POF in AR^–/– female mice. (a–d) Histology of AR^+/+ and AR^–/– ovaries at 3 weeks, 8 weeks, 32 weeks, and 40 weeks of age. All sections were stained with hematoxylin and eosin. An asterisk marks the atretic follicle. CL, corpus luteum. (e–h) Relative follicle counts at 3 weeks (e), 8 weeks (f), 32 weeks (g), and 40 weeks (h) of age. Numbers represent total counts of every fifth section from serially sectioned ovaries (n = 4 animals per genotype). (i) Immunohistochemical study for activated, cleaved caspase-3 revealed increased positive cells (apoptotic cells) in AR^–/– ovaries. Sections were counterstained with hematoxylin. An asterisk marks the caspase-3-positive cell. CL, corpus luteum. (j) Age-dependent reduction in the number of pups per litter in AR^–/– female mice. A continuous breeding assay was started at 24 weeks of age (n = 6–10 animals per genotype). For all panels, data are shown as mean ± SEM and were analyzed by using Student's t test.

Genome-wide microarray analysis and semiquantitative RT-PCR revealed that expression of the oocyte–granulosa cell regulator loop was down-regulated in AR^–/– ovaries. (a) Microarray analysis of AR^–/– compared with AR^+/+ ovaries at 3 and 8 weeks of age. Data obtained from microarray analysis as described in Materials and Methods were used to generate a cluster analysis. Each vertical line represents a single gene. The ratios of gene expression levels in AR^–/– ovaries compared with wild type are presented. (b and c) Semiquantitative RT-PCR analysis of AR-regulated genes identified from the microarray study. Results shown are representative (using one ovary per genotype in each experiment) of five independent experiments. Data are shown as mean ± SEM and were analyzed by using Student's t test. (d) Comparison of Kitl gene expression by Northern blot analysis among placebo-, DHT-, and flutamide (FL)-treated AR^+/+ mouse ovaries. (e) Induction of KITLG gene expression by DHT treatment in KGN cells. (f and g) Androgen responsiveness in the mouse and human kit ligand promoters by a luciferase assay performed by using KGN cells. Data are shown as mean ± SEM and were analyzed by using Student's t test.

Phenotypic characterization of AR knockout female mice. (a) Diagram of the wild-type Ar genomic locus (+), floxed AR L3 allele (L3), and AR allele (L-) obtained after Cre-mediated excision of exon 1. K, KpnI; E, EcoRI; H, HindIII; B, BamHI. LoxP sites are indicated by arrowheads. The targeting vector consisted of a 7.6-kb 5′ homologous region containing exon 1, a 1.3-kb 3′ homologous region, a single loxP site, and the neo cassette with two loxP sites. (b) Detection of the Y chromosome-specific Sry gene in AR^–/Y mice by PCR. (c) Absence of AR protein in AR^–/– mice ovaries by Western blot analysis using a specific C-terminal antibody. (d) Normal weight gain in AR^–/– females. (e) Histology of pituitary, uterus, and bone tissues in AR^+/+ and AR^–/– females at 8 weeks of age. (f) Female reproductive organs were macroscopically normal in AR^–/– mice. (g) Serum hormone levels at the proestrus stage in AR^–/– mice were not significantly altered. Serum 17β-estradiol, progesterone, testosterone, luteinizing hormone (LH), and follicle-stimulating hormone (FSH) levels in AR^+/+ (n = 13) and AR^–/– (n = 10) females at 8–10 weeks of age are shown. (h) Lobuloalveolar development is impaired in AR^–/– mammary glands. Whole mount of inguinal mammary glands (Left) and its higher magnification (Right) were prepared on day 3 of lactation. (i) Average number of pups per litter is markedly reduced in AR^–/– mice at 8 weeks of age. Data are shown as mean ± SEM and analyzed by using Student's t test. (j) AR immunocytochemistry in AR^+/+ and AR^–/– ovaries. Sections were counterstained with eosin.

No significant alterations in mRNA levels of several major regulators in folliculogenesis. Shown is semiquantitative RT-PCR of LH receptor (Lhr), FSH receptor (Fshr), p450 side chain cleavage enzyme (Cyp11a1), 17-α-hydroxylase (Cyp17a1), Aromatase (Cyp19), estrogen receptor-β (Esr2), cyclin D2 (Ccnd2), insulin-like growth factor 1 (Igf1), cyclooxygenase 2 (Ptgs2), or progesterone receptor (Pgr) gene expression in AR^+/+ and AR^–/– ovaries. Results shown were representative (using one ovary per genotype in each experiment) of five independent experiments.