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Androgen-responsive microRNAs in mouse Sertoli cells.

Panneerdoss S, Chang YF, Buddavarapu KC, Chen HI, Shetty G, Wang H, Chen Y, Kumar TR, Rao MK - PLoS ONE (2012)

Bottom Line: This is in part because only a few androgen-responsive genes have been definitively identified in the testis.Here, we propose that microRNAs--small, non-coding RNAs--are one class of androgen-regulated trans-acting factors in the testis.Our results reveal that several of these microRNAs are preferentially expressed in the testis and regulate genes that are highly expressed in Sertoli cells.

View Article: PubMed Central - PubMed

Affiliation: Greehey Children's Cancer Research Institute, The University of Texas Health Science Center at San Antonio, San Antonio, Texas, United States of America.

ABSTRACT
Although decades of research have established that androgen is essential for spermatogenesis, androgen's mechanism of action remains elusive. This is in part because only a few androgen-responsive genes have been definitively identified in the testis. Here, we propose that microRNAs--small, non-coding RNAs--are one class of androgen-regulated trans-acting factors in the testis. Specifically, by using androgen suppression and androgen replacement in mice, we show that androgen regulates the expression of several microRNAs in Sertoli cells. Our results reveal that several of these microRNAs are preferentially expressed in the testis and regulate genes that are highly expressed in Sertoli cells. Because androgen receptor-mediated signaling is essential for the pre- and post-meiotic germ cell development, we propose that androgen controls these events by regulating Sertoli/germ cell-specific gene expression in a microRNA-dependent manner.

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Related in: MedlinePlus

Androgen-dependent expression of FoxD1 in Sertoli cells.(A) Immunohistochemical analysis on testis sections from Sham, flutamide-acyline (Flut+Acy) and flutamide-acyline testosterone-replacement (Flut+Acy+T) mice, using antibodies against Foxd1 (1∶50) and Sox9 (1∶500). Insets show Sertoli cell-specific expression of Foxd1 in magnified testicular section from Sham, Flut+Acy, and Flut+Acy+T mice. (B) Real-time PCR analysis of Foxd1 transcript levels in total cellular RNA isolated from the purified Sertoli cells from Sham, Flut+Acy, and Flut+Acy+T mice testes. All values are normalized against RNU19 levels. (C) Western blot analysis on total testicular lysates from Sham, Flut+Acy, and Flut+Acy+T mice by using anti-Foxd1 antibody (1∶1000). Tubulin was used as a loading control. Values below the gel were quantified using the Image J software. Foxd1 protein level for the control was set to 1. Arrowheads indicate Sertoli cells. All images were taken at magnification of 600×.
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pone-0041146-g006: Androgen-dependent expression of FoxD1 in Sertoli cells.(A) Immunohistochemical analysis on testis sections from Sham, flutamide-acyline (Flut+Acy) and flutamide-acyline testosterone-replacement (Flut+Acy+T) mice, using antibodies against Foxd1 (1∶50) and Sox9 (1∶500). Insets show Sertoli cell-specific expression of Foxd1 in magnified testicular section from Sham, Flut+Acy, and Flut+Acy+T mice. (B) Real-time PCR analysis of Foxd1 transcript levels in total cellular RNA isolated from the purified Sertoli cells from Sham, Flut+Acy, and Flut+Acy+T mice testes. All values are normalized against RNU19 levels. (C) Western blot analysis on total testicular lysates from Sham, Flut+Acy, and Flut+Acy+T mice by using anti-Foxd1 antibody (1∶1000). Tubulin was used as a loading control. Values below the gel were quantified using the Image J software. Foxd1 protein level for the control was set to 1. Arrowheads indicate Sertoli cells. All images were taken at magnification of 600×.

Mentions: Next we sought to determine whether testosterone indeed controls expression of Foxd1 and Dsc1 in Sertoli cell in vivo. We performed immunohistochemical analysis on testicular sections from sham-treated control, flutamide-acyline-treated and flutamide-treated testosterone-replacement mice. In comparison with control mice, flutamide treatment significantly lowered Foxd1 and Dsc1, whereas testosterone-replacement rescued their expressions in the Sertoli cells (Figure 6A and Figure 7A). In contrast to Foxd1 and Dsc1, expression of Sertoli cell-specific protein Sox9, which is not regulated by the androgen in the adult testis, showed no change in flutamide-acyline-treated or testosterone-supplemented groups when compared with the sham-treated control group. Supporting this, real-time RT-PCR analysis on the purified Sertoli cells from control, flutamide-acyline-treated and testosterone-supplementation mice testes showed significantly lower Foxd1 and Dsc1 levels in the flutamide-acyline-treated mice when compared to control mice, while testosterone-supplementation rescued their transcripts levels comparable to the control mice (Figure 6B and Figure 7B). Western blot analysis on testicular lysates from control, flutamide-acyline-treated and flutamide-treated androgen-replacement mice further substantiated these findings (Figure 6C and Figure 7C). Taken together, these findings suggest that testosterone-responsive miR-471 regulate expression of Foxd1 and Dsc1, which are expressed in a testosterone-dependent manner in the mouse Sertoli cells. Future studies manipulating the levels of miR-471 in the Sertoli cells in vivo will further validate these findings.


Androgen-responsive microRNAs in mouse Sertoli cells.

Panneerdoss S, Chang YF, Buddavarapu KC, Chen HI, Shetty G, Wang H, Chen Y, Kumar TR, Rao MK - PLoS ONE (2012)

Androgen-dependent expression of FoxD1 in Sertoli cells.(A) Immunohistochemical analysis on testis sections from Sham, flutamide-acyline (Flut+Acy) and flutamide-acyline testosterone-replacement (Flut+Acy+T) mice, using antibodies against Foxd1 (1∶50) and Sox9 (1∶500). Insets show Sertoli cell-specific expression of Foxd1 in magnified testicular section from Sham, Flut+Acy, and Flut+Acy+T mice. (B) Real-time PCR analysis of Foxd1 transcript levels in total cellular RNA isolated from the purified Sertoli cells from Sham, Flut+Acy, and Flut+Acy+T mice testes. All values are normalized against RNU19 levels. (C) Western blot analysis on total testicular lysates from Sham, Flut+Acy, and Flut+Acy+T mice by using anti-Foxd1 antibody (1∶1000). Tubulin was used as a loading control. Values below the gel were quantified using the Image J software. Foxd1 protein level for the control was set to 1. Arrowheads indicate Sertoli cells. All images were taken at magnification of 600×.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3401116&req=5

pone-0041146-g006: Androgen-dependent expression of FoxD1 in Sertoli cells.(A) Immunohistochemical analysis on testis sections from Sham, flutamide-acyline (Flut+Acy) and flutamide-acyline testosterone-replacement (Flut+Acy+T) mice, using antibodies against Foxd1 (1∶50) and Sox9 (1∶500). Insets show Sertoli cell-specific expression of Foxd1 in magnified testicular section from Sham, Flut+Acy, and Flut+Acy+T mice. (B) Real-time PCR analysis of Foxd1 transcript levels in total cellular RNA isolated from the purified Sertoli cells from Sham, Flut+Acy, and Flut+Acy+T mice testes. All values are normalized against RNU19 levels. (C) Western blot analysis on total testicular lysates from Sham, Flut+Acy, and Flut+Acy+T mice by using anti-Foxd1 antibody (1∶1000). Tubulin was used as a loading control. Values below the gel were quantified using the Image J software. Foxd1 protein level for the control was set to 1. Arrowheads indicate Sertoli cells. All images were taken at magnification of 600×.
Mentions: Next we sought to determine whether testosterone indeed controls expression of Foxd1 and Dsc1 in Sertoli cell in vivo. We performed immunohistochemical analysis on testicular sections from sham-treated control, flutamide-acyline-treated and flutamide-treated testosterone-replacement mice. In comparison with control mice, flutamide treatment significantly lowered Foxd1 and Dsc1, whereas testosterone-replacement rescued their expressions in the Sertoli cells (Figure 6A and Figure 7A). In contrast to Foxd1 and Dsc1, expression of Sertoli cell-specific protein Sox9, which is not regulated by the androgen in the adult testis, showed no change in flutamide-acyline-treated or testosterone-supplemented groups when compared with the sham-treated control group. Supporting this, real-time RT-PCR analysis on the purified Sertoli cells from control, flutamide-acyline-treated and testosterone-supplementation mice testes showed significantly lower Foxd1 and Dsc1 levels in the flutamide-acyline-treated mice when compared to control mice, while testosterone-supplementation rescued their transcripts levels comparable to the control mice (Figure 6B and Figure 7B). Western blot analysis on testicular lysates from control, flutamide-acyline-treated and flutamide-treated androgen-replacement mice further substantiated these findings (Figure 6C and Figure 7C). Taken together, these findings suggest that testosterone-responsive miR-471 regulate expression of Foxd1 and Dsc1, which are expressed in a testosterone-dependent manner in the mouse Sertoli cells. Future studies manipulating the levels of miR-471 in the Sertoli cells in vivo will further validate these findings.

Bottom Line: This is in part because only a few androgen-responsive genes have been definitively identified in the testis.Here, we propose that microRNAs--small, non-coding RNAs--are one class of androgen-regulated trans-acting factors in the testis.Our results reveal that several of these microRNAs are preferentially expressed in the testis and regulate genes that are highly expressed in Sertoli cells.

View Article: PubMed Central - PubMed

Affiliation: Greehey Children's Cancer Research Institute, The University of Texas Health Science Center at San Antonio, San Antonio, Texas, United States of America.

ABSTRACT
Although decades of research have established that androgen is essential for spermatogenesis, androgen's mechanism of action remains elusive. This is in part because only a few androgen-responsive genes have been definitively identified in the testis. Here, we propose that microRNAs--small, non-coding RNAs--are one class of androgen-regulated trans-acting factors in the testis. Specifically, by using androgen suppression and androgen replacement in mice, we show that androgen regulates the expression of several microRNAs in Sertoli cells. Our results reveal that several of these microRNAs are preferentially expressed in the testis and regulate genes that are highly expressed in Sertoli cells. Because androgen receptor-mediated signaling is essential for the pre- and post-meiotic germ cell development, we propose that androgen controls these events by regulating Sertoli/germ cell-specific gene expression in a microRNA-dependent manner.

Show MeSH
Related in: MedlinePlus