Yoshinobu Moritoki1, Yutaro Hayashi2, Kentaro Mizuno1, Hideyuki Kamisawa1, Hidenori Nishio1, Satoshi Kurokawa1, Shinya Ugawa3, Yoshiyuki Kojima4, Kenjiro Kohri1. 1. Department of Nephro-Urology, Graduate School of Medical Sciences, Nagoya City University, Nagoya, Japan. 2. Department of Nephro-Urology, Graduate School of Medical Sciences, Nagoya City University, Nagoya, Japan. Electronic address: yutaro@med.nagoya-cu.ac.jp. 3. Department of Neurobiology and Anatomy, Graduate School of Medical Sciences, Nagoya City University, Nagoya, Japan. 4. Department of Urology, Fukushima Medical University, Fukushima, Japan.
Abstract
PURPOSE: We elucidated the mechanism of spermatogonial stem cell disturbance of cryptorchidism and investigated the expression of miRNAs and their target genes in undescended testes. MATERIALS AND METHODS: Using microarray analysis we compared total miRNA expression in unilateral undescended testes with that in contralateral descended and normal testes in a rat model of cryptorchidism. The model was derived by administering flutamide to pregnant Sprague Dawley® rats. We identified mRNA targets of miRNAs by bioinformatic analysis, followed by in situ hybridization and immunohistochemistry to localize candidate miRNAs and mRNAs, respectively. We also investigated whether miRNAs could inhibit target protein expression in vitro. RESULTS: Microarray analysis and subsequent quantitative reverse transcriptase-polymerase chain reaction showed that only miR-135a was expressed at a lower level in undescended testes. We identified its target as FoxO1, which is essential for stem cell maintenance. miR-135a and FoxO1 localized to spermatogonial stem cells. FoxO1 localized to the spermatogonial stem cell nucleus less frequently in undescended testes, indicating that the activity of FoxO1, which acts as a transcription factor, is altered in undescended testes. Finally, miR-135a transfection into spermatogonia in vitro resulted in down-regulation of FoxO1 expression. CONCLUSIONS: In cryptorchid testes there is a decreased number of spermatogonial stem cells in which FoxO1 is activated, indicating that failure of spermatogonial stem cell maintenance results in spermatogenesis alteration. We also noted interaction between miR-135a and FoxO1, and propose that miR-135a contributes to spermatogonial stem cell maintenance through modulation of FoxO1 activity.
PURPOSE: We elucidated the mechanism of spermatogonial stem cell disturbance of cryptorchidism and investigated the expression of miRNAs and their target genes in undescended testes. MATERIALS AND METHODS: Using microarray analysis we compared total miRNA expression in unilateral undescended testes with that in contralateral descended and normal testes in a rat model of cryptorchidism. The model was derived by administering flutamide to pregnant Sprague Dawley® rats. We identified mRNA targets of miRNAs by bioinformatic analysis, followed by in situ hybridization and immunohistochemistry to localize candidate miRNAs and mRNAs, respectively. We also investigated whether miRNAs could inhibit target protein expression in vitro. RESULTS: Microarray analysis and subsequent quantitative reverse transcriptase-polymerase chain reaction showed that only miR-135a was expressed at a lower level in undescended testes. We identified its target as FoxO1, which is essential for stem cell maintenance. miR-135a and FoxO1 localized to spermatogonial stem cells. FoxO1 localized to the spermatogonial stem cell nucleus less frequently in undescended testes, indicating that the activity of FoxO1, which acts as a transcription factor, is altered in undescended testes. Finally, miR-135a transfection into spermatogonia in vitro resulted in down-regulation of FoxO1 expression. CONCLUSIONS: In cryptorchid testes there is a decreased number of spermatogonial stem cells in which FoxO1 is activated, indicating that failure of spermatogonial stem cell maintenance results in spermatogenesis alteration. We also noted interaction between miR-135a and FoxO1, and propose that miR-135a contributes to spermatogonial stem cell maintenance through modulation of FoxO1 activity.
Authors: Eljo Y van Battum; Marieke G Verhagen; Vamshidhar R Vangoor; Yuki Fujita; Alwin A H A Derijck; Eoghan O'Duibhir; Giuliano Giuliani; Thijs de Gunst; Youri Adolfs; Daphne Lelieveld; David Egan; Roel Q J Schaapveld; Toshihide Yamashita; R Jeroen Pasterkamp Journal: J Neurosci Date: 2017-12-01 Impact factor: 6.167