Subfertility and defective folliculogenesis in female mice lacking androgen receptor

Abstract

The roles of the androgen receptor (AR) in female fertility and ovarian function remain largely unknown. Here we report on the generation of female mice lacking AR (AR(-/-)) and the resulting influences on the reproductive system. Female AR(-/-) mice appear normal but show longer estrous cycles and reduced fertility. The ovaries from sexually mature AR(-/-) females exhibited a marked reduction in the number of corpora lutea. After superovulation treatment, the AR(-/-) ovaries produced fewer oocytes and also showed fewer corpora lutea. During the periovulatory period, an intensive granulosa apoptosis event occurs in the AR(-/-) preovulatory follicles, concurrent with the down-regulation of p21 and progesterone receptor expression. Furthermore, the defective conformation of the cumulus cell-oocyte complex from the AR(-/-) females implies a lower fertilization capability of the AR(-/-) oocytes. In addition to insufficient progesterone production, the diminished endometrial growth in uteri in response to exogenous gonadotropins indicates that AR(-/-) females exhibit a luteal phase defect. Taken together, these data provide in vivo evidence showing that AR plays an important role in female reproduction.

Fig. 1.

RT-PCR analysis of AR transcripts and macroscopic comparison of the reproductive organs of female AR^+/+ and AR^–/– mice. (A) Schematic diagram of the primer design that distinguishes AR^–/– from AR^+/+ transcripts. The size of RT-PCR products from AR^+/+ and AR^–/– mRNAs is 305 and 153 bp, respectively. The splicing of exon 1 and 3 in AR^–/– mRNA causes a translational frameshift, and two additional stop codons occur within exon 3. N, N-terminal domain; DBD, DNA-binding domain; LBD, ligand-binding domain. (B) The RT-PCR analyses revealed that ovaries and uteri from AR^–/– females carried the exon 2-deleted transcripts. (C) The genital tracts of 8-week-old AR^+/+ and AR^–/– females at the estrus stage were compared. The estrus stages of the mice were determined by vaginal smears that showed hundreds of cornified cells. AR^+/+ uteri are wider and thicker than AR^–/– uteri. O, ovary; U, uterus; V, vagina.

Fig. 2.

Morphological comparison of ovaries from AR^+/+ and AR^–/– females. (A and B) Ovaries from 4-week-old AR^+/+ and AR^–/– females were histologically similar. (C) Statistical analysis of the number of the follicular compartments in AR^+/+ and AR^–/– ovaries (n = 2). (D and E) The ovaries from sexually mature, 16-week-old AR^+/+ females, compared with their AR^–/– counterparts. The granulosa layers in the antral follicles were locally thin (open arrowheads) in the AR^–/– ovaries, whereas they were even in thickness in the AR^+/+ ovaries (arrows). An asterisk marks the zona pellucida remnants. (F) Statistical analysis of the number of the follicular compartments in AR^+/+ and AR^–/– ovaries (n = 3 for each genotype). Statistical significance determined by using Student's unpaired and two-tailed t test is indicated. Representative sections are shown. P, primordial and primary follicle; PF, preantral follicle; APF, atretic primordial, primary, and preantral follicle; A, antral follicle; AF, atretic antral follicle; CL, corpus luteum. (Bar: 200 μm.)

Fig. 3.

Comparison of morphology and gene expression in ovaries of AR^+/+ and AR^–/– females after superovulation treatment. Animals were injected with PMSG and then with hCG 48 h later and were killed after another 22 h. (A and B) Superovulated ovaries from 4.5-week-old AR^+/+ and AR^–/– females (n = 5 for each genotype) were compared. Many CLs were induced by superovulation in the AR^+/+ ovaries, compared with fewer and smaller CLs in the AR^–/– ovaries. (C) Statistical analysis of the number and size of CLs in both genotypes. *, P < 0.05; **, P < 0.01. (D) Real-time RT-PCR analysis of the expression levels of the functional markers for luteinization. Statistical significance determined by using Student's unpaired and two-tailed t test is indicated. **, P < 0.01; ***, P < 0.001. (E and F) Histological comparison at high magnification. In the AR^–/– ovaries, the large antral follicles show decreased and disorganized granulosa cells, although the oocyte showed no sign of apoptosis. The granulosa layers were locally thin (open arrowhead) in the AR^–/– ovaries, whereas they were even in thickness in the AR^+/+ ovaries (arrowhead). (G) The cumulus cells were disassociated from the oocyte during ovulation in the AR^–/– ovaries. The possibility that this phenomenon may also occur in the AR^+/+ ovaries needs to be further investigated. O, oocyte; C, cumulus oophorus; G, granulosa cells. (H) The AR^+/+ CLs contain large lutea cells, whereas the AR^–/– CLs contain early, small luteal cells, indicating delayed or impaired granulosa luteinization. (I) AR^+/+, but not AR^–/–, CLs (white dashed circles) show luteolysis (apoptosis) when TUNEL staining is used. Representative sections are shown. (Bars: A and B, 200 μm; E–G, 50 μm; H, 20 μm; I, 100 μm.)

Fig. 4.

Detection of apoptotic cells in the ovaries of AR^+/+ and AR^–/– females treasted with exogenous gonadotropins by using TUNEL assay. The treatment conditions are as indicated. (A and B) Preovulatory follicles from both genotypes were compared. The AR^–/– preovulatory follicle (B) shows slight apoptotic signals, whereas the AR^+/+ preovulatory follicle (A) shows no apoptosis. Note that after TUNEL staining and photographing (Upper), the sections were subjected to hematoxylin and eosin staining (Lower). (C and D) Preovulatory follicles during the periovulatory stage from both genotypes were compared. The AR^–/– preovulatory follicle (D) shows intensive granulosa apoptosis and reduced size, whereas the AR^+/+ counterpart (C) shows no apoptosis. (E) The DNA fragmentation assay using ligation-mediated PCR. DNA ladders were amplified from 50 ng of genomic DNAs (Upper). At Lower, 250 ng of genomic DNAs (5×) is shown as loading control. (F and G) Superovulated ovaries from AR^+/+ and AR^–/– females were compared. In the AR^+/+ ovaries, apoptotic granulosa cells were scattered in many follicles but not in CLs, whereas intensive granulosa apoptosis was seen in many preantral and antral follicles in the AR^–/– ovaries. The Inset shows high magnification of the indicated follicles (arrowheads). Apoptotic signals are shown in bright green. Representative sections are shown. (Bar: A–D, 50 μm; F and G, 200 μm.)

Fig. 5.

Real-time RT-PCR analysis for gene expression. Whereas one ovary was processed for histological examination, the other one from the same mouse was subjected to real-time RT-PCR. (A) Comparison of ovarian morphology between AR^+/+ and AR^–/– mice (n = 2 for each genotype) at 10 days of age. The expression levels of FSHR and insulin-like growth factor-I receptor mRNA were quantitated by using real-time RT-PCR. (B) Comparison of ovarian morphology between AR^+/+ and AR^–/– mice at 4.5 weeks of age. The conditions of exogenous gonadotropin treatments were as indicated. The expression levels of indicated genes were quantitated by using real-time RT-PCR. FSHR, follicle-stimulating hormone receptor; IGF-IR, insulin-like growth factor-I receptor; PR, progesterone receptor; HAS2, hyaluronan synthase 2; TSG6, tumor necrosis factor-α-stimulated gene 6. (Bar: A, 50 μm; B, 200 μm.)

Fig. 6.

Comparison of uterine response to superovulation treatment in 4.5-week-old AR^+/+ and AR^–/– females. (A) Comparison of ovary size in both genotypes (Upper). The AR^+/+ uteri were wider in diameter and showed apparent thicker uterine walls, whereas AR^–/– uteri did not respond properly (Lower). O, ovary; U, uterus. (B and C) Comparison of longitudinal sections shows that AR^+/+ (B), but not AR^–/– (C) uteri showed the luteal response, as evident by the massive uterine hypertrophy and endometrial growth. The double arrowheads show the diameter of the muscular layer. (Bar: 200 μm.)