Gadd45g Is Essential for Primary Sex Determination,Male Fertility and Testis Development

In humans and most mammals, differentiation of the embryonic gonad into ovaries or testes is controlled by the Y-linked gene SRY. Here we show a role for the Gadd45g protein in this primary sex differentiation. We characterized mice deficient in Gadd45a, Gadd45b and Gadd45g, as well as double-knockout mice for Gadd45ab, Gadd45ag and Gadd45bg, and found a specific role for Gadd45g in male fertility and testis development. Gadd45g-deficient XY mice on a mixed 129/C57BL/6 background showed varying degrees of disorders of sexual development (DSD), ranging from male infertility to an intersex phenotype or complete gonadal dysgenesis (CGD). On a pure C57BL/6 (B6) background, all Gadd45g^−/− XY mice were born as completely sex-reversed XY-females, whereas lack of Gadd45a and/or Gadd45b did not affect primary sex determination or testis development. Gadd45g expression was similar in female and male embryonic gonads, and peaked around the time of sex differentiation at 11.5 days post-coitum (dpc). The molecular cause of the sex reversal was the failure of Gadd45g^−/− XY gonads to achieve the SRY expression threshold necessary for testes differentiation, resulting in ovary and Müllerian duct development. These results identify Gadd45g as a candidate gene for male infertility and 46,XY sex reversal in humans.     

BAX-mediated cell death affects early germ cell loss and incidence of testicular teratomas in Dnd1 mice

A homozygous nonsense mutation (Ter) in murine Dnd1 (Dnd1^Ter/Ter) results in a significant early loss of primordial germ cells (PGCs) prior to colonization of the gonad in both sexes and all genetic backgrounds tested. The same mutation also leads to testicular teratomas only on the 129Sv/J background. Male mutants on other genetic backgrounds ultimately lose all PGCs with no incidence of teratoma formation. It is not clear how these PGCs are lost or what factors directly control the strain-specific phenotype variation. To determine the mechanism underlying early PGC loss we crossed Dnd1^Ter/Ter embryos to a Bax-null background and found that germ cells were partially rescued. Surprisingly, on a mixed genetic background, rescued male germ cells also generated fully developed teratomas at a high rate. Double-mutant females on a mixed background did not develop teratomas, but were fertile and produced viable off-spring. However, when Dnd1^Ter/Ter XX germ cells developed in a testicular environment they gave rise to the same neoplastic clusters as mutant XY germ cells in a testis. We conclude that BAX-mediated apoptosis plays a role in early germ cell loss and protects from testicular teratoma formation on a mixed genetic background.     

Effects of androgen receptor mutation on testicular histopathology of patient having complete androgen insensitivity

Androgens are required for normal male sex differentiation and development of male secondary sexual characteristics. Mutations in AR gene are known to cause defects in male sexual differentiation. In current study, we enrolled a 46,XY phenotypically female patient bearing testes in inguinal canal. DNA sequencing of the AR gene detected a missense mutation C.1715A?>?G (p. Y572C) in exon 2 which is already known to cause complete androgen insensitivity syndrome (CAIS). We focused on the effects of this mutation on the testicular histopathology of the patient. Surface spreading of testicular tissues showed an absence of spermatocytes while H&E staining showed that seminiferous tubules predominantly have only Sertoli cells. This meiotic failure is likely due to the effect of the AR mutation which ultimately leads to Sertoli cell only syndrome. Tubules were stained with SOX9 and AMH which revealed Sertoli cells maturation arrest. Western blot and realtime PCR data showed that patient had higher levels of AMH, SOX9 and inhibin-B in the testis. Therefore, we suggest that the dysfunctioning of AR by mutation enhances AMH expression which ultimately leads to the failure in maturation of Sertoli cells.     

Pathology of 46, XY pure gonadal dysgenesis: absence of testis differentiation associated with mutations in the testis-determining factor

Abstract. Individuals with 46, XY pure gonadal dysgenesis present with a completely female phenotype. These individuals develop bilateral streak gonads and have normal Müllerian structures. The apparent absence of testicular tissue in these individuals suggests a mutation in the initial steps of the male sex-determining pathway. A candidate gene for the primary signal in this pathway was recently cloned ( SRY ) which encodes a protein with a DNA-binding capacity. In a study of 14 XY females with pure gonadal dysgenesis harbouring SRY , we analysed the histology of the gonads and compared it to the presence or absence of mutations in the SRY open reading frame ( SRY -orf). The histological analysis revealed two distinct groups of streak gonads. In the first group, the gonad was composed of exclusively ovarian-like stroma, with sclero-hyaline nodules in some areas. No tubules were observed. The gonads in the second group were composed of undifferentiated stroma harbouring either tubules or a rete structure. This suggests that in the latter group some differentiation (towards testis formation) has occurred, whereas in the first group ovarian differentiation has been interrupted. Individuals with mutations in the SRY -orf were found to have streak gonads of the first group, whereas most of the remaining XY females without detectable mutation in the SRY -orf had streak gonads belonging to the second group.
On the basis of histology, it may be possible to distinguish between mutations in the sex-determining or sex-differentiation pathways. We suggest that SRY may play a role in rete testis formation. We also present arguments favouring the mesonephros as the origin of testicular somatic cells in humans.     

Mutations in Hedgehog Acyltransferase (Hhat) Perturb Hedgehog Signaling, Resulting in Severe Acrania-Holoprosencephaly-Agnathia Craniofacial Defects

Holoprosencephaly (HPE) is a failure of the forebrain to bifurcate and is the most common structural malformation of the embryonic brain. Mutations in SHH underlie most familial (17%) cases of HPE; and, consistent with this, Shh is expressed in midline embryonic cells and tissues and their derivatives that are affected in HPE. It has long been recognized that a graded series of facial anomalies occurs within the clinical spectrum of HPE, as HPE is often found in patients together with other malformations such as acrania, anencephaly, and agnathia. However, it is not known if these phenotypes arise through a common etiology and pathogenesis. Here we demonstrate for the first time using mouse models that Hedgehog acyltransferase (Hhat) loss-of-function leads to holoprosencephaly together with acrania and agnathia, which mimics the severe condition observed in humans. Hhat is required for post-translational palmitoylation of Hedgehog (Hh) proteins; and, in the absence of Hhat, Hh secretion from producing cells is diminished. We show through downregulation of the Hh receptor Ptch1 that loss of Hhat perturbs long-range Hh signaling, which in turn disrupts Fgf, Bmp and Erk signaling. Collectively, this leads to abnormal patterning and extensive apoptosis within the craniofacial primordial, together with defects in cartilage and bone differentiation. Therefore our work shows that Hhat loss-of-function underscrores HPE; but more importantly it provides a mechanism for the co-occurrence of acrania, holoprosencephaly, and agnathia. Future genetic studies should include HHAT as a potential candidate in the etiology and pathogenesis of HPE and its associated disorders.     

H-Y antigen in 46,XY gonadal dysgenesis

Summary Presence of H-Y antigen has been correlated with testicular differentiation, and absence of H-Y with failure of testicular differentiation, in a variety of mammalian species. To determine more precisely the relationship between expression of H-Y antigen and development of the testis, we studied the cells of phenotypic females with the 46,XY male karyotype. Blood leukocytes were typed H-Y⁺ in five XY females with gonadal dysgenesis, although in other studies blood leukocytes from XY females with gonadal dysgenesis were typed H-Y^-. Thus mere presence of H-Y antigen is not sufficient to guarantee normal differentiation of the testis. In the present paper we review evidence for an additional factor in gonadal organogenesis, the H-Y antigen receptor. We infer that testicular development requires engagement of H-Y and its receptor. It follows that XY gonadal dysgenesis is the consequence of functional absence of the H-Y testis inducer as in the following conditions: failure of synthesis of H-Y or failure of specific binding of H-Y.     

The decoupling of Smoothened from Galphai proteins has little effect on Gli3 protein processing and Hedgehog-regulated chick neural tube patterning

The Hedgehog (Hh) signal is transmitted by two receptor molecules, Patched (Ptc) and Smoothened (Smo). Ptc suppresses Smo activity, while Hh binds Ptc and alleviates the suppression, which results in activation of Hh targets. Smo is a seven-transmembrane protein with a long carboxyl terminal tail. Vertebrate Smo has been previously shown to be coupled to Gα_i proteins, but the biological significance of the coupling in Hh signal transduction is not clear. Here we show that although inhibition of Gα_i protein activity appears to significantly reduce Hh pathway activity in Ptc^−/− mouse embryonic fibroblasts and the NIH3T3-based Shh-light cells, it fails to derepress Shh- or a Smo-agonist-induced inhibition of Gli3 protein processing, a known in vivo indicator of Hh signaling activity. The inhibition of Gα_i protein activity also cannot block the Sonic Hedgehog (Shh)-dependent specification of neural progenitor cells in the neural tube. Consistent with these results, overexpression of a constitutively active Gα_i protein, Gα_i2QL, cannot ectopically specify the neural cell types in the spinal cord, whereas an active Smo, SmoM2, can. Thus, our results indicate that the Smo-induced Gα_i activity plays an insignificant role in the regulation of Gli3 processing and Shh-regulated neural tube patterning.     

Sexually dimorphic gene expression in the developing mouse gonad

Over the course of a few days, the bipotential embryonic mouse gonad differentiates into either a testis or an ovary. Though a few gene expression differences that underlie gonadal sex differentiation have been identified, additional components of the testicular and ovarian developmental pathways must be identified to understand this process. Here we report the use of a PCR-based cDNA subtraction to investigate expression differences that arise during gonadal sex differentiation. Subtraction of embryonic day 12.5 (E12.5) XY gonadal cDNA with E12.5 XX gonadal cDNA yielded 19 genes that are expressed at significantly higher levels in XY gonads. These genes display a variety of expression patterns within the embryonic testis and encode a broad range of proteins. A reciprocal subtraction (of E12.5 XX gonadal cDNA with E12.5 XY gonadal cDNA) yielded two genes, follistatin and Adamts19, that are expressed at higher levels in XX gonads. Follistatin is a well-known antagonist of TGFbeta family members while Adamts19 encodes a new member of the ADAMTS family of secreted metalloproteases.     

Dynamic phosphorylation of the kinesin Costal-2 in vivo reveals requirement of fused kinase activity for all levels of hedgehog signalling

Hedgehog (Hh) proteins are secreted molecules that play an essential role in development and tumorigenesis. In Drosophila cultured cells, phosphorylation of the kinesin-like Costal2 (Cos2) protein at Ser572 is triggered by the kinase fused (Fu) upon Hh pathway activation. Here, we validate the first phospho-antibody for one of the Hh pathway components, Cos2, as a universal in situ readout of Hh signal transduction. For the first time, this tool allows the visualisation of a gradient of signalling activity and therefore the range of the activating Hh ligand in different tissues. We also show that, in vivo, Fu kinase is activated by and necessary to transduce all levels of intracellular Hh signalling. Our study fills a gap in the understanding of the Hh pathway by showing that the molecular cascade leading to Cos2 phosphorylation is conserved in all cells activated by Hh. Therefore, we propose that the extracellular Hh information is conveyed to an intracellular signal through graded Fu kinase activity.     

Improvement of Mercuric Chloride-Induced Testis Injuries and Sperm Quality Deteriorations by Spirulina platensis in Rats

The present study was undertaken to investigate the protective effect of the filamentous cyanobacterium Spirulina platensis (S. platensis) on mercury (II) chloride (HgCl₂)-induced oxidative damages and histopathological alterations in the testis of Wistar albino rats. The animals were divided into four equal groups, i ) control, ii ) HgCl₂, iii ) S. platensis and iv ) combination of HgCl₂+S. platensis. Oxidative stress, induced by a single dose of HgCl₂ (5 mg/kg, bw; subcutaneously, s.c.), substantially decreased (P<0.01) the activity level of testicular key enzymatic antioxidant biomarkers (superoxide dismutase, SOD; catalase, CAT and glutathione peroxidase, GPx), oxidative stress makers (blood hydroperoxide; testicular reduced glutathione, GSH and malondialdehyde, MDA), and testicular mercury levels. Moreover, HgCl₂ administration resulted in a significant (P<0.01) increase in the number of sperms with abnormal morphology and decrease in epididymal sperm count, motility, plasma testosterone level and testicular cholesterol. Furthermore, HgCl₂ exposure induced histopathological changes to the testis including morphological alterations of the seminiferous tubules, and degeneration and dissociation of spermatogenic cells. Notably, oral pretreatment of animals with Spirulina (300 mg/kg, bw) lowered the extent of the observed HgCl₂-mediated toxicity, whereby significantly reducing the resulting lipid peroxidation products, mercury accumulation in the testis, histopathological changes of the testes and spermatozoal abnormalities. In parallel, the pretreatment with Spirulina also completely reverted the observed Hg-Cl₂-induced inhibition in enzymatic activities of antioxidant biomarkers (SOD, CAT and GPx) back to control levels. The pretreatment of rats with S. platensis significantly recovered the observed HgCl₂-mediated decrease in the weight of accessory sex organs. Taken together, our findings clearly highlight the role of S. platensis as a protective modulator of HgCl₂-induced testicular injuries and suggest some therapeutic potential in mammals. Further investigation of therapeutic strategies employing Spirulina against heavy metals toxicity in humans is therefore warranted.