Mol. Hum. Reprod. Advance Access originally published online on February 16, 2004
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Molecular Human Reproduction, Vol. 10, No. 4, pp. 247-252, 2004
© European Society of Human Reproduction and Embryology 2004
Expression of carcinoembryonic antigen-related cell adhesion molecule-1 (CEACAM1) in normal human Sertoli cells and its up-regulation in impaired spermatogenesis
1Institute of Anatomy I, 2Medical Clinic I and 3Department of Clinical Chemistry, University Hospital Hamburg–Eppendorf and 4Department of Urology, Armed Forces Hospital Hamburg, Germany
5 To whom correspondence should be addressed at: Institute of Anatomy I, University Hospital Hamburg–Eppendorf, Martinistr. 52, D-20246 Hamburg, Germany
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Carcinoembryonic antigen-related cell adhesion molecule-1 (CEACAM1) is usually expressed at the luminal surface of different epithelia and is up-regulated in endothelial cells during angiogenesis. Here, we demonstrate evidence of morphogenetic effects of CEACAM1 in spermatogenesis. CEACAM1 is detectable in normal testicular tissue and seminal fluid. It is present in the adluminal part of Sertoli cells extending only as far as the tight junctions between them. CEACAM1 immunostaining is significantly increased and extends to the basal part of Sertoli cells in the presence of carcinoma in situ. Also, in vitro-induced spermatogenetic disturbance leads to an enhanced CEACAM1 expression in Sertoli cells after 3 days of culture. Remarkably, seminiferous tubules containing exclusively Sertoli cells do not exhibit any CEACAM1 expression. CEACAM1 staining was absent in vascular endothelial cells of normal testicular tissue, but present in small blood vessels of seminomas. These data suggest that CEACAM1 expression in Sertoli cells depends on the presence of germ cells and plays a role in adhesive interactions between Sertoli and differentiating germ cells. Its up-regulation in Sertoli cells accompanying spermatogenic damage may contribute to reconstruction and maintenance of the tubular structure of seminiferous tubules. Additionally, CEACAM1 is apparently involved in the angiogenesis of germ cell tumours.
Key words: Key words: angiogenesis/CEACAM1/germ cell tumour/human spermatogenesis
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Introduction |
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Carcinoembryonic antigen-related cell adhesion molecule-1 (CEACAM1), formerly known as biliary glycoprotein (BGP) or CD66a, is a member of the CEA family which belongs to the immunoglobulin superfamily (Obrink, 1991). It can bind homophilically to itself as well as heterophilically to the other CEA family members (Rojas et al., 1990). Up to now, 11 alternative splicing forms of the CEACAM1 gene are known (Obrink, 1991; Kuroki et al. 1991; Barnett et al., 1993). CEACAM1 is normally expressed at luminal surfaces of epithelia of different organs and in leukocytes (Prall et al., 1996). It has been shown that mRNA expression of CEACAM1 is down-regulated in some tumours such as colorectal and prostate carcinomas (Neumaier et al., 1993; Nollau et al., 1997; Lin and Pu, 1999). Based on such results, a tumour-suppressive role has been postulated. Also, the increased expression of CEACAM1 in an experimental tumour model of prostate cancer suppressed the tumour growth (Lin and Pu, 1999). It has been reported that the tumour inhibitory function of CEACAM1 depends on the cis-determinants in its cytoplasmic domain (Izzi et al., 1999). Recently, it was demonstrated that CEACAM1 is differently expressed in proliferating and quiescent epithelial cells and that this influences their proliferation (Singer et al., 2000). On the other hand it has been shown that CEACAM1 is expressed in endothelial cells of small tumour blood vessels whereas it is absent in endothelial cells of quiescent blood vessels of normal tissues. Moreover, it could be demonstrated that soluble CEACAM1 exhibits pro-angiogenic effects and functions as a morphogenic effector for vascular endothelial growth factor (Ergun et al., 2000; Wagener and Ergun, 2000).
The localization of CEACAM1 at luminal surfaces of epithelia has been shown for different organs such as prostate glands, colon, renal proximal tubules and mammary glands (Prall et al., 1996). These findings implicate a role for CEACAM1 in differentiation and polarization of the epithelial cells as well as for cell–cell adhesion at this part of the epithelial lining in glandular or tubular organized structures. Cell adhesion also plays a crucial role in the formation of seminiferous tubules and in the organization of germinal epithelium which is of importance for spermatogenesis. Since the spermatocytes and immature spermatids remain adherent to the Sertoli cells until the release of mature spermatids, and intercellular tight junctions between Sertoli cells serve as a basis for construction and maintenance of the blood–testis barrier (Setchell and Main, 1975), we asked whether CEACAM1 is involved in the intercellular adhesive processes between Sertoli cells themselves as well as between Sertoli and spermatogenic cells.
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Materials and methods |
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Tissue samples
Normal human testicular tissue and normal human epididymal tissues were obtained from patients who had been orchiectomized because of prostate cancer. They were untreated prior to orchiectomy. Tumour tissues were obtained from patients who underwent orchiectomy because of solid germ cell tumours. In 10 cases of normal human testicular and in five cases of normal human epididymal tissues, the localization and expression of CEACAM1 was studied using Western blot analyses and immunohistochemistry. In comparison to normal testicular tissue, CEACAM1 expression was studied in testicular tumours such as seminomas and teratomas. Five specimens exhibiting early germ cell tumours were included in the studies of CEACAM1 expression where tumour cells are still localized in the seminiferous tubules without invasion of the interstitium. This stage of tumour development is usually accompanied by damage of spermatogenesis. In order to create a progressive disruption of spermatogenesis in vitro, additionally, fragments of seminiferous tubules were isolated from normal testicular tissue and cultured as described below. For the studies on these human tissues, permission was obtained by the university ethics committee.
Samples of human ejaculates
The samples of human ejaculates assessed as normal according to the World Health Organization (2000) criteria were obtained from the Department of Andrology of our University Hospital. Only the samples with normal sperm counts and normal motility of sperm were used for Western blot analyses.
Tissue culture of human seminiferous tubules
For functional analyses, an organotypic culture of seminiferous tubules was used as an in vitro assay. For details see Seidl and Holstein (1990) and Lauke et al. (1991). In the present study, basal cell culture medium (Dulbecco’s modified Eagle’s medium/Ham’s F-12) supplemented with 0.1% bovine serum albumin, 1% penicillin/streptomycin, 50 µg/ml gentamycin, but without fetal calf serum, was used. In this assay, isolated fragments of human seminiferous tubules were cultured from 0 to 9 days, as a result of which a progressive damage of spermatogenesis occurred. In order to study this process immunohistologically, tubules were removed from the culture daily and embedded in paraffin. In these series of cultured tubules the expression of CEACAM1 was studied.
SDS–PAGE and Western blot analysis
Frozen human testicular tissue pieces, which had been stored at –70°C, were thawed and used for protein extraction by lysis buffer containing 100 mmol/l Tris–HCl, pH 7.8 + 500 mmol/l sucrose. After determination of the total protein amount, the samples were prepared for sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) using Laemmli buffer (Laemmli, 1970). Onto each lane, 50 µg of total protein was loaded and separated by SDS–PAGE. Subsequently, proteins were transferred to a polyvinylidene difluoride membrane (Immunobilon Millipore, USA) by a semi-dry electrophoretic transfer procedure for 1.5 h at 1 mA/cm2. After blocking the membrane with 1% blocking reagent (Boehringer-Mannheim, Germany) it was incubated with monoclonal CEACAM1 antibody (4D1/C2) (2 µg/ml) as described by (Drzeniek et al., 1991; Stoffel et al., 1993). Using the same antibody, Western blot analyses were performed to detect CEACAM1 in human ejaculates. Frozen ejaculate samples from healthy individuals were thawed and subsequently denatured using Laemmli buffer as mentioned above without treatment with lysis buffer. In each lane, 25 µl of each sample was loaded and separated by SDS–PAGE. The subsequent steps of immunoblotting were carried out as described above. To exclude any cross-reactivity, the CEACAM1 antibody 4D1/C2 was preincubated with the purified CEACAM1 from human granulocytes (1x4D1/C2:6xCEACAM1 protein) overnight and used in the immunoblotting analyses instead of the antibody 4D1/C2. The antibody as well as the purified human CEACAM1 was kindly provided by the group of Professor C. Wagener, Department of Clinical Chemistry, University Hospital Hamburg–Eppendorf. In each lane, 25 µg of total protein was added. In order to determine the protein amount on the membrane, the detection of vimentin (Dako, Germany; final dilution 1:50) in the same blots was used as an internal control.
Immunohistochemistry
Immunohistochemical staining for CEACAM1 was performed using paraffin-embedded tissue sections of normal testicular and epididymal tissue, germ cell tumours (seminomas and teratomas), as well as seminiferous tubules after different times in culture. Tissue specimens had been fixed in Bouin’s fixative (24 h) at room temperature and processed further as described previously (Ergun et al., 1997; Kilic and Ergun, 2001). Immunolocalization of CEACAM1 was performed using the monoclonal antibody 4D1/C2 (final dilution: 2.5 µg/ml). The following controls were performed: (i) primary antibody 4D1/C2 was incubated with CEACAM1 protein for 4 h prior to use; (ii) primary and/or secondary antibodies were replaced by phosphate-buffered saline; (iii) instead of the primary antibody, sections were incubated with normal mouse serum (Sigma) at dilutions in the range of 0.1–0.01%.
Immune electron microscopy
First, CEACAM1 was visualized using the immunostaining on paraffin sections as described above. Subsequently, the sections were dehydrated in alcohol and xylol. A pill capsule filled with unpolymerized Epon was inverted over each section and the glass slides with the capsules attached were incubated at 60°C overnight. Usually, the capsule with the embedded tissue specimens can be easily detached from the glass slides. Fine sections were prepared from these tissue blocks for electron microscopic studies (Kilic and Ergun, 2001).
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Results |
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CEACAM1 is detectable in human male reproductive organs
To study whether CEACAM1 is expressed in human testicular and epididymal tissues, we performed Western blot analyses using the antibody 4D1/C2 which recognizes human CEACAM1 specifically. Immunoblotting of normal testicular and epididymal tissues as well as of testicular tumours such as seminomas and teratomas revealed two bands, one at 160 kDa, the corresponding size of CEACAM1, and an additional band at 120 kDa (Figure 1A). Note that the amount of both 160 and 120 kDa forms is higher in tumour tissues than in normal testicular and epididymal tissues. In order to exclude possible cross-reactivity, we performed Western blot analyses using preabsorption controls. As demonstrated in Figure 1C, the preabsorption of the CEACAM1 antibody 4D1/C2 with the purified CEACAM1 from human granulocytes overnight was able to abolish the specific bands at 160 and 120 kDa detected in normal human testicular tissue as well as in a seminoma. The detection of vimentin as internal control in the same blots revealed that equal amounts of protein were loaded into the lanes (Figure 1C).
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CEACAM1 is detectable in human ejaculates
We then examined normal human ejaculates using Western blot analyses to study whether CEACAM1 is detectable in seminal fluid. Similar to the testicular tissue, these analyses revealed two bands at 160 and 120 kDa (Figure 1B).
CEACAM1 is localized at the adluminal part of Sertoli cells of normal germinal epithelium
To localize CEACAM1 in normal human testicular and epididymal tissues, we carried out immunohistochemical staining using the antibody for CEACAM1 that was also used in immunoblotting. In normal human testicular tissues, CEACAM1 was found in the adluminal compartment of normal germinal epithelium of seminiferous tubules, probably localized in the contact zones between Sertoli cells as well as between Sertoli cells and germ cells of the adluminal compartment of seminiferous tubules (Figure 2A). In contrast, germ cells themselves remained unstained (Figure 2A). CEACAM1 was often expressed heterogeneously within the germinal epithelium. Note that in the interstitial compartment of normal testicular tissue, Leydig cells and blood vessels were negative (Figure 2A). Correspondingly, post-embedding immune electron microscope studies of human testicular tissue revealed a specific staining only at the adluminal part of Sertoli cells (Figure 2B). There was no specific immunostaining found in the germ cells, neither in spermatogonia nor in differentiating germ cells (Figure 2B). Note that the staining extends to the luminal surface of seminiferous tubules and surrounds the area around the head of a mature spermatid as shown in higher electron microscopic magnification (insert in Figure 2B). No specific staining was observed in the control (data not shown).
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CEACAM1 is found at the luminal surface of the epithelium of the epididymal duct
An apical localization of CEACAM1 could also be found in the epithelium of the human epididymal duct (Figure 2C), whereas basal cells, lamina propria and blood vessels in the interstitium were negative. There was no specific staining in the control sections (data not shown).
CEACAM1 is not expressed in Sertoli cells of seminiferous tubules with Sertoli cells only (SCO)
To study whether CEACAM1 expression in Sertoli cells depends on the presence of germ cells, we performed immunohistochemical staining on paraffin sections of human testicular tissues which contained areas of seminiferous tubules with only Sertoli cells as an in vivo model. These studies revealed that Sertoli cells of such tubules exhibit no CEACAM1 staining (Figure 2D) while Sertoli cells of neighbouring seminiferous tubules containing both Sertoli and germ cells are positive for CEACAM1. Spermatogenic tubules viewed under higher magnification revealed CEACAM1 staining at the adluminal part of Sertoli cells (Figure 2E), whereas no specific staining was visible in neighbouring seminiferous tubules containing only Sertoli cells (Figure 2F). No specific staining was found in the control sections (data not shown).
CEACAM1 expression in Sertoli cells is enhanced and extends up to the basal cell side in presence of CIS
In comparison to delicate CEACAM1-staining in Sertoli cells of seminiferous tubules with normal spermatogenesis, an enhanced CEACAM1 expression was observed in Sertoli cells of adjacent tubules exhibiting impaired spermatogenesis but the strongest increase of CEACAM1 expression in Sertoli cells was found in some CIS tubules (Figure 3A). In these tubules, CEACAM1 staining reached the basement membrane, but tumour cells remained negative (Figure 3B and C). In this early stage of germ cell tumours, blood vessels in the nearby interstitium also remained negative for CEACAM1 whereas blood vessels of solid germ cell tumours exhibited CEACAM1 immunostaining (Figure 3D). There was no specific staining in the control sections treated only with secondary antibody (Figure 3E).
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CEACAM1 expression in Sertoli cells is enhanced and extends up to the cell basal side in case of damaged spermatogenesis
Extension of CEACAM1 staining to the basal side of Sertoli cells could be observed in areas of seminiferous tubules exhibiting damaged spermatogenesis in paraffin sections of normal testicular tissue. To further examine the role of CEACAM1 in human germinal epithelium, isolated fragments of seminiferous tubules were cultured for up to 9 days (Figure 4A, B), successively removed and embedded in paraffin for histological evaluation via haematoxylin–eosin (HE) staining (Figure 4C–E) and for immunohistochemical studies (Figure 4F–H). Histological examinations of HE-stained seminiferous tubules demonstrated a significant progressive disturbance of the germinal epithelium, in particular of the differentiating germ cells localized in the adluminal compartment. This damage was first visible on culture days 2–3 (Figure 4D). Interestingly, between day 1 and day 3 there was no apparent change of CEACAM1 expression in Sertoli cells when compared with normal germinal epithelium. In contrast, it was markedly enhanced in these cells under culture conditions in a time-dependent manner from day 3 to day 9 (Figure 4G, H). CEACAM1 staining intensity increased along the progression of spermatogenic damage and the staining was not only localized in the apical, but extended also to the basal, part of Sertoli cells. There was no specific staining in the control sections (data not shown).
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Discussion |
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The present findings demonstrate the expression of CEACAM1 in male reproductive organs. Briefly, our results show: (i) CEACAM1 expression in the adluminal part of Sertoli cells of normal human germinal epithelium, but its absence in Sertoli cells of tubules lacking germ cells; (ii) an increase of CEACAM1 expression in Sertoli cells and its extension up to the basement membrane in various cases of impaired spermatogenesis, i.e. hypospermatogenic tubules, spermatogenic damage accompanying CIS, and experimentally induced spermatogenic disturbance; (iii) CEACAM1 expression in endothelial cells of seminoma blood vessels but not in those of normal testicular tissue; (iv) CEACAM1 expression at the luminal epithelial surface of the epididymal duct; (v) CEACAM1 detection in normal seminal fluid.
The detection of CEACAM1 in Sertoli cells in the presence but not in the absence of germ cells lets us assume that CEACAM1 expression depends on the interaction between Sertoli and germ cells. Since CEACAM1 is strictly localized at the adluminal part of Sertoli cells, it may have a role for polarization and differentiation of these cells. This differentiation of Sertoli cells serves as the basis for a functioning blood–testis barrier that is constituted of specialized inter-Sertoli cell tight junctions (Schulze, 1984) and is a prerequisite for spermatogenesis. Only type I spermatocytes pass through these junctions and migrate from the basal to the adluminal compartment. All differentiating germ cells adhere to the adluminal membrane of Sertoli cells until the release of mature spermatids, and this strictly conforms with the CEACAM1 expression pattern. The data suggest that CEACAM1 particularly is involved in Sertoli–germ cell interactions during differentiation and maturation of germ cells. As a cell–cell adhesion molecule, CEACAM1 is prone to be involved in the adherence of spermatogenic cells to Sertoli cells. These processes do not seem to be mediated by homotypic binding, because germ cells themselves do not exhibit CEACAM1. Unknown factors expressed on differentiating germ cells may interact with CEACAM1 present on Sertoli cells.
Presumably, during all stages of spermatogenic maturation, Sertoli cells and germ cells communicate through direct cell–cell contacts but the underlying molecular mechanisms are poorly understood. However, recent intriguing data on mouse testes showed that an N-glycan carbohydrate structure present on differentiating germ cells plays a crucial role for germ cell survival through interaction with Sertoli cells. In knockout mice for this carbohydrate the spermatogenic cells failed to adhere to Sertoli cells (Akama et al., 2002). These data correspond to a great extent to the adluminal expression pattern of CEACAM1 in human germinal epithelium as shown here. Postulating an adhesive starting point from Sertoli cells, CEACAM1 might be a good candidate for anchoring differentiating germ cells as a major carrier of carbohydrate determinants such as Lewisx Galß(1,4)[Fuc
(1,3)]GlcNAc), Sialyl-Lewisx NeuNAc2,3Galß1,4(Fuc(1,3)GlcNAc and other glycans, for instance high-mannose. Whether these carbohydrate epitopes of CEACAM1 are involved in cell–cell adhesion between germ cells and Sertoli cells via interaction with those glycans as presented by Akama and colleagues remains to be verified. However, we assume that the presence of two different forms of CEACAM1 at 160 and 120 kDa in testicular and epididymal tissues may be caused by different glycosylation of CEACAM1, which in turn might be involved in Sertoli–Sertoli cell adhesion events. Transfection studies in CHO and HEK293 cells for CEACAM1 resulted also in the production of the 120 kDa form of CEACAM1. Mass spectrometric sequencing of this form confirmed the presence of the whole CEACAM1 protein without Sialyl-Lewisx, indicating a different glycosylation (Prof. C.Wagener, Dept. of Clinical Chemistry, University Hospital Hamburg–Eppendorf, personal communication). Further studies are needed to characterize the 120 kDa form of CEACAM1 in human testicular tissue.
Sertoli cell-only (SCO) tubules as well as hypoplastic tubules revealing solid cords of Sertoli cells are an occasional finding in testicular tissue. It is believed that such segments of seminiferous tubules have not been colonized by germ cells during testicular development (Huber et al., 1968; Knecht, 1976). Other SCO tubules represent the endpoint of complete germ cell degeneration. The absence of CEACAM1 in SCO tubules implies that CEACAM1 expression in Sertoli cells may be initiated through cell–cell interaction or by unknown factors released from germ cells which may act in a paracrine manner on Sertoli cells and regulate CEACAM1 expression in them. Further studies are needed to identify such potential factors and mechanisms regulating these processes.
An early effect of CIS is a massive loss of germ cells, particularly of differentiating germ cells of the adluminal compartment. Our findings demonstrate a significant up-regulation of CEACAM1 in Sertoli cells of seminiferous tubules revealing impaired spermatogenesis, as well as in tubules containing CIS in comparison to Sertoli cells of normal germinal epithelium. The presented extension of CEACAM1 staining to the basal part of Sertoli cells is obviously related to the destruction of the blood–testis barrier. It is generally assumed that the cells in the adluminal compartment are immunoprotected, which is achieved by the blood–testis barrier mediated by Sertoli–Sertoli cell contacts. Whether and how CEACAM1 is involved in the construction of the blood–testis barrier remains unanswered and needs further studies. To study whether the presence of tumour cells or the loss of germ cells caused by the tumour cells led to the increased CEACAM1 expression in Sertoli cells, we used an organotypic culture of isolated fragments of human seminiferous tubules which was already established in our laboratory (Seidl and Holstein, 1990; Lauke et al., 1991). The culture of normal seminiferous tubules in the present study led to a targeted spermatogenetic disturbance that was paralleled by a thickening of the lamina propria within 2–3 days of culture. On culture day 3, CEACAM1 immunostaining increased in Sertoli cells in comparison to those of culture day 0. This up-regulation of CEACAM1 in Sertoli cells in in vitro-induced spermatogenic disturbance underlines our hypothesis that differentiating germ cells may be involved in the regulation of CEACAM1 expression in Sertoli cells. It is notable that the intensity of CEACAM1 staining increased and reached its highest level between culture days 7 and 8, although after culture day 2 the differentiating germ cells were progressively degenerating and around day 7 spermatogonia as well as Sertoli cells were present exclusively. Thus, spermatogonia may secrete factors which act on Sertoli cells and lead to a further increase of CEACAM1 expression. Because Sertoli cells serve as the basis for the proper internal organization of the germinal epithelium, the loss of spermatocytes and immature spermatids probably induces mechanisms which lead to severe structural disturbances of the germinal epithelium, including the disturbance of the blood–testis barrier. Therefore, the high amount of CEACAM1 may rather reflect a morphogenic influence to maintain or to reconstitute the tubular structure of germinal epithelium than a direct influence of CIS. However, there is an obvious and intriguing relationship of germ cell tumours to CEACAM1 as a potent angiogenic factor. Its absence in vascular endothelial cells of normal testicular tissue, but its presence in small blood vessels of seminomas, indicates a pivotal role for angiogenesis of germ cell tumours.
Since the increased amount of CEACAM1 in Sertoli cells in cases of impaired spermatogenesis is not distributed homogeneously but is localized focally on sites of Sertoli cells, which seem to be the adhesive cell–cell contacts, we postulate a role for CEACAM1 in the maintenance of adhesive processes between Sertoli cells and germ cells which may be of importance for maintenance of structural tubular organization. The finding that CEACAM1 expression pattern in normal germinal epithelium frequently covers particular segments of cross-sectioned tubules suggests that CEACAM1 is probably involved in the regulation of spermatogenesis in a stage-dependent manner. The detection of CEACAM1 in ejaculates by Western blot analyses indicates the presence of soluble forms in the seminal fluid. They may be released by Sertoli cells but also by the epithelium of the epididymal duct. However, there are no indications for an action of CEACAM1 on released sperm until now. Whether CEACAM1 levels in seminal fluid may have any relationship to fertility remains unknown.
In summary, our results demonstrate novel functional findings which show that the cell adhesion molecule CEACAM1 is expressed in Sertoli cells depending on the presence of germ cells and plays a role in morphogenic processes such as organization of the germinal epithelium as well as regulation of spermatogenesis. Additionally, our data support the idea that CEACAM1 may have a role in the internal reorganization of seminiferous tubules in cases of damaged spermatogenesis as well as in angiogenesis of germ cell tumours. Further studies are necessary to evaluate whether the detection of CEACAM1 in testicular tissue and ejaculates can be used as a parameter in fertility diagnostics.
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Acknowledgements |
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The authors are grateful to Mrs C.Kretzschmar and K.Miethe for their excellent technical assistance. We are also grateful to Professor Dr M.Davidoff for his continuous advice. We also thank Professor W.Schulze for the providing the ejaculate samples. We are thankful to the ‘Verein zur Förderung der Forschung auf dem Gebiete der Fortpflanzung e.V.’ for the support of this work. The authors are grateful to Prof. C.Wagner for his support and advice.
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Submitted on November 3, 2003; accepted on November 10, 2003.
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