Male Sexual Differentiation Research Articles

Is There a Place for AMH Testing in Canada?

Anti-müllerian hormone (AMH) is a glycoprotein traditionally known for its role in male sexual differentiation. More recently, AMH has been studied for its role in ovarian folliculogenesis and as a potential marker of ovarian reserve. A number of studies have suggested that measurement of serum AMH is a superior test of ovarian reserve because it is highly associated with the number of antral follicles, has little cycle variability, and declines throughout the reproductive lifespan. There is also evidence supporting the ability of AMH levels to predict age of menopause. Other studies have shown that measurement of serum AMH can predict responsiveness to ovarian follicular stimulation and the risk for ovarian hyperstimulation syndrome in women undergoing in vitro fertilization. Given the variety of potential applications and favourable test characteristics, it is reasonable to ask whether there is a place for providing universal AMH testing in Canada. RésuméL’hormone antimüllérienne (HAM) est une glycoprotéine traditionnellement connue pour son rôle dans la différenciation sexuelle masculine. Plus récemment, l’HAM a été étudiée en fonction de son rôle dans la folliculogenèse ovarienne et à titre de marqueur potentiel de la réserve ovarienne. Un certain nombre d’études ont laissé entendre que la mesure du taux sérique d’HAM constitue un test supérieur de la réserve ovarienne, puisqu’il est fortement associé au nombre de follicules antraux, qu’il présente peu de variabilité cyclique et qu’il décline tout au long de la durée de vie génésique. Certaines données soutiennent également la capacité des taux d’HAM de prédire l’âge de la ménopause. D’autres études ont indiqué que la mesure du taux sérique d’HAM pouvait prédire la réactivité à la stimulation folliculaire ovarienne et le risque de syndrome d’hyperstimulation ovarienne chez les femmes se soumettant à la fécondation in vitro. Compte tenu de la variété des applications potentielles et des caractéristiques favorables du test, il est raisonnable de se demander si l’offre d’un dépistage universel de l’HAM s’avère souhaitable au Canada.

Androgen Receptor Expression and Sperm Function in Male Infertility

The androgen receptor (AR), a member of the nuclear receptor superfamily, playsimportant role in male reproductive functions. A functional androgen receptor isrequired for male embryonic sexual differentiation, pubertal development andregulation of spermatogenesis. The role of AR during spermatogenesis has been thesubject of intense interest for many years. Several reports have shown that ARfunction is required for the completion of meiosis and the transition of spermatocytesto haploid round spermatids. The aim of the present study was to determine theassociation between androgen receptor expression and fertilization ability of humansperm in different andrological diseases. Eighty semen samples obtained from menattending the Andrology Outpatient Clinic, Mansoura University Hospital wereincluded in the study .The samples were grouped into control (n=20), accessory sexgland inflammation (MAGI) ( n=18), varicocele ( n=25) and idiopathic infertility(n=17). The semen samples analyses were done according to the recommendation ofthe World Health Organization. Computer assisted semen analysis (Autosperm) wasperformed. The acrosome reaction of spermatozoa recovered from each sample wasassessed before and after stimulation with the calcium ionophore A23187 (Sigma, St.Louis, Missouri, USA) by the pisum sativum (Sigma, St. Louis, Missouri, USA)fluorescence method with simultaneous vitality stain (Hoechst 33258, Germany).RNA was extracted from sperm of all cases and controls. cDNA and amplification ofa gene region from 1648 to 2055 bp corresponding to the DNA binding domain plusthe hinge region of human androgen receptor was carried out by one step RT-PCR.The PCR product size of 400 bp was electrophoresed on agarose gel 2%electrophoresis. Androgen receptor expression was significantly decreased in MAGI,varicocele and idiopathic groups compared with the control (P<0.01). Furthermore,AR expression was positively correlated with grade A+B motility (r=0.476,P<0.0001, velocity (r=0.362, P<0.001), linear velocity (r=0.454, P<0.0001), α-glucosidase (r= 0.420, P<0.0001) and acrosome reaction (r=0.532, P<0.0001).

Prenatal testosterone and dihydrotestosterone exposure disrupts ovine testicular development

Androgens play important roles during the first trimester of intrauterine life, coinciding with genital tract differentiation, during virilization and maintenance of secondary male characteristics, and during initiation of spermatogenesis. Little is known about the impact of inappropriate exposure to excess androgens during fetal development on male sexual maturation and reproduction. The objectives of this study were to determine the effects of prenatal 5α-dihydrotestosterone (DHT) and testosterone treatment during ovine sexual differentiation on post-pubertal testicular formation and subsequent potential for fertility as assessed by epididymal sperm characteristics. Rams prenatally treated with testosterone exhibited increased testicular weight relative to age-matched controls and prenatal DHT-treated rams (P<0.05), as well as elevated total and free testosterone concentrations compared with DHT-treated rams (P=0.07 and P<0.05 respectively). The percentage of progressively motile sperm from the epididymis was significantly reduced in prenatal DHT-treated but not testosterone-treated rams compared with control rams (P<0.05). The testosterone-treated rams had a greater number of germ cell layers than DHT-treated rams, but comparable to the controls. Prenatal testosterone-treated rams had significantly larger seminiferous tubule diameter and lumen diameter compared with prenatal DHT-treated (P<0.05). Significantly, more prenatal DHT- and testosterone-treated rams (P<0.05) had occluded tubule lumen than control rams. Findings from this study demonstrate that exposure to excess testosterone/DHT during male fetal sexual differentiation have differential effects on post-pubertal testicular size, seminiferous tubule size and function, sperm motility, and testosterone concentrations.

β-Catenin is essential for Müllerian duct regression during male sexual differentiation

During male sexual differentiation, the transforming growth factor-β (TGF-β) signaling molecule anti-Müllerian hormone (AMH; also known as Müllerian inhibiting substance, MIS) is secreted by the fetal testes and induces regression of the Müllerian ducts, the primordia of the female reproductive tract organs. Currently, the molecular identity of downstream events regulated by the AMH signaling pathway remains unclear. We found that male-specific Wnt4 expression in mouse Müllerian duct mesenchyme depends upon AMH signaling, implicating the WNT pathway as a downstream mediator of Müllerian duct regression. Inactivation of β-catenin, a mediator of the canonical WNT pathway, did not affect AMH signaling activation in the Müllerian duct mesenchyme, but did block Müllerian duct regression. These data suggest that β-catenin mediates AMH signaling for Müllerian duct regression during male sexual differentiation.

Disruption of androgen receptor signaling in males by environmental chemicals

Androgen-disruptors are environmental chemicals in that interfere with the biosynthesis, metabolism or action of endogenous androgens resulting in a deflection from normal male developmental programming and reproductive tract growth and function. Since male sexual differentiation is entirely androgen-dependent, it is highly susceptible to androgen-disruptors. Animal models and epidemiological evidence link exposure to androgen disrupting chemicals with reduced sperm counts, increased infertility, testicular dysgenesis syndrome, and testicular and prostate cancers. Further, there appears to be increased sensitivity to these agents during critical developmental windows when male differentiation is at its peak. A variety of in vitro and in silico approaches have been used to identify broad classes of androgen disrupting molecules that include organochlorinated pesticides, industrial chemicals, and plasticizers with capacity to ligand the androgen receptor. The vast majority of these synthetic molecules act as anti-androgens. This review will highlight the evidence for androgen disrupting chemicals that act through interference with the androgen receptor, discussing specific compounds for which there is documented in vivo evidence for male reproductive tract perturbations.This article is part of a Special Issue entitled ‘Endocrine disruptors’.

Expression of an Androgenic Gland-Specific Insulin-Like Peptide during the Course of Prawn Sexual and Morphotypic Differentiation

The crustacean male-specific androgenic gland (AG) regulates sexual differentiation. In the prawn Macrobrachium rosenbergii, silencing an AG-specific insulin-like encoding transcript (Mr-IAG) inhibited the development of male sexual characters, suggesting that Mr-IAG is a key androgenic hormone. We used recombinant pro-Mr-IAG peptide to generate antibodies that recognized the peptide in AG cells and extracts, as verified by mass spectrometry. We revealed the temporal expression pattern of Mr-IAG and studied its relevance to the timetable of sex differentiation processes in juveniles and after puberty. Mr-IAG was expressed from as early as 20 days after metamorphosis, prior to the appearance of external male sexual characters. Mr-IAG expression was lower in the less reproductively active orange-clawed males than in both the dominant blue-clawed males and the actively sneak mating small males. These results suggest a role for Mr-IAG both in the timing of male sexual differentiation and in regulating reproductive strategies.

Molecular cloning of anti-Müllerian hormone from the American alligator, Alligator mississippiensis

Anti-Müllerian hormone (AMH) plays an important role in male sex differentiation in vertebrates. AMH produced by Sertoli cells of the fetal testis induces regression of the Müllerian duct in mammalian species. In alligators, sexual differentiation is controlled by the temperature during egg incubation, termed temperature-dependent sex determination (TSD). The TSD mechanism inducing sex differentiation is thought to be unique and different from that of genetic sex determination as no gene such as the SRY of mammals has been identified. However, many of the genes associated with gonadal differentiation in mammals also are expressed in the developing gonads of species exhibiting TSD. To clarify the molecular mechanisms associated with gonad formation during the temperature-sensitive period (TSP), we have cloned the full length AMH gene in the alligator, and quantitatively compared mRNA expression patterns in the gonad–adrenal–mesonephros (GAM) complex isolated from alligator embryos incubated at male and female producing temperatures. The deduced amino acid sequence of the alligator AMH cDNA showed high identity (59–53%) to avian AMH genes. AMH mRNA expression was high in the GAM of male alligator embryos at stage 24 (immediately after sex determination) and hatchlings, but suppressed in the GAM of estrogen-exposed hatchlings incubated at the male-producing temperature. In the alligator AMH proximal promoter, a number of transcriptional factors (for SF-1. GATA, WT-1 and SOX9) binding elements were also identified and they exhibit a conserved pattern seen in other species. SOX9 up-regulates transcriptional activity through the amAMH promoter region. These results suggested that AMH and SOX9 play important roles in TSD of the American alligator.

A Sexual Shift Induced by Silencing of a Single Insulin-Like Gene in Crayfish: Ovarian Upregulation and Testicular Degeneration

In sequential hermaphrodites, intersexuality occurs naturally, usually as a transition state during sexual re-differentiation processes. In crustaceans, male sexual differentiation is controlled by the male-specific androgenic gland (AG). An AG-specific insulin-like gene, previously identified in the red-claw crayfish Cherax quadricarinatus (designated Cq-IAG), was found in this study to be the prominent transcript in an AG cDNA subtractive library. In C. quadricarinatus, sexual plasticity is exhibited by intersex individuals in the form of an active male reproductive system and male secondary sex characters, along with a constantly arrested ovary. This intersexuality was exploited to follow changes caused by single gene silencing, accomplished via dsRNA injection. Cq-IAG silencing induced dramatic sex-related alterations, including male feature feminization, a reduction in sperm production, extensive testicular degeneration, expression of the vitellogenin gene, and accumulation of yolk proteins in the developing oocytes. Upon silencing of the gene, AG cells hypertrophied, possibly to compensate for low hormone levels, as reflected in the poor production of the insulin-like hormone (and revealed by immunohistochemistry). These results demonstrate both the functionality of Cq-IAG as an androgenic hormone-encoding gene and the dependence of male gonad viability on the Cq-IAG product. This study is the first to provide evidence that silencing an insulin-like gene in intersex C. quadricarinatus feminizes male-related phenotypes. These findings, moreover, contribute to the understanding of the regulation of sexual shifts, whether naturally occurring in sequential hermaphrodites or abnormally induced by endocrine disruptors found in the environment, and offer insight into an unusual gender-related link to the evolution of insulins.

Characterization of Androgen Receptor Structure and Nucleocytoplasmic Shuttling of the Rice Field Eel

Androgen receptor (AR) plays a critical role in prostate cancer and male sexual differentiation. We have identified AR from a primitive vertebrate with a sex reversal characteristic, the rice field eel. AR of this species (eAR) is distinct from human AR, especially in the ligand binding domain (LBD), and its expression in gonads shows an increasing tendency during gonadal transformation from ovary via ovotestis to testis. eAR has a restricted androgen-dependent transactivation function after a nuclear translocation upon dihydrotestosterone exposure. A functional nuclear localization signal was further identified in the DNA binding domain and hinge region. Although nuclear export is CRM1-independent, eAR has a novel nuclear export signal, which is negatively charged, indicating that a nuclear export pathway may be mediated by electrostatic interaction. Further, our studies have identified critical sequences for ligand binding in the C terminus. A structure of three α-helices in the LBD has been conserved from eels to humans during vertebrate evolution, despite a distinct amino acid sequence. Mutation analysis confirmed that the LBD is essential for dihydrotestosterone-induced nuclear import of eAR and following transactivation function in the nucleus. In addition, eAR interacts with both Sox9a1 and Sox9a2, and their interaction regulates transactivation of eAR. Our data suggest that the primitive species conserves and especially acquires key novel domains, the nuclear export signal and LBD, for the eAR function in spite of a rapid sequence evolution.

A Functional Polymorphism c.-712C&gt;T in the FGF9 Promoter Is Associated with Sertoli Cell-Only Syndrome.

Fibroblast growth factor 9 (FGF9) is an autocrine/paracrine growth factor considered to be important for a variety of biological processes, including proliferation, differentiation and cell survival activities in embryogenesis and adult. Recent studies from Fgf9 knockout mice emphasized the importance of FGF9 in male sex determination and differentiation. The reproductive system phenotypes of Fgf9-/- null mice range from testicular hypoplasia to male-to-female sex reversal and gonadal dysgenesis, suggesting the crucial role for FGF9 signaling in sex determination and testicular development. The RNA and protein expressions of FGF9 in the testes of Sertoli cell-only syndrome (SCOS) were significantly decreased, compared to controls. We further systematically examined the sequences of FGF9 gene in 29 SCOS infertile men and 79 unrelated healthy controls to test whether sequence variants of FGF9 gene play a pathological role in SCOS phenotype. A single nucleotide polymorphism and two polymorphic microsatellites were detected. The allelic and genotypic frequencies of the c.-712C>T in the promoter region of FGF9 were significantly different between SCOS patients and controls (P = 0.005 and 0.0008, respectively), while the microsatellites c.-124dupT(8_10) in the 5'UTR and c.*275_76TG(13_17) in the 3'UTR did not show any difference between these two groups. Reporter assay demonstrated that the polymorphism c.-712C>T dramatically attenuated promoter activity. By transcription element search system (TESS), E2F transcription factor 1 (E2F-1) was predicted to bound to -712C and this binding was confirmed by chromatin immuno-precipitation (ChIP) assay. Moreover, in electrophoretic mobility shift assay (EMSA), substitutional mutation of E2F-1 binding site (i.e. -712C>T) reduced DNA-protein complex formation. Collectively, our data suggest the functional polymorphism c.-712C>T within the promoter region of FGF9 gene, which results in decreased FGF9 expression and may contribute to one of the causes of SCOS. (poster)

Bilateral eyestalk ablation of the blue swimmer crab, Portunus pelagicus, produces hypertrophy of the androgenic gland and an increase of cells producing insulin-like androgenic gland hormone

The androgenic glands (AG) of male decapod crustaceans produce insulin-like androgenic gland (IAG) hormone that controls male sex differentiation, growth and behavior. Functions of the AG are inhibited by gonad-inhibiting hormone originating from X-organ-sinus gland complex in the eyestalk. The AG, and its interaction with the eyestalk, had not been studied in the blue swimmer crab, Portunus pelagicus, so we investigated the AG structure, and then changes of the AG and IAG-producing cells following eyestalk ablation. The AG of P. pelagicus is a small endrocrine organ ensheathed in a connective tissue and attached to the distal part of spermatic duct and ejaculatory bulb. The gland is composed of several lobules, each containing two major cell types. Type I cells are located near the periphery of each lobule, and distinguished as small globular cells of 5–7 μm in diameter, with nuclei containing mostly heterochromatin. Type II cells are 13–15 μm in diameter, with nuclei containing mostly euchromatin and prominent nucleoli. Both cell types were immunoreactive with anti-IAG. Following bilateral eyestalk ablation, the AG underwent hypertrophy, and at day 8 had increased approximately 3-fold in size. The percentage of type I cells had increased more than twice compared with controls, while type II cells showed a corresponding decrease.

Expression of gonadal soma derived factor (GSDF) is spatially and temporally correlated with early testicular differentiation in medaka

In the teleost fish, medaka ( Oryzias latipes), the sex is genetically determined at the time of fertilization. The males are heterogametic with XY chromosome composition, while females are of XX chromosome composition. The male sexual differentiation is initiated in XY embryos of medaka by the sex-determining gene Dmy. In this study, we have cloned the gonadal soma derived factor ( Gsdf) from medaka and characterized its expression pattern during the initiation of morphological testicular differentiation. By real-time PCR, an XY-specific up-regulation was detected in the expression levels of Gsdf in the whole embryos of medaka at 6 days post fertilization (dpf), coincident with the initiation of testicular differentiation in the XY gonads. Whole mount and section in situ hybridizations reaffirmed that Gsdf was expressed exclusively in primordial gonads of only the genetic males at 6 dpf. Conversely, the expression of Gsdf was found to be very weak in the XX gonads during embryogenesis. Importantly, Gsdf and Dmy were found to be co-localized in the same somatic cells in the XY gonads. When the XY embryos were treated with estradiol-17β, in order to reverse their phenotypic sex, a decline was observed in the expression of Gsdf in these embryos by real-time PCR.

Epigenetic abnormality of SRY gene in the adult XY female with pericentric inversion of the Y chromosome

In normal ontogenetic development, the expression of the sex-determining region of the Y chromosome (SRY) gene, involved in the first step of male sex differentiation, is spatiotemporally regulated in an elaborate fashion. SRY is expressed in germ cells and Sertoli cells in adult testes. However, only few reports have focused on the expressions of SRY and the other sex-determining genes in both the classical organ developing through these genes (gonad) and the peripheral tissue (skin) of adult XY females. In this study, we examined the gonadal tissue and fibroblasts of a 17-year-old woman suspected of having disorders of sexual differentiation by cytogenetic, histological, and molecular analyses. The patient was found to have the 46,X,inv(Y)(p11.2q11.2) karyotype and streak gonads with abnormally prolonged SRY expression. The sex-determining gene expressions in the patient-derived fibroblasts were significantly changed relative to those from a normal male. Further, the acetylated histone H3 levels in the SRY region were significantly high relative to those of the normal male. As SRY is epistatic in the sex-determination pathway, the prolonged SRY expression possibly induced a destabilizing effect on the expressions of the downstream sex-determining genes. Collectively, alterations in the sex-determining gene expressions persisted in association with disorders of sexual differentiation not only in the streak gonads but also in the skin of the patient. The findings suggest that correct regulation of SRY expression is crucial for normal male sex differentiation, even if SRY is translated normally.

Androgen Receptor Mutations Associated with Androgen Insensitivity Syndrome: A High Content Analysis Approach Leading to Personalized Medicine

Androgen insensitivity syndrome (AIS) is a rare disease associated with inactivating mutations of AR that disrupt male sexual differentiation, and cause a spectrum of phenotypic abnormalities having as a common denominator loss of reproductive viability. No established treatment exists for these conditions, however there are sporadic reports of patients (or recapitulated mutations in cell lines) that respond to administration of supraphysiologic doses (or pulses) of testosterone or synthetic ligands. Here, we utilize a novel high content analysis (HCA) approach to study AR function at the single cell level in genital skin fibroblasts (GSF). We discuss in detail findings in GSF from three historical patients with AIS, which include identification of novel mechanisms of AR malfunction, and the potential ability to utilize HCA for personalized treatment of patients affected by this condition.

Sex differentiation and sex change in the protandrous black porgy, Acanthopagrus schlegeli

Protandrous black porgy fish, Acanthopagrus schlegeli, have a striking life cycle with a male sex differentiation at the juvenile stage and male-to-female sex change at 3 years of age. We had characterized the sex differentiation and sex change in this species by the integrative approaches of histology, endocrine and molecular genetics. The fish differentiated in gonad at the age around 4-months and the gonad further developed with a bisexual gonad for almost for 3 years and sex change at 3 year of age. An antagonistic relationship in the testicular and ovarian tissues was found during the development of the gonadal tissue. Male- (such as sf-1, dmrt1, dax-1 and amh) and female- (such as wnt4, foxl2 and cyp19a1a) promoting genes were associated with testicular and ovarian development, respectively. During gonadal sex differentiation, steroidogenic pathway and estrogen signaling were also highly expressed in the brain. The increased expression of sf-1 and wnt4, cyp19a1a in ovarian tissue and decreased expression of dax-1 in the ovarian tissue may play important roles in sex change from a male-to-female. Endocrine factors such as estradiol and luteinizing hormone may also involve in the natural sex change. Estradiol induced the expression of female-promoting genes and resulted in the precocious sex change in black porgy. Our series of studies shed light on the sex differentiation and sex change in protandrous black porgy and other animals.

Synergistic Disruption of External Male Sex Organ Development by a Mixture of Four Antiandrogens

BackgroundBy disrupting the action of androgens during gestation, certain chemicals present in food, consumer products, and the environment can induce irreversible demasculinization and malformations of sex organs among male offspring. However, the consequences of simultaneous exposure to such chemicals are not well described, especially when they exert their actions by differing molecular mechanisms.ObjectivesTo fill this gap, we investigated the effects of mixtures of a widely used plasticizer, di(2-ethylhexyl) phthalate (DEHP); two fungicides present in food, vinclozolin and prochloraz; and a pharmaceutical, finasteride, on landmarks of male sexual development in the rat, including changes in anogenital distance (AGD), retained nipples, sex organ weights, and malformations of genitalia. These chemicals were chosen because they disrupt androgen action with differing mechanisms of action.ResultsStrikingly, the effect of combined exposure to the selected chemicals on malformations of external sex organs was synergistic, and the observed responses were greater than would be predicted from the toxicities of the individual chemicals. In relation to other hallmarks of disrupted male sexual development, including changes in AGD, retained nipples, and sex organ weights, the combined effects were dose additive. When the four chemicals were combined at doses equal to no observed adverse effect levels estimated for nipple retention, significant reductions in AGD were observed in male offspring.ConclusionsBecause unhindered androgen action is essential for human male development in fetal life, these findings are highly relevant to human risk assessment. Evaluations that ignore the possibility of combination effects may lead to considerable underestimations of risks associated with exposures to chemicals that disrupt male sexual differentiation.

Cumulative Effects of Administration Mixtures of "Antiandrogens" in Rats: A New Framework Based upon Common Systems Rather Than Common Mechanisms.

Since humans and wildlife are exposed to more than one chemical at a time, concern has arisen about the effects of complex mixtures on reproduction and development. To date, different regulatory groups have not yet developed consistent approaches to conducting assessments of the risks of mixtures to human health. One commonly used approach is based upon the assumption that chemicals that act via common mechanisms of toxicity will act jointly (Dose addition) whereas chemicals that act via disparate mechanisms will act independently (Response addition). The consequence of the assumption that Response addition rather than Dose addition modeling accurately predicts the effects of mixtures of chemicals with different mechanisms of toxicity is that response addition models predict that there is no effect of the mixture if the level of each chemical is low (0+0+0+...= 0% affected) whereas Dose addition models may predict that serious effects will occur even though the level of each chemical is too low to produce any effects individually (0+0+0+...= 100% affected). Over the last ten years our laboratory as been conducting studies to determine how mixtures of endocrine disrupting chemicals behave when administered during sexual differentiation of rats. The approach has been to use chemicals with 1) well characterized mechanisms of toxicity and 2) known dose-related reproductive effects. The effects of the mixtures are then compared to the effects predicted by Response- and Dose addition modeling to determine which model more accurately predicts the effects of mixture of chemicals with similar and disparate mechanisms of toxicity. The chemicals used in these studies include pesticides that are androgen receptor antagonists (procymidone, vinclozolin, prochloraz, linuron), and/or inhibitors of steroid hormone synthesis (linuron and prochloraz), plasticizers like the phthalates that inhibit fetal Leydig cell hormone synthesis and the potent Ah receptor agonist 2,3,7,8-tetrachlorodibenzodioxin (TCDD). In the mixture studies we combined pairs of AR antagonists, pairs of phthalates, phthalates with AR antagonists and seven chemicals (four pesticides and four phthalates). In the new, unpublished studies, presented herein, we examined the effects of in utero exposure to a mixture of TCDD plus a phthalate on epididymal differentiation and a complex mixture of ten chemicals (four pesticides and six phthalates) on several androgen-dependent endpoints in the male rat. Results of these studies clearly demonstrate that Dose addition models more accurately predict the effects of these mixtures on male rat sexual differentiation. For example, when the mixture of ten chemicals was administered in utero, 100% of the males displayed reproductive tract malformations. The Dose addition model predicted this (0+0+...0 = 100% affected) whereas the Response addition predicted that none of the males would be malformed (0+0+...0 = 0% affected). Based upon these results and similar results from other laboratories we propose that the regulatory framework for cumulative risk assessments should not be based upon common mechanisms of toxicity, as this under-predicts the effects of mixtures of chemicals with dissimilar mechanisms of toxicity. Rather, the framework should be based upon the disruption of common fetal targets or systems during development regardless of the mechanism of toxicity. Furthermore, a recently National Academy of Science Panel, formed at the request of the USEPA to advise the Agency on how to conduct a cumulative risk assessment on the phthalates came to the same conclusion, stating that the framework for cumulative risk assessments should be based upon common targets and not narrowly on common mechanisms of toxicity. This abstract does not necessarily reflect EPA policy. Supported in part by NTP, NIEHS/EPA Interagency Cooperative Research Agreement HHS Y1-ES-8014-01; EPA RW75922855-01-0. (poster)

A case of complete androgen insensitivity syndrome presenting with incarcerated inguinal hernia: an immunohistochemical study

To present a case of complete androgen insensitivity syndrome that initially presented as incarcerated inguinal hernia and to investigate the expression of antimüllerian hormone (AMH), SOX-9 (SRY-box-containing gene 9), prostaglandin D synthase (PGDS), and androgen receptors (AR). Case report. District hospital. A 12-year-old girl with negative past medical and family history, who presented with a 6-hour history of progressive left groin pain. Open gonadectomy. Immediate surgery, diagnosis, and referral to a multidisciplinary team for further management. Pathologic analysis revealed a left twisted and infarcted testicle, and a normal right one containing immature seminiferous tubules with a decreased number of spermatogonia. Immunohistochemical analysis revealed strong cytoplasmic and nuclear expression of AMH and SOX-9, respectively, by the Sertoli cells as well as focal weak cytoplasmic PGDS expression. The spermatogonia showed focal weak nuclear PGDS expression. The Leydig cells showed no immunoreactivity at all. No AR immunoreactivity was observed. Negative AR immunostaining could either reflect Sertoli-cell immaturity or a mutation resulting in no protein production. The AMH immunodetection, consistent with its high serum levels, could potentially reflect Sertoli-cell immaturity. The SOX-9 nuclear detection in the Sertoli cells was consistent with its role, inducing male sex differentiation, including AMH expression. The nuclear localization of PGDS in the spermatogonia needs further investigation.

The PGD2 pathway, independently of FGF9, amplifies SOX9 activity in Sertoli cells during male sexual differentiation

Activation by the Y-encoded testis determining factor SRY and maintenance of expression of the Sox9 gene encoding the central transcription factor of Sertoli cell differentiation are key events in the mammalian sexual differentiation program. In the mouse XY gonad, SOX9 upregulates Fgf9, which initiates a Sox9/Fgf9 feedforward loop, and Sox9 expression is stimulated by the prostaglandin D2 (PGD2) producing lipocalin prostaglandin D synthase (L-PGDS, or PTDGS) enzyme, which accelerates commitment to the male pathway. In an attempt to decipher the genetic relationships between Sox9 and the L-Pgds/PGD2 pathway during mouse testicular organogenesis, we found that ablation of Sox9 at the onset or during the time window of expression in embryonic Sertoli cells abolished L-Pgds transcription. By contrast, L-Pgds(-/-) XY embryonic gonads displayed a reduced level of Sox9 transcript and aberrant SOX9 protein subcellular localization. In this study, we demonstrated genetically that the L-Pgds/PGD2 pathway acts as a second amplification loop of Sox9 expression. Moreover, examination of Fgf9(-/-) and L-Pgds(-/-) XY embryonic gonads demonstrated that the two Sox9 gene activity amplifying pathways work independently. These data suggest that, once activated and maintained by SOX9, production of testicular L-PGDS leads to the accumulation of PGD2, which in turn activates Sox9 transcription and nuclear translocation of SOX9. This mechanism participates together with FGF9 as an amplification system of Sox9 gene expression and activity during mammalian testicular organogenesis.

Conditional Inactivation of Androgen Receptor Gene in the Nervous System: Effects on Male Behavioral and Neuroendocrine Responses

Testosterone (T) profoundly influences central sexual differentiation and functions. In the brain, T signals either directly through androgen receptor (AR) or indirectly through estrogen receptor (ER) following aromatization into E2 (17-beta-estradiol). As T, through AR, also controls peripheral male sexual differentiation, the relative contribution of central AR in T-mediated regulation of behavioral and neuroendocrine responses still remains unclear. To address this question, we generated, by using Cre-loxP technology, mice selectively lacking AR expression in the nervous system. The mutant male urogenital tract was normally developed, and mice were able to produce offspring. Nonetheless, sexual motivation and performance as well as aggressive behaviors were affected. Only a low percentage of males displayed a complete sexual behavior and offensive attacks. The latency to show masculine behaviors was increased and copulation length prolonged. Erectile activity during mating was also altered. These alterations occurred despite increased levels of T and its metabolites, and an unaffected number of ERalpha-immunoreactive cells. Olfactory preference and neuronal activation, mapped by Fos immunoreactivity, following exposure to estrus female-soiled bedding were also normal. At comparable T levels, greater differences in masculine behaviors were observed between gonadectomized control and mutant males. AR invalidation in the nervous system also disrupted the somatotropic axis since mutant males exhibited growth retardation and decreased serum levels of insulin-like growth factor I. Our findings show that central AR is required in T-induced regulation of male-typical behaviors and gonadotrope and somatotropic axes. This genetic model offers a unique opportunity in the understanding of AR's role in cerebral functions of T.