Hum. Reprod. Advance Access originally published online on April 22, 2004
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Human Reproduction, Vol. 19, No. 6, 1338-1344,
June 2004
© 2004 European Society of Human Reproduction and Embryology
Developmental expression of POU5F1 (OCT-3/4) in normal and dysgenetic human gonads*
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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BACKGROUND: To investigate how long fetal germ cells retain pluripotency, which may be linked to their ability to transform into histologically variable tumours, we examined the expression of OCT-3/4 (POU5F1), a transcription factor essential for the maintenance of totipotency in embryonic stem cells. METHODS: The ontogeny of expression of OCT-3/4 was studied in 74 specimens of normal human gonads during development and in 58 samples of gonads from cases with testicular dysgenesis syndrome (TDS), including disorders of sex differentiation and malignant changes. RESULTS: OCT-3/4 expression was found in primordial germ cells during migration to the gonadal ridges and in the indifferent gonad. The expression in testes gradually decreased until
20 weeks of gestation, and thereafter it was more rapidly down-regulated, but persisted in a few cells until 3–4 months of postnatal age, which coincides with the final differentiation of gonocytes into infantile spermatogonia. Subsequently, OCT-3/4 was not detected in normal testes. In the ovaries, OCT-3/4 was expressed in primordial oogonia, but was down-regulated in oocytes that formed primary follicles. The pattern of expression was heterogeneous in dysgenetic and intersex cases, with OCT-3/4-positive gonocytes detected in this series until 14 months of age. Visibly neoplastic gonadoblastoma and carcinoma in situ (CIS) expressed abundant OCT-3/4 regardless of the age. CONCLUSIONS: In the human ovary, OCT-3/4 is silenced at the onset of the first meiotic prophase, whereas in the testis, down-regulation of OCT-3/4 is a gradual process associated with differentiation of gonocytes. This normal pattern of expression is disturbed in dysgenetic gonads, especially in rare intersex cases, thus increasing the risk of malignant transformation. The high abundance of OCT-3/4 in CIS cells is consistent with their early fetal origin and pluripotency.
Key words: germ cell differentiation/OCT-3/OCT-4/POU5F1/testicular carcinoma in situ
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Introduction |
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Self-renewal and pluripotency with a tightly controlled ability to differentiate are the hallmarks of stem cells. The mechanisms involved in this process have only recently begun to be unravelled. One of the few transcription factors known to be involved in the self-renewal of embryonic stem cells is an octamer-binding protein, OCT-4, also known as OCT-3 (Pan et al., 2002
). This factor is a member of the POU family of transcription factors, therefore its gene is designated as POU5F1 (Schöler et al., 1989; Okamoto et al., 1990; Rosner et al., 1990). Murine Oct-3/4 is expressed very early in embryogenesis, initially in all blastomeres, and later in the totipotent inner cell mass only (Palmieri et al., 1994; Brehm et al., 1998; Pan et al., 2002). The same pattern of expression was also observed in human blastocysts (Hansis et al., 2000). After implantation, the expression is progressively down-regulated and remains active only in primordial germ cells (Rosner et al., 1990; Schöler et al., 1990). During gonadal development, the expression of Oct-4 in mouse remains high until the onset of meiosis, with marked differences between males and females (Pesce et al., 1998). The ontogenesis of fetal expression of OCT-3/4 in humans has not been systematically studied, except for a recent report of the immunoreactivity of fetal germ cells between 17 and 37 weeks of gestation, examined as a part of an extensive study of germ cell tumours and other tumour types (Looijenga et al., 2003).
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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Tissue samples
The series included 52 normal fetal tissue samples (36 testicular specimens and 16 ovaries) from tissue archives of the Copenhagen University Hospital. The specimens were obtained after induced or spontaneous abortions and stillbirths, mainly due to placental or maternal problems. The developmental age was calculated from the date of the last menstrual bleeding, supported by the foot size of the fetus. The sex of the undifferentiated gonad was determined by in situ hybridization. Normal postnatal testicular samples (n = 22) were obtained either from infants who died suddenly of causes unrelated to the reproductive system or as testicular biopsies performed in boys with acute leukaemia for monitoring the spread of disease. Pathological specimens comprised a series of 35 dysgenetic gonads from individuals with intersex disorders or TDS. The 22 intersex cases included mixed gonadal dysgenesis (45,X/46,XY) with and without gonadoblastoma, the androgen insensitivity syndrome (AIS), true hermaphroditism, Prader–Willi syndrome and the adrenogenital syndrome (congenital adrenal hyperplasia). The remaining specimens (n = 21) included testicular biopsies with some dysgenetic features (e.g. undifferentiated tubules and/or microliths) with or without CIS, obtained from young adult men with subfertility or with contralateral germ cell tumours. Finally, 12 samples of overt testicular germ cell tumours, including classical seminoma, non-seminomas and spermatocytic seminomas, were examined as controls. Diagnosis of the tumours was confirmed by morphological criteria and immunohistochemical staining for placental-like alkaline phosphatase (PLAP) (Jacobsen and Norgaard-Pedersen, 1984
). All specimens are listed in Table I. Most of the fetal and pre-pubertal tissues were described in detail in our previous studies (Jørgensen et al., 1995; Rajpert-De Meyts et al., 1996, 1999). The use of these samples for immunohistochemical studies was reviewed and approved by a regional ethics committee.
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Immunohistochemistry
The tissue samples were fixed in Stieve’s fluid, buffered formalin or Cleland’s fluid (few) and subsequently embedded in paraffin. Commercially available antibodies were used, including a polyclonal goat anti-OCT-3/4 antibody (C-20, sc 8629; Santa Cruz Biotechnology Inc., USA), raised against an epitope mapped to the C-terminus of the protein, and validated on Western blots by the manufacturer. In addition, several other antibodies were used as controls, either for diagnostic purposes to detect neoplastic cells (e.g. anti-PLAP, a monoclonal antibody from DakoCytomation; anti-KIT, a polyclonal antibody from Santa Cruz Biotechnology) or for the assessment of tissue preservation in the autopsy specimens, e.g. anti-Müllerian hormone (AMH), a marker for immature Sertoli cells which are present in pre-pubertal testes, both normal and dysgenetic, throughout development (Rajpert-De Meyts et al., 1999
). A monoclonal anti-AMH antibody was kindly provided by R. Cate from Biogen, USA. The immunohistochemical staining was performed using a standard indirect peroxidase method, as previously described for other antibodies (Rajpert-De Meyts and Skakkebæk, 1994; Rajpert-De Meyts et al., 1999). Briefly, the dewaxed and rehydrated sections fixed in Cleland’s fluid were heated in a microwave oven to unmask the antigen, while sections fixed in formalin or Stieve’s fixative were not heated. Subsequently, the sections were incubated with H2O2 to inhibit the endogenous peroxidase, followed by diluted human serum to block non-specific binding sites. The incubation with the primary anti-OCT-3/4 antibody diluted 1:200–1:400 in a background-decreasing buffer (DakoCytomation, Denmark) was carried out overnight at 4°C. For negative control, a serial section from each block was incubated with non-immune goat serum or a dilution buffer. Subsequently, a biotinylated rabbit–anti-goat link antibody was applied (Zymed; USA), followed by the horseradish peroxidase–streptavidin complex (Zymed). Between all steps the sections were thoroughly washed. The bound antibody was visualized using acetyl carbazole (DakoCytomation). Some sections were lightly counterstained with Mayer’s haematoxylin to mark unstained nuclei. 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 10–50% of germ cells; + –: staining in 1–10% 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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The ontogeny of OCT 3/4 expression in the normal human testes and ovaries
The results of the immunohistochemical stainings are listed in Table I. The intensity of staining was slightly stronger in specimens fixed in formaldehyde in comparison to those fixed in Stieve’s or Clelan’s fixatives (as illustrated in a specimen with CIS in Figure 1I, J). In all tissue specimens the staining was present exclusively in germ cells, and was confined to the cell nucleus, with the exception of faint cytoplasmic staining observed in some oocytes in meiotic prophase.
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The strongest OCT-3/4 expression was observed in the earliest stages of gonadal development. The specimen isolated from a genetically male fetus at 8 weeks of gestation (
6 weeks of development) with gonadal ridges in the process of the gonad formation, showed clearly OCT-3/4-positive primordial germ cells, some still on their migration route (Figure 1A). Primordial germ cells were also strongly positive for OCT-3/4 in a male gonad at 10 weeks of gestation when the sex cords just begun to form (Figure 1C). Subsequently, for 3–4 following weeks, virtually all gonocytes in the developing testes displayed a strong OCT-3/4 expression. From 15 weeks of gestation, both the number of OCT-3/4-positive gonocytes and the intensity of staining began to decline gradually. In a specimen of 19 weeks gestational age, 50% of gonocytes were positive. Around 20 weeks of gestation, the expression of OCT-3/4 started to decrease more rapidly, with only 5–10% positive gonocytes observed at week 22, and 1–2% at week 26. Subsequently, only single weakly stained gonocytes were observed; some specimens required several serial sections to detect just one positive nucleus. Such a pattern persisted until the first postnatal months; the oldest specimen in our series with a few nuclei weakly positive for OCT-3/4 was from a 4 month old infant. Thereafter, all pre-pubertal, peri-pubertal and adult testicular samples in our series were consistently negative. No staining was observed in spermatogonia, or any other stage of spermatogenesis.
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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The present study established the ontogeny of expression of OCT-3/4 in normal human testes and ovaries during fetal development and investigated putative aberrations of this expression pattern in gonads of subjects with various forms of intersex or TDS. The results showed a marked difference between the chronology of OCT-3/4 expression in the testis versus the ovary, and demonstrated that the high expression of OCT-3/4 is indeed specific for embryonic stem cells, primordial germ cells in both sexes and early testicular gonocytes. Consequently, the strong OCT-3/4 expression in CIS and gonadoblastoma provides evidence that these cell types are likely to be derived from embryonic/early fetal germ cells.
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 2–4 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 11–12 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 cells—which 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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The authors wish to thank Ms Lene Andersen for excellent technical assistance. The study was supported by grants from The Danish Cancer Society, The Svend Andersen Foundation and The Danish Medical Research Council.
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FOOTNOTES |
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* Preliminary data were presented at the 42nd Annual ESPE Meeting, Ljubljana, Slovenia, September 2003.
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Submitted on February 10, 2004; accepted on March 23, 2004.
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R.T. Mitchell, G. Cowan, K.D. Morris, R.A. Anderson, H.M. Fraser, K.J. Mckenzie, W.H.B. Wallace, C.J.H. Kelnar, P.T.K. Saunders, and R.M. Sharpe Germ cell differentiation in the marmoset (Callithrix jacchus) during fetal and neonatal life closely parallels that in the human Hum. Reprod., August 11, 2008; (2008) den295v1. [Abstract] [Full Text] [PDF]
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D. M. Kristensen, J. E. Nielsen, N. E. Skakkebaek, N. Graem, G. K. Jacobsen, E. R.-D. Meyts, and H. Leffers Presumed pluripotency markers UTF-1 and REX-1 are expressed in human adult testes and germ cell neoplasms Hum. Reprod., April 1, 2008; 23(4): 775 - 782. [Abstract] [Full Text] [PDF]
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C. L. Kerr, C. M. Hill, P. D. Blumenthal, and J. D. Gearhart Expression of pluripotent stem cell markers in the human fetal ovary Hum. Reprod., March 1, 2008; 23(3): 589 - 599. [Abstract] [Full Text] [PDF]
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M. F. Gjerstorff, K. Kock, O. Nielsen, and H. J. Ditzel MAGE-A1, GAGE and NY-ESO-1 cancer/testis antigen expression during human gonadal development Hum. Reprod., April 1, 2007; 22(4): 953 - 960. [Abstract] [Full Text] [PDF]
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 A. M. Wikstrom, C. E. Hoei-Hansen, L. Dunkel, and E. Rajpert-De Meyts Immunoexpression of Androgen Receptor and Nine Markers of Maturation in the Testes of Adolescent Boys with Klinefelter Syndrome: Evidence for Degeneration of Germ Cells at the Onset of Meiosis J. Clin. Endocrinol. Metab., February 1, 2007; 92(2): 714 - 719. [Abstract] [Full Text] [PDF]
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C.E. Hoei-Hansen, E. Carlsen, N. Jorgensen, H. Leffers, N.E. Skakkebaek, and E. Rajpert-De Meyts Towards a non-invasive method for early detection of testicular neoplasia in semen samples by identification of fetal germ cell-specific markers Hum. Reprod., January 1, 2007; 22(1): 167 - 173. [Abstract] [Full Text] [PDF]
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 J. Houldsworth, J. E. Korkola, G. J. Bosl, and R. S. K. Chaganti Biology and Genetics of Adult Male Germ Cell Tumors J. Clin. Oncol., December 10, 2006; 24(35): 5512 - 5518. [Abstract] [Full Text] [PDF]
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 B. Aflatoonian and H. Moore Germ cells from mouse and human embryonic stem cells. Reproduction, November 1, 2006; 132(5): 699 - 707. [Abstract] [Full Text] [PDF]
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 D. Ferrara, N. Hallmark, H. Scott, R. Brown, C. McKinnell, I. K. Mahood, and R. M. Sharpe Acute and Long-Term Effects of in Utero Exposure of Rats to Di(n-Butyl) Phthalate on Testicular Germ Cell Development and Proliferation Endocrinology, November 1, 2006; 147(11): 5352 - 5362. [Abstract] [Full Text] [PDF]
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 M. Cools, S. L. S. Drop, K. P. Wolffenbuttel, J. W. Oosterhuis, and L. H. J. Looijenga Germ Cell Tumors in the Intersex Gonad: Old Paths, New Directions, Moving Frontiers Endocr. Rev., August 1, 2006; 27(5): 468 - 484. [Abstract] [Full Text] [PDF]
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 T.-Y. Ling, M.-D. Kuo, C.-L. Li, A. L. Yu, Y.-H. Huang, T.-J. Wu, Y.-C. Lin, S.-H. Chen, and J. Yu Identification of pulmonary Oct-4+ stem/progenitor cells and demonstration of their susceptibility to SARS coronavirus (SARS-CoV) infection in vitro PNAS, June 20, 2006; 103(25): 9530 - 9535. [Abstract] [Full Text] [PDF]
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M. Cools, H. Stoop, A.-M. F. Kersemaekers, S. L. S. Drop, K. P. Wolffenbuttel, J.-P. Bourguignon, J. Slowikowska-Hilczer, K. Kula, S. M. H. Faradz, J. W. Oosterhuis, et al. Gonadoblastoma Arising in Undifferentiated Gonadal Tissue within Dysgenetic Gonads J. Clin. Endocrinol. Metab., June 1, 2006; 91(6): 2404 - 2413. [Abstract] [Full Text] [PDF]
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 E. Rajpert-De Meyts Developmental model for the pathogenesis of testicular carcinoma in situ: genetic and environmental aspects Hum. Reprod. Update, May 1, 2006; 12(3): 303 - 323. [Abstract] [Full Text] [PDF]
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K. Pauls, H. Schorle, W. Jeske, R. Brehm, K. Steger, N. Wernert, R. Buttner, and H. Zhou Spatial expression of germ cell markers during maturation of human fetal male gonads: an immunohistochemical study Hum. Reprod., February 1, 2006; 21(2): 397 - 404. [Abstract] [Full Text] [PDF]
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A. Sutcliffe, H. A. Spoudeas, D. Nair, P. Bouloux, T. Oliver, P. Sambrook, W. Bannister, C. B. Lambalk, and T. Spector Comparison of serum FSH and Inhibin B levels between adult male dizygotic and monozygotic twins Hum. Reprod., February 1, 2006; 21(2): 447 - 450. [Abstract] [Full Text] [PDF]
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M. Cools, K. van Aerde, A.-M. Kersemaekers, M. Boter, S. L. S. Drop, K. P. Wolffenbuttel, E. W. Steyerberg, J. W. Oosterhuis, and L. H. J. Looijenga Morphological and Immunohistochemical Differences between Gonadal Maturation Delay and Early Germ Cell Neoplasia in Patients with Undervirilization Syndromes J. Clin. Endocrinol. Metab., September 1, 2005; 90(9): 5295 - 5303. [Abstract] [Full Text] [PDF]
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 C. E. Hoei-Hansen, E. Rajpert-De Meyts, G. Daugaard, and N. E. Skakkebaek Carcinoma in situ testis, the progenitor of testicular germ cell tumours: a clinical review Ann. Onc., June 1, 2005; 16(6): 863 - 868. [Abstract] [Full Text] [PDF]
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H. Stoop, F. Honecker, M. Cools, R. de Krijger, C. Bokemeyer, and L.H.J. Looijenga Differentiation and development of human female germ cells during prenatal gonadogenesis: an immunohistochemical study Hum. Reprod., June 1, 2005; 20(6): 1466 - 1476. [Abstract] [Full Text] [PDF]
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 C. E. Hoei-Hansen, J. E. Nielsen, K. Almstrup, S. B. Sonne, N. Graem, N. E. Skakkebaek, H. Leffers, and E. R.-D. Meyts Transcription Factor AP-2{gamma} Is a Developmentally Regulated Marker of Testicular Carcinoma In situ and Germ Cell Tumors Clin. Cancer Res., December 15, 2004; 10(24): 8521 - 8530. [Abstract] [Full Text] [PDF]
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