFBP-3 expression was increased in mitochondria after treatment compared with control, which was confirmed by an IGFBP-3 enzyme-linked immunosorbent assay. Dot blot studies further validated the BAX-IGFBP-3 binding in vitro. IGFBP-3 as well as BAX induced release of cytochrome c and DIABLO from isolated testicular mitochondria in vitro. IGFBP-3, when combined with an ineffective dose of BAX, triggered release of these proteins from isolated mitochondria at a 4-fold lower dose than IGFBP-3 alone. Our data demonstrate that the IGFBP-3 and BAX interaction activates germ cell apoptosis via the mitochondria-dependent pathway. This represents a novel pathway regulating germ call homeostasis that may have significance for male fertility and testicular disease.

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.

Insights into the pathogenesis of XXY phenotype from comparison of the clinical syndrome with an experimental XXY mouse model.

Klinefelter syndrome (47, XXY male) is the most common sex chromosome disorder in men. To study the underlying mechanisms of XXY phenotypes and design specific and novel therapeutic regimens for KS men, an experimental mouse model (41, XXY) was established. This manuscript compares the phenotypes of XXY men and mice and discusses the possible contributions of low androgen levels and extra X chromosome genes to the XXY phenotypes. The phenotypic similarities between XXY mouse and men suggest that the common genes that escape the X inactivation between XXY mouse and men may be responsible for the clinical manifestations in men with Klinefelter syndrome.

transient scrotal hyperthermia and levonorgestrel enhance testosterone-induced spermatogenesis suppression in men through increased germ cell apoptosis.

CONTEXT: In rodents and monkeys, a combination of hormonal and physical agents accelerates germ cell death. OBJECTIVE: A "proof of concept" study was performed to investigate whether addition of heat exposure or a progestin to an androgen induces germ cell death and more complete and rapid spermatogenesis suppression. DESIGN AND SETTINGS: A randomized clinical trial was performed at academic medical centers. PARTICIPANTS: We treated four groups of healthy male volunteers (18 per group) for 18 wk: 1) testosterone undecanoate (TU) 1000 mg im (first dose), followed by 500 mg im every 6 wk; 2) submersion of scrota at 43 C in water for 30 min/d for 6 consecutive days; 3) TU plus heat; and 4) TU plus oral levonorgestrel (LNG) 250 microg/d. MAIN OUTCOME MEASURES: Semen parameters, testicular histology, and germ cell apoptosis were the main outcome measures. RESULTS: Heat alone and TU plus heat suppressed sperm counts more than TU alone by wk 6. By wk 9, recovery began in the heat only group, whereas spermatogenesis remained suppressed in the TU plus heat group. Oral LNG plus TU suppressed spermatogenesis earlier and more severely than TU alone. At wk 2, significantly greater germ cell apoptosis occurred in heat and heat plus TU subjects, but not in subjects without heat treatment, compared with pretreatment subjects. By 9 wk, markedly smaller seminiferous tubule diameters and fewer spermatocytes and spermatids were noted in all 12 biopsies from men receiving TU, TU plus LNG, with most dramatic differences for the TU plus heat group, whereas no differences from pretreatment biopsies were observed in men who received heat treatment only. CONCLUSIONS: Heat causes a rapid and transient suppression of spermatogenesis. TU plus heat resulted in low-sperm output that was maintained by continuous treatment with TU. Addition of an oral progestin accelerated spermatogenesis suppression by TU alone. Increased germ cell apoptosis contributed to suppression of spermatogenesis.

Dedifferentiation of adult monkey Sertoli cells through activation of extracellularly regulated kinase 1/2 induced by heat treatment.

Sertoli cells play a key role in triggering and regulating the process of spermatogenesis. Failure of a Sertoli cell to mature functionally will presumably render it incapable of supporting germ cell survival and development that appeared after puberty. Expression of cytokeratin 18 (ck-18) intermediate filaments indicates a state of undifferentiation usually observed in Sertoli cells of prepubertal testis. In this study we demonstrated that local testicular heat treatment of adult monkey with water at 43 C for 30 min once daily for 2 consecutive days was capable of activating reexpression of ck-18 in Sertoli cells, which was coincident with activation of ERK1/2 and Akt kinases. Using primary Sertoli cell culture isolated from adult monkey testis, we further confirmed that the heat treatment of the cells at 43 C could also induce ck-18 reexpression, which was similar to the in vivo treatment. ERK MAPK was also induced by the heat treatment in a time- and protein kinase A (PKA)-dependent manner. After blocking the ERK MAPK signaling pathway, an inhibition of ck-18 expression in the cultured Sertoli cells was observed, and this inhibitory effect was also detected by blocking the PKA activation. However, ck-18 activation in Sertoli cells remained unaltered when the phosphatidylinositol 3-kinase/Akt pathway was blocked. In conclusion, the heat treatment of adult monkey Sertoli cells are capable of inducing a reversible change in the Sertoli cells from an adult differentiated state to an immature-like dedifferentiated state through PKA-ERK MAPK-dependent pathways but not via the phosphatidylinositol 3-kinase/Akt pathway.

Expression of orphan receptors TR2, TR3, TR4, and p53 in heat-treated testis of cynomolgus monkeys (Macaca fascicularis).

To investigate the possible role of testicular orphan receptors (TR) TR2, TR3, and TR4 in the process of germ cell apoptosis in the heat-treated testis of monkey, we have examined the spatiotemporal expression of the 3 TR mRNAs in relation to p53 mRNA levels in the monkey testis by in situ hybridization and reverse transcription polymerase chain reaction techniques. The results showed that TR2 mRNA was confined to spermatocytes; TR4 and TR3 mRNAs were expressed in both spermatocytes and spermatids. The heat treatment did not change TR2 mRNA level but significantly reduced TR4 mRNA expression in spermatocytes on days 3 and 8 after the heat treatment. TR3 mRNA expression was affected by the heat treatment in a time-dependent manner, with the lowest level on day 30 after the heat shock. Low to moderate signal for p53 mRNA was detected in spermatocytes before treatment, which increased dramatically on days 3, 8, and 30 after the heat shock. The coincident expression of the testicular TR3 and p53 mRNA, spatially and time dependently, implied that the decrease in TR3 expression in the heat-treated testis might be closely related to the p53 signal pathway, whereas the temporal decrease in TR4 production in the testis at the early stage indicated that this orphan receptor might be also involved in germ cell apoptosis. The data suggest that TR3, TR4, and p53 could be important regulators of germ cell apoptosis induced by the heat treatment, whereas TR2 might not be a key regulator in this process.

Male reproductive ageing: using the brown Norway rat as a model for man.

The Brown Norway (BN) rat is an excellent model for male reproductive ageing. We and others have shown that with ageing, the BN rat exhibits low serum testosterone, low Leydig cell steroidogenic capacity, decreased Sertoli cell function and number, marked reduction in seminiferous tubule volume and sperm content, and accelerated germ cell apoptosis. These testicular changes are the result of a combination of a primary testicular defect and a secondary hypothalamic dysfunction. Leydig cell dysfunction results from decreased activities of the steroidogenic enzymes and Leydig cell secretory capacity and is not corrected by daily administration of replacement luteinizing hormone (LH), suggesting a primary testicular defect. However ageing in male BN rats is associated with decreased LH pulse amplitude, reduced gonadotropin releasing hormone (GnRH) and gonadotropin responsiveness to excitatory amino acids, and decreased GnRH mRNA and peptide in the hypothalamus. We have further shown in the hypothalamus of ageing BN rats that while the excitatory amino acid receptor content is reduced, nitric oxide synthase (NOS) activity is increased which is due to increased inducible (iNOS) but not neuronal NOS (nNOS). The increased iNOS protein in the hypothalamus is associated with increased peroxynitrite formation and neuronal cell apoptosis. We conclude that increased hypothalamic levels of iNOS may result in neurotoxicity in the hypothalamus leading to loss of hypothalamic GnRH secretory cells and impaired GnRH pulsatile secretion that contributes to the abnormal Leydig cell function characteristic of male reproductive ageing.

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

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

Pharmacokinetics and tissue distribution of humanin and its analogues in male rodents.

Humanin (HN) is a novel 24-amino acid mitochondrial-derived peptide that has demonstrated diverse cytoprotective effects, including an emerging role in diabetes. The purpose of this study was to examine the pharmacokinetics of humanin analogues, which show great potential as therapeutic agents (HNG and the non-IGFBP-3 binding, HNGF6A). 11-week-old male IGFBP-3(-/-) and wild type (WT) mice were divided into 3 groups: WT mice treated with HNG, WT mice treated with HNGF6A, and IGFBP-3(-/-) mice treated with HNG. Plasma was obtained from mice following ip injection with HN analogues, and HN levels were measured with ELISA. WT mice treated with HNGF6A and IGFBP-3(-/-) mice treated with HNG displayed a longer half-life of HN compared with WT mice treated with HNG. Following HNG injection, both IGF-1 and IGFBP-3 levels decreased over time. Adult male Sprague Dawley rats were also ip injected with HNG, and HN levels were measured in various tissues (plasma, liver, heart, and brain) by ELISA. The half-life of HN was found to be longer in rats compared with mice. In rats, HN levels were found to be highest in plasma, present in liver, and undetectable in brain or heart. The current study provides evidence of HN and IGFBP-3 association in the circulation and suggests that native HN may modulate the distribution of IGF-1 and IGFBP-3. The results also demonstrate varying kinetic profiles of HN analogues and interspecies variation in rodents. Sustainable levels of circulating HN measured in plasma underline the potential value of HN analogues as a new therapeutic intervention in the treatment of diabetes.

Comparison of tamsulosin with extracorporeal shock wave lithotripsy in treating distal ureteral stones.

BACKGROUND: Tamsulosin, an alpha-1 receptor antagonist, has been demonstrated effective in promoting distal ureteral stone passage and in reducing pain associated with stone expulsion. This study aimed to evaluate the effect of tamsulosin in comparison with nifedipine and extracorporeal shock wave lithotripsy (ESWL) on the expulsion rate of distal ureteral stones at different sizes. METHODS: We assigned 314 patients to three categories: I, the stone with maximal diameter of 4.0 - 5.9 mm; II, 6.0 - 7.9 mm, and III, 8.0 - 9.9 mm. Patients in each category were randomly subdivided into three treatment subgroups: group A (nifedipine group), group B (tamsulosin group), and group C (ESWL group). Stone-free rate and the dose of analgesics were recorded weekly during the 4-week follow-up period. RESULTS: Three hundred and three patients completed the study. The results showed that nifedipine and tamsulosin treatments promoted a small (4 - 8 mm, categories I and II) stone expulsive rate that was comparable with ESWL treatment. Nonetheless, when the stone diameter was 8.0 - 9.9 mm, ESWL showed a greater stone free rate than nifedipine and tamsulosin treatments; no significant difference existed between the latter two therapies. Although the ESWL treatment group required the least analgesics, tamsulosin treatments required less pain medication than nifedipine (P < 0.05). CONCLUSIONS: Tamsulosin treatment is recommended for patients with the stone diameter smaller than 8 mm because of its feasibility, effectiveness and safety. ESWL is more appropriate than tamsulosin therapy for the patients whose stones are larger than 8 mm.

Expression of nitric oxide synthase during germ cell apoptosis in testis of cynomolgus monkey after testosterone and heat treatment.

This study investigates the possible involvement of nitric oxide synthase (NOS) in activating germ cell death in monkeys after mild testicular hyperthermia and/or hormonal deprivation. Groups of 8 adult male monkeys received 1 of the following treatments for 12 weeks: 1) 2 empty Silastic implants, 2) 2 testosterone (T) implants, 3) daily exposure of testes to heat (43 degrees C for 30 minutes) for 2 consecutive days, or 4) 2 T implants plus testicular heat exposure. Testicular biopsies were performed before and on days 3, 8, 28, and 84 of the treatment. In control monkey testes, endothelial NOS (eNOS) was observed mainly in Sertoli cells and spermatogonia. No obvious alteration in eNOS levels was detected in any of the treatment group as assessed by Western blotting. Induction of inducible NOS (iNOS) in testes of the 3 treated groups was detected by immunoblotting as early as day 3 after treatment compared with that of controls. Immunocytochemistry further revealed a small increase in iNOS expression in both germ cells and Sertoli cells after T treatment. However, treatment of heat or heat in combination with T markedly induced iNOS expression in germ cells. These data suggest that iNOS, but not eNOS, may be involved in monkey testicular germ cell death after heat and/or T treatment.

Role of caspase 2 in apoptotic signaling in primate and murine germ cells.

This study investigates the role of caspase 2 in apoptotic signaling of nonhuman primate male germ cells triggered by mild testicular hyperthermia, testosterone (T(e)) implants, or by combined interventions. Mean incidence of germ cell apoptosis increased significantly by Day 3 in the heat (H(e)) alone group and by Day 8 in the Te alone group but peaked at Day 3 in H(e) + T(e) group. We found activation of caspase 2 in both germ cells and Sertoli cells after induction of apoptosis. Most notably, active caspase 2 immunoreactivity was detected only in those germ cells susceptible to apoptosis compared with controls, where little or no such staining is detected. To further explore the role of caspase 2 in regulating male germ cell death, we next evaluated the efficacy of caspase 2 inhibition in preventing or attenuating heat-induced germ cell apoptosis in rats. Caspase 2 inhibition significantly (P < 0.05) prevented such heat-induced germ cell apoptosis. The protection offered by the caspase 2 inhibitor occurred upstream of mitochondria, involving suppression of mitogen-activated protein kinase (MAPK) 14 activation and inducible nitric oxide synthase (NOS2) induction and, in turn, suppression of cytochrome c-mediated death pathway. Together, our results show that caspase 2 is activated in male germ cells undergoing apoptosis in nonhuman primates after heat stress, hormonal deprivation, or after combined interventions. Blockade of caspase 2 activation prevents heat-induced germ cell apoptosis in rats by suppressing the MAPK14- and NO-mediated intrinsic pathway signaling.

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.

Signaling pathways for germ cell death in adult cynomolgus monkeys (Macaca fascicularis) induced by mild testicular hyperthermia and exogenous testosterone treatment.

Male contraception has focused, to a great extent, on approaches that induce azoospermia or severe oligospermia through accelerated germ cell apoptosis. Understanding the specific steps in the germ cell apoptotic pathways that are affected by male contraceptives will allow more specific targeting in future contraceptive development. In this study, we have used a nonhuman primate model to characterize the key apoptotic pathway(s) in germ cell death after mild testicular hyperthermia, hormonal deprivation, or combined interventions. Groups of 8 adult (7- to 10-year-old) cynomolgus monkeys (Macaca fascicularis) received one of the following treatments: 1) two empty silastic implants; 2) two 5.5-cm testosterone (T) implants; 3) daily exposure of testes to heat (43 degrees C for 30 min) for 2 consecutive days; and 4) two T implants plus testicular heat exposure for two consecutive days. Testicular biopsies were performed before and at Days 3, 8, and 28 of treatment. Treatment with T, heat, or both led to sustained activation of both mitogen-activated protein kinase (MAPK) 1/3 and MAPK14. Activation of MAPK1/3 and MAPK14 were accompanied by an increase in B-cell leukemia/lymphoma (BCL) 2 levels in both cytosolic and mitochondrial fractions of testicular lysates (BAX levels remained unaffected) and cytochrome c and DIABLO release from mitochondria. These treatments also resulted in inactivation of BCL2 through phosphorylation at serine 70, thereby favoring the death pathway. We conclude that the serine phosphorylation of BCL2 and activation of the MAPK14-mediated mitochondria-dependent pathway are critical for male germ cell death in monkeys.

Delayed testicular aging in pituitary adenylate cyclase-activating peptide (PACAP) null mice.

Age-related decline in male sex hormones is a direct consequence of testicular aging. These changes in the hormonal complement cause physiological disturbances affecting the quality of life for millions of aging men. To assess the influence on testicular aging of pituitary adenylate cyclase-activating peptide (PACAP), a polypeptide that regulates testicular steroidogenesis in vitro, we compared the testicular structure and function between C57BL/6 wild-type and PACAP-/- male mice, at 4 and 15 months of age. We show that, in 4-month-old PACAP-/- mice, steroidogenesis (evaluated by levels of testosterone, steroidogenic acute regulatory protein, 3beta-hydroxysteroid dehydrogenase, and P450c17) was impaired. However, the testicular structure of these animals was not affected. At 15 months of age, wild-type testis displayed typical signs of aging (patchy seminiferous tubules, germ cell depletion, and vacuolization), whereas testicular structure was remarkably well conserved in PACAP-/- animals. The depletion of germ cells found in wild-type animals was associated with a higher content of peroxynitrites, a marker of reactive oxygen species, and a higher number of apoptotic cells compared with PACAP-/- mice. Our results show that testicular aging is delayed in PACAP-/- animals. Because the expression levels of steroidogenic factors are low and constant over time in knockout animals, a proposed mechanism for the protection against testicular degeneration is that production of reactive oxygen species, a byproduct of steroidogenesis that induces apoptosis, is down-regulated in PACAP-/- animals.

The role of the ubiquitin-proteasome pathway in the formation of mallory bodies.

The dynamics of Mallory body (MB) formation are difficult to follow in vivo. Because of the lack of an in vitro mouse hepatocyte culture model, a cellular extract approach was developed. In this model an immunoprecipitate was obtained using an antibody to cytokeratin-8 (CK-8). The isolate contained a large number of compounds: CK-8, ubiquitin, a frameshift mutation of ubiquitin (UBB(+1)), proteasomal subunits beta5 (a catalytic subunit of the 20S proteasome) and Tbp7 (an ATPase subunit of the 26S proteasome), transglutaminase, tubulin, heat shock proteins 90 and 70, and MBs. In Western blots, CK-8 immunoprecipitates showed colocalization of these components in a complex of proteins colocalized in a high-molecular-weight smear. When the CK-8 immunoprecipitate was incubated with the isolate of proteasomes and an energy generating source (ATP), the components of the ubiquitinated protein smear increased. These observations taken together with the in vivo observation that these proteins colocalized at the edge of the MB shown in the present study suggest that these proteins form aggregates through covalent binding of CK-8, ubiquitin, and the proteasomes. Covalent aggregation is suggested by the fact that the protein complex found in the high-molecular-weight smear that forms in vitro fails to dissociate in SDS. This protein complex is present in the CK-8 immunoprecipitates of livers forming MBs but not in control livers. In conclusion, the results support the concept that Mallory bodies are aggresomes which form as the result of the failure of the ubiquitin-proteasome complex to adequately eliminate cytokeratins destined for proteolysis.

Molecular misreading of the ubiquitin B gene and hepatic mallory body formation.

BACKGROUND & AIMS: Molecular misreading of the ubiquitin B gene has been documented in the cerebral cortex of patients with Alzheimer's disease and Down syndrome. This novel process consists of the unfaithful conversion of genomic information into aberrant transcripts and its subsequent translation into +1 proteins. METHODS: Because Mallory bodies (MBs) also contain ubiquitinated proteins, we stained 11 autopsied and 6 biopsied MB-containing livers from patients with steatohepatitis with an antibody to ubiquitin(+1) to look for the presence of mutant (ubiquitin(+1)) protein. Antibodies to wild-type ubiquitin were used to document the presence of MBs in all cases. RESULTS: Ubiquitin(+1) immunoreactivity was detected in all MB-containing livers with steatohepatitis; no ubiquitin(+1) immunoreactivity was found in 13 MB-free liver controls. A subpopulation (about one third of the MBs) of the MB-containing hepatocytes in autopsied livers showed ubiquitin(+1) immunoreactivity (i.e., ubiquitin and ubiquitin(+1) colocalized in MBs). MB-containing liver biopsy specimens showed colocalization of ubiquitin and ubiquitin(+1) in every MB. Western blot analysis showed an ubiquitin(+1) band of 11 kilodaltons. Molecular misreading of the ubiquitin B gene (DeltaGU) was shown in one of the livers, which contained numerous MBs using an expression cloning strategy. CONCLUSIONS: The results showed that molecular misreading of the ubiquitin B gene occurred in hepatocytes in virtually all of the MB-containing livers tested. Ubiquitin(+1) protein was only found within the MBs and therefore may act by interfering with the degradation of the MBs because ubiquitin(+1) may inhibit proteolytic function of the proteasome.