REPRODUCTIONRESEARCH Expression of the rhesus glycoproteins, ammonia transporter family members, RHCG and RHBG in male reproductive organs Hyun-Wook Lee1, Jill W Verlander1, Mary E Handlogten1, Ki-Hwan Han2, Paul S Cooke3 and I David Weiner1,4 1Department of Medicine, University of Florida College of Medicine, PO Box 100224, Gainesville, Florida 32610, USA, 2Anatomy Department, Ewha Womans University, Seoul, Korea, 3Department of Physiological Sciences, University of Florida College of Veterinary Medicine, Gainesville, Florida, USA and 4Nephrology and Hypertension Section, NF/SGVHS, Gainesville, Florida, USA Correspondence should be addressed to I D Weiner; Email: [email protected]fl.edu Abstract The rhesus glycoproteins, Rh B glycoprotein (RHBG) and Rh C glycoprotein (RHCG), are recently identified ammonia transporters. Rhcg expression is necessary for normal male fertility, but its specific cellular expression is unknown, and Rhbg has not been reported to be expressed in the male reproductive tract. This study sought to determine the specific cellular expression of Rhcg, to determine whether Rhbg is expressed in the male reproductive tract, and, if so, to determine which cells express Rhbg using real-time RT-PCR, immunoblot analysis, and immunohistochemistry. Both Rhbg and Rhcg were expressed throughout the male reproductive tract. In the testis, high levels of Rhbg were expressed in Leydig cells, and Rhcg was expressed in spermatids during the later stages of their maturation (steps 13–16) in stages I–VIII of the seminiferous epithelium cycle. In the epididymis, basolateral Rhbg was present in narrow cells in the initial segment, in principal cells in the upper corpus, and in clear cells throughout the epididymis. Apical Rhcg immunolabel was present in principal cells in the caput and upper corpus epididymidis and in clear cells in the middle and lower corpus and cauda epididymidis. In the vas deferens, apical Rhcg immunolabel and basolateral Rhbg immunolabel were present in some principal cells and colocalized C with H -ATPase immunolabel. We conclude that both Rhbg and Rhcg are highly expressed in specific cells in the male reproductive tract where they can contribute to multiple components of male fertility. Reproduction (2013) 146 283–296 Introduction kidneys, where ammonia formation and transport are central to renal regulation of acid–base homeostasis The ammonia transporter family, which includes Mep (Eladari et al. 2002, Quentin et al. 2003, Verlander et al. proteins in yeast, AMT proteins in many bacteria and 2003, Weiner & Verlander 2011). In liver, Rh glyco- plants, and rhesus (Rh) glycoproteins in mammalian proteins are expressed in perivenous hepatocytes, which species, is a recently identified extended family of mediate high-affinity ammonia metabolism (Weiner integral membrane proteins that mediate critical roles et al. 2003). Sweat contains ammonia in concentrations in transmembrane ammonia transport (Winkler 2005, w100-fold greater than in plasma (Brusilow & Gordes Weiner 2006, Knepper 2008, Weiner & Verlander 2010). 1964, 1968, Liu et al. 2001), and Rh glycoproteins are Mammals express three ammonia transporter family highly expressed in sweat glands (Liu et al. 2001). members, Rh A glycoprotein (RHAG), RHBG, and Another tissue that expresses Rh glycoproteins is the RHCG (Liu et al. 2000, 2001, Marini et al. 2000, Weiner male reproductive tract. RHCG is expressed in both the & Verlander 2010). Rh glycoproteins are integral testis and epididymis (Liu et al. 2000), and its expression, membrane proteins with 12 transmembrane segments. at least as determined by global knockout studies, is They are present in plasma membranes in a homo- necessary for normal male fertility (Biver et al. 2008). trimeric form and enable specific transport of ammonia, However, both the testis and epididymis contain but no other known solutes, across plasma membranes. multiple cell types with distinct cellular functions, and Rh glycoproteins are expressed specifically in tissues the specific cells in these organs that express Rhcg have in which ammonia transport is necessary for normal not been identified. In the studies reporting the initial biological functioning. They are highly expressed in the cloning of mammalian RHBG, northern blots did not q 2013 Society for Reproduction and Fertility DOI: 10.1530/REP-13-0154 ISSN 1470–1626 (paper) 1741–7899 (online) Online version via www.reproduction-online.org Downloaded from Bioscientifica.com at 09/29/2021 10:24:30PM via free access 284 H-W Lee and others identify RHBG mRNA expression in male reproductive transcribed using SuperScript First Strand Synthesis System organs (Liu et al. 2001). However, we have shown that for RT-PCR (Invitrogen) and random hexamer primers. For RHBG is expressed in several tissues in which it was real-time PCR amplification of Rhbg, we used forward 0 0 not identified in the initial cloning report (Handlogten primer, 5 -GCCTGCAGAGTGTGTTTCCA-3 ; reverse primer, 0 0 et al. 2005, Han et al. 2009). Thus, whether RHBG is 5 -GAGCTGATACACGGCCTGAGA-3 ; and fluorescent probe, expressed in the male reproductive tract is unclear. 6Fam-TGGCACTCCGCTGACCCTTGG-Tamra. For Rhcg, the forward primer was 50-GGATACCCCTTCTTGGACTCTTC-30, In view of the importance of RHCG in male fertility, but 0 0 lack of knowledge regarding its specific cellular the reverse primer was 5 -TGCCTTGGAACATGGGAAAT-3 , expression, and the uncertainty regarding whether and the fluorescent probe was 6Fam-AGCCTCCGCCTGCTCC- RHBG is expressed in the male reproductive organs, the CCAAC-Tamra. Real-time RT-PCR was performed on an ABI purpose of the current study was to perform a detailed Prism GeneAmp 7500 Sequence Detection System. We used a examination of RHCG and RHBG mRNA and protein two-step cycle protocol including an initial 95 8C denaturation 8 expression in the testis and epididymis. Our results show step for 15 s and then 60 C for 1 min and then 40–50 cycles of alternating temperatures. 18S RNA was amplified in all distinct cell-specific RHCG and RHBG expression in the experiments as an internal control using commercially testis and cell-specific expression combined with axial available primers and probes (Applied Biosystems). All heterogeneity in the epididymis and vas deferens. These experiments included samples not treated with RT and samples observations indicate that RHCG and RHBG are likely to which RNA was not added as internal controls. All results to be involved in multiple components of male fertility. C were confirmed by repeating the experiments on specimens Ammonia exists in two molecular forms, NH4 and from at least three different mice. NH3, in aqueous solutions. In this manuscript, we use the term ‘ammonia’ to refer to the combination of these two Protein preparation molecular forms. When referring specifically to one of C these molecular forms, we use the terms ‘NH3’ or ‘NH4 .’ Animals were anesthetized with inhalant isoflurane, and the testes and epididymides were rapidly removed. The epididy- mides were dissected cleaned of connective tissues and Materials and methods divided into caput, corpus, and cauda regions. The tissues were frozen in liquid nitrogen and stored at K70 8C until used. Animals The tissues were homogenized using microtube pestles (USA Adult wild-type C57Bl/6 mice were used in these studies. All Scientific, Ocala, FL, USA), and proteins were extracted using animal studies were approved by the University of Florida T-PER Tissue Protein Extraction Reagent (Pierce Biotechnology, College of Medicine and the North Florida/South Georgia Rockford, IL, USA) according to the manufacturer’s rec- Veterans Health System IACUC. ommended procedures. An aliquot was obtained for protein determination using a BCA assay, and the remainder was stored frozen at K70 8C until used. Antibodies Affinity-purified antibodies to RHCG and RHBG generated in Immunoblotting procedure our laboratory have been characterized previously (Verlander Ten micrograms of protein were electrophoresed on 10% PAGE et al. 2003, Weiner et al. 2003, Mak et al. 2006). Antibodies to C ReadyGel (Bio-Rad). Gels were then transferred electrophoreti- the a4 subunit of H -ATPase were kindly provided by Dr Fiona cally to nitrocellulose membranes, blocked with 5 g/dl nonfat Karet (University of Cambridge, UK) and rabbit polyclonal dry milk, and incubated at 4 8C overnight with primary antibody for glutamine synthetase (cat #AB73593) was antibody diluted in Blotto buffer (50 mM Tris, 150 mM NaCl, purchased from Abcam (Cambridge, MA, USA). 5mMNa2EDTA, and 0.05% Tween 20, pH 7.6) with 5 g/dl nonfat dry milk. Loading and transfer equivalence were mRNA extraction assessed with Ponceau S staining. After washing, membranes were exposed to secondary antibody goat anti-rabbit IgG Adult C57Bl/6 male mice were anesthetized with inhalant (Millipore, Billerica, MA, USA) conjugated to HRP at a dilution isoflurane. After the mice were killed, the kidney, testis, caput, of 1:5000. Sites of antibody–antigen reaction were visualized corpus and cauda epididymidis, and vas deferens were using ECL (SuperSignal West Pico Substrate, Pierce) and a removed rapidly, placed into RNAlater (Qiagen), and stored Kodak Image Station 440CF digital imaging system. Band in a K70 8C freezer until used. Total RNA was extracted from density was quantified using Kodak 1D, version 5.0, Software kidney, testis, and epididymis using RNeasy Mini Kit (Qiagen) (Kodak Scientific Imaging, New Haven, CT, USA). Absence of and stored in a K70 8C freezer until used. saturation was confirmed by examining pixel intensity distribution in all immunoblots. Real-time RT-PCR Immunohistochemistry Real-time RT-PCR amplification of Rhbg and Rhcg was performed as described in detail previously (Weiner et al. Following isoflurane anesthesia, testis and epididymis were 2003, Handlogten et al. 2005). Briefly, total RNA was reverse preserved by in vivo cardiac perfusion with PBS (pH 7.4) Reproduction (2013) 146 283–296 www.reproduction-online.org Downloaded from Bioscientifica.com at 09/29/2021 10:24:30PM via free access RHCG and RHBG expression in testis and epididymis 285 followed by periodate–lysine–2% paraformaldehyde and then (Kim et al. 2009).