Male specific expression of lipocalin-type prostaglandin D synthase (cPTGDS) during chicken gonadal differentiation: relationship with cSOX9.

As in mammals, the SOX9 gene (cSOX9) is specifically expressed in male differentiating Sertoli cells during chicken gonadal development. Recent studies in mouse have shown that the prostaglandin D(2) (PGD(2))/lipocalin-type prostaglandin D synthase (PTGDS) pathway is functionally associated with the regulation of SOX9 and is specifically expressed in male developing gonads. In this study, we have shown that, as in mammals, cPTGDS is a male specific gene during chicken testicular development and is temporally expressed in the same window as cSOX9 in Sertoli cells. Using a culture of gonadal explants, we have shown that exogenous PGD(2) enhances cSOX9 expression in male, and activates its ectopic expression in female gonads without up-regulating cAMH. These data indicate a conserved PGD(2) modulation of SOX9 expression during testicular differentiation between birds and mammals.

Steroidogenic factor-1 contributes to the cyclic-adenosine monophosphate down-regulation of human SRY gene expression.

In mammals, male sex determination is initiated by SRY (sex-determining region of the Y chromosome) gene expression and followed by testicular development. This study describes specific down-regulation of the human SRY gene transcription by cAMP stimulation using reverse transcription-polymerase chain reaction experiments. Using transfection experiments, conserved nuclear hormone receptor (NHR1) and Sp1 consensus binding sites were identified as essential for this cAMP transcriptional response. Steroidogenic factor-1 (SF-1), a component of the sex-determination cascade, binds specifically to the NHR1 site and activates the SRY promoter. Activation of SF-1 was abolished by cAMP pretreatment of the cells, suggesting a possible effect of cAMP on the SF-1 protein itself. Indeed, human SF-1 protein contains at least two in vitro cAMP-dependent protein kinase (PKA) phosphorylation sites, leading after phosphorylation to a modification of both DNA-binding activity and interaction with general transcription factors such as Sp1. Taken together, these data suggest that cAMP responsiveness of human SRY promoter involves both SF-1 and Sp1 sites and could act via PKA phosphorylation of the SF-1 protein itself.

Muscle differentiation is antagonized by SOX15, a new member of the SOX protein family.

SOX proteins belong to a multigenic family characterized by a unique DNA binding domain, known as the high mobility group box, that is related to that of the testis determining gene SRY. cDNA sequences for more than 30 SOX genes have been identified, and some are known to have diverse roles in vertebrate differentiation and development. Here, we report the isolation and characterization of mouse Sox15 that was uncovered during a screen for high mobility group box containing transcription factors that are expressed at different levels during skeletal muscle differentiation. Sox15 cDNAs were found at a much higher frequency in myoblasts prior to their differentiation into myotubes. Electrophoretic mobility shift assays indicated that recombinant SOX15 protein was capable of binding to a consensus DNA binding site for SOX proteins. When overexpressed in C2C12 myoblasts, wild type SOX15, but not a C-terminal truncated form or the related protein SOX11, specifically inhibited activation of muscle-specific genes and expression of the basic helix-loop-helix myogenic factors myogenin and MyoD, resulting in a failure of the cells to differentiate into myotubes. These results suggest a specific and repressive role for SOX15, requiring the C-terminal domain, during myogenesis.

Expression and subcellular localization of SF-1, SOX9, WT1, and AMH proteins during early human testicular development.

Many transcription factors have been identified and implicated in male sex determination pathway. Specifically involved in Sertoli cell differentiation and subsequent anti-Müllerian hormone (AMH) secretion in eutherian mammals, they include steroidogenic factor-1 (SF-1), SOX9 (SRY HMG box related gene 9), WT1 (Wilms' tumor 1), and GATA-4 (a zinc finger transcription factor). These factors have been described to execute their function in the male sex determination pathway by controlling AMH transcriptional expression. To understand the hierarchies of these factors and their involvement in the developing testis, for the first time we show the expression and subcellular localization of these factors by immunohistochemistry in the early human testis during embryogenesis compared with AMH expression. If these studies do not refute their possible synergistic interaction to control AMH expression in human embryo, they also reveal a new sexual dimorphism in SOX9 expression during the sex determination process. We show that SOX9 sex specifically shifts from the cytoplasmic to the nuclear compartment at the time of testis differentiation and AMH expression. Putative models for this subcellular distribution are discussed.

Male specific expression suggests role of DMRT1 in human sex determination.

Sex determination in mammals is controlled by various transcription factors. Following the identification of SRY on the Y chromosome, several other factors have been identified. They can normally be identified as being involved in sex determination by the identification of sex reversal mutations or deletions, functional studies, and also by male-specific expression patterns in embryos. Here, it is shown that DMRT1, recently demonstrated to be deleted in 9p monosomies associated with sex reversal, is specifically expressed during sex determination in the genital ridge of human male, but not female, embryos, similar to SRY.

[Sex determination in mammals: state of the art]. / Détermination du sexe chez les mammifères: état des lieux.

Sex determination relies on the translation of chromosomal sex established at fertilisation into gonadal sex (testis or ovary), and later into somatic sex (male or female) under the control of gonadal hormone secretions. The aim of the current review will be to highlight our knowledge of the key events which, in the presence of a Y chromosome, induce the organisation of the developing epithelial cells located inside the genital ridges into testicular cords. Many groups have tried to define the molecules relevant to this process, with a double goal: unravelling a molecular pathway which leads to cell fate decision (Sertoli cell in this particular case) during development; improving the establishment of a diagnosis and subsequent medical management in cases where chromosomal, gonadal and then somatic sexes are discordant. Recent progress made in this area will be depicted, with the introduction of several pieces to this developmental jigsaw puzzle.

Anosmin-1 is a regionally restricted component of basement membranes and interstitial matrices during organogenesis: implications for the developmental anomalies of X chromosome-linked Kallmann syndrome.

Kallmann syndrome is a developmental disease characterized by gonadotropin-releasing hormone (GnRH) deficiency and olfactory bulb hypoplasia. The gene underlying the X chromosome-linked form, KAL-1, has been identified for several years, yet the pathogenesis of the disease is not understood. By immunohistofluorescence and immunoelectron microscopy, we establish that the KAL-1 encoded protein, anosmin-1, is a transient and regionally restricted component of extracellular matrices during organogenesis in man. Anosmin-1 was detected in the basement membranes and/or interstitial matrices of various structures including bronchial tubes, mesonephric tubules and duct, branches of the ureteric bud, muscular walls of the digestive tract and larger blood vessels, precartilaginous models of skeletal pieces, muscle tendons, head mesenchymes, inner ear, and forebrain subregions. Our results suggest that this protein acts as a local, rather than a long-range, cue during organogenesis. In the olfactory system, anosmin-1 was detected from week 5 onward. The protein was restricted to the olfactory bulb presumptive region and later, to the primitive olfactory bulbs. We therefore suggest that the genetic defect underlying X-linked Kallmann syndrome disrupts the terminal navigation of the early olfactory axons or directly affects the initial steps of olfactory bulb differentiation. The mechanism of the GnRH deficiency is also discussed, relying on the evidence that anosmin-1 is present in the medial walls of the primitive cerebral hemispheres, along the rostro-caudal migratory pathway of the GnRH-synthesizing neurons, at 6 weeks. Finally, the present results strongly suggest that the renal aplasia observed in about one third of the affected individuals results from primary failure of the collecting duct system.

Direct interaction of SRY-related protein SOX9 and steroidogenic factor 1 regulates transcription of the human anti-Müllerian hormone gene.

For proper male sexual differentiation, anti-Müllerian hormone (AMH) must be tightly regulated during embryonic development to promote regression of the Müllerian duct. However, the molecular mechanisms specifying the onset of AMH in male mammals are not yet clearly defined. A DNA-binding element for the steroidogenic factor 1 (SF-1), a member of the orphan nuclear receptor family, located in the AMH proximal promoter has recently been characterized and demonstrated as being essential for AMH gene activation. However, the requirement for a specific promoter environment for SF-1 activation as well as the presence of conserved cis DNA-binding elements in the AMH promoter suggest that SF-1 is a member of a combinatorial protein-protein and protein-DNA complex. In this study, we demonstrate that the canonical SOX-binding site within the human AMH proximal promoter can bind the transcription factor SOX9, a Sertoli cell factor closely associated with Sertoli cell differentiation and AMH expression. Transfection studies with COS-7 cells revealed that SOX9 can cooperate with SF-1 in this activation process. In vitro and in vivo protein-binding studies indicate that SOX9 and SF-1 interact directly via the SOX9 DNA-binding domain and the SF-1 C-terminal region, respectively. We propose that the two transcription factors SOX9 and SF-1 could both be involved in the expression of the AMH gene, in part as a result of their respective binding to the AMH promoter and in part because of their ability to interact with each other. Our work thus identifies SOX9 as an interaction partner of SF-1 that could be involved in the Sertoli cell-specific expression of AMH during embryogenesis.

Steroidogenic factor-1 regulates transcription of the human anti-müllerian hormone receptor.

Anti-müllerian hormone type II receptor (AMHRII) is a serine/threonine receptor and a member of type II receptors of the transforming growth factor beta superfamily. AMHRII has been recently identified in humans, mice, rats, and rabbits. In the male embryo, the AMHRII gene has been shown to be expressed in Sertoli's cells, in Leydig's cells and in the mesenchymal cells surrounding the müllerian duct. To determine the functional region of the AMHRII promoter as well as the factors controlling AMHRII gene expression, we used a 1.1-kilobase DNA fragment from the 5'-flanking region of the human AMHRII gene to generate a series of deletion or mutation and analyzed the resulting transcriptional activities after transfection of the NT2/D1 teratocarcinoma cell line. Our results indicate that maximal expression of the AMHRII promoter in this particular cell line, a cell line positive for endogenous AMHRII expression, requires a conserved estrogen receptor half-site element (AGGTCA) identical to the binding element for steroidogenic factor-1 (SF-1). Studies of this SF-1 binding element using gel mobility shift, antibody supershift assays, and transient transfections of reporter constructs indicate that SF-1 can bind and transactivate the AMHRII promoter. Finally, SF-1 protein expression in human male embryos was shown to display a good coincidence with the previously reported AMHRII expression profile. We then propose that SF-1 may be a key transcriptional regulator of AMHRII gene expression during early human development.

Phosphorylation of an N-terminal motif enhances DNA-binding activity of the human SRY protein.

Of the several strategies that eukaryotes have evolved to modulate transcription factor activity, phosphorylation is regarded as one of the major mechanisms in signal-dependent transcriptional control. To conclusively demonstrate that the human sex-determining gene SRY is affected by such a post-translational control mechanism, we have analyzed its phosphorylation status in living cells. In the present study, we show that the cyclic AMP-dependent protein kinase (PKA) phosphorylates the human SRY protein in vitro as well as in vivo on serine residues located in the N-terminal part of the protein. This phosphorylation event was shown to positively regulate SRY DNA-binding activity and to enhance the ability of SRY to inhibit a basal promoter activity located downstream of an SRY DNA-binding site concatamer. Together these results strongly support the hypothesis that human SRY is a natural substrate for PKA in vivo and that this phosphorylation significantly modulates its major activity, DNA-binding, thereby possibly altering its biological function.

The human testis determining factor SRY binds a nuclear factor containing PDZ protein interaction domains.

The human Y-linked testis determining gene SRY encodes a protein with a DNA binding domain from the high mobility group box family. To date, no function has been assigned to amino acid sequences located outside this DNA binding motif. Here, we identify in a yeast two-hybrid screen a PDZ protein termed SIP-1, as an interacting protein with human SRY. In vitro, biochemical analysis, immunoprecipitation experiments, as well as expression of SIP-1 in human embryonic testis confirm that the two proteins can interact together. Interacting domains were mapped to the C-terminal seven amino acids of SRY and to the PDZ domains of SIP-1, respectively. We hypothesize that SIP-1 could connect SRY to other transcription factors providing SRY for its missing trans-regulation domain.

Distribution of calretinin mRNA in the vestibular nuclei of rat and guinea pig and the effects of unilateral labyrinthectomy: a non-radioactive in situ hybridization study.

We examined the localization of neurons expressing mRNA for calretinin, a cytosolic EF hand calcium-binding protein, throughout the vestibular nuclei of rat and guinea pig by non-radioactive in situ hybridization, using an alkaline phosphatase labeled oligonucleotide probe. Labeled cells were particularly numerous in the medial vestibular nucleus (mVN) and their distribution was similar in rat and guinea pig, and presented a characteristic rostrocaudal and mediolateral pattern. The effects of hemilabyrinthectomy were assessed at various times post lesion from 10 h to 30 days by comparison of the pattern of labeling in the ipsi- and contra-lateral vestibular nuclei of guinea pig. After up to 48 h no modification in the calretinin mRNA distribution was detected. After 3 to 30 days of survival, there was a decrease (about 30%) of the calretinin expressing neurons in the nucleus on the side of the lesion. The unilateral sensory deprivation seemed to induce a permanent asymmetry in the expression of calretinin which was not abolished after vestibular compensation. These results suggested that the calretinin expression in these neurons depends upon the integrity and activity of sensorineuronal peripheral vestibular influences.

Localization of calretinin mRNA in rat and guinea pig inner ear by in situ hybridization using radioactive and non-radioactive probes.

The localization of calretinin mRNA was studied in the rat and guinea pig inner ear by in situ hybridization, and compared to the distribution of the protein previously examined by immunocytochemistry. Radioactive and non-radioactive in situ hybridization (ISH) were performed using oligonucleotide probes labelled with 35S or digoxigenin. Radioactive ISH was more sensitive than non-radioactive ISH. In cochlear and vestibular ganglia, calretinin mRNA was localized in subpopulations of neurons with patterns of distribution similar to those shown by immunocytochemistry. By contrast, the observations in the sensory epithelia differed with the two techniques, ISH revealing less positive structures than immunocytochemistry. Rat inner hair cells and guinea pig inner hair cells, Hensen's cells and Deiters cells, which had been described strongly immunoreactive, appeared positive with radioactive but not with non-radioactive ISH. On the other hand, rat vestibular type II hair cells and guinea pig interdental cells of the spiral limbus which were faintly immunoreactive were not positive with both ISH techniques.

Detection of calbindin-D 28k mRNA in rat vestibular ganglion neurons by in situ hybridization.

The cellular distribution of calbindin-D 28k mRNA in the rat vestibular ganglion was examined by in situ hybridization. Using a [35S]cDNA probe a neuronal subpopulation expressing calbindin-D 28k mRNA with a strong intensity has been identified. These findings confirm the presence of a subclass of calbindin-immunoreactive neurons in the rat vestibular ganglion.

Glutamate-like immunoreactivity in the peripheral vestibular system of mammals.

Using a specific antibody raised against glutamate (Glu) conjugated to bovine serum albumin with glutaraldehyde, the distribution of Glu-like immunoreactivity was studied by postembedding staining in semithin sections of nonosmicated or osmicated tissue through the vestibular sensory epithelia and ganglia of different mammalian species (mouse, rat and cat). Strong immunoreactive staining was found in all ganglion neurons and their peripheral and central nerve processes as well as in the two types of sensory hair cells whereas, in contrast, supporting cells were devoid of immunoreactivity. Glu-like immunoreactivity found in vestibular fibers and ganglion neurons, is in good agreement with the proposition of glutamate as the neurotransmitter involved in vestibular nerve transmission. In sensory hair cells, glutamate, apart from its metabolic function, may play a role in synaptic transmission between the sensory cells and the vestibular afferent fibers.

Immuno-electronmicroscopic localization of 'vitamin D-dependent' calcium-binding protein (CaBP-28k) in the vestibular hair cells of the cat.

The PAP immunohistochemical method was used to carry out a light- and electronmicroscopic study of the distribution of the vitamin D-dependent calcium-binding protein (CaBP-28k, calbindin, cholecalcin) in the vestibule of the young cat. It was found that the two types of hair cells, types I and II, were stained differently. Type II cells were intensely immunoreactive and their staining did not vary with the location of the cells within the crista ampullaris. Type I cells at the top of the cristae were lightly stained, or unstained, while the type I cells laterally or basally were frequently intensely stained. The nerve fibers arriving at the top of the cristae are highly immunoreactive while the fibers of the base are not stained. Immunostaining for CaBP was correlated with differences in the innervation of hair cells at the top and base of the cristae. This differential CaBP-immunostaining may reflect differences in the physiological activity of the cells. The electronmicroscopic study showed that CaBP is present throughout the cytoplasm of the hair cells but that its concentration was particularly high in the cuticular plate and stereocilia. This specific intracellular distribution of CaBP is discussed with the possible role of Ca2+ in the physiology of the vestibular hair cells.