Spermatogenesis arrest caused by conditional deletion of Hsp90α in adult mice

Abstract

It is controversial whether a functional androgen receptor (AR) on germ cells, including spermatogonia, is essential for their development into sperm and, thus, initiation and maintenance of spermatogenesis. It was recently shown that many spermatocytes underwent apoptosis in the testes of Hsp90α KO mice. We had generated Hsp90α KO mice independently and confirmed this phenotype. However, the important question of whether Hsp90α is required to maintain spermatogenesis in adult mice in which testicular maturation is already completed could not be addressed using these conventional KO mice. To answer this question, we generated a tamoxifen-inducible deletion mutant of Hsp90α and found that conditional deletion of Hsp90α in adult mice caused even more severe apoptosis in germ cells beyond the pachytene stage, leading to complete arrest of spermatogenesis and testicular atrophy. Importantly, immunohistochemical analysis revealed that AR expression in WT testis was more evident in spermatogonia than in spermatocytes, whereas its expression was aberrant and ectopic in Hsp90α KO testis, raising the possibility that an AR abnormality in primordial germ cells is involved in spermatogenesis arrest in the Hsp90α KO mice. Our results suggest that the AR, specifically chaperoned by Hsp90α in spermatogonia, is critical for maintenance of established spermatogenesis and for survival of spermatocytes in adult testis, in addition to setting the first wave of spermatogenesis before puberty.

Keywords: Androgen receptor; Hsp90α; Spermatogenesis; Spermatogonia.

Fig. 1.. Strategy for production of Hsp90α KO mice.

(A) Generation of Hsp90α floxed mice and Hsp90α KO mice. Homologous recombination of the targeting construct into the Hsp90aa1 gene resulted in production of Hsp90α^neo mice. By mating with EIIa-Cre mice, Hsp90α^flox and Hsp90α^Ex9−10− mice were obtained. The primers (red boxes) used for PCR detection of the recombination status were on exon 8 and 11, respectively. (B) Generation of Hsp90α conditional KO mice. Hsp90α^flox/flox/CreER^T2 and Hsp90α^flox/+/CreER^T2 were generated by the indicated breeding procedures. Administration of tamoxifen resulted in production of Hsp90α^−/− and Hsp90α^−/+ mice, respectively. (C) Hsp90α KO adult mice (8 weeks) underwent progressive testicular atrophy. (D) H&E staining and a TUNEL assay of the testes of day 17 and 8-week-old WT and Hsp90α KO mice. Scale bars = 100 µm.

Fig. 2.. Tamoxifen-induced conditional deletion of Hsp90α results in testicular atrophy.

(A) The scheme of oral administration of tamoxifen and removal of the testes for assay. (B) Genome analysis after administration of tamoxifen. There were some non-specific bands in lanes 3 and 4 in each panel. (C) Expression levels of Hsp90α and Hsp90β in the indicated tissues tested at day 6 and day 30 after administration of tamoxifen. Hsp90α but not Hsp90β is largely downregulated at day 30. (D) Testes obtained at day 6 and 30 after treatment with tamoxifen are shown. The testis recovered from f/f but not f/+ mice underwent atrophy at day 30. (E) Weight (g) of the testes recovered from f/f, f/+, and +/+ mice at day 30 are shown as bar graphs. (n = 6 from 3 mice of each genotype; error bars indicate SD).

Fig. 3.. Immunohistochemical analysis of testes recovered from tamoxifen-treated f/+ and f/f mice at day 30.

The testes derived from f/f but not f/+ mice showed severe apoptosis of germ cells beyond the pachytene stage and spermatogenesis arrest, as indicated by H&E staining (upper panel) and the TUNEL assay (lower panel). Scale bar = 100 µm.

Fig. 4.. Aberrant AR expression in Hsp90α KO testes.

H&E staining of WT and knockout testes (Top row). Expression of ERα, (2^nd row), ERβ (3^rd row) and AR (4^th row) in testes derived from WT and Hsp90α KO mice (8 weeks) was tested with specific antibodies. ERβ and AR mostly stained spermatogonia of WT testis. On the other hand, spermatogonia of Hsp90α KO testis were mostly negative and many spermatocytes stained positive, in marked contrast to the pattern in WT testes. Scale bars = 100 µm.

Fig. 5.. SCP3 and AR staining in mirror sections of testes.

Expression of SCP3 and AR in testes derived from WT and Hsp90α KO mice ((A) 8 weeks after birth, (B) 17 days after birth) was tested with specific antibodies. SCP3 stained spermatocytes with meiosis, not spermatogonia, not Sertoli cells. AR stained spermatogonia and spermatocytes of 8 weeks WT testis (A). On the other hand, spermatogonia of Hsp90α KO testis were mostly negative and many spermatocytes stained positive, in marked contrast to the pattern in WT testes (A). AR stained only spermatocytes in both WT and Hsp90α KO testis (17 days) (B). The marked cells are indicated as representative spermatogonia (solid lines), spermatocyte (dashed lines), and Sertoli cells (dotted lines). Scale bars = 50 µm, except top row of A where scale bar = 100 µm.