- Email: [email protected]
Expression of the CD68 glycoprotein in the rat epididymis
Biochimie 118 (2015) 221e224
Contents lists available at ScienceDirect
Biochimie journal homepage: www.elsevier.com/locate/biochi
Short communication
Expression of the CD68 glycoprotein in the rat epididymis Giovanna Liguori, Valeria De Pasquale, Rossella Della Morte, Luigi Avallone, Anna Costagliola, Alfredo Vittoria, Simona Tafuri* Department of Veterinary Medicine and Animal Productions, University of Naples Federico II, Via F. Delpino n. 1, 80137 Naples, Italy
a r t i c l e i n f o
a b s t r a c t
Article history: Received 3 June 2015 Accepted 28 September 2015 Available online xxx
The 110 kDa trans-membrane glycoprotein CD68 is highly expressed by human monocytes and tissue macrophages. However, in addition to the monocyte/macrophage system, CD68 has been also found in normal and tumor cells with no macrophagic activity such as lymphocytes, fibroblasts, endothelial cells, small intestinal epithelial cells, and neoplastic cells of different origins. Here, for the first time we demonstrate the immunohistochemical localization of CD68 in the principal cells of the cranial and caudal segments of rat epididymis. These results were confirmed by biochemical analyses showing the expression of CD68 mRNA transcripts and the protein in the epididymis tissues. Our findings, while providing further evidence that CD68 expression is not restricted to the monocyte/macrophage system, suggest that the glycoprotein might be involved in the functions of epididymal principal cells that contribute to spermatozoa maturation process. © 2015 Published by Elsevier B.V.
Keywords: CD68 Epididymis Rat Immunohistochemistry
1. Introduction The glycoprotein CD68 is a member of lamp/lgp lysosomal plasma membrane shuttling protein family which includes mouse macrosialin [1], human lamp 1 [2], chicken lep 100 [3], and rat Igp 120 [4]. Due to their particular localization, lamp glycoproteins are found as components of the plasma membrane of the endoexocytic vesicular complex characteristic of the lysosomal activity of living animal cells. The CD68 protein is more abundant than other lamp components on the membrane of the monocyte/ macrophage system, thus representing a molecular marker for it [5]. However, in addition to the monocyte/macrophage system, CD68 has been also found in a wide range of normal and malignant cells with no macrophagic activity, including lymphocytes, fibroblasts, endothelial cells, small intestinal epithelial cells, and neoplastic cells of myeloid and non-myeloid origin [6e9]. The intracellular domain of CD68 protein contains a 10 amino acid sequence involved in the processing of the antigenic material internalized by pino-phagocytosis or, alternatively, in protecting the vesicular membrane of the lysosomal vacuoles from hydrolase attack [1,10]. On the other hand, the trans-membrane domain of CD68 is composed by 25 hydrophobic residues and an extracellular portion of 298 amino acids showing a mucin-like
* Corresponding author. E-mail address: [email protected] (S. Tafuri). http://dx.doi.org/10.1016/j.biochi.2015.09.034 0300-9084/© 2015 Published by Elsevier B.V.
structure for the presence of numerous side-chain carbohydrates O- and N-linked to the aminoacidic backbone. The mucin-like structure is typically present in a group of hematopoietic glycoproteins such as leuco-sialin/CD43, the lymph node endothelial ligand for L-selectin GlyCAM-1, and the stem cell antigen CD34 [10e13]. A glycoprotein of the granulocytic membrane showing a molecular structure similar to that of CD68 is involved in the cellular binding to P-selectins of the vascular endothelium inducing extra-vascular diapedesis [14]. The epididymis is the genital segment where the immature spermatozoa acquire fertilizing capability, their maturation being influenced by the aqueous environment of the epididymal tubules. The mono/pluri-layered epithelium of the epididymis is formed by six different cytotypes: principal, narrow, clear, apical, basal and halo cells [15]. The principal cells are rich in organelles for synthesis, secretion, and absorption, whereas basal cells abutting the basal lamina have little cytoplasm and sparse organelles. In an ultrastructural and immunohistochemical study of the human epididymis, lymphocytes and macrophages were observed between the epididymal epithelial cells and in the interstitial tissue. However, immunohistochemical reactions with CD68 antibodies were positive mainly in cells located in the interstitial tissue [16]. The evidence that CD68 binding to organ specific lectin or selectins is probably responsible for the shifting movements of the macrophages on cellular layers and their homing in particular tissues and organs prompted us to better investigate the expression of
222
G. Liguori et al. / Biochimie 118 (2015) 221e224
CD68 in mammalian epididymis where the glycoprotein could play a role during spermatozoa transit. To this purpose, we carried out a study aimed to establish the localization of CD68 in the epithelial cells of the cranial and caudal portions of rat epididymis by immunohistochemistry. We also investigated the expression of CD68 mRNA and the protein in these tissues by reversetranscription polymerase chain reaction (RT-PCR) and Western blotting analysis, respectively.
following specific primers were used: forward 50 - CATCCTTCACGGAGACACCT-30 and reverse 50 -GGGAATGAGAGAGCCAAGTG-30 for CD68; forward 50 -GAGGCTCAGAGCAAGAGAGG-30 and reverse 50 TGACATCTCGCACAATCTCC-30 for b-actin. Amplified PCR products were sequenced by Primm [GenBank accession numbers: NM001031638 for rat CD68, and NM-031144 for rat b-actin]. For Western blotting analysis, testis samples were homogenized and processed as previously described [17e20].
2. Material and methods
3. Results and discussion
2.1. Antibodies and chemicals
Immunohistochemical analysis demonstrated the presence of CD68 immunoreactivity in cells belonging to the principal cytotype which lined the mucosa of many tubules of the cranial (Fig. 1 a and b) and caudal (Fig. 1 c and d) portions of the epididymal epithelium (Fig. 1). In some tubules, the number of labeled cells was very high so that almost the entire profile of the lining epithelium appeared intensely stained. Few negative cells belonging to cytotypes other than the principal one were observed. The shape of the immunoreactive material was finely granular, and the granules appeared to be grouped in clusters filling the apical portion of the principal cells. Although no quantitative analysis was carried out, the amount of labeled cells observed in the cranial segment of the epididymis was approximately as high as that observed in the caudal portion. Control sections, obtained by replacing the CD68 antibody with phosphate saline buffer or pre-absorbing the CD68 antibody with an excess of its control peptide, resulted always negative (data not shown). Our immunohistochemical analysis demonstrates that CD68 glycoprotein is mainly localized in the tubules of both the cranial and caudal portions of the organ. The intense staining evidenced in the segments of the tubular complexes might be related to the elevated presence of lysosomes rendering such zones functionally more active than other areas. Indeed, the principal cells of the cranial and caudal tracts of the epididymis internalize from the lumen tracers which soon after are found in endocytic pits and vesicles, and later appear in multivesicular bodies and lysosomes [21]. The immunohistochemical detection of the glycoprotein CD68 in the rat epididymis was confirmed by the RT-PCR and immunoblotting results. The RT-PCR analysis for the presence of CD68 mRNA in the cranial and caudal portions of rat epididymis resulted in the amplification of 484 bp DNA fragments in all tested samples (Fig. 2A, top). A 469 bp transcript was obtained from the amplification of b-actin cDNA in all tested samples (Fig. 2A, bottom). The presence of the glycoprotein in the epididymis tissues was confirmed by immunoblotting using a goat polyclonal antibody raised against a peptide mapping near the C-terminus of CD68 of mouse origin. A band with a molecular size of approximately 110 kDa, corresponding to the known molecular size of the glycoprotein [1,22], was detected in all the tissue samples (Fig. 2B, upper blot, lane 1). The specificity of the response was confirmed by preincubation of the CD68 antibody with its blocking peptide. There was no expression of CD68 in these preparations (Fig. 2B, upper blot, lane 2). The stripping of the blot and its re-probing with a mouse monoclonal anti-tubulin antibody demonstrated equal loading of proteins in all lanes (Fig. 2B, lower blot). The presence of CD68 mRNA transcripts and the protein in both cranial and caudal portions of epididymis samples suggests that the glycoprotein is locally synthesized in the rat epididymis. The localization of CD68 in the cytoplasm of the principal cells of mammalian epididymis, while extends previous evidence that CD68 expression is not restricted to the monocyte/macrophage system, suggests that the glycoprotein might play a role in the functions of these epididymal cells.
Horseradish peroxidase conjugated anti-goat IgG (A-5420), and the mouse anti-tubulin monoclonal antibody (T6557) were purchased from Sigma Chemical Co. (St. Louis, MO, USA); the biotinylated secondary antibodies and avidin-biotin complex (PK6101) from Vector Laboratories (Burlingame, CA, USA); Triazol from Invitrogen (Carlsbad, CA, USA); the DC protein assay kit from BioRad Laboratories (Hercules, CA, USA); the chemioluminescence (ECL) kit, the GFX PCR DNA and gel purification kit (code 27-960201) from Amersham (Little Chalfont, UK); . The primers were provided by Primm (Milan, Italy) and the kit for the PCR and RT-PCR by Promega (Madison, WI, USA). 2.2. Animals and sampling Nine healthy adult Wistar male rats, kept in groups of three animals per cage, were housed in the vivarium of the Department of Veterinary Medicine and Animal Productions (University of Naples Federico II, Naples, Italy). The rats had free access to food and tap water, and were kept under constant condition of light, temperature (22 C) and humidity. Experimental procedures were approved by the Ethical Committee for Animal Testing of our University, and were conducted in accordance with the European Union normative for the care and use of experimental animals. The animals were sacrificed by decapitation, and the epididymes were collected. For immunohistochemical analysis, cranial (caput) and caudal portions of the epididymes were fixed in Bouin's fluid for 18e24 h. For biochemical analyses, samples from the epididymal segments were frozen in liquid nitrogen, and stored at 80 C. 2.3. Immunohistochemistry Immunohistochemical analysis was performed by the procedure previously described [17e19]. Briefly, after fixation, the samples were dehydrated through an ascending series of ethanol and embedded in Paraplast. Microtomical sections (6 mm thick) were deparaffinised, rehydrated, and stained by the avidin-biotin complex. In the specific step, the anti-CD68 antibody was applied on sections overnight. The 3,30 -diaminobenzidine (DAB) was used as a final staining. The sections were examined under a light microscope (Nikon E600), and microphotographs were taken by using a digital camera (Colpix 8400). In this study, two anti-CD68 antibodies were used: the goat polyclonal anti-CD68 antibody (M-20, sc-7084) from Santa Cruz Biotechnology and the mouse monoclonal anti-human CD68 (Clone KP1, M 0814) from DAKO. They gave similar results. 2.4. RT-PCR and immunoblotting analyses Total RNA extraction from testis samples, the synthesis of cDNA, and RT-PCR for the detection of CD68 transcripts were performed according to the procedure previously described [17,18]. The
G. Liguori et al. / Biochimie 118 (2015) 221e224
223
Fig. 1. Immunohistochemical localization of CD68 in the cranial (a, b) and caudal (c, d) portions of rat epididymis. (a) The lining epithelium of three adjacent tubules is composed of principal cells containing CD68-immunoreactive material. (b) The CD68 staining appears to be always localized in the apical cytoplasm of the cells. (c, d) Five tubules of the caudal epididymis almost entirely lined by CD68 positive principal cells. Negative basal cells closely in contact with the basal membrane are visible. Avidin-biotin immunohistochemical staining. Bars: a, c: 30 mm; b, d: 20 mm.
molecular complexes from the tubular compartment of the epididymis and secrete them into the lumen of the organ after metabolic rearrangements [25]. The secretion of high hydrophobic proteins associated with luminal membrane vesicles are liberated from the surface of the principal cells as vesicular blebs known as epididymosomes or as soluble high mass protein complexes [24]. This study provides the first evidence for the expression of CD68 in the principal cells of rat epididymis. Whether the glycoprotein is present in the epididymosomes and in the gametes is under investigation in order to establish the physiological role of CD68 in the mammalian epididymis. Fig. 2. Expression of CD68 mRNA and the protein in the rat epididymis. A. RT-PCR analysis of CD68 mRNA in the caudal portion of rat epididymis (lane 2). Lane 1 corresponds to the DNA ladder. Negative control (distilled water) is shown in lane 3. Bottom: Expression of b-actin mRNA transcripts in the same tissues of the top (RT-PCR internal control). The non-specific bands at the basis of lanes 2e3 correspond to the primer dimers. Similar results were obtained when cranial portions of rat epididymis were subjected to RT-PCR analysis. B. Western blotting analysis by using antiserum directed against CD68 (Santa Cruz). Lane 1: homogenate from the caudal portion of rat epididymis; lane 2: negative control (homogenate treated with the CD68 antiserum pre-treated with its control peptide). The upper blot was stripped and re-probed with an anti-tubulin monoclonal antibody to ensure equal loading of proteins in all lanes. On the left, molecular weight markers are indicated. Similar results were obtained when cranial portions of rat epididymis were subjected to Western blotting analysis. The results reported in panels A and B are representative of four separated experiments of identical design.
The epididymal epithelium is responsible for creating a luminal environment conducive to the acquisition of fertilizing capacity by the spermatozoa within it [23,24]. In particular, the composition of the fluid suitable for spermatozoa survival and maturation depends on the receptoresecretory activity of the principal cells of the epididymal epithelium. The principal cells internalize fluids and
Conflict of interest None. Acknowledgments This study was supported by the U.BI.CO. 11941 grant from the University of Naples Federico II. The technical assistance of Mr. Antonio Calamo is gratefully acknowledged. We thank Dr. Emma Cirillo for administrative work. References [1] C.L. Holness, R.P. da Silva, J. Fawcett, S. Gordon, D.L. Simmons, Macrosialin, a mouse macrophage-restricted glycoprotein, is a member of the lamp/lgp family, J. Biol. Chem. 268 (1993) 9661e9666. [2] M. Fukuda, Lysosomal membrane glycoproteins. Structure, biosynthesis, and intracellular trafficking, J. Biol. Chem. 266 (1991) 21327e21330. [3] A.S. Zot, D.M. Fambrough, Structure of a gene for a lysosomal membrane glycoprotein (LEP100). Housekeeping gene with unexpected exon organization, J. Biol. Chem. 265 (1990) 20988e20995. [4] C.L. Howe, B.L. Granger, M. Hull, S.A. Green, C.A. Gabel, A. Helenius, I. Mellman, Derived protein sequence, oligosaccharides, and membrane insertion of the
224
[5]
[6]
[7]
[8]
[9]
[10] [11]
[12]
[13]
[14]
G. Liguori et al. / Biochimie 118 (2015) 221e224 120-kDa lysosomal membrane glycoprotein (lgp120): identification of a highly conserved family of lysosomal membrane glycoproteins, Proc. Natl. Acad. Sci. U.S.A. 85 (1988) 7577e7581. L. Martinez-Pomares, N. Platt, A.J. McKnight, R.P. da Silva, S. Gordon, Macrophage membrane molecules: markers of tissue differentiation and heterogeneity, Immunobiology 195 (1996) 407e416. K.A. Pulford, A. Sipos, J.L. Cordell, W.P. Stross, D.Y. Mason, Distribution of the CD68 macrophage/myeloid associated antigen, Int. Immunol. 2 (1990) 973e980. L.A. Kunz-Schughart, A. Weber, M. Rehli, E. Gottfried, G. Brockhoff, S.W. Krause, R. Andreesen, M. Kreutz, The “classical” macrophage marker CD68 is strongly expressed in primary human fibroblasts, Verh. Dtsch. Ges. Pathol. 87 (2003) 215e223. X.P. Lin, N. Almqvist, E. Telemo, Human small intestinal epithelial cells constitutively express the key elements for antigen processing and the production of exosomes, Blood Cells Mol. Dis. 35 (2005) 122e128. E. Gottfried, L.A. Kunz-Schughart, A. Weber, M. Rehli, A. Peuker, A. Müller, M. Kastenberger, G. Brockhoff, R. Andreesen, M. Kreutz, Expression of CD68 in non-myeloid cell types, Scand. J. Immunol. 67 (2008) 453e463. C.L. Holness, D.L. Simmons, Molecular cloning of CD68, a human macrophage marker related to lysosomal glycoproteins, Blood 81 (1993) 1607e1613. C.S. Shelley, E. Remold-O'Donnell, A.E. Davis 3rd, G.A. Bruns, F.S. Rosen, M.C. Carroll, A.S. Whitehead, Molecular characterization of sialophorin (CD43), the lymphocyte surface sialoglycoprotein defective in Wiskott-Aldrich syndrome, Proc. Natl. Acad. Sci. U. S. A. 86 (1989), 2819-2823. Erratum in: Proc. Natl. Acad. Sci. U.S.A. 86 (1989) 4689. J. Cyster, C. Somoza, N. Killeen, A.F. Williams, Protein sequence and gene structure for mouse leukosialin (CD43), a T lymphocyte mucin without introns in the coding sequence, Eur. J. Immunol. 20 (1990) 875e881. L.A. Lasky, M.S. Singer, D. Dowbenko, Y. Imai, W.J. Henzel, C. Grimley, C. Fennie, N. Gillett, S.R. Watson, S.D. Rosen, An endothelial ligand for Lselectin is a novel mucin-like molecule, Cell 69 (1992) 927e938. K.L. Moore, N.L. Stults, S. Diaz, D.F. Smith, R.D. Cummings, A. Varki, R.P. McEver, Identification of a specific glycoprotein ligand for P-selectin
(CD62) on myeloid cells, J. Cell Biol. 118 (1992) 445e456. [15] L. Hermo, B. Robaire, Epididymal cell types and their functions, in: B. Robaire, B.T. Hinton (Eds.), The Epididymis: from Molecules to Clinical Practice, Raven Press Ltd., New York, 2002, pp. 81e102. [16] M. Marchlewicz, Localization of immunocompetent cells in the human epididymis, Folia Histochem. Cytobiol. 39 (2001) 173e174. [17] S. Tafuri, L.M. Pavone, R. Lo Muto, M. Basile, E. Langella, E. Fiorillo, L. Avallone, N. Staiano, A. Vittoria, Expression of orexin A and its receptor 1 in the rat epididymis, Regul. Pept. 155 (2009) 1e5. [18] S. Tafuri, R. Lo Muto, L.M. Pavone, S. Valiante, A. Costagliola, N. Staiano, A. Vittoria, Novel localization of orexin A in the tubular cytotypes of the rat testis, Regul. Pept. 164 (2010) 53e57. [19] L.M. Pavone, S. Tafuri, R. Lo Muto, A. Spina, A.E. Lychkova, V. Mastellone, A. Vittoria, L. Avallone, N. Staiano, Expression of the serotonin transporter SERT in the genital tract of cattle, Regul. Pept. 154 (2009) 1e4. [20] G. Cerulo, S. Tafuri, V. De Pasquale, S. Rea, S. Romano, A. Costagliola, R. Della Morte, L. Avallone, L.M. Pavone, Serotonin activates cell survival and apoptotic death responses in cultured epithelial thyroid cells, Biochimie 105 (2014) 211e215. [21] H.D. Moore, J.M. Bedford, The differential absorption activity of epithelial cells of the rat epididymis before and after castration, Anat. Rec. 193 (1979) 313e327. [22] K. Micklem, E. Rigney, J. Cordell, D. Simmons, P. Stross, H. Turley, B. Seed, D. Mason, A human macrophage-associated antigen CD68 detected by six different monoclonal antibodies, Br. J. Haematol. 73 (1989) 6. [23] C.H. Yeung, T.G. Cooper, Developmental changes in signaling transduction factors in maturing sperm during epididymal transit, Cell. Mol. Biol. (Noisy-legrand) 49 (2003) 341e349. [24] J.L. Dacheux, F. Dacheux, New insights into epididymal function in relation to sperm maturation, Reproduction 147 (2013) R27eR42. [25] D.G. Cyr, B. Robaire, L. Hermo, Structure and turnover of junctional complexes between principal cells of the rat epididymis, Microsc. Res. Tech. 30 (1995) 54e66.
Contents lists available at ScienceDirect
Biochimie journal homepage: www.elsevier.com/locate/biochi
Short communication
Expression of the CD68 glycoprotein in the rat epididymis Giovanna Liguori, Valeria De Pasquale, Rossella Della Morte, Luigi Avallone, Anna Costagliola, Alfredo Vittoria, Simona Tafuri* Department of Veterinary Medicine and Animal Productions, University of Naples Federico II, Via F. Delpino n. 1, 80137 Naples, Italy
a r t i c l e i n f o
a b s t r a c t
Article history: Received 3 June 2015 Accepted 28 September 2015 Available online xxx
The 110 kDa trans-membrane glycoprotein CD68 is highly expressed by human monocytes and tissue macrophages. However, in addition to the monocyte/macrophage system, CD68 has been also found in normal and tumor cells with no macrophagic activity such as lymphocytes, fibroblasts, endothelial cells, small intestinal epithelial cells, and neoplastic cells of different origins. Here, for the first time we demonstrate the immunohistochemical localization of CD68 in the principal cells of the cranial and caudal segments of rat epididymis. These results were confirmed by biochemical analyses showing the expression of CD68 mRNA transcripts and the protein in the epididymis tissues. Our findings, while providing further evidence that CD68 expression is not restricted to the monocyte/macrophage system, suggest that the glycoprotein might be involved in the functions of epididymal principal cells that contribute to spermatozoa maturation process. © 2015 Published by Elsevier B.V.
Keywords: CD68 Epididymis Rat Immunohistochemistry
1. Introduction The glycoprotein CD68 is a member of lamp/lgp lysosomal plasma membrane shuttling protein family which includes mouse macrosialin [1], human lamp 1 [2], chicken lep 100 [3], and rat Igp 120 [4]. Due to their particular localization, lamp glycoproteins are found as components of the plasma membrane of the endoexocytic vesicular complex characteristic of the lysosomal activity of living animal cells. The CD68 protein is more abundant than other lamp components on the membrane of the monocyte/ macrophage system, thus representing a molecular marker for it [5]. However, in addition to the monocyte/macrophage system, CD68 has been also found in a wide range of normal and malignant cells with no macrophagic activity, including lymphocytes, fibroblasts, endothelial cells, small intestinal epithelial cells, and neoplastic cells of myeloid and non-myeloid origin [6e9]. The intracellular domain of CD68 protein contains a 10 amino acid sequence involved in the processing of the antigenic material internalized by pino-phagocytosis or, alternatively, in protecting the vesicular membrane of the lysosomal vacuoles from hydrolase attack [1,10]. On the other hand, the trans-membrane domain of CD68 is composed by 25 hydrophobic residues and an extracellular portion of 298 amino acids showing a mucin-like
* Corresponding author. E-mail address: [email protected] (S. Tafuri). http://dx.doi.org/10.1016/j.biochi.2015.09.034 0300-9084/© 2015 Published by Elsevier B.V.
structure for the presence of numerous side-chain carbohydrates O- and N-linked to the aminoacidic backbone. The mucin-like structure is typically present in a group of hematopoietic glycoproteins such as leuco-sialin/CD43, the lymph node endothelial ligand for L-selectin GlyCAM-1, and the stem cell antigen CD34 [10e13]. A glycoprotein of the granulocytic membrane showing a molecular structure similar to that of CD68 is involved in the cellular binding to P-selectins of the vascular endothelium inducing extra-vascular diapedesis [14]. The epididymis is the genital segment where the immature spermatozoa acquire fertilizing capability, their maturation being influenced by the aqueous environment of the epididymal tubules. The mono/pluri-layered epithelium of the epididymis is formed by six different cytotypes: principal, narrow, clear, apical, basal and halo cells [15]. The principal cells are rich in organelles for synthesis, secretion, and absorption, whereas basal cells abutting the basal lamina have little cytoplasm and sparse organelles. In an ultrastructural and immunohistochemical study of the human epididymis, lymphocytes and macrophages were observed between the epididymal epithelial cells and in the interstitial tissue. However, immunohistochemical reactions with CD68 antibodies were positive mainly in cells located in the interstitial tissue [16]. The evidence that CD68 binding to organ specific lectin or selectins is probably responsible for the shifting movements of the macrophages on cellular layers and their homing in particular tissues and organs prompted us to better investigate the expression of
222
G. Liguori et al. / Biochimie 118 (2015) 221e224
CD68 in mammalian epididymis where the glycoprotein could play a role during spermatozoa transit. To this purpose, we carried out a study aimed to establish the localization of CD68 in the epithelial cells of the cranial and caudal portions of rat epididymis by immunohistochemistry. We also investigated the expression of CD68 mRNA and the protein in these tissues by reversetranscription polymerase chain reaction (RT-PCR) and Western blotting analysis, respectively.
following specific primers were used: forward 50 - CATCCTTCACGGAGACACCT-30 and reverse 50 -GGGAATGAGAGAGCCAAGTG-30 for CD68; forward 50 -GAGGCTCAGAGCAAGAGAGG-30 and reverse 50 TGACATCTCGCACAATCTCC-30 for b-actin. Amplified PCR products were sequenced by Primm [GenBank accession numbers: NM001031638 for rat CD68, and NM-031144 for rat b-actin]. For Western blotting analysis, testis samples were homogenized and processed as previously described [17e20].
2. Material and methods
3. Results and discussion
2.1. Antibodies and chemicals
Immunohistochemical analysis demonstrated the presence of CD68 immunoreactivity in cells belonging to the principal cytotype which lined the mucosa of many tubules of the cranial (Fig. 1 a and b) and caudal (Fig. 1 c and d) portions of the epididymal epithelium (Fig. 1). In some tubules, the number of labeled cells was very high so that almost the entire profile of the lining epithelium appeared intensely stained. Few negative cells belonging to cytotypes other than the principal one were observed. The shape of the immunoreactive material was finely granular, and the granules appeared to be grouped in clusters filling the apical portion of the principal cells. Although no quantitative analysis was carried out, the amount of labeled cells observed in the cranial segment of the epididymis was approximately as high as that observed in the caudal portion. Control sections, obtained by replacing the CD68 antibody with phosphate saline buffer or pre-absorbing the CD68 antibody with an excess of its control peptide, resulted always negative (data not shown). Our immunohistochemical analysis demonstrates that CD68 glycoprotein is mainly localized in the tubules of both the cranial and caudal portions of the organ. The intense staining evidenced in the segments of the tubular complexes might be related to the elevated presence of lysosomes rendering such zones functionally more active than other areas. Indeed, the principal cells of the cranial and caudal tracts of the epididymis internalize from the lumen tracers which soon after are found in endocytic pits and vesicles, and later appear in multivesicular bodies and lysosomes [21]. The immunohistochemical detection of the glycoprotein CD68 in the rat epididymis was confirmed by the RT-PCR and immunoblotting results. The RT-PCR analysis for the presence of CD68 mRNA in the cranial and caudal portions of rat epididymis resulted in the amplification of 484 bp DNA fragments in all tested samples (Fig. 2A, top). A 469 bp transcript was obtained from the amplification of b-actin cDNA in all tested samples (Fig. 2A, bottom). The presence of the glycoprotein in the epididymis tissues was confirmed by immunoblotting using a goat polyclonal antibody raised against a peptide mapping near the C-terminus of CD68 of mouse origin. A band with a molecular size of approximately 110 kDa, corresponding to the known molecular size of the glycoprotein [1,22], was detected in all the tissue samples (Fig. 2B, upper blot, lane 1). The specificity of the response was confirmed by preincubation of the CD68 antibody with its blocking peptide. There was no expression of CD68 in these preparations (Fig. 2B, upper blot, lane 2). The stripping of the blot and its re-probing with a mouse monoclonal anti-tubulin antibody demonstrated equal loading of proteins in all lanes (Fig. 2B, lower blot). The presence of CD68 mRNA transcripts and the protein in both cranial and caudal portions of epididymis samples suggests that the glycoprotein is locally synthesized in the rat epididymis. The localization of CD68 in the cytoplasm of the principal cells of mammalian epididymis, while extends previous evidence that CD68 expression is not restricted to the monocyte/macrophage system, suggests that the glycoprotein might play a role in the functions of these epididymal cells.
Horseradish peroxidase conjugated anti-goat IgG (A-5420), and the mouse anti-tubulin monoclonal antibody (T6557) were purchased from Sigma Chemical Co. (St. Louis, MO, USA); the biotinylated secondary antibodies and avidin-biotin complex (PK6101) from Vector Laboratories (Burlingame, CA, USA); Triazol from Invitrogen (Carlsbad, CA, USA); the DC protein assay kit from BioRad Laboratories (Hercules, CA, USA); the chemioluminescence (ECL) kit, the GFX PCR DNA and gel purification kit (code 27-960201) from Amersham (Little Chalfont, UK); . The primers were provided by Primm (Milan, Italy) and the kit for the PCR and RT-PCR by Promega (Madison, WI, USA). 2.2. Animals and sampling Nine healthy adult Wistar male rats, kept in groups of three animals per cage, were housed in the vivarium of the Department of Veterinary Medicine and Animal Productions (University of Naples Federico II, Naples, Italy). The rats had free access to food and tap water, and were kept under constant condition of light, temperature (22 C) and humidity. Experimental procedures were approved by the Ethical Committee for Animal Testing of our University, and were conducted in accordance with the European Union normative for the care and use of experimental animals. The animals were sacrificed by decapitation, and the epididymes were collected. For immunohistochemical analysis, cranial (caput) and caudal portions of the epididymes were fixed in Bouin's fluid for 18e24 h. For biochemical analyses, samples from the epididymal segments were frozen in liquid nitrogen, and stored at 80 C. 2.3. Immunohistochemistry Immunohistochemical analysis was performed by the procedure previously described [17e19]. Briefly, after fixation, the samples were dehydrated through an ascending series of ethanol and embedded in Paraplast. Microtomical sections (6 mm thick) were deparaffinised, rehydrated, and stained by the avidin-biotin complex. In the specific step, the anti-CD68 antibody was applied on sections overnight. The 3,30 -diaminobenzidine (DAB) was used as a final staining. The sections were examined under a light microscope (Nikon E600), and microphotographs were taken by using a digital camera (Colpix 8400). In this study, two anti-CD68 antibodies were used: the goat polyclonal anti-CD68 antibody (M-20, sc-7084) from Santa Cruz Biotechnology and the mouse monoclonal anti-human CD68 (Clone KP1, M 0814) from DAKO. They gave similar results. 2.4. RT-PCR and immunoblotting analyses Total RNA extraction from testis samples, the synthesis of cDNA, and RT-PCR for the detection of CD68 transcripts were performed according to the procedure previously described [17,18]. The
G. Liguori et al. / Biochimie 118 (2015) 221e224
223
Fig. 1. Immunohistochemical localization of CD68 in the cranial (a, b) and caudal (c, d) portions of rat epididymis. (a) The lining epithelium of three adjacent tubules is composed of principal cells containing CD68-immunoreactive material. (b) The CD68 staining appears to be always localized in the apical cytoplasm of the cells. (c, d) Five tubules of the caudal epididymis almost entirely lined by CD68 positive principal cells. Negative basal cells closely in contact with the basal membrane are visible. Avidin-biotin immunohistochemical staining. Bars: a, c: 30 mm; b, d: 20 mm.
molecular complexes from the tubular compartment of the epididymis and secrete them into the lumen of the organ after metabolic rearrangements [25]. The secretion of high hydrophobic proteins associated with luminal membrane vesicles are liberated from the surface of the principal cells as vesicular blebs known as epididymosomes or as soluble high mass protein complexes [24]. This study provides the first evidence for the expression of CD68 in the principal cells of rat epididymis. Whether the glycoprotein is present in the epididymosomes and in the gametes is under investigation in order to establish the physiological role of CD68 in the mammalian epididymis. Fig. 2. Expression of CD68 mRNA and the protein in the rat epididymis. A. RT-PCR analysis of CD68 mRNA in the caudal portion of rat epididymis (lane 2). Lane 1 corresponds to the DNA ladder. Negative control (distilled water) is shown in lane 3. Bottom: Expression of b-actin mRNA transcripts in the same tissues of the top (RT-PCR internal control). The non-specific bands at the basis of lanes 2e3 correspond to the primer dimers. Similar results were obtained when cranial portions of rat epididymis were subjected to RT-PCR analysis. B. Western blotting analysis by using antiserum directed against CD68 (Santa Cruz). Lane 1: homogenate from the caudal portion of rat epididymis; lane 2: negative control (homogenate treated with the CD68 antiserum pre-treated with its control peptide). The upper blot was stripped and re-probed with an anti-tubulin monoclonal antibody to ensure equal loading of proteins in all lanes. On the left, molecular weight markers are indicated. Similar results were obtained when cranial portions of rat epididymis were subjected to Western blotting analysis. The results reported in panels A and B are representative of four separated experiments of identical design.
The epididymal epithelium is responsible for creating a luminal environment conducive to the acquisition of fertilizing capacity by the spermatozoa within it [23,24]. In particular, the composition of the fluid suitable for spermatozoa survival and maturation depends on the receptoresecretory activity of the principal cells of the epididymal epithelium. The principal cells internalize fluids and
Conflict of interest None. Acknowledgments This study was supported by the U.BI.CO. 11941 grant from the University of Naples Federico II. The technical assistance of Mr. Antonio Calamo is gratefully acknowledged. We thank Dr. Emma Cirillo for administrative work. References [1] C.L. Holness, R.P. da Silva, J. Fawcett, S. Gordon, D.L. Simmons, Macrosialin, a mouse macrophage-restricted glycoprotein, is a member of the lamp/lgp family, J. Biol. Chem. 268 (1993) 9661e9666. [2] M. Fukuda, Lysosomal membrane glycoproteins. Structure, biosynthesis, and intracellular trafficking, J. Biol. Chem. 266 (1991) 21327e21330. [3] A.S. Zot, D.M. Fambrough, Structure of a gene for a lysosomal membrane glycoprotein (LEP100). Housekeeping gene with unexpected exon organization, J. Biol. Chem. 265 (1990) 20988e20995. [4] C.L. Howe, B.L. Granger, M. Hull, S.A. Green, C.A. Gabel, A. Helenius, I. Mellman, Derived protein sequence, oligosaccharides, and membrane insertion of the
224
[5]
[6]
[7]
[8]
[9]
[10] [11]
[12]
[13]
[14]
G. Liguori et al. / Biochimie 118 (2015) 221e224 120-kDa lysosomal membrane glycoprotein (lgp120): identification of a highly conserved family of lysosomal membrane glycoproteins, Proc. Natl. Acad. Sci. U.S.A. 85 (1988) 7577e7581. L. Martinez-Pomares, N. Platt, A.J. McKnight, R.P. da Silva, S. Gordon, Macrophage membrane molecules: markers of tissue differentiation and heterogeneity, Immunobiology 195 (1996) 407e416. K.A. Pulford, A. Sipos, J.L. Cordell, W.P. Stross, D.Y. Mason, Distribution of the CD68 macrophage/myeloid associated antigen, Int. Immunol. 2 (1990) 973e980. L.A. Kunz-Schughart, A. Weber, M. Rehli, E. Gottfried, G. Brockhoff, S.W. Krause, R. Andreesen, M. Kreutz, The “classical” macrophage marker CD68 is strongly expressed in primary human fibroblasts, Verh. Dtsch. Ges. Pathol. 87 (2003) 215e223. X.P. Lin, N. Almqvist, E. Telemo, Human small intestinal epithelial cells constitutively express the key elements for antigen processing and the production of exosomes, Blood Cells Mol. Dis. 35 (2005) 122e128. E. Gottfried, L.A. Kunz-Schughart, A. Weber, M. Rehli, A. Peuker, A. Müller, M. Kastenberger, G. Brockhoff, R. Andreesen, M. Kreutz, Expression of CD68 in non-myeloid cell types, Scand. J. Immunol. 67 (2008) 453e463. C.L. Holness, D.L. Simmons, Molecular cloning of CD68, a human macrophage marker related to lysosomal glycoproteins, Blood 81 (1993) 1607e1613. C.S. Shelley, E. Remold-O'Donnell, A.E. Davis 3rd, G.A. Bruns, F.S. Rosen, M.C. Carroll, A.S. Whitehead, Molecular characterization of sialophorin (CD43), the lymphocyte surface sialoglycoprotein defective in Wiskott-Aldrich syndrome, Proc. Natl. Acad. Sci. U. S. A. 86 (1989), 2819-2823. Erratum in: Proc. Natl. Acad. Sci. U.S.A. 86 (1989) 4689. J. Cyster, C. Somoza, N. Killeen, A.F. Williams, Protein sequence and gene structure for mouse leukosialin (CD43), a T lymphocyte mucin without introns in the coding sequence, Eur. J. Immunol. 20 (1990) 875e881. L.A. Lasky, M.S. Singer, D. Dowbenko, Y. Imai, W.J. Henzel, C. Grimley, C. Fennie, N. Gillett, S.R. Watson, S.D. Rosen, An endothelial ligand for Lselectin is a novel mucin-like molecule, Cell 69 (1992) 927e938. K.L. Moore, N.L. Stults, S. Diaz, D.F. Smith, R.D. Cummings, A. Varki, R.P. McEver, Identification of a specific glycoprotein ligand for P-selectin
(CD62) on myeloid cells, J. Cell Biol. 118 (1992) 445e456. [15] L. Hermo, B. Robaire, Epididymal cell types and their functions, in: B. Robaire, B.T. Hinton (Eds.), The Epididymis: from Molecules to Clinical Practice, Raven Press Ltd., New York, 2002, pp. 81e102. [16] M. Marchlewicz, Localization of immunocompetent cells in the human epididymis, Folia Histochem. Cytobiol. 39 (2001) 173e174. [17] S. Tafuri, L.M. Pavone, R. Lo Muto, M. Basile, E. Langella, E. Fiorillo, L. Avallone, N. Staiano, A. Vittoria, Expression of orexin A and its receptor 1 in the rat epididymis, Regul. Pept. 155 (2009) 1e5. [18] S. Tafuri, R. Lo Muto, L.M. Pavone, S. Valiante, A. Costagliola, N. Staiano, A. Vittoria, Novel localization of orexin A in the tubular cytotypes of the rat testis, Regul. Pept. 164 (2010) 53e57. [19] L.M. Pavone, S. Tafuri, R. Lo Muto, A. Spina, A.E. Lychkova, V. Mastellone, A. Vittoria, L. Avallone, N. Staiano, Expression of the serotonin transporter SERT in the genital tract of cattle, Regul. Pept. 154 (2009) 1e4. [20] G. Cerulo, S. Tafuri, V. De Pasquale, S. Rea, S. Romano, A. Costagliola, R. Della Morte, L. Avallone, L.M. Pavone, Serotonin activates cell survival and apoptotic death responses in cultured epithelial thyroid cells, Biochimie 105 (2014) 211e215. [21] H.D. Moore, J.M. Bedford, The differential absorption activity of epithelial cells of the rat epididymis before and after castration, Anat. Rec. 193 (1979) 313e327. [22] K. Micklem, E. Rigney, J. Cordell, D. Simmons, P. Stross, H. Turley, B. Seed, D. Mason, A human macrophage-associated antigen CD68 detected by six different monoclonal antibodies, Br. J. Haematol. 73 (1989) 6. [23] C.H. Yeung, T.G. Cooper, Developmental changes in signaling transduction factors in maturing sperm during epididymal transit, Cell. Mol. Biol. (Noisy-legrand) 49 (2003) 341e349. [24] J.L. Dacheux, F. Dacheux, New insights into epididymal function in relation to sperm maturation, Reproduction 147 (2013) R27eR42. [25] D.G. Cyr, B. Robaire, L. Hermo, Structure and turnover of junctional complexes between principal cells of the rat epididymis, Microsc. Res. Tech. 30 (1995) 54e66.