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Male Reproductive Tract |
Equipe "Gamète Mâle et Fertilité" UMR 6175 INRA-CNRS-Université,3 PRC, INRA, 37380 Nouzilly, France
CBM CNRS UPR 4301,4 45 071 Orléans cedex 2, France
Unitat de Bioquimica i Biologia Molecular,5 Departament de Biologia, Universitat de Girona, Spain
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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epididymis, male reproductive tract, sperm maturation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We have recently shown that Train A presented an RNase A family motif in its sequence (i.e., the eight conserved cysteine residues characteristic of this superfamily). The presence of an additional sequence before the RNase motif indicated that Train A represents a new group of proteins in the RNase A superfamily. The RNase A superfamily is actually composed of diverse groups of secretory proteins that share distinct structural features and enzymatic activity that have presumed different roles in vivo [7].
In the male genital tract, ribonucleolytic activity has been detected in bovine seminal vesicles [8], in the semen of different mammals such as the boar (for which activity was high and originated from the epididymal fluid [9]), and in the caudal epididymal region of different species such as the monkey [10]. In the latter, this activity was repressed by testosterone [10].
The aim of this work was to study the enzymatic activity and the regulation of this putative epididymal RNase A. The work was undertaken to determine: 1) A Train RNase activity by zymography, 2) A Train reabsorption in vitro by epididymal cells and its possible interaction with sperm using biochemical and immunogold-labeling techniques, and 3) A Train expression during postnatal development and in efferent duct-ligated animals, which were used as models to produce a sperm-free androgen-maintained epididymis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Dulbecco modified Eagle medium (DMEM) without methionine and cysteine, x-ray films (Kodak X-OMAT-XAR5; Eastman Kodak, Rochester, NY), CHAPS (3-([3-cholamidopropyl]dimethylammonio])-1-propanesulfonate), and mouse and goat anti-rabbit immunoglobulin G (IgG) coupled to horseradish peroxidase were purchased from Sigma Chemical Co. (St. Louis, MO). [35S]Methionine (Easy-Tag Express [35S]) was purchased from NEN (Les Ulis, France); [
-32P]dCTP was from Amersham (Les Ulis, France); acrylamide 2x, N-N'methylene bisacrylamide 2x powder (Serva), and ampholytes pH 2–11 (Servalytes) were from Serva (Heidelberg, Germany); ampholytes pH 3–10 (Ampholytes), Coomassie brilliant blue (Phastgel Blue R), Poly (cytidylic acid) [poly(C)] and poly (uridylic acid) [poly(U)] were from Pharmacia (Saclay, France). Isopropyl-ß-D-thiogalactopyranoside (IPTG) was from Promega (Charbonnières, France). All other chemicals were of molecular biology grade and were purchased from Sigma.
Animals and Organ Sampling
Ten adult boars and four animals aged 1, 2, 3, and 4 mo were used in this study. The efferent ducts of four adult boars were ligated with sterile braided silk for 3, 5, or 9 days, the ipsilateral epididymides of these animals being used as controls. Because Train A mRNA was also detected in rodents, four mice were efferent duct-ligated for 24 h. Four other boars were castrated at 15 days before slaughter. The testes were removed through a scrotal incision, and the epididymides were returned to the scrotum. Two castrated animals received testosterone propionate replacement for 30 days, at a dose of 90 mg/animal injected i.m. every 3 days, as previously described [11]. Epididymides and testes were removed from freshly killed animals at a local boar slaughterhouse. The epididymis was divided into 10 regions (0–9) for boars as previously described [4] and into 4 zones (initial segment, E0; caput, E1; corpus; and cauda) for mice. The luminal fluids from the different epididymal zones were obtained by perfusion for protein analysis as previously described [12, 13]. Testicular fluid was carefully collected by directly puncturing the rete testis. Spermatozoa were separated from the fluids by centrifugation (1500 x g for 15 min at 4°C). The fluids were removed and centrifuged again (15 000 x g for 10 min) and used directly or stored at –20°C until use. All procedures described within were reviewed and approved by the Ethical Animal Committee of INRA and were performed in accordance with the Guiding Principles for the Care and Use of Laboratory Animals.
Gel Electrophoresis, Fluorography, and Western Blotting
Gel and sample preparation and methods for isoelectric focusing have previously been described [4]. SDS 6%–16% polyacrylamide gel gradients or SDS 15% polyacrylamide gels were used for protein separation. For detection of radioactive proteins, the gels were exposed on a PhosphorImaging screen (Storm; Molecular Dynamics, Paris, France). For immunodetection, the proteins from polyacrylamide gels were electrotransferred (0.8 mA/cm2 for 2 h) by a semidry technique onto a 0.2-µm nitrocellulose membrane. The membranes were blocked overnight with Tris-buffered saline containing 0.5% (w/v) Tween 20 (TBST) and 5% (w/v) dry skimmed milk on a rocking platform at 4°C. The primary antibody, raised against an internal peptide (anti-Train-A peptide 1) [5] or anti-recombinant Train A (anti-rTrain A, see below), was added at 1:5000 dilution in TBST. The primary monoclonal antibody, anti-"His-Tag" was used at 1:5000 dilution in 5% (w/v) dry skimmed milk TBST. Blots were washed with the same buffer for all primary antibodies, and incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG diluted at 1:5000 in TBST. The peroxidase was revealed by chemiluminescent substrate (Western Blot Chemiluminescence Reagent Plus; NEN, Boston, MA) according to the manufacturer's instructions.
RNase Activity Assay
Two methods were used to measure epididymal fluid RNase activity. Zymography for RNAse activity was performed on one-dimensional (1D) gels following the method described by Bravo et al. [14] using epididymal proteins (20 µg) from various regions and on two-dimensional (2D) zymography. The immobilin pH gradient was between 3 and 10. After nondenaturing strip rehydration in 8 M urea, 2% CHAPS, and 0.5% IPG buffer (Amersham) overnight at 20°C, the sample (30 µg) was added to the strip and the program was used as provided by the manufacturer (500 V for 250 Vh, 1000 V for 500 Vh, and 8000 V for 8000 Vh). At the end, the strips were washed in equilibration solution (50 mM Tris/HCl pH 8.8, 6 M urea, 30% glycerol [v/v], 2% SDS [w/v]) for 10 min. Proteins were separated under nondenaturing conditions on 15% polyacrylamide-SDS (1D or 2D) gels containing 0.3 mg·ml–1 poly(C) or poly(U). The SDS was extracted from the gels by successive incubations in 10 mM Tris/HCl pH 8, 20% (v/v) isopropanol, and 10 mM Tris/HCl pH 8. RNase activity was revealed in 0.1 M Tris/HCl pH 8, and the reaction was monitored under UV light. The gel was then stained with 0.2% (w/v) toluidine blue in 10 mM Tris/HCl pH 8. Positive bands or spots appeared white against the blue background. To detect the presence of Train A in the same sample, the toluidine blue-stained zymogram of the sample was incubated in electrophoresis buffer for 30 min and then transferred onto a nitrocellulose membrane before detection with an antibody raised against an internal peptide.
The RNase activity of epididymal fluids was also determined by the release of acid-soluble nucleotides from bakers yeast total RNA after ribonuclease digestion, as previously described [15]. Briefly, 120 µg of bakers yeast total RNA (Fluka; Sigma Aldrich) were incubated with 5 µg of epididymal proteins in 50 mM Tris-HCl pH 8 in a final volume of 50 µl for 10 min at 37°C. To stop the reaction, 200 µl of 7% perchloric acid and 0.1% uranyl acetate were added. After 30 min on ice and 20 min of centrifugation, 200 µl of the supernatant were diluted to 800 µl with water, and the absorbance at 260 nm was measured to detect the presence of acid-soluble material.
In Vitro Secretion of [35S]Methionine-Cysteine-Labeled Proteins from Isolated Tubules
In the boar, in vitro secretion of [35S] methionine-cysteine-labeled proteins was estimated from all epididymal regions (isolated tubules) and from testis and efferent duct samples as previously described [4]. Briefly, one closed-end tubule for each epididymal region was incubated in a mixture of [35S]methionine and [35S]cysteine in DMEM solution. The medium was separated from the tissues at the end of the incubation period (5 h at 32°C), and the lumen fluid of each tubule was collected by microperfusion. All the incubation media were centrifuged at 2500 x g for 15 min. The pellet was used for sperm membrane extraction, and after a second centrifugation at 16 000 xg for 10 min, the supernatants were used or stored at –20°C.
Extraction of [35S]Methionine-Cysteine-Labeled Sperm Membrane Proteins
The sperm pellet obtained after centrifugation of the incubation media was resuspended in DMEM and then washed twice in Percoll (50% in PBS). The pellet was resuspended in PBS containing a protease inhibitor cocktail, mixed with an equal volume of 1% SDS, and incubated for 1 h. Sperm were centrifuged (15 000 x g for 1 h), and the supernatant was stored at –80°C until use.
In Vitro Incubation of Proteins in Isolated Epididymal Tubules
The [35S]cysteine-labeled proteins secreted into the tubule in region 0 (as described earlier) were collected by microperfusion and injected slowly into the tubules isolated from regions 0 to 6 to replace the native fluid. These closed-end tubules were then incubated for 7 h at 32°C. At the end of the incubation period, the contents of each tubule were collected by microperfusion and analyzed by fluorography and Western blotting (as described above).
Reverse Transcription-Polymerase Chain Reaction, DNA Probe, and Northern Blot Hybridization
After different times of ligation, total RNA from boar and mouse proximal caputs (regions 0 and 1) was extracted from 200 mg of frozen tissue using an RNAble kit extraction (Eurobio, Les Ulis, France). We amplified cDNA from Train A mRNA by reverse transcription-polymerase chain reaction (RT-PCR) using 2 µg of this total RNA.
Reverse transcription was performed by Superscript II reverse transcriptase RNase H– (Invitrogen, Cergy Pontoise, France) with oligo(dT) primers (Promega). For the boar, the specific reverse transcript Train A cDNA was then amplified by DNA polymerase (Goldstar; Eurogentec, Seraing, Belgium) with 30 pmol of a pair of specific primers (reverse primer, 5'-GCTCTGAGCATCTTGTTTCCTCC-3'; forward primer, 5'-GAGGAAAGTGATCAGCTACTGAGTGAG-3') corresponding to 185 base pairs (bp) (bases 160–344) of Train A cDNA [5]. For the mouse, the fragment was amplified with the primer corresponding to 500 bp of murine ribonuclease (forward primer, 5'-GGAGGATCAGCCACTGA-3'; reverse primer, 5'-CCAGTTGTCTCTTGTCA-3') at the same concentrations. PCR was performed for 30 cycles (94°C, 30 sec; 55°C, 30 sec; 72°C, 1 min) and a final elongation step at 72°C for 5 min).
DNA probes for Train A were obtained as above and labeled with [32P]dCTP using Megaprime from Amersham. Hybridization on Northern blots obtained with total RNA from epididymal tissues was performed using [32P]-labeled probes as previously described [5].
Train A Recombinant Protein
A recombinant protein corresponding to the complete sequence of the boar Train A protein was produced. The cDNA of Train A was amplified by RT-PCR on total RNA extracted from the proximal caput using the following primers: 5'-GCCTCGAGTGATCCAGACATTACCTGGAC-3' and 5'-CGGATCCGGGGTTGGGACTTCAGATGGCT-3' (Fig. 1). The PCR fragment of 521 bp encoding Train A (Fig. 2A) was inserted into the pET-22b(+) bacterial expression vector (Novagen, Madison, WI). DNA manipulation and bacterial cell transformations were performed as previously described [16]. The C600 bacterial strain was used for plasmid construction and the Rosetta DE3 strain was used for protein production. Most of the proteins produced by the bacteria were found in an insoluble fraction corresponding to cytoplasmic inclusion bodies. As expected, rTrain A was detected as a 28 kDa band (with a degradation fragment of 14 kDa) (Fig. 2B). Growth of the transformed bacteria did not change the type of recombinant protein produced (not shown).
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Purification of the recombinant proteins was performed on the solubilized fractions in urea. Elution from the affinity column was performed in the presence of arginine, which is known to enhance refolding. After progressive elimination of arginine, rTrain A could be obtained as a soluble protein. After 2D gel separation, purified rTrain A appeared as four spots with a molecular mass of 28 kDa and a pI between 7.3 and 7.7 (not shown).
The rTrain A recombinant protein was used directly to provide a specific polyclonal antibody in rabbits as previously described [5].
Immunocytochemical Analysis of Tissue Sections of Isolated Tubules
Following in vitro biosynthesis and secretion of RNase from isolated tubules (as described above) and in vitro incubation of the tubules of the different regions microperfused by proteins secreted into the tubule in region 0 (as described above), epididymal tubules from regions 0, 1, and 2 were fixed in Bouin solution, as previously described [5]. Tubules were then transferred to 70% ethanol, followed by standard embedding procedures, including dehydration in alcohols, clearing in toluene, and final paraffin embedding.
For immunolocalization of RNase, sections (5–6 µm) of paraffin-embedded tissues were deposited on slides precoated with 3-aminopropyltriethoxy-silane (Sigma). Sections were deparaffinized in toluene and rehydrated in ethanol with increasing concentrations of water. Endogenous peroxidase activity was quenched with 3% hydrogen peroxide for 30 min, and the nonspecific binding sites were blocked with 10% normal goat serum (NGS) for 1 h. Immunostaining was performed as previously described [5] using the avidin-biotin-peroxidase complex (ABC) technique (Vectastain Elite ABC kit, Vector Laboratories). Primary and secondary antibodies were diluted in a solution of PBS containing 10% NGS. Tissue sections were exposed to anti-RNase A antibody (diluted 1/1000 to 1/ 8000) overnight at 4°C. After several rinses in PBS, the sections were incubated with secondary antibody at room temperature for 1 h followed by incubation in ABC reagent. Peroxidase activity was revealed by a solution of diaminobenzidine (DAB) in 0.05 M Tris buffer containing H2O2. Specificity of staining was checked by incubation of sections with preimmune rabbit serum. The sections were counterstained with hematoxylin, then dehydrated, rinsed in toluene, and mounted in DePeX.
Immunogold Electron Microscopy
After in vitro incubation, the isolated tubules from the caput epididymal regions (regions 0 to 3) were cut into small pieces and immersed in a mixture of 0.5% glutaraldehyde and 4% paraformaldehyde in 0.1 M phosphate buffer for 2 h at 4°C. The tissues were then washed with PBS containing 4% sucrose and treated with 4% sucrose and 50 mM NH4Cl for 1 h at 4°C. The tissue was then washed, dehydrated in graded methanol up to 90%, infiltrated in Lowicryl K4M, and then left overnight in 100% Lowicryl. After being embedded in precooled Lowicryl-filled gelatin capsules, the tissue was polymerized using UV light at –30°C. Ultrathin sections were mounted on 300-mesh formvar-coated nickel grids. Each section was then floated for 30 min on a drop of 20 mM Tris-HCl buffered saline (TBS) containing 0.1% BSA, 5% NGS, and then incubated on a drop of anti-RNase A antibody (1/50 to 1/2000) diluted in the same buffer. After further washing in TBS, the sections were transferred for 10 min into TBS/5%NGS followed by colloidal gold (10 nm)-conjugated goat anti-rabbit IgG for 30 min. After several washes in TBS and then in distilled water, sections were counterstained with uranyl acetate and lead citrate. Specificity of staining was checked by incubation of sections with normal rabbit serum. Photographs were taken using a Philips CM10 transmission electron microscope.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A polyclonal antibody obtained from the purified rTrain A specifically immunodetected rTrain A protein and the native forms of Train A in E0 and E1 of the boar epididymis (Fig. 3A). All the isoforms of this protein (molecular mass from 26 to 33 kDa and pI from 5 to 8.5) were recognized by this antibody (Fig. 3B). However, no immunoprecipitation of Train A protein could be obtained with this antiserum. No positive immunodetection could be obtained for other species (bull, stallion, ram, or mouse) either by enzyme-linked immunosorbent assay or by Western blotting, regardless of the antiserum dilution or epididymal region (Fig. 3A).
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Ribonucleolytic Activities of Train A and rTrain A
For the recombinant protein, no supplementary RNase activity was detected by zymography in transformed bacteria. Ribonucleolytic activities observed in the supernatant of the culture and in the pellet (Fig. 2C) were not related to the production of the recombinant protein. For purified recombinant rTrain A protein, no ribonucleolytic activity was detected (not shown).
For testicular and epididymal fluid, RNase activity was estimated directly or after separation by gel zymography (Fig. 4B) followed by immunodetection after transfer onto nitrocellulose membrane (Fig. 4A) and direct enzymatic evaluation in the fluids (Fig. 4C). Enzyme activity was detected in several protein bands from 40 to 25 kDa by 1D gel zymography. Maximum activities were observed in the rete testis fluid by zymography and directly measured in fluids for similar amounts of total separated protein (Fig. 4, B and C). No supplementary activity was found in regions E0 and E1 where Train A protein was immunodetected (Fig. 4A).
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Many protein spots with ribonucleolytic enzyme activity were detected in region 0 (E0) by 2D gel zymography, with sizes ranging from 38 to16 kDa and pI from 5 to 9 (Fig. 5A). Although there were similarities in the number and distribution of these different activities detected (Fig. 5A) with the Train A isoforms (Fig. 5B), none of these activities could be attributed to Train A (Fig 5B).
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Factors Involved in mRNA Expression and Secretion of Train A
We investigated Train A expression and secretion according to postnatal development in 1-mo-old to 4-mo-old boars. The mRNA expression detected by Northern blot hybridization with a Train A probe was observed only in 4-mo-old boars in region E0 (Fig. 6A). At the same time, the secretion and presence of Train A protein in the luminal fluid of this region was detected by immunoblotting (Fig. 6B).
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Expression and secretion of the protein were also analyzed in castrated and castrated-testosterone-supplemented animals, and from ipsilateral-ligated efferent ducts. In boars, 5-day efferent duct ligation induced a decrease in mRNA expression (Fig. 7B) and protein concentration in the proximal caput (Fig. 7A). Train A mRNA disappeared entirely from the proximal caput after 9 days of ligation (Fig. 7B) or castration (data not shown), and the protein was no longer detected by Western blotting in the epididymal fluids (Fig. 7A). In comparing the results obtained in mice, the effect of efferent duct ligature was more rapid: Train A mRNA disappeared in the initial segment of the epididymis in less than 24 h (Fig. 7C).
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Train A mRNA expression and protein secretion did not reappear in the epididymis after castration and supplementation with testosterone (data not shown).
Factors Involved in the Disappearance of Train A from the Luminal Fluid
The Train A protein was present only in the luminal fluid of the tubules of regions E0 and E1, where it was secreted in abundance. The protein was undetectable in the nearest subsequent regions (E2 and E3) (Fig. 4A). To understand the origin of such a rapid disappearance of this protein from the fluid, its binding ability onto the sperm surface, and the capacity of the lining epithelium to absorb it were investigated.
Train A Binding on the Sperm Surface
Binding of Train A protein onto the sperm membrane surface was estimated after incubating the gametes for 5 h in the presence of [35S]-labeled neosynthesized epididymal proteins from regions E0 to E9 (see Materials and Methods) (Fig. 8A). No radioactive (Fig. 8B) or immunodetected Train A could be evidenced among the epididymal proteins retrieved from the sperm membrane. We conclude that the Train A protein has no or very low affinity for the sperm surface.
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Absorption of Train A by the Epididymal Tubule
Quantification of Train A protein absorption by the epididymal epithelium was performed by a microperfusion technique using [35S]-labeled E0 epididymal proteins in the lumen of tubules from different epididymal regions previously washed of their own luminal fluids. After 7 h of incubation, the luminal proteins were collected from each tubule, separated by SDS-PAGE, and analyzed by autoradiography and immunoblotting with anti-Train A antibody. Two experiments were performed: one set with epididymal tubules E0, E1, and E2 (Fig. 9, A–D); the second with epididymal tubules E0, E2, E4, and E6 (Fig. 9E).
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Most of the major proteins present in the E0 fluid after 7 h of incubation (i.e., hexosaminidase and glutathione peroxidase [GPX]), were retrieved at the same levels as before incubation, regardless of the origin of the perfused tubules (Fig. 9A). However, 35S-labeled Train A strongly decreased and disappeared from the luminal fluid of E2 (Fig. 9, A and D). Immunodetection of Train A in these incubated fluids confirmed its absence from E2 (Fig. 9, B and D) and its decrease in regions 4–6 (Fig. 9E), but showed its significant increase in regions E0 and E1 (Fig. 9, B, D, and E). This increase was related to the neosynthesis of Train A in these regions during incubation (Fig, 9C). No or few peptides from Train A proteolysis could be evidenced in electrophoresis separation after E2 tubule incubation (Fig. 9B).
The decrease and disappearance of Train A were related to specific absorption of Train A by the lining epithelium of the tubule, including regions E0 and E1, which actively secreted it.
Immunocytochemical Localization of Train A in Epididymal Cells: Secretion and Absorption Sites
Synthesis and secretion sites After 7 h of in vitro incubation, all the cells in the tubules of region E2 were negative for Train A (Fig. 10B). In contrast, in region E0, immunoreactive material was detected in all the epithelial cells, especially in the supranuclear region (Fig. 10A) (probably corresponding to the Golgi zone), and in the apical cytoplasm near or adhering to the plasma membrane (Fig. 10A).
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At the ultrastructural level, the gold-conjugated complexes were clearly present in the Golgi zone, in association with stacked arrays of Golgi membranes and in the lumen of some dilated saccules (Fig. 11B). Adjacent to the Golgi, several large vesicles (on the trans side of the stacks) were labeled, especially on their weakly electron-dense content (Fig. 11B). Many of these labeled secretory vesicles were present in the apical cytoplasm and close to the cell surface (Fig. 11A). In these large vesicles, gold particles were associated with patches of a weakly electron-dense material. Some gold particles were present near the microvilli in the duct lumen, or in association with the plasma membrane (Fig. 11A) and numerous gold particles appeared on the flocculent material present in the lumen. The spermatozoa were devoid of reaction product (Fig. 11C).
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Absorption sites The isolated tubules of region 2, which did not synthesize Train A, were always negative with the specific antisera (see Fig. 10B). When the tubules of region 2 were microperfused with luminal proteins secreted in region 0, conspicuous immunoreactive Train A material was present in the apical cytoplasm of the cells (Fig. 10C). The localization appeared different from the synthesis sites (Fig. 10A).
An ultrastructural immunogold labeling study was undertaken to define the intracellular sites of this endocytosis pathway. Immunogold labeling was not abundant in the lumen of the duct (Fig. 12A). Gold particles were found in association with the plasma membrane, especially in apical tubule profiles and in small irregular vesicles distributed in the apical cytoplasm close to the plasma membrane, some of which appeared to be connected to the cell surface (Fig. 12, A and B). The coated pits and coated vesicles, which were numerous in this region and faintly discernible due to weak fixation and nonosmication of Lowicryl-embedded tissue, appeared negative (Fig. 12, A and B). Gold particles were also present in some endosomes (vesicles from which thin tubules seemed to emanate, Fig. 12C) and in large vesicles, probably corresponding to multivesicular bodies (Fig. 12D). The numerous dense bodies characteristic of the porcine middle caput, and previously described as lysosomes and immunoreactive for hexosaminidase [17], appeared negative.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In terms of amino acid sequences, the homology between Train A proteins and ribonuclease A is principally linked to the presence of eight conserved cysteines that are characteristic of this superfamily and therefore, to a putative homologous three-dimensional (3D) structure formed by an alpha+beta fold, with a long curved beta sheet and three helices.
The potential ribonuclease activity of Train A was evaluated in the fluid of all the epididymal regions. Regardless of the nature of this protein, no endonucleolytic cleavage was obtained with either poly(C) or poly(U) substrates. The RNase A consensus pattern C-K-x(2)-N-T-F in position C131 and the H-K-H triad (expected in positions: 103, 132, 211) were not present in the Train A amino acid sequence. The absence of enzymatic activity found in this study confirmed that these key amino acids are essential for RNase activity [18]. Train A is the first complete structurally inactive RNase to be described. Enzymatic activities are variable in the RNase family and biological functions for some RNases are not directly related to their potential ribonucleolytic activity. The term RISBAses, which denotes RIbonucleases with Special (i.e., noncatalytic) biological actions, has been proposed to classify several RNases [19].
In spite of this enzymatic inactivity, Train A is a new member of this RNase A family; its gene is located in the same chromosome cluster as the other RNases (Fig. 13A). Phylogenic analysis between the members of the human RNase family showed that Train A should be close to angiogenin and a new putative RNase (Fig. 13B). The RNase activity of these two proteins was weak for the angiogenin and unknown for the new putative RNase, but in the latter compound only one amino acid of the catalytic triad was present. The loss of RNase activity for Train A could induce a new and unknown function, as for some mammalian proteins such as lactalbumin. Despite possessing the lysozyme fold, this protein lacks the active site to hydrolyze the bond between NAG subunits in bacterial cell walls, and has a novel activity in lactose synthesis [20, 21]. The lack of effect on bacterial growth when the two Train A recombinant proteins were expressed does not preclude an antibacterial activity of Train A, observed for other members of the RNase A superfamily (RNase 3 [22], RNase 7 [23]).
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Several RNase activities were present in the testicular fluid and in fluids throughout the epididymis (unpublished data). Such activity was previously detected in seminal vesicles of the male bovine genital tract [8], in semen of different mammals such as the boar (for which activity was high and originated from the epididymal fluid) [9], and in the caudal epididymal region of different species such as the monkey epididymis (where this activity is repressed by testosterone) [10]. New epididymis-specific RNases such as RNase 9 and RNase 10 have also recently been identified in mice, by an "in silico" approach [24]; the latter being probably the Train A protein previously found in mice [5, 25], although the authors described this mouse RNase10 as a membrane protein according to nucleotide sequence but without evidence of the protein.
The presence of Train A protein in the epididymal fluid is the result of a steady state between secretion and reabsorption. It was very surprising that Train A was found in the epididymal lumen exclusively in the region where this protein was secreted. As previously shown by 2D electrophoresis analysis [4] and immunohistochemistry [5], Train A was entirely absent in the regions adjacent to those that secreted it (region 2). Train A did not bind to the sperm membrane, because this protein was never found on testicular or epididymal spermatozoa after incubation with radioactive-labeled Train A or in vivo by immunogold labeling.
Our immunocytochemical studies clearly showed that the Train A protein is synthesized and secreted exclusively by the epithelial cells of the first part of the epididymis where a conspicuous reaction was detected throughout the classical secretory pathway (Golgi saccules, secretory vesicles, and in the lumen). These results and the presence of a peptide signal in the sequence [5] suggest a merocrine secretion. The absence of this protein in the adjacent region indicates an intense reabsorption mechanism.
We showed by an in vitro biochemical study of its absorption, that this protein is specifically reabsorbed in the first part of the epididymis, mostly by the epithelium that secreted it. This is the first report of such an efficient secretion-absorption combination in the epididymis. Most of the other major proteins that have been studied are still present in luminal fluid in the subsequent epididymal regions, even if they were partially reabsorbed (e.g., clusterin [3]). Because absorption of Train A takes place in the secretory region, we can speculate that secretory cells are able to absorb the protein. However, our specific antisera detected all the protein present in the secretory cells (region 0), both in secretory vacuoles and in endocytotic vesicles. In order to define the intracellular sites of the endocytosis pathway we used region 2 (where the protein was not secreted) after perfusion of the secreted protein of region 0. Uptake of Train A by epididymal cells seemed to correspond to a clathrin-independent endocytotic mechanism, including tubular structures, endosomes, and multivesicular elements resembling the endosomal carrier vesicles/multivesicular bodies of the degradation pathway. However, the absence of Train A in lysosomes seems to exclude this hypothesis. Recent results have shown that molecules that are internalized independently from clathrin may be associated with microdomains on the plasma membrane [26] or functional rafts [27], and then delivered to early endosomes where efficient sorting occurs [28]. A potential role of these rafts/microdomains has been suggested in endocytosis and intracellular transport, and the possibility of differential sorting at the plasma membrane predetermines the ensuing intracellular fate of a given molecule as well as its physiological function [29].
Recent reports on the internalization of secretory ribonucleases have shown that RNase A is internalized by a dynamin-independent endocytotic pathway and reaches the cytosol probably by translocation from an endosome. This does not require an acidic environment or transport to the endoplasmic reticulum and is different from other proteins [30]. Angiogenin RNase A is processed via a specific receptor on the surface of the endothelial cell and then the protein is translocated to the nucleus [31]. However, the Train A protein does not contain any localization sequences such as the nuclear localization signal or the KFERQ pentapeptide sequence, which is required to target cytosolic proteins for lysosomal degradation, such as in cytosolic RNase A [30].
Following internalization, ribonucleases are responsible for diverse biological activities [32, 33]. For example, bovine seminal ribonuclease has antitumor, antiviral, aspermatogenic, and immunosuppressive activity [33]; angiogenin can undertake neovascularization [34]; eosinophil-derived toxin (RNase 2) develops neurotoxicity [35]; and eosinophil cationic protein (RNase 3) and RNase 7 have an antibacterial activity [22, 23]. However, RNase A itself does not have marked antitumor, antiviral, or immunosuppressive activity, and its intracellular action is related to the cleavage of cellular RNA [33].
The potential activities of Train A should involve action on the cells that secreted it. Such an autocrine pathway for a secretory protein has not been described in the epididymis to date. We could also speculate that the secreted protein has to be modified by luminal components before its reabsorption, or be bound to unknown luminal components. However, the number of isoforms of Train A in the fluid did not change with its secretion by the epithelium, or the presence or absence of spermatozoa in the lumen, or in the presence of rete testis fluid (unpublished data).
The presence of this protein only in the restricted region E0 was linked in this study to the entry of testicular fluid into the epididymis and was not dependent on systemic androgen. After efferent duct ligation, Train A mRNA and the protein disappeared completely from the epithelium and from the lumen of E0 region, respectively. Train A was the most sensitive of all proteins secreted in this epididymal region to efferent duct ligation. The other proteins such as GPX and hexosaminidase were still present, although they were reduced in expression and secretion. Furthermore, Train A mRNA and protein were found to be in region E0 only in 4-mo-old boars, when the first spermatozoa and testicular fluid enter the organ. The other proteins, GPX, and hexosaminidase were found to be already secreted into the lumen [11]. Several other proteins localized in the first part of the epididymis of different species have been reported to be regulated by testicular factors such as gamma-glutamyl transpeptidase mRNA-IV [36], polyomavirus enhancer activator 3 mRNA [37], CRES mRNA [38], and aquaporine-9 in the principal cells of the initial segment [39]. However, in contrast to the other proteins, the effect of efferent duct ligature on Train A appeared faster. Thus, Train A mRNA disappeared completely from mouse epididymal cells after less than 24 h of efferent duct ligature, indicating that the active testicular factor disappears more rapidly in mice than in the boar, the latter having a greater luminal fluid content in the epididymal caput tubule.
In all the species studied, mRNA expression of this protein was always found localized to the initial part of the epididymis [5]. However, the protein remains to be characterized in any other species because antisera obtained from synthetic peptides or from recombinant Train A protein from the pig (not shown) did not recognize epitopes in any species studied, in spite of the high degree of homology between species. This species-specificity of the antiserum is probably linked to the fact that the homologous amino acid sequences are localized inside the core of the molecule, which is involved in the conserved 3D structure and consequently poorly accessible. New species-specific antisera have to be raised.
In conclusion, the initial part of the epididymis is a specific region for the expression and secretion of a new form of RNase A, Train A. This protein is actively reabsorbed by the cell that secreted it, and lacks an active enzymatic site. The functions of this protein are unknown: it does not seem to interact directly with the sperm. As for other RNase family members (e.g., angiogenin), its biological activity may be expressed after its cellular reabsorption. Thus, this newly described protein, which is under testicular control, could act by an autocrine pathway. Thus, it may participate in an unknown function in the epithelial cells of this first part of the epididymis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Françoise Dacheux, UMR INRA-CNRS 6073, 37380 Nouzilly, France. FAX: 33 02 47 42 77 43; dacheux{at}tours.inra.fr
Received: 5 May 2004.
First decision: 25 May 2004.
Accepted: 7 July 2004.
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