1 Department of Pathology, Josephine Nefkens Institute, Erasmus MC-University Medical Center Rotterdam, Daniel den Hoed Cancer Center, Rotterdam, The Netherlands and 2 Department of Hematology/Oncology, University of Tübingen, Tübingen, Germany
3 To whom correspondence should be addressed: Department of Pathology, Erasmus MC-University Medical Center Rotterdam, Josephine Nefkens Institute, Room 430b, P.O.Box 1738, 3000 DR Rotterdam, The Netherlands. Email: l.looijenga{at}erasmusmc.nl
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Abstract |
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Key words: differentiation/fetal ovary/germ cells/immunohistochemistry/proliferation
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Introduction |
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In order to further investigate the distribution and timing of proliferation and differentiation, we undertook an extensive study on the presence of a number of markers involved in either proliferation or maturation of normal female germ cells spanning the intrauterine period between week 15.5 gestational age and the neonatal period. In the human ovary, this crucial period includes the transition from oogonial replication by mitosis to primordial follicle formation. In this study we investigated the maturation process seen in normal development of the ovary and assess its potential as a reference for future identification of pathological processes of maturation.
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Materials and methods |
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Histochemical and immunohistochemical stainings
Stainings were performed as described before (Stoop et al., 2001). For immunohistochemistry, sections were incubated with the primary antibody overnight at 4 °C [placental/germ-like cell alkaline phosphatases (PLAP), c-KIT, VASA,
-catenin, E-cadherin] or 2 h at room temperature (OCT3/4, Ki-67). The primary and secondary antibodies used are indicated in Table I. All slides were counterstained with haematoxylin. For PLAP,
-catenin and E-cadherin, positive staining of the Fallopian tube, and for c-KIT, the presence of mast cells were used as an internal positive control. Negative controls were performed by omitting the primary antibody, resulting in complete absence of signal.
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To semiquantitatively assess expression of the markers investigated, cells showing a positive signal were counted in representative visual fields (magnification x200) containing surface areas of both the medullary and the cortical region of the ovary in comparable proportions in all cases. In addition, cells positive for PLAP, OCT3/4 and Ki-67 were counted in three representative high power fields (magnification x400) of both the medullary and cortical regions of seven cases (gestational ages 15.5, 20, 23, 28, 33, 35 and 36 weeks) to assess regional differences. For quantitative analysis of OCT3/4, the percentage of positive cells among the total number of germ cells (as identified by histology) was assessed in three cases (gestational ages 15.5, 28 and 36 weeks) counting three representative visual fields (magnification x200). The counting was performed independently by three different observers (H.S., F.H. and M.C.) who were unaware of the gestational age at the time-point of the investigation.
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Results |
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The proliferation index was assessed using Ki-67, which showed a nuclear staining in both early germ cells and interstitial cells throughout the whole period of ovarian development investigated in this series. A marked decrease in the number of positive cells was observed after weeks 2224 of gestational age, mainly due to the cessation of mitosis of germ cells. This indicates that proliferating germ cells contribute significantly to the overall number of Ki-67 positive cells in fetal ovaries. In germ cells, Ki-67 was observed mainly in oogonia (see Figure 2A). Yet, whereas the majority of oocytes engaged in folliculogenesis did not show Ki-67 expression, it was occasionally observed in oocytes of primordial follicles, where it was strictly confined to the nucleolus. Perinatally, only a few cells, mainly interstitial cells or granulosa cells, were positive.
The highest number of germ cells positive for PLAP was seen in the earliest stages of fetal development examined, and decreased sharply after 25 weeks gestational age (Figure 1). PLAP was detected in early germ cells predominantly located in the cortical region (Figure 2B and Table II). After birth, PLAP positive germ cells were hardly ever seen, with a maximum of one to three positive cells per visual field. PLAP expression was not restricted to germ cells, but was also seen in the Fallopian tube and occasionally in the epithelial lining of the ovary.
Nuclear staining for OCT3/4 was restricted to germ cells and the overall staining pattern was comparable to PLAP, yet total numbers of germ cells positive for OCT3/4 were somewhat higher (Figure 1 and Table II). In addition to oogonia, OCT3/4 was occasionally seen in early oocytes, but was never detectable in cells involved in folliculogenesis (Figure 2C). Similar to the expression pattern of Ki-67 and PLAP, a decline in the expression of OCT3/4 in fetal ovaries was seen at 24 weeks of gestation. At term and in ovaries of neonates, hardly any positive germ cells were detectable.
Immunohistochemical detection of c-KIT, -catenin/E-cadherin and VASA
A comparable staining pattern was seen for c-KIT, -catenin and E-cadherin (Figure 3AF). Both in early, immature germ cells (oogonia) and at all later stages of folliculogenesis, these factors were predominantly localized at the membrane, but sometimes also showed a cytoplasmic localization (see also Figure 1). In primordial follicles, all three factors showed strong signal intensity at sites of cellcell interaction (Figure 3AF). As
-catenin and E-cadherin were expressed in the same cells in adjacent slides in similar localization, these factors could be linked, forming an E-cadherincatenin complex in germ cells. In addition to their presence in germ cells, these factors were also seen in epithelial cells of the Fallopian tube, serving as an internal positive control.
VASA was observed in the cytoplasm of germ cells, both oogonia and oocytes, at all gestational ages and after birth (Figure 1G and H). In addition to oogonia and early oocytes, all stages of follicle maturation showed presence of VASA. Signal intensity varied with maturation of germ cells: early germ cells showed only weak staining for VASA, and signal intensity increased with maturation and was strongest in primordial follicles (Figure 3G). The total number of germ cells, determined by VASA, decreased with gestational age: whereas at 15.5 weeks 600 germ cells, mostly oogonia and early oocytes, were seen per visual field, the average number of germ cells at term was
100200 per visual field.
Results of double-stainings
Double-stainings were performed to assess correlations or differences of markers at different stages of maturation. A total of nine cases of 15.5, 18, 24 (three different cases), 28, 33, 36 weeks gestational age, and a neonate of 2 weeks, born at term, were stained for six different combinations: OCT3/4 and PLAP, PLAP and c-KIT, OCT3/4 and c-KIT, OCT3/4 and VASA, OCT3/4 and -catenin, and PLAP and Ki-67 (Figure 2DH respectively). The results of all double-staining experiments were in accordance with the results of the respective single-staining experiments. Staining for OCT3/4 and PLAP revealed that a higher number of germ cells were positive for OCT3/4 than for PLAP, with a constant ratio of 1.4:1 observed for all ages investigated (Figure 2D). PLAP was never detected in germ cells negative for OCT3/4, and expression of OCT3/4 was still observed at later stages of development, when PLAP was already undetectable. Double-staining for PLAP and c-KIT showed that at all gestational ages more germ cells were positive for c-KIT than for PLAP. PLAP was never detected in oogonia without expression of c-KIT, but in contrast to PLAP, c-KIT remained positive throughout maturation, including different stages of folliculogenesis (Figure 2E). In accordance with these findings, OCT3/4 and c-KIT were found in comparable numbers only in early germ cells, whereas during folliculogenesis, c-KIT remained positive in germ cells that had already lost OCT3/4 expression (Figure 2F). Similar staining patterns were observed for the combinations OCT3/4 and VASA, and OCT3/4 and
-catenin (not shown), where co-expression was restricted to early germ cells, whereas expression of OCT3/4 was no longer seen at later stages of maturation. Interestingly, an inverse correlation was found between the presence of OCT3/4 and the staining intensity of VASA. Oogonia were positive for OCT3/4, but only weakly positive for VASA, whereas primordial follicles staining strongly for VASA no longer showed OCT3/4 (Figure 2G). Double-staining for PLAP and Ki-67 revealed that significantly more cells were positive for Ki-67 than for PLAP at all ages. Furthermore, not all early germ cells (identified by PLAP expression) were proliferating at the same time (Figure 2H). A proportion of germ cells of between 50% at earlier and 37% at later gestational ages showed co-expression of both markers. This indicates that in addition to a decrease in the absolute number of early germ cells, these cells are also less likely to proliferate at later stages of ovarian development. During the third trimester, expansion of the pool of early germ cells (indicated by co-expression of Ki-67 and PLAP) has basically ceased.
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Discussion |
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In the following paragraphs, the most interesting findings of the individual markers included in this study will be discussed in more detail.
To assess proliferation, we used Ki-67, a nuclear protein that is present in all phases of the cell cycle, but is absent in G0-phase cells (Gerdes et al., 1984). Overall expression of Ki-67 in both germ cells and cells of non-germ cell origin (mostly interstitial cells and granulosa cells) was highest in the earliest case investigated (15.5 weeks), and decreased steadily throughout the second and third trimester. Around term, almost all germ cells (mostly oocytes in primordial follicles) and the majority of cells of non-germ cell origin have entered a quiescent phase. Expression of Ki-67 was markedly higher in the cortex than in the medulla at earlier ages, whereas no differences between the two regions were found during the third trimester (Table II). To assess the proportion of germ cells of overall Ki-67 positive cells and examine the percentage of proliferating germ cells, we performed double-stainings using a combination of PLAP and Ki-67. Of all cells positive for Ki-67,
50% were early germ cells. The percentage of proliferating versus quiescent germ cells was 45 to 50% at 15.5 and 20 weeks, and between 37 and 40% of the few early germ cells found at week 28 gestation and 3 weeks after birth respectively. This indicates that immature germ cells at early stages show strong proliferation around weeks 15.5 to 20, whereas at later ages both the number and the fraction of proliferating germ cells decreases. At term, the expansion of germ cells has almost ceased. This finding is in line with earlier reports on germ cell numbers during the prenatal period (for review, see Rabinovici and Jaffe, 1990
). Interestingly, a small number of oocytes in primordial follicles showed presence of Ki-67 in the nucleolus. Similar findings have been reported previously, suggesting a role of this factor not only during mitosis, but also during meiosis (Wrobel et al., 1996
; Traut et al., 2002
). The number of positive meiotic cells was too small to influence the overall conclusion, and did not allow conclusions regarding possible physiological meaning.
Alkaline phosphatases are regarded as archetypal onco-fetal proteins. They are detectable at the mammalian blastocyst stage and have been described in germ cells of human embryos of <4 weeks gestational age (Pinkerton et al., 1961). PLAP expression has been described in primordial germ cells at 813 weeks and at later ages in human oogonia not yet engaged in cellcell interactions with somatic cells, whereas ovaries of neonates and infants were devoid of any immunopositivity (Hustin et al., 1990
; Gould et al., 2000
). Our finding that PLAP expression is restricted to early germ cells, most likely oogonia, is in line with these previous reports. The highest expression was seen in the cortex, where the number of positive cells decreased steadily with advancing age. In the medulla, differences at different ages were less pronounced after week 20. In our study, PLAP was still occasionally detected in germ cells of neonates shortly after birth, albeit at very low numbers.
OCT3/4 is a transcriptional regulator, expressed exclusively in pluripotent human embryonic stem cells and germ cells, including PGC (Goto et al., 1999; Hansis et al., 2000
). In addition, presence of OCT3/4 has been described in germ cell tumour cells with pluripotent potential such as embryonal carcinoma and seminomas (Looijenga et al., 2003
). In fetal human testes, OCT3/4 has been found to be highly expressed in PGC between weeks 17 and 24 and to a lesser extent at later stages (Looijenga et al., 2003
). Expression of OCT3/4 in normal and dysgenetic human ovaries has been reported recently (Rajpert-De Meyts et al., 2004
). In our analysis, OCT3/4 was occasionally seen in early oocytes, but never in germ cells involved in folliculogenesis, indicating that human female germ cells irreversibly lose pluripotency once they progress to meiosis and engage in a close cellcell interaction with granulosa cells. It is therefore tempting to speculate that both the processes of cellcell interaction and down-regulation of OCT3/4 are important for germ cell maturation. It should be noted that this is different from the situation found in mouse follicles, where germ cells after completion of meiotic prophase I express OCT3/4 (see Table IV) (Pesce et al., 1998
). However, the exact mechanisms of interaction are not yet understood, and deserve further investigation. In this context it is interesting that ovarian teratomas originate from germ cells at different stages of meiosis (Surti et al., 1990
). The finding that these tumours show a restricted potential to differentiate and exclusively demonstrate somatic differentiation is in line with previous reports that the presence of OCT3/4 in embryonic stem cells is crucial to keep these cells in an undifferentiated state (Niwa et al., 2000
). We therefore speculate that ovarian teratomas arise from germ cells that have already lost expression of OCT3/4. Our findings demonstrate the value of both PLAP and OCT3/4 for the characterization of immature germ cells in the female gonad and indicate that these markers can be useful for the identification of developmentally arrested germ cells in pathological conditions, e.g. in dysgenetic gonads, as has been described recently (Rajpert-De Meyts et al., 2004
).
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c-KIT, -catenin, and E-cadherin are all involved in cell signalling and cellcell interaction. c-KIT is a type III receptor tyrosine kinase found on early germ cells, whereas the ligand stem cell factor (SCF or KIT-L) is present in granulosa cells (Matzuk et al., 2002
, for review). The c-KIT/SCF system has been found to be involved in survival and proliferation of migrating germ cells in mice (McLaren, 1992
). In human intrauterine gonadogenesis, c-KIT expression has been described in female PGC at the period of arrival of PGC at the gonadal ridges at week 7 and later between 13 and 21 weeks of gestational age (Horie et al., 1993
; Jørgensen et al., 1995
; Robinson et al., 2001
). During folliculogenesis, c-KIT has been postulated to play a role in germ cell survival, possibly by up-regulating Mcl-1, an anti-apoptotic member of the Bcl-2 family (Hartley et al., 2002
).
Whereas some data are available on the cadherincatenin complex in mouse PGC development and oocyte maturation (Ohsugi et al., 1999; Di Carlo and De Felici, 2000
), little is known about the role of these factors in human germ cell development. In an earlier analysis, the presence of E-cadherin on human oocytes has been demonstrated (Campbell et al., 1995
). Here we show that c-KIT,
-catenin and E-cadherin are all present in germ cells throughout all stages of female intrauterine development. The presence of these factors not only in maturing oocytes and germ cells during folliculogenesis, but also in oogonia could be demonstrated by double-stainings combining markers for early germ cells such as OCT3/4 and PLAP with c-KIT and
-catenin. As oogonia are often found in clusters, these factors seem to be involved in signalling between neighbouring immature germ cells. In primordial follicles, expression of these factors was predominantly seen in oocytes at the site of cellcell interaction with granulosa cells. This suggests that cellcell contacts play an important role, both for immature germ cells and later between oocytes and surrounding granulosa cells. In fact, E-cadherin has been reported to be required for germ cell determination in PGC (Okamura et al., 2003
), and in oocyte growth and acquisition of meiotic competence in mice (Carabatsos et al., 2000
). The functional importance of E-cadherin has further been demonstrated by dissociation/reassociation experiments in a mouse model: dissociated cells from gonads were unable to reform their initial contacts when cultured in the presence of an antibody to E-cadherin (Mackay et al., 1999
).
The total numbers of germ cells seen in one visual field, as assessed by either c-KIT, -catenin or VASA at different gestational ages (see Figure 1), are in accordance with previous reports, where a steady decrease of germ cells from around weeks 1820 gestational age resulted in numbers that were only one-sixth of peak values at term (Baker, 1963
). We therefore conclude that assessment of cell numbers in one representative visual field at magnification x200 and including comparable surface areas of cortical and medullary areas is a reliable method for semiquantitative analyses. Our results allow some interesting comparisons with data on mouse germ cell maturation (see Table IV).
Taken together, our data provide a number of interesting findings regarding the development and differentiation of human germ cells in the fetal ovary. Immature germ cells are characterized by expression of PLAP and show pluripotent potential, as is illustrated by the expression of OCT3/4. During the first part of the second trimester included in our study (gestational ages of weeks 15.520), the number of immature germ cells is high, and the pool of these cells decreases significantly after weeks 2224. In accordance with previous reports, regional differences in the number of immature germ cells were found when comparing the cortex and medulla of the ovary. This finding supports the model of compartmentalization, in which the cortex is the area where expansion of immature germ cells take place, the medulla providing the environment needed for germ cell maturation. Accompanying the gradual shift from oogonia and early oocytes to germ cells engaged in formation of primordial follicles, loss of expression of first PLAP and consecutively OCT3/4 can be observed. Interestingly, in male fetal germ cells loss of pluripotency as judged by loss of OCT3/4 is observed at the time when gonocytes become attached to the basal membrane of the seminiferous tubules and come into close contact with nurturing Sertoli cells (Honecker et al., 2004). As down-regulation of OCT3/4 in female germ cells occurs at a time when oocytes become surrounded by granulosa cells and start to form primordial follicles, a major role of cellcell interactions in the maturation of germ cells in both sexes can be postulated. Expression of c-KIT,
-catenin, and E-cadherin in germ cells at all stages of germ cell development points towards a role of these factors for germ cell survival and maturation. Our findings help to define normal germ cell development and maturation in the human fetal ovary and will serve as a reference for further analyses investigating both normal and pathological processes in germ cell maturation, i.e. in dysgenetic gonads or in individuals showing chromosomal aberrations (Kersemaekers et al., 2005
, M.Cools et al., unpublished data).
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Acknowledgements |
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Notes |
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Submitted on February 19, 2004; resubmitted on December 24, 2004; accepted on January 20, 2005.