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Expression analyses to identify genes involved in testis-specific vascular development

ABSTRACT: One of the earliest morphological changes during testicular differentiation is the establishment of an XY specific vasculature. The testis vascular system is derived from mesonephric endothelial cells that migrate into the gonad. In the XX gonad, mesonephric cell migration and testis vascular development is inhibited by WNT4 signaling. In Wnt4 mutant XX gonads, endothelial cells migrate from the mesonephros and form a male-like coelomic vessel. Consequently, the XX Wnt4 mutant mice presented an opportunity to focus a gene expression screen on the processes of mesonephric cell migration and testicular vascular development. We compared differences in gene expression between XY Wnt4+/+ and XX Wnt4+/+ gonads and between XX Wnt4-/- and XX Wnt4+/+ gonads to identify genes similarly upregulated in wildtype XY gonads and XX mutant gonads that might play important roles in testis-specific vascular development. Keywords: genetic sex and phenotypic comparisons

ORGANISM(S): Mus musculus

PROVIDER: GSE3650 | GEO | 2005/11/24

SECONDARY ACCESSION(S): PRJNA93775

REPOSITORIES: GEO

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Project description:Sry is sufficient to induce testis formation and subsequent male development of internal and external genitalia in chromosomally female mice and humans. In XX sex-reversed males such as XX/Sry-transgenic (XX/Sry) mice, however, testicular germ cells always disappear soon after birth due to germ cell autonomous defects. Therefore, it remains unclear whether or not Sry alone is sufficient to induce a fully functional testicular soma capable of supporting complete spermatogenesis in the XX body. Here we demonstrated that the testicular somatic environment of XX/Sry males is defective in the later phases of spermatogenesis. Spermatogonial transplantation analyses using XX/Sry male mice revealed that donor XY spermatogonia are capable of proliferating, entering meiosis and differentiating into the round spermatid stage. XY donor-derived round spermatids, however, were frequently detached from the XX/Sry seminiferous epithelia and underwent cell death, thereby preventing further progress beyond the elongated spermatid stage. In contrast, immature XY seminiferous tubule segments transplanted under XX/Sry testis capsules clearly displayed proper differentiation into elongated spermatids in the transplanted XY donor tubules. Microarray analysis of seminiferous tubules isolated from XX/Sry testes confirmed missing expression of several Y-linked genes and alterations in the expression profile of genes associated with spermatogenesis. Therefore, our findings indicate dysfunction of the somatic tubule components, probably Sertoli cells, of XX/Sry testes, supporting our hypothesis that Sry alone is insufficient to induce a fully functional Sertoli cell in XX mice. Experiment Overall Design: Whole testes and seminiferous tubules of XX/Sry and W/Wv males were used for microarray expression analysis using the Affymetrix GeneChip system (Affymetrix, CA). In order to isolate the seminiferous tubules, the tunica was carefully removed from the testes which were then incubated in the medium with 5 mg/ml collagnease at 37oC for 40 min. The remaining seminiferous tubules were washed several times with PBS using a 70-Âºm cell strainer to remove interstitial cells. After total RNA was extracted using a RNeasy Mini Kit (Qiagen, Germantown, MD), double-stranded cDNA and biotin-labeled cRNA were synthesized using One-Cycle cDNA Synthesis and IVT Labeling kits (Affymetrix, CA), respectively. Twenty micrograms of fragmented biotin-labeled cRNA was hybridized to the Affymetrix Mouse Expression Array MOE 430A for 16 hr at 45oC. The chips were washed, stained, and then scanned with the GeneArray Scanner (Hewlett Packard, CA) in accordance with the manufacturer's standard protocols. Finally, the microarray data were analyzed using Microarray Suite ver. 5.0 (Affymetrix). Differential expression was defined as a difference of 2-fold or more in both whole testis and seminiferous tubule samples between two recipient males. Mouse 430A Affymetrix Genome Array IDs were used to query the NetAffx data mining tool for gene annotations.

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Expression analyses to identify genes involved in testis-specific vascular development

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