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Nandrolone decanoate interferes with testosterone biosynthesis altering blood-testis barrier components.

ABSTRACT: The aim of this study was to investigate whether nandrolone decanoate (ND) use affects testosterone production and testicular morphology in a model of trained and sedentary mice. A group of mice underwent endurance training while another set led a sedentary lifestyle and were freely mobile within cages. All experimental groups were treated with either ND or peanut oil at different doses for 6 weeks. Testosterone serum levels were measured via liquid chromatography-mass spectrometry. Western blot analysis and quantitative real-time PCR were utilized to determine gene and protein expression levels of the primary enzymes implicated in testosterone biosynthesis and gene expression levels of the blood-testis barrier (BTB) components. Immunohistochemistry and immunofluorescence were conducted for testicular morphological evaluation. The study demonstrated that moderate to high doses of ND induced a diminished serum testosterone level and altered the expression level of the key steroidogenic enzymes involved in testosterone biosynthesis. At the morphological level, ND induced degradation of the BTB by targeting the tight junction protein-1 (TJP1). ND stimulation deregulated metalloproteinase-9, metalloproteinase-2 (MMP-2) and the tissue inhibitor of MMP-2. Moreover, ND administration resulted in a mislocalization of mucin-1. In conclusion, ND abuse induces a decline in testosterone production that is unable to regulate the internalization and redistribution of TJP1 and may induce the deregulation of other BTB constituents via the inhibition of MMP-2. ND may well be considered as both a potential inducer of male infertility and a potential risk factor to a low endogenous bioavailable testosterone.

SUBMITTER: Barone R

PROVIDER: S-EPMC5542904 | biostudies-other | 2017 Aug

REPOSITORIES: biostudies-other

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Project description:The intricate interaction between protein endocytosis, transcytosis, recycling and endosome- or ubiquitin-mediated protein degradation determines the junction integrity in epithelial cells including Sertoli cells at the blood-testis barrier (BTB). Studies have shown that androgens and cytokines (e.g., TGF-β3) that are known to promote and disrupt BTB integrity, respectively, accelerate protein endocytosis at the BTB. We hypothesized that testosterone-induced endocytosed proteins are transcytosed and recycled back to the Sertoli cell surface, whereas cytokine-induced endocytosed proteins are degraded so that androgens and cytokines have opposing effects on BTB integrity. Herein, we report that both testosterone and TGF-β3 induced the steady-state level of clathrin, an endocytic vesicle protein. Testosterone and TGF-β3 also induced the association between internalized occludin (a BTB integral membrane protein) and clathrin, as well as early endosome antigen-1 (EEA-1). Interestingly, testosterone, but not TGF-β3, also induced the levels of proteins that regulate protein transcytosis (e.g., caveolin-1) and recycling (e.g., Rab11), and their association with internalized occludin and N-cadherin from the cell surface. In contrast, TGF-β3, but not testosterone, induced the level of ubiquitin-conjugating enzyme E2 J1 (Ube2j1), a protein crucial to the intracellular protein degradation pathway, and its association with internalized occludin. Based on these findings and recent reports in the field, we hypothesize that the concerted effects of testosterone and TGF-β3 likely facilitate the transit of preleptotene spermatocytes at the BTB while maintaining the immunological barrier in that testosterone induces the assembly of "new" tight junction (TJ)-fibrils below migrating spermatocytes via protein transcytosis and recycling before cytokines induce the disassembly of "old" TJ-fibrils above spermatocytes via endocytic vesicle-mediated degradation of internalized proteins. This thus provides a unique mechanism in the testis to facilitate the transit of preleptotene spermatocytes, many of which are connected in "clones" via cytoplasmic bridges, at the BTB while maintaining the immunological barrier during stage VIII of the seminiferous epithelial cycle of spermatogenesis.

Project description:During spermatogenesis in the mammalian testis, preleptotene/leptotene spermatocytes differentiate from type B spermatogonia and traverse the blood-testis barrier (BTB) at stage VIII of the seminiferous epithelial cycle for further development. This timely movement of germ cells involves extensive junction restructuring at the BTB. Previous studies have shown that these events are regulated by testosterone (T) and cytokines [e.g., the transforming growth factor (TGF) -betas], which promote and disrupt the BTB assembly, respectively. However, the mechanisms underlying the "opening" of the BTB above a migrating preleptotene/leptotene spermatocyte and the "resealing" of the barrier underneath this cell remain obscure. We now report findings on a novel mechanism utilized by the testes to regulate these events. Using cell surface protein biotinylation coupled with immunoblotting and immunofluorescent microscopy, we assessed the kinetics of endocytosis and recycling of BTB-associated integral membrane proteins: occludin, JAM-A, and N-cadherin. It was shown that these proteins were continuously endocytosed and recycled back to the Sertoli cell surface via the clathrin-mediated but not the caveolin-mediated pathway. When T or TGF-beta2 was added to Sertoli cell cultures with established functional BTB, both factors accelerated the kinetics of internalization of BTB proteins from the cell surface, perhaps above the migrating preleptotene spermatocyte, thereby opening the BTB. Likewise, T also enhanced the kinetics of recycling of internalized biotinylated proteins back to the cell surface, plausibly relocating these proteins beneath the migrating spermatocyte to reassemble the BTB. In contrast, TGF-beta2 targeted internalized biotinylated proteins to late endosomes for degradation, destabilizing the BTB. In summary, the transient opening of the BTB that facilitates germ cell movement is mediated via the differential effects of T and cytokines on the kinetics of endocytosis and recycling of integral membrane proteins at the BTB. The net result of these interactions, in turn, determines the steady-state protein levels at the Sertoli-Sertoli cell interface at the BTB.

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Nandrolone decanoate interferes with testosterone biosynthesis altering blood-testis barrier components.

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