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Endocrinology Vol. 140, No. 3 1459-1469
Copyright © 1999 by The Endocrine Society

ARTICLES

Testes Exhibit Elevated Expression of Calcitonin Gene-Related Peptide Receptor Component Protein1

Wayne Balkan, Edward L. Oates, Guy A. Howard and Bernard A. Roos

Geriatric Research, Education, and Clinical Center, Veterans Affairs Medical Center, and the Departments of Medicine (W.B., E.L.O., G.A.H., B.A.R.), Biochemistry and Molecular Biology (G.A.H.), and Neurology (B.A.R.), University of Miami School of Medicine, Miami, Florida 33101

Address all correspondence and requests for reprints to: Wayne Balkan, Ph.D., Veterans Affairs Medical Center, 1201 NW 16th Street, Geriatric Research, Education, and Clinical Center (11GRC), Miami, Florida 33125. E-mail: wbalkan{at}mednet.med.miami.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Calcitonin gene-related peptide (CGRP) receptor component protein (RCP) is a novel protein that modulates CGRP responsiveness in a variety of cell types. Using probes based on the isolation of CGRP-RCP complementary DNA (cDNA) from a guinea pig organ of Corti cDNA library, we cloned human (h) and mouse (m) CGRP-RCP cDNAs, both of which encode 148-residue proteins that at the amino acid levels are approximately 88% identical to each other and to the 146-residue guinea pig CGRP-RCP. Northern blot analysis confirmed the presence of CGRP-RCP messenger RNA in all of the human and mouse tissues tested. In these human tissues, hCGRP-RCP messenger RNA (major band at ~3.1 kb, minor band at ~7.5 kb) was most prevalent in the testis. In the mouse, the highest abundance of CGRP-RCP RNA was clearly in the testis (major band at ~1.6 kb, minor band at ~1.1 kb). Based on this tissue distribution of RNA, we sought to identify the cells in the murine testis that contained CGRP-RCP protein. Numerous antisera generated against hCGRP-RCP, including one to recombinant hCGRP-RCP, exhibited strong immunoreactivity localized to the head region of spermatozoa. No CGRP-RCP immunoreactivity was observed in other cells at less mature stages of sperm maturation, in Sertoli or interstitial (Leydig) cells, or in human spermatozoa. Murine epididymal (mature) spermatozoa exhibited CGRP-RCP immunoreactivity identical to that of testicular spermatozoa. Spermatozoa that underwent an experimentally induced acrosome reaction (acrosomal discharge) lost their CGRP-RCP immunoreactivity. Therefore, it appears that CGRP-RCP is associated with the acrosome, suggesting that it may play an important role in reproduction.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CALCITONIN gene-related peptide (CGRP) is a 37-amino acid neuropeptide with widespread expression and a wide array of biological effects (see Refs. 1, 2, 3 for reviews), including neuromodulation (3, 4), vasodilation (5), and bone anabolism (6, 7, 8, 9). In addition, a role for CGRP in reproduction is becoming increasingly evident. CGRP influences many aspects of mammalian development (10, 11, 12, 13), affects the function of male (14, 15, 16, 17, 18) and female (19, 20, 21, 22, 23) reproductive tissues, and plays a critical role during parturition (19, 20, 21).

The major cellular response to CGRP is an increase in the levels of intracellular cAMP (1, 2, 3). This response inspired an expression-cloning strategy based on the Xenopus laevis oocyte expression system, which led to the initial identification of the CGRP receptor component protein (CGRP-RCP) by its ability to confer CGRP responsiveness to these cells (24). A similar cloning strategy was recently used to identify the receptor activity-modifying proteins (RAMPs), a family of proteins that affect the membrane presentation, glycosylation, and ligand specificity of the calcitonin receptor-like receptor (a CGRP receptor) and the endogenous oocyte CGRP receptor (25). Although the exact relationship among CGRP-RCP, CGRP receptors, and RAMPs has not been elucidated, the discovery of these two accessory proteins illustrates the complexity associated with CGRP responsiveness.

Our initial cloning of the complementary DNA (cDNA) for CGRP-RCP was from a guinea pig organ of Corti cDNA library (24). Unlike the reported CGRP receptors (26, 27) that belong to the family of seven-transmembrane-spanning, G protein-coupled receptors, or RAMPs (25), CGRP-RCP has no obvious membrane-spanning domain. Its structure suggests that rather than directly binding CGRP, this factor, in combination with the endogenous CGRP receptors present in the Xenopus oocyte (28), enables the oocytes to respond to CGRP via a stimulation of cAMP and/or an increase in protein kinase A activity (24). Additional evidence that CGRP-RCP functions in CGRP signaling derives from two observations. First, cells of the guinea pig cerebellum and cochlea that synapse with CGRP-containing neurons and presumably contain CGRP receptors also contain CGRP-RCP mRNA by in situ hybridization (24). Second, a functional assay in murine uterus established a correlation between the presence of CGRP-RCP and CGRP responsiveness (19).

When we began this work, there was little information on the tissue distribution of CGRP-RCP despite virtually every tissue being a target for CGRP (1, 2, 3). Therefore, in seeking more relevant and convenient models for studying CGRP-RCP, we cloned human and mouse versions of CGRP-RCP and ascertained their tissue distributions. We found that human (h) and mouse (m) CGRP-RCP are highly homologous to each other and to the guinea pig (gp) CGRP-RCP at the amino acid level and that they were expressed in all tissues examined. Particularly striking were the high levels of expression in murine testis, where our CGRP-RCP antisera reacted strongly with the head region of spermatozoa, specifically in the acrosome. Although the roles of CGRP-RCP and CGRP in the functioning of spermatozoa are not understood, the high concentration of CGRP-RCP in acrosomes of murine spermatozoa suggests that this protein plays an important function in reproduction.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Screening of cDNA libraries
Cloning of hCGRP-RCP cDNA. A human cerebellum (CLONTECH {lambda}-DR2, CLONTECH Laboratories, Inc., Palo Alto, CA) library was screened with a gpCGRP-RCP cDNA probe of 594 bp encompassing the entire CGRP-RCP-coding region. The probe was synthesized by PCR using the following primers: 5'-GCGGGATCCGGTGGCAGAGCGTGAC-3' and 5'-GGCGAATTCAGGCAGTTGGGATTGAGGC-3' (24). Plaques were adsorbed onto six DuPont colony/plaque-screening filters (DuPont, Wilmington, DE). Hybridization conditions were 65 C in 3.5% SDS, 0.5 M sodium phosphate (pH 7.2), 1.0 mM EDTA, 0.5% BSA, and 200 µg/ml denatured salmon sperm DNA. Final washes were performed at 50 C in 1 x SET (150 mM NaCl, 2 mM EDTA, 30 mM Tris, pH 8.0) and 0.1% SDS. This and all other hybridizations were carried out in a Techne HB-1 hybridization oven (Techne, Princeton, NJ), and Kodak AR film (Eastman Kodak Co., Rochester, MN) was used for all autoradiograms. Five clones (hCGRP-RCP-1 to -5; Fig. 1AGo) were plaque purified by rescreening, rescued as pDR2 (CLONTECH Laboratories, Inc.) plasmids, and sequenced by an automated fluorescent-dideoxy (29) technique using vector insert-flanking pDR2 forward and reverse primers (CLONTECH Laboratories, Inc.) and deduced internal primers. All clones have an XbaI site at their 3'-ends that must be present in the gene because XbaI linkers and XbaI digestion were used in the production of the cDNA library (CLONTECH Laboratories, Inc.). All subsequent work with hCGRP-RCP was carried out using clone hCGRP-RCP-3 (GenBank accession no. U51134) because it encodes a full-length (148-residue) CGRP-RCP.



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  		Figure 1. Schematic diagram of the cDNA clones isolated. A, The five hCGRP-RCP clones. B, The three mCGRP-RCP clones. Open and closed boxes represent putative introns and exons, respectively. Numbers above the clones represent codons. In hCGRP-RCP-2 the numbers beneath ASL represent the number of the exon (intron shaded) of the ASL gene included in this CGRP-RCP clone.

 
Cloning of murine CGRP-RCP cDNA. Initially we screened a mouse brain {lambda}gt11 cDNA library (CLONTECH Laboratories, Inc.) with the PCR-amplified, full-length gpCGRP-RCP cDNA as described above. Membranes containing 1 x 106 plaques were hybridized at 42 C in 50% formamide, 1% SDS, 5 x SSC (standard saline citrate), 10% dextran sulfate, 50 x Denhardt’s solution, and 50 µg/ml denatured calf thymus DNA. The final wash was at 55 C in 0.2 x SSC and 1% SDS. From this initial screening we obtained a single cDNA clone, which was subcloned into pGEM7zf(+) plasmid (Promega Corp., Madison, WI) and sequenced as described above using SP6 and T7 primers. We used a 352-bp fragment of this cDNA clone to rescreen the cDNA library and obtained two more clones from 2 x 106 plaques. These subsequent two clones were sequenced (as described above), initially using {lambda}gt11 primers and subsequently with internal primers derived from this sequencing (Fig. 1BGo).

Concurrent with our isolation of these clones, we observed that a newly deposited GenBank expressed sequence tag (EST) from a murine 13.5- and 14.5-day postcoitum (dpc) total fetal cDNA library was highly homologous to the mouse and human clones we had isolated. Plasmid DNA from this clone (GenBank accession no. W99936; American Type Culture Collection, Manassas, VA) was isolated using the Qiagen plasmid isolation kit (Qiagen, Valencia, CA), sequenced (GenBank accession no. AF118271), and subcloned into the pcDNA3 vector (Invitrogen, San Diego, CA).

Computer comparisons to genetic database sequences
The Wisconsin GCG package of programs (Wisconsin Package Versions to 9.1, Genetics Computer Group, Madison, WI) was used for DNA sequencing (FAS) and DNA and amino acid database similarity searching and motif matching (fasta, blast, motifs, profilesearch) (30). Comparisons of CGRP-RCP clones to EST clones were made from NCBI dbEST release from May 5, 1998.

RNA analysis
Human RNA. A commercially prepared Northern blot (CLONTECH Laboratories, Inc.) containing approximately 2 µg human polyadenylated [poly(A)+] RNA/lane on positively charged nylon membranes was hybridized with a probe consisting of the full-length insert of hCGRP-RCP clone 3. The probe was PCR labeled with [{alpha}-32P]deoxy-CTP, denatured, and hybridized to the blot at 42 C in 5 x SSPE (150 mM NaCl, 10 mM sodium phosphate, 1 mM EDTA, pH 7.4), 10 x Denhardt’s solution, 100 µg/ml denatured herring sperm DNA, 1.4 x SDS, and 50% formamide. The final wash of the blot was performed at 60 C in 2 x SET and 0.1% SDS. It was stripped and reprobed with a 2-kb human ß-actin cDNA supplied with the Northern blot (CLONTECH Laboratories, Inc.). Densitometry was performed by scanning on a Scanmaker III (Microtek, Redondo Beach, CA) and analyzing with the ImageCalc software package (available from Dr. T. H. van Kuppevelt, University of Nijmegen, Nijmegen, The Netherlands; 31). The densities of hCGRP-RCP and ß-actin RNA bands in each tissue were compared.

Mouse RNA. All animal work was conducted in accordance with the Guide for the Care and Use of Laboratory Animals and was approved by our institutional animal care and use committee. Mouse tissues were dissected out and immediately homogenized in Trizol reagent (Life Technologies, Gaithersburg, MD) to isolate total RNA. Thirty micrograms of total RNA were loaded per well and electrophoresed through a formaldehyde-containing 1% agarose gel. RNA was transferred to Nytran membranes (Schleicher & Schuell, Inc., Keene, NH), and prehybridization, hybridization, and washing were carried out as described by the manufacturer in a solution containing 50% formamide, 2.5 x SSPE, and 200 µg/ml herring sperm DNA at 42 C overnight. The final wash was performed at 65 C in 0.1 x SSPE and 1% SDS. Blots were probed with [{alpha}-32P]deoxy-CTP random prime (Boehringer Mannheim, Indianapolis, IN) labeled fragments of the murine CGRP-RCP cDNA generated by PCR amplification of the W99936 cDNA with primers 5'-CTGGGCAGCAGAACTTGAACGCC-3' and 5'-GGATCCGAGA-GAGGGGGTCAGGC-3' (which also adds a BamHI restriction site to the 3'-end of the amplimer), resulting in the formation of a 447-bp amplimer. Blots were stripped and reprobed with a random prime labeled, 250-bp fragment of the mouse ß-actin cDNA. The CGRP-RCP and actin blots were exposed for 48 and 16 h, respectively, and densitometry was performed as described above.

Bacterially expressed recombinant hCGRP-RCP
Full-length hCGRP-RCP was expressed and purified from Escherichia coli as a maltose-binding protein-hCGRP-RCP fusion protein (MBP-RCP) using the pMAL-c2 expression vector (32, 33) and purification system kit (New England Biolabs, Inc., Beverly, MA). Protocols were essentially as described for the kit. Briefly, hCGRP-RCP cDNA that began with the initiator methionine was synthesized by PCR from pcDNA3-subcloned, sequenced cDNA using 5'-ATGGAAGTGAAGGATGCC-3' as the sense primer and an SP6 primer (5'-GATTTAGGTGACACTATAG-3') as the reverse primer. This DNA was cut at the 3'-end with XbaI and ligated into the pMAL-c2 vector that had been treated with the restriction enzymes XmnI and XbaI. The resulting construct was introduced into E. coli (TB1 strain) by electroporation. The fidelity of the insert was confirmed by DNA sequencing. Bacteria were grown overnight, and production of the MBP-RCP fusion protein (~60-kDa) was induced by 2-h growth in medium containing 0.3 mM isopropyl-ß-D-thiogalactoside (IPTG). Bacteria were lysed in column binding buffer [CB; 20 mM Tris-Cl (pH 7.4), 200 mM NaCl, and 1 mM EDTA] containing 0.5% Tween-20 by a combination of freeze-thawing (four times) and sonication on ice. Soluble protein (14,500 x g, 10 min, 4 C) was diluted in CB, applied to a composite amylose/agarose bead affinity column (New England Biolabs, Inc.), washed with CB, and eluted with CB containing 10 mM maltose according to the manufacturer’s protocol. Synthesis and purification of recombinant CGRP-RCP were monitored by electrophoresis on Tris-glycine and 10–20% acrylamide Ready Gels (Bio-Rad Laboratories, Inc., Hercules, CA). Samples were denatured by boiling (3 min) in urea containing SDS sample buffer [8 M Ultrapure urea, 120 mM Tris-HCl (pH 6.8), 4% ß-mercaptoethanol, 2% SDS, and 1.6% saturated bromophenol blue solution]. Gels not subjected to Western blot analysis were stained with Coomassie blue.

Antisera
CGRP-RCP antisera. Three antigens were used to generate anti-CGRP-RCP polyclonal antisera in New Zealand White rabbits. For two antisera we used coupling of peptides to keyhole limpet hemocyanin with either hCGRP-RCP peptide-1 (LKEQRKESGKNKHSSGQQ; RCP-1 in Fig. 2Go) for antiserum MU57 or hCGRP-RCP peptide-2 (TLKYISKTPCRHQSPEIV; RCP-2 in Fig. 2Go) for antiserum MU59 and injected the peptides into individual rabbits (Covance Research Products, Inc., Denver, PA) using conventional methodology (13). The third antiserum (MU66) was generated by separate injections of the recombinant human MBP-hCGRP-RCP (MBP-RCP) protein and of the same protein denatured by boiling in 1.0% SDS. The human CGRP-RCP was used to generate antisera because of the high degree of homology of this protein in all mammals studied. Antibody titer was monitored by enzyme-linked immunosorbent assay (34). Ninety-six-well plates (Immulon II, Dynex Technologies, Inc., Chantilly, VA) were coated by incubation with peptide overnight at 4 C or with fusion protein (50 µl containing 10–20 ng/well peptide) in carbonate coating buffer and were blocked for 1 h at 37 C with Dulbecco’s PBS containing 0.05% Tween-20 (PBST), 0.25% BSA, and 1% normal goat serum (Life Technologies). Rabbit sera were diluted in this blocking solution and incubated in the wells (50 µl) for 1 h at 37 C. Plates were washed three times in PBST and then incubated with affinity-purified horseradish peroxidase-labeled goat antirabbit IgG (Kirkegaard & Perry Laboratories, Gaithersburg, MD) diluted 1:500 in blocking solution. After three or four washes with PBST, plates were developed with 2,2'-azino-di-[3-ethylbenzthiazoline sulfonate (6)] diammonium salt (Boehringer Mannheim), and absorptions were measured at 405 nm.



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  		Figure 2. Comparison of CGRP-RCP amino acid sequences from different species. Human (humCGRP-RCP; U51134), mouse (musCGRP-RCP; W99936), rat kidney EST (ratCGRP-RCP; aa848339), guinea pig (gpCGRP-RCP; U50188), and chicken lens EST (chkCGRP-RCP; D26313) cDNAs and CGRP-RCP-like sequences predicted from genomic DNA of C. elegans (celCGRP-RCPl; Z46935) and yeast (ystCGRP-RCPl; Z49286) are shown. Single letter abbreviations are capitalized where residues are identical in four of five vertebrate sequences. {wedge}, Location of putative introns. The number of base pairs removed from C. elegans putative introns is indicated below the sequence. Synthetic human peptides RCP-1 and RCP-2, used for the production of antisera MU57 and MU59, respectively, are shown above the sequence. The C. elegans and yeast CGRP-RCP-like sequences are 40.4% (51/126) and 33.1% (41/124) identical to the vertebrate matching consensus in overlapping regions.

 
CGRP antisera. Antiserum MU33 (13, 22, 24) was used to detect the presence of immunoreactive CGRP in mouse and rat epididymi. This antiserum is directed against the amidated carboxyl-terminal portion of CGRP. It was used at a dilution of 1:500.

MBP antiserum. The antiserum directed against bacterial MBP was included with the bacterial fusion protein expression kit (New England Biolabs, Inc.). It was diluted to concentrations of 1:500 and 1:1000 for immunohistochemistry and 1:8000 for Western blot analysis.

Western blot analysis
Bacterially expressed recombinant human MBP-RCP fusion protein previously digested with factor Xa and mouse testis extract [obtained by homogenizing individual testes in sample buffer in a Polytron (Brinkmann Corp., Westbury, NY)] was electrophoresed on 15% SDS-PAGE Ready Gel minigels (Bio-Rad Laboratories, Inc.) and electrotransferred (18 V, 16 h) to Immobilon P (Millipore Corp., Bedford, MA) membranes. The membranes were air-dried, prewet in methanol, and transferred to PBST. Blots were serially incubated at room temperature for 1 h each in Western blocking solution (PBST and 5% nonfat dry milk), primary antibodies [rabbit sera diluted 1:5000 (1:8000 for MBP antiserum) in blocking solution], or preadsorbed primary antibodies (1:5000 final dilution), and then secondary antibody (affinity-purified horseradish peroxidase-labeled goat antirabbit IgG (0.1 µg/ml; Kirkegaard & Perry Laboratories) diluted in blocking solution, followed by three or four washes in PBST after each antibody incubation. Rabbit antibodies were preadsorbed by diluting serum in blocking solution (1:200) and incubating with 25 µg of either 60-kDa MBP-RCP or 100 µg recombinant MBP (MBP2; New England Biolabs, Inc.) at room temperature for 1 h and discarding the pelleted material (14,500 x g, 10 min). The blots were developed with LumiGLO (Kirkegaard & Perry Laboratories), a luminol-based chemiluminescent horseradish peroxidase substrate, and exposed to x-ray film. Kaleidoscope prestained standards (Kaleidoscope, Bio-Rad Laboratories, Inc.) were used as protein mol wt standards.

Immunohistochemistry
Mouse testes and rat epididymi were dissected out and immediately frozen in isopentane cooled in liquid nitrogen. Six- to 10-µm sections were cut on a Bright-Hacker cryostat (Bright-Hacker Corp., Fairfield, NJ), allowed to air-dry, and stored at -20 C. Freshly ejaculated human semen was diluted in PBS, and smears were obtained. Murine testis and epididymal sections and mouse and human sperm smears were fixed in HC Tissue Fixative (Amresco, Solon, OH) for 15 min at room temperature, washed in PBS, and then incubated overnight at 4 C with a 1:500 to 1:2500 dilution of antiserum MU57, MU59, or MU66 (to assess CGRP-RCP immunoreactivity); a 1:500 dilution of antiserum MU33 (for CGRP immunoreactivity (13, 22, 24)); a 1:500 or 1:1000 dilution of MBP antiserum; or the equivalent concentration of preimmune antiserum. Sera were diluted in 1% normal swine or goat serum (Vector Laboratories, Inc., Burlingame, CA), as appropriate, in PBS containing 0.3% Triton X-100 (PBSTx). Slides were washed in PBS and incubated for 2–4 h at room temperature with either swine antirabbit fluorescein isothiocyanate (FITC)-conjugated secondary antibody (Dakopatt, Dako Corp., Carbindale, CA) diluted 1:50 in 1% normal swine serum in PBSTx or FITC-conjugated goat antirabbit secondary antibody (Vector Laboratories, Inc.) diluted 1:500 in 1% normal goat serum in PBSTx (both secondary antibodies produced identical results). Slides were washed in PBS, and then mounting medium (Vector Laboratories, Inc.) and coverslips were applied. The slides were subsequently examined on a Nikon FXA fluorescence microscope (Nikon, Melville, NY). For immunoadsorption studies, antibody MU66 was preincubated with either the MBP-RCP fusion protein or MBP2 as described above.

Acrosome reaction
Mouse epididymal sperm were dissected out and cultured in fertilization medium (35) containing 30 mg/ml BSA. To induce the acrosome reaction (acrosomal discharge), medium was supplemented with 15 µM A23187, a calcium ionophore (36) (Sigma Chemical Co., St. Louis, MO) in dimethylsulfoxide (1:150 dilution) and 2 µM CaCl2. The spermatozoa were added and left for 15–30 min at 37 C in 5% CO2. Control spermatozoa were incubated similarly in dimethylsulfoxide (without A23187) and an equivalent concentration of CaCl2. Spermatozoa were centrifuged at 3200 x g for 10 min at 4 C, resuspended in PBS, and air-dried onto slides. Slides were stained for CGRP-RCP immunoreactivity as described above, followed by a 20-min incubation with 0.5 µg/ml rhodamine-conjugated lectin derived from the peanut A. hypogaea (Sigma Chemical Co.) to assess the presence of an acrosome (37). The slides were washed in PBS, and mounting medium and coverslips were applied. The slides were then examined on a Nikon FXA fluorescence microscope.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Human and mouse CGRP-RCP cDNAs
Our screening of a human cerebellum library ({lambda}-DR2) identified five cDNAs (Fig. 1AGo) homologous to gpCGRP-RCP (24). One of these cDNAs (1048 bp; hCGRP-RCP-3; GenBank accession no. U51134) appeared to contain the entire hCGRP-RCP-coding sequence, including 61 bp of 5'-noncoding and 540 bp of 3'-noncoding sequences. The open reading frame encodes a 148-residue protein with 88% homology to the gpCGRP-RCP (Fig. 2Go). One of the other cDNA clones (hCGRP-RCP-5) appears to be incomplete at the 5'-end, whereas the other three (hCGRP-RCP-1, -2, and -4) contain insertions that reflect possible alternative RNA splicing (Fig. 1AGo). One of these clones (hCGRP-RCP-2) had 964 bp of an alternatively spliced argininosuccinate lyase (ASL) gene at its 5'-end. Otherwise, corresponding regions in these clones were identical in sequence.

We also screened a mouse {lambda}gt11 mouse brain library and identified three incomplete clones, one in each of three screens of 106 plaques (Fig. 1BGo). Clone mCGRP-RCP-1 (532 bp long) represented the 5'-most sequence, but was missing the first three codons compared with the human and guinea pig cDNAs (Fig. 2Go). In place of these codons (eight nucleotides) was a 49-bp insert whose sequence is the same as the last 49 bp of an 84-bp insert found in a mouse 19.5-dpc EST (GenBank accession no. W12651). This location is also identical to the 80-bp insert in hCGRP-RCP-1 (Fig. 1AGo). Because the insert in hCGRP-RCP-1 causes a frame shift that puts a stop codon after the fourth amino acid, the mouse insert has a stop codon instead of the fourth amino acid, and neither has a new start codon, it appears that alternative RNA splicing has occurred. Clone mCGRP-RCP-1 also contained a deletion of 95 nucleotides that begins in codon 48 and resumes in codon 81 (Fig. 1BGo), resulting in a frame-shift mutation that encodes a stop sequence in 76 nucleotides. Clone mCGRP-RCP-2 (1416 bp long) begins at codon 28 and contains the 95 nucleotides that are deleted in mCGRP-RCP-1 and 1045 bp of the 3'-untranslated region, including a putative polyadenylation site located at the extreme 3'-end of this clone. The 227-bp segment at the 5'-end of the 1787-bp clone mCGRP-RCP-3 appears to be part of an intron. Beginning at codon 79, this clone encodes exonic and 3'-untranslated sequences. Its 3'-untranslated region ends just after a second putative polyadenylation site (downstream of that in clone mCGRP-RCP-2), suggesting the possibility of alternative splicing at the 3'-end.

During the progression of this work, a search of the GenBank EST database revealed a clone (GenBank accession no. W99936) from a 14.5-dpc mouse fetal cDNA library that was highly homologous to the guinea pig (24), human, and mouse CGRP-RCP clones that we have already isolated. Our complete sequencing of this clone revealed that it was 1501 bp long. The 94 bp of 5'-untranslated sequence upstream of the initiator methionine codon included a Kozak translation initiation consensus sequence (38). This cDNA encoded an open reading frame for a protein containing 148 residues, whose sequence was approximately 88% identical to both the guinea pig (24) and human (Fig. 2Go) CGRP-RCP [and included the first three codons (MEV) based on the human and guinea pig sequences missing from clone mCGRP-RCP-1]. The 3'-untranslated region contains a putative polyadenylation signal (AATAAA). The last 100 bases of this clone, upstream of the polyadenylase addition and a stretch of 25 A nucleotides, is a B1 repetitive DNA sequence (39) that was also found in the mCGRP-RCP-2 and -3 clones. More recently, a murine CGRP-RCP cDNA was isolated from mouse uterus (19) that is identical to clone W99936 except for minor sequence differences, including 10 and 23 fewer bp at the 5'- and 3'-ends, respectively (Fig. 2Go). The 3'-untranslated regions of clones mCGRP-RCP-2 and -3 are significantly longer than those of the reported uterine (19) and W99936 clones, suggesting the possibility of multiple polyadenylation sites.

Further searches of DNA, protein, and protein functional motif databases found significant matches of these CGRP-RCPs with human EST cDNA entries from uterus; neuroepithelium; a mixture of fetal lung, testis, and B cell clones; and HeLa cell chromosome 7 libraries (GenBank accession no. aa035173, aa206208, aa91375, and aa077758, respectively); with 28 mouse cDNA EST entries from 10 libraries; with a chicken lens cDNA EST (GenBank accession no. CHKESTPCSC; D26313); and most recently from a rat kidney cDNA EST (GenBank accession no. aa848339; Table 1Go). In addition, a Caenorhabditis elegans genomic DNA entry (GenBank accession no. CEM106;Z46935) and an open reading frame in Saccharomyces cerevisiae genomic DNA (GenBank accession no. Z49286) are 40% and 33% identical, respectively, to the matching vertebrate consensus CGRP-RCP sequences (Fig. 2Go). No matches were obtained with any known protein or defined functional motif. We deduced the positions of five putative introns (Fig. 2Go) by aligning these many homologous sequences with the insertions and deletions found in three of our five human cDNA clones (hCGRP-RCP-1, -2, and -4) and in our three mouse clones and from our partial sequencing of a mouse genomic clone (data not shown).


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  		Table 1. GenBank expressed sequence Tag libraries with entries that contain significant homologies to CGRP-RCP (from NCBI dbEST release 050198)

 
Tissue distribution of human and mouse CGRP-RCP
We performed Northern blot analysis to determine the size of the CGRP-RCP messenger RNA(s) [mRNA(s)] and to assess the tissue distribution of CGRP-RCP in humans and mice. A commercially prepared Northern blot containing 2 µg poly(A)+ RNA from human tissues was probed stringently for the presence of CGRP-RCP mRNA. We observed a major hybridizing mRNA species of about 3.1 kb in length and a fainter band of about 7.4 kb (Fig. 3AGo). The blot was rehybridized with human ß-actin as a control for RNA loading. The amount of CGRP-RCP mRNA varied among the eight tissues tested. The highest level of the 3.1-kb RNA was seen in testis. Next highest levels were in prostate, ovary, small intestine, and spleen, which all contained similar amounts of CGRP-RCP mRNA, whereas thymus, colon, and polymorphic blood monocytes exhibited barely detectable levels of CGRP-RCP mRNA.



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  		Figure 3. A, Northern blot analysis of human tissues. Two micrograms of poly(A)+ RNA from human spleen, thymus, prostate, testis, ovary, small intestine (Sm Intest), colon, and polymorphic blood monocytes (PBMC) were examined for the presence of CGRP-RCP mRNA. Numbers to the left represent RNA size markers (in kilobases); numbers to the right represent the sizes of the CGRP-RCP messages (in kilobases). Top blot, hCGRP-RCP; bottom blot, ß-actin. B, Northern blot analysis of murine tissues. Thirty micrograms of total RNA from mouse brain, heart, lung, liver, kidney, spleen, testis, ovary, uterus, and skeletal muscle (Sk Muscle) were analyzed for the presence of CGRP-RCP RNA. Numbers to the left represent RNA size markers; numbers to the right represent the sizes of CGRP-RCP messages. Top blot, mCGRP-RCP; bottom, blot ß-actin.

 
In mouse tissues there was a major band of RNA at approximately 1.6 kb in Northern blots containing 30 µg total RNA (Fig. 3BGo). This size is equivalent to that reported recently for CGRP-RCP mRNA (GenBank accession no. AF028242) in mouse uterus (19). By densitometry, the amount of CGRP-RCP mRNA in mouse testis was at least 3- to 10-fold greater than that in the other mouse tissues (Fig. 3BGo). In testis we also saw a minor species at about 1.1 kb, which may reflect RNA degradation or represent a shorter CGRP-RCP mRNA due to alternative splicing or a shorter 3'-untranslated region. Such differences in RNA have been observed in the expression of the testicular versions of somatic genes (40). It is also possible that other tissues contain the 1.1-kb CGRP-RCP RNA, but that it was not detectable in these Northern blots. RT-PCR analysis of a variety of mouse tissues confirmed that mCGRP-RCP mRNA expression is widespread (data not shown). From GenBank data we calculated a frequency of 3/862,615 (1/287,538) clones for human and 29/230,000 (1/7,931) clones for mouse in the EST databases.

Production of recombinant hCGRP-RCP
As a prelude to generating antibodies to CGRP-RCP, hCGRP-RCP was synthesized in E. coli as a recombinant protein fused to the carboxyl end of MBP. SDS-PAGE analysis of the lysed bacteria shows that before induction no fusion protein is apparent (Fig. 4AGo, lane 2). After IPTG induction a major band (~18% of total protein by densitometric scanning of gels) of about 60 kDa was observed (Fig. 4AGo, lane 3). Approximately equivalent amounts of this 60-kDa protein were present in both the soluble (Fig. 4AGo, lane 4) and insoluble (Fig. 4AGo, lane 5) fractions after lysis of the bacteria. Affinity purification of the soluble material resulted in a highly purified 60-kDa fusion protein (Fig. 4AGo, lane 6) that could be specifically and fully digested with factor Xa to the approximately 40-kDa (MBP) and the approximately 20-kDa hCGRP-RCP bands (Fig. 4AGo, lane 7). Therefore, production of hCGRP-RCP as a recombinant protein fused to MBP in E. coli was apparently successful and gave high yields of pure material in a single step.



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  		Figure 4. A, Purification of recombinant hCGRP-RCP. Extracts from the steps in the process of isolation of recombinant hCGRP-RCP were examined by SDS-PAGE and Coomassie blue staining. Lane 1, Mr markers; lane 2, control bacteria; lane 3, bacteria induced to produce the MBP-RCP fusion protein by a 2-h exposure to IPTG; lane 4, soluble fraction obtained after lysis of induced bacteria; lane 5, insoluble fraction (pellet) after induction; lane 6, material from soluble fraction eluted from an amylose affinity column; lane 7, material obtained after digestion of the 60-kDa MBP-RCP fusion protein with factor Xa. B, Western blot with antiserum MU66. Thirty micrograms of mouse testis protein (lanes 1, 3, 5, 7, and 9) and 30 ng factor Xa-digested 60-kDa MBP-RCP fusion protein material (lanes 2, 4, 6, 8, and 10) were electrophoresed on a 15% SDS-PAGE minigel. Lanes 1 and 2, Preimmune serum for antiserum MU66; lanes 3 and 4, antiserum MU66; lanes 5 and 6, antiserum MU66 preadsorbed with 60-kDa MBP-RCP fusion protein; lanes 7 and 8, antiserum MU66 preadsorbed with MBP, lanes 9 and 10, MBP antiserum.

 
The 60-kDa fusion protein was isolated and used for the production of rabbit polyclonal antisera, from which we selected antiserum MU66. To test the specificity of this antiserum and to identify CGRP-RCP in testis, factor Xa-digested 60-kDa fusion protein and mouse testicular protein were subjected to SDS-PAGE and Western blot analysis using MU66, preadsorbed MU66, anti-MBP, and preimmune antisera (Fig. 4BGo, even-numbered lanes). The preimmune serum (Fig. 4Go, lane 2) served as a negative control. Antiserum MU66 stained both the 20-kDa (hCGRP-RCP) and 40-kDa (MBP) bands strongly (Fig. 4BGo, lane 4). Preadsorption of the antiserum with the 60-kDa fusion protein (Fig. 4BGo, lane 6) eliminated staining of the CGRP-RCP band, whereas preadsorption with the MBP (Fig. 4BGo, lane 8) did not. Both preadsorptions resulted in decreased staining of the MBP band. Anti-MBP antisera stained only the MBP band (Fig. 4BGo, lane 10), indicating that the CGRP-RCP band observed with antiserum MU66 is not due to cross-reactivity with anti-MBP antibodies.

Staining of mouse testicular proteins with antiserum MU66 revealed a diffuse band(s) extending from 17–20 kDa (presumably CGRP-RCP), an intense 35-kDa band, and a weaker 75-kDa band (Fig. 4BGo, lane 3). It appears that the 35- and 75-kDa bands are nonspecific, because they are dramatically reduced when antiserum MU66 is immunoadsorbed with MBP protein, whereas the staining of the diffuse band is not lost (Fig. 4BGo, lane 7). It is difficult to be sure that the diffuse approximately 20-kDa testis band is competed away by preadsorption with the 60-kDa fusion protein (Fig. 4BGo, lane 5), similar to the bacterially produced 20-kDa CGRP-RCP (Fig. 4BGo, lane 6), because of the consistently higher background found with this preadsorbed serum. With the MBP antiserum, only the 75-kDa band was stained (Fig. 4BGo, lane 9).

CGRP-RCP present at high levels in murine spermatozoa
Based on the high level of CGRP-RCP mRNA found in mouse testis, we focused our immunohistochemical studies on this tissue. Serial sections of testis were obtained and exposed to antisera derived from rabbits immunized with either peptide RCP-1 (see Fig. 2Go; antiserum MU57; Fig. 5AGo) or peptide RCP-2 (see Fig. 2Go; antiserum MU59; data not shown). These two antisera directed against peptide fragments of CGRP-RCP produced the same staining pattern as that seen with antiserum MU66, which was directed against the intact protein (Fig. 5BGo). The immunoreactivity was limited to a sickle-shaped staining pattern seen only at the apical aspect of Sertoli cells adjacent to the lumen of the seminiferous tubules. This staining pattern, apparently located in the acrosome, is indicative of the heads of murine spermatozoa. Neither other cells in the testis, including cells at less mature phases of spermatogenesis (Fig. 5Go), nor other mouse tissues (data not shown) exhibited immunoreactivity with any of the three antisera. In no case was staining observed with any of the preimmune sera (Fig. 5Go, D and E and data not shown). To verify that the observed immunoreactivity was due to the presence of CGRP-RCP in murine spermatozoa, antiserum MU66 was immunoadsorbed with MBP, which had no effect on staining (Fig. 5CGo), or with MBP-RCP fusion protein (Fig. 5FGo), which completely eliminated immunoreactivity. To verify further that this staining was not due to cross-reactivity with MBP, tissue sections were exposed to MBP antiserum. This treatment of the tissue sections produced no immunoreactivity (Fig. 5GGo).



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  		Figure 5. Immunostaining of mouse testis with CGRP-RCP antiserum. Cross-sections through a seminiferous tubule(s) of adult mouse testis were examined for the presence of CGRP-RCP immunoreactivity. A, Antiserum MU57 directed against peptide 1 of hCGRP-RCP (see Fig. 2Go); B, antiserum MU66; C, antiserum MU66 preadsorbed with MBP; D, preimmune serum for MU57; E, preimmune serum for MU66; F, antiserum MU66 preadsorbed with MBP-RCP fusion protein; G, MBP antiserum; H, antiserum MU66 staining of testis from a 30-day-old mouse (sexually immature); I, antiserum MU66 staining of testis from a 35-day-old mouse (corresponding to the initial appearance of spermatozoa in the testis). The bar = 80 µm for all panels, except B, where it represents 160 µm.

 
To confirm that the CGRP-RCP immunoreactivity was associated exclusively with spermatozoa, we examined the testes of sexually immature mice. With all three antisera, testicular CGRP-RCP immunoreactivity was observed only in mice aged 35 days and older (Fig. 5Go, H and I). This time of onset of CGRP-RCP immunoreactivity corresponds to the initial appearance of spermatozoa in the mouse testis (41).

We also examined mouse cauda epididymi to determine whether these more mature spermatozoa continued to exhibit CGRP-RCP immunoreactivity. As expected, the CGRP-RCP immunoreactivity that we observed in these spermatozoa was identical to the staining seen in spermatozoa in the murine testis (Fig. 6Go). Although human testis contains elevated levels of CGRP-RCP RNA (Fig. 3AGo), ejaculated spermatozoa failed to exhibit CGRP-RCP immunoreactivity with these hCGRP-RCP antisera (data not shown).



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  		Figure 6. Immunostaining of murine epididymis with CGRP-RCP antiserum MU66. Frozen sections of mouse epididymis were exposed to preimmune serum (A) or MU66 antiserum (B and C). The bar = 80 µm for A and B and 40 µm for C. L, Lumen; E, epithelium.

 
CGRP staining in epididymis
Leung et al. (14) showed that human and rat epididymi contain CGRP immunoreactivity located in the epithelial cells surrounding the lumen of tubules. Based on the association between CGRP-RCP and CGRP, we investigated the questions of whether mouse epididymi also contained CGRP immunoreactivity and whether we could confirm the presence of CGRP immunoreactivity in rat epididymi. As expected, the epididymi of both species stained positively for CGRP (Fig. 7Go); however, no staining was seen in the epithelium. Instead, a high level of CGRP immunoreactivity was observed in the stroma in both species (Fig. 7Go). This immunoreactive CGRP was apparently localized to nerves.



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  		Figure 7. Immunostaining of rat and mouse cauda epididymis for CGRP. Cross-sections through mouse (A–C) and rat (D) cauda epididymi. The presence of CGRP immunoreactivity was assessed by staining with CGRP antiserum MU33 (16 22 24 ) (A, B, and D) compared with staining with preimmune serum (C). Staining of rat epididymis with preimmune serum was similarly negative as in C (data not shown). Bar = 160 µm for A and C and 80 µm for B and D. L, Lumen; E, epithelium; S, stroma.

 
Acrosome-reacted spermatozoa
Based on the apparent location of CGRP-RCP immunoreactivity (Figs. 5Go and 6Go), we examined whether CGRP-RCP immunoreactivity was limited to the acrosomal region of murine spermatozoa. Thus, the acrosome reaction (acrosomal discharge) was induced by incubating murine epididymal spermatozoa in medium containing the calcium ionophore A23187 (36) (Fig. 8Go, A–C) or vehicle (Figs. 8Go, D–F, and 9), and spermatozoa were examined for CGRP-RCP immunoreactivity by FITC immunofluorescence (Fig. 8Go, B and E) and for the presence of an acrosome as indicated by intense fluorescent staining with rhodamine-conjugated lectin derived from the peanut A. hypogaea (37) (Fig. 8Go, C and F). We observed that CGRP-RCP immunoreactivity correlated with the presence of an acrosome. In the absence of acrosomal staining, no CGRP-RCP immunoreactivity was seen (Figs. 8Go and 9Go), although occasionally an acrosome-reacted sperm had some residual bright lectin staining that also costained with the CGRP-RCP antibody in the same regions (data not shown).



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  		Figure 8. Correlation between the presence of an acrosome and CGRP-RCP immunoreactivity. Murine epididymal sperm were exposed to control medium (D–F) or the calcium ionophore A23187 (A–C) to induce the acrosome reaction. Sperm were then dried onto slides (A and D are phase contrast micrographs) and assayed for CGRP-RCP immunoreactivity (B and E) and the presence of an acrosome (C and F). Bar = 80 µm for all panels. Closed arrows point to acrosome intact sperm heads. Open arrows point to acrosome-reacted sperm heads.

 


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  		Figure 9. Fluorescent double staining of murine spermatozoa. Murine epididymal sperm were stained with CGRP-RCP antiserum MU66 (FITC-conjugated secondary antibody) and with an acrosome-specific binding lectin (rhodamine-labeled peanut agglutinin) and photographed separately. Shown are the superimposed (Corel Photo-Paint) images. Bright yellow costaining is visible in intact acrosomes, but not in spontaneously acrosome-reacted sperm (arrow).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Certain receptors require the presence of additional components (receptor complex) for signal transduction to proceed in response to ligand. Examples of such receptors include type I interferon (42), ciliary neurotropic factor (43), and interleukin-6 (44). It appears that the receptor for the neuropeptide CGRP requires additional factors, such as CGRP-RCP (24) and RAMPs (25). In the X. laevis oocyte expression system, CGRP-RCP (24) or RAMP-1 (25) is required to elicit a response to CGRP, illustrating the complexity associated with CGRP signaling.

There is additional evidence that CGRP-RCP functions in CGRP signaling. The postsynaptic hair cells in the guinea pig inner ear that respond to CGRP and presumably contain CGRP receptors also contain CGRP-RCP mRNA (24). Guinea pig cerebellum, a region of the brain that contains numerous CGRP-binding sites (2), also contains CGRP-RCP mRNA. Moreover, the presence of CGRP-RCP correlates with CGRP responsiveness in mouse myometrium (19). In mice, acetylcholine is a potent inducer of uterine contractions (19, 21). CGRP is capable of inhibiting these contractions (19, 21). At parturition, CGRP loses this inhibitory effect, and this loss is correlated with a dramatic decrease in the amount of immunoreactive CGRP-RCP present in the myometrium (19). Perhaps the dramatic endocrine changes that occur at parturition influence the expression of CGRP-RCP in this tissue. Changes in some of these same hormones early in pregnancy are known to alter the expression of calcitonin (45, 46), a peptide hormone produced by alternative splicing of the same (calcitonin) gene as CGRP (47, 48). The presence of calcitonin in the uterine epithelium appears to be required for mouse embryos to implant (45, 46).

The cloning of the human, mouse (19), guinea pig (24), and rat (GenBank accession no. aa848339) CGRP-RCPs illustrates the high conservation of the protein in all of these species. It is composed of 146–148 residues, and it has no obvious homologies to other proteins in the database. The frequency of CGRP-RCP-like cDNA clones in the EST database, in our cloning of human and mouse CGRP-RCP, and in our Northern blot analyses suggests that this gene is not highly expressed in most tissues, but in the mouse is greatest in the testis. The EST database includes a large number of inserted and deleted CGRP-RCP clones compared with the number of total clones. Although small numbers of unusually spliced RNA are often found in cDNA libraries, this increased frequency for the CGRP-RCP gene suggests some important regulation of RNA splicing. It is unclear whether the human cerebellar clone (hCGRP-RCP-2) that contained an aberrantly spliced ASL cDNA at its 5'-end is an artifact. However, this result and the finding of an EST for CGRP-RCP (GenBank accession no. aa07758) in a library of human chromosome 7-specific cDNAs suggest that the hCGRP-RCP gene is located downstream of ASL on chromosome 7q21.3-q22 (49).

We observed high levels of CGRP-RCP immunoreactivity in murine spermatozoa using antisera directed against peptides within hCGRP-RCP (MU57 and MU59) and bacterially expressed MBP-RCP fusion protein (MU66). In Western blots, recombinant hCGRP-RCP ran as a single band at 20-kDa, whereas mouse testis extracts exhibited a diffuse band of approximately the same size that presumably is CGRP-RCP. Further characterization of this protein will be necessary to determine whether differences such as posttranslational modifications, proteolysis, or different antibody epitopes account for the lack of a sharp 20-kDa band as was seen in guinea pig uterine extracts (19).

We localized CGRP-RCP to the acrosome of murine spermatozoa by the observation that when these spermatozoa were induced to undergo the acrosome reaction in vitro, they no longer exhibited CGRP-RCP immunoreactivity. This loss indicates that the release or breakdown of this protein occurs upon acrosomal discharge. The acrosome is located in the anterior part of the sperm head and functions in sperm-egg interaction and penetration. The form, function, and contents of acrosomes are highly polymorphic between and sometimes within species (50). After binding of the spermatozoon to ZP3, the only one of three major glycoproteins in the zona pellucida (ZP) that possesses both sperm-binding and acrosome reaction-inducing capabilities, the acrosome reaction occurs, resulting in the release of hydrolytic enzymes and the exposure of new membrane domains (50, 51). These enzymes are thought to facilitate sperm penetration through the ZP; however, the role of CGRP-RCP in this process is not understood.

The signal transduction cascade that begins after sperm-ZP binding and that culminates in the acrosome reaction exhibits numerous similarities to receptor-mediated exocytic responses in somatic cells (50, 51), including an increase in cAMP (52), presumably due to an increase in the activity of adenyl cyclase (53). As a similar role has been proposed for CGRP-RCP based on its actions in the Xenopus oocyte expression system (24), the acrosomal location of CGRP-RCP suggests that CGRP may similarly affect the acrosome.

The elevated amounts of CGRP-RCP RNA in testis, particularly in mice, the amount of CGRP-RCP immunoreactivity in murine spermatozoa, and the action of CGRP-RCP in mediating CGRP signaling (19, 24) suggest a role for CGRP in sperm function in mice. CGRP is present in many regions of the male reproductive tract, including prostate (15), seminal vesicles (16), and epididymis (14), where it appears to function as a modulator of secretion (14, 15, 16). In response to CGRP, certain human prostate cancer cell lines exhibit an increase in cAMP (54). CGRP administered to the basal aspect of rat and human epididymal cell monolayer culture can regulate anion secretion (14), whereas administration to the apical aspect of these cells had no effect. Our observation of CGRP’s localization to the stroma of mouse and rat epididymi is consistent with this action of CGRP. CGRP may also be present in semen (55), which would place it in close association with sperm. Although the presence of CGRP in female reproductive fluid has never been confirmed, CGRP is also found in many tissues of the female reproductive tract (19, 20, 21, 22, 23), where it functions in counteracting acetylcholine-induced (19, 21) and substance P-induced (20) contractions.

Although the role of CGRP in sperm is unclear, other neuropeptides are known to affect the function of these cells. Seminal plasmin, which is homologous to neuropeptide YY, regulates calcium transport in bovine sperm and promotes the acrosome reaction in bovine spermatozoa (56). Fertilization-promoting peptide, a tripeptide structurally related to TRH, stimulates capacitation of and fertilization by both mouse and human spermatozoa (57). High concentrations of gastrin-releasing peptide (GRP) in combination with ionophore treatment enhance sperm function, including the acrosome reaction, although physiological levels of GRP have no effect (58).

There is also some evidence that calcitonin affects sperm function. Semen contains a high concentration of calcitonin (59), and human spermatozoa possess calcitonin receptors (60). Although human calcitonin has no effect on the motility of human sperm (61, 62), salmon calcitonin does inhibit motility (62). The status of CGRP receptors on spermatozoa remains to be evaluated; however, the high degree of homology between CGRP-RCPs in different species, CGRP-RCP’s documented function in vitro (24) and in vivo (19), and its expression during gestation suggest that this protein plays an important role during many different stages of life.


   Acknowledgments
 
We thank Ms. Monika Genehr and Ms. Wendy Tahuico for expert technical assistance.


   Footnotes
 
1 This work was supported in part by the Department of Veterans Affairs. Part of this work was conducted during the tenure of an Initial Investigatorship Award (to W.B.) from the American Heart Association, Florida Affiliate, Inc. Back

Received July 15, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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