Tenuous Transcriptional Threshold of Human Sex Determination. I. SRY and Swyer Syndrome at the Edge of Ambiguity.

Male sex determination in mammals is initiated by SRY, a Y-encoded transcription factor. The protein contains a high-mobility-group (HMG) box mediating sequence-specific DNA bending. Mutations causing XY gonadal dysgenesis (Swyer syndrome) cluster in the box and ordinarily arise de novo. Rare inherited variants lead to male development in one genetic background (the father) but not another (his sterile XY daughter). De novo and inherited mutations occur at an invariant Tyr adjoining the motif's basic tail (box position 72; Y127 in SRY). In SRY-responsive cell lines CH34 and LNCaP, de novo mutations Y127H and Y127C reduced SRY activity (as assessed by transcriptional activation of principal target gene Sox9) by 5- and 8-fold, respectively. Whereas Y127H impaired testis-specific enhancer assembly, Y127C caused accelerated proteasomal proteolysis; activity was in part rescued by proteasome inhibition. Inherited variant Y127F was better tolerated: its expression was unperturbed, and activity was reduced by only twofold, a threshold similar to other inherited variants. Biochemical studies of wild-type (WT) and variant HMG boxes demonstrated similar specific DNA affinities (within a twofold range), with only subtle differences in sharp DNA bending as probed by permutation gel electrophoresis and fluorescence resonance-energy transfer (FRET); thermodynamic stabilities of the free boxes were essentially identical. Such modest perturbations are within the range of species variation. Whereas our cell-based findings rationalize the de novo genotype-phenotype relationships, a molecular understanding of inherited mutation Y127F remains elusive. Our companion study uncovers cryptic biophysical perturbations suggesting that the para-OH group of Y127 anchors a novel water-mediated DNA clamp.

Transcriptional analysis of the multiple Sry genes and developmental program at the onset of testis differentiation in the rat.

BACKGROUND: The commonly used laboratory rat, Rattus norvegicus, is unique in having multiple Sry gene copies found on the Y chromosome, with different copies encoding amino acid variations that influence the resulting protein function. It is not clear which Sry genes are expressed at the onset of testis differentiation or how their expression correlates with that of other genes in testis-determination pathways. METHODS: Here, two independent E11-E14 developmental RNAseq datasets show that multiple Sry genes are expressed at E12-E13. RESULTS: The identified copies expressed during testis initiation include Sry4A, Sry1, and Sry3C, which are conserved in every strain of Rattus norvegicus with genomes sequenced to date. CONCLUSIONS: This work represents a first step in defining the complex environment of rat testis differentiation that can open the door for generating sex reversal model systems using embryo manipulation techniques that have been available in the mouse but not the rat.

Wnt signaling in testis development: Unnecessary or essential?

Testis development is a fundamental process in sexual development and reproduction. It is under the regulation of multiple factors. Wnt signaling pathway is a classical pathway, which plays an essential role during early development. From a traditional view, Wnt signaling serves as a key regulator of female reproductive system. However, its role in testis development is relatively controversial. This paper reviews Wnt signaling's part in the major events during testis development, including primordial germ cell specification, proliferation and migration, testis determination, spermatogenesis and somatic cell regulation, and summarizes Wnt signaling's impact on testis-related disorders. We evaluate the outcomes of current studies in the field and suggest future research directions.

The novel p.E89K mutation in the SRY gene inhibits DNA binding and causes the 46,XY disorder of sex development

Male sex determination in humans is controlled by the SRY gene, which encodes a transcriptional regulator containing a conserved high mobility group box domain (HMG-box) required for DNA binding. Mutations in the SRY HMG-box affect protein function, causing sex reversal phenotypes. In the present study, we describe a 19-year-old female presenting 46,XY karyotype with hypogonadism and primary amenorrhea that led to the diagnosis of 46,XY complete gonadal dysgenesis. The novel p.E89K missense mutation in the SRY HMG-box was identified as a de novo mutation. Electrophoretic mobility shift assays showed that p.E89K almost completely abolished SRY DNA-binding activity, suggesting that it is the cause of SRY function impairment. In addition, we report the occurrence of the p.G95R mutation in a 46,XY female with complete gonadal dysgenesis. According to the three-dimensional structure of the human SRY HMG-box, the substitution of the conserved glutamic acid residue by the basic lysine at position 89 introduces an extra positive charge adjacent to and between the positively charged residues R86 and K92, important for stabilizing the HMG-box helix 2 with DNA. Thus, we propose that an electrostatic repulsion caused by the proximity of these positive charges could destabilize the tip of helix 2, abrogating DNA interaction.