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Biology of Reproduction 61, 1146-1151 (1999)
©Copyright 1999 Society for the Study of Reproduction, Inc.

Articles

Differentiation of Murine Premigratory Primordial Germ Cells in Culture1

Ami J. Richardsa, George C. Endersb, and James L. Resnick2,a

a Department of Molecular Genetics and Microbiology, University of Florida College of Medicine, Gainesville, Florida 32610-0266 b Department of Anatomy and Cell Biology, School of Medicine, University of Kansas Medical Center, Kansas City, Kansas 66160-7400


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In the mouse embryo, primordial germ cells first appear in the extraembryonic mesoderm and divide rapidly while migrating to the fetal gonad. Shortly after their arrival in the gonad, germ cells sexually differentiate as proliferation ceases. Previous studies have established that primordial germ cells proliferate and migrate in feeder layer culture. To explore cellular regulation of fetal germ cell development, we have used germ cell nuclear antigen 1 (GCNA1), a marker normally expressed only in postmigratory germ cells, to investigate the developmental potency of both pre- and postmigratory cells in this culture system. We found that explanted premigratory germ cells will initiate expression of this marker and are, therefore, capable of undertaking some aspects of gonocyte differentiation without intimate exposure to the fetal gonad. We have also tested whether postmigratory gonocytes are stable in culture. As detected by either alkaline phosphatase or GCNA1, we did not detect long-term survival of either prospermatogonia or oogonia under conditions that support the survival, proliferation, and differentiation of earlier premigratory cells. These observations are consistent with an autonomous cellular mechanism governing the initial stages of gonocyte differentiation, and suggest that differentiation towards gonocytes is accompanied by a change in requirements for cell survival.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
During mammalian embryogenesis, primordial germ cells (PGCs) undergo a highly ordered pattern of allocation, migration, proliferation, and continuing differentiation. The germline of the mouse embryo is first detectable at 7–8 days postcoitus (dpc) as a small population of surface alkaline phosphatase-expressing cells in the extraembryonic tissue near the base of the allantois [1]. Over the next several days, the PGCs migrate to the genital ridge, the gonadal anlage. Upon arrival in the genital ridge, germ cells are referred to as gonocytes, or as oogonia and prespermatogonia after sexual differentiation. Migration temporally overlaps the period of PGC proliferation, so that by embryonic Day 12 the PGCs are located in the genital ridge, and by about 13.5 dpc germ cell proliferation is complete [2–5]. Prospermatogonia mitotically arrest awaiting reactivation as spermatogonia after birth. In female embryos, the oogonia directly enter prophase I of meiosis. By the time of birth, most oogonia have reached the diplotene stage [6].

Although the patterns of PGC migration, proliferation, and differentiation have been known for many years, the regulation of fetal germ cell development is still poorly understood. Mutations at several growth factor and receptor loci indicate the importance of intercellular signaling in the PGC development. The Sl and W genes encode steel factor (SLF, stem cell factor) and its cognate receptor c-kit, respectively. Mutations at either locus result in decreased PGC numbers and increased frequency of ectopic germ cells [4, 7–9]. Another mutant, gcd, also exhibits decreased PGC numbers [10]. Mapping of this mutation to chromosome 11 near several members of the leukemia inhibitory factor (LIF) cytokine family suggests a role for this ligand family in germ cell development [11].

The roles of these and other factors on PGCs have been primarily tested in a transient cell culture assay. In this assay, germ cell-bearing embryo fragments are disrupted to a single cell suspension and plated on mitotically inactivated feeder layers. As several cell types are present in these primary cultures, PGCs are detected and quantitated by their surface alkaline phosphatase reaction. Under these culture conditions, PGCs survive, proliferate, and migrate [12]. While all functions of the feeder layer are not known, presentation of a transmembrane-bound form of SLF is necessary for optimal PGC survival [13–15].

PGCs explanted into this culture system exhibit an unusual pattern of accumulation. Regardless of the age of the embryo at the time of explant, the PGCs will accumulate in vitro until the time corresponding to about embryonic Day 12–13 in vivo. After this peak, PGC numbers, as measured by their alkaline phosphatase reaction, unfailingly decline over several days. Because PGC proliferation in vivo also ceases by 13.5 dpc, several laboratories have speculated that PGC proliferation is autonomously programmed [12, 15, 16]. Ohkubo et al. [17] tested this hypothesis by observing the behavior of PGCs in mixed cultures of 8.5-dpc premigratory and 11.5-dpc postmigratory germ cells. Consistent with an autonomous program of PGC proliferation, these investigators did not detect a proliferation-inhibiting activity of postmigratory cells upon premigratory cells, nor a proliferation-extending activity of premigratory cells upon postmigratory cells. Finally, an autonomous program of fetal germ cell development is also suggested by the observation that XX or XY germ cells, ectopically located in the adrenal cortex, stop mitotic proliferation and enter meiosis in near synchrony with oogonia in the ovary [18].

Most previous in vitro studies have used surface alkaline phosphatase to quantitate PGC numbers. Since PGCs lose their alkaline phosphatase activity at about 14.5 dpc in vivo [19], we sought to quantitate germ cell number by additional means, in case culture conditions depressed surface alkaline phosphatase expression, and therefore did not accurately represent germ cell numbers. If depression of surface alkaline phosphatase expression did occur, we predicted it would lead to under-representation of older germ cells. We used germ cell nuclear antigen 1 (GCNA1) as an additional marker of germ cell numbers. GCNA1 is a nuclear antigen expressed exclusively in germ cells of either sex. GCNA1 first becomes detectable at 10.5 to 11.5 dpc in mouse gonadal germ cells, and it is expressed throughout the remaining embryonic period [20].

Here we report for the first time evidence that cultured mouse PGCs express GCNA1. Furthermore, we have compared the expression of GCNA1 and surface alkaline phosphatase to explore the developmental potential of PGCs. We found that both XX and XY premigratory 8.5-dpc PGCs initiate GCNA1 expression in culture. This result provides the first direct evidence of continuing differentiation of PGCs in vitro, and further indicates that neither completion of PGC migration nor exposure to the genital ridge is necessary for differentiation towards gonocytes. While these culture conditions support the proliferation and differentiation of migratory PGC for several days in vitro, gonadal germ cell survival is not similarly supported by current culture conditions.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Germ Cell Isolation and Culture

All experiments were performed on embryos obtained from timed matings of B6C3F1 mice. Germ cell suspensions were plated on irradiated (approximately 500 rads) Sl/Sl4 m220 feeder layers [21] in 96-cell wells in QBSF-58 (Quality Biological, Gaithersburg, MD) supplemented with 100 U penicillin, 50 µg streptomycin, 2 µmol glutamine, and 1000 U LIF (ESGRO; Life Technologies, Gaithersburg, MD) per milliliter. To obtain germ cells, either the caudal sections of 8.5-dpc embryos encompassing the allantois or the urogenital ridges of 12.5-dpc embryos were pooled and disrupted to a homogeneous suspension by exposure to 0.05% (w:v) trypsin/0.53 mM EDTA for 5 min at 37°C and subsequent trituration through a 200-µl pipette tip. The trypsin was then quenched by addition of a small volume of fetal bovine serum (FBS), and the cells were recovered by centrifugation at 1500 x g for 2 min. After resuspension in culture medium, the cells were aliquoted into wells. In some experiments, gelatinized 96-cell wells were treated with 50 µg/ml poly-D-lysine before plating feeder layer cells. One hundred percent of the culture medium was replaced daily. Each data point represents the average and standard deviation of at least 5 wells. The general pattern of results was observed in numerous repetitions, and a representative result is shown. The results shown here were obtained in serum-free media, which we have found to result in higher levels of PGC proliferation (unpublished observations). We have observed similar results regarding gonocyte differentiation and survival in medium containing FBS.

Immunocytochemical Methods

Endogenous alkaline phosphatase Alkaline phosphatase was detected with a naphthol AS-MX phosphate kit (Sigma, St. Louis, MO) after paraformaldehyde fixation as previously described [22].

GCNA1 Immunocytochemistry for GCNA1 was performed using the 10D9G11 monoclonal antibody [20]. Cultures were fixed overnight at 4°C in methanol:dimethyl sulfoxide (4:1), rehydrated by 15-min incubations in 50% and 30% methanol, rinsed in PBS, and blocked at 4°C for 1 h in PBSMT (PBS, 2% w:v nonfat dry mild, 0.1% Triton X-100) and in PBSMT containing 0.1% normal goat serum (Sigma). Cultures were then incubated overnight in 10D9G11 supernatant diluted 1:50 in PBSMT at 4°C. After two 10- to 30-min washes in PBSMT at 4°C and 3 washes at room temperature, alkaline phosphatase-conjugated goat anti-rat IgM (Pierce, Rockford, IL) diluted 1:1000 in PBSMT was added overnight at 4°C. The PBSMT washes were repeated and followed by 3 room-temperature washes in NTMTL (0.1 M NaCl, 0.1 M Tris HCl pH = 9.2, 50 mM MgCl2, 0.1% Triton X-100, 2 mM levamisole). Color development was performed with 175 µg/ml BCIP: 250 µg/ml NBT (BCIP is 5-bromo-4-chloro-3-indolyl phosphate; NBT is nitroblue tetrazolium [Boehringer-Mannheim, Indianapolis, IN]) in NTMTL, and usually required approximately 10–20 min at room temperature. We have occasionally observed rare cells that exhibit cytoplasmic staining. This staining pattern also occurs when isotype control antibody is substituted for 10D9G11.

Stage-specific embryonic antigen 1 All procedures were performed at room temperature. Cultures were fixed in 4% neutralized paraformaldehyde in PBS and washed 3 times for 5 min in PBS before blocking for 10 min in 10% normal rabbit serum (Sigma) diluted in PBS. Supernatant from the monoclonal antibody producing cell line TG1 [23] was diluted 1:20 in PBS and added for 1 h. After 3 washes, 5 min each in PBS, 10% normal rabbit serum was again added for 5 min. Horseradish peroxidase-conjugated rabbit anti-mouse IgM (Zymed, San Francisco, CA), diluted 1:1000 in PBS, was added for 30 min. After 3 washes in PBS, color development was performed with an AEC (3-amino-9-ethylcarbazole) kit as recommended by the supplier (Sigma).

Determination of Sex of Premigratory PGCs

To determine the sex of premigratory germ cells, DNA was prepared from the anterior fraction of the embryo and subjected to polymerase chain reaction (PCR) amplification of the Y chromosome Zfy-1 gene exactly as described [24].


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Premigratory Primordial Germ Cells Differentiated in Culture

We anticipated that GCNA1 expression could provide an assay to quantitate continuing PGC differentiation in vitro. GCNA1 is not detected in germ cells until completion of migration to the genital ridge [20], suggesting that this antigen could provide a positive marker of PGC differentiation towards gonocytes in culture. First, we tested whether GCNA1 could be detected in gonocytes explanted into the PGC culture system. A suspension of 12.5-dpc genital ridges was plated onto Sl/Sl4 m220 feeder layers and immunocytochemically stained for GCNA1 two days later. Prominently staining nuclei were readily found both as isolated cells and in small colonies (Fig. 1A). The appearance of small colonies of GCNA1-positive cells is consistent with fetal germ cells retaining cytoplasmic bridges during their proliferative period. GCNA1-positive cells were not detected either in feeder cell-bearing wells lacking germ cells, nor in germ cell-bearing wells stained with isotype control antibody. On rare occasions, weak cytoplasmic staining in isolated cells was detected. This unusual pattern could also be detected with isotype control antibody and may represent a specificity of the secondary antibody (not shown).



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  		FIG. 1. Detection of GCNA1-expressing cells in culture. All photographs were taken at x400 magnification (published at 85%). A) Gonocytes from 12.5 dpc were plated on feeder layers and stained for GCNA1 two days later. A small colony of expressing cells is shown. Isolated GCNA1-expressing cells were also observed. Nonstaining cells arise from either the feeder layer or from somatic cells of the urogenital ridge. B) Premigratory PGCs were cultured for 4 days and sequentially stained for GCNA1 and SSEA1. GCNA1-positive nuclei (blue black) are located within SSEA1-expressing cells (reddish). Arrowheads indicate cells with both markers; arrows indicate cells expressing only SSEA1. C) A rare GCNA1-positive cell found one day after plating 8.5-dpc PGCs. The positive nucleus is located within an SSEA1-expressing cell. D) GCNA1 staining of a 4-day culture of 12.5-dpc urogenital ridges. The frequency of disrupted nuclei increased as the cultures progressed

The ability to detect GCNA1-expressing cells provided an opportunity to investigate whether premigratory PGCs would differentiate to express this antigen in culture. PGCs from 8.5 dpc were cultured on feeder cells, and cultures were sequentially assayed for both alkaline phosphatase- and GCNA1-expressing cells. Under these culture conditions, the number of alkaline phosphatase-expressing PGCs increased several-fold over 4 days of culture before a decline. GCNA1-expressing cells were also readily detected, with significant numbers first appearing by 3–4 days of culture (Fig. 2).



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  		FIG. 2. Differentiation of cultured primordial germ cells. Premigratory 8.5-dpc PGCs were seeded on feeder layers at a density of approximately 0.6 pooled embryo equivalents per well and stained for endogenous alkaline phosphatase (open circles) and GCNA1 (closed circles) on successive days of culture

We tested whether GCNA1-positive cells represent a subpopulation of cultured premigratory PGCs. Although cultures that have been stained for the PGC endogenous alkaline phosphatase may readily be stained for GCNA1, the pink alkaline phosphatase stain is lost from the majority of cells during the GCNA1 immunocytochemical procedure, and few cells expressing both markers were detectable. Therefore, to confirm that GCNA1 expression in culture is initiated only by germ cells, we took advantage of another PGC specific marker, TG1. The monoclonal antibody TG1 recognizes an SSEA1-like (stage-specific embryonic antigen) surface carbohydrate antigen distinctive for proliferative-phase PGCs [19]. A 4-day culture of 8.5-dpc PGCs previously stained for GCNA1 was stained for TG1 using a horseradish peroxidase-conjugated secondary antibody and subsequent AEC cytochemistry, thereby staining PGCs reddish. Figure 1B shows that GCNA1-positive nuclei are localized within TG1-positive cells, confirming that in culture, as in vivo, GCNA1 expression is limited to germ cells. Interestingly, TG1 staining also showed that some GCNA1-expressing cells may have elongated cytoplasmic extensions characteristic of migratory PGCs.

In several experiments, a small number of GCNA1-expressing cells were detected as early as one day after plating of 8.5-dpc PGCs. Again, we found that these rare cells were TG1-positive, confirming their germ cell identity (Fig. 1C). In vivo, GCNA1 is not detectable until 10.5–11.5 dpc. It is not clear whether these rare cells present in our cultures resulted from 1) a difference in mouse strains used, 2) a small population of early GCNA1-positive cells that was not detected in the original description of the GCNA1 expression pattern, or 3) induction by explant or culture procedures. The appearance of GCNA1 in cultured 8.5-dpc premigratory germ cells as shown in Figure 2 was primarily due to differentiation and not simply rapid proliferation of a pre-existing GCNA1 population, because at the plating densities used here many wells were initially free of GCNA1 at the earliest time points, while at later times all wells harbored numerous GCNA1-positive cells.

In vivo, both male and female germ cells present the GCNA1 antigen by 11.5 dpc. The previous experiments were performed with PGCs obtained from pooled embryos and were thus likely to contain a mixture of male and female germ cells. To determine whether both XX and XY germ cells present this marker in culture, the caudal portions of single 8.5-dpc embryos were plated in individual 96-cell wells and analyzed for GCNA1 expression 4 days later. The sex of each embryo was determined by PCR amplification of the Y chromosome-specific gene Zfy-1 using DNA obtained from the remaining embryo fragment (not shown). While the absolute numbers of both alkaline phosphatase- and GCNA1-expressing germ cells varied significantly from embryo to embryo, both male and female germ cells initiated expression of this gonocyte marker (Fig. 3).



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  		FIG. 3. Differentiation of premigratory primordial germ cells in cultures of individual embryos. The caudal portions of individual 8.5-dpc embryos were plated on feeder cells in single 96-cell wells, and the number of alkaline phosphatase- (open bars) and GCNA1-positive (closed bars) cells were determined 4 days later. The sex of each embryo was determined by PCR for the Y chromosome gene Zfy-1. Embryos A–E were female. Embryos F–L were male

Postmigratory Gonocytes In Culture

In culture, the number of alkaline phosphatase-expressing PGCs declines after the time corresponding to 12.5–13.5 dpc in vivo. Because alkaline phosphatase expression is normally lost from germ cells in vivo at about this time, we questioned whether germ cells might survive in culture but remain undetected due to absence of the alkaline phosphatase marker. As GCNA1 is expressed by gonocytes throughout the embryonic period, it provides an ideal marker to investigate the viability of postmigratory germ cells in culture. Suspensions of 12.5-dpc urogenital ridges were plated on feeder layers, and the numbers of alkaline phosphatase- and GCNA1-expressing cells were subsequently determined. As is routinely observed, the numbers of alkaline phosphatase-positive cells declined precipitously over 4 days of culture. In the same cultures, we also found that the number of GCNA1-expressing cells declined at a similar rate. The behavior of both male and female germ cells was similar (Fig. 4). The decline in germ cell numbers was accompanied by the appearance of a GCNA1 staining pattern that may indicate nuclear breakdown and fragmentation (Fig. 1D). Because several nuclei appeared to be included in each of these disrupted nuclear centers, we were unable to accurately determine the number of cells affected. However, the frequency of these disrupted nuclei increased as these cultures progressed.



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  		FIG. 4. Postmigratory germ cell culture. Gonocytes from 12.5-dpc urogenital ridges were plated at 0.15 embryo equivalents per well and subsequently scored for both alkaline phosphatase- (open circles) and GCNA1- (closed circles) expressing cells. Embryos were sex-segregated by inspection for the presence of testis cords. Only clearly distinguishable, intact nuclei were enumerated


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The feeder layer-based PGC culture system has been widely used to investigate factors regulating germ cell survival and proliferation. That the timing of PGC proliferation observed in vitro mimics that observed in vivo [12, 15, 25] has led to the hypothesis that germ cell proliferation is autonomously programmed [17]. A prediction of this hypothesis is that PGCs will recapitulate several aspects of their in vivo program after explant into culture. Our results demonstrate that cultured premigratory PGCs do undergo differentiation towards gonocytes as they complete their proliferative phase. These results also indicate that neither completion of migration nor exposure to the genital ridge are necessary for this differentiation event. These results are consistent with the observation that the genital ridge is not essential for expression of GCNA1, as rare ectopic germ cells in the adrenal cortex express this marker [26].

An examination of the growth patterns of alkaline phosphatase-expressing PGCs in clonal cultures, and in cultures containing more than one age of germ cell, led Ohkubo et al. [17] to predict that cessation of proliferation is a cell-autonomous event. The appearance of GCNA1-expressing cells in culture is consistent with this model. Furthermore, the small colonies of GCNA1-expressing cells that are frequently observed in both pre- and postmigratory germ cell cultures (Fig. 1A) may either arise by aggregation [27] or indicate incomplete cytokinesis following germ cell division [28].

Ohkubo et al. [17] further suggested that cessation of proliferation occurs stochastically, rather than after a set number of cell divisions. The timing of the appearance of GCNA1 in PGC culture is also consistent with a stochastic model of differentiation. While a few GCNA1-positive cells are detectable shortly after plating PGCs from 8.5-dpc embryos, the frequency of expressing cells increases significantly between the second and third day of culture. Because primordial germ cell division in culture occurs at a lower rate than that observed in vivo [2], our data also suggest that differentiation could be stochastic, regulated more by the passage of time than after a preset number of cell doublings.

While these data suggest that some aspects of gonocyte differentiation can occur without exposure to the urogenital ridge, a great deal of evidence points to important roles for the urogenital ridge in regulating early stages of gonocyte development. Genital ridge-conditioned media stimulate PGC proliferation and exert a chemotropic effect upon cultured PGCs [29]. Using organ cultures, Byskov and colleagues found that female germ cell entry into meiosis may depend upon the presence of the mesonephros [30]. More recently, McLaren and Southee [31] demonstrated that commitment of male PGCs to spermatogenesis occurs between 11.5 and 12.5 dpc, and may require the presence of nascent testis cords. We cannot rule out that critical inductive factors shared by the genital ridge are also available in the PGC cell culture system. These factors could be presented by the feeder layer, the somatic cells of the embryo that accompany germ cells into culture, or the culture medium. In cultures of 8.5-dpc germ cells, GCNA1 becomes detectable in a smaller fraction of the germ cells than that initially observed in 12.5-dpc germ cells (compare Figs. 2 and 4), perhaps indicating that a critical factor is limiting in the culture system. Expression of GCNA1 in premigratory PGCs should provide a direct assay for such gonocyte differentiation-inducing factors. Alternatively, an analysis of cellular factors regulating induction of the GCNA1 antigen may provide insight into mechanisms governing gonocyte differentiation.

We did not detect long-term persistence of GCNA1-positive gonocytes in these cultures. Two observations argue that most gonocytes are dying in this culture system, rather than surviving without maintaining GCNA1 expression. First, premigratory cells initiate and appear to maintain GCNA1 expression, indicating that GCNA1 expression could be maintained under these conditions. Second, the decline in GCNA1-positive cells is also accompanied by increased frequency of disrupted nuclei, a characteristic of apoptotic death [32, 33]. Interestingly, in vivo both male and female germ cells are also susceptible to apoptotic death at about 12.5–13.5 dpc [34–38]. It is not clear whether this in vivo deletion of germ cells is due to activation of a death receptor or absence of a trophic factor, or is a cell autonomous event. The GCNA1 culture assay may also prove useful in understanding regulation of gonocyte death.


   ACKNOWLEDGMENTS
 
We gratefully acknowledge the technical expertise of Mike Ziemak. We thank Vicky Kelley for irradiating feeder cells and Mark Potter for help with photomicroscopy. We thank Dr. Richard Moyer for valuable advice. We are especially grateful to Dr. Cami Brannan for encouragement, support, and critical reading of the manuscript.


   FOOTNOTES
 
1 This work was supported by a New Investigator award from the Cooperative States Research Service, U.S. Department of Agriculture (93–37205–9074), and by a College Incentive Fund award from the University of Florida College of Medicine to J.R. Back

2 Correspondence: James Resnick, Department of Molecular Genetics and Microbiology, University of Florida, 1600 SW Archer Road, Gainesville, FL 32610–0266. FAX: 352 392 3133; resnick{at}college.med.ufl.edu Back

Accepted: June 4, 1999.

Received: March 22, 1999.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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