Male Sexual Differentiation Research Articles

MEF2 Is Restricted to the Male Gonad and Regulates Expression of the Orphan Nuclear Receptor NR4A1

Leydig cell steroidogenesis is controlled by the pituitary gonadotropin LH that activates several signaling pathways, including the Ca(2+)/calmodulin kinase I (CAMKI) pathway. In other tissues, CAMKI regulates the activity of the myocyte enhancer factor 2 (MEF2) transcription factors. MEF2 factors are essential regulators of cell differentiation and organogenesis in numerous tissues but their expression and role in the mammalian gonad had not been explored. Here we show that MEF2 factors are expressed in a sexually dimorphic pattern in the mouse gonad. MEF2 factors are present in the testis throughout development and into adulthood but absent from the ovary. In the testis, MEF2 was localized mainly in the nucleus of both somatic lineages, the supporting Sertoli cells and the steroidogenic Leydig cells. In Leydig cells, MEF2 was found to activate the expression of Nr4a1, a nuclear receptor important for hormone-induced steroidogenesis. In these cells MEF2 also cooperates with forskolin and CAMKI to enhance Nr4a1 promoter activity via two MEF2 elements (-318 and -284 bp). EMSA confirmed direct binding of MEF2 to these elements whereas chromatin immunoprecipitation revealed that MEF2 recruitment to the proximal Nr4a1 promoter was increased following hormonal stimulation. Modulation of endogenous MEF2 protein level (small interfering RNA-mediated knockdown) or MEF2 activity (MEF2-Engrailed active dominant negative) led to a significant decrease in Nr4a1 mRNA levels in Leydig cells. All together, our results identify MEF2 as a novel testis-specific transcription factor, supporting a role for this factor in male sex differentiation and function. MEF2 was also positioned upstream of NR4A1 in a regulatory cascade controlling Leydig cell gene expression.

46, XX Sry(+) Male Sexual Differentiation Disorder with Metabolic Syndrome: A Case Report

46, XX Sry(+) Male Sexual Differentiation Disorder with Metabolic Syndrome: A Case Report

Normal phenotype in conditional androgen receptor (AR) exon 3-floxed neomycin-negative male mice

Androgens (testosterone and dihydrotestosterone) acting via the androgen receptor (AR) are required for male sexual differentiation, and also regulate the development of many other tissues including muscle, fat and bone. We previously generated an ARlox mouse line with exon 3 of the AR gene targeted by loxP sites. The deletion of exon 3 is in-frame, so only the DNA binding-dependent actions of the AR are deleted, but non-DNA binding-dependent actions are retained. This line also contained an antibiotic resistance selection cassette, neomycin (neo) in intron 3, which was also flanked by loxP sites. Hemizygous ARlox male mice demonstrated a phenotype of hyperandrogenization, with increased mass of androgen-dependent tissues. We hypothesized that this hyperandrogenization was likely to be due to the presence of the neo cassette. In this study, we have generated an ARlox neo-negative mouse line, using the EIIa-cre deleter mouse line to remove the neo cassette. Hemizygous ARlox neo-negative male mice have a normal phenotype, with normal body mass and normal mass of androgen-dependent tissues including the testis, seminal vesicles, kidney, spleen, heart and retroperitoneal fat. This neo-negative exon 3-targeted mouse line is the only floxed AR mouse line available to study the DNA binding-dependent actions of the AR in a tissue-specific manner, and is suitable for investigation in all tissues. This study demonstrates the importance of removing the selection cassette, which can potentially alter the phenotype of floxed mouse lines even in the absence of detectable effects on target gene expression.

Mind the gap: can we explain declining male reproductive health with known antiandrogens?

Several countries have experienced rises in cryptorchidisms, hypospadias and testicular germ cell cancer. The reasons for these trends are largely unknown, but Skakkebaek has proposed that these disorders form a testicular dysgenesis syndrome and can be traced to androgen insufficiency in foetal life. This suggests that antiandrogenic chemicals might contribute to risks, but few chemicals have been linked to these diseases in epidemiological studies. In animal studies with p,p ′-dichlorodiphenyldichloroethylene, effects typical of disruptions of male sexual differentiation became apparent when the foetal levels of this androgen receptor (AR) antagonist approached values associated with responses in in vitro assays. This prompted us to analyse whether the 22 chemicals with AR antagonistic properties would produce mixture effects in an in vitro AR antagonism assay when combined at concentrations found in human serum. Other antiandrogenic modalities could not be considered. Two scenarios were investigated, one representative of average serum levels reported in European countries, the other in line with levels towards the high exposures. In both situations, the in vitro potency of the 22 selected AR antagonists was too low to produce combined AR antagonistic effects at the concentrations found in human serum, although the high exposure scenario came quite close to measurable effects. Nevertheless, our analysis exposes an explanation gap which can only be bridged by conjuring up as yet undiscovered high potency AR antagonists or, alternatively, high exposures to unknown agents of average potency.

Androgen action.

Androgens are important for male sex development and physiology. Their actions are mediated by the androgen receptor (AR), a ligand-dependent nuclear transcription factor. The activity of the AR is controlled at multiple stages due to ligand binding and induced structural changes assisted by the foldosome, compartmentalization, recruitment of coregulators, posttranslational modifications and chromatin remodeling, leading to subsequent transcription of androgen-responsive target genes. Beside these short-term androgen actions, there is phenomenological and experimental evidence of long-term androgen programming in mammals and in the human during sensitive programming time windows, both pre- and postnatally. At the molecular level, research into androgen insensitivity syndrome has unmasked androgen programming at the transcriptome level, in genital fibroblasts and peripheral blood mononuclear cells, and at the epigenome level. Androgens are crucial for male sex development and physiology during embryogenesis, at puberty and in adult life. Testosterone and its more potent metabolite, dihydrotestosterone, which is converted from testosterone within the target cell by 5α-reductase II, are the main androgens involved in male sex differentiation. Androgen action is mediated by a single AR. The AR belongs to the nuclear receptor 3 group C, composed of the glucocorticoid receptor (NR3C1), mineralocorticoid receptor (NR3C2), progesterone receptor (NR3C3) and AR (NR3C4), and acts as a ligand-dependent transcription factor.

Differential expression of the MHM region and of sex-determining-related genes during gonadal development in chicken embryos

The chicken (Gallus gallus) embryo has been used as a classic model system for developmental studies because of its easy accessibility for surgical manipulation during embryonic development. Sex determination in birds is chromosomally based (ZZ for males and ZW for females); however, the basic mechanism of sex determination is still unknown. Here, the dynamics of expression of candidate genes implicated in vertebrate sex determination and differentiation were studied during embryonic chicken gonadal development. Gene expression profiles were obtained before, during, and after gonadal sex differentiation in females and males for DMRT1, SOX3, SOX9, DAX1, SCII, HINTZ, HINTW, and the male hypermethylated (MHM) region. Transcripts for the HINTZ, DMRT1, DAX1, SCII, and SOX9 genes were observed in both sexes, but expression was higher in male gonads and may be correlated with testicular differentiation. The expression patterns of HINTW, SOX3, and MHM suggest that they may act in ovary development and may be involved in meiosis entry. MHM was upregulated and DMRT1 was downregulated in females at the same developmental stage. This may indicate a regulation of DMRT1 by MHM ncRNA. Similar dynamics were observed between HINTW and HINTZ. This study reports on the MHM expression profile during gonadal development and its correlation with the expression of genes involved in vertebrate sex determination.

Similar causes of various reproductive disorders in early life

During the past few decades, scientific evidence has been accumulated concerning the possible adverse effects of the exposure to environmental chemicals on the well-being of wildlife and human populations. One large and growing group of such compounds of anthropogenic or natural origin is referred to as endocrine-disrupting chemicals (EDCs), due to their deleterious action on the endocrine system. This concern was first focused on the control of reproductive function particularly in males, but has later been expanded to include all possible endocrine functions. The present review describes the underlying physiology behind the cascade of developmental events that occur during sexual differentiation of males and the specific role of androgen in the masculinization process and proper organogenesis of the external male genitalia. The impact of the genetic background, environmental exposures and lifestyle factors in the etiology of hypospadias, cryptorchidism and testicular cancer are reviewed and the possible role of EDCs in the development of these reproductive disorders is discussed critically. Finally, the possible direct and programming effects of exposures in utero to widely use therapeutic compounds, environmental estrogens and other chemicals on the incidence of reproductive abnormalities and poor semen quality in humans are also highlighted.

Endocrine disrupter effects on the gonads and pubertY

Evidence accumulating the past decades indicates that human populations are exposed to environmental chemicals interfering with endocrine systems. There is growing concern of the potential adverse impact of exposure to such endocrine disrupting chemicals (EDCs) on human health, based on observations in wildlife, animal model systems and reports of yet unexplained increased incidences of hormone related disorders in humans. Exposure in fetal and neonatal age may be associated with developmental and reproductive disturbances due to interference with the programming of normal hormone signaling and metabolic pathways. Boys are conceived to be more vulnerable than girls due to their more complicated and androgen driven prenatal sex differentiation. Male prenatal sex differentiation is crucially dependent on the functioning of fetal Leydig cells. Exposure of these cells to EDCs at critical time windows of development may result in undermasculinization and disorder (s) of sex development (DSD), such as hypospadias, cryptorchidism and ambiguous genitalia. Furthermore, a novel trend of earlier start of puberty has been found during the past few decades, at least in some regions and mostly affecting girls. Among plausible causes behind this phenomenon it has been suggested that exposure to EDCs affecting the programming or triggering of the pubertal clock could result in premature activation of gonadotropin secretion. Another hypothesis is that gonadotropin independent effects of EDCs are associated with an earlier start of puberty. Proof of principle of such potential roles of EDCs has been obtained from a multitude of studies in experimental animals and gained some support by case studies, exposure data and epidemiological investigations in humans. This presentation will review recent data and ongoing studies on the mechanism(s) of action of EDCs, including effects of mixtures, on the hypothalamuspituitary-gonadal axis in experimental animals and humans. The impact of genetic susceptibility on the degree of disrupting activity caused by certain EDCs will be discussed on the basis of ongoing animal studies.

Müllerian Inhibiting Substance in the Caudate Amphibian Pleurodeles waltl

Müllerian inhibiting substance (MIS, also known as anti-Müllerian hormone), is a key factor of male sex differentiation in vertebrates. In amniotes, it is responsible for Müllerian duct regression in male embryos. In fish, despite the absence of Müllerian ducts, MIS is produced and controls germ cell proliferation during gonad differentiation. Here we show for the first time the presence of MIS in an amphibian species, Pleurodeles waltl. This is very astonishing because in caudate amphibians, Müllerian ducts do not regress in males. Phylogenetic analysis of MIS P. waltl ortholog revealed that the deduced protein segregates with MIS from other vertebrates and is clearly separated from other TGF-β family members. In larvae, MIS mRNA was expressed at higher levels in the developing testes than in the ovaries. In the testis, MIS mRNA expression was located within the lobules that contain Sertoli cells. Besides, expression of MIS was modified in the case of sex reversal: it increased after masculinizing heat treatment and decreased after estradiol feminizing exposure. In addition to the data obtained recently in the fish medaka, our results suggest that the role of MIS on Müllerian ducts occurred secondarily during the course of evolution.

Maternal treatment with picrotoxin in late pregnancy improved female sexual behavior but did not alter male sexual behavior of offspring

Previous studies from our laboratory investigated the effects of picrotoxin (PT), a γ-aminobutyric acid receptor antagonist administered during several perinatal periods, on the sexual behavior of male and female rats. We observed that the time of perinatal exposure to PT is critical to determine either facilitation or impairment of sexual behavior. The present study evaluated the effects of prenatal administration of a single dose of PT on gestation day 18 of dams (the first critical period of male brain sexual differentiation) on sexual behavior of male and female offspring. Thus, female Wistar rats were mated with males and, on gestation day 18, received 0.6 mg/kg of PT or 0.9% saline solution subcutaneously. On postnatal day 1, the offspring were weighed and several measures of sexual development were assessed. The sexual behaviors and the general activity in the open field of adult male and ovariectomized, hormone-treated female rats were observed. On comparison with the control group, maternal PT treatment: (i) did not alter the maternal weight, pup weight, anogenital distance, or male and female general activity; (ii) increased female sexual behavior, that is, decreased the latencies to first mount, first lordosis, and tenth lordosis, and the percentage of females presenting lordosis; and (iii) did not alter male sexual behavior. It is suggested that prenatal PT exposure interfered with epigenetic mechanisms related to the development of sex differences in the brain, leading to the observed sexually dimorphic effects on sexual behavior.

Serum anti-Müllerian hormone (AMH) levels correlate with infrarenal aortic diameter in healthy older men: is AMH a cardiovascular hormone?

Anti-Müllerian hormone (AMH) is a gonadal hormone present in the blood in men and pre-menopausal women. AMH regulates male sexual differentiation but has no putative function in adulthood. In recent studies, high AMH levels are associated with absence of cardiovascular disease in men and smaller atherosclerotic burden in monkeys. Mechanistically, AMH has downstream convergence with known regulators of the cardiovascular system, while the specific receptor for AMH is present in murine aorta and the human heart. Our primary objective was to examine whether AMH levels in healthy men correlated with the physical characteristics of their aorta. Our secondary aim was to document whether men with distinct vascular disorders expressed different levels of AMH. Serum AMH assayed by ELISA in 153 men (54-93 years) free from vascular disease inversely correlated with the ultrasonographic diameters of the distal- (r=-0.22, P=0.006) and mid-infrarenal aorta (r=-0.26, P=0.008). This association was similar in magnitude but opposite to that of body surface area (largest known determinant of aortic diameter) and independent of known cardiovascular risk factors. This relationship is specific to AMH, as inhibin B, a Sertoli cell hormone-like AMH, did not correlate with aortic diameter (r=-0.04, P=0.66) despite partially correlating with AMH. Among men with known vascular disease, higher AMH levels were associated with varicose vein disease, while men with higher levels of AMH were under-represented in the abdominal aortic aneurysm relative to the healthy cohort. These findings identify AMH as a novel putative regulator of the cardiovascular system.

Dose‐dependent alterations in gene expression and testosterone production in fetal rat testis after exposure to di‐n‐hexyl phthalate

In utero exposure to the phthalate ester plasticizer di-n-hexyl phthalate (DnHP) is known to affect the development of the male reproductive system and induce alterations in androgen-dependent tissues of male rat offspring. Male reproductive malformations produced by several phthalates have been causally linked to decreased testosterone production during the gestational period. This study was designed to evaluate the dose-response relationship for the effects of DnHP on the synthesis and production of testosterone in the fetal rat testis. Pregnant Sprague-Dawley rats were administered the vehicle (olive oil) and either DnHP (5 to 625 mg kg(-1) per day) or diethylhexyl phthalate (DEHP) (50 or 625 mg kg(-1) per day), by gavage, from gestation day (GD) 12 to19. Fetal testes were assessed on GD 19. DnHP reduced ex vivo testosterone production and down-regulated the expression of several genes required for cholesterol transport and steroid synthesis (i.e. SR-B1, StAR, P450scc, 3βHSD and P450c17). These inhibitions were dose dependent. A no-effect level was established at 5 mg kg(-1) per day and a lowest-effect level at 20 mg kg(-1) per day. mRNA levels of SR-B1, StAR, P450scc and 3βHSD were not similarly decreased in the adrenals. In conclusion, DnHP shares the same mode of action as DEHP in disrupting fetal testicular androgen synthesis. Alterations in testosterone production and in key steroidogenic gene expressions were apparent at lower doses than those causing postnatal reproductive malformations after gestational exposure during the critical period of male sexual differentiation. This suggests that they can be considered early biomarkers of DnHP-induced fetal testicular effects in rats.

MiR-124 controls male reproductive success in Drosophila

Many aspects of social behavior are controlled by sex-specific pheromones. Gender-appropriate production of the sexually dimorphic transcription factors doublesex and fruitless controls sexual differentiation and sexual behavior. miR-124 mutant males exhibited increased male-male courtship and reduced reproductive success with females. Females showed a strong preference for wild-type males over miR-124 mutant males when given a choice of mates. These effects were traced to aberrant pheromone production. We identified the sex-specific splicing factor transformer as a functionally significant target of miR-124 in this context, suggesting a role for miR-124 in the control of male sexual differentiation and behavior, by limiting inappropriate expression of the female form of transformer. miR-124 is required to ensure fidelity of gender-appropriate pheromone production in males. Use of a microRNA provides a secondary means of controlling the cascade of sex-specific splicing events that controls sexual differentiation in Drosophila. DOI:http://dx.doi.org/10.7554/eLife.00640.001.

Aberrant transcription of the LHCGR gene caused by a mutation in exon 6A leads to Leydig cell hypoplasia type II

Introduction: Male fetal sex differentiation is dependent on the androgen production of fetal testicular Leydig cells, which is induced by placental human chorionic gonadotropin. Later during gestation it is maintained by luteinizing hormone, secreted by the fetal pituitary gland. As the action of both glycoprotein hormones is mediated by the luteinizing hormone/chorionic gonadotropin receptor (LHCGR), this receptor is essential for male sex differentiation as well as for normal reproductive function in the adult.

Der alternative Androgensyntheseweg des Menschen

During the last year, alternative androgen synthesis pathways have been discovered in humans. This review article highlights these new concepts of androgen synthesis.We performed a selective literature research using PubMed.After the discovery of a new androgen synthesis pathway in marsupials, this new path-way of androgen synthesis could be established in humans during the last year from two independent studies. One of them could demonstrate that two pathways of androgen synthesis are needed for male sexual differentiation in humans; the other study established that the new pathway is an important source of androgen synthesis in congenital adrenal hyperplasia due to 21-hydroxylase deficiency. Additionally, it has been shown that an alternative androgen synthesis pathway that bypasses testosterone drives castration resistant prostate cancer.New and alternative androgen path-ways occur in humans. Importantly, these path-ways remain cryptic for the clinician, because the androgen synthesis circumvents classical intermediates like dehydroepiandrosterone, androstenedione and testosterone.

Prevalence of Undescended Testis and its Associated Factors among under-fives seen at Reproductive and Child Health Clinic in Ifakara, Tanzania

Background : The diagnosis of undescended testis/cryptorchidism is missed and ignored by most clinicians following tendency of not performing genital examination in children unless asked by parents. The male sexual differentiation and development is important for the normal reproductive life span. Similarly, risk of carcinoma of testis will be prevented, if early diagnosis of undescended testis (UDT) is made. To aim of the study was to determine the prevalence of undescended testis, its characteristics and associated risk factors among children underfive. Methods : This was a hospital-based cross sectional study that was conducted at the Reproductive and Child Health Clinic (RCHC) of the St. Francis Referral Hospital (SFRH) from October 2011 to May 2012. The district hospital is located in Ifakara, southeast Tanzania, a city of 49,528 people. Among the estimated 4500 under-five male children in the district attending the RCHC for routine services, 616 children were physically examined their genitalia after consent from their caretakers. Results : The prevalence of the undescended testis (UDT) was 2.1% (13/616) with 12 children having unilateral UDT and one child with bilateral UDT. About 85% of families of recruited children own durable assets. A small proportion of children, 6.8% (49/616) were of low birth weight and 2.6% (16/616) were born prematurely. Among mothers who had preeclampsia, only 2.4% (1/41) of the children had UDT and none of eclamptic mothers‟ children had UDT. None of the children with UDT had exhibited evidence of birth asphyxia. In case of mothers who had been smoking or exposed to second hand smoking 5.4% (33/616) and those who have been binge drinking alcohol, 0.6% (4/616) of their children had UDT. Among mothers exposed to herbs during pregnancy only 3.2% (1/31) had UDT. None of the mothers had gestational diabetes or existing diabetes mellitus prior to conception. Conclusion : The prevalence of UDT in this rural setting has a pattern similar to that observed in previous studies in other areas. Efforts should be done to do genital examination by all clinicians. The associated factors exist but no statistically significant association was found and a long term follow up study is needed. Key words : Cryptorchidism/undescended testis, underfives, Reproductive Child Health Clinic, small for gestation age.

Prevalence and Clinical Significance of Acellular Mucin in Locally Advanced Rectal Cancer Patients Showing Pathologic Complete Response to Preoperative Chemoradiotherapy

Occasionally, patients with locally advanced rectal adenocarcinoma who receive preoperative chemoradiotherapy (CRT) show acellular mucin in resection specimens that had shown pathologic complete response (pCR), but the clinical and prognostic significance of this finding has been controversial. This study analyzed data from 217 consecutive patients showing pCR to preoperative CRT followed by resection to evaluate the clinicopathologic features and prognostic significance of acellular mucin. Patients were categorized according to the presence of acellular mucin, as identified by pathologic analysis. The clinicopathologic findings and oncologic results were compared. Acellular mucins were identified in 35 (16.1%) of 217 pCR patients. Acellular mucins were found predominantly in male patients (20.8% vs. 9.8%, P=0.039) and in those with mucinous/signet ring cell differentiation (66.7% vs. 15.1%, P=0.008). The presence of acellular mucin was more frequent in patients with a shorter (<42 d) CRT-operation interval (22.6% vs. 10.3%, P=0.017). With a mean follow-up of 41 months (range, 2 to 119 mo), the 3-year overall survival (96.8% with mucin vs. 95.9% without mucin, P=0.314) and the 3-year disease-free survival (97.0% with mucin vs. 93.0% without mucin, P=0.131) did not differ between the groups. The presence of acellular mucin in rectal cancer patients showing pCR to preoperative CRT is associated with male sex and mucinous differentiation and does not have a significant impact on oncologic outcomes. Acellular mucins are also associated with the CRT-operation interval as a phenomenon of time-dependent response to CRT.

Klinefelter’s Syndrome –Unusual Associations

Klinefelter’s Syndrome –Unusual Associations Klinefelter’s syndrome (KFS) is the most common male sex chromosomal disorder. KFS patients are usually tall and have complete male sexual differentiation. In this report, we present two interesting cases of KFS. Our first patient had genital ambiguity and second child had short stature due to growth hormone deficiency.

An androgenic gland membrane-anchored gene associated with the crustacean insulin-like androgenic gland hormone

Crustacean male sexual differentiation is governed by the androgenic gland (AG) and specifically by the secreted insulin-like AG hormone (IAG), thus far identified in several decapod species including the Australian red claw crayfish Cherax quadricarinatus (termed Cq-IAG). While a few insulin-like AG genes have been identified in crustaceans, other AG-specific genes have not been documented until now. In the present study, we describe the recent identification of a non-IAG AG-specific transcript obtained from the C. quadricarinatus AG cDNA library. This transcript, termed C. quadricarinatus membrane-anchored AG-specific factor (Cq-MAG), was fully sequenced and found to encode a putative product of 189 amino acids including a signal anchoring peptide. Expression of a recombinant GFP fusion protein lacking the signal anchor encoding sequence dramatically affected recombinant protein localization pattern. While the expression of the deleterious fusion protein was observed throughout most of the cell, the native GFP::Cq-MAG fusion protein was observed mainly surrounding the periphery of the nucleus, demonstrating an endoplasmic reticulum (ER)-like localization pattern. Moreover, co-expression of the wild-type Cq-MAG (fused to GFP) and the Cq-IAG hormone revealed that these peptides indeed co-localize. This study is the first to report a protein specifically associated with the insulin-like AG hormone in addition to the finding of another AG-specific transcript in crustaceans. Previous knowledge suggests that insulin/insulin-like factor secretion involves tissue-specific transcripts and membrane-anchored proteins. In this regard, Cq-MAG's tissue specificity, anchoring properties and intracellular co-localization with Cq-IAG suggest that it may play a role in the processing and secretion of this insulin-like AG hormone.

A Case of Glomerulonephritis Associated with Klinefelter&amp;apos; Syndrome

&lt;p&gt;Klinefelter' syndrome is a disorder of sexual differentiation in males, characterized by the presence of two or more X-chromosomes, hypogonadism, and lack of secondary sexual characteristics. The association between Klinefelter' syndrome and glomerulonephritis has been reported, while cases of glomerulonephritis associated with Klinefelter' syndrome are rare. We report the Korean case: a 31-year-old man with Klinefelter' syndrome who developed glomerulonephritis. The patient's urine analysis shows microscopic hematuria and the result of kidney biopsy was minimal change disease. The onset and course of his disease might have been influenced by the sex hormone imbalance.&lt;/p&gt;