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Knockout of the Histone Demethylase Kdm3b Decreases Spermatogenesis and Impairs Male Sexual Behaviors Zhaoliang Liu1, 2, Mario G

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Knockout of the Histone Demethylase Kdm3b Decreases Spermatogenesis and Impairs Male Sexual Behaviors Zhaoliang Liu1, 2, Mario G Int. J. Biol. Sci. 2015, Vol. 11 1447 Ivyspring International Publisher International Journal of Biological Sciences 2015; 11(12): 1447-1457. doi: 10.7150/ijbs.13795 Research Paper Knockout of the Histone Demethylase Kdm3b Decreases Spermatogenesis and Impairs Male Sexual Behaviors Zhaoliang Liu1, 2, Mario G. Oyola1, Suoling Zhou1, Xian Chen1, Lan Liao1, Jean Ching-Yi Tien1, Shailaja K. Mani1, Jianming Xu1, 3 1. Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX, USA. 2. Institute of Cancer Research, Harbin Medical University, Harbin, China. 3. Institute for Cancer Medicine and College of Basic Medical Sciences, Sichuan Medical University, Luzhou, Sichuan, China Corresponding author: Jianming Xu, PhD, Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030. Phone: 713-798-6199; Fax: 713-798-3017; E-mail: [email protected] © 2015 Ivyspring International Publisher. Reproduction is permitted for personal, noncommercial use, provided that the article is in whole, unmodified, and properly cited. See http://ivyspring.com/terms for terms and conditions. Received: 2015.09.08; Accepted: 2015.10.04; Published: 2015.11.25 Abstract Kdm3b is a JmjC domain-containing histone H3 (H3) demethylase and its physiological functions are largely unknown. In this study, we found that Kdm3b protein is highly expressed in multiple cell types in the mouse testes, including Leydig cells, Sertoli cells, spermatogonia and spermatocytes at different differentiation stages. We also observed Kdm3b protein in the epithelial cells of the caput epididymis, prostate and seminal vesicle. Breeding tests revealed that the number of pups pro- duced by the breeding pairs with Kdm3b knockout (Kdm3bKO) males and wild type (WT) females was reduced 68% because of the decreased number of litters when compared with the breeding pairs with WT males and females. Further analysis demonstrated that Kdm3bKO male mice produced 44% fewer number of mature sperm in their cauda epididymides, displaying significantly reduced sperm motility. No significant differences in the circulating concentration of testosterone and the expression levels of androgen receptor and its representative target genes in the testis were observed. However, the circulating levels of 17β-estradiol, a modulator of sperm maturation and male sexual behaviors, was markedly reduced in Kdm3bKO male mice. Strikingly, abrogation of Kdm3b in male mice significantly increased the latencies to mount, intromit and ejaculate and decreased the number of mounts and intromissions, largely due to their loss of interest in female odors. These findings indicate that Kdm3b is required for normal spermatogenesis and sexual behaviors in male mice. Key words: Kdm3b, knockout mice, testis, reproductive function, sexual behavior Introduction The male reproductive system consists of multi- genesis in mouse, the preleptotene germ cells derived ple organs, including testis, epididymis, prostate and from spermatogonia proliferation and differentiation seminal vesicle. In the testis, Leydig cells located out- undergo the first meiosis step and sequentially de- side of the seminiferous tubules produce testosterone, velop into leptotene, zygotene, pachytene and diplo- the essential steroid hormone required for spermato- tene germ cells as well as the secondary spermato- genesis and male sexual behaviors. Sertoli cells an- cytes. The haploid spermatids formed following the chored on the inner face of the seminiferous basement rapid second meiosis step eventually develop into membrane support all germ cells in the seminiferous spermatozoa with a condensed nucleus, an acrosome, epithelium and nourish the proliferation and differ- and a flagellum via the round and elongating sper- entiation of these germ cells [1]. During spermato- matid phases [1]. The spermatozoa travel to the lumen http://www.ijbs.com Int. J. Biol. Sci. 2015, Vol. 11 1448 of cauda epididymis for motility acquisition and normal spermatogenesis and male sexual behaviors. storage. Sperm development and maturation require steroid hormones, especially testosterone and estro- Materials and Methods gen. It has been shown that disruption of the andro- Mice gen receptor (AR) abolishes spermatogenesis and knockout of either the aromatase or the estrogen re- The wild type (WT) control mice and the heter- ceptor inhibits spermatogenesis and spermatozoa ozygous and homozygous Kdm3b knockout maturation [2, 3]. (Kdm3bKO) mice were produced from the heterozy- Recent studies revealed that epigenetic regula- gous breeding pairs as we described previously [16]. tion is also a key mechanism governing spermato- All mice used in this study had a mixed C57BL/6J and genesis male germ cell development [4-6]. Epigenetic 129SvEv/j strain background. All mice were housed regulation can be implicated in changes of the histone in our facility with a 12 hours light and 12 hours dark and DNA modification enzyme activities and/or al- cycle and allowed free access to water and food. An- terations of the epigenetic codes and readers of both imals used in the reproductive behavior studies were common histones and testis-specific histone variants, housed under a reverse light cycle (12 hours dark/12 all of which can be important for spermatogenesis [7]. hours light) with lights off at 6:00 am. Behavior as- Among all the epigenetic histone codes, the methyla- sessments were performed between 9:00 am and 3:00 tion status of the histone H3 lysine 9 (H3K9) appears pm. All the animal protocols were approved by the to be one of the most important epigenetic modifica- Institutional Animal Care and Use Committee of tions in the testis. We have shown that the mono-, di- Baylor College of Medicine and were conducted in and tri-methylation levels of H3K9 (H3K9me1/2/3) in accordance with the National Institutes of Health the germ cells undergo dynamic changes during Guidelines. spermatogenesis [4]. Each one of the several histone Histological analysis and immunohistochem- methyltransferases and demethylases including istry (IHC) Suv39h1/2, G9a, ESET, Kdm3a, and Jmjd1c can mod- The testes, epididymides, prostates and seminal ify the methylation of H3K9 and appears to be indis- vesicles were isolated from sacrificed WT and pensable during spermatogenesis [4, 8-11]. However, Kdm3bKO male mice and fixed in 4% paraformalde- the expression pattern and role of Kdm3b, a close hyde overnight at 4oC. Paraffin sections were pre- family member of Kdm3a and Jmjd1c [12], in the testis pared from these tissues as described previously [16, and spermatogenesis have not been studied. 17]. These sections were stained with Hematoxylin Kdm3b is a histone H3 demethylase that specif- and Eosin (H&E) and then, examined and imaged ically catalyzes the demethylation of H3K9me1/2 under a microscope. IHC was performed as described [12]. Several studies have associated the role of previously [4, 18]. Briefly, the de-paraffinized and Kdm3b with cell growth and transformation. Specifi- re-hydrated tissue sections were treated in the 10 µM cally, Kdm3b plays a potential tumor suppressor role sodium citrate buffer at 95°C for 10 minutes for anti- in myeloid leukemia, myelodysplasia and breast gen retrieval. The treated tissue slides were further cancer, but it may promote acute promyelocytic leu- incubated in the methanol solution with 3% H2O2 for kemia [12-15]. We have recently generated Kdm3b quenching the endogenous peroxidase. After blocking knockout (Kdm3bKO) mice. We found that knockout with 10% goat serum and 1% bovine serum albumin of Kdmb3 restricted the postnatal somatic growth of (BSA), the tissue slides were incubated with the pri- these mice. At the molecular level, Kdm3bKO mice mary antibody and then the biotin-conjugated sec- exhibited decreased levels of the insulin growth factor ondary antibody. The bound secondary antibody was binding protein-3 (IGFBP-3) expression, resulting in a visualized by sequentially incubating with the Avi- significantly decreased IGF-1 stability in the blood din-conjugated horseradish peroxidase (HRP) circulation and a restriction of postnatal somatic (AK-5200, Vector Laboratories) and its substrate dia- growth [16]. We also found that Kdm3bKO female minobenzidine (SK-4100, Vector Laboratories). The mice were infertile because of irregular estrous cycles tissue slides were counter-stained with hematoxylin, and decreased ovulation, fertilization, and uterine sealed in Permount and examined by bright field mi- decidual response [16]. croscopy. In this study, we defined the expression patterns of the Kdm3b protein in the male reproductive organs Antibodies and the physiological functions of Kdm3b in the male This study used antibodies against Kdm3b reproductive system through characterizing the re- (2621S, Cell signaling), histone H3 (H3) (ab1791, productive phenotypes of the male Kdm3bKO mice. Abcam), H3K9me1 (ab9045, Abcam), H3K9me2 Our results demonstrate that Kdm3b is required for (07-441, Upstate), H3K9me3 (07-442, Upstate), β-actin http://www.ijbs.com Int. J. Biol. Sci. 2015, Vol. 11 1449 (A2228, clone ac-74, Sigma-Aldrich). 45˚) was used to allow for ventral viewing. Once the female was introduced, males were allowed 1800 Quantitative RT-PCR (QPCR) seconds to mount, intromit or ejaculate. Once ejacula- RNA samples were prepared from the testes of tion occurred the test was terminated. If the male WT and Kdm3bKO mice by using the Trizol Reagent neither intromitted nor ejaculated within1800 sec- (15596-018, Invitrogen).
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