Alexis Parada-Bustamante

Androgen Receptor Gene CAG and GGN Repeat Polymorphisms in Chilean Men With Primary Severe Spermatogenic Failure

There is ample documentation supporting the fact that androgens are required for normal spermatogenesis. A minority of infertile men have abnormal testosterone blood levels or mild androgen receptor mutations. We investigated the androgen receptor CAG and GGN repeat lengths in Chilean men with spermatogenic impairment. We studied 117 secretory azoospermic/oligozoospermic men (93 idiopathic and 24 excryptorchidic), without Y-chromosome microdeletions, and 121 controls with normal spermatogenesis (42 obstructive and 79 normozoospermic men). Peripheral blood was drawn to obtain genomic DNA for polymerase chain reaction and automated sequencing of CAG and GGN repeats. Testicular characterization included hormonal studies, physical evaluation, and seminal and biopsy analysis. The CAG and GGN polymorphism distributions were similar among idiopathic men, excryptorchidic men, and controls and among the different types of spermatogenic impairment. However, the proportion of the CAG 21 allele was significantly increased in idiopathic cases compared to controls (P = .012 by Bonferroni test, odds ratio = 2.99, 95% confidence interval, 1.27-7.0) and the CAG 32 allele only was observed in excryptorchidic patients (P < .0002, Bonferroni test). Idiopathic cases with Sertoli cell-only syndrome showed the highest proportion of the CAG 21 allele (P = .024, χ(2) test). On the other hand, in idiopathic cases and controls the most common GGN allele was 23, followed by 24, but an inverse relation was found among excryptorchidic cases. The joint distribution of CAG and GGN in control, idiopathic, and excryptorchidic groups did not show an association between the 2 allele repeat polymorphisms (P > 0.05, χ(2) test). Our results suggest that the CAG 21 allele seems to increase the risk of idiopathic Sertoli cell-only syndrome. Moreover, the GGN 24 allele could be contributing to deranged androgen receptor function, associated with cryptorchidism and spermatogenic failure.

Testis development in the absence of SRY: chromosomal rearrangements at SOX9 and SOX3

Duplications in the ~2 Mb desert region upstream of SOX9 at 17q24.3 may result in familial 46,XX disorders of sex development (DSD) without any effects on the XY background. A balanced translocation with its breakpoint falling within the same region has also been described in one XX DSD subject. We analyzed, by conventional and molecular cytogenetics, 19 novel SRY-negative unrelated 46,XX subjects both familial and sporadic, with isolated DSD. One of them had a de novo reciprocal t(11;17) translocation. Two cases carried partially overlapping 17q24.3 duplications ~500 kb upstream of SOX9, both inherited from their normal fathers. Breakpoints cloning showed that both duplications were in tandem, whereas the 17q in the reciprocal translocation was broken at ~800 kb upstream of SOX9, which is not only close to a previously described 46,XX DSD translocation, but also to translocations without any effects on the gonadal development. A further XX male, ascertained because of intellectual disability, carried a de novo cryptic duplication at Xq27.1, involving SOX3. CNVs involving SOX3 or its flanking regions have been reported in four XX DSD subjects. Collectively in our cohort of 19 novel cases of SRY-negative 46,XX DSD, the duplications upstream of SOX9 account for ~10.5% of the cases, and are responsible for the disease phenotype, even when inherited from a normal father. Translocations interrupting this region may also affect the gonadal development, possibly depending on the chromatin context of the recipient chromosome. SOX3 duplications may substitute SRY in some XX subjects.

DAX-1 and DAX-1A expression in human testicular tissues with primary spermatogenic failure

DAX-1 [dosage-sensitive sex reversal-adrenal hypoplasia congenital (AHC) critical region on the X chromosome gene 1; NR0B1] is an orphan nuclear receptor that acts as a transcriptional repressor in adrenal/gonadal development, steroidogenesis and probably spermatogenesis. An alternatively spliced form called DAX-1A (NR0B1A) has been described in several tissues including the testis, and in vitro studies have shown an inhibitory effect on DAX-1 transcriptional function. We aimed to study the mRNA and protein expression of DAX-1 in testicular tissues of 65 men with primary spermatogenic failure [complete Sertoli cell only syndrome (SCOS), focal SCOS, maturation arrest and mixed atrophy] compared with 33 controls with normal spermatogenesis. As a novel finding, we observed intense immunostaining, not only in the nucleus of Sertoli cells, but also in pachytene spermatocytes and round spermatids. The quantitative mRNA expression of DAX-1 and DAX-1A was similar between cases and controls and was not associated with the levels of gonadotrophins and steroids. Moreover, DAX-I transcript expression level was ∼750-fold higher than DAX-1A, and there was a strong positive correlation between them (r = 0.52; P< 0.001). We conclude that, in addition to Sertoli cells, DAX-1/DAX-1A is expressed in germ cells from spermatogonia to round spermatids. Besides, the similar mRNA expression of DAX-I and DAX-IA in testicular tissues from cases and controls does not support the involvement of DAX-1 in the etiology of primary spermatogenic failure. Finally, the low level of expression of the alternative transcriptional variant DAX-1A would not support its putative inhibitory function in vivo.

Androgen receptor CAG and GGN polymorphisms in boys with isolated hypospadias.

Abstract Background: The etiology of hypospadias is multifactorial. Abnormal androgenic secretion and/or action during the development of external genitalia may be involved in the etiology of this congenital malformation. This study explored CAG and GGN polymorphisms in the androgen receptor (AR) gene, which may affect its transcriptional activity, in patients with isolated hypospadias. Methods: The length of the CAG/GGN polymorphisms was determined in 44 boys with non-severe (glandular) or severe (penile or penoscrotal) isolated hypospadias and with a normal hormonal evaluation. In addition, 79 healthy men, as controls, were studied. Results: Mean CAG repeats were significantly higher in total and severe cases compared to controls (24.4±2.8 and 24.7±3.1 vs. 22.7±3.3, respectively; p<0.05, Student’s t and Bonferroni test). In addition, a frequency of CAG alleles >23 was significantly different in total and severe cases compared to controls (70.5% and 74.1% vs. 39.2%, respectively, p<0.05, χ2 and Bonferroni test). The median number and the distribution of GGN polymorphisms were similar in cases and controls. Conclusion: Boys with isolated hypospadias have longer CAG alleles in their AR, which may be related with the development of this congenital malformation.

Analysis of 6 single-nucleotide polymorphisms in the androgen receptor gene in Chilean patients with primary spermatogenic failure.

Androgens are essential for spermatogenesis. It has been postulated that androgen activity is modulated directly or indirectly by genetic variability in the androgen receptor gene sequence, including CAG/GGN polymorphisms and single-nucleotide polymorphisms (SNP). In this study, the frequency of 6 SNPs that constitute a haplotype in the androgen receptor sequence was determined by enzyme restriction assays and allele-specific polymerase chain reactions in 117 secretory azo/oligozoospermic men (93 idiopathic and 24 excryptorchidic), and in 121 controls with normal spermatogenesis (42 obstructive and 79 normozoospermic men) whose hormonal measurements and length of CAG/GGN polymorphisms were previously determined. The frequency of these 6 SNPs was not different between patients and controls. A total of 10 haplotypes (HAPs 1-10) formed by these 6 SNPs were found, and one of these haplotypes was observed with high frequency in the total population (HAP1, 83.2%; P < .001, χ(2) test). The frequency of the 10 haplotypes was not different between patients and controls, except for HAP5, which was only detected in one patient with a history of bilateral cryptorchidism (P = 0.014, Bonferroni test). On the other hand, no associations were found between the haplotypes studied and shorter or longer CAG or GGN polymorphisms. Interestingly, we found that the CAG 21 allele, which was previously correlated with an increased risk of idiopathic spermatogenic impairment, was more frequently found among the less common haplotypes that have higher follicle-stimulating hormone serum levels. In summary, we did not find an increased frequency of particular haplotypes in infertile men with idiopathic spermatogenic impairment compared with control men; however, we found that the CAG 21 allele, which appears to be associated with male infertility, is observed at a significantly higher proportion among the less common androgen receptor haplotypes.

Role of 2-methoxyestradiol, an Endogenous Estrogen Metabolite, in Health and Disease

Estradiol (E2) is a steroid hormone whose physiological actions are mainly mediated by its interaction with intracellular estrogen receptors (ER) leading to modification on the mRNA and protein synthesis in its target cells. However, estrogens can also activate several intracellular signal transduction cascades by non-genomic mechanisms. Estrogens must be inactivated and removed from blood through its conversion to soluble compounds with an apparent low estrogenic activity and decreased affinity for ER. In this context, 2-methoxyestradiol (2ME2) is generated by a sequential hydroxylation of E2 via the enzyme cytochrome P450 isoform 1A1 to produce 2-hydroxyestradiol (2OHE2) followed by a conjugation reaction catalyzed by the enzyme Catechol-O-Methyltransferase generating 2ME2 from 2OHE2. Recent evidence indicates that physiological concentration of 2ME2 may regulate several biological processes while high concentrations of this metabolite may induce pathophysiological alterations in several tissues. In the last years, 2ME2 has also been described as a promising anticancer drug although its cellular and molecular mechanisms are still being disclosed. Herein, we will review the available literature concerning the role of 2ME2 in health and disease. We will focus on to describing the intracellular mechanisms by which 2ME2 exerts its effects on reproductive and non-reproductive tissues. The promising anticancer effects of 2ME2 and its synthetic derivatives will also be discussed. Finally, a group of 2ME2-target genes that could be used as biomarkers of 2ME2 under physiological or pathophysiological conditions will be reviewed.

46,XX/SRY-negative true hermaphrodite

OBJECTIVE To describe genetic evaluation and response to surgery and letrozole therapy of a 46,XX/SRY-negative true hermaphrodite. DESIGN Case report. SETTING University Medical Center. PATIENT(S) Nineteen-year-old male with penile hypospadias, micropenis, and crytorchidism at the time of birth. INTERVENTION(S) Unilateral gonadectomy, and contralateral conservative gonadal surgery, followed by therapy with letrozole. MAIN OUTCOME MEASURE(S) Histopathologic, genetic and hormonal studies. RESULT(S) Genetic analysis showed that the subject was 46,XX/SRY-negative. Gonadectomy of the left gonad was performed at 16 years. The gonad resected was an ovotestes. The patients estradiol was high (492±25 pmol/L), whereas the testosterone was low (4.2±0.5 nmol/L). Nineteen months later, conservative gonadal surgery of the contralateral gonad was performed to resect ovarian tissue, and treatment with letrozole was started. During letrozole treatment, testosterone was significantly increased (8±0.7 nmol/L), but estradiol was not changed (323±118 pmol/L). After letrozole withdrawal, testosterone did not decreased significantly (6.9±0.4 nmol/L), estradiol showed an oscillating pattern and a gonadal ultrasound showed an ovoid structure, which appeared to correspond to a follicle. At that time, estradiol was elevated (393 pmol/L). CONCLUSION(S) We present the case of a 46,XX/SRY-negative phenotypic male with bilateral ovotestes. Conservative gonadal surgery should be performed only when all ovarian tissue can be resected. Our results suggest that letrozole is not an adequate treatment for 46,XX true hermaphrodite males with ovotestes.

Overexpression of CYP19A1 aromatase in Leydig cells is associated with steroidogenic dysfunction in subjects with Sertoli cell-only syndrome

Several observational studies have showed a combination of lower testosterone (T) to LH ratio and higher estradiol (E2) 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® Green‐based qPCR. In addition, testicular T and E2 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®). Moreover, Rq data for CYP19A1 had a direct correlation with testicular levels of E2 and the E2/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 E2. Our results suggest that increased expression of aromatase in Lc leads to higher E2 production and may account for the functional impairment of the Lc in patients with SCOS.

Disturbed testicular expression of the estrogen‐metabolizing enzymes CYP1A1 and COMT in infertile men with primary spermatogenic failure: possible negative implications on Sertoli cells

Estradiol (E2) 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 E2 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.

Mating decreases plasma levels of TGFβ1 and regulates myosalpinx expression of TGFβ1/TGFBR3 in the rat.

Mating shuts down a 2‐methoxyestradiol (2ME)‐dependent, non‐genomic activity that is responsible for accelerating egg transport in the rat oviduct. The aims of this work were to investigate the role of TGFβ1 in this 2ME‐reduced activity and to determine the effect of mating on the expression and distribution of TGFβ1 and its receptor TGFBR3 in the rat oviduct. We determined the level of TGFβ1 in the plasma and oviductal fluid at 1, 3, or 6 hr during Day 1 of the oestrous cycle in unmated or mated animals. We then examined if 2ME accelerates oviductal egg transport in unmated rats that were previously treated with a neutralizing TGFβ1 antibody. The expression of Tgfb1 and Tgfbr3 mRNA and the level and distribution of TGFBR3 protein in the oviduct were also determined at these time points. Mating decreased TGFβ1 in the plasma, but not in the oviductal fluid, whereas antibody neutralization of circulating TGFβ1 did not prevent the effect of 2ME on egg transport. Mating decreased Tgfb1 and hastened the increase in TGFBR3 abundance in the myosalpinx. These results indicate that mating decreased circulating levels of TGFβ1 without shutting down the non‐genomic 2ME response that normally accelerates egg transport. Levels of Tgfb1 transcript and TGFBR3 protein, however, changed in the myosalpinx of mated rats, suggesting a role for mating‐associated factors in the autocrine and paracrine effects of TGFβ in the oviduct. Mol. Reprod. Dev. 81: 1053–1061, 2014.