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Bone Mineral Density and Testicular Failure: Evidence for a Role of Vitamin D 25-Hydroxylase in Human Testis - pdf attached
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published online on January 26, 2011
The Journal of Clinical Endocrinology & Metabolism
Carlo Foresta, Giacomo Strapazzon, Luca De Toni, Lisa Perilli, Antonella Di Mambro, Barbara Muciaccia, Leonardo Sartori and Riccardo Selice
Department of Histology (C.F., G.S., L.D.T., L.P., A.D.M., R.S.), Microbiology, and Medical Biotechnologies, Section of Clinical Pathology and Centre for Male Gamete Cryopreservation, University of Padova, 35128 Padova, Italy; Department of Histology (B.M.) and Medical Embryology, University of Rome "La Sapienza," 00161 Rome, Italy; Clinica Medica I (L.S.), Department of Medical and Surgical Sciences, University of Padova, 35128 Padova, Italy; and EURAC Institute of Mountain Emergency Medicine (G.S.), 39100 Bozen/Bolzano, Italy Address all correspondence and requests for reprints to: Prof. Carlo Foresta, Department of Histology, Microbiology, and Medical Biotechnologies, Section of Clinical Pathology and Centre for Male Gamete Cryopreservation, University of Padova, Via Gabelli 63, 35121 Padova, Italy. E-mail:
Working Hypothesis: Mutations in the CYP2R1 gene, highly expressed in the testis and encoding vitamin D 25-hydroxylase, result in a vitamin D deficiency and a defective calcium homeostasis leading to rickets.
Objective: Our aim was to investigate CYP2R1 expression in pathological testis samples and relate this to vitamin D metabolism in testiculopathic patients.
Design, Patients, Setting: Testis samples for in vitro study and 98 young men were transversally evaluated at Padova's Center for Male Gamete Cryopreservation.
Methods: CYP2R1 mRNA expression and protein production were evaluated by quantitative RT-PCR, Western blot analysis, and immunofluorescence. Hormonal and bone-marker levels, and bone densitometry by dual-energy x-ray absorptiometry, were determined in patients with Sertoli-cell-only syndrome and severe hypospermatogenesis.
Results: We found a lower gene and protein expression of CYP2R1 in samples with hypospermatogenesis and Sertoli-cell-only syndrome (P < 0.05) and a colocalization with INSL-3, a Leydig cell marker, at immunofluorescence. In all testiculopathic patients 25-hydroxyvitamin D levels were significantly lower and PTH levels higher compared to controls (P < 0.05). Furthermore, testiculopathic patients showed osteopenia and osteoporosis despite normal testosterone levels compared with controls both with increased bone-marker levels and altered dual-energy x-ray absorptiometry in the femoral neck and lumbar spine (for all parameters, P < 0.05).
Conclusions: Our data show an association between testiculopathy and alteration of the bone status, despite unvaried androgen and estrogen levels and no other evident cause of vitamin D reduction. Further studies in larger cohorts are needed to confirm our results.
The balance between bone remodeling and bone resorp- tion is essential for normal skeletal development and maintenance during adult life (1, 2). Vitamin D is a key reg- ulatory factor of bone mineralization and of calcium ho- meostasis in both men and women, being activated in a two- step tightly regulated process at positions 25 and 1 (3).
Even if 25-hydroxyvitamin D concentration is regarded as the most reliable indicator of vitamin D status, the iden- tity of 25-hydroxylase and its regulation is still poorly understood. Both mitochondrial and microsomal subcel- lular fractions have 25-hydroxylase activity (4-7). The mitochondrial enzyme CYP27A1 (8) is a low-affinity high-capacity enzyme, whose deficiency or loss of enzymatic activity has an important effect on cholesterol levels but poor influence on vitamin D metabolism (9, 10). Interestingly, a known inactivating mutation in the micro- somal form, CYP2R1 (4, 10), results in a deficiency of vitamin D, defective calcium homeostasis, and classical bone lesions referred to as rickets (4, 11-14). In human adult and fetal tissues CYP2R1 mRNA is ubiquitous, but its expression reaches the highest levels in the testis (3, 15-17).
Steroid sex-hormone status reflects bone mass, such that hormonal deficiency leads to progressive bone loss (2, 18) and to a sexually-dimorphic pattern. In fact, men are relatively protected from the development of osteoporosis by a higher peak bone mass compared with women, whereas bone-loss rates increase in women after the menopause (19). Nevertheless, a loss of bone mass already starts in men during early adult life, increasing with age (20), but the pathophysiological mechanisms linking male osteo- porosis and bone pathophysiology are not completely understood.
In this study we investigated the expression of CYP2R1 mRNA and protein expression in normal and pathological male human tissues. Furthermore, we aimed to determine vitamin D status and bone mass in a cohort of normotes- tosteronemic young men with severe hypospermatogenesis or idiopathic Sertoli-cell-only syndrome (SCOS).
Thermal cycling included initial steps at 95 C for 10 min, followed by 40 cycles at 95 C for 15 s and at 60 C for 1 min. The fluorescence intensity of the double-stranded products was monitored in real time. cDNA was amplified and quantified using the iQ5 real-time PCR detection system (Bio-Rad, Milan, Italy). Commercial liver cDNA library (Clontech, Mountain View, CA) was used as positive control for gene expression. Data were normalized to beta-actin as internal housekeeping gene (4326315E, Applied Biosystems). Data elaboration was performed as relative quantification analysis using the delta-Ct method. Results are reported as the mean value of three independent experiments in triplicate.
Hypogonadism represents one of the most important causes of male osteoporosis. Testosterone regulates male bone metabolism both indirectly by aromatization to estrogens and directly through the AR on osteoblasts, promoting periosteal bone formation during puberty and reducing bone resorption during adult life (18). Furthermore, recent studies suggest a possible relationship between vitamin D metabolism and testis function (17).
It has been demonstrated that the male reproductive tract expresses most of the enzymes involved in vitamin D activation through 25-hydroxylation (17). Inactivating mutations of CYP27A1 gene, previously assumed to be the main vitamin D-activating enzyme, are actually associated with impairment of cholesterol metabolism (10). On the other hand, an inactivating mutation of the human CYP2R1 gene has been shown to give rise to a bone phenotype (4, 10). Furthermore, a recent genome-wide association study, involving 33,996 individuals of European descent from 15 cohorts, evidenced that common variants at the CYP2R1 locus are associated with alterations in circulating 25(OH)D concentrations (27) and our group demonstrated that bilateral orchiectomy results in 33% of reduction in 25(OH)D levels despite adequate testoster- one-replacement therapy (28). Thus, our data are consistent with a role of CYP2R1 and testis in vitamin D activation, showing a progressive decrease of CYP2R1 expression in relationship to the severity of testiculopathy and suggesting that the Leydig cell could play a major role in this process.
It could be argued that high expression levels of CYP2R1 in the testis in vitro do not reflect the 25(OH)D reduction observed in vivo in our orchiectomized (28) and testiculopathic patients. Vicarious effects of the remaining pool of both CYP2R1 and of the other 25-hydroxylating enzymes, expressed in other tissues (3, 15-17), could explain this apparent discrepancy. Furthermore, the direct involvement of the Leydig cell is suggested by the expression of CYP2R1 as we highlighted in the immunofluores- cence assay (26).
Our patients displayed significantly reduced levels of 25(OH)D compared with controls. In particular, 43.8% of testiculopathic patients showed levels of 25(OH)D lower that 40 nmol/liter (25), despite the absence of nutritional derangements. Moreover, patients showed significant secondary increases in PTH levels compared to control values. A previous study demonstrated a central role of 25(OH)D in regulating PTH secretion (29), whose implication in the pathogenesis of osteoporosis is well established. Our results confirm their negative correlation and the pathogenetic link between 25(OH)D, PTH and BMD in altered bone metabolism.
Our patients affected by idiopathic SCOS and severe hypospermatogenesis were characterized by absence or reduced germ cells in the testis, normal testosterone, and increased LH and FSH levels. The altered LH levels depict a pattern of long-lasting sufferance of Leydig cells, par- tially compensated by a hyperactivation of the hypothalamic-gonadotropic-gonadal axis (30) that holds testosterone production at normal levels. Despite bone turnover is strictly affected by sex hormones, in our testiculopathic patients total testosterone and estrogens levels were in the physiological range. Moreover, we could not find AR mu- tations or altered CAG repeat polymorphism lengths (data not shown). However, Karin at al. reported how FSH and LH were better predictors of BMD than testosterone in osteoporotic men (31) and, accordingly, we found a significant and inverse correlation between FSH and femur BMD values in the cohort of testiculopathic patients. Another main actor in bone metabolism is 25(OH)D, which was positively correlated with both femur and lumbar BMD values in our population. Common risk factors for vitamin D deficiency include inadequate sun exposure, limited oral intake, malabsorption syndrome, kidney and proliferative diseases, and drugs (32). These conditions were excluded in our population, underlying a possible involvement of testiculopathy in bone metabolism.
Our study suffers limitations because a CYP2R1 knock-out model is still lacking (33), however a clear pattern of low-circulating levels of 25(OH)D was described in a human model of CYP2R1-inactivating mutation (4). Furthermore, we did not have direct evidence of the pro- duction of 25(OH)D by Leydig cells. Such investigation is difficult because of the lack of a well-established experi- mental model of human mature Leydig cell culture (34). Finally, an analysis on a larger cohort could better disclose the involvement of gonadotropins and weight load on bone density modulation.
This is the first study, to the best of our knowledge, showing an association between testiculopathy, altered 25(OH)D levels, and alteration of the bone status, despite unvaried androgen and estrogen levels and no other evident cause of vitamin D reduction. Even if testicular-cell expression of CYP2R1 suggests a possible influence on 25(OH)D levels, further studies on larger cohorts are needed to confirm our results.
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