title: Gene expression profile during testicular development in patients with SRY-negative 46,XX testicular disorder of sex development.
abstract: OBJECTIVE
  To elucidate alternative pathways in testicular development, we attempted to clarify the genetic characteristics of SRY-negative XX testes.
  
  
  METHODS
  We previously reported 5 cases of SRY-negative 46,XX testicular disorders of sex development and demonstrated that coordinated expression of genes such as SOX9, SOX3, and DAX1 was associated with testicular development. We performed a case-control study between the aforementioned boy with 46,XX testicular disorders of sex development and an age-matched patient with hydrocele testis (46,XY). During their consecutive surgeries, testicular biopsy specimens were obtained. Genes with differential expression compared with XY testis were identified using polymerase chain reaction (PCR)-based subtractive hybridization and sequencing. For validation of differential gene expression, real-time RT-PCR was performed using gene-specific primers. The distribution of candidate proteins in the testicular tissue was clarified by immunohistochemistry in human and rodent specimens. Moreover, in vitro inhibitory assays were performed.
  
  
  RESULTS
  We identified 13 upregulated and 7 downregulated genes in XX testis. Among the candidate genes, we focused on ROCK1 (Rho-associated, coiled-coil protein kinase 1) in the upregulated gene group, because high expression in XX testis was validated by real-time RT-PCR. ROCK1 protein was detected in germ cells, Leydig cells, and Sertoli cells by immunohistochemistry. Moreover, the addition of specific ROCK1 inhibitor to Sertoli cells decreased SOX9 gene expression.
  
  
  CONCLUSIONS
  On the basis of in vitro inhibitory assay, it is suggested that ROCK1 phosphorylates and activates SOX9 in Sertoli cells. Testes formation might be initiated by an alternative signaling pathway attributed to ROCK1, not SRY, activation in XX testes.
authors.affiliation: Department of Nephro-Urology, Nagoya City University Graduate School of Medical Sciences, Nagoya, Aichi, Japan. Electronic address: kmizuno@med.nagoya-cu.ac.jp.
authors.forename: Kentaro
authors.initials: K
authors.lastname: Mizuno
authors.forename: Yoshiyuki
authors.initials: Y
authors.lastname: Kojima
authors.forename: Hideyuki
authors.initials: H
authors.lastname: Kamisawa
authors.forename: Yoshinobu
authors.initials: Y
authors.lastname: Moritoki
authors.forename: Hidenori
authors.initials: H
authors.lastname: Nishio
authors.forename: Kenjiro
authors.initials: K
authors.lastname: Kohri
authors.forename: Yutaro
authors.initials: Y
authors.lastname: Hayashi
mesh_terms: D058531:46, XX Testicular Disorders of Sex Development / Q000235:genetics* / Q000378:metabolism; D015536:Down-Regulation; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental; D006801:Humans; D017403:In Situ Hybridization; D008297:Male; D010766:Phosphorylation; D060888:Real-Time Polymerase Chain Reaction; D055758:SOX9 Transcription Factor / Q000378:metabolism; D012708:Sertoli Cells / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D006848:Testicular Hydrocele / Q000235:genetics / Q000378:metabolism; D013737:Testis / Q000254:growth & development* / Q000473:pathology; D054460:rho-Associated Kinases / Q000378:metabolism*
chemical_list: D055758:SOX9 Transcription Factor; C527932:SOX9 protein, human; C515851:ROCK1 protein, human; D054460:rho-Associated Kinases