Biochimica et Biophysica Acta 1760 (2006) 1482–1488 www.elsevier.com/locate/bbagen Vitamin K deficiency reduces testosterone production in the testis through down-regulation of the Cyp11a a cholesterol side chain cleavage enzyme in rats ⁎ Hitoshi Shirakawa a, ,1, Yusuke Ohsaki a,1, Yoshihiko Minegishi a, Naofumi Takumi a, Kousaku Ohinata a, Yuji Furukawa a, Takeo Mizutani b, Michio Komai a a Laboratory of Nutrition, Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555, Japan b ALA Research Center, Yokohama 246-0002, Japan Received 5 March 2006; received in revised form 17 May 2006; accepted 30 May 2006 Available online 6 June 2006 Abstract Vitamin K (K) is an essential factor for the posttranslational modification of blood coagulation factors as well as proteins in the bone matrix (Gla proteins). It is known that K is not only distributed in the liver and bones but also abundantly distributed in the brain, kidney, and gonadal tissues. However, the role of K in these tissues is not well clarified. In this study, we used DNA microarray and identified the genes whose expression was affected in the testis under the K-deficient (K-def) state. The expression of genes involved in the biosynthesis of cholesterol and steroid hormones was decreased in the K-def group. The mRNA levels of Cyp11a – a rate-limiting enzyme in testosterone synthesis – positively correlated with the menaquinone-4 (MK-4) concentration in the testis. Moreover, as compared to the control (Cont) and K-supplemented (K-sup) groups, the K-def group had decreased testosterone concentrations in the plasma and testis. These results suggested that K is involved in steroid production in the testis through the regulation of Cyp11a. © 2006 Elsevier B.V. All rights reserved. Keywords: Vitamin K; Testis; Testosterone; Steroidogenesis; Cyp11a 1. Introduction papers have reported that K is not only distributed in the liver and bone tissues but also distributed abundantly in the brain and Vitamin K (K) is an essential factor for maintaining blood gonadal tissues [3–5]. Some amount of the K present in the coagulation and bone metabolism in mammals [1,2]. There are brain and gonadal tissues may play a role in the process of two types of naturally occurring K, namely, phylloquinone (K1) protein γ-carboxylation. However, it has been suggested that K and menaquinone (MK-n or K2). K1 is synthesized in plants and might have some functions other than those known thus far has a phytyl side chain at position 3 of 2-methyl naphthoqui- because the GGCX enzymatic activity and the amount of none. K2 is mainly produced by microorganisms and has a long substrates for GGCX in the brain and gonadal tissues are isoprenyl side chain (n=4–15). It is well known that both K1 considerably lower than those in the liver and bones. Recently, and K2 function as cofactors of the K-dependent carboxylase K has been reported to have other functions that are not related (γ-glutamyl carboxylase, GGCX) that converts the glutamic to protein γ-carboxylation. For example, menaquinone-4 (MK- acid residues in the blood coagulation factors and proteins in the 4), which is one of the K2s, inhibits osteoclast differentiation bone matrix into γ-carboxyglutamic acid (Gla) [2]. Several [6–11] and induces apoptosis in osteoclasts [7,12] and tumor- derived cells [13–16]. On the other hand, it inhibits apoptosis in human osteoblasts [17] and neuronal cells induced by oxidative ⁎ Corresponding author. Tel.: +81 22 717 8812; fax: +81 22 717 8813. stress [18]. Furthermore, MK-4 functions as a ligand for the E-mail address: [email protected] (H. Shirakawa). nuclear receptor SXR and regulates the expression of certain 1 These authors have equally contributed to this work. genes [19]. 0304-4165/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2006.05.008 H. Shirakawa et al. / Biochimica et Biophysica Acta 1760 (2006) 1482–1488 1483 In this study, we identified genes with altered expressions in K1 and MK-4 concentrations were determined by measuring the relative the K-deficient (K-def) state in the testis. In rats, the testis is one fluorescent intensity using menaquinone-3 (Eisai, Japan) as an internal standard [3]. of the organs that has almost the same K concentration as that in the liver [5]. Therefore, we hypothesized that the K-def state 2.3. RNA preparation and DNA microarray experiment directly influences gene expression in the testis. In the screening using the DNA microarray technique, the K-def group showed Total RNA was isolated from the whole testis by using the guanidine– decreased levels of mRNA encoding cholesterol and steroid isothiocyanate based-reagent Isogen (Nippon Gene, Japan) according to the synthesis enzymes. Furthermore, the mRNA levels of Cyp11a – instruction manual. In each dietary group, equivalent amounts of RNA from μ a rate-limiting enzyme in steroid hormone synthesis – positively individual animals were pooled, and 10 g of the pooled RNA was used as a template to synthesize cDNA that was then hybridized with probes on a DNA correlated with the K concentrations (MK-4 concentrations) in microarray. RNA was denatured with oligo-dT (2 μg, Amersham Biosciences, the testis. In addition, the testosterone concentration in the testis UK) at 65 °C for 5 min and then incubated in 50 mM Tris–HCl (pH 8.3); 75 mM and plasma was significantly decreased in the K-def group when KCl; 2.5 mM MgCl2; 10 mM dithiothreitol (DTT) containing 1 mM dATP, compared with those in the control (Cont) and MK-4- dGTP, and TTP; 200 units of SuperscriptII reverse transcriptase (Invitrogen, α 33 supplemented (MK-4-sup) groups. These results suggest that Carlsbad, CA); 60 units of RNaseOUT RNase inhibitor (Invitrogen); and - P dCTP (1.85 MBq, 110 TBq/mmol, Amersham Biosciences) at 42 °C for 50 min. K could participate in normal steroid hormone production in the After the cDNA synthesis, free nucleotides were separated using a BioRad gel testis and that K has additional functions that are not related to filtration spin column (Hercules, CA). Labeled cDNAs were hybridized to the protein γ-carboxylation. GeneFilter membrane (Research Genetics, Huntsville, AL) in the ExpressHyb hybridization solution (Clontech, Palo Alto, CA) containing mouse Cot-1 DNA 2. Materials and methods (1 μg/ml, Invitrogen) for 16 h at 65 °C. Following hybridization, the filters were washed according to GeneFilter's instruction manual, exposed to a Fuji imaging 2.1. Animals and diets plate (Fuji Photo Film, Japan), and then analyzed using a Bioimage analyzer FLA-2000 (Fuji Photo Film). K-def diet was obtained from Harlan-Teklad (Madison, WI, CAT #TD97053). Cont and K1-supplemented (K1-sup) diets were prepared by 2.4. Quantitative RT-PCR adding K1 (Eisai, Japan) to the K-def diet (final concentrations of 0.75 and 75 mg/kg diet, respectively). In addition, MK-4-sup diet was prepared by adding For cDNA synthesis, 2 μg of total RNA was used as a template. RNA was MK-4 (Eisai, Japan) to the K-def diet (final concentration of 75 mg/kg diet). All incubated in RT buffer (50 mM Tris–HCl pH 8.3, 75 mM KCl, and 5 mM DTT) diets were sterilized by irradiation with γ-rays (50 k Gy). containing 50 units of SuperscriptIII reverse transcriptase, 40 units of Germ-free rats were originally obtained from the ALA Research Center RNaseOUT RNase inhibitor, 1 mM dNTP, and 1 μg of oligo-dT at 50 °C for (Yokohama, Japan) and self bred. The animals (male, 10–12 weeks of age) were 50 min. An aliquot of cDNA was used as a template for quantitative PCR using housed at 23 °C±2 °C under a 12:12 light:dark cycle (lights switched on at Applied Biosystems Sequence Detection System 7000 (Foster City, CA). The 8:00 AM) in a sterilized plastic isolator and fed the experimental diets for 9 days. gene specific primers that are listed in Table 1 were used for the amplification of Stool examinations for aerobic and anaerobic bacteria, parasites, and fungi were certain cDNAs using the Syber Premix Ex Taq solution (Takara Bio, Japan). The negative both before and after the experiment. DNA fragments amplified by each primer set were confirmed by DNA The experimental plan of the present study was approved by the Animal sequencing. The relative expression level of each mRNA was normalized by Research-Animal Care Committee of the Graduate School of Agricultural using the glyceraldeyde-3-phosphate dehydrogenase mRNA level. Science, Tohoku University. The entire experiment was performed under the guidelines framed by this committee in accordance with the Japanese 2.5. Western blot analysis governmental legislation (1980). The same committee supervised the care and use of the rats in this study. The testis was homogenized in 5 volumes of phosphate-buffered saline and centrifuged at 12,000×g at 4 °C for 5 min. The collected supernatant was 2.2. HPLC measurement of the K content in the testis denatured in sodium dodecyl sulfate (SDS) gel loading buffer (62.5 mM Tris–HCl pH 6.8, 2% SDS, 50 mM DTT, and 6% glycerol, final) and Tissue samples were homogenized in 5 volumes of 66% 2-propanol. K electrophoresed on a 12.5% SDS-polyacrylamide gel. The protein bands were was extracted from the homogenate by using 6 volumes of n-hexane, as transferred onto Immobilon-P membranes (Millipore, Billerica, MA). After previously described [3], and measured using a fluorescence-HPLC system blocking in TBS-T (10 mM Tris–HCl pH 7.4, 150 mM NaCl, and 0.1% (Waters 600E system; Puresil 5C18 column, Waters, Milford, MA; RC 10-3 Tween 20) containing 5% dried milk, the membranes were reacted with anti- PtO2 column, IRICA, Japan; Hitachi F-1000 fluorescence detector, excitation rat Cyp11a antibody (1/5000 dilution in TBS-T containing 5% dried milk, at 240 nm, emission at 430 nm; Hitachi D-2000 data processor, Japan).