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Department of Physiology, Institute of Biomedicine,3
Turku Graduate School of Biomedical Sciences,4 University of Turku, FIN-20520 Turku, Finland
Imperial College London,5 Faculty of Medicine, London W12 0NN, United Kingdom
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Crisp, cysteine-rich secretory protein, epididymis, EST, male reproductive tract, sperm maturation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In addition to the changes occurring in the epididymis, sperms undergo several additional processes to attain the capacity for successful fertilization. After ejaculation into the female reproductive tract, sperms undergo a capacitation reaction. Several changes occur during capacitation, including an increase in the amount of cAMP and in intracellular pH, a loss of certain sperm surface components, increased fluidity of sperm membrane on cholesterol removal [5], and phosphorylation of some proteins [6]. Thereafter, the capacitated sperm bind to zona pellucida, and an acrosome reaction is initiated. Acrosome is a sac-like structure filled with hydrolytic enzymes that are released during the acrosome reaction, and they are believed to affect the ability of sperm to penetrate into zona pellucida and the fusion of gametes. When the acrosome reaction is completed, the second step of binding occurs, leading to fusion of the spermatozoon with the ovum vitelline membrane. Gamete fusion is believed to occur through interaction of the complementary molecules localized on specific domains of the sperm and egg plasma membranes. The proteins involved in this event, however, remain largely unknown. Candidate proteins include fertilin beta (ADAM1), cyritestin (ADAM3), and CRISP1 (AEG-1, DE, ARP) [7–9].
CRISP1 was first described over two decades ago as a protein produced by rat epididymis under androgen control, and it has been shown to attach to the sperm surface in the epididymal lumen [10–12]. Later, a homologue of rat CRISP1 was discovered in human [13], mouse [14], rhesus monkey [15], and horse [16]. Studies where mouse, rat, or human gametes have been incubated in the presence of recombinant or native CRISP1 protein have shown that the protein is able to bind to complementary binding sites on the egg surface and inhibit gamete fusion [17–20]. CRISP1 did not affect the binding of sperm to the egg, suggesting a role for the protein in an event subsequent to sperm-egg binding and leading to fusion [18–20].
There exist two populations of CRISP1 proteins: one released from the sperm membrane during capacitation and the other remaining on the sperm surface and migrating to the equatorial surface during the acrosome reaction [18, 21]. It is believed that the sperm proteins involved in sperm-egg fusion are localized in the equatorial segment of acrosome-reacted sperm since sperm fuses with oolemma through this region [7].
To date, three members of the cysteine-rich secretory protein (Crisp) family have been characterized (Crisp1, Crisp2, and Crisp3), and the proteins have been shown to contain 16 conserved cysteine residues [22, 23]. Crisp2, also known as Tpx-1, is expressed in testis by haploid male germ cells [24], and the protein is thought to be involved in the adhesion of spermatogenic cells to Sertoli cells [25]. Crisp3 was first characterized as a gene expressed in male salivary glands under the regulation of androgens [14]; further studies indicated that expression was also present in pancreas and prostate and, at low levels, in epididymis, ovary, thymus, and colon [13, 26, 27]. Based on its homology with pathogenesis-related proteins from plants and on the tissue distribution of its expression, CRISP3 may be involved in the innate immune defense. Crisp3 expression has been shown to be increased in chronic pancreatitis, chronic pancreatitis-like lesions in pancreatic cancer, and prostate cancer [28, 29]. However, the possible role of Crisp3 in the development of cancer or in the pathophysiology of chronic pancreatitis has not been confirmed.
In the present study, we describe the discovery and characterization of a member of the Crisp family that is expressed exclusively in mouse epididymis, named Crisp4.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The expressed sequence tag (EST) clusters from the UniGene RIKEN epididymal EST library deposited at the National Center for Biotechnology Information (NCBI) database (http://www.ncbi.nlm.nih.gov) with more than three EST copies were explored. The peptide sequences and putative functions of the unknown genes were deduced using the Expasy Translate tool (http://us.expasy.org), Interproscan (http://www.ebi.ac.uk/Tools/), and the protein-protein blast (http://www.ncbi.nlm.nih.gov/BLAST/). Using this in silico approach, we identified an EST cluster representing an unknown gene containing the Crisp family domain. The cDNA sequence of the gene was deduced from the EST sequences and confirmed by comparing it to the mouse genomic DNA sequence available in the NCBI database (http://www.ncbi.nlm.nih.gov/genome/guide/mouse/). Primers for reverse transcriptionase-polymerase chain reaction (RT-PCR) amplification of the cDNA were designed based on the deduced sequence. The latter was also blasted against the nucleotide sequences available in the NCBI database (http://www.ncbi.nlm.nih.gov/BLAST/) to reveal possible homologous DNA sequences.
Bioinformatics
The nucleotide sequence was translated into the corresponding peptide sequence, and several bioinformatic tools were used to analyze the obtained sequence. TMHMM (http://www.cbs.dtu.dk/services/TMHMM-2.0/) was used to predict transmembrane helices in the peptide, and PROSITE (http://us.expasy.org/tools/scanprosite/) and InterProScan (http://www.ebi.ac.uk/Tools/) were used to predict various protein patterns and profiles. PSORT II (http://psort.nibb.ac.jp) was used to predict protein-sorting signals and intracellular localization. Hoverprot (http://pbil.univ-lyon1.fr/databases/hovergen.html) was used to generate a phylogenetic tree of the protein family.
Mouse Models and RNA Extraction
Wild-type (WT) FVB/N male mice were used throughout the study, and all mice were handled in accordance with the institutional animal care policies of the University of Turku (Turku, Finland). The mice were specific pathogen-free and were fed with complete pelleted chow and tap water ad libitum in a room with controlled light (12L:12D) and temperature (21 ± 1°C). To analyze the tissue-specific expression of the gene, 2– 3-mo-old mice were used for RNA extraction. For these studies, the mice were killed by cervical dislocation, and selected tissues (hypothalamus, brain, heart, muscle, spleen, pancreas, liver, kidney, adrenal gland, bladder, seminal vesicle, testis, vas deferens, prostate, lung, intestine, mammary gland, and the initial segment and caput, corpus, and cauda epididymis) were dissected out, frozen in liquid nitrogen, and stored at –70°C. Total RNA from the tissues was isolated using the single-step method [30]. To characterize the androgen dependency of gene expression, 24 sexually mature male mice were gonadectomized under anesthesia. Thereafter, epididymides were collected 1, 3, 7, and 14 days after gonadectomy (4 mice/ group). Furthermore, two additional groups of male mice were treated with a supraphysiological dose of testosterone for 7 and 14 days (4 mice/group), starting immediately after gonadectomy. The testosterone replacement therapy was administered by inserting a silastic tube subcutaneously into the dorsal region of the mouse. One-centimeter-long capsules were prepared of silastic tubing (inner diameter 1.98 mm, outer diameter 3.18 mm; Doe Corning, Midland, MI), filled with testosterone powder (Fluka Chemie AG, Buchs, Switzerland), and sealed at both ends with silastic adhesive (Elastosil RTV-1 Silicone Rubber, Wacker-Chemie GmbH, Munich, Germany). For the developmental study, adult and juvenile animals were killed by decapitation or cervical dislocation, and the epididymides were dissected out, frozen immediately in liquid nitrogen, and stored at –70°C.
RT-PCR
For RT-PCR, 1 µg of total RNA was treated with deoxyribonuclease I (Invitrogen, CH Groningen, The Netherlands) and reverse-transcribed using avian myeloblastosis virus reverse transcriptase (Promega Corp., Madison, WI). The gene-specific primers used to obtain the cDNA sequence of the gene were CrixFw2: 5'-TGCCTTTGTTCCTGTTGTGA-3' and CrixRev1: 5'TTAGGGTTGTTGGGAAGTGG-3'. The RT-PCR products were visualized on agarose gel electrophoresis. The amplified cDNA was cloned into a pCR 4-TOPO vector (Invitrogen).
Sequencing
The cDNA clone obtained was sequenced with an ABI PRISM 377-XL DNA Sequencer by using the ABI PRISM BigDye Terminators v3.0 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) and the M13Fw and M13Rev primers from the vector sequence.
5' and 3'-RACEs
The 5' and 3'-RACE approaches were used to obtain full-length cDNAs. Hence, the SMART RACE cDNA Amplification Kit (Clontech, Palo Alto, CA) was used according to the manufacturer's protocols. The gene-specific primers used in the RT-PCR reaction were CrixR3: 5'-TGTCACAGTACCTCGCCAAGATTC-3' and CrixF3: 5'-CCATTCTGCAAGGCTTCTTGTCTG-3'.
Quantitative Real-Time RT-PCR
The expression of the gene was analyzed by quantitative RT-PCR with the primers CrixFw2 and CrixR3 (see previous discussion). The quantitative real-time RT-PCR analysis was performed using the DNA Engine Opticon system (MJ Research, Inc., Waltham, MA) with continuous fluorescence detection. One milligram of total RNA was treated with deoxyribonuclease I (Invitrogen), and the PCR reaction was performed using the QuantiTect SYBR Green RT-PCR Kit (Qiagen, Valencia, CA). Fifty nanograms of RNA were used in each reaction, and triplicate reactions were run from all samples and standards. The expression levels were analyzed in proportion to the ß-actin level. ß-Actin was used as an endogenous control to equalize for the amounts of RNA.
In Situ Hybridization
Five-micrometer-thick paraffin sections of adult mouse epididymides were used for in situ hybridization. The cDNA of the gene in the pCR 4-TOPO vector was used as a template for sense and antisense [35S]-
UTP-labeled probes generated by in vitro transcription with T3 and T7 RNA polymerases using the Riboprobe system II kit (Promega). Pretreatment and hybridization of the sections were performed as described previously [31]. Finally, the slides were processed for liquid emulsion autoradiography using NTB-2 emulsion (Eastman Kodak, Rochester, NY) and exposed in the dark at 4°C for 1–5 days. The slides were developed using Dektol developer (Eastman Kodak) and Kodak Fixer (Eastman Kodak). The slides were stained further with hematoxylin and Hoechst 33258 (Sigma, St. Louis, MO) and mounted thereafter with DAKO fluorescent mounting medium (DAKO Corporation, Carpinteria, CA). Hybridization with a sense probe was used as a control, and no hybridization signals were detected in any of the slides analyzed.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We recently reported that the UniGene RIKEN epididymal EST library deposited at the NCBI database is a good source for identifying novel epididymal genes [31]. Using this in silico approach, we identified an EST cluster representing an unknown mouse gene containing the Crisp family domain. The cDNA of the gene was obtained through RT-PCR, which produced a single band of the predicted size. The cDNA clone obtained was sequenced, and the sequence obtained was aligned with the EST sequences and the mouse genomic sequence available at the NCBI database. The sequence of the obtained cDNA was identical with the sequences available in the public databases, except for two nucleotide variations (nucleotide 581 t/c, nucleotide 744 a/g). While analyzing the predicted sequence of the gene, we noticed that there exists a putative exon 8.8 kb upstream of the initiation site of the sequence obtained by RT-PCR. However, we were not able to amplify this possible exon by conventional RT-PCR, nor were we able to obtain the sequence by the 5'-RACE approach. 3'-RACE did not reveal any additional sequence, either. Thus, we concluded that we had obtained the sequence of the full cDNA and that the sequence of the predicted first exon is not transcribed. The sequence has been submitted to the GenBank and MGI databases, and the GenBank accession number of the sequence is AY705449.
InterProScan showed the presence of the SCP (sperm-coating protein) domain characteristic of the members of the Crisp family in the carboxy terminus of the predicted protein. In some databases the SCP domain is referred to as the Allergen V5/Tpx1 family domain, as it has also been found in venom allergens. Based on bioinformatic analyses, the protein has a signal peptide that is putatively cleaved between the amino acids 20 and 21. Furthermore, the protein presented with putative sites for N-glycosylation, tyrosine sulfation, and several different phosphorylation sites (Fig. 1).
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The comparison of nucleotide sequences revealed homology between the novel mouse cDNA and the Crisp family members of several different species. The peptide sequence comparison (Fig. 2A) showed even higher identity to the members of the Crisp protein family than was obtained by the nucleotide comparison of the cDNAs. Thus, the gene was concluded to belong to the Crisp family and was named Crisp4. The mouse (m) CRISP4 was found to have the highest homology with human (h) CRISP1. The homology between the two peptide sequences was 59%, whereas the homology between mCRISP4 and mCRISP1 was 42%. Interestingly, the identity between hCRISP1 and mCRISP1 (40%) was markedly lower than the identity between hCRISP1 and the novel mCRISP4 (59%).
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A phylogenetic tree of the members of the Crisp protein family (Fig. 3) from the Hoverprot database further reveals the homologies and evolutionary relationships between the proteins. The family consists of three groups of proteins. The first group consists of the rhesus macaque, human, and horse CRISP1 proteins and the newly identified mouse CRISP4. The second group comprises the cysteine-rich proteins of snake and frog. The third group contains most of the known mammalian Crisp proteins, including mCRISP1, -2, and -3 and hCRISP2 and -3. Based on this graph, the Crisp proteins seem to have evolved differently in different species. mCRISP4 is the only known mouse protein in the subfamily that includes hCRISP1.
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Structure and Chromosomal Localization of the Gene
When Crisp4 cDNA was aligned with the mouse genome, the gene appeared to be located on mouse chromosome 1A4. The gene consists of seven exons, the first six of which are rather short, whereas the seventh exon is 566 bp long. The exon-intron structure of the gene is very similar to that of mouse and human Crisp1 (Fig. 2B), which consist of eight exons. mCrisp1 is located on chromosome 17 in a cluster with mCrisp2 and mCrisp3, whereas hCrisp1, -2, and -3 are located on chromosome 6 (http://www.ncbi.nlm.nih.gov). Further comparison of the peptide sequences encoded by the exons reveals a high degree of structural similarity between the four mouse Crisp proteins and human CRISP1 (Fig. 2A). Almost all the intron-exon borders are located at precisely the same part of the coding sequence, but the length of the introns varies between the genes.
The novel cysteine-rich secretory protein described in this paper is not located in the well-known cluster of the other members of the Crisp family on chromosome 17. Crisp4 on chromosome 1 is located on the minus strand. On the plus strand, 350 kb away from Crisp4, is the Cocoacrisp protein (NP 113579), and Protease inhibitor 15 is located 150 kb upstream of it. These two proteins contain a SCP protein domain also shared by the members of the Crisp protein family. Interestingly, Defb41 (another cysteine-rich protein recently discovered by us, unpublished results) was found to locate 120 kb upstream of the Crisp4 gene, also on the minus strand.
Tissue-Specific Expression of the Crisp4 Gene
The expression level of Crisp4 in various tissues was studied by quantitative RT-PCR (Q-RT-PCR). The results (Fig. 4A) show that Crisp4 is highly expressed in the initial segment and caput epididymis. Some expression was detected in corpus epididymis and barely detectable levels in cauda, pancreas, liver, kidney, seminal vesicle, and prostate. The expression in cauda could not be confirmed by in situ hybridization, and the barely detectable signal in Q-RT-PCR from several tissues was concluded to be caused by the very sensitive detection method used. In situ hybridization showed, furthermore, that the gene is highly expressed in the epithelial cells of caput epididymis throughout the segments III–IV (Fig. 4B), with a lower level of expression in the initial segment and corpus epididymis.
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To determine whether the expression of Crisp4 is regulated by androgens or other factors secreted by the testis, we studied mRNA expression in different regions of the epididymis after gonadectomy (1, 4, 7, and 14 days after gonadectomy) and after testosterone replacement therapy, initiated immediately after gonadectomy (administered for 7 or 14 days). The data indicated that the expression of Crisp4 decreased rapidly after gonadectomy, and mRNA was hardly detectable 3 days later (Fig. 5A). Treatment with testosterone retained the expression close to the level seen in nongonadectomized mice. These results are in line with the finding showing that the expression of Crisp4 is initiated around the age of 30 days, concomitant with the pubertal increase in testosterone production (Fig. 5B).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Crisp4 belongs to the Crisp protein family that previously consisted of three members: Crisp1, Crisp2, and Crisp3. Crisp genes have been found in several mammals, including mouse [14, 23, 24, 32, 33], human [33], rat [10, 25], and horse [16, 32]. Mouse (m) Crisp1 has been generally concluded to represent the mouse ortholog of human (h) Crisp1, and the proteins coded by both genes have been shown to participate in the sperm-egg fusion process in vitro [19, 20]. It has been indicated, however, that the homologies between hCrisp1 and mCrisp1 cDNAs and proteins are rather low, suggesting that hCrisp1 is not the human ortholog of rodent Crisp1 but rather a novel member of the Crisp family [33]. This hypothesis is in line with the present data indicating that mCrisp4 cDNA presents with 59% identity with the hCrisp1, which is markedly higher than the homology between mCrisp1 and hCrisp1 (40%). The phylogenetic tree presented here further indicates that, of the mouse Crisp proteins, CRISP4 is most closely related to human CRISP1. However, the homology between these two is not as high as the homology between many of the other family members, indicating that Crisp4 might not be a true ortholog of human Crisp1. Nevertheless, both mCrisp1 and mCrisp4 might be functional analogs of hCrisp1.
Human Crisp1 is expressed mainly in distal efferent ductules and corpus epididymis [33], mCrisp1 is expressed in corpus and cauda epididymis [34], whereas mCrisp4 shows highest expression in caput epididymis. Comparison of the expression patterns between mouse and human epididymides is complicated, as the structure of the human epididymis differs from that of rodents. However, based on the structure and site of expression, it is possible that Crisp4 is the equivalent of hCrisp1 expressed in distal efferent ducts and that mCrisp1 is the equivalent of hCrisp1 expressed in corpus epididymis.
The mCrisp4 and mCrisp1 genes are structurally very similar. Surprisingly, mCrisp4 was found to be located on chromosome 1 and not on chromosome 17 together with the other members of the family. However, at this chromosomal region (1A4), other cysteine-rich proteins were also identified, indicating that this chromosomal area contains a novel cluster of cysteine-rich proteins.
In a position identical for human and mouse Crisp1 genes, the computer analysis identified a putative additional exon upstream from the confirmed exon 1 of Crisp4 gene. The putative exon also contains an ATG start codon in frame with the actual peptide. However, our analyses indicate that the putative exon has a very low transcription frequency or is not transcribed at all. Unused ATGs upstream of the presumed translation initiation codons are common, for example, in the Crisp2 genes of several species [32] (http://www.ncbi.nlm.nih.gov). Mostly, these additional ATGs are not in frame or have very short reading frames, except for the equine Crisp3 [32]. Furthermore, rat Crisp1 has two transcription start sites [35], and there is evidence that mCrisp1 might also have an additional exon with low transcription frequency upstream from exon 1 [36]. Thus, there is heterogeneity in the 5' ends of the transcripts and peptide sequences of various Crisp family genes. However, the putative heterogeneity does not affect the sequence of the mature peptides, produced after the signal sequence cleavage.
Spermatozoa undergo a series of changes before and during fertilization. These changes are regulated by the activation of the intracellular signaling systems that control protein phosphorylation [37]. Serine, threonine, and tyrosine phosphorylations occur in spermatozoa. Of these, tyrosine phosphorylation has been studied most widely, and it is known to be associated with capacitation, hyperactivated motility, acrosome reaction, and sperm-oocyte fusion [37, 38]. mCRISP4 does not contain tyrosine phosphorylation sites but does contain two threonine and five serine phosphorylation sites. Whether mCRISP4 is a sperm-binding protein and whether these phosphorylation sites have a role in the regulation of sperm function remains to be resolved.
The promoter region of mCrisp1 contains several androgen response elements with different binding affinities for the androgen receptor [38], and the expression of the gene is shown to be androgen dependent [39]. Similarly, the expression of mCrisp4 is also regulated by androgens. In addition, the expression of mCrisp3 in the salivary gland has been shown to diminish in animals treated with gonadotropin-releasing hormone antagonist [39]. Mouse CRISP1 and mCRISP2 have been detected as early as 22 days after birth in epididymis and testis, respectively, and a sharp increase of mCRISP1 levels was noted on Day 40 [39]. Mouse Crisp4 expression was observed to start later, by the age of 30 days, and to increase toward sexual maturity. Similarly, the expression of mCRISP-3 in salivary gland starts around Day 30 [39].
In conclusion, CRISP4 is a mouse protein especially expressed in caput epididymis and putatively secreted into the epididymal lumen. Based on its high homology with human CRISP1, the protein might be involved in the processes leading to the fertilization of an oocyte, similar to the suggested role of human and mouse CRISP1.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Matti Poutanen, Department of Physiology, Institute of Biomedicine, University of Turku, Kiinamyllynkatu 10, FIN-20520 Turku, Finland. FAX: 35 82 250 2610; matti.poutanen{at}utu.fi
Received: 9 November 2004.
First decision: 27 November 2004.
Accepted: 18 January 2005.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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