Subfertility and defective folliculogenesis in female mice lacking androgen receptor Yueh-Chiang Hu*†, Peng-Hui Wang*†‡, Shuyuan Yeh*, Ruey-Sheng Wang*§, Chao Xie*, Qingquan Xu*, Xinchang Zhou*, Hsiang-Tai Chao‡, Meng-Yin Tsai¶, and Chawnshang Chang*ʈ *George Whipple Laboratory for Cancer Research, Departments of Pathology and Urology, University of Rochester, Rochester, NY 14642; ‡Department of Obstetrics and Gynecology, Taipei Veterans General Hospital and National Yang-Ming University, Taipei 112, Taiwan; §Department of Obstetrics and Gynecology, Taipei Medical University, Taipei 110, Taiwan; and ¶Reproductive Medicine Institute, Chang Gung University, Kaohsiung 833, Taiwan Communicated by Henry Lardy, University of Wisconsin, Madison, WI, June 18, 2004 (received for review August 16, 2003) The roles of the androgen receptor (AR) in female fertility and FSH, resulting in an enhancement of follicular proliferation (13). In ovarian function remain largely unknown. Here we report on the contrast, many other reports indicate that androgen͞AR can inhibit generation of female mice lacking AR (AR؊/؊) and the resulting follicular development by increasing follicular atresia (14, 15). This influences on the reproductive system. Female AR؊/؊ mice appear discrepancy is partly attributed to lack of animal models to unravel normal but show longer estrous cycles and reduced fertility. The the physiological roles of the AR in females. Because the AR gene ovaries from sexually mature AR؊/؊ females exhibited a marked is located on the X chromosome and males lacking functional AR reduction in the number of corpora lutea. After superovulation are infertile, homozygous female offspring lacking functional AR treatment, the AR؊/؊ ovaries produced fewer oocytes and also cannot be generated. In this study, we overcame this difficulty by showed fewer corpora lutea. During the periovulatory period, an using the Cre-lox conditional knockout strategy (7) to generate the intensive granulosa apoptosis event occurs in the AR؊/؊ preovu- female mice lacking functional AR, and we characterized the in vivo latory follicles, concurrent with the down-regulation of p21 and roles of the AR in the female reproductive system. progesterone receptor expression. Furthermore, the defective con- formation of the cumulus cell–oocyte complex from the AR؊/؊ Materials and Methods females implies a lower fertilization capability of the AR؊/؊ oo- Generation of Female AR؊/؊ Mice. Animal care, construction of the cytes. In addition to insufficient progesterone production, the targeting vectors, and the strategy of generation of ARϪ/Ϫ females diminished endometrial growth in uteri in response to exogenous are described in refs. 7 and 16. Briefly, male mice carrying floxed gonadotropins indicates that AR؊/؊ females exhibit a luteal phase AR on the X chromosome (fAR͞Y) were mated with females defect. Taken together, these data provide in vivo evidence show- genotyped with AR͞ar ACTB-Cre to produce female ARϪ/Ϫ mice ing that AR plays an important role in female reproduction. carrying the genotype ar͞ar ACTB-Cre. ACTB-Cre is a transgene containing the ␤-actin promoter to drive Cre recombinase cDNA he androgen receptor (AR) exerts its biological function expression that excises the floxed AR fragment (entire exon 2) on Tthrough activation of target gene expression via a sequence of the genome. Genotyping was performed by using PCR on the processes, including ligand binding, homodimerization, nuclear genomic DNA isolated from the tails of 3-week-old mice, as translocation, DNA binding, and complex formation with coregu- described in refs. 7 and 16, and was confirmed by RT-PCR on the lators and general transcription factors (1–3). While the AR has RNAs extracted from ovaries and uteri. The primers used were Ј Ј Ј long been known to play a central role in the development of male 5 -AATGGGACCTTGGATGGAGAAC-3 and 5 -TCCCT- Ј sex organs, secondary sexual characteristics, and the initiation and GCTTCATAACATTTCCG-3 , as illustrated in Fig. 1A. ,progression of prostate cancer (4–6), its physiological roles in Fertility of Female AR؊/؊ Mice. Ten- to 11-week-old female ARϩ/ϩ females remain unclear. To this end, we recently generated female ϩ/Ϫ Ϫ/Ϫ ϭ Ϫ Ϫ AR , and AR mice (n 8 for each genotype) were subjected / BIOCHEMISTRY AR knockout (AR ) mice and demonstrated that the loss of AR to a continuous mating study. Two female mice were housed with Ϫ/Ϫ retards mammary gland development (7). Also, the study of AR one 8- to 10-week-old known fertile male mouse, and male mice MCF-7 breast cancer cells has indicated the requirement of AR in were rotated weekly. Cages were monitored daily, and the number breast cancer growth (7). of pups and litters was recorded. Ovarian folliculogenesis is a process of the follicular development starting from the smallest primordial follicles recruited into the Superovulation and Oocyte Count. Superovulation was carried out on ϩ ϩ ϩ Ϫ Ϫ Ϫ growth pool through primary, preantral, and antral stages to the female AR / ,AR / , and AR / mice at the ages indicated in the largest Graafian or preovulatory follicles (POFs) that ovulate in figures. The protocol consisted of a single i.p. injection with 5 units response to the luteinizing hormone (LH) surge. After ovulation, of pregnant mare serum gonadotropin (PMSG) (Sigma) for 48 h, the remaining granulosa cells and theca cells within the POFs followed by 5 units of human chorionic gonadotropin (hCG) differentiate into luteal cells to form the corpus luteum (CL) (8). (Sigma) for an additional 18 h or indicated times. The animals were However, in each cycle, only one or very few of the primordial then killed and the genital tracts were excised for histological follicles initially recruited are destined to become POFs; most of examination or RNA extraction. The oocyte͞cumulus masses were them undergo apoptotic death (atresia), mainly at the early antral surgically harvested from the oviducts and cultured in Dulbecco’s stage (9). Follicle-stimulating hormone (FSH) is the major survival modified Eagle’s medium (Invitrogen) supplemented with 20% factor that rescues the early antral follicles from atresia (9). The FCS and antibiotics. After treating with 0.3% hyaluronidase entire folliculogenesis process involves a complex network of para- crine, autocrine, and endocrine signals, including sex hormones, in Abbreviations: AR, androgen receptor; CL, corpus luteum; COC, cumulus cell–oocyte a stage-dependent manner. The AR is present in the ovary in complex; hCG, human chorionic gonadotropin; FSH, follicle-stimulating hormone; FSHR, almost all stages of folliculogenesis and has been suggested to play FSH receptor; LH, luteinizing hormone; PMSG, pregnant mare serum gonadotropin; POF, a proliferative role for follicular development (10, 11). In addition, preovulatory follicle; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick androgen treatment induces the expression of insulin-like growth end labeling. factor-I and insulin-like growth factor-I receptors and enhances the †Y.-C.H. and P.-H.W. contributed equally to this work. growth of the immature follicles in the primate ovary (12). Andro- ʈTo whom correspondence should be addressed. E-mail: [email protected]. gen treatment also augments the granulosa cell responsiveness to © 2004 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0404372101 PNAS ͉ August 3, 2004 ͉ vol. 101 ͉ no. 31 ͉ 11209–11214 Downloaded by guest on September 28, 2021 performed RT-PCR analyses, which revealed that RNAs from those organs completely lacked the AR exon 2 and were 153 bp in length, as compared with 305 bp in length for their ARϩ/ϩ coun- terparts, when we used a pair of primers located on AR exon 1 and exon 3, respectively (Fig. 1B). Genital Tract Phenotype of AR؊/؊ Females. Next, we examined the reproductive organs from age-matched female mice. Under mac- roscopic examination, the ARϪ/Ϫ reproductive system appeared normal; however, when we compared the genital tracts from 8-week-old sexually mature ARϩ/ϩ and ARϪ/Ϫ females at the estrus stage, ARϩ/ϩ uteri transformed to the receptive status and exhibited obvious thick uterine walls, whereas ARϪ/Ϫ uteri exhibited less response to ovulation and remained thin (Fig. 1C). The difference was more significant between 16-week-old ARϩ/ϩ and ARϪ/Ϫ female mice (data not shown). To determine whether this defective uterine response to ovulation in ARϪ/Ϫ mice affects their repro- duction, we examined the pregnant uteri of female ARϩ/ϩ and ARϪ/Ϫ mice and found that pregnant ARϪ/Ϫ mice bore fewer embryos (5.3 Ϯ 1.5) than ARϩ/ϩ mice (9.8 Ϯ 2.2), suggesting that ARϪ/Ϫ females have reduced fertility. -Subfertility of Female AR؊/؊ Mice. To further evaluate the repro ductive performance of female ARϪ/Ϫ mice, we conducted a continuous mating study using sexually mature female mice (n ϭ 8 for each genotype) at 10 to 11 weeks of age mating with known fertile male mice. After 12 weeks of mating, female ARϪ/Ϫ mice Fig. 1. RT-PCR analysis of AR transcripts and macroscopic comparison of the consistently exhibited reduced fertility by showing a significant ϩ ϩ Ϫ Ϫ Ϯ Ͻ reproductive organs of female AR / and AR / mice. (A) Schematic diagram of decrease of litter size (4.5 2.5 pups per litter, P 0.0001) ϩ ϩ ϩ Ϫ the primer design that distinguishes ARϪ/Ϫ from ARϩ/ϩ transcripts. The size of compared with either AR / or AR / females, at 9.8 Ϯ 2.5 and RT-PCR products from ARϩ/ϩ and ARϪ/Ϫ mRNAs is 305 and 153 bp, respectively. 8.0 Ϯ 2.4 pups per litter, respectively (Table 1). Nevertheless, female The splicing of exon 1 and 3 in ARϪ/Ϫ mRNA causes a translational frameshift, and ARϪ/Ϫ mice had an average of 2.3 Ϯ 0.7 litters in a 12-week period, two additional stop codons occur within exon 3.