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BOR - Papers in Press, published online ahead of print February 8, 2006.
Biol Reprod 2006, 10.1095/biolreprod.105.048298
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BIOLOGY OF REPRODUCTION 74, 984–991 (2006)
DOI: 10.1095/biolreprod.105.048298
© 2006 by the Society for the Study of Reproduction, Inc.

Research Article

Identification of Rat Cysteine-Rich Secretory Protein 4 (Crisp4) as the Ortholog to Human CRISP1 and Mouse Crisp4

Michael A. Nolan 1 2, Leeying Wu 3, Hyun J. Bang 5, Scott A. Jelinsky 4, Kenneth P. Roberts 7,8, Terry T. Turner 56 , Gregory S. Kopf 2, and Daniel S. Johnston 2

Contraception,2 Women's Health and Musculoskeletal Biology, Wyeth Research, Collegeville, Pennsylvania 19426 Bioinformatics3 and Molecular Profiling Biomarker Discovery,4 Biological Technologies, Wyeth Research, Cambridge, Massachusetts 02104 Departments of Urology5 and Cell Biology,6 University of Virginia Health Science System, Charlottesville, Virginia 22908 Departments of Urologic Surgery7 and Physiology,8 University of Minnesota, Minneapolis, Minnesota 55455

ABSTRACT

Cysteine-rich secretory proteins (CRISPs) are present in a diverse population of organisms and are defined by 16 conserved cysteine residues spanning a plant pathogenesis related-1 and a C-terminal cysteine-rich domain. To date, the diversification of mammalian CRISPs is evidenced by the existence of two, three, and four paralogous genes in the rat, human, and mouse, respectively. The current study identifies a third rat Crisp paralog we term Crisp4. The gene for Crisp4 is on rat chromosome 9 within 1 Mb of both the Crisp1 and Crisp2 genes. The full-length transcript for this gene was cloned from rat epididymal RNA and encodes a protein that shares 69% and 91% similarity with human CRISP1 and mouse CRISP4, respectively. Expression of rat Crisp4 is most abundant in the epididymis, with the highest levels of transcription observed in the caput and corpus epididymis. In contrast, rat CRISP4 protein is most abundant in the corpus and cauda regions of the epididymis. Rat CRISP4 protein is also present in caudal sperm extracts, appearing as a detergent-soluble form at the predicted MWR (26 kDa). Our data identify rat Crisp4 as the true ortholog to human CRISP1 and mouse Crisp4, and demonstrate its interaction with spermatozoa in the epididymis.

epididymis, male reproductive tract, sperm maturation

INTRODUCTION

Members of the cysteine-rich secretory protein (CRISP) family of genes encode proteins conserved throughout evolution that are highly expressed in the mammalian male reproductive tract. Sixteen cysteine residues span the three characteristic regions common to all CRISP family members: a plant pathogenesis-related domain (PR-1) and a cysteine-rich domain (CRD) connected by a short hinge region [1]. Conservation of the PR-1 domain spans from genes induced in plants when responding to infection or stress to a class of venom proteins found in vespid wasps and is the major domain comprising the mammalian glioma and pathogenesis-related proteins [1, 2]. The complete hinge and CRD regions are found less frequently in nature, where the 10 conserved cysteines are present only in mammalian CRISPs and select snake and lizard venom proteins [3, 4]. One or more of the mammalian CRISP family members have been detected in the testis, epididymis, seminal vesicles, or prostate of a wide variety of mammals, suggesting that these extracellular proteins play a crucial role in male fertility [5–9].

Posttesticular maturation of spermatozoa mediated by the protein components of the epididymal lumen environment is required for the production of fertilization-competent gametes. Spermatozoa acquire the ability to both swim in a forward direction and undergo capacitation as they pass through the epididymis [10, 11]. As these maturational events occur without de novo protein synthesis in the gametes, the epididymal epithelium is a key source of proteins that bring about these changes [12]. The composition of the epididymal lumen compartment is a dynamic one, wherein epithelial cells produce a continuum of microenvironments through their regulated expression of secreted factors [13]. In rats, CRISP1 is one of the most abundantly expressed genes in the epididymis, resulting in CRISP1 protein comprising 5%–10% of the cauda epididymal fluid [14, 15].

Although CRISP proteins are abundantly expressed in the male reproductive tract, the molecular basis of their activity and their requirement for fertility remain unknown. As sperm pass through the epididymis, CRISP1 binds to spermatozoa, and most of this protein is shed during in vitro capacitation [16]. Inclusion of exogenous CRISP1 in the capacitation medium blocks rat sperm capacitation in a dose-dependent and reversible manner, suggesting the protein acts as an anticapacitation factor [17]. However, a population of CRISP1 maintains a prolonged interaction with sperm during capacitation [18, 19], and blocking of CRISP1 binding sites on rat oocytes before in vitro fertilization blocks sperm-egg fusion [20, 21]. CRISP2 proteins (originally termed Tpx-1) are synthesized directly by the developing male germ cells during spermatogenesis. This CRISP family member also remains in sperm following capacitation, and has been proposed to mediate both germ cell–Sertoli cell and sperm-egg interactions [22, 23]. CRISP3 proteins are more widely expressed throughout the body, and have been found in the seminal vesicles, salivary glands, and neutrophilic granulocytes [8, 24, 25]. Based on these expression patterns, CRISP3 family members have been suggested to play a role in innate immunity [24–26]. Although a unifying function for CRISP family members remains elusive, their physiological roles in reproduction and conservation and expression across vertebrate species has led to significant investigation of the CRISP family members in the past 20 years.

The rat Crisp1 gene was the first CRISP identified (termed Protein D/E, and later acidic epididymal glycoprotein, AEG), and together with its ortholog mouse Crisp1 (originally termed Aeg1) it is the most widely studied mammalian CRISP protein [27–30]. Although the original description of human CRISP1 (first termed AEG-Like-1) noted that it was not the ortholog of the murine (mouse and rat) Crisp1 genes, its abundant expression in the epididymis and lack of a true ortholog led to its eventual acceptance as CRISP1 [31]. This nomenclature denoting the most-similar relationship of Crisp1s remained acceptable until the recent identification of mouse Crisp4 as the gene most similar to human CRISP1 [32]. Mouse Crisp4 exhibits androgen-dependent expression that is highest in the proximal regions of the epididymis [32]. This is consistent with the expression profile of human CRISP1, wherein Western blot analyses detected protein in all regions of the epididymis, including the caput [31].

The current study identifies the rat ortholog of human CRISP1 and mouse Crisp4, and although it is the third rat Crisp gene to date, we have termed it Crisp4. This assignment was made to reflect the relatedness among the murine Crisp4 and human CRISP1 genes until the proper orthology and functionality of the mammalian Crisps are determined. The rat Crisp4 locus is in a region of chromosomal synteny with the known human CRISP genes (CRISP1, CRISP2, and CRISP3) and is located on the same chromosome as the presently known rat Crisps (Crisp1 and Crisp2). Rat Crisp4 also shows nearly exclusive expression in the epididymis, and its segment-dependent expression profile within this organ is consistent with that of mouse Crisp4 and human CRISP1. Finally, the current study identifies rat CRISP4 protein in the corpus and cauda epididymis, as well as the protein bound to caudal spermatozoa. Our findings demonstrate that the murine Crisp4 genes are the true orthologs of human CRISP1, and demonstrate that rat CRISP4 directly interacts with spermatozoa in the epididymis.

MATERIALS AND METHODS

Identification of Predicted Gene Sequence

The mouse Crisp4 gene in the Ensembl database (http://www.ensembl.org; Q9D259_MOUSE, ENSMUSG00000025774) was used to initiate a syntenic alignment comparing the contig sequences of mouse chromosome 1 to rat chromosome 9. The novel Ensembl-predicted gene ENSRNOG00000013612 was encountered as the Unique Best Reciprocal Hit in the rat genome, as was CRISP1 in the human genome.

Animal Use and RNA Extraction

Adult mice (C57BL6/J; Taconic) and rats (Sprague-Dawley; Taconic) maintained and handled in accordance with the Institutional Animal Care and Use policies at Wyeth Research were used throughout the study for tissue collection. Animals were fed ad libitum and housed in a room with a controlled light cycle (12L:12D). RNA samples were either purchased (FirstChoice Total RNA; Ambion) or extracted from tissues that were dissected and immediately placed in RNA Later (Ambion). Following homogenization in Trizol (Invitrogen) and phase separation by the addition of chloroform (1:5, v/v), an equal volume of 100% ethanol was added to the aqueous phase. These samples were immediately applied to an RNeasy column (Qiagen), and purified according to the manufacturer's protocol, including the DNase digestion step.

Microdissection of Epididymal Segments

Mouse epididymal segements were microdissected as previously described [13]. Similar techniques were employed to determine the natural boundaries defining the 19 segments of the rat epididymis. Due to the relative size of rat segment 12, it was divided into proximal (12p) and distal (12d) portions for RNA extraction and subsequent quantitiative RT-PCR (qRT-PCR) analyses. Segments from the bilateral epididymides of an individual rat were pooled to generate each sample, and three unique samples were analyzed for each gene.

5' and 3' RACE and cDNA Sequencing

The full-length cDNA sequences of mouse and rat Crisp4s were obtained from total epididymal RNA using the GeneRacer kit (Invitrogen) according to the manufacturer's protocols for 3' and 5' rapid amplification of cDNA ends (RACE). The gene-specific primers used were based on the predicted genomic sequence: RnCrisp4 3'GSP, 5'-GCC TTC AGG AGA AAC GTG TCC CCG CCA GCC AGG AAC ATG C-3'; RnCrisp4 5'GSP, 5'- GCA TGA TGC AAC ATC ACA GCC AAT GAG GTA GGA AGA GGC CCA GAC-3'; MmCrisp4 3'GSP, 5'-ACC CAT AAC GCC TTC AGG AGA AAA GTG TCC CCG CCA GCC AGG AAC-3'; MmCrisp4 5'GSP, ACA TCA CAG CCA ACG AGG TAG GTA GAG GCC CAG ACC ATC TGA G-3'. RACE products were cloned into the pCR4-TOPO vector using the TOPO TA Cloning Kit (Invitrogen) and sequenced using the BigDye Terminator v3.1 Sequencing Kit in an Applied Biosystems 3700 with the M13Fw and M13Rev primers from the vector sequence.

Bioinformatic Analyses

The obtained cDNA sequences for mouse and rat Crisp4s were aligned to genomic sequence to demonstrate the gene structure using Sequencher (Gene Codes Corp.). Multiple alignment of the cDNA sequences, percentage similarity analyses of the encoded peptides, and phylogenetic comparison of the rat, mouse, and human CRISPs were performed using the CLUSTAL W multiple alignment tool (http://www.ebi.ac.uk/clustalw).

Quantitative RT-PCR

Relative levels of gene expression were determined by quantitative real-time RT-PCR using the Qiagen Quantitect Reagents (Qiagen) and the following Taqman probe and primer sets for each gene: MmCrisp1F, 5'- TAT ACA CAC CTT ACA CTG CAG GAG AA; MmCrisp1Probe, 5'-CGT GTG CCA GTT GTC CTG ATC A; MmCrisp1R, 5'-GGT GCA TAG CCC ATC TTC ACA; MmCrisp4F, 5'-ACT CAG ATG GTC TGG GCC TCT; MmCrisp4Probe, 5'-CTA CCT CGT TGG CTG TGA TGT TGC AG; MmCrisp4R, 5'-CTG CCT TTT GTC TGC GAC AA; RnCrisp1F, 5'- ACT CAG GTT GTT TGG AAT TCA ACT T; RnCrisp1Probe, 5'-CCT GGT TGC ATG TGG AGT TGC; RnCrisp1R, 5'-CAA TGG TTG GTC AGG GCA TT; RnCrisp4F, 5'-ATT GGC TGT GAT GTT GCA TCA; RnCrisp4Probe, 5'-CCG CAG GCA AAA GGC AGC TAC GTA TC; RnCrisp4R, 5'-CCC CTC ATG GCA ATA GTG ACA. Fluorescence detection was performed on an Applied Biosystems 7900HT, and standard curves were generated using concentrations of total epididymal RNA ranging from 500 to 0.001 ng/µl. Each unknown sample was analyzed at the concentrations of 100 and 10 ng/µl, and relative expression levels were determined using the SDS2.2.1 software provided with the instrument. Relative levels of expression among the epididymal segments were determined by normalizing the level of expression within each segment to that detected in RNA extracted from the whole epididymis.

Protein Extraction and SDS-PAGE

Homogenization of rat epididymal regions (caput, corpus, and cauda) containing present spermatozoa was performed by physical disruption in 2 ml of a homogenization buffer (250 mM sucrose, 0.1 mM EDTA, 0.5 mM EGTA, and 20 mM TRIS-HCl, pH 7.8) supplemented with protease inhibitors (Protease Inhibitor Cocktail Set III; Calbiochem) followed by sonication (10 x 0.5 sec). The samples were clarified by centrifugation (15 min, 14000 rpm), and the aqueous fraction ("No Detergent Soluble") was removed. The insoluble material was resuspended in 1 ml of the homogenization buffer supplemented with 1% Triton X-100, sonicated, and clarified by centrifugation as before. After removing the aqueous phase ("1% Triton X-100 Soluble"), the insoluble material was resuspended in 0.5 ml of homogenization buffer supplemented with 0.5% SDS, sonicated, and clarified by centrifugation as before. After removing the aqueous phase ("0.5% SDS Soluble"), the insoluble material was resuspended in 0.5 ml of 1x LDS Sample Buffer (Invitrogen), heated to 95°C for 5 min, sonicated, supplemented with 3% beta-mercaptoethanol, and clarified by centrifugation. The aqueous phase ("Soluble in Sample Buffer") was removed. Rat cauda sperm samples were collected by swim-out from the cauda epididymis in 2 ml HS medium (135 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 20 mM HEPES, 5 mM glucose, 1 mM pyruvate, and 10 mM lactic acid, pH 7.4), followed by two washes in 4x volumes of fresh HS medium to remove epididymal fluid from the sperm. Samples enriched for rat testicular sperm were collected from minced seminiferous tubules by gentle shaking in ice-cold 1x PBS supplemented with protease inhibitors (Protease Inhibitor Cocktail Set III; Calbiochem) for 15 min. Tissue debris was removed by three gentle centrifugations (500 x g, 1 min each), and the enrichment of testicular spermatozoa in the sample was confirmed by light microscopy. Caudal and testicular spermatozoa cell lysis was performed in homogenization buffer by sonication (10 x 0.5 sec). The subsequent sequential detergent extraction was performed as per the protocol for the epididymal samples described above. After analysis of the No Detergent Soluble fractions using the Bradford Method (BioRAD Protein Assay Kit) 30 µg of protein was loaded into the lanes for SDS-PAGE. Proportional volumes of the detergent-soluble (0.5x for 1%Triton X-100 Soluble and 0.25x for 0.5% SDS Soluble and Soluble in Sample Buffer) fractions were loaded to account for volumes of solubilization buffer used and maintain relevant protein loading amounts. All samples were adjusted to 1x Sample Buffer, heated to 95°C for 5 min, and reduced with 3% beta-mercaptoethanol before SDS-PAGE (4%–12% Bis-Tris gels in NuPAGE MES Buffer; Invitrogen). Proteins were transferred to nitrocellulose membranes (0.2-µm pore size; Invitrogen), and the membranes were blocked in PBS with 5% (w/v) BSA for 30 min.

Polyclonal Antibody Generation and Western Blotting

Antipeptide rabbit polyclonal antibodies raised against rat CRISP1 (EEIINKHNQLRRTVSPSGSD, generated and affinity-purified as described in [33]) and CRISP4 (DEYNNCDKQVKL, custom production by Open Biosystems) were diluted 1:1000 in 1x PBS 5% BSA and incubated with the membranes for up to 10 h at 4°C. CRISP4 antibodies were affinity purified using the antigenic peptide immobilized on a HighTrap NHS-activated column (GE Healthcare/Amersham Biosciences), and specificity was confirmed by the absence of immunoreactive bands in blots probed with the preimmune serum (1:500 dilution in 1x PBS 5% BSA) from the immunized rabbit. Blots were washed 3 x 5 min in PBS with 0.05% Tween-20, followed by incubation with peroxidase-conjugated mouse anti-rabbit IgG (Calbiochem; 1:10 000 in PBS, 0.05% v/v Tween-20, 5% w/v nonfat dry milk) for 1 h. Following another 3 x 5 min wash, detection of immunoreactive proteins was performed using chemilluminescence reagents (SuperSignal; Pierce Biotechnology, Inc.) and Kodak X-Omat LS film.

RESULTS

Identification of Rat Crisp4 cDNA and Peptide Sequences

To assist in the identification of orthologous CRISP genes conserved among the rat, mouse, and human, we performed a comparison of chromosomal synteny among these species [34]. The loci for CRISP1, CRISP2, and CRISP3 are present on human chromosome 6p12.3 in a location that is syntenic with mouse chromosomes 17B3/C (Crisp1, Crisp2, and Crisp3) and 1A4 (Crisp4) and rat chromosome 9q12 (Crisp1 and Crisp2) [31, 32, 35]. Using in silico sequence analysis, we identified the novel Ensembl-predicted gene ENSRNOG00000013612 as a potential third member of the rat Crisp gene family within this syntenic region (Fig. 1A). The current rat genomic sequence (Ensembl build 3.1) contains large gaps in this region of chromosome 9. We obtained a more complete coverage through assembly of shotgun genomic sequencing fragments from the NCBI and Celera databases, although a gap remained within intron 4 of the rat Crisp4 gene (Fig. 1B).


Figure 1
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  		FIG. 1. The murine Crisp4 genes are human CRISP1 orthologs. A) Comparison of the CRISP gene family cluster on human chromosome 6 to their syntenic locations on mouse chromosomes 17 (–), 1 (=), and rat chromosome 9 ({equiv}). B) Exon structures of murine Crisp4 and human CRISP1 genes showing the predicted coding regions for each gene, including the alternate in-frame translation initiation codons (*) resulting in potential 5' extensions in the coding regions. C) Phylogenetic analysis of the known murine and human Crisp cDNAs, demonstrating murine Crisp4 genes as the orthologs to human CRISP1.

The full-length mRNA encoded by the predicted gene was obtained by RT-PCR from total RNA extracted from the rat epididymis followed by 5' and 3' RACE, and the sequence was deposited in the NCBI database as gi:DQ200907. This sequence was different than the Ensembl-predicted gene in exons 3 and 4, the region in which the rat genomic sequence contains gaps. The coding sequence deduced from the full-length RACE product is 87% identical to that of mouse CRISP4, and in consultation with the Mouse Genome Informatics Nomenclature group (www.informatics.jax.org, we termed this gene rat Crisp4. Unlike the reported mRNA encoding human CRISP1, our RACE analyses identified multiple translation initiation sites in-frame with the full-length cDNA for both rat and mouse Crisp4 (gi:DQ2009008). Although it remains to be determined whether translation occurs from these alternate codons, the complete transcripts reveal the similarity in gene structures among rat Crisp4, mouse Crisp4, and human CRISP1 (Fig. 1B). Phylogenetic analysis of the known human, mouse, and rat Crisp cDNAs also strongly suggests that rat and mouse Crisp4 are the murine orthologs of human CRISP1 (Fig. 1C). This is further supported by comparison of the percentage similarity among the 10 known mammalian CRISP proteins, wherein rat and mouse CRISP4 are the most similar to each other (91%) and to human CRISP1 (69%, Table 1).


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  		TABLE 1. Percentage of similarity of human, rat, and mouse CRISP proteins.

Epididymis-Selective Expression of Rat Crisp4

Although not a requirement in the definition of orthology, comparison of tissue-dependent expression patterns among organisms may provide insight into the functional relationships among highly similar genes. For example, the orthologous human CRISP1 and mouse Crisp4 genes are reported to be expressed almost exclusively in the epididymis, whereas the paralogous murine Crisp1 genes are also expressed in the salivary glands [9, 32, 36]. To confirm the reported human CRISP1 tissue distribution, we queried both the GeneLogic and Wyeth Research expression databases for tiling 207032_s_at on the HG-U133 Affymetrix genechip. Expression of human CRISP1 was present only in the epididymis, vas deferens, and seminal vesicles (Fig. 2A). To compare this to the pattern of rat and mouse Crisp4 tissue distribution, we used qRT-PCR to establish their relative levels of expression among 25 tissues. Rat Crisp4 expression was found to be limited to the epididymis and vas deferens (Fig. 2B), while mouse Crisp4 also was present in the seminal vesicles (Fig. 2C). For comparison to previous studies and validation of our methodology, rat and mouse Crisp1 expression were determined in the same tissue panels (Fig. 2, D and E). While Crisp1 expression is also highest in the murine epididymides, significant expression was also detected in the submandibular salivary glands. Subtle differences in tissue distribution exist between rat Crisp1 and mouse Crisp1 (see Fig. 2, D and E, prostate and vas deferens), but both showed expression in submandibular salivary glands. These tissue distribution experiments demonstrate that both mouse and rat Crisp4 are most abundantly expressed in the epididymis and are absent from the salivary glands, an expression pattern very similar to that observed for their ortholog, human CRISP1 [31].


Figure 2
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  		FIG. 2. Murine Crisp4 tissue distribution parallels human CRISP1. A) Expression profiling results of human CRISP1 as determined by Affymetrix genechip studies (n ≥ 3; source data from GeneLogic and Wyeth Research). Relative levels of rat and mouse Crisp4 (B and C) and Crisp1 (D and E) mRNA as determined by qRT-PCR analysis of total RNA isolated from 25 mouse and rat tissues (n ≥ 3 animals). BAT, Brown adipose tissue, not present in human source data; WAT, white adipose tissue.

Segment-Dependent Expression of Crisp4 in Murine Epididymides

The conserved anatomical morphology of rat and mouse epididymides allows for comparative studies of epididymal segment-dependent gene expression. Consistent with human CRISP1 being not orthologous to murine Crisp1s, the region-dependent expression in the epididymis is not conserved. CRISP1 expression is present in the proximal regions of the human epididymis (head/caput), but is not present until the distal regions in the mouse and rat (corpus/cauda) [33, 36]. To investigate the conservation of mouse and rat Crisp gene regulation within the epididymis, we compared the segment-dependent expression pattern of Crisp1 and Crisp4 in both species. A schematic representation of the segmentation of murine epididymides (10 segments in mouse [13], 19 in rat) is provided, with approximations of caput, corpus, and cauda regions noted (Fig. 3A). Crisp1 and Crisp4 genes exhibit different patterns of segment-dependent expression that are conserved between mouse and rat (Fig. 3, B and C). Crisp4 transcripts were abundant in the segments corresponding to the caput and corpus epididymis (also see Supplemental Fig. 1, available online at http://www.biolreprod.org) whereas Crisp1 had higher expression in the cauda region. Both Crisp1 and Crisp4 had similar levels of expression in the corpus (mouse segment 7 and rat segments 8–13). Segment-dependent expression of both Crisp1 and Crisp4 is conserved between mouse and rat, and these findings provide further evidence that murine Crisp4 genes are the true orthologs of human CRISP1.


Figure 3
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  		FIG. 3. Differential segment-dependent expression profiles of Crisp1 and Crisp4 mRNA in the mouse and rat epididymis. Murine epididymides are divided into naturally occurring segments schematically represented in A. Quantitative RT-PCR analysis of Crisp4 and Crisp1 expression from total RNA isolated from each of the 10 mouse (B) and 19 rat (C) segments. Expression levels are normalized to the level of expression of the gene in RNA isolated from the whole epididymis (W) from each species; data bars represent mean ± SD. n = 3.

Expression of Rat CRISP4 Protein

The segment-dependent regulation of transcription observed for rat Crisp1 and Crisp4 suggests that spermatozoa are exposed to CRISP4 protein in the lumen before they encounter CRISP1 protein. To test this hypothesis, we performed Western blot analyses on rat protein extracts sequentially solubilized from the insoluble fraction by detergents of increasing strength from the caput, corpus, or cauda regions of the epididymis (Fig. 4). The increasing abundance of rat CRISP1 protein (present at two MWR, ~28 kDa and ~26 kDa) from the proximal to distal regions of the epididymis (Fig. 4A) is consistent with the segment-dependent transcriptional profile seen in Figure 3. In contrast, the major immunoreactive CRISP4 protein (MWR ~32 kDa) expressed in the epididymis (Fig. 4B) does not parallel its segment-dependent transcriptional profile, particularly in the caput region. The consistent level of CRISP4 immunoreactivity observed when comparing the corpus and cauda regions suggests that the protein persists in the luminal compartment, even in the absence of transcription. These findings provide evidence that rat spermatozoa are exposed to both CRISP1 and CRISP4 in the lumen of the corpus and cauda epididymis.


Figure 4
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  		FIG. 4. Similar region-dependent expression profiles of CRISP1 and CRISP4 proteins in the rat epididymis. Western blot analysis of rat epididymal proteins sequentially extracted from the insoluble fraction by sonication in increasingly harsh detergents probing for either CRISP1 (CAP-A antibody) (A) or CRISP4 (B). Prior to the initial homogenization in the absence of detergent (No Det.), the epididymis was divided into the caput, corpus, and cauda regions, representing approximate segmentation as noted. Detergent solubility and proportional loading parameters of each lane are described in Materials and Methods. MWR standards are as indicated to the left of each image.

Both CRISP4 and CRISP1 display more than one immunoreactive band in the rat epididymal extracts, having differences in both gel mobility and detergent solubility (Fig. 4). To determine which of these forms are associated with mature spermatozoa, Western blotting of proteins extracted from rat caudal sperm (washed to remove epididymal fluid) was performed (Fig. 5). Multiple immunoreactive bands with varying detergent solubility were observed for both CRISP1 and CRISP4 in caudal sperm (Fig. 5, A and B). To account for nonspecific immunoreactivity of the anti-CAP-A and anti-CRISP4 antibodies, testicular spermatozoa were subjected to the same protein extraction protocol. The CAP-A antibody cross-reacts with two bands in the 26–28 kDa range of the Triton X-100 Soluble and 0.5% SDS Soluble fractions of testicular sperm (Fig. 5C). The CRISP4 antibody also cross-reacts with two bands in testicular sperm, however these are in the 0.5% SDS- and 1x sample buffer-soluble lanes at ~65 kDa (Fig. 5D). From these experiments, it appears that the CRISP1 that is present on sperm corresponds to the lowest MWR form seen in the cauda epididymal extracts (Fig. 4A, approximately 26 kDa), and is soluble in the absence of detergent (Fig. 5A). In contrast, the majority of the ~26 kDa form of rat CRISP4 present in cauda sperm requires detergent for its extraction from the insoluble material (Fig. 5B). Although a CRISP4 band at 26 kDa MWR is not apparent in the epididymal extracts (Fig. 4B), the band at ~32 kDa exhibits a similar detergent extraction pattern and may represent CRISP4 that is binding to sperm. It remains to be determined whether this discrepancy in MWR is an artifact of sample preparation or is physiologically relevant; however, it is not resolved by N-glycanase treatment of the epididymal No Detergent Soluble fraction (see Supplemental Fig. 2, available online at http://www.biolreprod.org). Finally, the lowest MWR immunoreactive CRISP4 band seen in the epididymis (~24 kDa, fully soluble in no detergent, Fig. 4B) is not present in the sperm extracts, suggesting that this form of CRISP4 either does not bind to or has a labile association with sperm.


Figure 5
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  		FIG. 5. Rat CRISP1 and CRISP4 exhibit different associations with caudal spermatozoa. Western blot analysis of caudal (A and B) and testicular (C and D) rat sperm proteins sequentially extracted from the insoluble fraction by sonication in increasingly harsh detergents probing for either CRISP1 (CAP-A antibody) (A and C) or CRISP4 (B and D). Detergent solubility and proportional loading parameters of each lane are described in Materials and Methods. MWR standards are as indicated to the left of each image.

DISCUSSION

The current study describes the identification of a novel member of the mammalian CRISP family from its in silico predicted sequence through its transcription and translation in the rat epididymis. Genomic, cDNA, and predicted amino acid sequences demonstrate that rat Crisp4 is orthologous to mouse Crisp4 and human CRISP1. The tissue-specific transcription pattern in the proximal regions of the epididymis further supports these orthologous relationships. However, this study was not an exhaustive search for all of the rat, mouse, and human CRISP family members. For this reason, we have chosen to designate this third identified rat Crisp gene as Crisp4 to maintain some degree of consistency in the nomenclature: murine Crisp4 genes are the true orthologs of human CRISP1. Until the search for more orthologous genes is completed and more is known about the molecular activity of CRISP proteins, complete revision of the current nomenclature system would be premature. In the meantime, however, we suggest that the murine Crisp4 gene products are the most appropriate model system for studying human CRISP1 functions in reproduction.

In light of the identification of the murine Crisp4 genes, the evolutionary relationships of the mouse, rat, and human Crisps need to be revisited. For example, a human ortholog of the murine Crisp1 genes remains to be identified, and it is possible that these genes are murine-specific Crisp homologs. The murine and human Crisp2 genes have more appropriate nomenclature, because the mouse, rat, and human proteins are >75% similar to one another and they appear to be solely expressed in the testis. However, the CRISP3 genes again show disparity between nomenclature and orthology. First, the mouse Crisp3 gene appears to have arisen as a gene duplication event, as it encodes a protein 80% similar to mouse CRISP1. Similarly, human CRISP3 may be a paralog arising from gene duplication, as the encoded protein is 77% similar to human CRISP2 and lacks an obvious murine ortholog. The fact that all of these genes already have at least two alternate names contributes to our hesitation in renaming the CRISP family until a thorough search for more mammalian CRISP genes is completed.

The present characterization of the rat Crisp4 gene has already provided clarification of reported inconsistencies between murine and human CRISP1 expression patterns, and may aid in determining the role of CRISPs in reproduction. Murine CRISP1 proteins are expressed in the salivary gland, a tissue in which human CRISP1 has not been observed [36, 37]. Within the epididymis, the murine CRISP1 proteins are abundantly found in the distal two thirds of the organ. However, human CRISP1 protein is abundant in the distal efferent ductules and all three regions of the epididymis [31]. Rat and mouse Crisp4s are almost identical in their transcriptional profiles, both in tissue distribution (Fig. 2) and in spatial expression within the epididymis (Fig. 3). The presence of human CRISP1 protein in the proximal regions of the epididymis is consistent with the murine Crisp4 transcriptional profiles and suggests that they may act as functional orthologs to human CRISP1. A significant hurdle to testing this hypothesis is that the specific molecular activity of mammalian CRISPs remains unknown. However, the distinct expression of both Crisp1 and Crisp4 in the murine epididymides suggests that they perform different functions, and provides a system in which direct comparisons between CRISP paralogs may be made.

The Western blotting experiments in the current study have identified multiple immunoreactive bands in epididymal and sperm extracts using the CAP-A and CRISP4 antibodies, some of which are nonspecific. By performing Western blots on testicular spermatozoa, we were able to determine which immunoreactive bands are present in these cells before they are exposed to CRISP1 and CRISP4. For example, the CRISP4 antibody detects nonspecific bands in caudal and testicular sperm (Fig. 5, B and D) that exhibit a significantly higher MWR than do CRISP family members and display detergent solubility inconsistent with secreted or membrane-associated proteins. Also of note are the distinct detergent-soluble bands present in caudal and testicular sperm using the CAP-A antibody (Fig. 5, A and C). These reveal the likely cross-reactivity with rat CRISP2, a conclusion supported by both the fact that the peptide antigen to which the CAP-A antibody was raised [33] contains significant similarity to the rat CRISP2 protein and the recent demonstration that CRISP2 in human sperm requires detergent for its extraction [38].

Although the cross-reactivity of the CAP-A antibody detracts from our ability to make conclusive comparisons between CRISP1 and CRISP4 in spermatozoal extracts, we are confident that the CAP-A antibody does not cross-react with CRISP4, and that the CRISP4 antibody does not detect CRISP1. These conclusions are supported both by the N-glycanase treatments (see Supplemental Fig. 2, available online at http://www.biolreprod.org) and the differences in MWR observed in the epididymal extracts upon reprobing of the same blot (data not shown). Thus, we were able to use these reagents to compare CRISP1 and CRISP4 proteins in epididymal extracts, revealing an interesting difference in the expression of these two gene products. CRISP1 protein expression is consistent with its transcriptional profile, with increasing expression of the protein in a distal progression through the epididymis (Fig. 4A). In contrast, posttranscriptional events appear to regulate the steady state levels of rat CRISP4, as evidenced by the low levels of protein despite high levels of transcription in the caput epididymis (Figs. 4B and 3C). It is unlikely that CRISP4 protein levels rise in the corpus from the use of an alternate transcript, because both detected transcripts for Crisp4 are expressed at the same ratio in the caput, corpus, and cauda (see Supplemental Fig. 1, available online at http://www.biolreprod.org). There are numerous potential explanations for the apparent posttranscriptional suppression of CRISP4 expression in the caput, including epitope masking specific to this region of the epididymis, and further experimentation is required to test these hypotheses. It is interesting, however, that the synthesis of CRISP1 and CRISP4 coincide with one another, an observation that may provide insight to the mechanism of posttranscriptional regulation of CRISP4 in future studies.

In conclusion, this work has identified rat Crisp4 as a novel ortholog of mouse Crisp4 and human CRISP1. Because the rat is the historical model system for studying CRISP function in reproduction, the identification and characterization of this gene and its encoded protein significantly change the context for past and future studies. Rat Crisp4 is highly expressed in the caput and corpus regions of the epididymis, although its encoded protein is not abundant until the corpus. However, this CRISP4 protein remains in the cauda epididymis and interacts with spermatozoa. Future work is required to characterize the biochemistry and activity of mammalian CRISP proteins with the aim of understanding the role of the epididymis in the production of fertilization-competent spermatozoa.

ACKNOWLEDGMENTS

The authors would like to thank David Hamilton and Lois Maltais for helpful discussions in preparation of this manuscript.

FOOTNOTES

1 Correspondence: Michael A. Nolan, Wyeth Research, Contraception, Women's Health and Musculoskeletal Biology, N3166, 500 Arcola Rd, Collegeville, PA 19426. FAX: 484 865 9367; nolanma{at}wyeth.com Back

Received: 5 October 2005.

First decision: 1 November 2005.

Accepted: 3 February 2006.

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