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From * Centro de Microscopía
Electrónica, Facultad de Ciencias Médicas, Universidad Nacional
de Córdoba, Argentina; and The Lankenau
Institute for Medical Research, Wynnewood, Pennsylvania.
| Correspondence to: Alejandro Peralta Soler, Lankenau Institute for Medical Research, 100 Lancaster Avenue, Wynnewood, PA 19096 (e-mail: rossinol{at}mlhs.org). |
| Received for publication April 21, 2004; accepted for publication July 19, 2004. |
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Abstract |
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Key words: Cadherins, seminal fluid, cell-cell adhesion
P-cadherin is a 118-kd transmembrane glycoprotein first described in the mouse placenta (Nose and Takeichi, 1986). In epithelial tissues, P-cadherin expression appears limited to basal and myoepithelial cells (Daniel et al, 1995). In the mammary gland, P-cadherin gene knockout causes precocious development and lactation in virgin females (Radice et al, 1997). Aberrant P-cadherin expression in breast cancers is associated with aggressive tumor behavior (Peralta Soler et al, 1999). However, P-cadherin overexpression in the mouse mammary epithelium does not cause significant change in mammary phenotype or development (Radice et al, 2003).
The proteolytic cleavage of the extracellular domain of cadherins results in soluble fragments that can be released into fluids, including serum (Knudsen et al, 2000). In the lactating breast, P-cadherin is secreted into the milk as a soluble 80-kd fragment (Peralta Soler et al, 2002). In this study, we present the first report of the presence of readily detectable levels of an 80-kd, soluble P-cadherin fragment in human semen.
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Materials and Methods |
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Semen was centrifuged at 1000 rpm for 20 minutes, seminal fluid was separated from sperm, and total protein concentration was measured with a BioRad kit (Bio-Rad Protein Assay, Bio-Rad Laboratories, Hercules, Calif). The expression of soluble P-cadherin in seminal fluid was determined by Western blot analysis. Thirty micrograms of sperm-free seminal fluid protein were run in a 15% acrylamide gel (Sigma Chemical Co, St Louis, Mo). A cell lysate from a human epidermoid carcinoma cell line (BD Transduction Laboratories, San Jose, Calif) was used as positive control. Molecular mass was assessed with the use of a Full Range Rainbow Molecular Weight Marker (Amersham-Life Science, Bucks, United Kingdom). The proteins were transferred to a nitrocellulose membrane (Hybond ECL, Amersham-Pharmacia-Biotech, Bucks, United Kingdom). Nonspecific sites were blocked with phosphate-buffered saline (PBS) containing 5% low-fat milk powder and 0.1% Tween-20 at room temperature. The membranes were incubated for 1 hour at 37°C with anti-P-cadherin mouse monoclonal antibody (Transduction Laboratories) diluted 1:100 in blocking buffer. After extensive washing with PBS and 0.1% Tween-20, the samples were incubated with a peroxidase-conjugated (HRP) goat anti-mouse secondary antibody (Pierce, Rockford, Ill), diluted 1:5000 in blocking buffer. The blots were thoroughly rinsed in PBS and 0.1% Tween-20 and detected with an ECL Western blotting detection system (Amersham Biosciences, Bucks, United Kingdom) on Hyperfilm (Amersham-Pharmacia-Biotech) following the manufacturer's instructions. Scanning and quantification was performed with Scion Image software (V.4.0.2, Scion Image Corp, Frederick, Md) at 3 different exposure times. P-cadherin levels were statistically analyzed with unpaired t tests, and the results were expressed as means ± SEM. Significance was reported at P < .05.
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Results |
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Western blots revealed a single 80-kd band corresponding to soluble P-cadherin. Densitometric analysis showed no significant differences between infertile and fertile patients (Figure).
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Parameters evaluated in the infertile group included count, motility, morphology, bacterial culture, and leukocyte count (Table). The limited number of cases analyzed in this study did not allow us to correlate P-cadherin levels with these parameters.
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Discussion |
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The size of soluble P-cadherin (80 kd) in semen is identical to the fragment found in milk, suggesting a common proteolytic cleavage mechanism. Soluble P-cadherin in ejaculated semen might result from release of the extracellular domain of P-cadherin from spermatozoa because previous data showed P-cadherin in ejaculated spermatozoa as a 90-kd protein (Rufas et al, 2000). However, the lack of correlation between P-cadherin expression in semen and the fertility status of the patients suggests that sperm might not be the source of P-cadherin in ejaculated seminal fluid. In the prostate and seminal vesicles, P-cadherin expression is largely limited to prostate-specific antigen-negative cells, including the basal cells of the prostate and seminal vesicle epithelium (Peralta Soler et al, 1997). Basal cells in the prostate are secluded from contact with the prostatic luminal secretion. However, P-cadherin was occasionally seen as a secretory protein in prostatic glands and in seminal vesicles (Peralta Soler et al, 1997), similar to the expression pattern observed in lactating breast (Peralta Soler et al, 2002), suggesting that those tissues could be the source of P-cadherin in seminal fluid.
Although the density of the bands was heterogeneous, P-cadherin expression and the various morphological parameters analyzed in the infertile patients were not correlated. However, the small number of samples limited the statistical value of the data.
The function of released soluble cadherin fragments in fluids is unknown. Interestingly, the extracellular domain of P-cadherin contains sequences identical to the Listeria-binding site of E-cadherin, which can act as decoy receptors for the bacteria (Cossart et al, 2003; da Silva et al, 2003; Pizarro-Cerda et al, 2004). But in contrast to E-cadherin-soluble fragments, which can disassemble epithelial tissues by binding to the cellular E-cadherin, soluble P-cadherin fragments cannot bind to cellular P-cadherin because of its secluded location in basal cells. Perhaps the soluble P-cadherin fragments in fluids serve as an alternative bacterial binding peptide without the deleterious effects that E-cadherin soluble fragments could have on the integrity of epithelial tissues. Another possibility is that the soluble seminal P-cadherin has an effect on P-cadherin-expressing female genital tract tissues. This possibility is supported by previous data showing expression of P-cadherin in normal and neoplastic uterine tissues, including normal myometrium and uterine leiomyomas (Tai et al, 2003), glandular tumors of the cervix (Han et al, 2000), and small cell carcinomas of the cervix (Zarka et al, 2003). Another possible role for P-cadherin could be the heterotypic interaction between the P-cadherin ectodomain and the E-cadherin extracellular domain within the male or female genitourinary tract. A recent report showed differences in E-cadherin expression in spermatozoa from fertile and infertile males (Purohit et al, 2004), suggesting a role for heterotypic cadherin interaction between spermatozoa and oocyte during fertilization.
Previous studies on the proteolytic processing of N-cadherin indicated critical specific cleavage sites for cell-cell interaction and for the assembly of the cadherin-catenin complex (Wahl et al, 2003). Interestingly, an 80-kd proteolytic fragment of E-cadherin resulting from metalloprotease-dependent cleavage was found as a potential biomarker for prostate cancer progression (Kuefer et al, 2003). Thus, identification of the cleavage site within P-cadherin will certainly be valuable in elucidating the mechanisms of P-cadherin ectodomain formation and its potential role in human reproduction and cancer.
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Acknowledgments |
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Footnotes |
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References |
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