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Restoration of spermatogenesis and male fertility using an androgen receptor transgene.

Walker WH, Easton E, Moreci RS, Toocheck C, Anamthathmakula P, Jeyasuria P - PLoS ONE (2015)

Bottom Line: The AR-EGFP transgene was expressed in a cyclical manner similar to that of endogenous AR in Sertoli cells and fertility was restored as offspring were produced in the absence of Sertoli cell AR.Thus, the AR-EGFP transgene under the control of AR regulatory elements is capable of rescuing AR function in a cell selective, AR- background.These initial studies provide proof of principle that a strategy employing the AR-EGFP transgene can be used to understand AR functions.

View Article: PubMed Central - PubMed

Affiliation: Center for Research in Reproductive Physiology, Department of Obstetrics, Gynecology and Reproductive Sciences, Magee Women's Research Institute, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America.

ABSTRACT
Androgens signal through the androgen receptor (AR) to regulate male secondary sexual characteristics, reproductive tract development, prostate function, sperm production, bone and muscle mass as well as body hair growth among other functions. We developed a transgenic mouse model in which endogenous AR expression was replaced by a functionally modified AR transgene. A bacterial artificial chromosome (BAC) was constructed containing all AR exons and introns plus 40 kb each of 5' and 3' regulatory sequence. Insertion of an internal ribosome entry site and the EGFP gene 3' to AR allowed co-expression of AR and EGFP. Pronuclear injection of the BAC resulted in six founder mice that displayed EGFP production in appropriate AR expressing tissues. The six founder mice were mated into a Sertoli cell specific AR knockout (SCARKO) background in which spermatogenesis is blocked at the meiosis stage of germ cell development. The AR-EGFP transgene was expressed in a cyclical manner similar to that of endogenous AR in Sertoli cells and fertility was restored as offspring were produced in the absence of Sertoli cell AR. Thus, the AR-EGFP transgene under the control of AR regulatory elements is capable of rescuing AR function in a cell selective, AR- background. These initial studies provide proof of principle that a strategy employing the AR-EGFP transgene can be used to understand AR functions. Transgenic mice expressing selective modifications of the AR-EGFP transgene may provide crucial information needed to elicit the molecular mechanisms by which AR acts in the testis and other androgen responsive tissues.

No MeSH data available.


Related in: MedlinePlus

Characterization of three BACs containing parts of the AR gene.(A) Top: a map of the AR gene and flanking region is shown. Below: maps of the BACs mAR BAC-196 (RP23-102011), mAR BAC-192 (RP24-352D1) and mAR BAC-185 (RP23-316P7) are shown. AR exons are denoted by rectangles on the lines. SalI restiction sites are denoted with an S. Boxes above exons 1 and 8 denote the probes used for Southern blotting. The BACs contained either extended 5’ sequence, the complete AR gene or extended 3’ sequence, respectively. The BACs were end sequenced to determine their insertion sites. (B) Restriction digests followed by PFGE and Southern blotting were performed to ensure that preperations of the BACs were correct. The SalI digested BACs were fractionated by PFGE and subjected to Southern blotting using 32P random primed labelled AR exon 1 and AR exon 8 probes. mAR BAC-192 was positive for both probes while mAR BAC-196 only probed positive for exon 8 and mAR BAC-185 probed positive only for exon1.
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pone.0120783.g001: Characterization of three BACs containing parts of the AR gene.(A) Top: a map of the AR gene and flanking region is shown. Below: maps of the BACs mAR BAC-196 (RP23-102011), mAR BAC-192 (RP24-352D1) and mAR BAC-185 (RP23-316P7) are shown. AR exons are denoted by rectangles on the lines. SalI restiction sites are denoted with an S. Boxes above exons 1 and 8 denote the probes used for Southern blotting. The BACs contained either extended 5’ sequence, the complete AR gene or extended 3’ sequence, respectively. The BACs were end sequenced to determine their insertion sites. (B) Restriction digests followed by PFGE and Southern blotting were performed to ensure that preperations of the BACs were correct. The SalI digested BACs were fractionated by PFGE and subjected to Southern blotting using 32P random primed labelled AR exon 1 and AR exon 8 probes. mAR BAC-192 was positive for both probes while mAR BAC-196 only probed positive for exon 8 and mAR BAC-185 probed positive only for exon1.

Mentions: Three BACs containing all or parts of the gene encoding AR were obtained from the BACPAC Resource Center (BPRC) at Children's Hospital Oakland Research Institute (CHORI, Oakland, CA). One BAC [mAR BAC-192 (RP24-352D1)] contains the entire gene (all exons) for AR. The other two BACs contain either 5’ [mAR BAC-185 (RP23-316P7)] or 3’ [mAR BAC-196 (RP23-102O11)] exons of the AR gene and either extensive 5’ or 3’ flanking regions (Fig. 1A). The BACs containing the AR genomic sequence were end sequenced to establish the positions of the inserts. Pulse field gel electrophoresis (PFGE) in combination with Southern blotting was used to verify the inserts by comparisons of restriction analysis of the BACs with known genomic sequence from the mouse genome project (Fig. 1B).


Restoration of spermatogenesis and male fertility using an androgen receptor transgene.

Walker WH, Easton E, Moreci RS, Toocheck C, Anamthathmakula P, Jeyasuria P - PLoS ONE (2015)

Characterization of three BACs containing parts of the AR gene.(A) Top: a map of the AR gene and flanking region is shown. Below: maps of the BACs mAR BAC-196 (RP23-102011), mAR BAC-192 (RP24-352D1) and mAR BAC-185 (RP23-316P7) are shown. AR exons are denoted by rectangles on the lines. SalI restiction sites are denoted with an S. Boxes above exons 1 and 8 denote the probes used for Southern blotting. The BACs contained either extended 5’ sequence, the complete AR gene or extended 3’ sequence, respectively. The BACs were end sequenced to determine their insertion sites. (B) Restriction digests followed by PFGE and Southern blotting were performed to ensure that preperations of the BACs were correct. The SalI digested BACs were fractionated by PFGE and subjected to Southern blotting using 32P random primed labelled AR exon 1 and AR exon 8 probes. mAR BAC-192 was positive for both probes while mAR BAC-196 only probed positive for exon 8 and mAR BAC-185 probed positive only for exon1.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4372537&req=5

pone.0120783.g001: Characterization of three BACs containing parts of the AR gene.(A) Top: a map of the AR gene and flanking region is shown. Below: maps of the BACs mAR BAC-196 (RP23-102011), mAR BAC-192 (RP24-352D1) and mAR BAC-185 (RP23-316P7) are shown. AR exons are denoted by rectangles on the lines. SalI restiction sites are denoted with an S. Boxes above exons 1 and 8 denote the probes used for Southern blotting. The BACs contained either extended 5’ sequence, the complete AR gene or extended 3’ sequence, respectively. The BACs were end sequenced to determine their insertion sites. (B) Restriction digests followed by PFGE and Southern blotting were performed to ensure that preperations of the BACs were correct. The SalI digested BACs were fractionated by PFGE and subjected to Southern blotting using 32P random primed labelled AR exon 1 and AR exon 8 probes. mAR BAC-192 was positive for both probes while mAR BAC-196 only probed positive for exon 8 and mAR BAC-185 probed positive only for exon1.
Mentions: Three BACs containing all or parts of the gene encoding AR were obtained from the BACPAC Resource Center (BPRC) at Children's Hospital Oakland Research Institute (CHORI, Oakland, CA). One BAC [mAR BAC-192 (RP24-352D1)] contains the entire gene (all exons) for AR. The other two BACs contain either 5’ [mAR BAC-185 (RP23-316P7)] or 3’ [mAR BAC-196 (RP23-102O11)] exons of the AR gene and either extensive 5’ or 3’ flanking regions (Fig. 1A). The BACs containing the AR genomic sequence were end sequenced to establish the positions of the inserts. Pulse field gel electrophoresis (PFGE) in combination with Southern blotting was used to verify the inserts by comparisons of restriction analysis of the BACs with known genomic sequence from the mouse genome project (Fig. 1B).

Bottom Line: The AR-EGFP transgene was expressed in a cyclical manner similar to that of endogenous AR in Sertoli cells and fertility was restored as offspring were produced in the absence of Sertoli cell AR.Thus, the AR-EGFP transgene under the control of AR regulatory elements is capable of rescuing AR function in a cell selective, AR- background.These initial studies provide proof of principle that a strategy employing the AR-EGFP transgene can be used to understand AR functions.

View Article: PubMed Central - PubMed

Affiliation: Center for Research in Reproductive Physiology, Department of Obstetrics, Gynecology and Reproductive Sciences, Magee Women's Research Institute, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America.

ABSTRACT
Androgens signal through the androgen receptor (AR) to regulate male secondary sexual characteristics, reproductive tract development, prostate function, sperm production, bone and muscle mass as well as body hair growth among other functions. We developed a transgenic mouse model in which endogenous AR expression was replaced by a functionally modified AR transgene. A bacterial artificial chromosome (BAC) was constructed containing all AR exons and introns plus 40 kb each of 5' and 3' regulatory sequence. Insertion of an internal ribosome entry site and the EGFP gene 3' to AR allowed co-expression of AR and EGFP. Pronuclear injection of the BAC resulted in six founder mice that displayed EGFP production in appropriate AR expressing tissues. The six founder mice were mated into a Sertoli cell specific AR knockout (SCARKO) background in which spermatogenesis is blocked at the meiosis stage of germ cell development. The AR-EGFP transgene was expressed in a cyclical manner similar to that of endogenous AR in Sertoli cells and fertility was restored as offspring were produced in the absence of Sertoli cell AR. Thus, the AR-EGFP transgene under the control of AR regulatory elements is capable of rescuing AR function in a cell selective, AR- background. These initial studies provide proof of principle that a strategy employing the AR-EGFP transgene can be used to understand AR functions. Transgenic mice expressing selective modifications of the AR-EGFP transgene may provide crucial information needed to elicit the molecular mechanisms by which AR acts in the testis and other androgen responsive tissues.

No MeSH data available.


Related in: MedlinePlus