Differential effects of spermatogenesis and fertility in mice lacking androgen receptor in individual testis cells Meng-Yin Tsai*†, Shauh-Der Yeh*‡, Ruey-Sheng Wang*‡, Shuyuan Yeh*, Caixia Zhang*, Hung-Yun Lin*, Chii-Ruey Tzeng‡, and Chawnshang Chang*§ *George H. Whipple Laboratory for Cancer Research, Departments of Urology and Pathology, University of Rochester, Rochester, NY 14642; †Graduate Institute of Clinical Medicine, Chang Gung University, Kaohsiung, Taiwan; and ‡Graduate Institute of Medical Sciences and Departments of Urology and Gynecology and Obstetrics, Taipei Medical University, Taipei 110, Taiwan Communicated by Henry Lardy, University of Wisconsin, Madison, WI, September 28, 2006 (received for review July 20, 2006) Using a Cre-Lox conditional knockout strategy, we generated a there is little AR staining in the germ cells (3, 9, 14–18). Interest- germ cell-specific androgen receptor (AR) knockout mouse (G- ingly, results from another study reveal that the expression of AR AR؊/y) with normal spermatogenesis. Sperm count and motility in in male germ cells is stage-specific, expressing only at the stage of epididymis from AR؊/y mice are similar to that of WT (G-AR؉/y) elongated spermatid (10). Previous animal studies conducted in rats mice. Furthermore, fertility tests show there was no difference in also found an AR-specific coregulator, SNURF͞RNF4, is ex- fertility, and almost 100% of female pups sired by G-AR؊/y males pressed in elongated spermatids (19). Together, these studies younger than 15 weeks carried the deleted AR allele, suggesting suggest that AR might play a direct role in germ cells at the the efficient AR knockout occurred in germ cells during meiosis. elongated spermatid stage. Together, these data provide in vivo evidence showing male mice Synaptonemal complex protein 1 (Sycp1) is expressed specifically without AR in germ cells can still have normal spermatogenesis and at the leptotene–zygotene stage of male germ cells (20). The fertility, suggesting the essential roles of AR during spermatogen- expression of the Sycp1-Cre transgene is able to excise the loxP- esis might come from indirect cell–cell communication in a para- floxed DNA fragment during meiosis in testes without disturbing crine fashion. We then compared the consequences of AR loss in fertility (20). Here, we report a germ cell ARϪ/y mouse model the spermatogenesis and fertility of G-AR؊/y mice with two other (G-ARϪ/y) through mating our floxed AR͞AR (fAR͞AR) female testicular cell-specific AR؊/y mice and total AR knockout male mice. mice with Sycp1-Cre transgenic male mice to study direct AR The results provide clear in vivo evidence that androgen͞AR sig- functions in male germ cells. We also compare the testis phenotype naling in Sertoli cells plays a direct important role in spermato- of G-ARϪ/y mice with our total AR knockout (T-ARϪ/y), Sertoli genesis and in Leydig cells plays an autocrine regulatory role to cell-specific AR knockout (S-ARϪ/y), and Leydig cell-selective AR modulate Leydig cell steroidogenic function. Total AR knockout knockout (L-ARϪ/y) mice to dissect the distinct roles of AR in male mice have the most severe defects among these mice. These different types of testicular cells involved in spermatogenesis. -contrasting data with G-AR؊/y mice suggest AR might have differ ent roles in the various cells within testis to contribute to normal Results -spermatogenesis and male fertility in mice. Generation of T-AR؊/y, S-AR؊/y, L-AR؊/y, and G-AR؊/y Mice. To gen erate total or cell-specific AR knockout mice, we have created the germ cell ͉ knockout ͉ Sertoli cells ͉ Leydig cells B6͞129SVEV loxP-fAR mice (21). According to testicular histol- ogy from testicular feminized (22, 23) or Actb-Cre T-ARϪ/y mice permatogenesis (exocrine) and androgen biosynthesis (endo- (21), the germ cells still can develop to the pachytene stage, Scrine) are major functions of mammalian testes. Both functions suggesting that meiosis after the pachytene stage might depend are complicated and highly regulated. Early studies indicated that highly on the function of androgen͞AR. Therefore, we designed an initiation, maintenance, and reinitiating of spermatogenesis depend experiment to excise the fAR gene in leptotene–zygotene sper- absolutely on androgen action (1). These androgen effects on matocytes by the Sycp1-Cre transgene (20). After mating Sycp1-Cre spermatogenesis were well documented in hypophysectomized and transgenic males with homozygous fAR females, we produced Ϫ Leydig cell-destroyed animals (2). The testis contains at least four G-AR /y mice (Fig. 1A), with ar͞Y in germ cells and fAR͞Yin different types of cells (Sertoli, Leydig, and peritubular myoid cells other cells. To examine AR recombination, we used PCR primers and germ cells at different spermatogenic stages), and spermato- ‘‘select and 2–3’’ for fAR allele and WT AR allele and primers CELL BIOLOGY genesis depends highly on autocrine and paracrine communication ‘‘select and 2–9’’ for deleted allele (ar) and WT AR allele, as shown Ϫ among different types of testicular cells. The role of the androgen (21). The genotype of G-AR /y was characterized in tail genomic receptor (AR) in each cell type within the testis remains unclear and DNA by PCR with primers ‘‘select and 2–3.’’ Because the tail of the Ϫ controversial. G-AR /y mouse does not express Cre recombinase protein, we Before the availability of AR-specific antibodies and AR cDNA expect our PCR results to show the fAR allele with Ϸ510 bp (Fig. probes, the distribution of AR was studied by using [3H]-labeled 1B). We also applied PCR to confirm the Cre transgene by using androgen-binding assays. These results found that androgen- binding activity was located in the germ cells and other testicular cells involved in spermatogenesis (3, 4). Since the isolation of AR Author contributions: M.-Y.T., S.-D.Y, and R.-S.W. contributed equally to this work; M.-Y.T., S.-D.Y., S.Y., C.-R.T., and C.C. designed research; M.-Y.T., S.-D.Y., S.Y., and H.-Y.L. performed cDNA (5) and the generation of AR-specific antibodies, much research; M.-Y.T., S.-D.Y., R.-S.W., and C.Z. analyzed data; and M.-Y.T., S.-D.Y., R.-S.W., S.Y., effort has been made to determine which cells in testes express C.Z., and C.C. wrote the paper. functional AR (6, 7). There is general agreement that AR can be The authors declare no conflict of interest. detected in Sertoli cells, peritubular myoid cells, and cells in the Abbreviations: AR, androgen receptor; fAR, floxed AR; T-ARϪ/y, total AR knockout; S-ARϪ/y, interstitial spaces, including Leydig cells (3, 6, 8–11). However, the Sertoli cell-specific knockout; L-ARϪ/y, Leydig cell-specific knockout; G-ARϪ/y, germ cell AR localization of AR using antibodies in male germ cells remains knockout. controversial. Several studies indicated that AR is present in germ §To whom correspondence should be addressed. E-mail: [email protected]. cells in different species (8, 10–13); however, other reports show © 2006 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0608565103 PNAS ͉ December 12, 2006 ͉ vol. 103 ͉ no. 50 ͉ 18975–18980 Downloaded by guest on September 25, 2021 Fig. 2. Determining the efficiency of AR knockout in germ cells by geno- typing of female offspring. (A) Two different genotypes of female offspring from G-ARϪ/y male mating with B6 ARϩ/ϩ female mice. (B and C) Designed primers, ‘‘select,’’ ‘‘2–9,’’ and ‘‘2–3’’ were used to determine genotype by PCR. -Examining the Knockout Efficiency of G-AR؊/y Mice by Female Off spring Genotype. Because the spermatocytes and spermatids are functionally syncytial and diploidy with the intercellular bridges (25), there might be a concern that if Sycpl-Cre AR knockout efficiency is not 100%, some rare spermatids produced from G-ARϪ/y male still get their functional AR by escaping from the Cre recombination through intercellular bridges. Therefore, it is im- portant to confirm our knockout efficiency to demonstrate the AR function in germ cells by Sycp1-Cre AR knockout strategy. The AR genotypes of 57 female pups, sired by male G-ARϪ/y mice mating with female ARϩ/ϩ (B6) mice, were checked by PCR to identify the Fig. 1. Generation and genotyping of germ cell-specific AR knockout (G-ARϪ/y) ratio of female mice possessing the deleted allele to total female mice. (A) Mating strategy and study design. The mouse marked by the gray box mice. The female genotype fertilized by sperm carrying deleted AR is a Sycp1-Cre conditional AR knockout male (G-ARϪ/y) mouse. (B) Genotyping of Ϫ allele would produce ar͞AR heterozygous female offspring, and tail genomic DNA of G-AR /y mice. The WT allele of the AR gene generated a PCR product Ϸ460 bp in size by primers ‘‘select and 2–3.’’ The fAR gene generated a those fertilized by sperm carrying fAR allele would produce the Ϸ510-bp product with the same primers. The presence of the Sycp1-Cre and fAR͞AR female genotype (Fig. 2). All 16 female progeny from Ϫ internal control IL-2 genes was confirmed by PCR. (C)(Upper) Genotyping of testis three individual G-AR /y male mice (mice A–C in Table 1) at not genomic DNA of G-ARϪ/y mice using primers ‘‘select and 2–3.’’ The PCR product more than 15 weeks of age, were genotyped as ar͞AR heterozygous amplified by ‘‘select and 2–3’’ can distinguish WT and fAR alleles with PCR genotype. Five of 10 female progeny were genotyped ar͞AR products Ϸ460 and Ϸ510 bp, respectively.