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From the * Granulocyte Research Laboratory,
Department of Hematology, Copenhagen University Hospital, Rigshospitalet,
Copenhagen, Denmark; and the Departments of
Urology and Laboratory Medicine, Division of
Clinical Chemistry and
Microbiology, Malmö University Hospital,
Lund University, Sweden.
| Correspondence to: Dr Lene Udby, Granulocyte Research Laboratory 9322, Department of Hematology, Copenhagen University Hospital, Rigshospitalet, Blegdamsvej 9, 2100 Copenhagen Ø, Denmark (e-mail: L.UDBY{at}rh.dk). |
| Received for publication August 16, 2004; accepted for publication December 3, 2004. |
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Abstract |
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Key words: CRISP-1, CRISP-2, SGP28, semen, prostate, fertilization
CRISPs are characterized by a high content of cysteine residues (16 of a total of 220-230 amino acids) that form 8 intramolecular disulfide bonds (Eberspaecher et al, 1995) and a high degree of similarity between the proteins, with 40% to 80% identity in primary structure (Krätzschmar et al, 1996; Magdaleno et al, 1997). The proteins are believed to have a 2-domain structure consisting of a large amino terminal domain and a smaller, compact cysteine-rich domain in the carboxy terminus containing 10 of the 16 cysteines (Eberspaecher et al, 1995). A domain with similarity to the amino terminal CRISP domain is also found in several other proteins from plants, yeasts, nematodes, insects, and vertebrates, but they are either devoid of or have different types of carboxy terminal domains (Szyperski et al, 1998; Henriksen et al, 2001). Little is known about the function of these proteins, and a common mode of action has not been described.
The latest years of research have revealed some aspects of mammalian CRISP function, especially in reproduction. Three different CRISPs are found in humans, horses, and mice, whereas only 2 have been described in rats. They are numbered according to similarities in tissue distribution, although they are not necessarily each other's orthologs, but are probably functional counterparts (Hayashi et al, 1996). CRISP-1, also known as protein DE in rats (Brooks, 1982), acidic epididymal glycoprotein (AEG) in mice (Kasahara et al, 1995), and AEG-related protein in humans (Hayashi et al, 1996), was the first CRISP to be discovered. Expression of CRISP-1 at both the mRNA and protein level is found predominantly in the epididymis, where the protein is secreted to seminal plasma and to some extent adheres to the surface of spermatozoa (Brooks and Tiver, 1983; Hayashi et al, 1996). CRISP-1 appears to be involved in posttesticular sperm maturation and inhibition of premature sperm capacitation and is necessary for the final fusion with the oocyte membrane (Cohen et al, 2000a; Roberts et al, 2003).
CRISP-2 is a testis-specific protein (also known as testis-specific protein 1) described in humans, rats, mice, horses, and guinea pigs, and it seems to be synthesized exclusively in the developing spermatids (Mizuki et al, 1992). Some CRISP-2 is secreted and is partly responsible for the adherence between spermatids and the Sertoli cells of the testis (Maeda et al, 1999), whereas some is stored in the acrosome and released, when sperm encounters the zona pellucida of the oocyte (Foster and Gerton, 1996; O'Bryan et al, 2001).
The third and least-studied member of the mammalian CRISP family, CRISP-3, has only been detected in humans, horses, and mice, but it has a wider tissue distribution than the other CRISPs, although transcripts are primarily found in salivary glands in all 3 species (Haendler et al, 1993; Krätzschmar et al, 1996; Schambony et al, 1998). In mice, mRNA transcripts have also been detected in bone marrow (apparently confined to preB lymphocytes) (Pfisterer et al, 1996) and the lacrimal gland (Haendler et al, 1999), but no transcripts have been detected by Northern blotting in the male reproductive tract (testis, epididymis, vas deferens, and prostate) (Haendler et al, 1993, 1997). In horses, however, CRISP-3 (also known as horse seminal plasma protein 3) is a major protein in seminal plasma (about 1 mg/mL) being secreted primarily from the ampulla vas deferens and seminal vesicles (Magdaleno et al, 1997; Schambony et al, 1998). Equine CRISP-3 seems to be attached to the sperm surface and to be positively correlated with fertility (Schambony et al, 1998). Human CRISP-3 (also known as specific granule protein of 28 kd; SGP28) was originally discovered at the protein level in neutrophilic granulocytes and was also cloned from a human bone marrow cDNA library (Kjeldsen et al, 1996). It was independently cloned from a human testis cDNA library, and mRNA expression was found primarily in salivary glands and the prostate, but also at lower levels in the epididymis, thymus, colon, and ovary. However, hardly any transcripts were detected in the testis (Krätzschmar et al, 1996).
We have recently developed immunological methods for the detection and quantification of human CRISP-3 protein in tissue samples and biological fluids and have demonstrated the presence of CRISP-3 protein, not only in specific and gelatinase granules of neutrophilic granulocytes, but also in blood plasma, saliva, sweat, and seminal plasma (Udby et al, 2002a,b). The function of CRISP-3 remains to be established, but apart from a probable role in reproduction, the localization in neutrophils and the presence in exocrine secretions indicate a role in innate immunity. In line with this, CRISP-3 seems to be up-regulated in chronic inflammation of salivary glands, including pancreas (Friess et al, 2001; Tapinos et al, 2002; Liao et al, 2003). Also, recent reports indicate a role of human CRISP-3 in prostate cancer, where the CRISP-3 gene appears to be highly up-regulated in the malignant prostatic epithelium (Asmann et al, 2002; Ernst et al, 2002; Kosari et al, 2002).
Apart from the gastrointestinal tract (Liao et al, 2003), tissue distribution of human CRISP-3 has never been reported at the protein level. To elucidate the origin of seminal plasma CRISP-3 and to help understand CRISP-3 function, we decided to investigate the tissue distribution of CRISP-3 in the human male reproductive tract by immunohistochemistry (IHC) and in situ hybridization (ISH) and to further examine the protein in seminal plasma.
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Materials and Methods |
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Spermatozoa were prepared by the swim-up procedure performed according to published recommendations (WHO, 1999) using Spermwash® (Cryos, Aarhus, Denmark).
Fresh tissue specimens of human prostate, seminal vesicles, vas deferens, epididymis, and testis (n = 4 from each location) were obtained by surgery from patients undergoing transurethral resection of the prostate or transvesical prostatectomy as a result of benign enlargement of the prostate, cystoprostatectomy as a result of invasive cancer of the urinary bladder, or radical prostatectomy or orchidectomy as treatment for prostate cancer. For IHC and ISH studies, the specimens were fixed within 30 minutes after removal in Bouin fixative (for 4-18 hours) or 4% buffered paraformaldehyde (overnight). For protein extraction, frozen tissue was sonicated (Soniprep 150; Tamro MedLab AB, Mölndal, Sweden) and incubated with agitation at 4°C for 2 to 3 hours in RIPA buffer (50 mM Tris, pH 7.5; 150 mM NaCl; 1% NP 40; 1 mM Na3PO4; 50 mM NaF; 2 mM EDTA), with one added Protein Inhibitor Cocktail Tablet (Complete Mini; Roche Applied Science, Mannheim, Germany) dissolved in 1.5 mL water and with 100 mM phenylmethylsulfonyl fluoride dissolved in DMSO. Samples were centrifuged at 19 000 x g for 30 minutes at RT. The supernatants were collected and stored at -70°C. All tissues were histopathologically normal according to hematoxylin-eosin staining. The Helsinki Declaration regarding the use of human tissues was followed.
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Antibodies |
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ELISA for CRISP-3
A previously described sandwich ELISA
(Udby et al, 2002b) was used
to quantify CRISP-3 in seminal plasma, protein extracts, and in fractions
after gel filtration. In short, the samples, pretreated with 2% sodium dodecyl
sulphate (SDS) and 4 mM dithiothreitol (DTT), were added to microtiter wells
coated with the IgG fraction of an anti-CRISP-3 antiserum. The immune
complexes were detected with biotinylated anti-CRISP-3 IgG followed by avidin
peroxidase (DAKO [P0347], Glostrup, Denmark) and visualized by color reaction
with o-phenylenediamine (Kem-En-Tec, Copenhagen, Denmark) and
hydrogen peroxide. Native CRISP-3 purified from human granulocytes was used as
standard.
Deglycosylation of Seminal Plasma
A threefold diluted sample of seminal plasma was denatured and incubated
with the enzyme N-Glycanase (10 U/mL) (Genzyme, Cambridge, Mass) or
buffer, as described previously (Udby et
al, 2002b). After reduction, the samples were subjected to
SDS-polyacrylamide gel electrophoresis (SDS-PAGE) in a 14% gel followed by
immunoblotting. An anti-CRISP-3 antiserum diluted 1:500 was used as primary
antibody, and after addition of peroxidase-conjugated swine anti-rabbit
immunoglobulin (DAKO [P0217]) (diluted 1:1000), color was developed using
DAB/metal concentrate and stable peroxide substrate buffer (Pierce, Rockford,
Ill).
Gel Filtration of Seminal Plasma
Seminal plasma was diluted in 1 volume of phosphate-buffered saline (PBS),
pH 7.4. A 200-µL sample was applied to a Superose 12 HR 10/30 column using
Äkta FPLC (Amersham Biosciences, Uppsala, Sweden). The gel filtration was
run under nondenaturing conditions using PBS as buffer. Fractions of 0.5 mL
were collected and analyzed for their content of CRISP-3 using the ELISA
described above.
The Superose 12 column was calibrated with a combination of high- and low-molecular weight markers in the same buffer as described by the manufacturer (Amersham Biosciences).
Immunoblotting of Protein Extracts
Protein extracts were diluted and boiled for 5 minutes in reducing SDS-PAGE
sample buffer and subjected to SDS-PAGE in 12% gels. The amount of protein
applied for each sample was chosen to assure similar levels of CRISP-3
reactivity (4-10 ng CRISP-3, as measured by the ELISA). Proteins were
transferred onto nitrocellulose membranes at 210 mA for 1 hour. Additional
binding sites were blocked by incubation for 1 hour in blocking solution (0.5%
skim milk and 3% bovine serum albumin [BSA] in PBS with 0.05% Tween-20
[PBS-T]), followed by incubation overnight at 4°C with anti-CRISP-3 IgG
(1.5 µg/mL) in blocking solution. Membranes were washed in PBS-T and
incubated for 30 minutes at RT in peroxidase-conjugated swine anti-rabbit
immunoglobulin (DAKO [P0217]) diluted 1:1000 in PBS-T with 5% skim milk.
Immune complexes were visualized with the enhanced chemoluminescence (ECL)
Western blotting detection kit (Amersham Biosciences UK Limited, Little
Chalfont, United Kingdom).
Protein Measurements
Total protein concentration in seminal plasma samples was determined by the
method described by Bradford
(1976) following the
instructions given by the manufacturer (Bio-Rad Laboratories, Hercules,
Calif). Bovine catalase (Sigma-Aldrich Co, St Louis, Mo) in concentrations
ranging from 0.05 to 0.5 mg/mL was used as standard.
In protein extracts from tissue specimens, the concentration was measured using a bicinchoninic acid protein assay kit with BSA as standard, according to the manufacturer's instructions (kit 23225; Pierce, Rockford, Ill).
Immunohistochemistry
The alkaline phosphatase anti-alkaline phosphatase method
(Cordell et al, 1984) was used
to immunostain sections of paraffin-embedded tissue specimens. The slides were
placed in a semiautomatic diagnostic system (Ventana ES, Ventana Inc, Tucson,
Ariz), and IHC was performed using a working concentration of 3 µg/mL of
the polyclonal antibodies against CRISP-3 (described above) and
Ventana-Enhanced Alkaline Phosphatase Red Detection Kit (cat number
760-031).
As a negative control, adjacent sections were processed by replacing the primary antibody with nonimmune rabbit IgG (DAKO [X0936]) used at the same concentration as the primary anti-CRISP-3 IgGs. In addition, anti-CRISP-3 IgGs were incubated overnight with 100x molar excess of recombinant CRISP-3 to verify that they specifically recognized CRISP-3 antigen.
Preparation of Probes for In Situ Hybridization
A CRISP-3 cDNA sequence comprising nucleotide 1204-1594 (X94323 in the
Entrez nucleotide database) from the 3' untranslated region (UTR) was
PCR amplified with the primers
5'-GCGCTCGAGGGCTAAGCATCTTCAAAGACG-3' and
5'-GCGACTAGTCCTGTAAAGTTACTATGTTTCC-3' using a plasmid containg the
3' UTR (Kjeldsen et al,
1996) as template. The PCR product was digested with XhoI
and SpeI and cloned in the plasmid pBluescript II KS+ (Stratagene, La
Jolla, Calif) restricted with the same enzymes. The correctness of the insert
was assured by sequencing. The selected region of the sequence is very
specific for the CRISP-3 mRNA according to BLAST searches in the nr
database, and a riboprobe corresponding to an adjacent part of the 3'
UTR has been used by others for successful ISH of CRISP-3 mRNA in prostate
cancer (Kosari et al,
2002).
Antisense and sense riboprobes were synthesized from the plasmid (pBluescript-CRISP-3-[3'UTR]) with T7 and T3 RNA polymerase following linearization of the plasmid with KpnI or XbaI, respectively. One microgram of linearized plasmid was incubated at 37°C for 2 hours with 40 U of T7 or T3 polymerase (Roche Applied Science, Mannheim, Germany) in 20 µL of 40 mM Tris-HCl, pH 8.0; 6 mM MgCl2; 10 mM DTT; 2 mM spermidine; and also containing 2 µL of DIG RNA labeling mix (Roche Applied Science) and 40 U of RNase inhibitor (Fermentas, Vilnius, Lithuania). The reaction was terminated by addition of 2 µL of 0.5 M EDTA, pH 8.0, and the RNA was precipitated at -20°C for 2 hours after addition of 2.2 µL of 4 M LiCl and 60 µL of 96% ethanol. The precipitate was harvested by centrifugation, washed with 70% ethanol, and dried under a gentle stream of nitrogen. The riboprobes were solved in 100 µL of ultrapure water, and 1 µL of RNase inhibitor (40 U) was added. The sizes and concentrations were estimated by comparison to a low-range RNA ladder (Fermentas), following electrophoresis in a 2.5% agarose gel and staining by ethidium bromide (1 µg/mL).
In Situ Hybridization
All reagents were purchased from Sigma-Aldrich and Amersham Pharmacia
Biotech. Tissue specimens were fixed, paraffin embedded, sectioned (4 µm),
dried for 2 hours at 65°C, and mounted on SuperFrostTM plus slides
(Menzel-Gläser) under RNase free conditions. The sections were
deparaffinized in xylene, rehydrated, and processed as described
(Paju et al, 2000). Briefly,
sections were pretreated with 0.2 M HCl to abolish endogenous enzyme activity
and digested with proteinase K (20 µg/mL in 20 mM Tris-HCl, 2 mM
CaCl2, pH 7.5) for 25 minutes at 37°C. After prehybridization
with 40 µL of hybridization buffer containing 50% (vol/vol) formamide, 10
mM Tris-HCl, pH 7.6, 1x Denhardt's solution (BSA, polyvinylpyrrolidone
and Ficoll, all at 0.2 mg/mL), 2x SSC, and 0.4 µg/mL salmon sperm DNA
at 55°C for 1 hour, the slides were hybridized with 40 µL of 250 ng/mL
antisense or sense probe in hybridization buffer first for 8 minutes at
85°C and then for 16 hours at 55°C. After hybridization, the slides
were washed at a stringency of 0.1x SSC at 60°C (4 x 15
minutes) and then equilibrated in TBS (100 mM Tris-HCl, 0.4 M NaCl, pH 7.5).
For detection of hybridization signals, tissue sections were first incubated
in blocking reagent and subsequently incubated with alkaline
phosphatase-conjugated Fab fragments of anti-digoxigenin and diluted 1:500 in
TBS containing 1% NSS and 0.03% Triton X-100 for 2 hours at RT. Hybridization
signals were visualized by levamisol, 4-nitroblue tetrazolium chloride, and
5-bromo-4-chloro-3-indolyl phosphate. The color reaction was stopped after 2-8
hours and the slides were coverslipped using FaramountTM mounting medium
(DAKO). For control purposes, adjacent sections were hybridized with the
corresponding digoxigenin-labeled sense probe, and hybridization buffer was
also included as an additional negative control.
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Results |
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The concentration of CRISP-3 in seminal plasma from 20 healthy individuals was measured by ELISA. A large variation in the concentration of CRISP-3 was found between donors, but this was not correlated to previous vasectomy (Table). Also, the total protein concentration and the specific CRISP-3 concentration ([CRISP-3]/[total protein]) in the samples varied between individuals but were not significantly different between the two groups (Table). To ensure that the contribution of organs upstream of the vas deferens was excluded in the samples from vasectomized donors, we also measured the content of hCAP-18, which in the male reproductive tract is expressed exclusively in the epididymis (Malm et al, 2000). The concentration of hCAP-18 in seminal plasma from vasectomized donors was below 5% of the concentration in nonvasectomized donors, as expected (data not shown).
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Gel filtration of seminal plasma from 2 different donors and subsequent evaluation of the CRISP-3 content in individual fractions by ELISA demonstrated that all the CRISP-3 present in seminal plasma eluted in a single peak according to the molecular weight of CRISP-3 itself, as shown in Figure 1B. This demonstrates that CRISP-3 is free in solution and not associated with prostasomes, in which case CRISP-3 would have eluted in the void volume (Andersson et al, 2002).
Detection of CRISP-3 in Protein Extracts
Immunoblotting of protein-enriched supernatants from homogenized tissue
samples obtained from a patient undergoing orchidectomy as treatment for
prostate cancer revealed 2 immunoreactive bands corresponding to glycosylated
and unglycosylated CRISP-3 in all regions examined, as shown in
Figure 2 (lanes 1-5). The
relative concentration of CRISP-3 was low in testis and also in caput and
corpus epididymis (approximately 0.025 µg CRISP-3 per mg protein), compared
to the concentration in cauda epididymis and vas deferens (approximately 4.5
µg CRISP-3 per mg protein), probably reflecting the intraluminal
accumulation of seminal plasma proteins downstream. The two forms of CRISP-3
were also detected in a sample of washed spermatozoa suspended in washing
medium (Figure 2, lane 7).
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Immunohistochemistry and In Situ Hybridization
CRISP-3 immunoreactivity in epithelial cells was detected in all tissue
sections examined, but the number of positive cells and staining intensity
varied markedly (Figure 3).
Immunostaining was restricted to the cytoplasm and was not identified in
nuclei. As expected, granulocytes occasionally observed in the stroma were
strongly immunoreactive. In testis, cells of the germinal epithelium, Sertoli
cells, and Leydig cells were immunostained with a weak intensity, but mature
spermatids showed an intense color reaction. A strong immunostaining was also
detected in most epithelial cells of epididymis, particularly in the cauda
region, and the luminal content with spermatozoa was heavily immunoreactive,
when present. A vast majority of the epithelial cells of vas deferens were
immunostained, with a strong immunoreaction found in the ampulla region,
whereas in the seminal vesicles, fewer epithelial cells were positive. In the
prostatic epithelium, luminal and basal cells were weakly immunostained.
Apically in luminal cells, a somewhat stronger immunoreaction was found in the
cytoplasm, as expected for secretory products. Scattered immunostained cells
were also found in the epithelium of the ejaculatory duct, as observed in
whole-mount tissue sections from the prostate gland. Freshly ejaculated
spermatozoa from healthy donors were immunostained in a similar fashion.
CRISP-3 immunoreactivity was commonly localized in the midpiece and tail, but
a few spermatozoa also displayed immunostaining in the acrosome.
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When the CRISP-3 IgG preparation was replaced by nonimmune IgG or buffer, or when recombinant, truncated CRISP-3 protein was added to anti-CRISP-3 IgGs before immunohistochemical processing, no immunostaining was detected in any of the tissue sections examined.
A digoxigenin-labeled, approximately 400-base-long antisense riboprobe specific to CRISP-3 was added to adjacent paraffin sections, generating hybridization signals with a distribution pattern in accordance with the immunohistochemical findings in the testis, epididymis (Figure 4), vas deferens, seminal vesicle, and in the prostate gland (not shown). All tissues were negative using the corresponding digoxigenin-labeled sense probe or when probe was omitted.
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Discussion |
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The tissue distribution of CRISP-3 protein in the human male genital tract has not been investigated previously. Our work demonstrates that CRISP-3 protein is present in the secretory epithelium throughout the tract from the epididymis to the prostate (Figure 3), although the staining was most prominent in the epididymis (especially cauda) and vas deferens. However, our measurements of CRISP-3 in seminal plasma from vasectomized donors, in whom contributions from the epididymis are eliminated, indicate that the epididymis is not the prime source of CRISP-3 in seminal plasma. This is in accordance with the previous observation by Krätzschmar et al, who found low CRISP-3 mRNA expression in the epididymis, compared to a high expression in the prostate. In the present study, we found a particular strong staining in cauda epididymis compared to other regions of this gland, and it must be pointed out that it is of utmost importance to define the dissected regions of the epididymis when material is collected for measurement of proteins and mRNA. We cannot exclude that the strong immunoreaction in cauda epididymis in part may reflect an uptake, but the ISH data argue against this. We found a strong immunoreaction in ampulla vas deferens. This, and the weaker staining of seminal vesicles and prostate gland from which the bulk of seminal fluid originates, may explain that vasectomy does not influence the level of CRISP-3 in seminal fluid. It should be kept in mind that immunohistochemistry only gives an instantaneous measure of the amount of protein ready for secretion and does not provide information about the amount of protein secreted over time.
The intense CRISP-3 immunoreactivity observed in mature spermatids and spermatozoa was a little surprising, as only very weak mRNA expression was originally described in testis (Krätzschmar et al, 1996). On the other hand, CRISP-3 was cloned from a testis cDNA library (Krätzschmar et al, 1996), and CRISP-3 transcripts have been detected in testis with multiple tissue dot blot assay using a specific probe corresponding to part of the 3' untranslated region (Kosari et al, 2002). Furthermore, our ISH data presented here, using a CRISP-3-specific riboprobe, show excellent concordance with IHC, which supports the presence of CRISP-3 protein and mRNA in testis. In humans, CRISP-3 is closely related to CRISP-2, with 72% amino acid sequence identity, and we cannot totally exclude the possibility that some of the immunoreactivity observed in testis or spermatozoa is due to cross-reactivity with CRISP-2, which is known to be present exclusively in these places. Human CRISP-2, however, is not glycosylated and thus exists in only one molecular-weight form, with a theoretical weight approximately 0.5 kd lower than the unglycosylated form of CRISP-3 (Kasahara et al, 1989; Cohen et al, 2001). No bands with this size were detected in immunoblotting of testis tissue or spermatozoa (Figure 2).
Some proteins are known to be secreted from the prostate in small
membrane-bound organelles called prostasomes, which to some extent fuse with
the sperm membrane but also are found freely in seminal plasma
(Ronquist et al, 1990). To
examine whether CRISP-3 is associated with these organelles, we performed gel
filtration of seminal plasma and examined the distribution of CRISP-3. These
experiments exclude this possibility. Furthermore, no CRISP-3 was
complex-bound by other proteins in seminal plasma, which is in contrast to the
behavior in blood plasma, where CRISP-3 is bound to
1B-glycoprotein and in gel filtration elutes in fractions
corresponding to a molecular weight of 100 kd
(Udby et al, 2004).
In mice, the expression of especially CRISP-1 but also CRISP-3 is regulated by androgens (Haendler et al, 1997). However, a differential androgen regulation of the human CRISP genes has not been reported. Human CRISP-3 is more closely related to murine CRISP-2, which is not regulated by androgens (Haendler et al, 1997), than to murine CRISP-1 and CRISP-3 (Hayashi et al, 1996). Also, the tissue distribution is quite different from murine CRISP-3, which has not been found in the male reproductive tract. These facts indicate that direct extrapolation from one species to another is not possible in this case. However, several sequences, with homology to the consensus androgen responsive element (Roche et al, 1992), can be found in the putative human CRISP-3 promoter region (personal observation), and we thus cannot exclude that human CRISP-3 could be under regulation by androgens, at least in some tissues.
As stated earlier, the function of human CRISP-3 is unknown. The equine counterpart with which it shares 65% amino acid sequence identity (Schambony et al, 1998) is a major seminal plasma protein produced in the accessory sexual glands, ampulla, and seminal vesicle. Equine CRISP-3 seems to be bound to the sperm head in large amounts, and it was suggested (Magdaleno et al, 1997) that equine CRISP-3 plays a role in sperm maturation or gamete interaction, similar to what was suggested and later confirmed for CRISP-1 in mice, rats, and humans (Cohen et al, 2000a,b, 2001). According to these findings, firmly attached CRISP-1 on the sperm surface binds to receptors on the oocyte and somehow mediates gamete fusion. A recent study indicates that loosely attached CRISP-1, which is lost after deposition in the female genital tract, acts as an inhibitor of capacitation and thus prevents premature activation of spermatozoa, as studied in rats (Roberts et al, 2003). Humans and horses are so far the only species in which CRISP-3 has been found in the male reproductive tract, and it may be that CRISP-3 has overlapping functions with CRISP-1 in the process of reproduction. The wider distribution of human CRISP-3 both in the male reproductive tract and also in exocrine secretions covering other parts of the body and in the major effector cell of the innate immune system (neutrophilic granulocytes) indicates that human CRISP-3 could also have a more generalized function in protection against microorganisms. This is supported by the fact that the amino terminal domain of CRISPs has similarities to a large group of plant proteins, the so-called pathogenesis-related proteins of group 1, which are produced in response to pathogen attack or any other type of stress and have fungicidal activity (Niderman et al, 1995).
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Acknowledgments |
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Footnotes |
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