1 Department of Growth & Reproduction and 2 Department of Pathology, Copenhagen University Hospital (Rigshospitalet), DK-2100 Copenhagen, Denmark and 3 Section of Molecular Genetics and Infertility, Department of Gynaecological Endocrinology & Reproductive Medicine, University of Heidelberg, D-69120 Heidelberg, Germany
4 To whom correspondence should be addressed at: Department of Growth & Reproduction, Section 5064, Copenhagen University Hospital (Rigshospitalet), Blegdamsvej 9, DK-2100 Copenhagen, Denmark. e-mail: erm{at}rh.dk
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Abstract |
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Key words: germ cell differentiation/OCT-3/OCT-4/POU5F1/testicular carcinoma in situ
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Introduction |
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Germ cell neoplasms may contain teratomatous elements of any somatic tissue type (Kleinsmith and Pierce, 1964). Despite this histological variability, both seminomas and non-seminomas, including teratomas, are derived from a common precursor cell, carcinoma in situ (CIS) (Skakkebæk, 1972
; Skakkebæk et al., 1987
). Subsequent studies of the phenotype of CIS provided growing evidence for a close similarity between CIS and fetal germ cells (Rajpert-De Meyts et al., 2003
, for review). CIS cells and CIS-derived classical seminoma and embryonal carcinoma retain a high expression of OCT-3/4, which is consistent with their fetal origin (Palumbo et al., 2002
; Looijenga et al., 2003
).
Based on observations of a frequent occurrence of germ cell neoplasia in intersex gonads and studies of expression patterns of selected genes, we proposed that a delay in fetal germ cell differentiation, most probably caused by an abnormal function of somatic cells in developmentally impaired gonads, may lead to the neoplastic transformation (reviewed in Rajpert-De Meyts et al., 1998). Epidemiological studies of an association between temporal and geographical trends in testicular cancer with trends for genital malformations and some forms of male infertility, led us to suggest that these disorders may be aetiologically linked within the testicular dysgenesis syndrome (TDS) (Skakkebæk et al., 2001
). The spectrum of TDS ranges from the presence of few tubules with slightly undifferentiated Sertoli cells to the severely malformed gonad, often with partial sex reversal, and all forms may contain neoplastic changes (Hoei-Hansen et al., 2003
; Skakkebæk et al., 2003
). CIS cells are usually found in the milder forms with retained testicular structure, whereas the severely dysgenetic testes frequently harbour gonadoblastoma, a CIS-like lesion growing in nests resembling primitive ovarian follicle-like structures.
The aims of this study were, first, to establish the ontogenesis of the OCT-3/4 expression as a marker of germ cell pluripotency in the normal fetal gonads of both sexes, and secondly, to examine whether there are any deviations in the normal pattern of expression in the dysgenetic gonads of patients with different forms of TDS, in order to shed some light on the timing of neoplastic transformation.
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Materials and methods |
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The sections were examined under a light microscope (Zeiss, Germany), and the staining was assessed using an arbitrary semiquantitative score: + +: staining in >50% of germ cells in the section; +: staining in 1050% of germ cells; + : staining in 110% of germ cells; +: staining in <1% of germ cells; / +: only single cells among serial sections positive or some specimens in the same age group negative; : no positive cells detected.
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Results |
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A different pattern of expression was observed in the fetal ovaries. Only the earliest specimen in our series, at 9 weeks of gestation (
7 weeks of development), immediately after ovarian differentiation took place, exhibited a strong OCT-3/4 expression in all oogonia (Figure 1B). Thereafter, the expression was retained only in oogonia, and was rapidly down-regulated in oocytes entering the first meiotic prophase in primary follicles. In addition, most of the specimens in the third trimester showed some weak OCT-3/4 staining in the cytoplasm of the primary follicles, which was not observed in the negative controls.
Expression of OCT-3/4 in dysgenetic gonads, including germ cell neoplasms
In the series of specimens isolated from individuals with various disorders of sexual differentiation and gonadal development (Table I), the pattern of expression of OCT-3/4 was roughly similar to that of the normal gonads, but with a few notable exceptions in the postnatal samples. Among the fetal samples, a low number of scattered OCT-3/4-positive gonocytes was detected in one 15 week old specimen with a mosaic isochromosome Y and one 20 week old with the androgen insensitivity syndrome (AIS). Among the postnatal specimens, increased expression of OCT-3/4 was observed in two specimens with complete AIS (Morris syndrome). While the presence of OCT-3/4-positive cells in the younger of the two, a 4 month old infant, can be considered normal, the number of positive cells was greater than in the normal infantile testes. In the second infantile AIS specimen, 9 months old, 10% of pre-spermatogonia were clearly stained, some of them intensely (Figure 1D) which is clearly outside of the normal window of expression. Among other dysgenetic specimens, an ovotestis isolated from a 14 month old genotypic and phenotypic female displayed a large number of OCT-3/4-positive gonocytes, while the adjacent oocytes were negative (Figure 1 F1, F2). All other postnatal intersex or AIS specimens did not contain germ cells positive for OCT-3/4, however, we might have observed more positive cases if a larger series of intersex cases were examined. The same was true for the post-pubertal testicular biopsies obtained from men with infertility, which displayed histological features of gonadal dysgenesis, such as immature tubules with undifferentiated Sertoli cells (Figure 1H). Regardless of the clinical diagnosis, in our series most of the cells positive for OCT-3/4 in specimens from individuals aged >1
years were those displaying clear signs of neoplastic transformation, e.g. gonadoblastoma (Figure 1G) or CIS (Figure 1I, J). The expression of OCT-3/4 in overt tumours (Table I) was high in classical seminoma and embryonal carcinoma but not detectable in spermatocytic seminoma and teratoma, in agreement with previous studies (Palumbo et al., 2002
; Looijenga et al., 2003
).
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Discussion |
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OCT-3/4 was the first marker of embryonic stem cells associated with their pluripotency detected in human blastocytes (Hansis et al., 2000). Here, we demonstrated that in an early human embryo (at 8 weeks of gestation), the expression of OCT-3/4 is no longer detectable in somatic tissues but is maintained at a high level in migrating primordial germ cells. This is consistent with the previously reported presence of the OCT-4 transcripts in human primordial germ cells isolated from male and female fetuses at 10 weeks of gestation (Goto et al., 1999
). In our series, the high expression of OCT-3/4 in fetal testes lasted only a few weeks, and subsequently both the number of positive cells and the intensity of staining rapidly decreased, and remained detectable only in a small proportion of gonocytes in the third trimester and the perinatal period. This decline was not due to the deterioration of specimen quality in older fetuses, which were autopsied, because the same specimens showed strong staining for AMH in Sertoli cells (Figure 1E2; Rajpert-De Meyts et al., 1999
). After birth, rare OCT-3/4-positive cells were observed in testicular specimens at 24
months of age. During this period, a transient increase in the production of testicular hormones occurs, known as the mini-puberty (Forest et al., 1973
), which coincides with the final stage of differentiation of gonocytes into infantile spermatogonia (Hadziselimovic et al., 1986
). Our observations in the human testes are largely in concert with the findings in mice (Schöler et al., 1989
; Palmieri et al., 1994
; Pesce et al., 1998
), with one notable difference; we have not detected any OCT-3/4 expression in either pre- or post-pubertal spermatogonia, whereas in the mouse, type A spermatogonia in the adult mice were clearly Oct-4-positive (Pesce et al., 1998
). Our results are in agreement with a recent study by Looijenga et al., (2003
) who have not observed OCT-3/4 staining in adult spermatogonia. We cannot, however, exclude a possibility that human type A (stem) spermatogonia express OCT-3/4 at a low level undetectable by immunohistochemistry.
In the fetal ovaries, the pattern of the OCT-3/4 expression was different. We noted a rapid decrease in the expression from 1112 weeks of gestation, due to a decline of the number of oogonia, which down-regulated OCT-3/4 while entering the first meiotic prophase. In oocytes, we did not detect OCT-3/4 except for a weak diffuse reaction in the cytoplasm of primordial follicles (seen only in paraformaldehyde-fixed tissues). A similar phenomenon was also observed in murine oocytes (Pesce et al., 1998
), therefore, it is possible that some OCT-3/4 molecules might be transported to the cytoplasm at the onset of meiosis. The biological significance of this phenomenon, if real, remains unknown.
Based on our observations in the human fetal gonads, we conclude that the regulation of the OCT-3/4 is different in male and female germ cells. In ovaries, the down-regulation of OCT-3/4 occurs already very early during fetal life, when the oocytes enter the first meiotic prophase, although OCT-3/4 may be detected in occasional primordial oogonia during later stages of pregnancy. In the male germ cells, this down-regulation is spread over a much longer period, and is associated with a gradual differentiation of primordial germ cells, first into gonocytes and later into infantile spermatogonia. In contrast to the ovary, the down-regulation of OCT-3/4 in the testis appears to occur a long time before the acquisition of meiotic competence. This differential expression illustrates the difference in germ cell development between the sexes, and may provide an explanation why the incidence of germ cell-derived cancer is higher in men than in women. Indeed, the number of OCT-3/4-positive cells after the first trimester is greater, and the window of expression longer in the testis than in the ovary. There may be, however, alternative explanations for a relative excess of germ cell tumours in male gonads, e.g. differences in the regulation of gene expression in immature Sertoli cellswhich are more abundant in the fetal testis than the granulosa cells in the ovary. Whatever the mechanism, we think that a delay in differentiation of germ cells, most probably caused by abnormalities in differentiation of somatic cells, is a key factor in the pathogenesis of malignant transformation of germ cells (Rajpert-De Meyts et al., 1998; Skakkebæk et al., 2001
; Sharpe et al., 2003
).
Our group was the first to notice the similarity between pre-invasive CIS cells and fetal gonocytes (Skakkebæk et al., 1987), and we and others have supported this hypothesis by investigating the pattern of developmental expression of a number of proteins, e.g. PLAP and KIT (Hustin et al., 1987
; Rajpert-De Meyts and Skakkebæk, 1994
; Jørgensen et al., 1995
). KIT, which was previously considered the best marker of stem cells in several lineages and which is highly expressed in CIS, gonadoblastoma and classical seminoma, was demonstrated in fetal germ cells in the testis until at least 19 weeks of development (Robinson et al., 2001
) and was found highly expressed beyond this point in some intersex cases (Rajpert-De Meyts et al., 1996
). As the presence of KIT may increase the survival of germ cells, this pathway was considered one of the possible mechanisms linking a delay in germ cell differentiation with malignant transformation (Rajpert-De Meyts et al., 1998
).
Another mechanism potentially facilitating malignant transformation of germ cells is retained pluripotency due to the presence of factors involved in the maintenance of undifferentiated state, such as OCT-3/4. OCT-3/4 is abundantly expressed only in cells that retain pluripotency, which is a hallmark of embryonic stem cells. The pluripotency may depend upon the precise quantitative expression of Oct-3/4 (Niwa et al., 2000). Furthermore, according to a recent experimental study investigating malignant transformation of embryonic stem cells into teratomas in nude mouse, the oncogenic potential of these cells may also be dependent on the Oct-3/4 activity in a dose-dependent manner (Gidekel et al., 2003
). Our study, therefore, provides additional evidence that human CIS cells, which express high levels of OCT-3/4, are most probably derived from primordial germ cells or early gonocytes that retain features of embryonic stem cells, including abundant OCT-3/4. A high expression of this factor in pre-invasive CIS is, therefore, consistent with its stem-cell-like ability to transform further to tumours which may contain various somatic tissue elements (Palumbo et al., 2002
; Gidekel et al., 2003
; Looijenga et al., 2003
; this study). Accordingly, gonocytes with low expression of OCT-3/4 at the end of gestation and in the infantile period would be less likely to transform into CIS. However, in some intersex cases which display a delay in the differentiation of the gonocytes, e.g. in a testis of a 9 month old girl with AIS or in an ovotestis of a 14 month old 46,XX female, we detected OCT-3/4-positive cells resembling early gonocytes. A transforming event in such persisting immature cells can perhaps occur in some cases postnatally. However, we believe that in the majority of cases, malignant transformation of germ cells occurs in early fetal life, and is not a post-pubertal phenomenon, with the possible exception of spermatocytic seminoma, an OCT-3/4-negative tumour of elderly men, which is not derived from CIS but most probably from spermatogonia (Skakkebæk et al., 1987
; Looijenga et al., 2003
). The outstanding questions concern the mechanisms involved in the regulation of OCT-3/4 expression in gonocytes, and factors which may disturb this regulation causing the arrest of gonocytes in a pluripotent stem cell-like stage prone to neoplastic transformation. The growing incidence of testicular germ cell cancer in recent decades highlights the need for further exploration of the mechanistic pathways of early germ cell differentiation.
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Acknowledgements |
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FOOTNOTES |
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Submitted on February 10, 2004; accepted on March 23, 2004.