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Biology of Reproduction 67, 147-153 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regular Article

Rabbit Epididymal Secretory Proteins. III. Molecular Cloning and Characterization of the Complementary DNA for REP381

Brett Nixona,b, Christopher M. Hardya, Russell C. Jonesb, Jason B. Andrewsa, and Michael K. Holland2,a

a Pest Animal Control Cooperative Research Centre, CSIRO Sustainable Ecosystems, Canberra, ACT 2601, Australia b Department of Biological Sciences, University of Newcastle, Callaghan, NSW 2308, Australia


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
REP38 is a rabbit epididymal secretory protein of 38 kDa that has recently been shown to interact with spermatozoa. A rabbit epididymal cDNA expression library was screened with a polyclonal antibody raised against REP38. A single clone (REP38-c1) with an open reading frame encoding a polypeptide of 666 amino acids was obtained. Cleavage of a 22-amino acid N-terminal signal peptide revealed a mature protein with a theoretical molecular mass of 74.5 kDa. Northern blot analysis revealed the presence of two cross-hybridizing transcripts of approximately 1.3 and 2.5 kilobases that appear to result from alternative mRNA splicing. This finding may explain the discrepancies between the observed (38 kDa) and deduced molecular mass of REP38. Expression of both transcripts was epididymis specific and was detected only in regions 2–6. During development, the expression of REP38-c1 mRNA was initiated between 1 and 2 mo postnatum and therefore precedes the appearance of sperm within the lumen of the epididymis. These findings are in agreement with the immunohistochemical localization of the REP38 protein. Androgen deprivation induced by orchidectomy reduced REP38-c1 mRNA levels below the limit of detection, an effect that was reversed by administration of exogenous testosterone. Although REP38-c1 mRNA was detected only in the rabbit epididymis, database searches indicated homology with two rat testis specific cDNAs, KTT4 and odf2, which encode sperm outer dense fiber proteins.

epididymis, gamete biology, sperm maturation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Neutralization of proteins that perform essential functions in sperm maturation could disrupt the process sufficiently to cause infertility [1]. Such proteins are thus potential antigens for contraceptive vaccines. REP38 is a rabbit epididymal-specific secretory protein that possesses many of the characteristics of a protein involved in sperm maturation: it is secreted from an epididymal region in which maturation takes place, it interacts with spermatozoa, its synthesis is regulated by androgen, and antisera to it blocks fertilization in vitro [2, 3]. However, there has been no information on its molecular role in the maturation process.

One approach to obtaining such information is to analyze the sequence of the cDNA encoding REP38. Here, we report the cloning and sequencing of REP38-cl and an analysis of potential functional motifs within this cDNA that was conducted to examine potential roles for REP38 in the process of maturation. In addition, we examined the ontogeny, tissue distribution, and androgen regulation of REP38 mRNA. We also assessed the potential of this antigen for inclusion in an immunocontraceptive vaccine intended for use in control of wild rabbits in Australia [4].


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

Male New Zealand White rabbits were obtained from a breeding colony housed at CSIRO, Sustainable Ecosystems, and were used with the approval of the Institute's Animal Care and Ethics Committee. Animals were maintained under a light regime of 16L:8D and were supplied with food and water ad libitum. The animals used in the development study were 1, 2, 3, 4, 6, and 8 mo of age (2 animals were used for each age group). Adult animals (>6 mo old) were used to study androgen dependence of REP38. Four animals were castrated, and 2 of these were given an exogenous testosterone implant 14 days after castration as previously described [2]. Rabbits were killed by an i.v. overdose of Valabarb (Jurox Pty. Ltd., Sydney, Australia) 14 days after castration (2 animals) or 14 days after implantation (2 animals).

Tissue Collection

The testis and epididymides were removed from rabbits (2 rabbits/treatment) at necropsy and immediately dissected free of fat and connective tissue. The epididymis was subdivided in accordance with the histomorphological criteria of Jones et al. [5]. All tissues for RNA analysis were snap-frozen in liquid nitrogen and subsequently stored at -70°C prior to being processed. Tissues for immunohistochemical analysis were immediately fixed by immersion in Bouin solution for 24 h then embedded in paraffin and sectioned at 4 µm.

Immunohistochemistry

Tissue sections were mounted on poly-L-lysine-coated slides, deparaffinized in xylene, and rehydrated through a series of ethanol solutions (100%, 95%, 75%) and PBS (137 mM NaCl, 20 mM Na2HPO4·12H2O, 1.5 mM KH2PO4, 2.5 mM KCl, pH 7.4). Nonspecific antibody binding was blocked by overnight incubation in 3% BSA/PBS at 4°C. Sections were then incubated with anti-REP38 IgG and fluorescein isothiocyanate-conjugated secondary antibody as previously described [3].

Construction and Immunological Screening of a Rabbit Epididymal cDNA Expression Library

An amplified epididymal cDNA expression library was prepared with a cDNA Synthesis Kit (Pharmacia Biotech, Uppsala, Sweden) using poly(A)+ RNA isolated from the epididymis of a mature male rabbit. The library was constructed in bacteriophage lambda gt11 in accordance with the manufacturer's instructions and titered using Escherichia coli Y1090 host cells. Approximately 2 x 106 plaques were screened [6] with murine anti-REP38 IgG [3]. Protocols described by Sambrook et al. [6] were adopted for the handling, maintenance, and manipulation of bacteria and bacteriophages throughout all experiments. Positive plaques were isolated, eluted in phage diluent, and rescreened to purity. Bacteriophages from purified positive clones were propagated in liquid culture, and the cDNA inserts were characterized by restriction analysis. On the basis of these results, a single insert, REP38-c1, was subsequently purified with Nucleotrap (Macherey-Nagel, Duren, Germany) and subcloned into the Bluescript plasmid transcription vector pBSII SK- (Stratagene, La Jolla, CA).

DNA Sequencing

A series of nested deletions encompassing the entire cDNA insert were generated in both directions using the Discrete Delete (Epicentre Technologies, Madison, WI) kit and the manufacturer's protocols. Plasmid DNA from overnight cultures was isolated using a FlexiPrep (Pharmacia Biotech) kit, and the double-stranded template was sequenced using the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction kit with AmpliTaq DNA polymerase FS (Perkin Elmer Applied Biosystems, Foster City, CA) and pUC/M13 reverse primers (Promega, Madison, WI). DNA sequences were analyzed using Geneworks software (IntelliGenetics, Mountain View, CA) and a suite of programs at the ExPASy Molecular Biology World Wide Web server (Swiss Institute of Bioinformatics, http://expasy.hcuge.ch/). Searches of the GenBank and EMBL databases for related sequences were performed using the Basic Alignment Search Tool program (BLAST [7]) at the National Centre for Biotechnology.

Preparation of DNA Probes

Two different probes were constructed from the cDNA insert specific for REP38. The first probe consisting of 2460 base pairs (bp) was constructed from the full length insert, and the second probe consisting of 171 bp was prepared from the 5' region of the transcript (nucleotides 199–370). The cDNA inserts were isolated from the vector by restriction enzyme digestion, resolved on 1% agarose slab gels, and purified with Nucleotrap (Macherey-Nagel). The probes were radiolabeled by random primer extension (Megaprime DNA labeling kit; Amersham International, Buckinghamshire, United Kingdom) in the presence of {alpha}32P-dCTP (Bresatec, Adelaide, Australia). Probes were denatured by heating to 100°C for 5 min immediately before being added to the hybridization solution.

Southern Blot Analysis

Genomic DNA was isolated from liver tissue of mature male rabbits by standard procedures [6]. The DNA was subsequently quantified spectrophotometrically (at 260 nm), and 10 µg was digested with restriction endonucleases. Digested DNA was separated on 1% (w/v) flatbed agarose gels using a Tris-borate-EDTA buffer system [6]. DNA was transferred by capillary blotting for 24 h under alkaline conditions to Hybond-N membranes (Amersham). Following transfer, filters were rinsed in 2x saline sodium citrate (SSC; 0.3 M NaCl, 0.03 M trisodium citrate) and prehybridized in Rapid-hyb buffer (Amersham) at 65°C for 1 h. The full-length {alpha}32P-labeled cDNA probe was added directly to the hybridization solution, and the filters were hybridized at 65°C for 1–2 h. The filters were then rinsed in 2x SSC/0.1% SDS at room temperature and subsequently washed in 1x SSC/0.1% SDS at 65°C for 30 min, followed by several changes of 0.2x SSC/1% SDS at 65°C over 30 min. After washing, the wet membranes were wrapped in plastic and exposed to autoradiographic film (X-OMAT X-ray film; Eastman Kodak Co., Rochester, NY) with a single intensifying screen for at least 12 h at -70°C.

RNA Extraction and Northern Blot Analysis

Tissue from male and female rabbits was frozen, and total RNA was extracted in the presence of Tri-reagent (Sigma Chemical Company, St. Louis, MO) in accordance with the manufacturer's protocol. The quantity and quality of RNA was assessed spectrophotometrically at 260 and 280 nm and on 1% denaturing agarose gels run in 1x MOPS buffer (10 mM 3-[N-morpholino]propanesulfonic acid, 1.1 mM sodium acetate, 0.25 mM EDTA) containing 1.85% (v/v) formaldehyde. Aliquots of 10 µg total RNA were resolved electrophoretically, stained by the addition of ethidium bromide directly to the loading/denaturing buffer (6.5% v/v formaldehyde, 50% v/v formamide, 0.05 µg/µl ethidium bromide, in 1x MOPS), and viewed under an ultraviolet transilluminator. Ribosomal 28S (4.71 kilobases [kb]) and 18S (1.87 kb) bands were used as internal standards to estimate the relative size of cross-hybridizing RNA species. Following electrophoresis, the RNA was transferred by capillary blotting for 24 h to Hybond-N membranes (Amersham). After transfer, the filters were treated as outlined above for Southern blots, except that the filters were first hybridized with a truncated probe and exposed to autoradiographic film and then stripped by boiling in 10% SDS until no signal could be detected. Filters were subsequently hybridized with a full-length cDNA probe and reexposed to autoradiographic film.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Complementary DNA Library Screening and Clone Isolation

Immunological screening of the rabbit epididymal cDNA library revealed nine clones (REP38-c1–REP38-c9) expressing an epitope recognized by anti-REP38 IgG. Restriction endonuclease analysis of the isolated clones indicated that a single population of clones had been selected (results not shown). On this basis, a single clone (REP38-c1) was selected for further analysis. Sequencing of this clone revealed that it was apparently full length and composed of 2443 bp up to the start of the poly(A) tail (Fig. 1). Analysis of the nucleotide sequence identified a consensus polyadenylation signal AATAAA [8] 13 bp upstream of the poly(A) tail and a single large open reading frame (orf) of 1998 bp. The translation initiation codon (ATG) of this orf was preceded by a 5' untranslated region of 342 nucleotides. The orf encodes a hypothetical protein of 666 amino acids. However, the initiation codon is flanked by a hydrophobic sequence of amino acids (Fig. 1) characteristic of an N-terminal signal peptide [9]. Cleavage of the signal peptide was predicted to occur between amino acids R22 and G23 (Fig. 1 [10]). The mature protein of 644 amino acids therefore has a theoretical molecular mass of 74 539 kDa and an isoelectric point of 6.03. The predicted protein encoded by REP38-c1 contains an amino acid sequence identical over seven amino acids (G465-I471; Fig. 1) to that obtained previously by microsequencing of a peptide generated by trypsin digestion of the REP38 protein [2].



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  		FIG. 1. Complete nucleotide and deduced amino acid sequence of REP38-c1. The amino acid sequence is listed below that of the corresponding nucleotide sequence. The numbering on the right corresponds to the position of the last nucleotides and amino acids, respectively. The translational start (start) and termination (stop) codons are underlined with a single line. The polyadenylation site is underlined with a double line, the predicted cleavage site of the hydrophobic N-terminal signal peptide is denoted by a downward double arrow (), putative leucine zipper motifs are underscored with a dashed line, the tripeptide LRE is underscored with pluses, the N-linked glycosylation site is indicated by asterisks, the myristylation sites are underscored with hats (), the protein kinase C phosphorylation sites are underscored with plus/minus signs (±±±), and the casein kinase II phosphorylation sites are underscored with similarity symbols (~~~). The amino acid sequence denoted by small bullets (••••) is identical to that of the tryptic peptide sequenced previously [3]

Molecular Characterization of REP38-c1 and Homology with Known Sequences

Hydropathy analysis using the Kyte-Doolittle algorithm [11] revealed that the mature protein is predominantly hydrophilic but possesses several alternating hydrophobic regions, raising the possibility that the protein may be globular. However, prediction of secondary structure using an adaptation of the Garnier-Osguthorpe-Robson (GOR) method (GOR II [12]) revealed that the putative protein is likely to assume an overall alpha helical structure (results not shown). Prediction of the transmembrane topology of the deduced protein using the methods described by Hofmann and Stoffel [13] failed to detect any obvious membrane spanning regions, as might be expected from a secreted protein.

A PROSITE motif search [14] identified a number of potential posttranslational modifications (Fig. 1) in the predicted amino acid sequence of the mature protein, including 4 myristylation sites, 1 ASN-glycosylation site, 9 protein kinase C phosphorylation sites, and 12 casein kinase II phosphorylation sites. Further notable features of the deduced amino acid sequence were 2 domains in the C-terminus that displayed consensus leucine zipper motifs and the presence of the tripeptide motif LRE, which is reported to be involved in cell adhesion [15, 16].

Searches of the GenBank database for related sequences identified KTT4 and rat odf2 (GenBank accessions X95272 and U62821, respectively). A computer-assisted alignment of the deduced amino acid sequence of REP38-c1 with that of KTT4 and rat Odf2 indicated that these sequences shared 78% and 85% (number of exact matches/number of total possible matches) identity, respectively (Fig. 2). Complete identity extended over large regions of the sequence. Areas of major divergence between the 3 sequences were associated with the N-terminus. In this respect, the initiation codon of REP38-c1 was located 333 and 75 nucleotides upstream of that postulated for KTT4 and rat odf2, respectively. The second predominant difference between the sequences was an apparent insertion of 23 amino acids between residues E179 and D204 of KTT4 that was absent in the corresponding region of either the REP38-c1 sequence (E280) or the odf2 sequence (E205). Examination of additional REP38 clones (REP38-c2–REP38-c5) failed to detect a similar insertion. Further differences were present in the form of 20 amino acid substitutions within the orf.



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  		FIG. 2. Global alignment of the deduced amino acid sequence of REP38-c1 with that of KTT4 and rat Odf2. The numbering on the right indicates the amino acid positions of the REP38-c1, KTT4, and rat Odf2, respectively. Identical amino acids are denoted by an asterisk. Areas lacking comparable sequence are denoted by a dashed line

Developmental Expression of REP38

REP38-c1 mRNA was expressed in a highly tissue specific pattern (Fig. 3). Under the high stringency conditions adopted in the present study, both REP38-c1 probes hybridized with transcripts in the caput and corpus epididymidis, whereas no positive signals were detected in any other tissue examined. Using the full-length REP38-c1 clone, two positive hybridization signals of approximately 2.5 kb and 1.3 kb were identified in epididymal regions 2–6. Both signals were of similar intensity. By contrast, the truncated probe constructed from the 5' region of REP38-c1 hybridized only with the signal of approximately 2.5 kb.



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  		FIG. 3. Epididymis-specific expression of REP38-c1 transcripts. Denaturing agarose gels were loaded with 10 µg of total RNA extracted from rabbit testis (T), epididymis (E), ovary (O), brain (B), spleen (S), kidney (K), liver (L), and muscle (M) (A, C, and E) and testis (T), epididymal regions 2–5 (2–5), epididymal region 6 (6), epididymal region 8 (8), vas deferens (V), seminal vesicles (S), ampulla (A), and prostate (P) (B, D, and F). Resolved RNA was viewed under an ultraviolet transilluminator (A and B) to ensure equal loading and RNA integrity and subsequently transferred to nylon membranes by capillary blotting. Northern blots were hybridized with a truncated probe constructed from the 5' region of REP38-c1 (nucleotides 199–370) and exposed to autoradiographic film (C and D). Membranes were then stripped, hybridized with a full-length REP38-c1 probe, and reexposed to autoradiographic film (E and F). Two gels were run per set of samples from each of 2 animals, and representative gels and blots are depicted. The positions of the internal molecular size markers, 28S (4.71 kb) and 18S (1.87 kb) rRNA, are shown on the left

The ontogeny and androgen regulation of expression of REP38-c1 in epididymal regions 2–5 is depicted in Figure 4. Using the full-length REP38-c1 probe, both transcripts were first detected in the rabbit epididymis between 1 and 2 mo postnatum. The relative signal intensity increased dramatically between 2 and 3 mo of age, after which time the intensity remained constant until 8 mo of age (the final time point examined). Similar patterns of developmental expression were observed in region 6 of the epididymis (data not shown). We did not observe any discernible differences in the size of transcripts throughout ontogenesis. Hormonal deprivation induced by castration for a period of 14 days led to an apparent abolition of transcription so that no positive signals were detected, and the response was reversed by androgen therapy for 14 days (Fig. 4).



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  		FIG. 4. Developmental and androgen regulation of REP38-c1 transcripts. Denaturing agarose gels were loaded with 10 µg of total RNA extracted from epididymal regions 2–5 of rabbits 1, 2, 3, 4, 6, and 8 mo of age and from mature rabbits that were orchidectomized (O) or orchidectomized and administered exogenous testosterone therapy (O + T). Resolved RNA was viewed under an ultraviolet transilluminator (A). Northern blots were hybridized with a truncated probe constructed from the 5' region of REP38-c1 (nucleotides 199–370) (B) and then stripped and reprobed with a full-length REP38-c1 probe (C). Two gels were run per set of samples from each of 2 animals, and representative gels and blots are depicted. The positions of the internal molecular size markers, 28S (4.71 kb) and 18S (1.87 kb) rRNA, are shown on the left

Immunohistochemical localization of REP38 was performed on tissue sections sampled during ontogenesis to evaluate any correlation between the age-related expression of REP38-c1 mRNA and the appearance of immunologically detectable protein within the epididymis (Fig. 5). Weak staining for REP38 was first detected in the principal cells of epididymal regions 5 and 6 in peripubertal 2-mo-old rabbits. At this age, cross-reactivity was also apparent surrounding the luminal border of epididymal region 8. Between 2 and 3 mo of age, there was a dramatic increase in the number of principal cells in regions 5 and 6 that stained for REP38. This increase was accompanied by an increase in the intensity of staining within individual cells. This trend continued until 4 mo of age, by which time staining patterns equivalent to those observed in adult animals (6 mo of age) were recorded (Fig. 5).



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  		FIG. 5. Immunocytochemical localization of REP38 within the epididymis during postnatal development. Localization of anti-REP38 IgG in epididymal region 8 of rabbits 1 (A), 2 (B), 3 (C), and 8 (D) mo of age. e, Epithelium; l, lumen. Bar = 50 µm

Southern Blot Analysis

Southern blot hybridizations were performed to investigate the number of REP38 genes. Rabbit genomic DNA digested with various restriction enzymes revealed simple hybridization patterns in which only 1 or 2 major hybridizing signals were detected. This pattern is consistent with that expected from DNA fragments produced by the restriction of a single gene. Thus, REP38 appears to be represented by a single-copy gene in the rabbit genome (Fig. 6).



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  		FIG. 6. Southern blot. Rabbit genomic DNA (20 µg) was digested with the restriction endonucleases EcoRI (E), BamHI (B), SacI (S), and HindIII (H) and electrophoretically separated on 0.8% agarose gels. Resolved DNA was viewed under an ultraviolet transilluminator (A). Southern blots were hybridized with a full-length REP38-c1 probe (B). Two gels were run per set of samples from each of 2 animals, and representative gels and blots are depicted. The size (kb) of the molecular markers (L) are shown on the left


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
A full-length cDNA clone (REP38-c1) encoding REP38, a major rabbit epididymal secretory protein, was isolated from a rabbit epididymal cDNA expression library. Northern blot analysis of total RNA isolated from the epididymis revealed 2 populations of mRNA (1.3 and 2.5 kb) that hybridize to the REP38-c1 clone. The hypothesis that both messages are translated in vivo has been supported by the fact that anti-REP38 IgG recognizes 2 proteins of 77 kDa (REP77) and 38 kDa (REP38) in epididymal luminal fluid [3]. The transcript of 2.5 kb encodes a protein with a theoretical molecular mass of 74.5 kDa, similar to that of REP77. Thus, the 38-kDa isoform (REP38) probably is encoded by the 1.3-kb transcript. The 2 mRNAs are apparently transcribed at comparable levels within the epididymis. This finding highlights the differences in the amount of protein detected in Western blots of epididymal tissue and luminal fluid [3]. The reason for this variability is unclear, but the rate of protein synthesis may be influenced by posttranscriptional mechanisms that are biased toward the production of REP38. Alternatively, the detection of REP77 may be less efficient than that of REP38.

Southern blots indicated that REP38 is expressed by a single-copy gene, which suggests that the 2 mRNA transcripts (and hence the 2 immunoreactive proteins) are produced by alternative splicing. Alternative splicing occurs frequently in differentiating tissues [17]; however, this phenomenon has not been explored in the epididymis. Nevertheless, at least 3 forms of alternate splicing (alternate exon inclusion, alternate exon skipping, and alternate splice acceptor usage) have been implicated in the control of genes specifically transcribed in the male reproductive tract [18, 19]. Although elucidation of the mechanism and site of REP38 gene splicing would require isolation of REP38 genomic clones, the truncated probe did hybridize to a single transcript of 2.5 kb, which suggests that the region recognized by this probe is absent in the smaller transcript.

The two REP38-c1 mRNA transcripts were found exclusively in the epididymis. Moreover, their expression was restricted to epididymal regions 2–5 and region 6. This distribution mirrors that of the REP38 protein [2, 3]. Minor differences reflect the fact that in the present study, regions 2–5 were grouped and processed as a single entity, whereas in previous immunohistochemical studies each region was examined separately. The restricted spatial distribution of REP38 gene expression argues against the protein performing a generalized "housekeeping" role and suggests that it may be involved in other events such as sperm maturation. This assumption is consistent with the fact that rabbit spermatozoa acquire the capacity to fertilize while they are in epididymal region 6 [20, 21].

The synthesis and secretion of REP38 has previously been reported to be androgen dependent [3], a property consistent with proteins implicated in sperm maturation [22, 23]. The results of the present study provide two lines of evidence to support the importance of androgens in the regulation of REP38 synthesis. First, androgen deprivation led to a rapid decline in REP38 mRNA expression, and administration of exogenous testosterone restored mRNA to precastration levels. Second, the pattern of REP38 gene expression followed very closely the developmental profile of circulating androgens in rabbits [24, 25]. These results are consistent with androgenic regulation of REP38 being exerted primarily through control of the steady-state level of cellular mRNA. Alteration of steady-state levels of mRNA in response to androgens can be brought about by changes in transcription rate, nuclear processing of primary transcripts, or degradation rate [26–28]. Recent evidence suggests that a combination of these factors are active in the mammalian epididymis [29, 30]. However, the mechanism by which androgens control the steady-state level of REP38 mRNA remains to be elucidated.

The nucleotide and derived amino acid sequence of REP38-c1 was highly homologous with that of 2 cDNAs, KTT4 and odf2, isolated independently from rat testicular cDNA expression libraries and reported to encode outer dense fiber (odf) proteins [31, 32]. The primary differences between the 2 clones were associated with divergent 5' sequences and an apparent insertion comprising a total of 69 bp in the KTT4 sequence that was absent in either the REP38-c1 or odf2 sequence. Aside from these differences the proteins appear to have retained a high degree of structural similarity based on the conservation of a number of posttranslational and functional motifs. The conservation of such motifs raises the possibility that REP38, odf2, and KTT4 may possess a common biological function. However, this assumption is not consistent with the fact that DNA probes constructed from REP38-c1 did not hybridize to transcripts within the rabbit testis. Instead, these findings encourage speculation that the proteins represent distinct members of an emerging family of proteins that fulfill a diverse range of functions within the male reproductive tract of different species. Such a conclusion is not without precedent. For instance, closely related members of the cysteine-rich secretory protein (CRISP) family have also been implicated in a number of central reproductive processes ranging from formation of odfs [33] and the acrosome during spermatogensis [34, 35] to the formation of sperm receptors for the oolemma [36, 37] during sperm maturation.

Hydropathy and membrane topology analysis of the deduced protein identified an N-terminal hydrophobic core sequence of 22 amino acids that was interpreted as a signal peptide [9]. By virtue of their hydrophobic nature, eukaryotic signal peptides direct the translocation of nascent polypeptides across the endoplasmic reticulum membrane prior to their transport to the Golgi apparatus and eventual secretion from the cell. The presence of this signal therefore implies that the protein encoded by REP38-c1 is a secretory protein and is in agreement with earlier demonstrations that REP38 is synthesized within principal cells and secreted to the lumen of the epididymis [3]. A number of sites for putative posttranslational modifications were identified within the predicted amino acid sequence of REP38-c1, including a potential asparagine-linked glycosylation site within the N-terminal region and several protein kinase C and casein kinase II phosphorylation sites.

Another notable feature of the deduced amino acid was the presence of 2 domains in the C-terminus that displayed consensus leucine zipper motifs. These motifs are present in many gene regulatory proteins and are commonly involved in DNA binding [38, 39]. The ability to bind DNA is not a feature expected in a protein involved in sperm maturation. However, the finding that leucine zippers are also involved in other forms of protein dimerization [40, 41] raises the interesting possibility that they may facilitate binding of REP38 to the sperm surface.

REP38 harbors the tripeptide motif LRE within an extended {alpha}-helical domain, predicted to be exposed on the surface of the protein. This motif is of considerable interest because it is the adhesive site of s-laminin, a glycoprotein involved in neuronal attachment [15, 16]. By analogy, it is tempting to speculate that this motif endows REP38, and hence maturing spermatozoa, with the ability to interact with complementary receptors on the oolemma as a prelude to gamete fusion. This view is supported, but not proven, by the demonstration that REP38 migrates to a sperm domain compatible with sperm-oolemma adhesion during capacitation and that anti-REP38 IgG is capable of compromising fertilization in vitro at the level of gamete interaction [3]. Future studies will be directed toward elucidation of the biological significance of the REP38 LRE motif.


   FOOTNOTES
 
First decision: 24 May 1999.

1 This work was supported by a grant from the Australian Research Council, the Research Management Committee, University of Newcastle, and the Pest Animal Control Cooperative Research Centre, Canberra, Australia. B.N. was supported by an Australian Postgraduate Award and a Pest Animal Control Cooperative Research Centre Student Award. Back

2 Correspondence: Michael Holland, Pest Animal Control Cooperative Research Centre, CSIRO Sustainable Ecosystems, P.O. Box 284, Canberra, ACT 2601, Australia. FAX: 61 2 6242 1511;michael.holland{at}csiro.au Back

Accepted: February 22, 2002.

Received: April 8, 1999.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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B. Nixon, R. C. Jones, and M. K. Holland
Molecular and Functional Characterization of the Rabbit Epididymal Secretory Protein 52, REP52
Biol Reprod, May 1, 2008; 78(5): 910 - 920.
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