Defects of prostate development and reproductive system in the estrogen receptor-alpha null male mice

Abstract

The estrogen receptor-alpha knockout (ERalphaKO, ERalpha-/-) mice were generated via the Cre-loxP system by mating floxed ERalpha mice with beta-actin (ACTB)-Cre mice. The impact of ERalpha gene deletion in the male reproductive system was investigated. The ACTB-Cre/ERalpha(-/-) male mice are infertile and have lost 90% of epididymal sperm when compared with wild-type mice. Serum testosterone levels in ACTB-Cre/ERalpha(-/-) male mice are 2-fold elevated. The ACTB-Cre/ERalpha(-/-) testes consist of atrophic and degenerating seminiferous tubules with less cellularity in the disorganized seminiferous epithelia. Furthermore, the ventral and dorsal-lateral prostates of ACTB-Cre/ERalpha(-/-) mice display reduced branching morphogenesis. Loss of ERalpha could also be responsible for the decreased fibroblast proliferation and changes in the stromal content. In addition, we found bone morphogenetic protein, a mesenchymal inhibitor of prostatic branching morphogenesis, is significantly up-regulated in the ACTB-Cre/ERalpha(-/-) prostates. Collectively, these results suggest that ERalpha is required for male fertility, acts through a paracrine mechanism to regulate prostatic branching morphogenesis, and is involved in the proliferation and differentiation of prostatic stromal compartment.

Fig. 1.

Breeding and genotyping of ACTB-Cre/ERα^−/− male mice via Cre-loxP strategy. A, Using the Cre-loxP strategy, exon III of mouse ERα gene was targeted and flanked by loxP sites. The schematic map shows floxed ERα allele and deleted ERα allele in the ACTB-ERα mutant mice. The primer set, P1 and P2, was used for genotyping. B, Breeding strategy to generate the ACTB-Cre/ERα^−/− males and genotyping of ACTB-Cre/ERα^−/− males using primer mixture: P1, P2, and Cre primer. Floxed ERα homozygous female mice were mated with ACTB-Cre transgenic male mice to obtain ACTB-Cre/ERα^+/− male mice (F1). Mating ACTB-Cre/ERα^+/− males with floxed ERα heterozygote females obtained the ACTB-Cre/ERα^+/+ (Wt) and ACTB-Cre/ERα^−/− (KO) mice (F2). The genotyping results were as follows: lane 1, ACTB-Cre/ERα^+/+ mice, and the size of Wt ERα and Cre was 741 and 411 bp, respectively; lane 2, ACTB-Cre/ERα^+/− mice, in which intervening DNA of floxed ERα allele was deleted by ACTB-Cre recombinase. The size of deleted ERα allele was reduced to 223 bp; lane 3, ACTB-Cre/ERα^−/− mice, in which the size of both floxed ERα alleles was reduced to 223 bp by ACTB-Cre recombinase and only the deleted ERα band was present. C, Western blot analysis of ERα protein in the testis, epididymis, and prostate of Wt and ACTB-Cre/ERα^−/− males using antibody against ERα C terminus. No ERα immunoreactivity was detected in ACTB-Cre/ERα^−/− male reproductive organs (lanes 2, 4, and 6). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) immunoblotted as an indication of equal loading. D, The serum hormone profiles in the 12- to 14-wk-old ACTB-Cre/ERα^−/− males. Results are presented as means ± sd (n = 5). *, P < 0.05 vs. Wt littermates (statistically significant by Student’s unpaired t test).

Fig. 2.

The male reproductive organ in the 12-wk-old Wt and ACTB-Cre/ERα^−/− males. A, The comparison of the anatomy of the genital tracts between adult ACTB-Cre/ERα^−/− and Wt littermates. B, The comparison of the reproductive organ weights between adult ACTB-Cre/ERα^−/− and Wt littermates. The organs from 12-wk-old males were weighed. *, P < 0.05 vs. Wt littermates (statistically significant by Student’s unpaired t test). Results are means ± sd (n = 8). C, The comparison of the sperm count between adult ACTB-Cre/ERα^−/− and Wt littermates. *, P < 0.01 vs. Wt littermates (statistically significant by Student’s unpaired t test). Results are means ± sd (n = 3).

Fig. 3.

Histological analyses and comparison of testes and epididymides in the 12-wk-old Wt, neo-ERα^−/−, and ACTB-Cre/ERα^−/− males. A, Histological analyses of testes from Wt and ACTB-Cre/ERα^−/− males. The adult Wt testes consist of compact seminiferous tubules (I and III). The adult ACTB-Cre/ERα^−/− testes consist of atrophic and degenerating seminiferous tubules (red line) (II). The seminiferous epithelial layer in the ACTB-Cre/ERα^−/− testes contained few spermatogenic cells with decreased germ cell number compared with Wt (IV vs. III, double-headed arrows), and the vacuoles were often present in the seminiferous epithelial layer (IV, arrowhead). B, Histological analyses of testes from Wt and neo-ERα^−/− males. The adult neo-ERα^−/− testes showed the defects with less extent compared with ACTB-Cre/ERα^−/− testes. Some normal seminiferous tubules could be observed in neo-ERα^−/− testes (II, arrow). C, Histological analyses of caudal epididymis from Wt, ACTB-Cre/ERα^−/−, and neo-ERα^−/− males. The adult Wt epididymis is full of sperm in the caudal lumen (I and IV). The caudal epididymis in the adult ACTB-Cre/ERα^−/− males contains much less sperm and appears pale in the histology staining (II and V). The epididymis in the adult neo-ERα^−/− males exhibits the heterogeneous phenotype. Some epididymal tubes contain very few sperm, but some are full of sperm (III and VI, arrows).

Fig. 4.

The defects of prostate development in the ACTB-Cre/ERα^−/− males. A, Photographs demonstrating the branching morphogenesis from a representative pair of adult ACTB-Cre/ERα^−/− and Wt littermates after microdissection of VP. B, The total number of ductal tips, branches, and ductal points in ACTB-Cre/ERα^−/− and Wt littermates. The VP and DLP, but not AP, of ACTB-Cre/ERα^−/− mice have significantly fewer ductal tips than those of WT littermates. *, P < 0.01 vs. Wt littermates (statistically significant by Student’s unpaired t test). Results are means ± sd (n = 5). C and D, The cell proliferation of VP from 1-wk-old ACTB-Cre/ERα^−/− and Wt littermate is determined by Ki-67 immunostaining (C). Arrows show positive staining in the epithelial cells. Arrowheads show positive staining in the stromal cells. The Ki-67 proliferation index is scored as the percentage of Ki-67-positive cells (D). Note that there was significantly reduced stromal proliferation in the ACTB-Cre/ERα^−/− VP. *, P < 0.05, compared with Wt VP cell proliferation, Student’s t test. The results are means ± sd of triplicate samples. E, Characterization of proliferating stromal cells in the ACTB-Cre/ERα^−/− and Wt VP. Immunofluorescence analyses were carried out by using sections from neonatal VP lobes. Each section was double stained by Ki-67 and vimentin (fibroblast cell marker) or desmin (smooth muscle cell marker) followed by counterstaining with 4′,6′-diamino-2-phenylindole (DAPI) to visualize the nuclei. Arrowheads show positive Ki-67 staining in the fibroblasts (c, g, k, and o). Arrows show positive Ki-67 staining in the epithelial cells (c and g). Long arrow shows positive Ki-67 staining in the smooth muscle cells (k). Note the fibroblasts are actively proliferating (c and g), but the smooth muscle cells rarely proliferate (k and o) in the stromal compartment. F, The comparison of the expression of the stromal marker between ACTB-Cre/ERα^−/− and Wt littermates by real-time PCR. Assays were performed on RNA from the VP of each individual mouse and then averaged for the gene expression. 18s acts as internal control. The results are means ± sd of six samples. Note that the expression of vimentin is decreased, but the expression of both smooth muscle α-actin and desmin is increased in the ACTB-Cre/ERα^−/− VP. G, The comparison of the expression levels of the genes involved in prostatic branching morphogenesis between ACTB-Cre/ERα^−/− and Wt littermates by real-time PCR. Assays were performed on RNA from the VP of each individual mouse and then averaged for the gene expression. 18s acts as internal control. The results are means ± sd of six samples. *, P < 0.05, compared with Wt gene expression, Student’s t test. Note that only BMP4 is significantly up-regulated in the ACTB-Cre/ERα^−/− VP.