Hum. Reprod. Advance Access originally published online on January 17, 2008
Human Reproduction 2008 23(3):589-599; doi:10.1093/humrep/dem411
Expression of pluripotent stem cell markers in the human fetal ovary
1 Institute for Cellular Engineering, Department of Gynecology and Obstetrics, Johns Hopkins University School of Medicine, Broadway Research Building Suite 771, 733 N. Broadway, MD 21205, USA 2 Department of Gynecology and Obstetrics, Stanford University, 300 Pasteur Drive, Stanford, CA 94305, USA
3Correspondence address. Tel: +1-410-614-3444; Fax: +1-410-955-7427; E-mail: ckerr{at}jhmi.edu
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Abstract |
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BACKGROUND: Human primordial germ cells (PGCs) can give rise to pluripotent stem cells such as embryonal carcinoma cells (ECCs) and embryonic germ cells (EGCs).
METHODS: In order to determine whether PGCs express markers associated with pluripotency in EGCs and ECCs, the following study cross examines the expression patterns of multiple pluripotent markers in the human fetal ovary, 5.5–15 weeks post-fertilizaton (pF) and relates this expression with the ability to derive pluripotent EGCs in vitro.
RESULTS: Specific subpopulations were identified which included OCT4+/Nanog+/cKIT+/VASA+ PGCs and oogonia. Interestingly, these cells also expressed SSEA1 and alkaline phosphatase (AP) and SSEA4 expression occurred throughout the entire gonad. Isolation of SSEA1+ cells from the gonad resulted in AP+ EGC colony formation. The number of OCT4+ or Nanog+ expressing cells peaked by week 8 and then diminished after week 9 pF, as oogonia enter meiosis. In addition, the efficiency of EGC derivation was associated with the number of OCT4+ cells. TRA-1-60 and TRA-1-81 were only detected in the lining of the mesonephric ducts and occasionally in the gonad.
CONCLUSIONS: These results demonstrate that PGCs, a unipotent cell, express most, but not all, of the markers associated with pluripotent cells in the human fetal ovary.
Key words: human/fetal/ovary/embryonic germ cells/primordial germ cells
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Introduction |
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In the developing fetal ovary, germ cells have been historically identified using morphological criteria as well as alkaline phosphatase (AP) and periodic acid Schiff staining. Based on these criteria, primordial germ cells (PGCs) have been identified in the hindgut at 4 weeks post-fertilization (pF) and observed to migrate to colonize the developing gonads by 7 weeks pF (Witschi, 1948
; Gondos et al., 1971). By the end of the fifth week or early in the sixth week of gestation, it has been estimated that 
1000 PGCs begin to actively migrate from the dorsal mesentery into the gonadal anlage (Baker, 1963; Pinkerton et al., 1961; Makabe et al., 1992; Motta et al., 1997). Between 7 and 9 weeks, gonadal differentiation occurs at which time germ cells are referred to as gonocytes and begin to develop into oogonia. During this time, germ cells along with somatic, granulosa cells arrange into cords and sheets without specific organization (Gondos, 1985). These oogonia then continue mitotic proliferation until the onset of meiosis as oocytes around 10–11 weeks pF which then extends into the second trimester (Skrzypczak et al., 1981, Gondos et al., 1986; Francavilla et al., 1990; Rabinovici and Jaffe, 1990). AP activity is lost just after germ cells enter meiosis (Baker and Franchi, 1967; Sun and Gondos, 1984; Makabe et al., 1992).
Although this knowledge provides the cornerstones of early human germ cell development, very little is known regarding their gene expression. The immergence of embryonic stem cells (ESCs) from human germ cells as well as the origin of germ cell tumors (GCTs) in women questions the pluripotent nature of the germ cells present in the developing gonad (Looijenga et al., 2003). Indeed, recent studies investigating GCTs in women have examined the expression of pluripotent stem cell markers in the germline of the fetal ovary. Based on OCT4 expression, these studies have suggested that the loss of pluripotency does not occur until late into the first trimester and occurs at the onset of the first meiotic prophase followed by an increase in VASA and cKIT expression during the second trimester (Rajpert-De Meyts et al., 2004; Stoop et al., 2005; Hoei-Hansen et al., 2007). However, these studies fall short in characterizing distinct populations of germ cells as they progress through early development, and this information could be critical for increasing our understanding of germ cell development and the role of pluripotency in germ cell fate. Thus, the purpose of this study is to distinguish these populations by comparing the expression of multiple germ cell and stem cell markers in the fetal ovary during its early development. Pluripotentiality of the germ cells are also investigated in terms of their ability to derive a pluripotent stem cell line, embryonic germ cells (EGCs).
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Collection of tissue
Gonadal tissues were obtained using a protocol approved by the Joint Committee on Clinical Investigation of the Johns Hopkins University School of Medicine from human fetuses 5–15 weeks pF as a result of termination of pregnancy. Gestational age was estimated through a comparison of anatomical markers including crown heel and crown rump measurements, limb and digit formation and also the first day of the last maternal menstrual cycle. Ages are discussed in terms of fetal development and not the age from the last menstrual period. After acquisition, all tissue was immediately prepared for either cryopreservation or paraffin embedding.
PGC isolation and EGC derivation
PGCs were isolated using magnetic cell sorting (MACS) technology using an indirect labeling of cells with magnetically-tagged goat anti-mouse IgM antibodies toward a mouse-anti SSEA1 antibody. Briefly, gonads were minced in 1 mg/ml collagenase and incubated at 37°C for 20 min, rinsed, incubated with 1:5 antibody for 30 min on ice. Afterwards the secondary antibody was applied at 1:100 dilution for another 30 min on ice. After a final rinse, cells were applied on magnetic columns and sorted off the column in culture media using manufacturer’s instructions (Miltenyi Biotech, Auburn, CA). PGCs were grown in culture and EGC derivation was propagated as described previously (Shamblott et al., 1998; Kerr et al., 2006). Briefly, PGCs are grown on gamma-irradiated STO feeder layers in growth media consisting of Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA) supplemented with 15% fetal bovine serum (Hyclone, Logan, UT), 0.1 mM nonessential amino acids (Invitrogen), 0.1 mM 2-mercaptoethanol (Sigma, St. Louis, MO), 2 mM glutamine (Invitrogen), 1 mM sodium pyruvate (Invitrogen), 100 U/ml penicillin (Invitrogen), 100 µg/ml streptomycin (Invitrogen), 1000 U/ml human recombinant LIF (Millipore, Billerica, MA), 1–2 ng/ml human recombinant FGF-2 (R&D Systems) and 10 µM forskolin (Sigma), prepared in DMSO. Media was changed daily and cultures were split 1:2 every 10 days onto new feeder layers. EGC colonies first appear around 10–15 days following subculture. Once established, EGC cultures can be maintained for several months under these conditions.
EGCs were efficiently produced from PGCs isolated from a single ovary. Depending on the age of the tissue, the number of AP+ PGCs isolated by MAC varied dramatically from around 20 000 cells isolated at 6 weeks pF to around 72 000 by 8 weeks pF per ovary consistent with the number of germ cells reported during this time period (Bendsen et al., 2006). PGCs are distinguished in cell culture by AP staining. AP does not stain somatic cells of the ovary during this time period or the feeder layers. PGCs present as single AP+ cells in culture which disappear between 2–3 weeks according to the loss of AP expression and as expected, the rate at which PGCs disappear in culture occurs more rapidly in older tissues. Post-meiotic cells have not been identified and only fibroblastic-spindle shaped cells appear to be present after this time.
Cryostaining
Fresh tissue was rinsed in Dulbecco’s PBS (DPBS), frozen in O.C.T. TissueTek freezing compound (Bayer Corp., Pittsburgh, PA) and stored at –80°C. For immunohistochemistry, tissue blocks were cut in 5 µm sections, placed on slides (ProbeOn Plus, Fisher Scientific, Waltham, MA) and immediately prepared for indirect immunofluorescent staining. Sections were either fixed in 4°C acetone for 10 min to detect cell surface markers, or in 4% paraformaldehyde for 10 min to detect nuclear markers. Antibodies and the concentrations used are summarized in Table I. Briefly, cell surface antibodies were diluted in 15% goat serum in DPBS and incubated on sections for 1 h at room temperature, and nuclear antibodies were diluted in 5% goat serum and incubated overnight at 4°C. All antibodies were detected by using fluorescently-labeled goat anti-mouse secondary antibodies (1:200 dilution; Molecular Probes, Eugene, OR) in 15% goat serum in DPBS for 1 h at room temperature. Sections were counterstained with DAPI (Sigma) and mounted using ProLong antifade mounting medium (Molecular Probes). Sections stained with a nuclear and cell surface antibody followed the protocol for nuclear staining. Negative controls were also performed on each gonad including incubations with secondary antibodies only and with mouse ascites fluid.
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Fluorescent in-situ hybridization
The sex of each gonad was determined using CEP X (SpectrumOrange)/CEP Y (SpectrumGreen) DNA probes (Vysis, Des Plaines, IL). Slides were prepared as described above, fixed in Carnoy’s fixative for 45 s, pretreated in 1 M sodium thiocyanate for 5 min at 75°C and post-fixed in 100% methanol for 1 min. Sections were then denatured in 60% formamide + 2x SSC (sodium chloride and citric acid) buffer, pH 5.3, at 75°C for 3 min, followed by 1 min in cold 70, 95 and 100% ethanol and then incubated with CEP X/Y DNA probe overnight at 37°C. The following day, sections underwent three post-hybridization washes in 60% formamide (Sigma) + 0.3% NP40 (Igepal, Sigma) +2x SSC, pH 5.3. Sections were then counterstained with DAPI and mounted as described previously.
Microscopic imaging
Fluorescent images were captured at 40–400x magnification using a Nikon Eclipse E800 microscope (Nikon, Inc., Melville, NY) equipped with a 4–40x Plan Apo lens. Alexa Fluor 594, Alexa Fluor 568 and CEP X SpectrumOrange probe fluorescence was detected using a G2ERHOD 541551 nm excitation filter, a 575 nm dichroic mirror and a barrier filter with a band width of 590. Alexa Fluor 488 and CEP Y SpectrumGreen probe was detected using a FITC excitation filter, a 505 nm dichroic mirror and a barrier filter with a band width of 515–555 nm. DAPI and ELF 97 was detected using a standard DAPI/Hoechst filter set, UV 2E/C 340–380 nm excitation filter, 400 nm dichroic mirror and a barrier filter with a band width of 435–485 nm. Barrier filters were manufactured by Chroma, Inc. (Burlington, VT). Images were captured with a Photometrics 20 MHz cooled interlined CCD camera and imported into Metamorph software, v.6.2 (Universal Imaging Corp., Downingtown, PA).
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Results |
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Stem cell marker expression was observed in 35 ovaries between 5.5 and 15 weeks pF using the antibodies summarized in Table I. Each ovary provided 100–150 full-length sagittal sections (at 5µ thickness) from which a karyotype and molecular signature of every marker was performed. Therefore, the characterization of each marker presented in this study was tested in 3–5 replicates from each age group, week pF. Importantly, expression presented here was consistent within similar age groups. The result shown in Fig. 1 is representative of Fluorescence in-situ hybridization in a female gonad showing typical red staining of the X chromosomes in the nuclei of cells compared with the green Y staining seen in a male gonad.
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Stage-specific embryonic antigens SSEA1 and SSEA4 are developmentally regulated during early embryogenesis and are widely used as markers to monitor the differentiation of pluripotent EGCs, ESCs and embryonic carcinoma cells (ECCs) (Henderson et al., 2002
). However, the developmental expression of these antigens during human germ cell development is unclear. Fig. 2 summarizes the co-expression of pluripotent cell surface antigens, SSEA1 and SSEA4 in the human fetal ovary at 5.5, 8, 11 and 15 weeks pF. At 5.5 weeks pF, SSEA1 expressing PGCs (arrows) are shown migrating from the mesonephric ducts into the undifferentiated gonadal anlage, whereas SSEA4 appears to be generally expressed by the gonad proper. By 8 weeks pF, there is a dramatic increase in the number of SSEA1 expressing PGCs throughout the medullary region along with an active migration into the cortex of the differentiating gonad. Following the onset of meiosis around 9 weeks pF as reported by others, a dramatic decrease in the number of SSEA1 expressing cells occurs such that by 11 weeks pF the SSEA1 expression is weaker and the remainder of expressing cells are predominately located in the cortex. While results showed that all cells that express SSEA1 are positive for SSEA4, SSEA4 expression also occurred throughout the gonad in somatic cells surrounding the cords. To ensure the staining of SSEA4 was not a procedural artifact, negative controls for SSEA4 were also included using secondary antibodies alone and with mouse ascites fluid. Furthermore, cells within the rete ovarii as well as in the mesonephros did not stain. Another marker of mouse PGCs, EMA1, was also studied and was indistinguishable from the SSEA1 staining (data not shown). Identification of SSEA1+ cells as germ cells is confirmed with the co-expression of SSEA1 and AP in Fig. 5.
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SSEA1 and SSEA4 expression has been demonstrated in mouse PGCs. Therefore to confirm that the expression observed in the gonad was occurring specifically in human PGCs, we compared this staining with other markers of germ cell development. During the first trimester, results showed that OCT4 and cKIT expression also demonstrated a pattern similar to SSEA1 (Figs 3 and 4, respectively). Reports that OCT4 expression is lost at meiosis and other reports that show the expression of cKIT decreases following the onset of meiosis may explain the higher number of cKIT+ cells compared with OCT4+ cells between 10 and 13 weeks as germ cells begin this transition. However, by 13 weeks pF, cKIT expression was no longer detected, whereas OCT4 expression remained robust in cells in the cortex (Fig. 4, inset). In fact, OCT4 expression was consistent up to 15 weeks pF in all second trimester gonads studied (n = 9). As differences in their expression suggested distinguishable populations, comparison expression profiles of these markers and others were performed to delineate the progression of these cells in the developing ovary. The results confirmed that SSEA1+ cells also expressed all three markers of pluripotency and early germ cell development, OCT4, cKIT and AP (Fig. 5). Importantly, most SSEA1+ cells consistently expressed all three markers between 7.5 and 8.5 weeks pF. However, some SSEA1+ cells did not appear to express OCT4 and cKIT in independent experiments suggesting that the cells are further along in differentiation. Identification of these cells warrant future investigations.
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Importantly, the majority of OCT4+ cells also expressed cKIT, NANOG and VASA between 8–9.5 weeks pF (Fig. 6). However, not all cKIT+ cells express OCT4, consistent with other reports that demonstrate cKIT expression is still present following meiosis (Horie et al., 1993
; Robinson, 2001; Stoop et al., 2005), while OCT4 expression is lost past the oogonia stage (Stoop et al., 2005). Taken together, these data suggest the presence of cKIT+OCT4– cells in the oogonia to oocyte transition. The number of OCT4–/cKIT+ cells increased between 8 and 10 weeks pF, followed by a sharp decline in cKIT+ cells by 13 weeks pF. OCT4 expression was also compared with another pluripotent stem cell marker, Nanog, as well as an early germ cell marker, VASA. Fig. 6 shows that while OCT4+ cells also expressed NANOG, most OCT4+ cells by 8 weeks pF also expressed a more mature marker of germ cell fate, VASA. This pattern of VASA expression was consistent with a previous study which demonstrated cytoplasmic staining in oogonia and maturing oocytes in 15 and 17 week pF ovaries (Castrillon et al., 2000). By 13 pF a substantial number of OCT4–/VASA+ cells were found, consistent with the development into oocytes at this stage.
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SSEA1+ PGCs is also supported in vitro, by the isolation of PGCs with the same antibody against SSEA1 used in MACS. After sorting, SSEA1+ cells grown in culture also expressed AP and produced EGC colonies after one week (Fig. 7). Evidence of AP expression in these cells suggest that they are not undergoing meiosis. Additional RT–PCR analysis also demonstrated that isolated SSEA1+ cells did not express markers of meiosis including SCP1 and SCP3 (data not shown). Collectively these data suggest that PGCs initially express SSEA1, SSEA4, OCT4, Nanog, cKIT, AP and VASA and that there remain some cells which are SSEA1+/SSEA4+/OCT4+ into the second trimester. Interestingly, the stem cell markers TRA-1-60 and TRA-1-81 showed intense staining in the lining of the mesonephric and Mullerian ducts, while TRA-1-60 expression was only found in a couple of cells from 2 out of the 35 specimens studied (Fig. 8). Since human EGCs express both tumor antigens in vitro, these results suggest that the expression of these two markers are induced in culture, proposing a possible association between their expression and the pluripotentiality of the EGCs in culture.
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Collectively these data demonstrate a population which express some but not all pluripotent stem cell markers that we propose to define PGCs. The rounded morphology and the large nucleus to cytoplasmic ratio in the cells which express OCT4 are consistent with PGC morphology. These PGCs maintain active in proliferation during gonadal differentiation and persist into the second trimester despite the loss of cKIT expression in the second trimester. Furthermore, when the number of OCT4+ cells was compared with the efficiency of deriving pluripotent EGC lines in vitro (Fig. 9), they were associated, suggesting that the initial population of cells founding EGC colonies comes from an OCT4+ PGC population. No EGC colonies were derived by 13 weeks pF.
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3± SEM) and (B) the number of OCT4 positive cells counted per microscopic field at the same age (closed square) (n |
Discussion |
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The present study provides a comprehensive evaluation of PGC development in the human ovary using comparative expression analysis of several markers associated with early germ cell and stem cell identity across multiple specimens from both the first and second trimester. This was accomplished using a combinatorial approach looking at biochemical and immunohistochemical characterization of PGCs in the early gonad as well as those isolated by the stem cell marker, SSEA1. Importantly, the study demonstrates the expression of multiple genes shown collectively in identical germ cells in vivo. Although other studies have also observed expression of some markers during fetal development, information from this study describes the co-expression of multiple pluripotent stem cell markers during early germ cell development to help evaluate the pluripotent nature of these cells.
Historically, studies attempting to identify PGCs in the human fetal gonad have been restricted to AP activity and morphological characteristics, including rounded morphology and the large eccentric nuclei, to separate PGCs from somatic cells (Witschi, 1963; Baker and Franchi, 1967; Fukuda et al., 1975). Since then, stem cell surface markers have been identified that are expressed by mouse PGCs and as such inferred to be expressed by human PGCs as well. These markers include: EMA1, SSEA1, SSEA4, TRA-1-60 and TRA-1-81 (Solter and Knowles, 1979; Kannagi et al., 1983, Andrews et al., 1984; Hahnel and Eddy, 1987). However, except for a few reports of TRA-1-60 expression, none of these markers have been shown to bind to human PGCs in the developing ovary. These markers represent stage-specific embryonic antigens that are developmentally regulated in the inner cell mass (ICM) during early embryogenesis in mouse and human and as such are widely used as markers to monitor the differentiation of ESCs, EGCs and ECCs. However, it is not clear whether differences in expression patterns found among these lines indicates significant alterations in their pluripotent state, or whether this corresponds to a distinction in their progenitor cell types. For instance, human ESCs are characterized by the expression of the cell surface antigens SSEA3, SSEA4, TRA-1-60, TRA-1-81, and by the lack of SSEA1, while human EGCs also express SSEA1 (Shamblott et al., 1998; Henderson et al., 2002). This study demonstrates for the first time the expression of SSEA1 and SSEA4 in human PGCs of the fetal ovary. While SSEA1 appeared to be exclusively expressed by only AP+ PGCs, SSEA4 was also expressed throughout the gonadal anlage. Importantly, specific staining of the SSEA4 antibody was conferred by absence of staining in the rete ovarii, dorsal mesentery and mesonephros as well as in negative controls. At the same time, SSEA1 and EMA1 demonstrated identical expression patterns, although since the antibodies to both antigens were the same isotype, their staining could not be studied on the same sections. Other investigations have also suggested that SSEA1 and EMA1 recognize similar antigens (De Felici et al., 2004). Upon further examination, SSEA1 and SSEA4 also co-expressed AP, OCT4, cKIT and VASA in gonocytes or preoogonia. Although it appeared that all SSEA1+ cells expressed AP, the majority but not all expressed OCT4 and cKIT suggesting populations which are further along in differentiation. After 13 weeks pF, most germ cells which were SSEA1–/OCT–/cKIT–/VASA+ were found in the medullary region which we suggest are developing oocytes, while SSEA1+/OCT+/cKIT–/VASA– oogonia remained in the cortex.
Oct4, or Pou5f, is a transcriptional regulator found in ESCs, in cancer cells and in germ cells following embryonic development (Goto et al., 1999; Hansis et al., 2000; Looijenga et al., 2003). Information regarding the expression of OCT4 in germ cell development during the first trimester has been limited to two studies each based on only one specimen using either an RT–PCR analysis on a 10 week pF specimen (Goto et al., 1999) or by using an immunohistochemical approach on one 9.5 week pF and several second trimester ovaries (Rajpert-De Meyts et al., 2004). Another report also studied several specimens from the second and third trimester (Stoop et al., 2005). Together, these reports suggested that the cells expressing OCT4 were oogonia and early oocytes not involved in folliculogenesis and that these germ cells irreversibly loose pluripotency once meiosis occurs.
In this report, we describe for the first time the expression of multiple pluripotent markers in the earliest specimens evaluated at 5.5 weeks pF, as well as in multiple specimens throughout the first trimester. In the earliest specimen examined, 5.5 weeks pF, OCT4 expression was limited to a small number of cells located at the cranial end of the undifferentiated gonad. When serial gonadal sections were stained and the number of OCT4+ cells counted, the results were consistent with the number of PGCs reported by Baker (1963) (1000 germ cells) at this age (Baker, 1963). The identity of these cells was further confirmed by their expression of Nanog, cKIT and VASA. In fact, this study has shown for the first time a developmental pattern of expression of Nanog in the fetal ovary during the first trimester. One study has reported on the lack of Nanog expression in the second trimester (Hoei-Hansen et al., 2007). However, in this study Nanog was consistently expressed in all fetal ovaries examined from 5.5 to 15 weeks pF albeit at weaker levels of expression in the later stages.
By 7 weeks pF, OCT4+/Nanog+ cells could be seen throughout the gonad, dispersed evenly in the medullary and cortex regions. By 8 weeks pF, during which time the gonad is undergoing sexual differentiation into a committed ovary, the number of cells expressing OCT4 increased and began occupying the cortex. By 11 weeks pF, the number of OCT4+/Nanog+ cells had decreased and were primarily located in the outer cortex. This pattern of OCT4/Nanog expression supports the compartmentalization theory of ovarian development in which the cortex supports the expansion of immature germ cells, whereas the medulla supports germ cell maturation. The number of OCT4+/Nanog+ germ cells present in the beginning of the second trimester is consistent with a previous report which also demonstrated high numbers of OCT4+ cells in the cortex at 13 and 15 weeks pF (Stoop et al., 2005).
In addition to OCT4 and Nanog, the expression of SSEA1 and SSEA4 was also compared with the stem cell marker, cKIT. cKIT is a type III receptor tyrosine kinase involved in cell signaling and cell–cell interaction and is found in early germ cells and in most stem cells. In particular, cKIT has also been found to be involved in survival and proliferation of migrating mouse PGCs and to be expressed in various cells in the human fetal ovary (Molyneaux and Wylie, 2003). In this study, cells expressing cKIT also expressed OCT4 and SSEA1 suggesting a population of PGCs which expressed all three markers up to 13 weeks pF. Subsequently, cKIT staining was greatly diminished, whereas OCT4 and SSEA1 persisted in cells between 13–15 pF. While the results from the first trimester ovaries are consistent with other reports demonstrating cKIT expression alone (Jorgensen et al., 1995; Hoyer, 2005), the dramatic reduction of cKIT expression in the gonads during the beginning of the second trimester suggested a newly defined population of germ cells which reside in the outer cortex during this period of time. Indeed, several other papers investigating the expression of cKIT in the early second trimester had also suggested a decrease in cKIT that was attributed to meiotic-associated transitions of oogonia into oocytes (Horie et al., 1993; Robinson et al., 2001; Stoop et al., 2005). While this does not address the dramatic reduction reported here, it may explain the inability of cells isolated from the gonad during this time to become EGCs in culture. Importantly, to our knowledge this is the first report demonstrating the co-expression of cKIT with multiple pluripotent identifiers during the first trimester.
To further investigate differences between the two SSEA1+/OCT4+ populations which either expressed cKIT during colonization or did not express cKIT after the onset of meiosis, comparisons with another marker of PGC maturation, VASA was also studied. VASA is a member of the DEAD-box family of RNA helicases and is exclusively expressed in the germ cell lineage (Castrillon et al., 2000). In humans, VASA expression has been detected by immunohistochemistry in migratory PGCs at 4–5 weeks pF (gestational age 6 and 7 weeks) but is most abundantly expressed in mature oocytes (Castrillon et al., 2000). In this study, both OCT4+/VASA+ and OCT4+/VASA– cells could be found up to 11 weeks pF even though most cells expressed both OCT4 and VASA at 8 weeks pF. Additionally, VASA expression progressively increased between 8–15 weeks pF such that by 13 weeks pF most of the VASA staining was localized to OCT4–/Nanog–/cKIT– germ cells in the inner cortex and medullary regions, consistent with higher expression in maturing oocytes than in oogonia (Castrillon et al., 2000; Stoop et al., 2005). Thus, a population of OCT4+/SSEA1+/SSEA4+ cells persist in the ovarian cortex by the second trimester which do not express cKIT or VASA. This population we propose are oogonia which express OCT4, but can not produce EGCs in culture.
Interestingly, when the expression of TRA-1-60 and TRA-1-81 was investigated, it was not generally observed in the gonad, but on the surface epithelium lining the mesonephric and Mullerian ducts. Although the (Andrews, Banting, Damjanov, Arnaud and Avner) original report on these antibodies stated that TRA-1-60 and TRA-1-81 were not expressed in gonadal tissue of various ages, this is the first comprehensive report of TRA-1-81 expression in the early gonad. In contrast, one previous report demonstrated in paraffin sections the expression of TRA-1-60 in the majority of germ cells in the first-trimester gonads (Jorgensen et al., 1995). As differences in immunohistochemical procedures and sensitivity of the antibodies can result in these discrepancies, further investigation is warranted. However, it is interesting to postulate the differences in the expression of these antigens in EGCs in culture versus the nature of PGCs in the gonad. Despite the pluripotent markers found in PGCs in this study, the lack of expression of the TRA antigens questions whether these antigens have roles in a conversion between PGCs into EGC in culture and ECC in vivo and what role this may have in pluripotency. Interestingly, expression of several tumor antigens expressed in various types of tumors and in the germs cells of normal adult testis have now been reported in the ovary as early as the second trimester (Nelson et al., 2007). It has been suggested that these cancer/testis antigens are not expressed in early PGCs, as are TRA-1-61 and TRA-1-80, but are expressed later in development, primarily in oogonia and spermatogonia (Takahashi et al., 1995; Jungbluth et al., 2000). Therefore, although TRA-1-61 and TRA-1-80 are not expressed in the adult (Andrews et al., 1984), a more detailed study of their expression into the late second and third trimester is warranted.
In summary, our data provide a number of interesting findings regarding the early development and differentiation of human germ cells in the fetal ovary. PGCs are characterized by expression of OCT4, Nanog, cKIT, AP, SSEA1, SSEA4 and VASA. These cells also show pluripotent potential in culture as is demonstrated by the ability of MACS-sorted SSEA1+ cells to develop EGCs in vitro. It is interesting to note that this study provides several novel insights into differences between early germ cell development and pluripotency. First, EGC derivation does not occur after 11 weeks pF concomitant with the loss of cKIT expression despite the expression of OCT4. Second, this study has suggested both the pluripotentiality of PGCs in expressing OCT4 and Nanog as well as in terms of a more differentiated state. For instance, PGCs did not express the tumor antigens TRA-1-60 and TRA-1-81 associated with pluripotent human EGCs, ECCs and ESCs. Noteworthy also is the the expression of SSEA1 in PGCs and EGCs compared with the lack of expression in ICM, ESCs and ECCs, suggesting a unique role of SSEA1 in germ cell fate compared with mouse in which both ESCs and EGCs express this antigen. Taken together, these differences in gene expression between PGCs, EGCs and ESCs illustrate the importance of defining the pluripotent state in terms of not only the gene expression of a few markers but also the understanding the heterogeneity in gene expression within progressively diverse populations with pluripotent potential. Particularly, it emphasizes the necessity of future studies in defining the multiple steps or pathways involved in demonstrating the ability of a stem cell to retain or acquire pluripotentiality. Based on an extensive dataset, 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 the pluripotentiality of human germ cells in early development.
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The Institute for Cellular Engineering, Johns Hopkins University. Baltimore, MD.
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We would like to thank Drs Ann Burke, Rameet Singh, Roxanne Jamshidi and Ann Lawler as well as the Birth Defects Laboratory at University of Washington for their assistance in acquiring tissue. We would also like to thank Joyce Axelman for her unwavering support and assistance in cell culture. Their enthusiasm and patience throughout this endeavor is greatly appreciated.
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References |
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Submitted on August 1, 2007; resubmitted on October 31, 2007; accepted on December 5, 2007.
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