A Sertoli cell-selective knockout of the androgen receptor causes spermatogenic arrest in meiosis

Abstract

Androgens control spermatogenesis, but germ cells themselves do not express a functional androgen receptor (AR). Androgen regulation is thought to be mediated by Sertoli and peritubular myoid cells, but their relative roles and the mechanisms involved remain largely unknown. Using Cre/loxP technology, we have generated mice with a ubiquitous knockout of the AR as well as mice with a selective AR knockout in Sertoli cells (SC) only. Mice with a floxed exon 2 of the AR gene were crossed with mice expressing Cre recombinase ubiquitously or selectively in SC (under control of the anti-Müllerian hormone gene promoter). AR knockout males displayed a complete androgen insensitivity phenotype. Testes were located abdominally, and germ cell development was severely disrupted. In contrast, SC AR knockout males showed normal testis descent and development of the male urogenital tract. Expression of the homeobox gene Pem, which is androgen-regulated in SC, was severely decreased. Testis weight was reduced to 28% of that in WT littermates. Stereological analysis indicated that the number of SC was unchanged, whereas numbers of spermatocytes, round spermatids, and elongated spermatids were reduced to 64%, 3%, and 0% respectively of WT. These changes were associated with increased germ cell apoptosis and grossly reduced expression of genes specific for late spermatocyte or spermatid development. It is concluded that cell-autonomous action of the AR in SC is an absolute requirement for androgen maintenance of complete spermatogenesis, and that spermatocyte/spermatid development/survival critically depends on androgens.

Fig. 1.

Generation of an AR allele with a floxed exon 2. (A) Schematic overview of the followed strategy. LoxP sites are indicated by triangles. Genomic fragments (5.6 and 2.5 kb) (bold) were used to allow homologous recombination of a region containing a floxed AR exon 2 and neo cassette in the targeting vector with the WT AR locus. Probes A, B, and C were used to screen for correct homologous recombination by Southern blotting. The neo cassette was excised by transient transfection of a Cre expression plasmid (pOG231) in a correctly recombined ES clone. (B) Southern blot analysis of EcoRV-digested DNA from ES cells transfected with the targeting vector by using probe A. The presence of a 4.6-kb fragment (AR^neo/Y) vs. a WT band of 9.3 kb (AR⁺/Y) revealed correct homologous recombination. (C) Southern blot analysis of DNA isolated from pOG231 transfected recombined ES cells. DNA was digested with KpnI and hybridized with probe A. All possible recombinations are shown: complete excision (AR⁰/Y); excision of exon 2 only (AR^del2/Y); no excision (AR^neo/Y); and the desired excision of the neo cassette only (AR^flox/Y) showing a 4.4-kb fragment.

Fig. 2.

Dissection of urogenital tracts of WT, ARKO, and SCARKO male mice at the age of 50 days. dd, ductus deferens; sv, seminal vesicles; t, testis; e, epididymis; ft, fat tissue.

Fig. 3.

Comparison between the testes obtained from adult WT, ARKO, and SCARKO mice. In WT mice (a), AR are immunolocalized to nuclei of SC (arrowheads), PT (arrow), and LC (L); no immunopositive staining was detected in ARKO (b), and PT were multilayered (arrows). In SCARKO (c), LC (L) and PT (arrows) were immunopositive for AR, but no SC staining was detected. Hematoxylin/eosin staining of testes from day 50 WT (d), ARKO (e), and SCARKO (f) revealed clear differences in germ cell complement. Complete spermatogenesis including round (R) and elongate (E) spermatids was observed in WT, but spermatogenic arrest occurred in both AR mutants (e and f). Apotag-positive germ cells (*) were rare in WT (g) but were observed in many tubules in ARKO (h) and SCARKO (i). (Bar = 50 μm.)