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Transgelin: An androgen-dependent protein identified in the seminal vesicles of three Saharan rodents
Theriogenology 80 (2013) 748–757
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Transgelin: An androgen-dependent protein identified in the seminal vesicles of three Saharan rodents Naïma Kaci-Ouchfoun a, *, Djamila Izemrane a, Abdelkrim Boudrissa b, Thérèse Gernigon a, Farida Khammar a, Jean Marie Exbrayat c a
Laboratory of Arid Areas, Biological Sciences Institute, USTHB, Algiers, Algeria Department of Parasitology, Algerian Pasteur Institute, Algiers, Algeria University of Lyon, UMRS 449, Laboratory of General Biology, Catholic University of Lyon (UCLy), Reproduction and Comparative Development EPHE, Lyon Cedex, France
b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 22 October 2012 Received in revised form 22 June 2013 Accepted 23 June 2013
During the breeding season, a major androgen-dependent protein with an apparent molecular weight of 21 kDa was isolated and purified from the seminal vesicles of three Saharan rodents (MLVSP21 from Meriones libycus, MSVSP21 from Meriones shawi, and MCVSP21 from Meriones crassus). The 21-kDa protein was isolated and purified from soluble seminal vesicle proteins of homogenate by one-dimensional polyacrylamide gel electrophoresis (SDS-PAGE). Using polyclonal antibodies directed against POSVP21 (Psammomys obesus seminal vesicles protein of 21 kDa), a major androgen-dependent secretory protein from sand rat seminal vesicles, identified previously as transgelin, we showed an immunological homology with POSVP21 by immunoblotting. These three major androgen-dependent proteins with a same apparent molecular weight of 21 kDa designated as MLVSP21 (Meriones libycus seminal vesicles protein of 21 kDa), MSVSP21 (Meriones shawi seminal vesicles protein of 21 kDa), and MCVSP21 (Meriones crassus seminal vesicles protein of 21 kDa) were localized by immunohistochemistry and identified by applying a proteomic approach. Our results indicated that the isolated proteins MLSVP21, MSSVP21, and MCSVP21 seem to correspond to the same protein: the transgelin. So that transgelin can be used as a specific marker of these rodent physiological reproduction mechanisms. Ó 2013 Elsevier Inc. All rights reserved.
Keywords: Transgelin Androgen-dependent proteins Rodent Desert Reproduction Seminal vesicles
1. Introduction Several proteins secreted by the seminal vesicles [1–5], prostate[6–8], epididymis [9–11], and vas deferent [12] of adult males have been studied extensively as markers of testosterone action. It has been shown in several species of mammals that the majority of the secreted components from seminal vesicles are proteins that are rapidly synthesized in the presence of androgens [13]. In fact, androgens play an important role in the development, growth, and maintenance of differentiated functions of
* Corresponding author. Tel./fax: þ213 72201282. E-mail address: [email protected] (N. Kaci-Ouchfoun). 0093-691X/$ – see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2013.06.014
seminal vesicles [14,15], and the expression of some of these proteins is dependent on the presence of testosterone [16]. In addition, in order to be regulated by steroid sex hormones, seminal vesicle and prostate are dependent on reciprocal stromal/epithelial interactions mediated by several molecules such as growth factors [17,18]. In a series of investigations, the rodent has been proved to be a good experimental model for the study, and some of the protein components of rat and mouse seminal vesicle secretions have been identified in several species: for example, the guinea pig [19,20] and the mouse [21,22]. In the mouse, the seminal vesicles secrete a similar group of androgen-regulated proteins [4,22,23], and the mechanism by which testosterone regulates the expression of these proteins has been investigated. It has been shown
N. Kaci-Ouchfoun et al. / Theriogenology 80 (2013) 748–757
that androgens acted mainly on gene transcription of a secretory protein of 99 amino acids (MSVSP99) and that this transcriptional regulation was exerted by steroid-receptor complexes interacting with enhancer sequences adjacent to the gene [24]. In the rat seminal vesicles, the physiological roles of the SVS proteins are still not yet well-defined; however, it has been proposed that the lower molecular weight SVS IV-VI proteins serve as proteinase inhibitors in the vaginal and uterine tract and help maintain a suitable environment for sperm motility and fertilization [25]. The SVSI-II proteins appear to be components of the rat copulatory plug, with the SVS II protein being the major component [26]. Clotting of the SVS II protein with itself and other SVS proteins is catalyzed by a transglutaminase that produces extensive covalent cross-linking between glutamine and lysine and the formation of g-glutamyl-ε-lysine [27]. Finding the structure and function of these proteins has been attempted in order to understand their roles in seminal vesicle physiology and their effects on gamete activity. We previously reported that in sand rat Psammomys obesus seminal vesicles, one major secretory androgen-dependent protein designated as POSVP21 (i.e., P. obesus seminal vesicles protein of 21 kDa) was abundantly synthesized when the androgen level increased. It accounts for over 22% of soluble proteins from homogenate of seminal vesicles during the breeding season. During the nonbreeding season, very large part of this protein was greatly reduced. Gernigon et al. have reported that the expression of this protein is depended on the presence of testosterone [28]. More recently, this protein was purified and characterized. Its site of synthesis was determined and its peptidic sequence was analyzed. POSVP21 was identified, such as transgelin [29,30]. The present study sought to expand our investigations to other Saharan rodents, such as Libyan jird Meriones libycus, Meriones shawi, and desert jird Meriones crassus. They are also characterized by a seasonal reproduction cycle. Transgelin is a 22-kDa protein identified as SM22, WS3-10, or mouse p27. The DNA and protein sequences of SM22 protein are highly conserved across species, and homologues have been found in invertebrate species, as distant as Caenorhabtidis elegans (unc-87) [31] and Drosophila melanogaster (mp20) [32]. Transgelin, also named SM22a, is a cytoskeleton-associated protein that is expressed abundantly in smooth muscle tissues of normal adult vertebrates [33]. Its expression is one of the earliest markers of smooth muscle differentiation during embryogenesis [34]. Several investigations suggested that SM22a might play a significant role in differentiation or cellular senescence by stabilizing the cytoskeleton through actin binding. [35]. Recent studies reported that the transgelin identified in smooth muscles and blood vessels was also found in the fibroblasts and in some epithelia such as the intestinal epithelium, the epithelium of the mammary channel [36], the glomerular epithelium [37], and the prostate epithelium [38].
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In this work, we explored the possible expression of transgelin in the seminal vesicles of three Saharan species: Meriones libycus, Meriones shawi, and Meriones crassus. 2. Materials and methods 2.1. Animals The sand rat P. obesus is a diurnal herbivorous rodent that lives around wadis in the Sahara desert. The Libyan jird Meriones libycus and the desert jird Meriones crassus are nocturnal herbivorous and granivore Saharan rodents belonging to the Gerbillidae family. They live in a superficial burrow arranged under the largest bushes. So, they benefy of shade procured by plants [39]. The adult animals were trapped in the wild in the regions of Béni Abbès (30 070 N, 2100 W) and M’sila (35 N, 4 E) (during the breeding season) and euthanized 24 to 48 hours later to study the proteins secreted by the seminal vesicles and other secretory organs of the male genital tract. Dissected tissues were fixed or stored at 80 C for RNA or protein extraction. 2.2. Extraction of proteins from adult sand rat seminal vesicles In normal adult males, proteins obtained either from secretions or from homogenized tissues were used. Frozen tissue samples were homogenized for 30 seconds at 4 C in 2 or 4 mL buffer A, in a glass-glass handheld homogenizer (Braun, Melsungen, Germany). After centrifugation at 12,000 g for 10 min at 4 C, the supernatant fluid was retained and used as “homogenate.” The concentration of soluble proteins was determined by the micro-method procedure of Bradford assay [40]. 2.3. Protein gel electrophoresis One-dimensional (1D) electrophoresis was performed under denaturing conditions [41] and carried out on slab gels (140 mm 120 mm 1.5 mm) using a Biorad model 220; sodium dodecyl sulfate protein samples (100 mg) were applied to 15% resolving gels with 4.5% stacking gel and run at 20 mA at room temperature until the tracking dye (bromophenol blue) reached the bottom of the gels. The gels were then stained with 0.25% (wt/vol) Coomassie Brilliant Blue in an aqueous solution containing 50% (vol/ vol) methanol and 10% (vol/vol) acetic acid for 45 minutes at room temperature and destained in a solution without dye. The apparent molecular weights of the proteins were calculated using the mobility of standard proteins as a reference. 2.4. Immunoblotting Western blotting of polyacrylamide gels onto nitrocellulose sheets was carried out according to Towbin et al. [42]. After 1D electrophoresis, proteins were electroblotted onto a polyvinylidene difluoride membrane (Immobilon-Psq, Millipore, Bedford, MA, USA) using a
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semidry transfer blotter (Bio-Rad) and visualized by Coomassie Brilliant Blue staining. 2.5. Immunohistochemistry Both seminal vesicles were quickly excised, carefully freed from surrounding fat and weighed. For immunochemistry, tissue samples (w5 mm thick) were fixed in 10% neutral buffered formalin during about 24 hours, dehydrated in a graded series of ethanol, cleared in cyclohexane, and embedded in paraffin. Serial sections (5 mm) were cut with a Leitz microtome and mounted with sterile watercoated superfrost glass slides. The paraffin sections were dewaxed and pretreated with microwave irradiation in citrate buffer at pH 6.0 for 15minutes to complete antigen unmasking. After blocking the endogenous peroxidase activity with 3% hydrogen peroxide for 15 minutes, the sections were washed in PBS (pH 7.4); nonspecific binding was blocked by incubation with normal goat serum (blocking reagent) (DakoCytomation) for 45 minutes. The sections were incubated overnight at þ4 C with primary antibody against POSVP21 at a dilution of 1:200 according to the avidin-biotin horseradish peroxidase complex method using the Vectastain kit (Vector Laboratories, Burlingame, USA). In control sections, the primary antibody was replaced with buffer. The slides were then rinsed in PBS three times for 5 minutes and treated with the secondary biotinyled antibody directed against the primary one for 30 minutes at ambient temperature. This step was followed by the application of streptavidin-peroxidase solution for 30 minutes, and the enzymatic reaction was revealed by the use of the substrate-chromogen solution diaminobenzidine for 10 to 15 minutes at ambient temperature. The sections were counterstained with aqueous hematoxylin to increase the contrast of specific staining, and they were then mounted in aqueous medium (crystal mount). Sections were studied with a Nikon microscope and images were captured by a digital Nikon camera (Nikon, Dusseldorf, Germany). Labeling results were categorized as weak, medium, and strong staining. 2.6. Mass spectrometry analysis The bands of interest were subjected to tryptic digestion using a procedure previously described [43]. Protein bands were excised from the gel and destained with a solution of 25 mM ammonium bicarbonate for 30 minutes and then with a 1:1 (vol/vol) solution of acetonitrile and 25 mM ammonium bicarbonate for 30 minutes. Washes were repeated until the blue color of Coomassie was removed. Bands were then dried by vacuum centrifugation and rehydrated in 10 mM DTT in 25 mM ammonium bicarbonate solution for 1 hour at 56 C. The supernatant was removed and the bands were incubated in the dark for 45 minutes with 55 mM iodoacetamide in 25 mM ammonium bicarbonate. Bands were washed as previously described, dried completely in a Speed Vac Plus (Savant, NY, USA) for 30 minutes, and enzymatically digested overnight using sequencing grade trypsin (Promega, Madison, WI, USA).
Coomassie Brilliant Blue, SDS, and salts were removed from the protein sample after passive elution using a ZipTipHPL (hydrophilic interaction chromatography) according to the manufacturer’s instructions (Millipore, Bedford, MA, USA). Briefly, the ZipTipHPL was rehydrated in buffer A (H2O/ ACN/AcOH: 50/50/0.1, pH 5.5) and washed with buffer B (H2O/ACN/AcOH: 10/90/0.1, pH 5.5). Protein eluates were diluted in 200 mL of buffer A, and proteins were eluted in 4 mL of H2O/ACN/TFA (50/50/0.1). NanoLC-MS/MS analyses were performed on an ion trap mass spectrometer (LTQ-OrbitrapXL, Thermo Fischer, USA) with nano-HPLC Ultimate 3000 (Dionex, Jouy en Josas, France). Peptide sequences were analyzed using Mascot software (Matrix Science, London, UK) and the SwissProt database. 2.7. Database searching MS/MS fragmentation was compared using Mascot software with sequences in the SwissProt database and the NCBInr database. The reliability of protein identifications was evaluated on the basis of multiple variables, including the Mascot score, the number of peptide matches, mass error (m/z accuracy), percent coverage of the matched protein, similarity of experimental and theoretical protein molecular weights (Mr) and isoelectric points (pIs), and degree of phylogenetic divergence of the species from which the sequence was matched. 3. Results 3.1. Comparative analysis of MLSVP21, MSSVP21, and MCSVP21 3.1.1. Comparative analysis of soluble proteins from seminal vesicle homogenates of P. obesus, Meriones libycus, Meriones crassus, and Meriones shawi During the breeding season in Libyan jird (Meriones libycus), Meriones shawi, and desert jird (Meriones crassus), the soluble protein compositions of seminal vesicle homogenates (Figs. 1b, 2d, and 2e) revealed by SDS-PAGE were similar. They appeared relatively abundant in comparison to the seminal vesicle homogenates of sand rat P. obesus (Fig. 3d), w30 polypeptides ranging in size from 10 to 200 kDa were observed. The major product of soluble androgen-dependent proteins from the homogenates of seminal vesicles of Meriones libycus, Meriones shawi, and Meriones crassus appears to be 12, 17, 24, 34, and 21 kDa (Figs. 1b, 2d, and 2e). The 21-kDa proteins corresponded to MLSVP21, MSSVP21, MCSVP21, and POSVP21 (Figs. 1b, 2d, 2e, and 3d). The proteins with an apparent molecular weight of 30, 41, 43, 45, and 67 kDa decreased during the breeding season. When compared with the components of Meriones libycus, Meriones shawi, and Meriones crassus seminal vesicle homogenates, eight major bands with an apparent molecular weight of 12, 17, 21, 24, 30, 41, 57, 67, and 78 kDa were matched to comparable bands in seminal vesicle homogenates of P. obesus. The proteins with an apparent molecular weight of 53, 45, 43, 32, 29, 27, and 25 kDa seemed to characterize the seminal vesicles of Meriones libycus, Meriones shawi, and Meriones crassus.
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Fig. 1. Polyacrylamide gel electrophoretic analysis of soluble proteins from the adult Libyan jird Meriones libycus seminal vesicles. Protein components of soluble proteins from homogenate (seminal vesicles and secretions). A suitable amount of protein was subjected to SDS-PAGE on a 15% slab gel (140 mm 120 mm 1.5 mm). Each lane was loaded with 100 mg proteins. Proteins were stained with Coomassie Brilliant Blue. The arrow indicates the position of the 21-kDa protein. (a) Protein standard: phosphorylase B (94 kDa); bovine serum albumin (67 kDa); ovalbumin (43 kDa); carbonic anhydrase (30 kDa); soybean trypsin inhibitor (20 kDa); lactalbumin (14 kDa). (b) Proteins from homogenate of the adult Libyan jird Meriones libycus seminal vesicles during the breeding season (early spring) caught in the yield. (c) Proteins from homogenate of the adult Libyan jird Meriones libycus seminal vesicles during the nonbreeding season (late summer) caught in the yield.
During the nonbreeding season, the very large part of these proteins, particularly the 21-kDa proteins MLSVP21, MSSVP21, MCSVP21, POSVP21 or transgelin, and the P78, were largely reduced. Conversely, the 30-, 41-, 43- and 45-kDa proteins increased (Figs. 1c, 2b, 2c, 3b, and 3c).
3.2. Detection of immunological homology between MLSVP21, MSSVP21, MCSVP21, and POSVP21 Proteins extracted from the seminal vesicles of Meriones libycus, Meriones shawi, and Meriones crassus were submitted to Western blot analysis using an anti-POSVP21–specific polyclonal antibody. Purified POSVP21 or transgelin was used as positive controls in these experiments. An immunoreactive signal at 21 kDa was detected in seminal vesicle homogenates from these Saharan species. On the basis of these results, we hypothesized that the three proteins
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Fig. 2. Polyacrylamide gel electrophoretic analysis of soluble proteins from the adult desert jird Meriones crassus and Meriones shawi seminal vesicles. Protein components of soluble proteins from homogenate (seminal vesicles and secretions). (a) Protein standard. (b) Proteins from homogenate of the adult Meriones shawi seminal vesicles during the nonbreeding season (early winter) caught in the yield. (c) Proteins from homogenate of the adult desert jird Meriones crassus seminal vesicles during the nonbreeding season (early winter) caught in the yield. (d) Proteins from homogenate of the adult Meriones shawi seminal vesicles during the breeding season (early spring) caught in the yield. (e) Proteins from homogenate of the adult desert jird Meriones crassus seminal vesicles during the breeding season (early spring) caught in the yield.
with a same apparent molecular weight share antigenic determinants with sand rat seminal vesicle proteins POSVP21. The positive immunoreactivity provides the immunological homology between MLSVP21, MSSVP21, MCSVP21, and POSVP21 or transgelin. In conclusion, it appears that antigens corresponding to POSVP21 or transgelin could be detected in the seminal vesicles from other Saharan rodents, such as Meriones libycus, Meriones shawi, and Meriones crassus. 3.3. Localization of MLSVP21, MSSVP21, and MCSVP21 in seminal vesicles of Meriones libycus, Meriones shawi, and Meriones crassus Sections from sand rat seminal vesicles during the breeding season (Fig. 4A) and the nonbreeding season (Fig. 4B) were used as positive and negative controls in these experiments. Immunohistochemical staining with the avidin-biotin complex technique using affinity-purified polyclonal antibodies against POSVP21 was performed to confirm the immunological homology between MLSVP21, MCSVP21 MSSVP21, and POSVP21, and to localize these proteins in sections of the seminal vesicles. The morphology of the sections was studied after staining with Mayer’s hematoxylin. Figure 5B–D showed a positive staining in the epithelial cells.
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These results reveal that antigens corresponding to POSVP21 or transgelin could be detected in seminal vesicles from other Saharan rodents, such as Meriones libycus, Meriones crassus, and Meriones shawi, with a same distribution. In the seminal vesicles of Meriones libycus, Meriones crassus, and Meriones shawi, POSVP21 immunostaining was observed in epithelial cells and in secretory products in the lumen (Figs. 5B, 5D, 6B, and 7). No other cells stained in this tissue, and thus synthesis of MLSVP21, MCSVP21, and MSSVP21 can be assigned to the vesicular epithelial cells.
3.4. Identification of MLSVP21, MSSVP21, and MCSVP21 The MLSVP21, MSSVP21, MCSVP21, and peptide sequence analysis reveal a strong homology with the Rattus norvegicus transgelin peptide sequences like for POSVP21. Indeed, all of the peptide sequences identified at the level of MLSVP21, MSSVP21, and MCSVP21 are also present at the level of the transgelin peptide sequences. They cover 54.73%, 54.23%, and 28.86% of the transgelin protein sequences, which accounts for two-thirds of the total sequence of transgelin (Fig. 8). We also noted that peptide sequence analysis of MLSVP21, MSSVP21, and MCSVP21 revealed a similar amino acid composition to POSVP21. Fig. 3. Polyacrylamide gel electrophoretic analysis of soluble proteins from the adult sand rat Psammomys obesus seminal vesicles. Protein components of soluble proteins from homogenate (seminal vesicles and secretions). A suitable amount of protein was subjected to SDS-PAGE on a 15% slab gel (140 mm 120 mm 1.5 mm). Each lane was loaded with 100 mg proteins. Proteins were stained with Coomassie Brilliant Blue. The arrow indicates the position of the 21-kDa protein. (a) Protein standard: phosphorylase B (94 kDa); bovine serum albumin (67 kDa); ovalbumin (43 kDa); carbonic anhydrase (30 kDa); soybean trypsin inhibitor (20 kDa); lactalbumin (14 kDa). (b, c) Proteins from homogenate of the adult sand rat seminal vesicles during the nonbreeding season (late summer) caught in the yield. (d) Proteins from homogenate of the adult sand rat seminal vesicles during the breeding season (early autumn) caught in the yield.
The connective tissue and nucleus had negative immunoreaction for POSVP21. The positive immunoreactivity provides the immunological homology between MLSVP21, MCSVP21, MSSVP21, and POSVP21 or transgelin (Fig. 5B–D). The polyclonal antibodies against POSVP21 reacted positively with MCSVP21 in sections of the Meriones crassus seminal vesicles. MCSVP21 immunostaining was observed in epithelial cells and no signal has been detected in smooth muscle cells and connective tissue (Fig. 6B). During the breeding season, immunoexpression from MSSVP21 was located in the seminal vesicle epithelial cells. The connective tissue and the smooth muscle cells were not at all immunomarked. The immunohistochemical analysis indicated a reactivity of anti-POSVP21 antibodies with respect to the MLSVP21, MCSVP21, and MSSVP21. It also indicates that these three proteins with a same apparent molecular weight of 21 kDa share antigenic determinants with sand rat seminal vesicle proteins POSVP21.
4. Discussion The sand rat P. obesus, the Libyan jird Meriones libycus, the Meriones shawi, and the desert jird Meriones crassus are the Saharan rodents under studies submitted to a seasonal cycle of reproduction. During the active period, the reproductive organs from these species report a considerable weight growth. The structure and the ultrastructure are developed and both testicular and plasma testosterone are high. In fact, it is known that the majority of the rodents living in the arid and semiarid regions breed according to a seasonal cycle and they are characterized by weight, structural, ultrastructural, and biochemical variations [44]. In P. obesus [28,45], Meriones libycus [46,47], Meriones crassus [48], or even Meriones shawi [49], the phase of quiescence is typically characterized by a reduction in the size of the reproductive organs (seminal vesicle and prostate) induced by a marked decrease in testicular and plasma testosterone levels. In the sand rat P. obesus, the seasonal, structural, ultrastructural, and biochemical cycles studied by Gernigon et al. [28] coincided with the hormonal seasonal cycle reported by Khammar et al. [45]. In the previous studies, we have reported that seminal vesicles of adult sand rat P. obesus contain a major secretory protein band (21 kDa) designated as POSVP21 (P. obesus seminal vesicles protein of 21 kDa). This protein accounts for over 22.3% of the soluble proteins from homogenate during the breeding season, 13.3% during the middle season, and 5.3% during the hormonal regression season. During the nonbreeding season, the amount of POSVP21 is greatly reduced. The expression of POSVP21 is induced by testosterone, and the levels of this protein decrease most dramatically after castration [28].
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Fig. 4. POSVP21 immunochemistry in the adult sand rat Psammomys obesus seminal vesicles during the breeding season (early autumn). Sections of sand rat seminal vesicle fixed with formalin and embedded in paraffin were incubated with the anti-POSVP21 polyclonal antibody diluted at 1:200 (A) or nonimmune serum diluted at 1:200 (B). Bound antibodies were detected using a peroxidase conjugated to anti-rabbits IgG at a dilution 1:200. L, lumen; E, epithelium; S, secretion; C, connective tissue; N, nucleus; Sn, supranuclear zone; St, smooth muscle cells.
Major efforts have been devoted to the characterization of rat seminal vesicle secretory proteins and their differential regulation by androgens [50]. Mouse seminal vesicles also secrete a similar group of androgen-regulated proteins [4,22,23]. However, because the absence of specific antibodies and proteomic approaches had not been developed, previous studies have been limited to the analysis of steady-state levels of their mRNAs [4,23,24]. In this work, the seminal vesicle proteins studied were the three major androgen-dependent proteins with a same apparent molecular weight of 21 kDa. They are designated as follows: MLSVP21 (Meriones libycus seminal vesicles protein of 21 kDa), MSSVP21 (Meriones shawi seminal vesicles
protein of 21 kDa), and MCSVP21 (Meriones crassus seminal vesicles protein of 21 kDa). The profile of expression of these proteins seems affected by seasonal fluctuations and by seminal vesicles sensitive to androgens. In fact, during the breeding season, MLSVP21, MSSVP21, and MCSVP21 were highly expressed. Contrarily, during the phase of quiescence, the very large amounts of these three 21-kDa proteins were greatly reduced. It is also know that in the Saharan rodent seminal vesicles, the mechanism of synthesis and regulation is adapted to the insecure and irregular food in desert. In other rodents (mouse, rat), the mechanism by which testosterone regulates the expression of androgen-dependent proteins has recently been investigated in the mouse. It has
Fig. 5. MLSVP21 immunochemistry in the adult Libyan jird Meriones libycus seminal vesicles during the breeding season (early spring). In sections of Libyan jird Meriones libycus seminal vesicles, bound antibodies were detected using a peroxidase conjugated to anti-rabbits IgG at a dilution 1:200 (B–D) or nonimmune serum diluted at 1:200 (A). The immunoreactivity to MLSVP21 staining was localized to the epithelial cells (B–D) and in the secretory products in the lumen (D). The smooth muscle cells, the connective tissue, and the nucleus had negative immunoreaction for POSVP21 (C and D). L, lumen; E, epithelium; S, secretion; C, connective tissue; N, nucleus; Sn, supranuclear zone; St, smooth muscle cells.
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Fig. 6. MCSVP21 immunochemistry in the adult desert jird Meriones crassus seminal vesicles during the breeding season (early spring). In sections of desert jird Meriones crassus seminal vesicles, bound antibodies were detected using a peroxidase conjugated to anti-rabbits IgG at a dilution 1:200 (B) or nonimmune serum diluted at 1:200 (A). Immunoreactivity to MCSVP21 staining was localized to the epithelial cells (B). The smooth muscle cells, the connective tissue, and the nucleus had negative immunoreaction for POSVP21 (B). L, lumen; E, epithelium; C, connective tissue; N, nucleus; Sn, supranuclear zone; St, smooth muscle cells.
been reported that androgens act mainly on MSVSP99 gene transcription. This transcriptional regulation is exerted by steroid-receptor complexes interacting with enhancer sequences adjacent to the gene [24,51]. It should be noted that the major androgen-dependent protein from sand rat seminal vesicles or POSVP21 shares many traits with proteins involved in reproduction mechanisms: it is abundant in secretions, regulated by androgens, and is also present in vaginal plug. Recently, POSVP21 is localized in the cytoplasm of seminal vesicle epithelial cells and in the lumen secretory products [29]. The peptide sequences reveal that POSVP21 seems to correspond to the R. norvegicus transgelin [30]. Using polyclonal antibodies directed against POSVP21, Western blot and the immunohistochemical analyses show a reactivity of anti-POSVP21 antibodies with respect to the MLSVP21, MSSVP21, and MCSVP21. These results indicate an immunological homology with this major secretory androgen-dependent protein, identified previously as transgelin. They indicate that these three proteins, with a
Fig. 7. MSSVP21 immunochemistry in the adult Meriones shawi seminal vesicles during the breeding season (early spring). In sections of Meriones shawi seminal vesicles, bound antibodies were detected using a peroxidase conjugated to anti-rabbits IgG at a dilution 1:200. Immunoreactivity to MSSVP21staining was localized to the epithelial cells; the smooth muscle cells, the connective tissue, and the nucleus had negative immunoreaction for POSVP21. E, epithelium; C, connective tissue; St, smooth muscle cells.
same apparent molecular weight, share antigenic determinants with sand rat seminal vesicle proteins POSVP21. The antigens corresponding to POSVP21 or transgelin could be detected in seminal vesicles from other Saharan rodents, such as Meriones libycus, Meriones crassus, and Meriones shawi, with a same distribution. In fact, during the breeding season, immunoexpression from MLSVP21, MSSVP21, and MCSVP21 were located in the seminal vesicle epithelial cells and in the lumen secretory products. In contrast, smooth muscle cells and connective cells do not possess proteins displaying any cross-reactivity with the antibodies directed against POSVP21 or transgelin. As to POSVP21, synthesis of MLSVP21, MSSVP21, and MCSVP21 can be assigned to the vesicular epithelial cells. Mainly, studies postulated the localization of most proteins was closely related to their physiological functions. In fact, in a series of investigations, several roles have been attributed to some components as follows: semen coagulation after ejaculation is caused by the semenogelin, a major protein expressed exclusively in the men seminal vesicles is the major coagulum-forming protein, and semen liquefaction is provoked by prostatic-specific antigen (PSA); this protease, secreted by the prostate, plays a prominent role in the process of liquefaction by causing semenogelin proteolysis [52]. Finding the structure and function of these proteins have been attempted in order to understand their roles in seminal vesicle physiology and their effects on gamete activity. The analysis of MLSVP21, MSSVP21, and MCSVP21 peptide sequences reveal a strong homology with the R. norvegicus transgelin peptide sequences like for POSVP21. Indeed, all of the peptide sequences identified at the level of MLSVP21, MSSVP21, and MCSVP21 are also present at the level of the transgelin peptide sequences that cover 54.73%, 54.23%, and 28.86 % of the transgelin protein sequences. It account for two-thirds of the total sequence of transgelin. We also noted that all of the peptide sequences identified from MLSVP21, MSSVP21, and MCSVP21 are found in the POSVP21 sequence. In our study, the proteomic approach shows that the isolated proteins, MLSVP21, MSSVP21, and MCSVP21, seem to correspond to the same protein, the transgelin. POSVP21 or transgelin was initially identified in seminal vesicles from sand rat P. obesus [30]. The role of POSVP21 remains unknown, but during the late spring, when the
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755
MANKGPSYGM
SREVQSKIEK
KYDEELEERL
VEWIVMQCGP
DVGRPDRGRL
TGLN R. n.
----------
- - - - - - - IEK
KYDEELEERL
VEWIVMQCGP
DVGRPDR- - L
MLSVP
----------
----------
KYDEELEERL
VEWIVMQCGP
DVGRPDR- - L
MSSVP
----------
- - - - - - - IEK
KYDEELEER-
VEWIVMQCGP
DVGRPDRGRL
MCSVP
GFQVWLKNGV
ILSKLVNSLY
PEGSKPVKVP
ENPPSMVFKQ
MEQVAQFLKA
TGLN R. n.
GFQVWLKNGV
ILSKLVNSLY
PEGSKPVKVP
ENPPSMVFKQ
MEQVAQFLKA
MLSVP
GFQVWLKNGV
----------
----------
---------Q
MEQVAQFLK -
MSSVP
GFQVWLKNGV
ILSKLVNSLY
PEGSKPVKVP
ENPPSMVFKQ
---------A
MCSVP
AEDYGVTKTD
MFQTVDLFEG
KDMAAVQRTV
MALGSLAVTK
NDGHYRGDPN
TGLN R. n.
AEDYGVTKTD
MFQTVDLFEG
K-
--------
----------
- - - - - - GDPN
MLSVP
- - - - - - - - TD
MFQTVDLFEG
K-
--------
----------
----------
MSSVP
AEDYGVTKTD
MFQTVDLFEG
KDMAAVQR-
-
----------
----------
MCSVP
WFMKKAQEHK
REFTDSQLQE
GKHVIGLQMG
SNRGASQAGM
TGYGRPRQII
TGLN R. n.
WFMK - - - - - -
----------
----------
----------
----------
MLSVP
----------
----------
----------
----------
----------
MSSVP
----------
----------
----------
----------
----------
MCSVP
S
TGLN R. n.
S
MLSVP
S
MSSVP
S
MCSVP
Fig. 8. Homologies between the amino acid sequences from MLSVP21, MSSVP21, and MCSVP21 fragment and the sequence from Rattus norvegicus transgelin. The identical amino acids are presented in red with other sequences aligned to show homology. TGLN R.n.: transgelin from Rattus norvegicus.
protein diminishes, the sand rat rarely undergoes pregnancy. This protein therefore might also be implicated in the fertilization process. The high expression of MLSVP21, MSSVP21, and MCSVP21 during the breeding season and the important reduction during the long phase of sexual quiescence suggest that these proteins may have an important specific function in reproduction. Transgelin has since been identified in many different species and has been given different names such as TAGLN [53], SM22 [54], WS3-10 [55], and mouse p27 [56]. It was first isolated from chicken gizzard as a transformation- and shape-change sensitive actin-binding protein, the expression of which is lost in virally transformed cells. Its expression is highly conserved across species, and homologs have been found in invertebrate species, as distant as Caenorhabtidis elegans (unc-87) [31] and Drosophila melanogaster (mp20) [32]. Although its physiological role remains uncertain, insights into its potential function are found in studies under its several different names [56,57] such as SM22a, transgelin, WS3-10, and mouse p27 [35]. It was also originally described as a predominant expression in smooth muscle cells to bind the actin, suggesting it is involved in cell differentiation and cytoskeletal rearrangement [58,59]. Its expression is one of the earliest markers of smooth muscle differentiation. Tissue distribution of transgelin is contentious. It is found throughout the smooth muscle tissues of normal adult
vertebrates [58]. Indeed, its mRNA levels are highest in adult human tissues containing large amounts of smooth muscle, such as uterus, bladder, stomach, and prostate. However, it is also expressed in several adult tissues containing little or no smooth muscle, including spinal chord, adrenal gland, and, notably, in the heart [53,60]. As we have shown in the epithelial cell seminal vesicles, from these Saharan rodents, P. obesus, Meriones libycus, Meriones shawi, and Meriones crassus, transgelin expression has also been identified recently in epithelial cells than in intestinal epithelial cells [36], glomerular epithelial cells [37], breast ductal epithelium [36,61], and prostate epithelium [38]. Transgelin is only sensitive to protein kinase C phosphorylation in vitro, which attenuates actin binding [62]. The cellular location and functional role of transgelin are at present unknown. It is unique in its composition and properties and in its amino acid sequencing. It is shown to resemble no other previously reported protein [63]. The physiological role of SM22a is still unclear, but recent studies reveal that SM22a may play a role in agonist-induced vascular smooth muscle contractility [64]. In prostate carcinoma cells, transgelin has been shown to block androgen-stimulated cell growth by preventing the binding of an androgen receptor coactivator with androgen receptor and subsequent translocation to the nucleus [38]. Transgelin also acts to suppress the expression of the metallo-matrix proteinase-9 [65]. Because we have found transgelin and localized this protein in the epithelium and in the secretion of these
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rodent species seminal vesicles, we suggest that there is a passage through an external medium that, we believe, is involved in the organization of the epithelial cell cytoskeleton. This allows it to maintain the structure and the contractile state of the seminal vesicle. In rodents, one of the functional properties of seminal vesicle secretion is to contribute to the clot formation of ejaculated semen. It also influences the metabolism, mobility, and surface properties of spermatozoa to induce capacitation and suppress the immunocompetence of the female genital tract [66,67]. Apart from their role in the male genital tract, the secretions of seminal vesicles have a role in the female genital tract, where they mediate stimulation of the motility of smooth muscles [23]. 4.1. Conclusion The present investigation shows the high expression of MLSVP21, MSSVP21, and MCSVP21 during the breeding season and the important reduction during the long phase of sexual quiescence. The specific polyclonal antibodies directed against POSVP21 reveal that MLSVP21, MSSVP21, and MCSVP21 are immunologically related to POSVP21, a major androgen-dependent secretory protein identified initially as transgelin in the seminal vesicles from sand rat P. obesus. The immunohistochemical study localized MLSVP21, MSSVP21, and MCSVP21 in the epithelial cells of adult Meriones libycus, Meriones crassus, and Meriones shawi seminal vesicles during the breeding season. The peptide sequence analysis reveal that MLSVP21, MSSVP21, and MCSVP21 correspond to the same protein, the transgelin. Transgelin identified in the seminal vesicle of Meriones libycus, Meriones shawi, and Meriones crassus with a same distribution can be used as a marker for physiological mechanisms of reproduction of these Saharan species. Our results made the first demonstration that the transgelin is expressed highly and distinctly in the epithelial seminal vesicles of three Saharan rodents and its transcription appears to be regulated by testosterone. Further studies are needed to clarify the mechanism by which transgelin is involved in the rodent physiological reproduction. References [1] Mansson PE, Sugino A, Harris SE. Use of a cloned double stranded cDNA coding for a major androgen dependent protein in rat seminal vesicle secretion: the effect of testosterone in gene expression. Nucleic Acids Res 1981;9:935–46. [2] Mc Donald C, Williams L, Mc Turk P, Fuller F, Mc Intosh E, Higgins S. Isolation and characterization of genes for androgen-responsive secretory proteins of rat seminal vesicles. Nucleic Acids Res 1983; 11:917–30. [3] Williams L, Mc Donald C, Jackson S, Mc Intosh E, Higgins S. Isolation and characterization of genomic and cDNA clones for androgenregulated secretory protein of rat seminal vesicles. Nucleic Acids Res 1983;11:5021–32. [4] Chen YH, Pentecost BT, Mc Lachlan JA, Teng CT. The androgendependent mouse seminal vesicle secretory protein IV: characterization and complementary deoxyribonucleic acid cloning. Mol Endocrinol 1987;1:707–16. [5] Higgins SJ, Hemingway AL. Effects of androgens on the transcription of secretory protein genes in rat seminal vesicle. Mol Cell Endocrinol 1991;76:55–61. [6] Parker MG, White R, Williams JG. Cloning and characterization of androgen-dependent mRNA from rat ventral prostate. J Biochem Chem 1980;255:6996–7001.
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Transgelin: An androgen-dependent protein identified in the seminal vesicles of three Saharan rodents Naïma Kaci-Ouchfoun a, *, Djamila Izemrane a, Abdelkrim Boudrissa b, Thérèse Gernigon a, Farida Khammar a, Jean Marie Exbrayat c a
Laboratory of Arid Areas, Biological Sciences Institute, USTHB, Algiers, Algeria Department of Parasitology, Algerian Pasteur Institute, Algiers, Algeria University of Lyon, UMRS 449, Laboratory of General Biology, Catholic University of Lyon (UCLy), Reproduction and Comparative Development EPHE, Lyon Cedex, France
b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 22 October 2012 Received in revised form 22 June 2013 Accepted 23 June 2013
During the breeding season, a major androgen-dependent protein with an apparent molecular weight of 21 kDa was isolated and purified from the seminal vesicles of three Saharan rodents (MLVSP21 from Meriones libycus, MSVSP21 from Meriones shawi, and MCVSP21 from Meriones crassus). The 21-kDa protein was isolated and purified from soluble seminal vesicle proteins of homogenate by one-dimensional polyacrylamide gel electrophoresis (SDS-PAGE). Using polyclonal antibodies directed against POSVP21 (Psammomys obesus seminal vesicles protein of 21 kDa), a major androgen-dependent secretory protein from sand rat seminal vesicles, identified previously as transgelin, we showed an immunological homology with POSVP21 by immunoblotting. These three major androgen-dependent proteins with a same apparent molecular weight of 21 kDa designated as MLVSP21 (Meriones libycus seminal vesicles protein of 21 kDa), MSVSP21 (Meriones shawi seminal vesicles protein of 21 kDa), and MCVSP21 (Meriones crassus seminal vesicles protein of 21 kDa) were localized by immunohistochemistry and identified by applying a proteomic approach. Our results indicated that the isolated proteins MLSVP21, MSSVP21, and MCSVP21 seem to correspond to the same protein: the transgelin. So that transgelin can be used as a specific marker of these rodent physiological reproduction mechanisms. Ó 2013 Elsevier Inc. All rights reserved.
Keywords: Transgelin Androgen-dependent proteins Rodent Desert Reproduction Seminal vesicles
1. Introduction Several proteins secreted by the seminal vesicles [1–5], prostate[6–8], epididymis [9–11], and vas deferent [12] of adult males have been studied extensively as markers of testosterone action. It has been shown in several species of mammals that the majority of the secreted components from seminal vesicles are proteins that are rapidly synthesized in the presence of androgens [13]. In fact, androgens play an important role in the development, growth, and maintenance of differentiated functions of
* Corresponding author. Tel./fax: þ213 72201282. E-mail address: [email protected] (N. Kaci-Ouchfoun). 0093-691X/$ – see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2013.06.014
seminal vesicles [14,15], and the expression of some of these proteins is dependent on the presence of testosterone [16]. In addition, in order to be regulated by steroid sex hormones, seminal vesicle and prostate are dependent on reciprocal stromal/epithelial interactions mediated by several molecules such as growth factors [17,18]. In a series of investigations, the rodent has been proved to be a good experimental model for the study, and some of the protein components of rat and mouse seminal vesicle secretions have been identified in several species: for example, the guinea pig [19,20] and the mouse [21,22]. In the mouse, the seminal vesicles secrete a similar group of androgen-regulated proteins [4,22,23], and the mechanism by which testosterone regulates the expression of these proteins has been investigated. It has been shown
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that androgens acted mainly on gene transcription of a secretory protein of 99 amino acids (MSVSP99) and that this transcriptional regulation was exerted by steroid-receptor complexes interacting with enhancer sequences adjacent to the gene [24]. In the rat seminal vesicles, the physiological roles of the SVS proteins are still not yet well-defined; however, it has been proposed that the lower molecular weight SVS IV-VI proteins serve as proteinase inhibitors in the vaginal and uterine tract and help maintain a suitable environment for sperm motility and fertilization [25]. The SVSI-II proteins appear to be components of the rat copulatory plug, with the SVS II protein being the major component [26]. Clotting of the SVS II protein with itself and other SVS proteins is catalyzed by a transglutaminase that produces extensive covalent cross-linking between glutamine and lysine and the formation of g-glutamyl-ε-lysine [27]. Finding the structure and function of these proteins has been attempted in order to understand their roles in seminal vesicle physiology and their effects on gamete activity. We previously reported that in sand rat Psammomys obesus seminal vesicles, one major secretory androgen-dependent protein designated as POSVP21 (i.e., P. obesus seminal vesicles protein of 21 kDa) was abundantly synthesized when the androgen level increased. It accounts for over 22% of soluble proteins from homogenate of seminal vesicles during the breeding season. During the nonbreeding season, very large part of this protein was greatly reduced. Gernigon et al. have reported that the expression of this protein is depended on the presence of testosterone [28]. More recently, this protein was purified and characterized. Its site of synthesis was determined and its peptidic sequence was analyzed. POSVP21 was identified, such as transgelin [29,30]. The present study sought to expand our investigations to other Saharan rodents, such as Libyan jird Meriones libycus, Meriones shawi, and desert jird Meriones crassus. They are also characterized by a seasonal reproduction cycle. Transgelin is a 22-kDa protein identified as SM22, WS3-10, or mouse p27. The DNA and protein sequences of SM22 protein are highly conserved across species, and homologues have been found in invertebrate species, as distant as Caenorhabtidis elegans (unc-87) [31] and Drosophila melanogaster (mp20) [32]. Transgelin, also named SM22a, is a cytoskeleton-associated protein that is expressed abundantly in smooth muscle tissues of normal adult vertebrates [33]. Its expression is one of the earliest markers of smooth muscle differentiation during embryogenesis [34]. Several investigations suggested that SM22a might play a significant role in differentiation or cellular senescence by stabilizing the cytoskeleton through actin binding. [35]. Recent studies reported that the transgelin identified in smooth muscles and blood vessels was also found in the fibroblasts and in some epithelia such as the intestinal epithelium, the epithelium of the mammary channel [36], the glomerular epithelium [37], and the prostate epithelium [38].
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In this work, we explored the possible expression of transgelin in the seminal vesicles of three Saharan species: Meriones libycus, Meriones shawi, and Meriones crassus. 2. Materials and methods 2.1. Animals The sand rat P. obesus is a diurnal herbivorous rodent that lives around wadis in the Sahara desert. The Libyan jird Meriones libycus and the desert jird Meriones crassus are nocturnal herbivorous and granivore Saharan rodents belonging to the Gerbillidae family. They live in a superficial burrow arranged under the largest bushes. So, they benefy of shade procured by plants [39]. The adult animals were trapped in the wild in the regions of Béni Abbès (30 070 N, 2100 W) and M’sila (35 N, 4 E) (during the breeding season) and euthanized 24 to 48 hours later to study the proteins secreted by the seminal vesicles and other secretory organs of the male genital tract. Dissected tissues were fixed or stored at 80 C for RNA or protein extraction. 2.2. Extraction of proteins from adult sand rat seminal vesicles In normal adult males, proteins obtained either from secretions or from homogenized tissues were used. Frozen tissue samples were homogenized for 30 seconds at 4 C in 2 or 4 mL buffer A, in a glass-glass handheld homogenizer (Braun, Melsungen, Germany). After centrifugation at 12,000 g for 10 min at 4 C, the supernatant fluid was retained and used as “homogenate.” The concentration of soluble proteins was determined by the micro-method procedure of Bradford assay [40]. 2.3. Protein gel electrophoresis One-dimensional (1D) electrophoresis was performed under denaturing conditions [41] and carried out on slab gels (140 mm 120 mm 1.5 mm) using a Biorad model 220; sodium dodecyl sulfate protein samples (100 mg) were applied to 15% resolving gels with 4.5% stacking gel and run at 20 mA at room temperature until the tracking dye (bromophenol blue) reached the bottom of the gels. The gels were then stained with 0.25% (wt/vol) Coomassie Brilliant Blue in an aqueous solution containing 50% (vol/ vol) methanol and 10% (vol/vol) acetic acid for 45 minutes at room temperature and destained in a solution without dye. The apparent molecular weights of the proteins were calculated using the mobility of standard proteins as a reference. 2.4. Immunoblotting Western blotting of polyacrylamide gels onto nitrocellulose sheets was carried out according to Towbin et al. [42]. After 1D electrophoresis, proteins were electroblotted onto a polyvinylidene difluoride membrane (Immobilon-Psq, Millipore, Bedford, MA, USA) using a
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semidry transfer blotter (Bio-Rad) and visualized by Coomassie Brilliant Blue staining. 2.5. Immunohistochemistry Both seminal vesicles were quickly excised, carefully freed from surrounding fat and weighed. For immunochemistry, tissue samples (w5 mm thick) were fixed in 10% neutral buffered formalin during about 24 hours, dehydrated in a graded series of ethanol, cleared in cyclohexane, and embedded in paraffin. Serial sections (5 mm) were cut with a Leitz microtome and mounted with sterile watercoated superfrost glass slides. The paraffin sections were dewaxed and pretreated with microwave irradiation in citrate buffer at pH 6.0 for 15minutes to complete antigen unmasking. After blocking the endogenous peroxidase activity with 3% hydrogen peroxide for 15 minutes, the sections were washed in PBS (pH 7.4); nonspecific binding was blocked by incubation with normal goat serum (blocking reagent) (DakoCytomation) for 45 minutes. The sections were incubated overnight at þ4 C with primary antibody against POSVP21 at a dilution of 1:200 according to the avidin-biotin horseradish peroxidase complex method using the Vectastain kit (Vector Laboratories, Burlingame, USA). In control sections, the primary antibody was replaced with buffer. The slides were then rinsed in PBS three times for 5 minutes and treated with the secondary biotinyled antibody directed against the primary one for 30 minutes at ambient temperature. This step was followed by the application of streptavidin-peroxidase solution for 30 minutes, and the enzymatic reaction was revealed by the use of the substrate-chromogen solution diaminobenzidine for 10 to 15 minutes at ambient temperature. The sections were counterstained with aqueous hematoxylin to increase the contrast of specific staining, and they were then mounted in aqueous medium (crystal mount). Sections were studied with a Nikon microscope and images were captured by a digital Nikon camera (Nikon, Dusseldorf, Germany). Labeling results were categorized as weak, medium, and strong staining. 2.6. Mass spectrometry analysis The bands of interest were subjected to tryptic digestion using a procedure previously described [43]. Protein bands were excised from the gel and destained with a solution of 25 mM ammonium bicarbonate for 30 minutes and then with a 1:1 (vol/vol) solution of acetonitrile and 25 mM ammonium bicarbonate for 30 minutes. Washes were repeated until the blue color of Coomassie was removed. Bands were then dried by vacuum centrifugation and rehydrated in 10 mM DTT in 25 mM ammonium bicarbonate solution for 1 hour at 56 C. The supernatant was removed and the bands were incubated in the dark for 45 minutes with 55 mM iodoacetamide in 25 mM ammonium bicarbonate. Bands were washed as previously described, dried completely in a Speed Vac Plus (Savant, NY, USA) for 30 minutes, and enzymatically digested overnight using sequencing grade trypsin (Promega, Madison, WI, USA).
Coomassie Brilliant Blue, SDS, and salts were removed from the protein sample after passive elution using a ZipTipHPL (hydrophilic interaction chromatography) according to the manufacturer’s instructions (Millipore, Bedford, MA, USA). Briefly, the ZipTipHPL was rehydrated in buffer A (H2O/ ACN/AcOH: 50/50/0.1, pH 5.5) and washed with buffer B (H2O/ACN/AcOH: 10/90/0.1, pH 5.5). Protein eluates were diluted in 200 mL of buffer A, and proteins were eluted in 4 mL of H2O/ACN/TFA (50/50/0.1). NanoLC-MS/MS analyses were performed on an ion trap mass spectrometer (LTQ-OrbitrapXL, Thermo Fischer, USA) with nano-HPLC Ultimate 3000 (Dionex, Jouy en Josas, France). Peptide sequences were analyzed using Mascot software (Matrix Science, London, UK) and the SwissProt database. 2.7. Database searching MS/MS fragmentation was compared using Mascot software with sequences in the SwissProt database and the NCBInr database. The reliability of protein identifications was evaluated on the basis of multiple variables, including the Mascot score, the number of peptide matches, mass error (m/z accuracy), percent coverage of the matched protein, similarity of experimental and theoretical protein molecular weights (Mr) and isoelectric points (pIs), and degree of phylogenetic divergence of the species from which the sequence was matched. 3. Results 3.1. Comparative analysis of MLSVP21, MSSVP21, and MCSVP21 3.1.1. Comparative analysis of soluble proteins from seminal vesicle homogenates of P. obesus, Meriones libycus, Meriones crassus, and Meriones shawi During the breeding season in Libyan jird (Meriones libycus), Meriones shawi, and desert jird (Meriones crassus), the soluble protein compositions of seminal vesicle homogenates (Figs. 1b, 2d, and 2e) revealed by SDS-PAGE were similar. They appeared relatively abundant in comparison to the seminal vesicle homogenates of sand rat P. obesus (Fig. 3d), w30 polypeptides ranging in size from 10 to 200 kDa were observed. The major product of soluble androgen-dependent proteins from the homogenates of seminal vesicles of Meriones libycus, Meriones shawi, and Meriones crassus appears to be 12, 17, 24, 34, and 21 kDa (Figs. 1b, 2d, and 2e). The 21-kDa proteins corresponded to MLSVP21, MSSVP21, MCSVP21, and POSVP21 (Figs. 1b, 2d, 2e, and 3d). The proteins with an apparent molecular weight of 30, 41, 43, 45, and 67 kDa decreased during the breeding season. When compared with the components of Meriones libycus, Meriones shawi, and Meriones crassus seminal vesicle homogenates, eight major bands with an apparent molecular weight of 12, 17, 21, 24, 30, 41, 57, 67, and 78 kDa were matched to comparable bands in seminal vesicle homogenates of P. obesus. The proteins with an apparent molecular weight of 53, 45, 43, 32, 29, 27, and 25 kDa seemed to characterize the seminal vesicles of Meriones libycus, Meriones shawi, and Meriones crassus.
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Fig. 1. Polyacrylamide gel electrophoretic analysis of soluble proteins from the adult Libyan jird Meriones libycus seminal vesicles. Protein components of soluble proteins from homogenate (seminal vesicles and secretions). A suitable amount of protein was subjected to SDS-PAGE on a 15% slab gel (140 mm 120 mm 1.5 mm). Each lane was loaded with 100 mg proteins. Proteins were stained with Coomassie Brilliant Blue. The arrow indicates the position of the 21-kDa protein. (a) Protein standard: phosphorylase B (94 kDa); bovine serum albumin (67 kDa); ovalbumin (43 kDa); carbonic anhydrase (30 kDa); soybean trypsin inhibitor (20 kDa); lactalbumin (14 kDa). (b) Proteins from homogenate of the adult Libyan jird Meriones libycus seminal vesicles during the breeding season (early spring) caught in the yield. (c) Proteins from homogenate of the adult Libyan jird Meriones libycus seminal vesicles during the nonbreeding season (late summer) caught in the yield.
During the nonbreeding season, the very large part of these proteins, particularly the 21-kDa proteins MLSVP21, MSSVP21, MCSVP21, POSVP21 or transgelin, and the P78, were largely reduced. Conversely, the 30-, 41-, 43- and 45-kDa proteins increased (Figs. 1c, 2b, 2c, 3b, and 3c).
3.2. Detection of immunological homology between MLSVP21, MSSVP21, MCSVP21, and POSVP21 Proteins extracted from the seminal vesicles of Meriones libycus, Meriones shawi, and Meriones crassus were submitted to Western blot analysis using an anti-POSVP21–specific polyclonal antibody. Purified POSVP21 or transgelin was used as positive controls in these experiments. An immunoreactive signal at 21 kDa was detected in seminal vesicle homogenates from these Saharan species. On the basis of these results, we hypothesized that the three proteins
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Fig. 2. Polyacrylamide gel electrophoretic analysis of soluble proteins from the adult desert jird Meriones crassus and Meriones shawi seminal vesicles. Protein components of soluble proteins from homogenate (seminal vesicles and secretions). (a) Protein standard. (b) Proteins from homogenate of the adult Meriones shawi seminal vesicles during the nonbreeding season (early winter) caught in the yield. (c) Proteins from homogenate of the adult desert jird Meriones crassus seminal vesicles during the nonbreeding season (early winter) caught in the yield. (d) Proteins from homogenate of the adult Meriones shawi seminal vesicles during the breeding season (early spring) caught in the yield. (e) Proteins from homogenate of the adult desert jird Meriones crassus seminal vesicles during the breeding season (early spring) caught in the yield.
with a same apparent molecular weight share antigenic determinants with sand rat seminal vesicle proteins POSVP21. The positive immunoreactivity provides the immunological homology between MLSVP21, MSSVP21, MCSVP21, and POSVP21 or transgelin. In conclusion, it appears that antigens corresponding to POSVP21 or transgelin could be detected in the seminal vesicles from other Saharan rodents, such as Meriones libycus, Meriones shawi, and Meriones crassus. 3.3. Localization of MLSVP21, MSSVP21, and MCSVP21 in seminal vesicles of Meriones libycus, Meriones shawi, and Meriones crassus Sections from sand rat seminal vesicles during the breeding season (Fig. 4A) and the nonbreeding season (Fig. 4B) were used as positive and negative controls in these experiments. Immunohistochemical staining with the avidin-biotin complex technique using affinity-purified polyclonal antibodies against POSVP21 was performed to confirm the immunological homology between MLSVP21, MCSVP21 MSSVP21, and POSVP21, and to localize these proteins in sections of the seminal vesicles. The morphology of the sections was studied after staining with Mayer’s hematoxylin. Figure 5B–D showed a positive staining in the epithelial cells.
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These results reveal that antigens corresponding to POSVP21 or transgelin could be detected in seminal vesicles from other Saharan rodents, such as Meriones libycus, Meriones crassus, and Meriones shawi, with a same distribution. In the seminal vesicles of Meriones libycus, Meriones crassus, and Meriones shawi, POSVP21 immunostaining was observed in epithelial cells and in secretory products in the lumen (Figs. 5B, 5D, 6B, and 7). No other cells stained in this tissue, and thus synthesis of MLSVP21, MCSVP21, and MSSVP21 can be assigned to the vesicular epithelial cells.
3.4. Identification of MLSVP21, MSSVP21, and MCSVP21 The MLSVP21, MSSVP21, MCSVP21, and peptide sequence analysis reveal a strong homology with the Rattus norvegicus transgelin peptide sequences like for POSVP21. Indeed, all of the peptide sequences identified at the level of MLSVP21, MSSVP21, and MCSVP21 are also present at the level of the transgelin peptide sequences. They cover 54.73%, 54.23%, and 28.86% of the transgelin protein sequences, which accounts for two-thirds of the total sequence of transgelin (Fig. 8). We also noted that peptide sequence analysis of MLSVP21, MSSVP21, and MCSVP21 revealed a similar amino acid composition to POSVP21. Fig. 3. Polyacrylamide gel electrophoretic analysis of soluble proteins from the adult sand rat Psammomys obesus seminal vesicles. Protein components of soluble proteins from homogenate (seminal vesicles and secretions). A suitable amount of protein was subjected to SDS-PAGE on a 15% slab gel (140 mm 120 mm 1.5 mm). Each lane was loaded with 100 mg proteins. Proteins were stained with Coomassie Brilliant Blue. The arrow indicates the position of the 21-kDa protein. (a) Protein standard: phosphorylase B (94 kDa); bovine serum albumin (67 kDa); ovalbumin (43 kDa); carbonic anhydrase (30 kDa); soybean trypsin inhibitor (20 kDa); lactalbumin (14 kDa). (b, c) Proteins from homogenate of the adult sand rat seminal vesicles during the nonbreeding season (late summer) caught in the yield. (d) Proteins from homogenate of the adult sand rat seminal vesicles during the breeding season (early autumn) caught in the yield.
The connective tissue and nucleus had negative immunoreaction for POSVP21. The positive immunoreactivity provides the immunological homology between MLSVP21, MCSVP21, MSSVP21, and POSVP21 or transgelin (Fig. 5B–D). The polyclonal antibodies against POSVP21 reacted positively with MCSVP21 in sections of the Meriones crassus seminal vesicles. MCSVP21 immunostaining was observed in epithelial cells and no signal has been detected in smooth muscle cells and connective tissue (Fig. 6B). During the breeding season, immunoexpression from MSSVP21 was located in the seminal vesicle epithelial cells. The connective tissue and the smooth muscle cells were not at all immunomarked. The immunohistochemical analysis indicated a reactivity of anti-POSVP21 antibodies with respect to the MLSVP21, MCSVP21, and MSSVP21. It also indicates that these three proteins with a same apparent molecular weight of 21 kDa share antigenic determinants with sand rat seminal vesicle proteins POSVP21.
4. Discussion The sand rat P. obesus, the Libyan jird Meriones libycus, the Meriones shawi, and the desert jird Meriones crassus are the Saharan rodents under studies submitted to a seasonal cycle of reproduction. During the active period, the reproductive organs from these species report a considerable weight growth. The structure and the ultrastructure are developed and both testicular and plasma testosterone are high. In fact, it is known that the majority of the rodents living in the arid and semiarid regions breed according to a seasonal cycle and they are characterized by weight, structural, ultrastructural, and biochemical variations [44]. In P. obesus [28,45], Meriones libycus [46,47], Meriones crassus [48], or even Meriones shawi [49], the phase of quiescence is typically characterized by a reduction in the size of the reproductive organs (seminal vesicle and prostate) induced by a marked decrease in testicular and plasma testosterone levels. In the sand rat P. obesus, the seasonal, structural, ultrastructural, and biochemical cycles studied by Gernigon et al. [28] coincided with the hormonal seasonal cycle reported by Khammar et al. [45]. In the previous studies, we have reported that seminal vesicles of adult sand rat P. obesus contain a major secretory protein band (21 kDa) designated as POSVP21 (P. obesus seminal vesicles protein of 21 kDa). This protein accounts for over 22.3% of the soluble proteins from homogenate during the breeding season, 13.3% during the middle season, and 5.3% during the hormonal regression season. During the nonbreeding season, the amount of POSVP21 is greatly reduced. The expression of POSVP21 is induced by testosterone, and the levels of this protein decrease most dramatically after castration [28].
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Fig. 4. POSVP21 immunochemistry in the adult sand rat Psammomys obesus seminal vesicles during the breeding season (early autumn). Sections of sand rat seminal vesicle fixed with formalin and embedded in paraffin were incubated with the anti-POSVP21 polyclonal antibody diluted at 1:200 (A) or nonimmune serum diluted at 1:200 (B). Bound antibodies were detected using a peroxidase conjugated to anti-rabbits IgG at a dilution 1:200. L, lumen; E, epithelium; S, secretion; C, connective tissue; N, nucleus; Sn, supranuclear zone; St, smooth muscle cells.
Major efforts have been devoted to the characterization of rat seminal vesicle secretory proteins and their differential regulation by androgens [50]. Mouse seminal vesicles also secrete a similar group of androgen-regulated proteins [4,22,23]. However, because the absence of specific antibodies and proteomic approaches had not been developed, previous studies have been limited to the analysis of steady-state levels of their mRNAs [4,23,24]. In this work, the seminal vesicle proteins studied were the three major androgen-dependent proteins with a same apparent molecular weight of 21 kDa. They are designated as follows: MLSVP21 (Meriones libycus seminal vesicles protein of 21 kDa), MSSVP21 (Meriones shawi seminal vesicles
protein of 21 kDa), and MCSVP21 (Meriones crassus seminal vesicles protein of 21 kDa). The profile of expression of these proteins seems affected by seasonal fluctuations and by seminal vesicles sensitive to androgens. In fact, during the breeding season, MLSVP21, MSSVP21, and MCSVP21 were highly expressed. Contrarily, during the phase of quiescence, the very large amounts of these three 21-kDa proteins were greatly reduced. It is also know that in the Saharan rodent seminal vesicles, the mechanism of synthesis and regulation is adapted to the insecure and irregular food in desert. In other rodents (mouse, rat), the mechanism by which testosterone regulates the expression of androgen-dependent proteins has recently been investigated in the mouse. It has
Fig. 5. MLSVP21 immunochemistry in the adult Libyan jird Meriones libycus seminal vesicles during the breeding season (early spring). In sections of Libyan jird Meriones libycus seminal vesicles, bound antibodies were detected using a peroxidase conjugated to anti-rabbits IgG at a dilution 1:200 (B–D) or nonimmune serum diluted at 1:200 (A). The immunoreactivity to MLSVP21 staining was localized to the epithelial cells (B–D) and in the secretory products in the lumen (D). The smooth muscle cells, the connective tissue, and the nucleus had negative immunoreaction for POSVP21 (C and D). L, lumen; E, epithelium; S, secretion; C, connective tissue; N, nucleus; Sn, supranuclear zone; St, smooth muscle cells.
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Fig. 6. MCSVP21 immunochemistry in the adult desert jird Meriones crassus seminal vesicles during the breeding season (early spring). In sections of desert jird Meriones crassus seminal vesicles, bound antibodies were detected using a peroxidase conjugated to anti-rabbits IgG at a dilution 1:200 (B) or nonimmune serum diluted at 1:200 (A). Immunoreactivity to MCSVP21 staining was localized to the epithelial cells (B). The smooth muscle cells, the connective tissue, and the nucleus had negative immunoreaction for POSVP21 (B). L, lumen; E, epithelium; C, connective tissue; N, nucleus; Sn, supranuclear zone; St, smooth muscle cells.
been reported that androgens act mainly on MSVSP99 gene transcription. This transcriptional regulation is exerted by steroid-receptor complexes interacting with enhancer sequences adjacent to the gene [24,51]. It should be noted that the major androgen-dependent protein from sand rat seminal vesicles or POSVP21 shares many traits with proteins involved in reproduction mechanisms: it is abundant in secretions, regulated by androgens, and is also present in vaginal plug. Recently, POSVP21 is localized in the cytoplasm of seminal vesicle epithelial cells and in the lumen secretory products [29]. The peptide sequences reveal that POSVP21 seems to correspond to the R. norvegicus transgelin [30]. Using polyclonal antibodies directed against POSVP21, Western blot and the immunohistochemical analyses show a reactivity of anti-POSVP21 antibodies with respect to the MLSVP21, MSSVP21, and MCSVP21. These results indicate an immunological homology with this major secretory androgen-dependent protein, identified previously as transgelin. They indicate that these three proteins, with a
Fig. 7. MSSVP21 immunochemistry in the adult Meriones shawi seminal vesicles during the breeding season (early spring). In sections of Meriones shawi seminal vesicles, bound antibodies were detected using a peroxidase conjugated to anti-rabbits IgG at a dilution 1:200. Immunoreactivity to MSSVP21staining was localized to the epithelial cells; the smooth muscle cells, the connective tissue, and the nucleus had negative immunoreaction for POSVP21. E, epithelium; C, connective tissue; St, smooth muscle cells.
same apparent molecular weight, share antigenic determinants with sand rat seminal vesicle proteins POSVP21. The antigens corresponding to POSVP21 or transgelin could be detected in seminal vesicles from other Saharan rodents, such as Meriones libycus, Meriones crassus, and Meriones shawi, with a same distribution. In fact, during the breeding season, immunoexpression from MLSVP21, MSSVP21, and MCSVP21 were located in the seminal vesicle epithelial cells and in the lumen secretory products. In contrast, smooth muscle cells and connective cells do not possess proteins displaying any cross-reactivity with the antibodies directed against POSVP21 or transgelin. As to POSVP21, synthesis of MLSVP21, MSSVP21, and MCSVP21 can be assigned to the vesicular epithelial cells. Mainly, studies postulated the localization of most proteins was closely related to their physiological functions. In fact, in a series of investigations, several roles have been attributed to some components as follows: semen coagulation after ejaculation is caused by the semenogelin, a major protein expressed exclusively in the men seminal vesicles is the major coagulum-forming protein, and semen liquefaction is provoked by prostatic-specific antigen (PSA); this protease, secreted by the prostate, plays a prominent role in the process of liquefaction by causing semenogelin proteolysis [52]. Finding the structure and function of these proteins have been attempted in order to understand their roles in seminal vesicle physiology and their effects on gamete activity. The analysis of MLSVP21, MSSVP21, and MCSVP21 peptide sequences reveal a strong homology with the R. norvegicus transgelin peptide sequences like for POSVP21. Indeed, all of the peptide sequences identified at the level of MLSVP21, MSSVP21, and MCSVP21 are also present at the level of the transgelin peptide sequences that cover 54.73%, 54.23%, and 28.86 % of the transgelin protein sequences. It account for two-thirds of the total sequence of transgelin. We also noted that all of the peptide sequences identified from MLSVP21, MSSVP21, and MCSVP21 are found in the POSVP21 sequence. In our study, the proteomic approach shows that the isolated proteins, MLSVP21, MSSVP21, and MCSVP21, seem to correspond to the same protein, the transgelin. POSVP21 or transgelin was initially identified in seminal vesicles from sand rat P. obesus [30]. The role of POSVP21 remains unknown, but during the late spring, when the
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755
MANKGPSYGM
SREVQSKIEK
KYDEELEERL
VEWIVMQCGP
DVGRPDRGRL
TGLN R. n.
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- - - - - - - IEK
KYDEELEERL
VEWIVMQCGP
DVGRPDR- - L
MLSVP
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KYDEELEERL
VEWIVMQCGP
DVGRPDR- - L
MSSVP
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- - - - - - - IEK
KYDEELEER-
VEWIVMQCGP
DVGRPDRGRL
MCSVP
GFQVWLKNGV
ILSKLVNSLY
PEGSKPVKVP
ENPPSMVFKQ
MEQVAQFLKA
TGLN R. n.
GFQVWLKNGV
ILSKLVNSLY
PEGSKPVKVP
ENPPSMVFKQ
MEQVAQFLKA
MLSVP
GFQVWLKNGV
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---------Q
MEQVAQFLK -
MSSVP
GFQVWLKNGV
ILSKLVNSLY
PEGSKPVKVP
ENPPSMVFKQ
---------A
MCSVP
AEDYGVTKTD
MFQTVDLFEG
KDMAAVQRTV
MALGSLAVTK
NDGHYRGDPN
TGLN R. n.
AEDYGVTKTD
MFQTVDLFEG
K-
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- - - - - - GDPN
MLSVP
- - - - - - - - TD
MFQTVDLFEG
K-
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MSSVP
AEDYGVTKTD
MFQTVDLFEG
KDMAAVQR-
-
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MCSVP
WFMKKAQEHK
REFTDSQLQE
GKHVIGLQMG
SNRGASQAGM
TGYGRPRQII
TGLN R. n.
WFMK - - - - - -
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MLSVP
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MSSVP
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MCSVP
S
TGLN R. n.
S
MLSVP
S
MSSVP
S
MCSVP
Fig. 8. Homologies between the amino acid sequences from MLSVP21, MSSVP21, and MCSVP21 fragment and the sequence from Rattus norvegicus transgelin. The identical amino acids are presented in red with other sequences aligned to show homology. TGLN R.n.: transgelin from Rattus norvegicus.
protein diminishes, the sand rat rarely undergoes pregnancy. This protein therefore might also be implicated in the fertilization process. The high expression of MLSVP21, MSSVP21, and MCSVP21 during the breeding season and the important reduction during the long phase of sexual quiescence suggest that these proteins may have an important specific function in reproduction. Transgelin has since been identified in many different species and has been given different names such as TAGLN [53], SM22 [54], WS3-10 [55], and mouse p27 [56]. It was first isolated from chicken gizzard as a transformation- and shape-change sensitive actin-binding protein, the expression of which is lost in virally transformed cells. Its expression is highly conserved across species, and homologs have been found in invertebrate species, as distant as Caenorhabtidis elegans (unc-87) [31] and Drosophila melanogaster (mp20) [32]. Although its physiological role remains uncertain, insights into its potential function are found in studies under its several different names [56,57] such as SM22a, transgelin, WS3-10, and mouse p27 [35]. It was also originally described as a predominant expression in smooth muscle cells to bind the actin, suggesting it is involved in cell differentiation and cytoskeletal rearrangement [58,59]. Its expression is one of the earliest markers of smooth muscle differentiation. Tissue distribution of transgelin is contentious. It is found throughout the smooth muscle tissues of normal adult
vertebrates [58]. Indeed, its mRNA levels are highest in adult human tissues containing large amounts of smooth muscle, such as uterus, bladder, stomach, and prostate. However, it is also expressed in several adult tissues containing little or no smooth muscle, including spinal chord, adrenal gland, and, notably, in the heart [53,60]. As we have shown in the epithelial cell seminal vesicles, from these Saharan rodents, P. obesus, Meriones libycus, Meriones shawi, and Meriones crassus, transgelin expression has also been identified recently in epithelial cells than in intestinal epithelial cells [36], glomerular epithelial cells [37], breast ductal epithelium [36,61], and prostate epithelium [38]. Transgelin is only sensitive to protein kinase C phosphorylation in vitro, which attenuates actin binding [62]. The cellular location and functional role of transgelin are at present unknown. It is unique in its composition and properties and in its amino acid sequencing. It is shown to resemble no other previously reported protein [63]. The physiological role of SM22a is still unclear, but recent studies reveal that SM22a may play a role in agonist-induced vascular smooth muscle contractility [64]. In prostate carcinoma cells, transgelin has been shown to block androgen-stimulated cell growth by preventing the binding of an androgen receptor coactivator with androgen receptor and subsequent translocation to the nucleus [38]. Transgelin also acts to suppress the expression of the metallo-matrix proteinase-9 [65]. Because we have found transgelin and localized this protein in the epithelium and in the secretion of these
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rodent species seminal vesicles, we suggest that there is a passage through an external medium that, we believe, is involved in the organization of the epithelial cell cytoskeleton. This allows it to maintain the structure and the contractile state of the seminal vesicle. In rodents, one of the functional properties of seminal vesicle secretion is to contribute to the clot formation of ejaculated semen. It also influences the metabolism, mobility, and surface properties of spermatozoa to induce capacitation and suppress the immunocompetence of the female genital tract [66,67]. Apart from their role in the male genital tract, the secretions of seminal vesicles have a role in the female genital tract, where they mediate stimulation of the motility of smooth muscles [23]. 4.1. Conclusion The present investigation shows the high expression of MLSVP21, MSSVP21, and MCSVP21 during the breeding season and the important reduction during the long phase of sexual quiescence. The specific polyclonal antibodies directed against POSVP21 reveal that MLSVP21, MSSVP21, and MCSVP21 are immunologically related to POSVP21, a major androgen-dependent secretory protein identified initially as transgelin in the seminal vesicles from sand rat P. obesus. The immunohistochemical study localized MLSVP21, MSSVP21, and MCSVP21 in the epithelial cells of adult Meriones libycus, Meriones crassus, and Meriones shawi seminal vesicles during the breeding season. The peptide sequence analysis reveal that MLSVP21, MSSVP21, and MCSVP21 correspond to the same protein, the transgelin. Transgelin identified in the seminal vesicle of Meriones libycus, Meriones shawi, and Meriones crassus with a same distribution can be used as a marker for physiological mechanisms of reproduction of these Saharan species. Our results made the first demonstration that the transgelin is expressed highly and distinctly in the epithelial seminal vesicles of three Saharan rodents and its transcription appears to be regulated by testosterone. Further studies are needed to clarify the mechanism by which transgelin is involved in the rodent physiological reproduction. References [1] Mansson PE, Sugino A, Harris SE. Use of a cloned double stranded cDNA coding for a major androgen dependent protein in rat seminal vesicle secretion: the effect of testosterone in gene expression. Nucleic Acids Res 1981;9:935–46. [2] Mc Donald C, Williams L, Mc Turk P, Fuller F, Mc Intosh E, Higgins S. Isolation and characterization of genes for androgen-responsive secretory proteins of rat seminal vesicles. Nucleic Acids Res 1983; 11:917–30. [3] Williams L, Mc Donald C, Jackson S, Mc Intosh E, Higgins S. Isolation and characterization of genomic and cDNA clones for androgenregulated secretory protein of rat seminal vesicles. Nucleic Acids Res 1983;11:5021–32. [4] Chen YH, Pentecost BT, Mc Lachlan JA, Teng CT. The androgendependent mouse seminal vesicle secretory protein IV: characterization and complementary deoxyribonucleic acid cloning. Mol Endocrinol 1987;1:707–16. [5] Higgins SJ, Hemingway AL. Effects of androgens on the transcription of secretory protein genes in rat seminal vesicle. Mol Cell Endocrinol 1991;76:55–61. [6] Parker MG, White R, Williams JG. Cloning and characterization of androgen-dependent mRNA from rat ventral prostate. J Biochem Chem 1980;255:6996–7001.
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