In mammals, the Y-linked sex-determining gene Sry cell-autonomously promotes Sertoli cell differentiation from bipotential supporting cell precursors through SRY-box containing gene 9 (Sox9), leading to testis formation. Without Sry action, the supporting cells differentiate into granulosa cells, resulting in ovarian development. However, how Sry acts spatiotemporally to switch supporting cells from the female to the male pathway is poorly understood. We created a novel transgenic mouse line bearing an inducible Sry transgene under the control of the Hsp70.3 promoter. Analysis of these mice demonstrated that the ability of Sry to induce testis development is limited to approximately 11.0-11.25 dpc, corresponding to a time window of only 6 hours after the normal onset of Sry expression in XY gonads. If Sry was activated after 11.3 dpc, Sox9 activation was not maintained, resulting in ovarian development. This time window is delimited by the ability to engage the high-FGF9/low-WNT4 signaling states required for Sertoli cell establishment and cord organization. Our results indicate the overarching importance of Sry action in the initial 6-hour phase for the female-to-male switching of FGF9/WNT4 signaling patterns.

Developmental gene expression is defined through cross-talk between the function of transcription factors and epigenetic status, including histone modification. Although several transcription factors play crucial roles in mammalian sex determination, how epigenetic regulation contributes to this process remains unknown. We observed male-to-female sex reversal in mice lacking the H3K9 demethylase Jmjd1a and found that Jmjd1a regulates expression of the mammalian Y chromosome sex-determining gene Sry. Jmjd1a directly and positively controls Sry expression by regulating H3K9me2 marks. These studies reveal a pivotal role of histone demethylation in mammalian sex determination.

Sox7, Sox17 and Sox18 constitute group F of the Sox family of HMG box transcription factor genes. Dominant-negative mutations in Sox18 underlie the cardiovascular defects observed in ragged mutant mice. By contrast, Sox18-/- mice are viable and fertile, and display no appreciable anomaly in their vasculature, suggesting functional compensation by the two other SoxF genes. Here, we provide direct evidence for redundant function of Sox17 and Sox18 in postnatal neovascularization by generating Sox17+/--Sox18-/- double mutant mice. Whereas Sox18-/- and Sox17+/--Sox18+/- mice showed no vascular defects, approximately half of the Sox17+/--Sox18-/- pups died before postnatal day 21 (P21). They showed reduced neovascularization in the liver sinusoids and kidney outer medulla vasa recta at P7, which most likely caused the ischemic necrosis observed by P14 in hepatocytes and renal tubular epithelia. Those that survived to adulthood showed similar, but milder, vascular anomalies in both liver and kidney, and females were infertile with varying degrees of vascular abnormalities in the reproductive organs. These anomalies corresponded with sites of expression of Sox7 and Sox17 in the developing postnatal vasculature. In vitro angiogenesis assays, using primary endothelial cells isolated from the P7 livers, showed that the Sox17+/--Sox18-/- endothelial cells were defective in endothelial sprouting and remodeling of the vasculature in a phenotype-dependent manner. Therefore, our findings indicate that Sox17 and Sox18, and possibly all three SoxF genes, are cooperatively involved in mammalian vascular development.

During the last decade, a novel mechanism of protein release has been recognized that involves small (30-100 nm) membrane vesicles termed exosomes. [1][2][3] Exosomal vesicles are secreted following the fusion of multivesicular late endosomes with the plasma membrane. While the range of exosomal proteins depends on cell type, these vesicles commonly carry cell-surface proteins and cytoskeletal proteins. Several physiologic roles have been assigned to exosomes, including the expulsion of obsolete membrane constituents, exchange of cellular material and intercellular communication. Exosome production has been observed in a variety of cell types in vitro, including reticulocytes, 4) cytotoxic T lymphocytes, 5)B lymphocytes, 6) dendritic cells 7) and neoplastic intestinal epithelial cells.8) Recent studies have reported that such vesicles are present in some physiological fluids, such as bronchoalveolar lavage 9) or urine. 10) However, there is still little evidence of whether exosomes are produced in vivo.Saliva is considered to provide the first line of oral cavity defense against bacterial and viral attack. Human whole saliva contains a potent mixture of diverse components such as mucin, immunoglobulin A (IgA), proline-rich proteins and defensins, which are produced in three major paired salivary glands (parotid, submadibular and sublingual) and several minor glands. Although comprehensive proteome analyses of whole saliva have been reported recently, 12,13) the protein constituents of saliva are not fully understood.The membrane-associated serine protease dipeptidyl peptidase IV (DPP IV), which is identical to the lymphocyte surface glycoprotein CD26, cleaves dipeptides from the N-terminus of peptides with a proline or alanine residue in the penultimate position.14) CD26/DPP IV is highly expressed on fibroblasts, epithelial and endothelial cells, and specific leukocyte subsets. The extracellular protease domain of CD26/DPP IV, which is produced by proteolytic cleavage of the membrane-bound form of CD26/DPP IV, also exists in a soluble form in plasma.15) Recently, it was reported that DPP IV is released from intestinal epithelial cells into the extracellular milieu as a constituent of exosome-like vesicles. 8)More recently, our previous report revealed that DPP IV is released into snake venom in an unprocessed form, 16) suggesting that the DPP IV is associated with membrane. In addition, we have found exosome-like vesicles that carry DPP IV in snake venom.17) DPP IV activity has been found in human saliva, 18) but the mechanism of its release has not been elucidated. Snake venom is highly modified saliva that is produced by special glands of certain species of snakes. Therefore, we hypothesized that exosome-like vesicles carrying DPP IV could be present in human saliva. We demonstrate for the first time a population of vesicles in human whole saliva similar in size to the previously described exosomes. We found that these vesicles contain DPP IV, galectin-3 and IgA. MATERIALS AND METHODS MaterialsGly-Pro-4-methyl...

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Abstract. Two different mRNA isoforms of the mouse Soxl 7 gene were isolated from adult mouse testis cDNAs. One form (referred to as form Sox17) encodes an Styrelated protein of 419 amino acids containing a single high mobility group box near the NH2 terminus, while the other form (referred to as form t-Sox17) shows a unique mRNA isoform of the Soxl 7 gene with a partial deletion of the HMG box region. Analysis of genomic DNA revealed that these two isoforms were produced at least by alternative splicing of the exon corresponding to the 5' untranslated region and NH2-terminal 102 amino acids. RNA analyses in the testis revealed that form Soxl7 is expressed in spermatogonia, and the expression clearly declines from the early pachytene spermatocyte stage onward. In contrast, expression of form t-Soxl7 began at the pachytene spermatocyte stage and was highly accumulated in round spermatids. Protein analyses revealed that t-Soxl7 isoforms, as well as Sox17 isoforms, were translated into the protein products in the testis, although the amount of t-Soxl7 products is lower in comparison to the high accumulation of t-Soxl7 mRNA. By the electrophoretic mobility-shift assay and the random selection assay using recombinant Soxl7 and t-Sox17 proteins, Soxl7 protein is a DNA-binding protein with a similar sequence specificity to Sry and the other members of Sox family proteins, while t-Soxl7 shows no apparent DNA-binding activity. Moreover, by a cotransfection experiment using a luciferase reporter gene, Soxl7 could stimulate transcription through its binding site, but t-Soxl7 had little effect on reporter gene expression. Thus, these findings suggest that Soxl7 may function as a transcriptional activator in the premeiotic germ cells, and that a splicing switch into t-Soxl7 may lead to the loss of its function in the postmeiotic germ cells.