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First published online December 30, 2003
doi: 10.1242/10.1242/dev.00957

Development 131, 459-467 (2004)
Published by The Company of Biologists 2004
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Androgen receptor function is required in Sertoli cells for the terminal differentiation of haploid spermatids

Robert W. Holdcraft and Robert E. Braun*

University of Washington School of Medicine, Department of Genome Sciences, Seattle, WA 98195, USA



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  		Fig. 1. Construction and characterization of Ar conditional allele. (A) Schematic of targeting construct (pyARdt1), wild-type and conditional hypomorphic (Arflox(ex1-neo)) alleles of Ar. Arrows over loxP sites indicate orientation of sites relative to one another. Probe used for Southern blots is indicated above exon 1. (B) EcoRV digest and Southern blot of ES cell DNAs. Probe detects presence of 12.5kb Ar fragment in wild-type FWB2 ES cells. Correctly targeted ES cell clones Ar126 and Ar206 contain only the 9 kb fragment representative of the correctly targeted allele. (C) Chimeric males transmit the X-linked Arflox(ex1-neo) allele exclusively to female offspring. (D) PCR genotyping detects the Ar inversion allele, inv(ex1-neo), specifically in the testes of Arflox(ex1-neo)/Y; Amh-cre animals. No product is observed in tails from the same animals or in testes or tails from Arflox(ex1-neo)/Y males. The non-recombinant hypomorphic allele, flox(ex1-neo), is detected in all samples. Te, testis; Ta, tail.

 


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  		Fig. 2. Analysis of protein and RNA levels in adult testes. (A) Western blot confirming that AR protein levels are reduced in testes of Arflox(ex1-neo)/Y and Arflox(ex1-neo)/Y; Amh-cre males. (B) Northern blot showing decrease in Prm1 and Pem mRNA expression in testes of Arflox(ex1-neo)/Y and Arflox(ex1-neo)/Y; Amh-cre males. (C) Quantification of mRNA levels from B. Values with the same superscript are significantly different from one another (a,c,d,f, P<0.05; b,e, P<0.001). n=4 for all groups.

 


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  		Fig. 3. Histological examination of epididymis and testis. (A-C) Transverse section through the epididymis. Spermatazoa are infrequent in Arflox(ex1-neo)/Y and absent from Arflox(ex1-neo)/Y; Amh-cre males. Pyknotic and vacuolated round spermatids populate the Arflox(ex1-neo)/Y; Amh-cre epididymis. Smooth muscle hyperplasia is apparent in Ar mutants. (D-F) Low magnification view of testis transverse section. Elongated spermatids are depleted from Arflox(ex1-neo)/Y; Amh-cre testes. (G-I) Stage VII tubules show decrease in (Arflox(ex1-neo)/Y) or complete absence of (Arflox(ex1-neo)/Y; Amh-cre) elongated spermatids. (J-L) Higher magnification of G-I. Black arrowhead indicates presence of degenerating sperm in Arflox(ex1-neo)/Y males.

 


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  		Fig. 4. Evaluation of somatic and germ cell protein expression. (A-F) MSY4 expression in pachytene spermatocytes and round spermatids is unaffected in Ar mutant males. Elongated spermatids are reduced in (B,E) and absent from (C,F) the tubules of Arflox(ex1-neo)/Y and Arflox(ex1-neo)/Y; Amh-cre, respectively. (G-I) PRM2-positive elongated spermatids are decreased in Arflox(ex1-neo)/Y males (H) and absent from Arflox(ex1-neo)/Y; Amh-cre males (I) at time of spermiation. (J-L) AR protein is expressed in somatic cells of the testis. AR is present in Sertoli cells of Arflox(ex1-neo)/Y males (K, blue arrowheads).

 

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© The Company of Biologists Ltd 2004