This work was supported by the National Fund for Scientific and Technological Development of Chile (FONDECYT), grant no. 1120176 (A.C.). The authors declare that no conflicting interests exist.
SUMMARYSeveral observational studies have showed a combination of lower testosterone (T) to LH ratio and higher estradiol (E 2 ) to T ratio in secretory infertile men compared to men with normal spermatogenesis, suggesting a steroidogenic dysfunction of Leydig cells (Lc) that may involve increased aromatase activity. Low T/LH ratio is associated with Lc hyperplasia, which together with LH hyperstimulation may represent compensation for impaired T production. Aromatase expression and oestrogen production are mainly detected in Lc of the testis, although Sertoli and germ cells also contribute to testicular aromatase activity. The aim of this study was to assess the transcriptional expression of CYP19A1 (aromatase) in isolated Lc of subjects with Sertoli cell-only syndrome (SCOS) and signs of Lc impairment. Nineteen patients with SCOS and 10 controls with normal spermatogenesis who had medical indication of testicular biopsy for sperm retrieval were studied. Leydig cells were isolated by Laser Capture Microdissection (LCM) and CYP19A1 mRNA expression was quantified by SYBR â Green-based qPCR. In addition, testicular T and E 2 and serum hormonal levels were measured. Relative to control group, CYP19A1 was overexpressed more than twofold in 10/19 cases (2.3-12.2-fold increase), showing a significant increment in cases with low T/LH ratio (T/LH < 2) compared to cases with T/LH ≥ 2 (p = 0.038, REST â ). Moreover, Rq data for CYP19A1 had a direct correlation with testicular levels of E 2 and the E 2 /T ratio (r = 0.869; p < 0.001 and r = 0.633; p = 0.005). In summary, Lc from infertile patients with signs of Lc dysfunction overexpressed aromatase and showed an increment of testicular E 2 . Our results suggest that increased expression of aromatase in Lc leads to higher E 2 production and may account for the functional impairment of the Lc in patients with SCOS.
Obesity is characterized by mild chronic inflammation that is linked with impaired iron homeostasis. Studies in human and murine show that there is a transgenerational epigenetic inheritance via the gametes in obesity; however, there is little information on changes in the expression of microRNAs related to inflammation and iron homeostasis in spermatozoa from obese subjects. The present study investigated the expression of microRNAs related to inflammation (miR-21 y miR-155) and iron nutrition (miR-122 and miR-200b) in plasma, peripheral blood mononuclear cells (PBMC) and spermatozoa from normozoospermic controls (Cn; n = 17; BMI: 24.6 ± 2.0) and obese (Ob; n = 17; BMI: 32.6 ± 4.4) men. To determine the inflammation levels, we measured IL-6, TNF-α, and monocyte chemoattractant protein-1 (MCP1) by Magnetic Luminex® Assay. mRNA expression of IL6, TNF-α, and hepcidin (HAMP) in PBMC were evaluated by RT-qPCR. The analysis of microRNAs was performed using the Taqman® assays. The iron content in PBMC, seminal plasma, and spermatozoa was determined by Inductively Coupled Plasma Mass Spectrometry (ICP-MS). High serum IL6, TNF-α, and MCP1 levels were observed in Ob group (p < 0.05). Gene expression analysis showed an increased abundance relative of TNF-α (p = 0.018), HAMP (p = 0.03), and IL6 (p = 0.02) in PBMC from obese subjects. Also, we observed high levels of serum ferritin (p = 0.03), iron content in seminal plasma (p = 0.04), and spermatozoa (p = 0.002), but lower serum Fe (p = 0.007) in obese subjects. In the Ob group, a high expression of miR-155 (p = 0.02) and miR-21 (p = 0.03) was observed in PBMC and miR-122 (p = 0.03) in plasma. In sperm, both miR-155 (p = 0.004) and miR-122 (p = 0.028) were high in the Ob group. Our results showed that obese subjects have increased expressions of miR-155 and miR-122, two microRNAs that were previously related with inflammation and iron metabolism, respectively, at both the systemic and sperm levels.
Estradiol (E ) is normally metabolized to hydroxyestradiols and methoxyestradiols by CYP1A1, CYP1B1 and COMT. However, an altered production of these metabolites by a disturbed expression of these enzymes is associated with reproductive and non-reproductive pathologies. In vitro studies suggest that increased hydroxyestradiols and methoxyestradiols intratesticular generation is related to male infertility, but no studies have explored whether infertile men have a disturbed testicular expression of the enzymes that generate these E metabolites. The aim of this study was to assess CYP1A1, CYP1B1 and COMT testicular expression at mRNA and protein level in men with spermatogenic impairment. Seventeen men with primary spermatogenic failure (13 with Sertoli cell-only syndrome and four with maturation arrest) and nine controls with normal spermatogenesis were subjected to testicular biopsy. mRNA was quantified using real-time RT-PCR and protein expression was evaluated using western blot and immunohistochemistry followed by integrated optic density analysis. Besides, the effects of hydroxyestradiols and methoxyestradiols on testosterone-induced transcriptional activity were evaluated in TM4 cells using a luciferase reporter assay system. Our results show that patients with Sertoli cell-only syndrome had significantly elevated COMT expression at the mRNA level, higher COMT immunoreactivity in their seminiferous tubules and increased protein expression of the soluble COMT isoform (S-COMT), whereas patients with maturation arrest had significantly elevated CYP1A1 mRNA levels and higher CYP1A1 immunoreactivity in interstitial space. Finally, 2-hydroxyestradiol decreased testosterone-induced transcriptional activity in Sertoli cells in vitro. In conclusion, male infertility is related to disturbed testicular expression of the enzymes responsible for producing hydroxyestradiols and/or methoxyestradiols. If these changes are related with increased intratesticular hydroxyestradiols and methoxyestradiols concentrations, they could elicit an impaired Sertoli cell function. Our results suggest CYP1A1 and COMT as new potential targets in treating male infertility.
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