Androgen-dependent Organ Weights Research Articles

Role of gonadotropin-releasing hormone-II and its receptor in swine reproduction.

The pig represents the only livestock mammal capable of producing a functional protein for the second mammalian form of gonadotropin-releasing hormone (GnRH-II) and its receptor (GnRHR-II). To examine the role of GnRH-II and its receptor in pig reproduction, we produced a unique swine line with ubiquitous knockdown of endogenous GnRHR-II levels (GnRHR-II knockdown[KD]), which is largely the focus of this review. In mature GnRHR-II KD males, circulating testosterone concentrations were 82% lower than littermate control boars, despite similar luteinizing hormone (LH) levels. In addition, nine other gonadal steroids were reduced in the serum of GnRHR-II KD boars, whereas adrenal steroids (except 11-deoxycortisol) did not differ between lines. Interestingly, testes from GnRHR-II KD males had fewer, hypertrophic Leydig cells and fewer, enlarged seminiferous tubules than control testes. As expected, downstream reproductive traits such as androgen-dependent organ weights and semen characteristics were also significantly reduced in GnRHR-II KD versus control boars. Next, we explored the importance of this novel ligand/receptor complex in female reproduction. Transgenic gilts had fewer, but heavier, corpora lutea with smaller luteal cells than littermate control females. Although the number of antral follicles were similar between lines, the diameter of antral follicles tended to be larger in GnRHR-II KD females. In regard to steroidogenesis, circulating concentrations of progesterone and 17β-estradiol were lower in transgenic compared to control gilts, even though serum levels of follicle-stimulating hormone and LH were similar. Thus, GnRH-II and GnRHR-II represent a potential avenue to enhance fertility and promote the profitability of pork producers.

The endocrine disrupting effects of sodium arsenite in the rat testis is not mediated through macrophage activation

Arsenic (As) is an endocrine disrupting chemical that can disturb the male reproductive system. In a previous study, it was suggested that testicular macrophages could display a role in endocrine disruption induced by As exposure. This work aimed to evaluate the effects of chronic As exposure in the testis function of Wistar rats and examine the participation of macrophage activation and inflammatory response in these processes. We examined gene expression of steroidogenic machinery and immunological markers by RT-QPCR, plasma testosterone concentrations, sperm count and morphology, and histomorphometrical parameters after 60-days exposure to 1 or 5 mg.kg−1.day−1 of sodium arsenite, combined or not with 50 μg.kg−1 of LPS administered one day before euthanasia. We have demonstrated that As exposure reduced the weight of androgen-dependent organs and induced changes in spermatogenesis, in particular at the highest dose. LPS and As co-exposure promoted a decrease in testosterone synthesis, but did not increase the overexpression of markers of macrophage activation seen in LPS-only rats. Our results suggest that As does not alter the testicular macrophage function, but under immunological challenges LPS and As can display a complex interaction, which could lead to endocrine disruption.

Baillonella toxisperma improves sexual performance and protects against stress-induced reproductive dysfunction in male Wistar rats

Baillonella toxisperma Pierre (Sapotaceae) is a medicinal plant widely used in Central Africa against several diseases including erectile dysfunction and male infertility. However, no study dealing with these male reproductive dysfunctions has been published until now. Accordingly, the present study was undertaken. It evaluated the capacity of an aqueous extract of stem bark of B. toxisperma (BT) at 75, 150 and 300 mg/kg/d to induce aphrodisiac effects and prevent the stress-induced reproductive dysfunction in males using normal non-experienced (an 8-day oral treatment) and stressed (a 6-h/day immobilization stress for 35 consecutive days)male Wistar rats. Both in normal and stressed animals, BT at the doses of 150 and 300 mg/kg decreased (p < 0.05) the mount and intromission latencies, and increased (p < 0.05) the number of total penile licking, mount and intromission as well as ejaculation frequencies. In stressed animals, a significant (p < 0.05) increase in sperm levels, sperm mobility as well as in the relative weight of androgen-dependent organs (testis, epididymis and seminal vesicles) was observed at the same doses compared with the stressed control. These results suggest that this aqueous extract of B. toxisperma might endow with aphrodisiac and androgenic properties in normal and stressed male Wistar rats.

Phosphorylation of GATA4 serine 105 but not serine 261 is required for testosterone production in the male mouse.

GATA4 is a transcription factor essential for male sex determination, testicular differentiation during fetal development, and male fertility in the adult. GATA4 exerts part of its function by regulating multiple genes in the steroidogenic enzyme pathway. In spite of these crucial roles, how the activity of this factor is regulated remains unclear. Studies in gonadal cell lines have shown that GATA4 is phosphorylated on at least two serine residues-serine 105 (S105) and serine 261 (S261)-and that this phosphorylation is important for GATA4 activity. The objective of the present study is to characterize the endogenous role of GATA4 S105 and S261 phosphorylation in the mouse testis. We examined both previously described GATA4 S105A mice and a novel GATA4 S261A knock-in mouse that we generated by CRISPR/Cas9 gene editing. The male phenotype of the mutants was characterized by assessing androgen-dependent organ weights, hormonal profiles, and expression of multiple testicular target genes using standard biochemical and molecular biology techniques. The fecundity of crosses between GATA4 S105A mice was reduced but without a change in sex ratio. The weight of androgen-dependent organs was smaller when compared to wild-type controls. Plasma testosterone levels showed a 70% decrease in adult GATA4 S105A males. This decrease was associated with a reduction in Cyp11a1, Cyp17a1, and Hsd17b3 expression. GATA4 S261A mice were viable and testis morphology appeared normal. Testosterone production and steroidogenic enzyme expression were not altered in GATA4 S261A males. Our analysis showed that blocking GATA4 S105 phosphorylation is associated with decreased androgen production in males. In contrast, S261 phosphorylation by itself is dispensable for GATA4 function. These results confirm that endogenous GATA4 action is essential for normal steroid production in males and that this activity requires phosphorylation on at least one serine residue.

Effects of clozapine and haloperidol treatment on plasma concentrations of androgen hormones and androgendependent organ changes in rats.

OBJECTIVES:Metabolic and endocrine adverse effects are among the most concerning unfavorable consequences of commonly used psychotropic drugs. The present research was planned to assess and determine the effects of haloperidol and clozapine on testosterone, cortisol, and corticosterone levels and also their influence on androgen-dependent organs in adult male Wistar rats.MATERIALS AND METHODS:Animals were casually distributed into three groups (n = 10 in each group). Drugs were administered intraperitoneally for 28 days. The control group received 2 mL of physiological saline, the second group received haloperidol (0.5 mg/kg), and the third group received clozapine (0.5 mg/kg). The subsequent testosterone, cortisol, and corticosterone plasma concentration levels were analyzed with chemiluminescent immunoassay.RESULTS:Clozapine and haloperidol treatments altered testosterone hormone levels. Testosterone mean values in both the clozapine (1.00–0.58) and haloperidol (0.65–0.62) groups were found to be lower than compared to controls (P = 0.003, P < 0.001). Histomorphometric analysis results also showed reduced testes size and reduced weight of androgen-dependent organs in drug-treated rats.CONCLUSION:It can be suggested that clozapine and haloperidol are effective in reducing the testosterone plasma concentration level and androgen-dependent organ sizes; therefore, clinicians should be aware of these effects when considering the use of antipsychotic drugs.

Abstract 1162: Castration-resistant prostate cancer bone metastasis model to assess new therapeutics

Abstract The role of androgens in the regulation of growth of primary prostate cancer is well established but less recognized in the context of advanced bone metastatic disease, which is the cause of high morbidity in patients. At advanced stage, prostate cancer cells typically lose their dependence on endogenous androgens leading to metastatic castration resistant prostate cancer (mCRPC). The aim of the study was to establish a novel mCRPC model resembling clinical aspects of the bone metastatic disease, which could be used for the evaluation of efficacy of new therapies. To obtain a model for castration resistant growth, 5-6 weeks old NOD.Scid male mice were divided into three study groups. Mice in two study groups were castrated either before inoculation of the cancer cells or at four weeks after inoculation when the tumors were already growing. The mice in one study group were left intact. All mice received an intratibial injection of 2x106 VCaP human prostate cancer cells originally derived from vertebral metastatic site. Tumor growth was followed biweekly for 16 weeks by PSA measurements and X-ray imaging of tumor induced bone changes (lesions). At endpoint, androgen dependent organs were weighed and tumor-bearing tibias were subjected to histological evaluation. At endpoint, a tumor take of 60%, 40% and 50% was observed in the intact mice and in the mice castrated one week before and four weeks after VCaP cell inoculation, respectively. Tumor take was assessed by PSA, X-ray and histology at endpoint. The PSA levels were higher in the intact mice and in the mice castrated at four weeks compared to the mice castrated before cancer cell inoculation. The PSA levels became detectable between 6-8 weeks after cancer cell inoculation in the intact mice and in the mice castrated at four weeks. In the mice castrated before cancer cell inoculation the PSA levels started to increase very late at 14-16 weeks. VCaP cells induced mainly new bone formation, an osteoblastic bone reaction typical for prostate cancer patients. In castrated mice, fewer bone lesions were observed compared to intact mice. The bone lesion areas quantified from X-ray images were larger in the intact mice compared to the mice castrated before cancer cell inoculation. As expected, the weight of androgen dependent organs was lower in the castrated mice compared to the intact mice. In conclusion, a model mimicking important clinical aspects of castration resistant prostate cancer bone metastases was established. Tumor take and growth rates indicated that early phases of tumor development into the bone are androgen dependent whereas tumor growth at later stage relies on the bone microenvironment. The results highlight the significance of the tumor microenvironment in establishing clinically relevant preclinical models for drug development. Citation Format: Tiina E. Kähkönen, Mari I. Suominen, Jussi M. Halleen, Jenni Bernoulli, Pascale Lejeune. Castration-resistant prostate cancer bone metastasis model to assess new therapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1162.

Abstract 5560: Characterization of a new non-steroidal small molecule inhibitor of CYP17 for castration-resistant prostate cancer.

Abstract Prostate cancer affects 1 in 6 men and is the second most common cause of cancer-related death. Since the majority of prostate cancers critically depend on androgens for growth, androgen deprivation therapy is a frontline treatment for advanced prostate cancer. The majority of prostate tumors initially respond to androgen deprivation therapy, but eventually the disease often progresses to castration-resistant prostate cancer. However, the current therapies that only reduce androgen produced in the testis do not affect androgen produced by adrenal glands can also produce androgens. This extra-testicular androgen production is thought to be a key factor in the development of castration resistant prostate cancer. CYP17 is a P450 enzyme that catalyzes the last step of androgen biosynthesis in both the testis and the adrenals. Inhibition of CYP17 therefore completely blocks androgen production and it is thus thought to be more effective to prevent prostate cancer progression than current therapies. Angion Biomedica has identified a promising series of proprietary non-steroidal CYP17 inhibitors. Our lead compound is potent, orally bioavailable, and significantly reduces serum testosterone levels in mice. Treatment of rats and mice with our lead CYP17 inhibitor significantly reduces the weights of androgen dependent organs. In two mouse xenograft models of androgen-dependent tumor growth our lead CYP17 inhibitor markedly reduces primary tumor growth, serum testosterone and PSA levels. Angion is currently further evaluating the therapeutic potential of this series of compounds as a possible therapy for castration resistant prostate cancer. Citation Format: Bert Oehlen, Siobhan McCormack, Gaifeng Ma, Dong Sung Lim, Bijoy Panicker, Itzhak D. Goldberg. Characterization of a new non-steroidal small molecule inhibitor of CYP17 for castration-resistant prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5560. doi:10.1158/1538-7445.AM2013-5560

Effect of a novel 17,20-lyase inhibitor, orteronel (TAK-700), on androgen synthesis in male rats

Endogenous androgens play a role in the development and progression of prostate cancer (PC), thus androgen suppression may offer an effective therapeutic strategy for this disease. Orteronel (TAK-700), 6-[(7S)-7-hydroxy-6,7-dihydro-5H-pyrrolo[1,2-c]imidazol-7-yl]-N-methyl-2-naphthamide, is a novel, non-steroidal, selective inhibitor of the 17,20-lyase activity of CYP17A – a key enzyme in the production of steroidal hormones – and is being developed as a therapy for PC. The purpose of this study was to elucidate the inhibitory activity of orteronel, in particular its specificity for androgen synthesis enzymes, in male rats – an androgen-synthesis model that largely reflects this pathway in humans.Orteronel inhibited 17,20-lyase activity in rats with an IC50 of 1200nM but did not inhibit 17α-hydroxylase or 11β-hydroxylase (CYP11B1) activity in rats at concentrations up to 10μM. In cellular steroidogenesis assays using rat testicular cells, orteronel suppressed testosterone and androstenedione production with an IC50 of 640nM and 210nM, respectively, but did not suppress either corticosterone or aldosterone production in rat adrenal cells at concentrations up to 30μM. In addition, serum testosterone and androstenedione levels in human chorionic gonadotropin-injected hypophysectomized rats were significantly reduced by single oral administration of orteronel at a dose of 30mg/kg (both p≤0.01); serum corticosterone and aldosterone levels in ACTH-injected hypophysectomized rats did not result in significant differences compared with controls, following orteronel administration at doses up to 300mg/kg. Serum testosterone levels in intact male rats were significantly reduced by orteronel 4h after dosing at 100mg/kg (p≤0.01); testosterone levels showed a tendency to recover afterward. In intact male rats, the weight of the prostate glands and seminal vesicles was decreased in a dose-dependent manner following multiple doses of orteronel at 37.5, 150, and 600mg/kg, TID for 4 days. The reversibility of orteronel was further confirmed using a human adrenocortical tumor cell line.In summary, orteronel is a selective and reversible 17,20-lyase inhibitor, and decreases the weight of androgen-dependent organs in male rats. Our data suggests that orteronel would therefore be effective for androgen-dependent disorders such as PC.

In vivo and in vitro estrogenic activity of the antidepressant fluoxetine

Recent years have seen an increase in the use of antidepressant drugs, especially fluoxetine (FLX), in sensitive populations, such as pregnant and lactating women. Although some evidence suggests a possible endocrine action of FLX, no specific studies have been performed to investigate this hypothesis. In the present study, we investigated the possible (anti)androgenic and (anti)estrogenic actions of FLX using Hershberger, uterotrophic (0.4, 1.7, and 17mg/kg), and reporter gene (7.6–129μM) assays. In the Hershberger assay, no differences were observed in androgen-dependent organ weights. However, the uterotrophic and gene reporter assays indicated a possible estrogenic action of FLX. Uterine weight increased in the 1.7 and 17mg/kg/day groups in the 3-day uterotrophic assay in immature rats. Additionally, noncytotoxic concentrations of FLX induced estrogenic responses and increased the estrogenic response of estradiol in MCF-7 breast cancer cells transfected with luciferase.

Relative sensitivity of developmental and immune parameters in juvenile versus adult male rats after exposure to di(2-ethylhexyl) phthalate

The developing immune system displays a relatively high sensitivity as compared to both general toxicity parameters and to the adult immune system. In this study we have performed such comparisons using di(2-ethylhexyl) phthalate (DEHP) as a model compound. DEHP is the most abundant phthalate in the environment and perinatal exposure to DEHP has been shown to disrupt male sexual differentiation. In addition, phthalate exposure has been associated with immune dysfunction as evidenced by effects on the expression of allergy. Male wistar rats were dosed with corn oil or DEHP by gavage from postnatal day (PND) 10–50 or PND 50–90 at doses between 1 and 1000mg/kg/day. Androgen-dependent organ weights showed effects at lower dose levels in juvenile versus adult animals. Immune parameters affected included TDAR parameters in both age groups, NK activity in juvenile animals and TNF-α production by adherent splenocytes in adult animals. Immune parameters were affected at lower dose levels compared to developmental parameters. Overall, more immune parameters were affected in juvenile animals compared to adult animals and effects were observed at lower dose levels.The results of this study show a relatively higher sensitivity of juvenile versus adult rats. Furthermore, they illustrate the relative sensitivity of the developing immune system in juvenile animals as compared to general toxicity and developmental parameters. This study therefore provides further argumentation for performing dedicated developmental immune toxicity testing as a default in regulatory toxicology.

Endocrine and testicular changes induced by olanzapine in adult Wistar rats

Olanzapine is an atypical antipsychotic drug that has been increasingly used in acute treatment of, and therapeutic support for, schizophrenia, bipolar disorder and other psychoses. Considering that olanzapine acts on the dopaminergic receptor and this receptor is detected in germ cells, the present study aims to investigate the effects of treatment with different doses of olanzapine on spermatogenesis, plasma testosterone and weight of androgen-dependent organs in rats. Results showed reduced plasma testosterone levels, and reduced testis, epididymis and prostate weights. Histopathologic and histomorphometric analysis of spermatogenesis indicated testicular degeneration. Furthermore, germ cell desquamation, syncytial multinucleated cells, Sertoli cell vacuolization and presence of necrotic and apoptotic germ cells wwew observed. Olanzapine treatment in rats promoted endocrinological changes and lesions in the testis, leading to a disturbance in spermatogenesis.

Cumulative Effects of Dibutyl Phthalate and Diethylhexyl Phthalate on Male Rat Reproductive Tract Development: Altered Fetal Steroid Hormones and Genes

Exposure to plasticizers di(n-butyl) phthalate (DBP) and diethylhexyl phthalate (DEHP) during sexual differentiation causes male reproductive tract malformations in rats and rabbits. In the fetal male rat, these two phthalate esters decrease testosterone (T) production and insulin-like peptide 3 (insl3) gene expression, a hormone critical for gubernacular ligament development. We hypothesized that coadministered DBP and DEHP would act in a cumulative dose-additive fashion to induce reproductive malformations, inhibit fetal steroid hormone production, and suppress the expression of insl3 and genes responsible for steroid production. Pregnant Sprague Dawley rats were gavaged on gestation days (GD) 14-18 with vehicle control, 500 mg/kg DBP, 500 mg/kg DEHP, or a combination of DBP and DEHP (500 mg/kg each chemical; DBP+DEHP); the dose of each individual phthalate was one-half of the effective dose predicted to cause a 50% incidence of epididymal agenesis. In experiment one, adult male offspring were necropsied, and reproductive malformations and androgen-dependent organ weights were recorded. In experiment two, GD18 testes were incubated for T production and processed for gene expression by quantitative real-time PCR. The DBP+DEHP dose increased the incidence of many reproductive malformations by >or=50%, including epididymal agenesis, and reduced androgen-dependent organ weights in cumulative, dose-additive manner. Fetal T and expression of insl3 and cyp11a were cumulatively decreased by the DBP+DEHP dose. These data indicate that individual phthalates with a similar mechanism of action, but with different active metabolites (monobutyl phthalate versus monoethylhexyl phthalate), can elicit dose-additive effects when administered as a mixture.

Prochloraz Inhibits Testosterone Production at Dosages below Those that Affect Androgen-Dependent Organ Weights or the Onset of Puberty in the Male Sprague Dawley Rat

Prochloraz (PCZ) is an imidazole fungicide that inhibits gonadal steroidogenesis and antagonizes the androgen receptor (AR). We hypothesized that pubertal exposure to PCZ would reduce testosterone production and delay male rat reproductive development. Sprague Dawley rats were dosed by gavage with 0, 31.3, 62.5, or 125 mg/kg/day of PCZ from postnatal day (PND) 23 to 42 or 51. There was a significant delay in preputial separation (PPS) at 125 mg/kg/day PCZ and several of the androgen-dependent organ weights were decreased significantly, but the significant organ weight effects were not consistent between the 2 necropsies (PND 42 vs. 51). At both ages, serum testosterone levels and ex vivo testosterone release from the testis were significantly decreased whereas serum progesterone and 17alpha-hydroxyprogesterone levels were significantly increased at dose levels below those that affected PPS or reproductive organ weights. The hormone results suggested that PCZ was inhibiting CYP17 activity. In a second pubertal study (0, 3.9, 7.8, 15.6, 31.3, or 62.5 mg/kg/day PCZ), serum testosterone levels and ex vivo testosterone production were significantly reduced at 15.6 mg/kg/day PCZ. In order to examine the AR antagonism effects of PCZ, independent of its effects on testosterone synthesis, castrated immature male rats were dosed with androgen and 0, 15.6, 31.3, 62.5, or 125 mg/kg/day PCZ for 10-11 days (Hershberger assay). In this assay, androgen-sensitive organ weights were only significantly decreased at 125 mg/kg/day PCZ. These data from the pubertal assays demonstrate that PCZ decreases testosterone levels and delays rat pubertal development, as hypothesized. However, the fact that hormone levels were affected at dosage eightfold below that which delayed the onset of puberty suggests that rather large reductions in serum testosterone may be required to delay puberty and consistently reduce androgen-dependent tissue weights.

Hershberger Assay to Investigate the Effects of Endocrine‐Disrupting Compounds with Androgenic or Antiandrogenic Activity in Castrate‐Immature Male Rats

The protocol described in this unit is designed to evaluate the effects of androgenic and antiandrogenic endocrine-disrupting compounds (EDCs) in the castrate-immature male rat. Continuous 10-day exposure of the prepubertal male to chemicals is used to identify androgenic or antiandrogenic activities based on the weights of several androgen-dependent tissues on the day after treatment is ended. Androgen-dependent organ weights and growth, along with kidney, liver, and adrenal weights are measured at necropsy. The androgen-dependent tissues include the ventral prostate, seminal vesicles (with fluid plus coagulating glands), levator ani plus bulbocavernosus muscles, Cowper's glands, and glans penis. Optional measures, which facilitate interpretation of the data when positive effects are detected, include serum testosterone and luteinizing hormone levels. Androgenic chemicals cause increases in one or all of the androgen-dependent tissue weights after being administered by oral gavage or subcutaneously (s.c.) to castrate-immature male rats. Antiandrogenic chemicals cause reductions in androgen-dependent tissue weights after being coadministered with testosterone propionate (s.c.).

Evaluation for reliability and feasibility of the draft protocol for the enhanced rat 28-day subacute study (OECD Guideline 407) using androgen antagonist flutamide

As part of an international validation project to establish a test protocol for the ‘Enhanced OECD Test Guideline no. 407’, a 28-day repeated dose study of flutamide was performed (1) to examine which of the current and/or additional parameters can detect endocrine effects of test chemicals most reliably and sensitively, (2) to investigate whether it is actually feasible to routinely include all additional parameters into the testing routine, and (3) to assess intra-laboratory variability by performing two identical studies (experiments A and B) in parallel using groups of five animals each per dose and sex. Groups of five male and five female CD(SD)IGS rats were treated by oral gavage with 0, 1, 10 and 100 mg flutamide/kg body weight for at least 28 days. The dose level considered to be around the MTD (100 mg/kg) exerted the expected antiandrogenic effects on androgen related tissues: significant decrease of the weights of androgen dependent organs and the sperm count and increase in histopathological lesions. At the middle dose (10 mg/kg), significant decrease of prostate weight (ventral and dorso-lateral parts combined) was observed and it was suggested that weight measurement of androgen dependent organs provides the most reliable and sensitive endpoint with this protocol. As for the feasibility, because of many items in this protocol, selection should be based on the sensitivity. From our data, addition of weight measurement of androgen dependent organs to the items of the existing OECD 407 guideline might allow accurate screening for endocrine disruptors. At the dose level considered to be around the MTD, the findings achieving statistical significance in one experiment with five animals/dose/sex could be reproduced in the second experiment, and evaluation with the small groups was consistent with findings using the combined groups of 10 animals/dose/sex. The results demonstrate that the protocol can reliably detect antiandrogenic effects of flutamide.

Evaluation for reliability and feasibility of the draft protocol for the enhanced rat 28-day subacute study (OECD Guideline 407) using androgen antagonist flutamide.

As part of an international validation project to establish a test protocol for the 'Enhanced OECD Test Guideline no. 407', a 28-day repeated dose study of flutamide was performed (1) to examine which of the current and/or additional parameters can detect endocrine effects of test chemicals most reliably and sensitively, (2) to investigate whether it is actually feasible to routinely include all additional parameters into the testing routine, and (3) to assess intra-laboratory variability by performing two identical studies (experiments A and B) in parallel using groups of five animals each per dose and sex. Groups of five male and five female CD(SD)IGS rats were treated by oral gavage with 0, 1, 10 and 100 mg flutamide/kg body weight for at least 28 days. The dose level considered to be around the MTD (100 mg/kg) exerted the expected antiandrogenic effects on androgen related tissues: significant decrease of the weights of androgen dependent organs and the sperm count and increase in histopathological lesions. At the middle dose (10 mg/kg), significant decrease of prostate weight (ventral and dorso-lateral parts combined) was observed and it was suggested that weight measurement of androgen dependent organs provides the most reliable and sensitive endpoint with this protocol. As for the feasibility, because of many items in this protocol, selection should be based on the sensitivity. From our data, addition of weight measurement of androgen dependent organs to the items of the existing OECD 407 guideline might allow accurate screening for endocrine disruptors. At the dose level considered to be around the MTD, the findings achieving statistical significance in one experiment with five animals/dose/sex could be reproduced in the second experiment, and evaluation with the small groups was consistent with findings using the combined groups of 10 animals/dose/sex. The results demonstrate that the protocol can reliably detect antiandrogenic effects of flutamide.

Effects of perinatal exposure to flutamide on sex hormone responsiveness in F1 male rats.

Pregnant rats were administered flutamide (0 and 10 mg/kg, p.o.) from gestation Day 14 to post-parturition Day 3 and effects on responsiveness to androgens (testosterone propionate, TP; dihydrotestosterone, DHT) in male offspring were examined with a Hershberger assay. Male pups of each group were assigned to 6 subgroups as follows: Group 1, castration and euthanized at postnatal Day 46 (PND 46); Group 2, castration + vehicle; Group 3, castration + TP; Group 4, castration + DHT; Group 5, vehicle; Group 6, DHT. After castrations were conducted at PND 36, animals were treated with TP (2 mg/kg in corn oil, s.c.) or DHT (1.25 mg/kg in corn oil, s.c.) once a day for 10 days, beginning at PND 46. At PND 56, the following organs/tissues were removed and weighed: ventral prostate, dorso-lateral prostate, seminal vesicles with coagulating glands, levator ani muscle plus bulbocavernosus muscle, Cowper's gland, and glands penis. Analysis of serum testosterone, LH and FSH in Groups 2, 3, 4, 5 and 6, and RT-PCR using prostate tissue from Groups 2, 3 and 4 were carried out. Perinatal exposure to flutamide caused decreased weights of androgen-dependent organs. Responses to androgens were recognized in organs of all castrated groups, with increased organ weights, especially in animals administered TP where values were essentially equal to or greater than those of intact animals in both the control and the 10 mg/kg group. On the other hand, the degree of weight increase of the ventral prostate and seminal vesicles with TP or DHT treatment in castrated animals was smaller in the flutamide administration group than in the controls. In hormone assays, castrated + vehicle animals showed higher serum LH than the other groups. Serum FSH was high in the castrated groups (Group 2>Group 4>Group 3), while in the noncastrated group a constant level was noted, with or without flutamide. No effect of flutamide administration was observed regarding sex hormone. RT-PCR using ventral prostate tissue revealed no significant differences in expression of AR, C3, VEGF, TGF-beta1, beta2, KGF and CK8 mRNA after androgen treatment between the control and flutamide treatment groups. C3 mRNA was increased in androgen-treated animals, whereas AR, TGF-beta and KGF mRNAs were decreased. Perinatal exposure to anti-androgen causes irreversible abnormalities in male pups. Concerning the responsiveness to TP and DHT, the degrees of weight changes in ventral prostate and seminal vesicles in castrated animals were decreased. However, the other organ weights, the sex hormone levels and androgen-reactive gene expression in the ventral prostate were not influenced by perinatal flutamide treatment in the present study.

Persistence of infertility in GnRH immunized male rats treated with subdermal implants of dihydrotestosterone (DHT)

Male hormonal contraception has been limited to date because two fundamental requirements have not been concurrently satisfied, these are, consistent and dependable azoospermia and infertility coupled with maintenance of libido. The objective of this study was to determine the extent to which implants of potent androgen (DHT) will restore androgenization and spermatogenesis in hypogonadotropic infertile male rats. Twenty-five sexually mature male rats of proven fertility were actively immunized against gonadotropin releasing hormone (GnRH) to induce azoospermia. After azoospermia was achieved, GnRH immunized rats received subdermal DHT-filled Silastic implants of 2, 4, 6, or 8 cm, or empty implants (n=5/group). Five untreated control rats received empty capsules. Eight weeks later, fertility was evaluated, sperm number was obtained from the testis, and weights of androgen-dependent organs were measured. The results indicate that immunoneutralization of GnRH induced complete azoospermia, and subsequent treatment with DHT implants of 2 or 4 cm for 8 wk restored accessory organ weights, but did not restore spermatogenesis or fertility. In addition, DHT implants of 6 to 8 cm partially restored spermatogenesis, but not fertility. We conclude that low-dose DHT supplementation of GnRH-immunized rats may be a suitable alternate therapy able to maintain androgenization in the face of persistent azoospermia in the rat. This may be an effective model for development of a male contraceptive.

Anti-androgen effects of the aromatase inhibitor, atamestane.

Prostatic hyperplasia can be induced in both intact and castrated dogs and in intact cynomolgus monkeys by the administration of androgenic steroids. Estrogenic steroids potentiate this effect in dogs. These changes also can be induced by androstenedione, which increases androgen and estrogen levels. Atamestane (ATA; 1-methyl-3,17-dione-androsta-1,4-diene), a potent aromatase inhibitor, inhibits some of the androstendione-induced effects; however, the nonsteroidal aromatase inhibitor, CGS-16949A, has been reported to decrease serum estradiol levels in adult rats but to have no effect on androgen-dependent organ weights. To examine the mechanisms by which ATA affects the rat prostate, in vivo and in vitro studies were conducted using adult rat ventral prostate (VP). Intact Sprague-Dawley rats were injected daily for 14 days with sesame seed oil, ATA (70 mg/kg/day), finasteride (FIN; 5 mg/kg/day), a 5 alpha-reductase inhibitor, or the combination of FIN plus ATA. A fifth group was castrated (CASTR) on day 1. The mean +/- standard error VP weight of the controls was 350 +/- 19 mg. It was reduced 17% (P < 0.05) by ATA, 29% (P < 0.001) by FIN, 48% (P < 0.001) by FIN plus ATA, and 86% (P < 0.001) by CASTR. The DNA/VP was reduced 22% (not significant) by ATA, 18% by FIN (not significant), 35% (P < 0.01) by FIN plus ATA, and 60% (P < 0.001) by CASTR. More significant changes were observed in RNA and protein. The mRNA for prostatein C3 was reduced by each of the treatments, but only CASTR increased the mRNA for TRPM-2, a marker of apoptosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Maintenance of sexual function with testosterone in the gonadotropin-releasing hormone-immunized hypogonadotropic infertile male rat.

We have previously shown that active immunization against GnRH in the mature male rat can predictably produce hypogonadotropic hypogonadism and azoospermia and, further, that normospermia and normal fertility can be restored by testosterone (T) administration alone. The objective of this study was to explore the hypothesis that GnRH-immunized azoospermic rats could be supplemented with T doses sufficient to restore sexual behavior, but insufficient to support adequate spermatogenesis or to allow restoration of fertility. Adult male rats of proven fertility were immunized against GnRH and supplemented with 2-, 4-, or 8-cm T implants or with empty implants. Eight weeks later, fertility was evaluated; concentrations of serum T, LH, FSH, growth hormone (GH), prolactin (PRL), and thyroid-stimulating hormone (TSH) were determined; sperm number was obtained from the testis; and weights of androgen-dependent organs were measured. GnRH immunization and T supplementation resulted in restoration of organ weights and of fertility in a dose-dependent manner. GnRH immunization with or without T supplementation resulted in the absence of circulating gonadotropins, but had no effect on serum GH, PRL, or TSH levels. Whereas all control animals were fertile, rats that received either empty or 2-cm T implants were completely infertile. Rats that received 4-cm or 8-cm T implants were fertile in 60% and 100% of cases, respectively. Sexual behavior of rats with empty and with 2-cm T implants was compared at 10-18 wk after immunization with GnRH. GnRH-immunized rats given empty implants displayed negligible sexual activity, but those with 2-cm T implants displayed sexual activity equivalent to that of untreated controls despite complete infertility.(ABSTRACT TRUNCATED AT 250 WORDS)