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.

Keywords: MMP-2; MMP-9; MUC1; TJP1; blood-testis barrier; nandrolone decanoate; testosterone.

Figure 1

Functional effects of endurance exercise on body weight. Changes in body weight over time. All mice were weighed every week. Horizontal axis: time of training (weeks). Vertical axis: body weight (g). (A): normal control mice (SED), sedentary low dose of ND (SED‐ND‐L), sedentary medium dose of ND (SED‐ND‐M), sedentary high dose of ND (SED‐ND‐H). (B): trained control mice (TR), trained low dose of ND (TR‐ND‐L), trained medium dose of ND (TR‐ND‐M) and trained high dose of ND (TR‐ND‐H). Data are presented as the mean ± S.D. # significantly different from TR first week mice (P < 0.05).

Figure 2

Effect of ND on testosterone secretion and steroidogenic gene/protein expression. (A): Measurement of testosterone level performed with liquid chromatography–mass spectrometry. Vertical axis: testosterone levels (ng/ml). Horizontal axis: mice groups. Normal control mice (SED), sedentary low dose of ND (SED‐ND‐L), sedentary medium dose of ND (SED‐ND‐M), sedentary high dose of ND (SED‐ND‐H), trained control mice (TR), trained low dose of ND (TR‐ND‐L), trained medium dose of ND (TR‐ND‐M) and trained high dose of ND (TR‐ND‐H). (B): qRT‐PCR evaluation of StAR,CYP11A1,HSD3B1 and CYP17A1 gene expression after ND administration and/or endurance training. The graphs show normalization with the reference genes, according to the Livak method (2^−∆∆CT). Vertical axis: 2^−∆∆CT. Horizontal axis: mice groups. (C): representative cropped blots for StAR (30 kDa), CYP11A1 (60 kDa), HSD3B1 (42 kDa) and CYP17A1 (55 kDa). The gels were run under the same experimental conditions and β‐actin was used as the internal control. (D): relative expression levels of StAR, CYP11A1, HSD3B1 and CYP17A1. Vertical axis: arbitrary units (AU). Horizontal axis: mice groups. Data are presented as the mean ± S.D. *P < 0.05; #P < 0.01.

Figure 3

Representative photomicrographs of testis sections stained with haematoxylin–eosin. Testis histology of normal control mice (SED), trained control mice (TR), low dose of ND (ND‐L), medium dose of ND (ND‐M) and high dose of ND (ND‐H). The photomicrographs showed degenerative changes and disorganization of the normal histology of testes with an incomplete germ cell maturation of seminiferous tubules (see arrows). Bar = 100 μm for all panels.

Figure 4

Effect of ND on gene expression levels of BTB components. qRT‐PCR evaluation of Junctional adhesion molecule A (JAM1), Dynamin‐2 (DNM2), Occludin (OCLN), Gap junction alpha‐1 (GJA1) and TJP1 gene expression after ND administration and/or endurance training. The graphs show normalization with the reference genes, according to the Livak method (2^−∆∆CT). Vertical axis: 2^−∆∆CT. Horizontal axis: mice groups. Normal control mice (SED), sedentary low dose of ND (SEDND‐L), sedentary medium dose of ND (SED‐ND‐M), sedentary high dose of ND (SED‐ND‐H), trained control mice (TR), trained low dose of ND (TR‐ND‐L), trained medium dose of ND (TRND‐M) and trained high dose of ND (TR‐ND‐H). Data are presented as the mean ± S.D. *vs SED, TR, TR‐ND‐L and SED‐ND‐M (P < 0.05); #vs SED, TR, TR‐ND‐L and SED‐ND‐H (P < 0.01); $vs SED (P < 0.05); &vs SED, TR, TR‐ND‐L, TR‐ND‐M and SED‐ND‐H (P < 0.01); †vs SED and SED‐ND‐M (P < 0.05); ¥vs SED and SED‐ND‐H (P < 0.05); Δvs SED‐ND‐M, SED‐ND‐H, TR‐ND‐H (P < 0.05); ◊vs TR and TR‐ND‐L (P < 0.05).

Figure 5

Representative images of immunofluorescence stain for TJP1 and MMP‐9 in testis sections. Testis histology of normal control mice (SED), trained control mice (TR), low dose of ND (ND‐L), medium dose of ND (ND‐M) and high dose of ND (ND‐H). The images above show TJP1 immunoreactivity distributed at the level of the BTB visibly as a line between adjacent Sertoli cells (see arrow) whereas arrow head indicates TJP1 signal in the cell cytoplasm at the basal and adluminal compartment of the seminiferous tubules. The images below show metalloproteinase‐9 (MMP‐9) immunoreactivity distributed pre‐eminently in the flagella of spermatocytes at the adluminal compartment of seminiferous tubules (see arrow). Bar = 100 μm for all panels.

Figure 6

Representative images of immunofluorescence stain for MMP‐2 and TIMP‐2 in testis sections. Testis histology of normal control mice (SED), trained control mice (TR), low dose of ND (ND‐L), medium dose of ND (ND‐M) and high dose of ND (ND‐H). The images above show MMP‐2 immunoreactivity distributed at the level of the basal lamina of the seminiferous tubules as well as in the cytoplasm and cell membrane of Leydig cells (see arrow) whereas arrow head indicates MMP‐2 distribution in the cytoplasm of Leydig cells. The images below show the tissue inhibitor of metalloproteinase‐2 (TIMP‐2) immunoreactivity distributed at the level of the basal lamina of the seminiferous tubules and in the cytoplasm of Leydig cells (see arrow). Bar = 100 μm for all panels.

Figure 7

Representative images of immunohistochemical stain for MUC1 in testis sections. Testis histology of normal control mice (SED), trained control mice (TR), low dose of ND (ND‐L), medium dose of ND (ND‐M) and high dose of ND (ND‐H). Moderate signal of mucin 1 (MUC1) immunoreactivity inside the cytoplasm of some germ cells (arrow). Intense signal of MUC1 immunoreactivity in the nuclei of spermatids of seminiferous tubules (arrow head). Bar = 100 μm for all panels.