ARID4A and ARID4B Regulate Male Fertility, a Functional Link to the AR and RB Pathways

ARID4A and ARID4B regulate male fertility, a functional link to the AR and RB pathways Ray-Chang Wua,1, Ming Jianga, Arthur L. Beaudetb, and Mei-Yi Wua,1 aDepartment of Biochemistry and Molecular Medicine, The George Washington University, Washington, DC 20037; and bDepartment of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030 Edited* by Bert W. O’Malley, Baylor College of Medicine, Houston, TX, and approved February 5, 2013 (received for review October 22, 2012) ARID4A and ARID4B are homologous members of the ARID (AT-rich AR functions as a ligand-dependent transcription factor, and interaction domain) gene family. ARID4A and ARID4B physically in- its transcriptional activity is known to require the interactions teract with each other. ARID4A is a retinoblastoma (RB)-binding with various coregulators that act in a sequential and combina- protein. Biological function of these interactions is still unknown. torial manner to regulate expression of the AR-responsive genes. Here, we report that mice with complete deficiency of Arid4a,com- Retinoblastoma (RB) has been reported to interact with AR and bined with haploinsufficiency of Arid4b (Arid4a−/−Arid4b+/−), functions as a coactivator to induce AR transcriptional activity fi showed progressive loss of male fertility, accompanied by hypogo- (11). The Sertoli cell-speci c Rb conditional knockout mice nadism and seminal vesicle agenesis/hypodysplasia. Arid4a and displayed progressive infertility in males (12). These infertile Arid4b are expressed mainly in Sertoli cells of testes, which implies male mice exhibited Sertoli cell dysfunction, leading to loss of that their roles in Sertoli cell function are to support spermatogen- elongating spermatids and spermatozoa, as well as loss of the integrity of the blood–testis barrier (12). esis and create the impermeable blood–testis barrier. In fact, eval- − − + − ARID4A/Arid4a and ARID4B/Arid4b are homologous members uation of germ cell development in the Arid4a / Arid4b / mice of the ARID (AT-rich interaction domain) gene family. Previously, showed spermatogenic arrest at the stages of meiotic spermato- ARID4A and ARID4B were known as RB-binding protein cytes and postmeiotic haploid spermatids. Analysis of the integrity 1(RBBP1, RBP1)(13)andRBBP1-like protein 1 (RBBP1L1) (14), – of the blood testis barrier showed increased permeability of semi- respectively. Biochemical analyses have suggested that ARID4A niferous tubules in the Arid4a−/−Arid4b+/− testes. Interestingly, − − + − and ARID4B are members of the chromatin-remodeling complex / / CELL BIOLOGY phenotypic Sertoli cell dysfunction in the Arid4a Arid4b mice, and function as transcriptional repressors upon recruitment by RB including spermatogenic failures and the impaired blood–testis bar- (15, 16). Recently, we generated the Arid4a and Arid4b knockout rier, recapitulated the defects found in the Sertoli cell-specifican- mouse models and demonstrated that ARID4A and ARID4B drogen receptor (AR) knockout mice and the Sertoli cell-specific physically and functionally interact with each other (17, 18). RB knockout mice. Investigation of the molecular mechanism iden- Studies of these mouse models provided in vivo evidence that tified several AR- and RB-responsive genes as downstream targets Arid4a and Arid4b play a role in genomic imprinting and function of ARID4A and ARID4B. Our results thus indicate that ARID4A and as leukemia suppressor genes through regulation of epigenetic ARID4B function as transcriptional coactivators for AR and RB and modifications (17, 18). play an integral part in the AR and RB regulatory pathways in- Although ARID4A and ARID4B are part of the RB complex, volved in the regulation of Sertoli cell function and male fertility. the biological relationship of ARID4A and ARID4B to RB has not been defined. In this study, using the Arid4a and Arid4b knockout male reproductive function | androgen receptor coactivator mouse model, we found that Arid4a and Arid4b play important roles in Sertoli cell function. Investigation of the transcriptional consequences after the Arid4a and Arid4b knockout identified their ale fertility is a complex process that involves growth and downstream targets overlapping the AR- and RB-responsive genes. Mformation of male reproductive organs as well as initiation We further demonstrated that ARID4A and ARID4B function as and maintenance of spermatogenesis. Spermatogenesis is a de- coactivators to enhance the AR and RB transcriptional activity. velopmental process in which mature spermatozoa are generated Our studies indicate that ARID4A and ARID4B not only physi- within seminiferous tubules of testes (reviewed in ref. 1). In cally interact with AR and RB, but also functionally link to the AR addition to the germ cells, testes contain three somatic cell types and RB pathways in regulation of male fertility. including Sertoli cells, peritubular myoid cells, and Leydig cells. Sertoli cells are the epithelium of the seminiferous tubules and Results provide both physical and nutritional supports for the developing Mice Deficiency for Arid4a and Haploinsufficiency for Arid4b Displayed germ cells (2). Between adjacent Sertoli cells, tight junctions are – Progressive Loss of Fertility in Males. To determine the involvement of formed to create the blood testis barrier, which segregates the Arid4a and Arid4b in male fertility, male mice with different Arid4a − − + − developing germ cells into the immunologically privileged adlu- and Arid4b genotypes, including Arid4a / (n = 25), Arid4b / + − + − − − + − minal compartment and prevents passage of cytotoxic substances (n = 21), Arid4a / Arid4b / (n = 19), Arid4a / Arid4b / (n = 32), into the seminiferous tubules (2, 3). and wild type (n = 15), were mated with adult wild-type females. Androgen receptor (AR), a member of the steroid hormone We monitored fertility of male mice from 2 to 12 mo of age. Adult − − + − + − + − receptor superfamily, mediates the androgen action and plays an Arid4a / , Arid4b / , Arid4a / Arid4b / , and wild-type males were − − + − important role in male fertility (4). Global AR knockout male fully fertile (Fig. 1A). In contrast, adult Arid4a / Arid4b / males mice exhibited infertility with strict defects in male reproductive organs and severe disruption of germ cell development (5). The fi fi cell-speci c AR knockout mouse models further clari ed the Author contributions: R.-C.W. and M.-Y.W. designed research; R.-C.W., M.J., and M.-Y.W. functions of AR in each specific type of testicular cells (6). performed research; R.-C.W. and A.L.B. contributed new reagents/analytic tools; R.-C.W. Sertoli cell-specific AR knockout affected the Sertoli cell func- and M.-Y.W. analyzed data; and R.-C.W. and M.-Y.W. wrote the paper. tions of nourishing and supporting the developing germ cells, The authors declare no conflict of interest. resulting in spermatogenesis arrest before first meiotic division *This Direct Submission article had a prearranged editor. and during the transition from round to elongated spermatids 1To whom correspondence may be addressed. E-mail: [email protected] or meiyiwu@gwu. (7–9). In addition, lack of AR in Sertoli cells causes impairment edu. – of the blood testis barrier (10). As expected, the Sertoli cell- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. specific AR conditional knockout male mice were infertile (7–9). 1073/pnas.1218318110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1218318110 PNAS Early Edition | 1of6 Downloaded by guest on October 2, 2021 displayed age-related reduced fertility. We found that 29 of 32 male mice were fertile at a younger age but lost their fertility at an − − + − (∼90%) Arid4a / Arid4b / males were fertile at 2 mo of age. The older age (from 3 to 12 mo of age) (Fig. 1A). These mice showed fertility decreased from 3 mo of age onward, with less than 40% of decreased size of testes with or without fused or hypotrophic − − + − the Arid4a / Arid4b / males remaining fertile at 1 y of age (Fig. seminal vesicles (Fig. 1C, mice 5 and 8). We also found that one 1A). These results showed that complete deficiency of Arid4a in male showed unilateral agenesis of seminal vesicles (Fig. 1C, combination with haploinsufficiency of Arid4b leads to progressive mouse 9), but remained fertile up to 12 mo old. infertility in male mice, suggesting that Arid4a collaborates with Measurements of testes revealed reduced testicular weights in the − − + − Arid4b to regulate male fertility. Despite the progressive loss of Arid4a / Arid4b / males compared with wild-type males at 2 and − − + − fertility, no significant difference in the litter size between the fertile 10 mo of age (Fig. 1 D, a). Testes of the infertile Arid4a / Arid4b / − − + − − − + − Arid4a / Arid4b / males and wild-type males was noted (Fig. 1B). mice weighed even less than the fertile Arid4a / Arid4b / mice − − + − Mice homozygous for Arid4b deficiency die early in embryogenesis (Fig. 1 D, a). The body weights of the Arid4a / Arid4b / mice between embryonic day 3 (E3) and E7 (17); therefore, conventional were also less than that of wild-type mice (17) (Fig. 1 D, b). It − − − − − − + − knockout mice with all four alleles mutated (Arid4a / Arid4b / ) should be noted that only the infertile Arid4a / Arid4b / males, − − + − are not available for the fertility assay. but not the fertile Arid4a / Arid4b / males, showed a significant We next examined the male reproductive organs for any mor- decrease in the ratios of testicular weight vs. body weight

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