Molecular Human Reproduction, Vol. 5, No. 10, 908-913,
October 1999
© 1999 European Society of Human Reproduction and Embryology
Molecular endocrinology |
Fetal antigen 1, an EGF multidomain protein in the sex hormone-producing cells of the gonads and the microenvironment of germ cells
1 Immunology and Microbiology, University of Southern Denmark, Odense University, Winsløwparken 21.1, DK-5000 Odense C and 2 The Fertility Clinic, Odense University Hospital, DK-5000 Odense C, Denmark
Abstract
Fetal antigen 1 (FA1), an epidermal growth factor (EGF) multidomain glycoprotein, was investigated in the human reproductive system. Immunohistochemical analysis of the male reproductive system revealed staining for FA1 in the Leydig cells only. Concentrations of FA1 in seminal plasma and serum were similar and significantly correlated in weekly samples from three men (P < 0.0065). The concentrations in seminal plasma from vasectomized men (n = 4) were not significantly different from those of normal men (n = 187). The concentration of FA1 in seminal plasma was significantly correlated with the sperm counts of normozoospermic men (P < 0.0001), and significantly higher in seminal plasma from men with sperm counts > 20x106/ml, compared with those with counts 
20x106/ml (P < 0.0001). Immunohistochemical analysis of the ovary showed the presence of FA1 in the theca interna and the hilus cells but not in the granulosa cells or the oocyte. In follicular fluid the concentration of FA1 (median; 73.3 ng/ml, n = 28) was three times higher than that of serum (median; 23.8 ng/ml). These data suggest a role for this novel member of the EGF superfamily in relation to human reproduction, but further experiments are needed to define the biological function and to elucidate its clinical potential.
delta-like/EGF-superfamily/fetal antigen 1/follicular fluid/seminal plasma
Introduction
Fetal antigen 1 (FA1) was originally isolated from second trimester human amniotic fluid and identified as a heterogenous glycoprotein with an apparent molecular mass of 32–38 kDa when analysed by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) (Fay et al., 1988
; Jensen et al., 1993). The amino acid sequence of human FA1 has been reported (Jensen et al., 1994) showing a heterogenous molecule of 225–260 amino acid residues containing six epidermal growth factor (EGF) motifs Protein Information Resource (PIR) accession nos: A44549 and S48713). FA1 contains 10 O- and N-glycosylation sites, six of which are differentially glycosylated, and the heterogeneity of the molecule is due to a ragged C-terminal part of the polypeptide backbone and differential glycosylation (Jensen et al., 1994). These observations indicate that FA1 is a member of the group of EGF multidomain proteins within the EGF superfamily.
Searches in translated DNA databases revealed 99% homology between the amino acid sequence of FA1 and translated human delta-like (dlk) mRNA (Laborda et al., 1993). Human dlk defines a membrane-associated protein of 357 amino acid residues and FA1 spans amino acid residues 24–294 of the translated dlk mRNA lacking the signal peptide and the membrane-associated C-terminal part (Jensen et al, 1994). From these data it was concluded that soluble FA1 in biological fluids is a cleavage product of a larger membrane-associated precursor defined by the mRNA, human dlk (Jensen et al., 1994).
A murine homologue to human FA1 (mFA1) has been isolated from murine amniotic fluid and the primary structure resolved (Bachmann et al., 1996; Krogh et al., 1997), and murine FA1 is defined by the mRNA referred to as mouse delta-like (mdlk) (Laborda et al., 1993), stromal cell derived protein-1 (SCP-1) (Genebank/D16847) and preadipocyte factor 1 (Pref-1) (Smas and Sul, 1993).
Immunohistochemical analysis has demonstrated a strong association between FA1 and endocrine tissues. In adult tissues, specific staining reactions are seen in the insulin-producing cells of the islets of Langerhans, in the adrenal gland with most pronounced reaction in zona glomerulosa and the somatotroph cells of the pituitary gland (Jensen et al., 1993, 1994; Tornehave et al, 1993; Larsen et al., 1996). FA1 has been demonstrated in endocrine and neuroendocrine tumours (Jensen et al., 1994; Tornehave et al., 1996) and increased serum concentrations have been demonstrated recently in patients with small cell lung carcinoma (Jensen et al., 1999a).
The distribution in fetal and adult tissues suggests that FA1 and/or its membrane-associated precursor may be involved in the development and maintenance of endocrine tissues. The expression of the mRNA dlk and Pref-1 have been discussed in the context of differentiation (Laborda et al., 1993; Smas and Sul, 1993). Smas et al. have also reported a biological effect of the soluble form of Pref-1 ie. mFA1 in preadipocyte differentiation (Smas et al., 1997) and, recently, FA1 has been suggested as a mediator of the antiadipogenic effect of growth hormone in primary rat preadipocytes (Hansen et al., 1998). Stromal cells expressing dlk seem to support the growth of repopulating haematopoietic stem cells in co-cultures as does soluble dlk/FA1 (Moore et al., 1997). In rat pancreas, the highest amounts of Pref-1 mRNA expression coincided with periods of maximum ß-cell proliferation (Carlsson et al., 1997).
An enzyme-linked immunosorbent assay (ELISA) technique for quantification of FA1 has been established and the application of this on normal healthy individuals revealed a serum FA1 reference interval of 12–47 ng/ml, with no diurnal variation, no variation during the menstrual cycle, no response during acute phase reaction and a high renal clearence of 11 ml/min (Jensen et al., 1997).
In order to investigate further the association between FA1 and endocrine tissue, the present report describes the application of immunohistochemistry and quantitative ELISA on tissues and biological fluids of the male and female reproductive systems.
Materials and methods
Biological fluids
All samples were kept at –20°C or –80°C until analysis. Samples were collected as follows:
Serum and corresponding seminal plasma
Serum was drawn weekly from three healthy male volunteers the same day as they delivered a semen sample. Two of the volunteers delivered four corresponding samples while three samples were obtained from the last.
Seminal plasma
Using World Health Organization sperm parameters (WHO, 1992), men attending the Fertility Clinic at Odense University Hospital, Denmark, were diagnosed as: normozoospermic (sperm density >20x106/ml, motile spermatozoa 
50%, n = 187), asthenozoospermic (sperm density >20x106/ml, motile spermatozoa <50%, n = 61), oligozoospermic (sperm density 20x106/ml, motile spermatozoa 50%, n = 27), oligoasthenozoospermic (sperm density 20x106/ml, motile spermatozoa <50%, n = 37) and azoospermic (no spermatozoa, n = 30) subjects; total number of men = 342. Semen samples were centrifuged for 20 min at 2000 g in order to pellet the spermatozoa. The seminal plasma was aspirated and the aliquots were stored at –80°C. Both seminal plasma and the clinical parameters of the semen were analysed.
Seminal plasma from four vasectomized men was kindly made available by Dr Palle Wang, Department of Clinical Biochemistry, Odense University Hospital, Denmark.
Follicular fluids
Multiple follicular fluids (FF) (four and two) were obtained from two women undergoing stimulated in-vitro fertilization (IVF) cycles (kindly provided by Professor J.G. Grudzinskas, Royal London Hospital, London, UK; no further details concerning the size or maturity of the follicles are available). FF from pre-ovulatory follicles and corresponding serum samples from 28 women undergoing IVF treatment were made available by the Fertility Clinic at Odense University Hospital, Denmark.
Immunohistochemistry
Formalin-fixed and paraffin-embedded specimens were cut into 5 µm sections, air-dried and subsequently deparaffinized and re-hydrated. Endogenous peroxidase activity was blocked with H2O2/methanol and antigen-retrieval was performed by incubation with 0.05% (w/v) protease (Sigma, St. Louis, USA, type XIV) in Tris-buffered saline (TBS) at 37°C for 10 min. The primary antibody (monospecific rabbit anti-human FA1) or control antibody (primary antibody liquid-phase absorbed with affinity purified human FA1 as described in Jensen et al., 1993) was diluted 1:100 and the secondary antibody, biotinylated goat anti-rabbit immunoglobulin (IgG; DAKO, Copenhagen, Denmark, E432), 1:200. Following incubation with primary and secondary antibodies, sections were incubated with horseradish peroxidase (HRP)-conjugated strepavidin (Dako P397) diluted 1:300 and developed using 3-amino-9-ethylcarbazol as chromogen. Counter-staining was performed with haematoxylin.
Spermatozoa
Two methods were employed for analysis of FA1 antigen in spermatozoa: (i) a smear of freshly ejaculated semen was allowed to air-dry and subsequently fixed in acetone for 10 min at room temperature before staining as described above; (ii) a freshly ejaculated semen sample was centrifuged in an Eppendorf tube at 1000 g for 20 min, followed by aspiration of the seminal plasma and washing spermatozoa twice with phosphate-buffered saline (PBS), pH 7.4. The resulting pellet was gently overlaid with phosphate-buffered 4% (v/v) formaldehyde and fixed for 24 h. The sperm pellet was carefully scraped onto and packed in filter paper before dehydration and paraffin embedding and processed as described for paraffin embedded tissues.
Tissue specimens
Paraffin-embedded specimens of normal fetal male (n = 1) and female gonads (n = 1) (gestational week 23) as well as adult testes (n = 3), prostate gland (n = 4), epididymis (n = 2), seminal vesicle (n= 2) and ovaries (n = 4) were obtained from the files at the Department of Pathology, Odense University Hospital, Denmark.
Quantification of FA1
FA1 was quantified using a sandwich ELISA technique based on polyclonal anti-FA1 antibodies purified by immunospecific affinity chromatography. The technique and assay parameters have been described in detail previously (Jensen et al., 1997). The assay does not cross-react with human EGF or human growth hormone (Sigma). During the study the inter-assay coefficients of variation, based on three quality controls, were <4%.
Statistical analysis
The Mann–Whitney U-test was used for comparison of differences between independent groups and correlations were tested with Spearman's Rank Correlation test.
Results
Male reproductive system
Tissue localization
Immunohistochemical analysis of adult testis revealed FA1 in the interstitial Leydig cells only (Figure 1A). No staining reaction was seen of the basement membranes, Sertoli cells or the intratesticular germ cells independent of the stage of maturity. Compared with the adult testis, the number of FA1-positive Leydig cells was higher in the fetal testis (Figure 1B). Rete testis, the ducts and the connective tissue as well as the epithelial cells of epididymis, the prostate gland and the seminal vesicle were found to be FA1-negative (not shown).
|
Immunohistochemical analysis of spermatozoa in smears of freshly ejaculated semen and of washed, formalin-fixed and paraffin-embedded spermatozoa revealed no FA1 on the surface of the cells.
FA1 in serum and corresponding seminal plasma
Weekly (3–4 times) serum and seminal plasma samples were obtained from three healthy volunteers. The results of FA1 quantification in serum and corresponding seminal plasma are shown in Figure 2. Both FA1 in seminal plasma and serum revealed titration curves which were parallel with the calibrator applied in the ELISA.
|
The coefficients of variation for weekly serum FA1 in the three volunteers were 0.177, 0.156 and 0.076 respectively. The corresponding coefficients of variation for seminal plasma were 0.336, 0.168 and 0.391. A significant correlation (P < 0.0065, Spearman's Rank Correlation test) was seen between the FA1 concentrations in serum and seminal plasma.
FA1 was also quantified in seminal plasma in men who had been vasectomized (n = 4). The FA1 concentration in these samples did not appear to differ from those obtained from normal subjects (3.4, 3.6, 19.2 and 24.8 ng/ml).
Seminal plasma FA1 and sperm quality
The FA1 concentration was analysed in normal plasma from 342 men attending the Odense Fertility Clinic. The semen samples were categorized into five groups according to WHO criteria, i.e. normozoospermic (n = 187), asthenozoospermic (n = 61), oligozoospermic (n = 27), oligoasthenozoospermic (n = 37) and azoospermic (n = 30). The FA1 concentration varied from 0.8 to 94.9 ng/ml. The seminal plasma concentration of FA1 in groups with low sperm counts, i.e. oligo-, oligoastheno- and azoospermic were significantly lower (P < 0.0001) in comparison with groups with sperm counts >20x106/ml (Figure 3). In the group of normozoospermic men, there was a significant correlation (P < 0.0001) between the concentration of FA1 and the sperm count; this correlation was not seen in the other groups. The distribution of FA1 concentrations in seminal plasma in relation to the groups of sperm quality is shown in Figure 4.
|
|
Female reproductive system
Tissue localization
Immunohistochemical analysis of normal adult ovaries revealed positive staining for FA1 in theca interna cells (Figure 1C
) and hilus cells (not shown), with the most pronounced staining reaction corresponding to the perinuclear Golgi zone. No staining of granulosa cells or oocytes was observed. In an ovary with corpus luteum (Figure 1D), FA1 staining was restricted to the luteinized cells. In an atrophic ovary (Figure 1E), pronounced FA1 staining was seen in cells rich in lipid vesicles. In two ovaries obtained from one fetus (gestational age 23 weeks), FA1 was only observed in a few unidentified cells.
FA1 concentrations in follicular fluids and corresponding serum samples
In order to examine the contribution of FA1 from the theca cells to the FF, FA1 was quantified in FF and corresponding serum samples obtained from 28 women during oocyte retrieval (Table I).
|
FA1 in FF revealed titration curves which were parallel with those of serum samples and the applied calibrator. All serum samples except one (7.1 ng/ml; reference interval >7.5 ng/ml) had FA1 concentrations within the reference interval. The median concentration in FF was 73.3 ng/ml – three times the median concentration of the corresponding serum samples. However, a significant correlation between FA1 in FF and serum was seen (P = 0.0073). In two patients, four and two FF samples were obtained and the FA1 concentrations were 67.9, 99.5, 121.8 and 177, and 39.3 and 30.8 ng/ml respectively.
Discussion
The results presented here further substantiate the connection between FA1, a novel EGF multidomain protein of the EGF superfamily, and the endocrine tissues. The presence of FA1 has been demonstrated previously in insulin secretory granules of the ß-cells within the islets of Langerhans (Jensen et al., 1994), in the somatrotroph cells of the adenopituitary gland (Larsen et al., 1996) and in the adrenal gland with the most pronounced staining reaction corresponding to zona glomerulosa (Jensen et al., 1993). The immunohistochemical technique was applied here to ovary and testis and the results revealed specific staining of the sex hormone producing cells, i.e. the interstitial Leydig cells of the testis and the theca interna and hilus cells of the ovary. These findings seem to support the hypothesis that FA1 is a biologically active protein involved in the development and/or maintenance of function of endocrine tissues (Jensen et al., 1994). Furthermore, the presence of FA1 has been demonstrated in endocrine and neuroendocrine tumours (Tornehave et al., 1996; Jensen et al., 1999a,b).
The presence of FA1 in the sex hormone producing cells of the gonads prompted the analysis of FA1 in the microenvironment of the germ cells, i.e. FF and seminal plasma in relation to the concentration of circulating FA1. This analysis was made possible following development and characterization of an ELISA for quantification of FA1 (Jensen et al., 1997). All biological fluids analysed showed titration curves parallel to that of the calibrator (second trimester amniotic fluid) which makes the given concentrations reliable and suggests immunological identity between FA1 of the calibrator and that of the biological fluids analysed.
The similar concentrations of FA1 observed in both seminal plasma and corresponding serum samples, and the fact that the FA1 concentration in the seminal plasma of vasectomized men was not different from that seen in normal men, suggest that the major source of FA1 in seminal plasma is the circulating pool of FA1. However, a local contribution that influences the sperm quality cannot be excluded. In normozoospermic men (n = 187), we found a significant correlation between the FA1 concentration in seminal plasma and the sperm count. The seminal plasma concentrations of FA1 were lower in groups with low sperm counts, compared with groups with counts of >20x106/ml. These data may indicate a biological function for FA1 in spermatogenesis, and the observation that 65% of the azoospermic patients had FA1 concentrations 5 ng/ml (in contrast to <10% of the groups of men diagnosed as normozoospermic) appear to support this suggestion. These observations indicate a biological role for FA1 in relation to male fertility, but the source of the biologically-active molecule and the mode of action remain to be elucidated.
In contrast to the observations on seminal plasma versus circulating FA1, the FA1 concentrations in FF were found to be three to four times higher than those observed in the corresponding serum samples. In spite of the significant correlation between the concentration of FA1 in FF and serum, the high concentration in FF suggests a major local contribution which may arise from the FA1-positive theca interna cells or, alternatively an active transport of FA1 from the circulation into the FF. The marked differences in FA1 concentrations found in multiple FF (30.8–39.3 and 67.9–177 ng/ml) from the same ovary seem to point to a local contribution rather than a facilitated transport from the circulating pool. It has to be taken into consideration that all women were stimulated and an effect of hormonal treatment cannot be excluded. As with the biological effect in relation to male fertility, the significance of FA1 in the FF and the mode of action also remain to be elucidated.
This novel member of the EGF superfamily, FA1 may be a new tool in the field of human reproduction, but more work is needed to define the biological role and elucidate its clinical potential.
Acknowledgments
This work was supported by grants from The Danish Medical Research Council, Novo Nordisk Foundation, A.J. Andersen og Hustru's Fond, Johanne og Aage Louis-Hansens Fond and The Danish Foundation for Advancement in Medical Sciences.
Notes
3 To whom correspondence should be addressed 
References
Bachmann, E., Krogh, T.N., Højrup, P. et al. (1996) Mouse fetal antigen 1 (mFA1), the circulating gene product of mdlk, pref-1 and SCP-1: isolation, characterization and biology. J. Reprod. Fertil., 107, 279–285.
Carlsson, C., Tornehave, D., Lindberg, K. et al. (1997) Growth hormone and prolactin stimulate the expression of rat preadipocyte factor-1/delta-like protein in pancreatic islets: molecular cloning and expression pattern during development and growth of the endocrine pancreas. Endocrinology, 138, 3940–3948.
Fay, T., Jacobs, I., Teisner, B. et al. (1988) Two fetal antigens (FA1 and FA2) and endometrial proteins (PP12 and PP14) isolated from amniotic fluid: characterization and distribution in fetal and maternal tissues. Eur. J. Obstet. Reprod. Biol., 29, 73–85.
Hansen, L.H., Madsen, B., Teisner, B. et al. (1998) Characterization of the inhibitory effect of growth hormone on primary preadipocyte differentiation. Mol. Endocrinol., 12, 1140–1149.
Jensen, C.H., Teisner, B., Højrup, P. et al. (1993) Studies on the isolation, structural analysis and tissue localization of fetal antigen 1 and its relation to a human adrenal specific cDNA, pG2. Hum. Reprod., 8, 635–641.
Jensen, C.H., Krogh, T.N., Højrup, P. et al. (1994) Protein structure of fetal antigen 1 (FA1) A novel circulating human epiderman-gowth-factor-like protein expressed in neuroendocrine tumors and its relation to the gene products of dlk and pG2. Eur. J. Biochem., 223, 83–92.
Jensen, C.H., Krogh, T.N., Støving, R.K. et al. (1997) Fetal antigen 1 (FA1), a circulating member of the epidermal growth factor (EGF) superfamily: ELISA development, physiology and metabolism in relation to renal function. Clin. Chim. Acta, 268, 1–20.[Web of Science][Medline]
Jensen, C.H., Drivsholm, L., Laursen, I. et al. (1999a) Elevated serum levels of Fetal antigen 1 (FA1), a member of the EGF-superfamily in patients with small cell lung cancer. Tumor Biol., 256–262.
Jensen, C.H., Schrøder, H.D., Teisner, B. et al. (1999b) Fetal antigen 1 (FA1) a member of the EGF superfamily in tissues and serum from patients with neurofibromatosis type 1 (NF-1) Br. J. Dermatol., 140, 1054–1059.[Web of Science][Medline]
Krogh, T.N., Bachmann, E., Teisner, B. et al. (1997) Glycosylation analysis and protein structure determination of murine fetal antigen 1 (mFA1)- the circulating gene product of the delta-like protein (dlk), preadipocyte factor 1 (Pref-1) and stromal-cell-derived protein 1 (SCP-1) cDNAs. Eur. J. Biochem., 244, 334–342.[Web of Science][Medline]
Laborda, J., Sausville, E.A., Hoffman, T. et al. (1993) Dlk, a putative mammalian homeotic gene differentially expressed in small cell lung carcinoma and neuroendocrine tumor cell line. J. Biol. Chem., 268, 3817–3820.
Larsen, J.B., Jensen, C.H., Schrøder, H.D. et al. (1996) Fetal antigen 1 and growth hormone in pituitary somatotroph cells. Lancet, 347, 191.
Moore, K.A., Pytowski, B., Witte, L. et al. (1997) Hematopoietic activity of a stromal cell transmembrane protein containing epidermal growth factor-like repeat motifs. Proc. Natl. Acad. Sci. USA, 94, 4011–4016.
Smas, C.M. and Sul, H.S. (1993) Pref-1, a protein containing EGF-like repeats, inhibits adipocyte differentiation. Cell, 73, 725–734.[Web of Science][Medline]
Smas, C.M., Chen, L. and Sul, H.S. (1997) Cleavage of membrane-associated pref-1 generates a soluble inhibitor of adipocyte differentiation. Mol. Cell. Biol., 17, 977–988.
Tornehave, D., Fay, T.N., Teisner, B. et al. (1989) Two fetal antigens (FA1 and FA2) and endometrial proteins (PP12 and PP14) isolated from amniotic fluid: localization in the fetus and adult female genital tract. Eur. J. Obstet. Gynecol. Reprod. Biol., 30, 221–232.[Web of Science][Medline]
Tornehave, D., Jansen, P., Teisner, B. et al. (1993) Fetal antigen 1 (FA1) in the human pancreas: Cell type expression topological and quantitative variations during development. Anat. Embryol., 187, 335–341.[Medline]
Tornehave, D., Jensen, C.H., Teisner, B. et al. (1996) FA1 immunoreactivity in endocrine tumours and during development of the human fetal pancreas; negative correlation with glucagon expression. Histochem. Cell. Biol., 106, 535–542.[Web of Science][Medline]
World Health Organization (1992) WHO Laboratory Manual for the Examination of Human Semen and Semen–Cervical Mucus Interaction. Cambridge University Press, Cambridge, UK, 107 pp.
Submitted on April 12, 1999; accepted on July 28, 1999.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  S. B. Sonne, K. Almstrup, M. Dalgaard, A. S. Juncker, D. Edsgard, L. Ruban, N. J. Harrison, C. Schwager, A. Abdollahi, P. E. Huber, et al. Analysis of Gene Expression Profiles of Microdissected Cell Populations Indicates that Testicular Carcinoma In situ Is an Arrested Gonocyte Cancer Res., June 15, 2009; 69(12): 5241 - 5250. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Li, M. Nair, D. R. Mackay, V. Bilanchone, M. Hu, M. Fallahi, H. Song, Q. Dai, P. E. Cohen, and X. Dai Ovol1 regulates meiotic pachytene progression during spermatogenesis by repressing Id2 expression Development, March 15, 2005; 132(6): 1463 - 1473. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. E. Shima, D. J. McLean, J. R. McCarrey, and M. D. Griswold The Murine Testicular Transcriptome: Characterizing Gene Expression in the Testis During the Progression of Spermatogenesis Biol Reprod, July 1, 2004; 71(1): 319 - 330. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. H. Jensen, E. I. Jauho, E. Santoni-Rugiu, U. Holmskov, B. Teisner, N. Tygstrup, and H. C. Bisgaard Transit-Amplifying Ductular (Oval) Cells and Their Hepatocytic Progeny Are Characterized by a Novel and Distinctive Expression of Delta-Like Protein/Preadipocyte Factor 1/Fetal Antigen 1 Am. J. Pathol., April 1, 2004; 164(4): 1347 - 1359. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Andersen, C. H. Jensen, R. K. Stoving, J. B. Larsen, H. D. Schroder, B. Teisner, and C. Hagen Fetal Antigen 1 in Healthy Adults and Patients with Pituitary Disease: Relation to Physiological, Pathological, and Pharmacological GH Levels J. Clin. Endocrinol. Metab., November 1, 2001; 86(11): 5465 - 5470. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||