Suppression Of Spermatogenesis Research Articles

Regenerative Potential of Spermatogonial Stem Cells, Endothelial Progenitor Cells, and Epithelial Progenitor Cells of C57Bl/6 Male Mice with Metabolic Disorders.

The properties of spermatogonial stem cells, endothelial progenitor cells, and the epithelial progenitors of C57Bl/6 mice under conditions of metabolic disorders were studied using the model of busulfan-induced suppression of spermatogenesis and in vitro culture technique. Spermatogonial stem cells CD117-CD90+ and epithelial progenitors CD45-CD31-Sca-1+CD49f+ derived from the testes of mice with metabolic disturbances demonstrated 17- and 28-fold increase in the respective cell mass and generated cell colonies in vitro. In contrast, spermatogonial stem cells with immune phenotype CD51-CD24+CD52+ had reduced selfrenewal capacity. Spermatogonial stem cells CD117-CD90+ and CD117+CD90+ as well as endothelial progenitors CD45-CD31+ derived from the testes of donor mice with metabolic disorders demonstrated high transplantation capacity in C57Bl/6 mouse testes damaged by cytostatic busulfan.

Ferulic acid prevents lead-induced testicular oxidative stress and suppressed spermatogenesis in rats.

Lead affects multiple organ systems including testis. We investigated the effects of ferulic acid (FA) on lead-induced oxidative stress and spermatogenesis suppression in rats. Animals received lead acetate (500mg/L in drinking water) and/or FA (50mg/kg, i.g.) for eight weeks. Lead increased testicular malondialdehyde (MDA) and nitrite levels and decreased glutathione (GSH) content and catalase (CAT) activity. Lead decreased testis weight and testosterone level. Sperm parameters decreased in lead group. FA ameliorated the decreased testis weight, serum testosterone as well as sperm count, viability, motility and normal morphology in lead group. FA improved antioxidant capacity as well as sperm count, viability, motility and normal morphology. FA decreased Johnsen's mean testicular biopsy score (MTBS) criteria by restoring degeneration, atrophy and tubular disarrangement. FA also normalised spermatogonia, spermatocytes and spermatids numbers in lead group and led to increases in number of Leydig and Sertoli cells. FA showed beneficial effects in lead-induced testicular oxidative stress and spermatological disorders, through inhibiting lipid peroxidation and enhancing antioxidant defence systems. The positive effects of FA on Leydig cells may be involved in restoring testosterone levels in lead group. FA can be considered a potential candidate to protect testis against the deleterious effect of lead intoxication.

The experimental and postmortem morphological characteristic of the chronic action of alcohol on the testicles

The objective of the present study was to analyze the dynamics of the morphological changes that manifest themselves in the testicles of mature rats exposed to chronic alcohol intoxication over a period up to 6 months and to compare them with the analogous changes in the testicles of men who had had the long history of alcohol dependence and died with the pronounced symptoms of alcohol intoxication. The study has demonstrated the strong resemblance between the changes in the testes of men and animals including such as the dystrophic alteration of the spermatogenic epithelium, diffuse overgrowth of the connective tissue, well apparent signs of disturbed blood circulation, and suppression of spermatogenesis. However, these morphological changes in human testicles were more pronounced than in the rat gonads. In certain cases they underwent further fibrous transformation with the formation of well apparent hyalinosis foci. It is worthwhile to note that such morphological changes in the humans were not the specific consequences of the excessive alcohol consumption but reflected the collective influence of various toxic agents, with alcohol being one of the leading intoxicants.

Regenerative Potential of Stem and Progenitor Cells from Ischemic Testes of C57Bl/6 Mice in Culture and in the Model of Spermatogenesis Suppression Caused by Busulfan.

The regenerative potential of stem and progenitor cells from ischemic testes of C57Bl/6 mice was studied in vitro (cell culture) and in vivo (mouse model of busulfan-induced suppression of spermatogenesis). Spermatogonial stem cells with phenotypes CD117-CD90+ and CD51-CD24+CD52+ from ischemic testes demonstrated 33-fold and 7-fold increments of cell mass and generated colonies in vitro. Epithelial (CD45-CD31-Sca-1+CD49f+) and endothelial (CD45-CD31+) precursors exhibited lower self-renewal capacity. On day 30 after injection of stem and progenitor cells from ischemic testes to the rete testis zone of the testes of busulfantreated animals, an increase in the count of CD117-CD90+ spermatogonial stem cells, total count, and mobile sperm count in the testes of recipient mice was observed. In addition, we observed an increase in Sca-1+ cell count, recovery of the spermatogenic epithelium in the seminiferous tubules, and appearance of immature Leydig cells in "busulfan" testes; the level of tissue testosterone and fertility index also increased.

Role of Tissue-Specific Stem and Progenitor Cells in the Regeneration of the Pancreas and Testicular Tissue in Diabetic Disorders.

Using the model of hypogonadism in C57Bl/6 male mice, we showed that injection of streptozotocin to newborn animals and high-fat diet induced serum IFN-γ and IL-17 elevation, glucose metabolism disturbances, insulin resistance, destructive changes of the Langerhans islets (deficit of PDX1+β cells), while the number of oligopotent β cell precursors (CD45-TER119-CD133+CD49flow) increased. Diabetes played the role of an inducer of testicular tissue inflammation (pan-hemopoietic cell infiltration, increase of IL-2, IL-17, and IL-23 content) and reproductive system disturbances in mice (decrease in free testosterone concentration, suppression of spermatogenesis, and infertility). The development of hypogonadism was paralleled by an increase in the count of spermatogonial stem cells (CD117+CD29+CD90+), multipotent mesenchymal stromal cells (CD45-CD31-CD90+CD106+), hemangiogenesis precursors (CD45-CD117+Flk1+), and epithelial cells (CD45-CD31-CD49f+CD326+).

Pentachlorophenol affected both reproductive and interrenal systems: In silico and in vivo evidence

The present study investigated the effects on reproductive and interrenal system by pentachlorophenol (PCP) using in silico and in vivo assays. Molecular docking results indicated interacting potency of PCP with steroid receptors (ERα, ERβ, AR, GR) but not Cytochrome P450 enzymes (CYPs). In the in vivo assay, sexually matured rare minnow (Gobiocypris rarus) was exposed to environmental relevant concentrations of PCP (0, 0.5, 5, 50 μg L−1). In male fish, 14-d exposure caused up-regulation of mRNA levels of hepatic erα, erβ, ar, gr, vtg and gonadal erα, vtg, ar, dmrt1, providing evidence for agonistic activities for steroid receptors by PCP. The up-regulated mRNA of gnrh, crf, pomc in the brain also indicated feed-forward responses of the hypothalamic-pituitary-gonadal/interrenal (HPG/I) axis. However, at 28th d the feed-forward response of the HPG axis seemed eased back and the HPI axis showed negative feedback responses. Corresponding changes including increases of plasma steroid hormones, inhibition of spermatogenesis, and decreased RSI were observed in male fish upon 28-d exposure to PCP. In the females, a transition from feed-forward responses to negative feedbacks of the HPG/I axis was also indicated by the transcriptional profiles at 14th and 28th day. Corresponding changes including increased E2, T and decreased C levels, degenerated ovaries, and decreased GSI and RSI were also observed. Overall, we concluded that PCP could interfere with steroid receptors, evoke responses of HPG/I axis, and finally result in adverse effects on reproductive and interrenal system in rare minnow at environmental relevant concentrations.

Response of Stem and Progenitor Cells to Testicular Ischemia.

Stem and progenitor cells were studied on mouse model of testicular ischemia. Testicular ischemia led to a decrease in free testosterone concentration. Hemodynamic changes, interstitial edema, and destruction of spermatogenic epithelium, Leydig, and Sertoli cells were observed in the testicular tissue. Accumulation of degenerative germ cells was accompanied by reduction in the count of spermatogonial stem cells with immunophenotype CD117(-)CD29(+)CD90(+) and CD117(+)CD29(+)CD90(+). Simultaneously with pathomorphological changes in the testes and suppression of spermatogenesis, ischemia reduced the count of hematopoietic progenitor cells, hematopoietic stem cells with immunophenotype Lin(-)CD117(+)Sca-1(+)c-kit(+)CD34(+) and Lin(-)CD117(+)Sca-1(+)c-kit(+)CD34(-), and multipotent mesenchymal stromal cells (CD45(-)CD31(-) CD90(+)CD106(+)) in the testicular tissue. The population of CD45(-)CD31(+)-endothelial cells in ischemic testicular tissue increased.

Efficacy of Curculigo orchioides in Deltamethrin Induced Reproductive System Impairment in Male Wistar Rats

Aim: To evaluate the ameliorating effect of Curculigo orchioides (CO) against deltamethrin (DLM) induced reproductive toxicity in male rats. Materials and Methods: A total of 42 adult male Wistar rats were divided into seven groups of six rats each. Group A (control) received normal diet and water ad-libitum, Group B (exposure) received DLM (2 mg/kg bw), orally in dimethylsulphoxide for 60 days, Group C (Treatment 1) received CO (100 mg/kg bw), orally dissolved in distilled water, Group D (Treatment 2) received CO at the dose of 200 mg/kg bw dissolved in distilled water, Group E (Combination 1) received DLM at the dose of 2 mg/kg with CO (100 mg/kg bw), Group F (Combination 2) received DLM at the dose of 2 mg/kg with CO (200 mg/kg bw), and Group G (metabolized) received DLM at the dose of 2 mg/kg bw for 60 days and DLM was left to metabolized for 60 days. Results and Discussion: DLM exposure caused significant reduction in body weight, reproductive organ weight, sperm head counts, sperm motility, reduced glutathione (GSH), catalase, superoxide dismutase (SOD), GSH-S-transferase (GST), GSH peroxidase (GPx), GSH reductase (GR), testosterone (T), follicles stimulating hormone (FSH), luteinizing hormone (LH) and steroidogenic enzyme 3β hydroxysteroid dehydrogenase (3β HSD), and 17β HSD. DLM exposure also showed increase in sperm abnormalities and level of lipid peroxidation. The treatment with CO improved the body weight, reproductive organ weight, sperm head count, sperm motility, and sperm abnormality. CO treatment also increased the level of GSH, SOD, GST, GPx, GR, T, FSH, LH and steroidogenic enzyme 3β HSD, and 17β HSD. Histological findings indicated vacuolation in lumen, disorganization of leydig cells and sertoli cells and inhibition of spermatogenesis in testicular tissue on DLM exposure, whereas CO treatment recovered normal histoarchitecture. Conclusion: The findings indicated that DLM damaged the reproductive system of male Wistar rats. CO treatment efficiently improved the reproductive system chiefly by increasing the level of important steroidogenic enzymes and hormones.

2, 4-Dichloro-6-nitrophenol, a photonitration product of 2, 4-dichlorophenol, caused anti-androgenic potency in Chinese rare minnows (Gobiocypris rarus)

2,4-Dichloro-6-nitrophenol (DCNP) is an environmental transformation product of 2,4-dichlorophenol that has been identified as widespread in effluent wastewater, but little is known about its toxicity because this compound is not regulated. Therefore, to investigate the endocrine disruption potency of DCNP in Chinese rare minnows (Gobiocypris rarus), adult and juvenile fish were exposed to various concentrations of DCNP (2, 20, and 200 μg/L) for 28 d. After 28 d exposure, the plasma vitellogenin (VTG) levels were reduced in females while increased in males and juvenile fish considerably, as compared with the control. These results suggested that DCNP affects the HPG-axis in a sex-dependent way. Testosterone (T) levels in the plasma were significantly lower in adult and juvenile fish and were accompanied by an increased estradiol (E2)/T ratio. Histopathological observation revealed hypertrophy of the hepatocytes and nuclear pyknosis in the liver, the inhibition of spermatogenesis in the testes, and the degeneration of oocytes in the ovaries after DCNP exposure. The expression pattern of selected genes indicated that the nuclear receptor, steroidogenesis and gonadotropin regulation pathways were perturbed after DCNP exposure. Above all, our results demonstrated that DCNP clearly had anti-androgenic activity in both adult and juvenile fish and can therefore be considered as an endocrine-disrupting chemical.

Antifertility efficacy of Coccinia indica in male mice and its possible mechanisms of action on spermatogenesis

The purpose of the present study was to investigate the antifertility efficacy of Coccinia indica and its possible mechanisms of action on testicular functions in Parkes male mice. Mice were orally administered 50% ethanolic leaf extract of Coccinia indica (200 and 500mgkg−1 body weight day−1) or distilled water (controls) for 35days. To assess reversibility, additional mice were treated with 500mgkg−1 body weight of Coccinia or distilled water for 35days and sacrificed 56days later. Several male reproductive parameters such as motility, viability, morphology and number of spermatozoa in the cauda epididymidis, histopathology, serum level of testosterone, and fertility indices were evaluated; further, activities of 3β- and 17β-hydroxysteroid dehydrogenases, western blot analyses of StAR protein, cytochrome P450scc enzyme and of caspase-3, germ cell apoptosis by TUNEL, and lipid peroxidation and antioxidant enzymes activities in the testis were assessed. Toxicological parameters were also examined. Histologically, testes in Coccinia-treated mice showed nonuniform diverse degenerative changes in the seminiferous tubules. Treatment had adverse effect on serum level of testosterone, steroidogenic markers in the testis and on sperm parameters in the cauda epididymidis. The treatment also affected oxidative status of the testis and induced germ cell apoptosis. Serum levels of alanine aminotransferase, aspartate aminotransferase and creatinine, and haematological parameters were, however, not affected in treated mice. Fertility of the extract-treated males was also suppressed, but their libido remained unaffected. By 56days of treatment withdrawal, the above parameters recovered to control levels, suggesting that the Coccinia treatment causes reversible suppression of spermatogenesis and fertility in P mice, without producing detectable signs of toxicity. Further, suppression of spermatogenesis may be caused by germ cell apoptosis resulting from deficiency of testosterone, which, in turn, may result from the adverse effect of C. indica treatment on steroidogenesis and oxidative status in the testis.

Regulation mechanisms of spermatogenesis in red-backed mice at different population phases

This paper gives morphological and functional analysis of the endocrine and germinal testicular sections in red-backed mice Myodes glareolus from a natural population at different population sizes. The decrease in the germinal testicular activity at the peak population is due to the reduced functional activity of the endocrine testicular section (Leydig and Sertoli cells), which leads to a decreased content of testosterone, androgen-binding protein, and inhibin that take part in the hormonal regulation of spermatogenesis. The trend towards suppression of spermatogenesis is noted to persist in animals at a low population size.

Reproductive endocrinology: Oral enclomiphene citrate in obese men with hypogonadism.

Testosterone supplementation therapy is an effective therapy for hypogonadism but can lead to the suppression of spermatogenesis. A new study investigated the effects of enclomiphene citrate, the trans isomer of clomiphene, on serum hormone levels and sperm counts in men with secondary hypogonadism.

Aqueous fruit extract of Mimusops elengi causes reversible suppression of spermatogenesis and fertility in male mice.

Antifertility efficacy of oral administration of aqueous fruit extract of Mimusops elengi (200, 400 and 600mgkg(-1) body weight/day for 35days) was evaluated in Parkes strain male mice. Various reproductive end points such as histopathology, sperm parameters, testosterone level, haematology, serum biochemistry and fertility indices were assessed; activities of 3β- and 17β-hydroxysteroid dehydrogenases, and immunoblot expressions of StAR and P450scc in the testis were also assessed. Histologically, testes in Mimusops-treated mice showed nonuniform and diverse degenerative changes in the seminiferous tubules; both affected and normal tubules were observed in the same sections of testis. The treatment had adverse effects on testicular hydroxysteroid dehydrogenases and StAR and P450scc, serum level of testosterone and on motility, viability and number of spermatozoa in cauda epididymis. However, serum levels of alanine aminotransferase, aspartate aminotransferase and creatinine, and haematological parameters were not affected by the treatment. Also, libido was not affected in treated males, but their fertility was markedly suppressed. By 56days of treatment withdrawal, the alterations caused in the above parameters recovered to control levels, suggesting that Mimusops treatment causes reversible suppression of spermatogenesis and fertility in Parkes mice. Further, there were no detectable signs of toxicity in treated males.

Suppression of sexual activity and reproduction in male small ruminants

Handling and husbandry of male small ruminants after sexual maturity often become difficult. Castration is currently the most reliable solution to this problem. Medicinal procedures for temporary inhibition of the gonad function could provide an alternative. Following a short overview of surgical castration, the current knowledge on the application of vaccines against gonadotropin-releasing hormone (GnRH) and GnRH agonist in rams and billy goats is presented in a literature overview. In rams, GnRH vaccination has been used successfully for temporary suppression of the reproduction function, regardless of an animal's age at the time of therapy initiation. Fewer investigations are available for the billy goat. A complete suppression of spermatogenesis was not achieved in all cases. Currently, treatment with GnRH agonists does not represent a relible method for the suppression of gonad function.

Oxidative stress and spermatogenesis suppression in the testis of cadmium-treated Bombyx mori larvae.

Bombyx mori L. (B. mori) were exposed to cadmium chloride (CdCl2) incorporated in an artificial diet (0, 6.25, 12.5, 25, and 50 mg kg(-1)) throughout the larval stage. Changes in malondialdehyde (MDA) and reduced glutathione (GSH) contents and activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px), as well as their corresponding messenger RNA (mRNA) levels in the testes of the fifth instar larvae were evaluated. Additionally, spermatozoon deformation in the testes was examined. Upon Cd treatment, the MDA content in the testes was significantly increased in a concentration-dependent manner. Cd-exposed larvae had increased levels of glutathione. Pearson's correlation analysis revealed that SOD and CAT activities were positively correlated (R (2) = 0.605, P = 0.017). The changing trends in the mRNA levels of these enzymes were not always consistent with those of enzymatic activities. Alterations in GSH-Px activities and mRNA levels were positively correlated (R (2) = 0.771, P < 0.01). Morphological analysis revealed that Cd deformed and affected the maturation of spermatozoa. Our results collectively support a relationship between Cd and alterations in the levels of antioxidant enzymes in B. mori testes.

Effect of polybrominated diphenyl ether (BDE-209) on testicular steroidogenesis and spermatogenesis through altered thyroid status in adult mice

Polybrominated diphenyl ethers (PBDEs), a class of brominated flame retardants (BFRs), have been widely used in many products to minimize the risk of fire, mainly by mixing in polymer products. BDE-209, a congener of PBDEs having structural similarity with thyroid hormones, acts as an endocrine disruptor by interfering with thyroid homeostasis. However, little is known about the effect of BDE-209 exposure on testicular steroidogenesis and spermatogenesis. This study was therefore conducted in adult mice to examine the effect of BDE-209 on testicular steroidogenesis and spermatogenesis in relation to thyroid status, and to explore possible mechanism(s) of its action. Adult Parkes strain male mice were orally gavaged with 750 and 950mg/kg BW of BDE-209 in corn oil for 35days. Significant reductions were noted in the levels of serum total T3, T4 and testosterone in mice treated with 950mg/kg BW of BDE-209 compared to controls; histologically, testes showed nonuniform degenerative changes in the seminiferous tubules as both affected and normal tubules were observed in the same section; further, number and viability of spermatozoa were also adversely affected in cauda epididymidis of these mice. Semiquantitative RT-PCR and western blot analyses also showed significant reductions in both testicular mRNA and protein levels of steroidogenic factor 1 (SF-1), steroidogenic acute regulatory (StAR) protein, cytochrome P450scc (CYP11A1), 3β-hydroxysteroid dehydrogenase (3β-HSD) and 17β-hydroxysteroid dehydrogenase (17β-HSD) in 950mg dose treated-mice compared to controls. Immunohistochemical and immunoblot analyses further revealed a marked decrease in proliferating cell nuclear antigen (PCNA) positive cells in testes of 950mg dose of BDE-209-treated mice. However, 750mg dose of BDE-209 had no effect on the above parameters. In conclusion, our results suggest that exposure of BDE-209 to adult mice causes reduction in serum levels of thyroid hormones and altered thyroid status may partly result into impairment of testicular steroidogenesis because of down-regulated expression of SF-1, thereby causing suppression of spermatogenesis.

Suppression of spermatogenesis by testosterone undecanoate-loaded injectable in situ-forming implants in adult male rats.

We have investigated the feasibility of administration of testosterone undecanoate (TU)-loaded injectable in situ-forming implant (ISFI) for contraception in adult male Sprague–Dawley rats. Male rats were treated with vehicle, TU-loaded ISFIs (540, 270 and 135 mg TU kg−1) or TU injections (45 mg TU kg−1 every 30 days) for 120 days. Fertility tests served for determining infertility or restoration of fertility in treated rats. Serum testosterone concentration, epididymal sperm count, motility, morphology, and histology of the testis were monitored. The TU-loaded ISFIs increased serum testosterone levels in rats steadily without fluctuation over 3 months. One month after TU administration, the epididymal sperm count decreased significantly in all experimental groups. After 3 months, the animals treated with 270 and 135 mg kg−1 TU-loaded ISFIs were 100% infertile, and no implantation sites were produced in the mated females. However, some of males treated with 540 mg kg−1 ISFI or TU injections were still fertile but numbers of implantation sites were also significantly lower than control values. TU-loaded ISFI at an appropriate dose has potential as a long-acting male contraceptive drug that suppresses spermatogenesis consistently over a period of 3 months.

Citrus limon extract: possible inhibitory mechanisms affecting testicular functions and fertility in male mice

The effect of oral administration of 50% ethanolic leaf extract of Citrus limon (500 and 1,000 mg/kg body weight/day) for 35 days on fertility and various male reproductive endpoints was evaluated in Parkes strain of mice. Testicular indices such as histology, 3β- and 17β-HSD enzymes activity, immunoblot expression of StAR and P450scc, and germ cell apoptosis by TUNEL and CASP- 3 expression were assessed. Motility, viability, and number of spermatozoa in the cauda epididymidis, level of serum testosterone, fertility indices, and toxicological parameters were also evaluated. Histologically, testes in extract-treated mice showed nonuniform degenerative changes in the seminiferous tubules. Treatment had adverse effects on steroidogenic markers in the testis and induced germ cell apoptosis. Significant reductions were noted in epididymal sperm parameters and serum level of testosterone in Citrus-treated mice compared to controls. Fertility of the extract-treated males was also suppressed, but libido remained unaffected. By 56 days of treatment withdrawal, alterations induced in the above parameters returned to control levels suggesting that Citrus treatment causes reversible suppression of spermatogenesis and fertility in Parkes mice. Suppression of spermatogenesis may result from germ cell apoptosis because of decreased production of testosterone. The present work indicated that Citrus leaves can affect male reproduction.

TiO2 Nanoparticle Exposure Decreases Spermatogenesis via Biochemical Dysfunctions in the Testis of Male Mice.

TiO2 nanoparticles (NPs) have been demonstrated to suppress spermatogenesis in animals, while there is little data related to the biochemical dysfunctions during spermatogenesis due to exposure to TiO2 NPs. In this study, male mice have been exposed to TiO2 NPs via intragastric administration for 60 consecutive days. The findings showed that TiO2 NP exposure resulted in lesions of testis and epididymis, deductions in sperm concentration and sperm motility, and an increase of the number of abnormal sperm in mice. Furthermore, TiO2 NP exposure with 2.5, 5, or 10 mg/kgbw decreased activities of lactate dehydrogenase (-11.59% to -39.84%), sorbitol dehydrogenase (-23.56% to -57.33%), succinate dehydrogenase (-27.04% to -57.85%), glucose-6-phosphate dehydrogenase (-28.3% to -56.42%), Na(+)/K(+)-ATPase (-15.59% to -53.11%), Ca(2+)-ATPase (-12.44% to -55.41%), and Ca(2+)/Mg(2+)-ATPase (-28.25% to -65.72%), and elevated activities of acid phosphatase (+10.48% to +40.0%), alkaline phosphatase (+20.65% to +64.07%), and total nitric oxide synthase (+0.68- to +2.3-fold) in the testes of mice, respectively. In addition, TiO2 NP exposure caused excessive production of reactive oxygen species (+16.15% to +110.62%), and increased malondialdehyde of lipid peroxidation product (+38.96% to +118.07%), carbonyl of protein oxidative product (+20.98% to +108.1%), and 8-hydroxydeoxyguanosine of DNA oxidative product (+0.9- to +1.83-fold) in the testes, respectively. It implied that spermatogenesis suppression caused by TiO2 NP exposure may be associated with alterations of testicular marked enzymes and oxidative stress in the testes.

Integrated assessment of wastewater treatment plant effluent estrogenicity in the Upper Murray River, Australia, using the native Murray rainbowfish (Melanotaenia fluviatilis).

The contamination of major continental river systems by endocrine-active chemicals (EACs) derived from the discharge of wastewater treatment plant (WWTP) effluents can affect human and ecosystem health. As part of a long-term effort to develop a native fish model organism for assessment of endocrine disruption in Australia's largest watershed, the Murray-Darling River Basin, the present study evaluated endocrine disruption in adult males of the native Australian Murray rainbowfish (Melanotaenia fluviatilis) exposed to effluent from an activated sludge WWTP and water from the Murray River during a 28-d, continuous-flow, on-site experiment. Analysis of the WWTP effluent and river water detected estrone and 17β-estradiol at concentrations up to approximately 25 ng L(-1) . Anti-estrogenicity of effluent samples was detected in vitro using yeast-based bioassays (yeast estrogen screen) throughout the experiment, but estrogenicity was limited to the first week of the experiment. Histological evaluation of the testes indicated significant suppression of spermatogenesis by WWTP effluent after 28 d of exposure. Plasma vitellogenin concentrations and expression of vitellogenin messenger RNA in liver were not significantly affected by exposure to WWTP effluent. The combination of low contaminant concentrations in the WWTP effluent, limited endocrine disrupting effects in the Murray rainbowfish, and high in-stream dilution factors (>99%) suggest minimal endocrine disruption impacts on native Australian fish in the Murray River downstream from the WWTP outfall.