Role of serotonin, estrogen, and TNF-α in the paroxetine-impaired steroidogenesis and testicular macrophages polarization.

BACKGROUND: Paroxetine, a selective serotonin reuptake inhibitor (SSRI) antidepressant, has caused male sexual dysfunction; however, the paroxetine mechanisms of action in testes are still unclear. OBJECTIVES: Paroxetine serotonergic effects in testes were evaluated, focusing on steroidogenesis and the correlation between macrophages population and possible TNF-α-derived oxidative stress. We also verified whether the changes are reversible following treatment interruption. MATERIALS AND METHODS: Adult rats received paroxetine (PG35 and PG65) or tap water (CG) for 35 days. PG65 was maintained without treatment for 30 more days. Intratesticular testosterone (IT), nitrite, and malondialdehyde concentrations were measured. To confirm serotonergic and estrogenic effects, Htr1b and Esr1 expressions were analyzed. The daily sperm production (DSP), frequency of abnormal seminiferous tubules (ST), SC number, ST area, and Leydig cells nuclear area (LCnu) were evaluated. TUNEL+ germ cells, M1 (CD68+ ), and M2 (Perls+ ) macrophages were quantified. 17ß-HSD7, CYP19A1, NDRG2, oxytocin, TNF-α, and iNOS were evaluated by immunoreactions. Oxytocin and NDRG2 protein levels as well as Tnfa mRNA expression were also analyzed. RESULTS: The Htr1b downregulation in testes confirmed the paroxetine serotonergic effect. The testicular sections showed abnormal ST frequency, ST atrophy and reduction of DSP, LCnu, SC number and Perls+ macrophages. TUNEL+ germ cells and LC were associated with strong NDRG2 immunoexpression. Paroxetine reduced IT levels and 17ß-HSD7 immunoexpression in parallel to increased CYP19A1, oxytocin, TNF-α and iNOS. Esr1 and Tnfa overexpression and increased number of CD68+ macrophages were also observed together with high nitrite and malondialdehyde levels. Most parameters were not recovered in PG65. CONCLUSIONS: Paroxetine serotonergic effect impairs LC steroidogenesis, via aromatization, increasing estrogen/testosterone ratio, which in turn upregulate NDRG2, promoting apoptosis, and impairing sperm production. Serotonin-estrogen pathways may be responsible for M2/M1 polarization, Tnfa upregulation, and induction of oxidative stress. The unrecovered testicular changes after treatment discontinuation are due to persistent paroxetine serotonin/estrogen effects.

Muscular atrophy, impaired epithelial autophagy and UCHL1 increase in androgen-deficient cauda epididymis.

In epididymis, cimetidine induces androgenic failure due to reduced sex hormone-binding globulin stromal levels and blockade of androgen receptor (AR) nuclear import. UCHL1, a hydrolase of ubiquitin-proteasome system (UPS), seems to play a role in autophagy and apoptotic pathway. However, the role of UPS and autophagy in epididymis has not been clarified. We evaluated UCHL1 and autophagy in epididymal cauda epithelium under androgenic deficiency induced by cimetidine, focusing on the interplay among these processes and apoptosis. The integrity of epididymal muscular layer was also evaluated. Male rats received cimetidine (CMTG) or saline (CG). Seminal vesicles were weighed, the expression of androgen-responsive genes Crisp1 and connexin 43 (Cx43) in cauda epididymis was evaluated, and cauda fragments were processed for light and transmission electron microscopy. The epithelium height and muscular thickness were measured. TUNEL, immunohistochemistry for caspase-3 and Cx43, and immunofluorescence for AR, Bcl-2, UCHL1, MAP LC3A, and p62/SQSTM1 (autophagic markers) were performed. Bcl-2, UCHL1, and Cx43 were detected by Western blot. In CMTG, the reduction in seminal vesicles weight accompanied by downregulation of Crisp1 and Cx43 confirmed epididymal androgenic failure. These results were associated with muscular atrophy, apoptosis and weak Cx43 and AR immunoexpression, supporting the androgenic dependence of muscular integrity. The high UCHL1 levels and reduction in Bcl-2 reinforce UCHL1 role in epithelial cells death. The intense immunoexpression of LC3A and p62/SQSTM1 indicates autophagic disturb, which in association with high UCHL1 levels, points to a role of UPS and autophagy in the regulation of epididymal epithelial cells viability under androgenic control.

Vitamin B12 Prevents Cimetidine-Induced Androgenic Failure and Damage to Sperm Quality in Rats.

Cimetidine, used as an anti-ulcer and adjuvant treatment in cancer therapy, causes disorders in the male reproductive tract, including steroidogenesis. However, its effect on sperm quality and male fertility has been poorly addressed. Since vitamin B12 has demonstrated to recover spermatogonia number and sperm concentration in cimetidine-treated rats, we evaluated the impact of cimetidine on sperm quality and fertility potential and whether vitamin B12 is able to prevent the harmful effect of this drug on steroidogenesis and sperm parameters. Adult male rats were treated for 52 consecutive days as follows: cimetidine group (100 mg/kg of cimetidine), cimetidine/vitamin B12 group (100 mg/kg of cimetidine + 3 µg vitamin B12), vitamin B12 group (3 µg vitamin B12) and control group (saline). Serum testosterone levels and immunofluorescence associated to western blot for detection of 17ß-HSD6 were performed. Sperm morphology and motility, mitochondrial activity, acrosome integrity, DNA integrity by Comet assay, lipid peroxidation as well as fertility potential were analyzed in all groups. Apoptotic spermatids were also evaluated by caspase-3 immunohistochemistry. In the cimetidine-treated animals, reduced serum testosterone levels, weak 17ß-HSD6 levels and impaired spermiogenesis were observed. Low sperm motility and mitochondrial activity were associated with high percentage of sperm tail abnormalities, and the percentage of spermatozoa with damaged acrosome and DNA fragmentation increased. MDA levels were normal in all groups, indicating that the cimetidine-induced changes are associated to androgenic failure. In conclusion, despite the fertility potential of rats was unaffected by the treatment, the sperm quality was significantly impaired. Therefore, considering a possible sperm-mediated transgenerational inheritance, the long term offspring health needs to be investigated. The administration of vitamin B12 to male rats prevents the androgenic failure and counteracts the damage inflicted by cimetidine upon sperm quality, indicating that this vitamin may be used as a therapeutic agent to maintain the androgenic status and the sperm quality in patients exposed to androgen disrupters.

Seminiferous epithelium damage after short period of busulphan treatment in adult rats and vitamin B12 efficacy in the recovery of spermatogonial germ cells.

Several different strategies have been adopted in attempt to recover from chemotherapy-damaged spermatogenesis that is often seen in oncologic patients. In this study, we have evaluated the impact of short period of exposure to busulphan on the haemogram and seminiferous epithelium of adult rats, focusing on spermatogonial depletion and Sertoli cell (SC) integrity. We then examined whether vitamin B12 supplementation improves the haematological parameters and spermatogonia number. The animals received 10 mg/kg of busulphan (BuG) or busulfan+vitamin B12 (Bu/B12 G) on the first and fourth days of treatment. In H.E.-stained testicular sections, the areas of the seminiferous tubule (ST) and seminiferous epithelium were measured. The number of spermatogonia in H.E-stained and PCNA-immunolabelled testicular sections was quantified. The frequency of tubules with abnormal SC nuclei or TUNEL-positive SC was evaluated. Vimentin immunofluorescence in ST was also evaluated. In BuG and Bu/B12 G, the animals showed leukopenia and thrombocytopenia, but the body weight reduced only in BuG. The areas of ST and seminiferous epithelium decreased in Bu/B12 G and BuG. In BuG, the number of H.E.-stained and PCNA-immunolabelled spermatogonia reduced significantly. The frequency of tubules containing abnormal SC nuclei and TUNEL-positive SC increased and the vimentin immunoexpression pattern changed. In Bu/B12 G, the number of H.E.-stained or PCNA-immunolabelled spermatogonia increased fourfold in comparison with BuG. The structural changes in ST after 6 days of busulphan exposure may be associated with the potential effect of this anti-neoplastic agent on SC. The increased number of spermatogonia in the busulphan-treated animals receiving vitamin B12 indicates that this vitamin can be an adjuvant therapy to improve the fertility in male cancer patients.

Primordial germ cells (spermatogonial stem cells) of bullfrogs express sex hormone-binding globulin and steroid receptors during seasonal spermatogenesis.

In vertebrate species, testosterone seems to inhibit spermatogonial differentiation and proliferation. However, this androgen can also be converted, via aromatase, into estrogen which stimulates spermatogonial differentiation and mitotic activity. During seasonal spermatogenesis of adult bullfrogs Lithobates catesbeianus, primordial germ cells (PGCs) show enhanced testosterone cytoplasm immunoexpression in winter; however, in summer, weak or no testosterone immunolabelling was observed. The aim of this study was to confirm if PGCs express stem cell markers - alkaline phosphatase (AP) activity and GFRα1 (glial-cell-line-derived neurotrophic factor) - and verify whether testosterone is maintained in these cells by androgen receptors (ARs) and/or sex hormone-binding globulin (SHBG) in winter. Furthermore, regarding the possibility that testosterone is converted into estrogen by PGCs in summer, the immunoexpression of estrogen receptor (ER)ß was investigated. Bullfrog testes were collected in winter and in summer and were embedded in glycol methacrylate for morphological analyses or in paraffin for the histochemical detection of AP activity. GFRα1, AR, SHBG and ERß expression were detected by Western blot and immunohistochemical analyses. The expression of AP activity and GFRα1 in the PGCs suggest that these cells are spermatogonial stem cells. In winter, the cytoplasmic immunoexpression of ARs and SHBG in the PGCs indicates that testosterone is maintained by these proteins in these cells. The cytoplasmic immunoexpression of ERß, in summer, also points to an ER-mediated action of estrogen in PGCs. The results indicate a participation of testosterone and estrogen in the control of the primordial spermatogonia during the seasonal spermatogenesis of L. catesbeianus.

Resposta testicular ao tratamento de ratos machos adultos com diferentes concentrações de etanol / Testicular response to the treatment of adult male rats with different concentrations of ethanol

É noção popular que o álcool é um afrodisíaco. No entanto, no seu consumo pode reduzir os níveis sanguíneos de testosterona, resultando em disfunção sexual. O objetivo deste trabalho foi investigar através da morfometria e estereologia a estrutura do testículo e o processo espermatogênico de ratos machos albinos de linhagem Wistar submetidos ao tratamento subcrônico com etanol. Os animais dos grupos experimentais receberam por gavage, 0,1ml de etanolo a cada 100g de peso corpóreo nas concentrações de 10%, 20% e 30%. Os animais dos grupos controles receberam água destilada. Etanol e água destilada foram administrados diariamente durante seis semanas. Os animais foram sacrificados 24 horas após o término do tratamento. Os testículos foram removidos, pesados e submetidos á rotina histológica usual para análise em microscopia de luz. Foram realizadas análiseshistológicas e histomorfométricas. Os resultados mostram que o tratamento com etanol não alterou o volume relativo das células de Leydig. Por outro lado, o volume correspondente aos túbulos seminíferos estava reduzido nos testículos dos animais alcoolizados em relação aos seus respectivos controles. Apesar desta diminuição no volume relativo, o diâmetro médio dos túbulos seminíferos não estava alterado. Nestas condições experimentais não foi observada nenhuma alteração na morfologia testicular. Nenhuma das funções testiculares foi, aparentemente, afetada embora tenha sido observada redução na freqüência de túbulos seminíferos contendo espermatogônias do tipo B, Intermediárias e espermátides naqueles animais que receberam as maiores concentrações de etanol.