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Glycoconjugate unique to migrating primordial germ cells differs with genera.

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THE ANATOMICAL RECORD 228:177-184 (1990)
Glycoconjugate Unique to Migrating Primordial
Germ Cells Differs With Genera
A.R. FAZEL, B.A. SCHULTE, AND S.S.
SPICER
Department of Anatomy, Mashhad University of Medical Sciences, Mashhad, Iran
(A.R.F.),and Department of Pathology and Laboratory Medicine, Medical University of
South Carolina, Charleston, South Carolina 29425 (B.A.S., S.S.S.)
ABSTRACT
Previous cytochemical studies showing that rat primordial germ
cells (PGCs) possess a unique surface glycoconjugate containing terminal a-Nacetylgalactosamine were extended in this study to determine whether a similar
distinctive glycoconjugate coats the surface of PGCs in the mouse. The results
showed t h a t mouse PGCs fail to react with peroxidase-conjugated lectins specific
for localizing glycoconjugate with terminal N-acetylgalactosamine. All available
lectin conjugates with affinity for other terminal sugars or internal sugar linkages
also failed to stain mouse PGCs except for the conjugates that bind to a-fucose. One
fucose-specific lectin conjugate stained only PGCs in the early mouse embryo but
stained additional sites in more mature embryos and lost reactivity with PGCs
after gestational day 14. Another fucose-specific conjugate stained PGCs until day
15, but with less selectivity, and a third such conjugate bound to several sites, but
not to PGCs. The results suggest that the developmental mechanisms mediating
cellular interaction, migration, and differentiation may be similar in different
genera, but the specific structure of the cell surface glycoconjugate involved in
these mechanisms differs.
Cell surface glycoconjugates and particularly certain
terminal residues such a s fucose, sialic acid, galactose,
and aminosugars play a n important role in developmental events during embryogenesis (for review, see
Gilbert, 1985). A unique surface glycoconjugate with
terminal a-N-acetylgalactosamine (a-GalNAc), for example, has been shown by previous histochemical studies to occur on the surface of the rat’s primoridal germ
cells (PGCs) exclusively and to exist there only during
their migration (Fazel et al., 1987). The appearance in
a specific histologic site of a glycoconjugate unique to
the embryo has potential importance in organogenesis.
Lectin histochemistry has demonstrated that carbohydrate-rich macromolecules in various cell types
within a n animal differ widely and that glycoconjugates in a given cell type differ between species (Spicer
et al., 1987). The goal of this study was to determine
whether the distinctive glycoconjugate on the surface
of rat PGCs occurs also on the surface of mouse PGCs
during their migration. The results show that PGCs in
mouse a s in rat contain a surface glycoconjugate that is
unique to the embryo but that the terminal sugar in
the mouse is cy-fucose rather than a-GalNAc. The glycoconjugate specific to migrating PGCs disappears
from the cell surface in the mouse as in the rat after the
cells gain residence in the developing male or female
gonad.
plug. At gestational stages of 10 through 18 days, pregnant mice were sacrificed by cervical dislocation and
the embryos were dissected from the uterus and intraembryonic membranes. Whole embryos or sections
from larger embryos were fixed overnight in a 1%sodium acetate, 6% mercuric chloride, 0.1% glutaraldehyde solution (Schulte and Spicer, 1983) or 3-5 h in
Carnoy’s fluid. After dehydration, embryos were embedded in paraffin blocks in three different orientations that permitted sagittal, frontal, and transverse
sectioning.
Lectin Histochemistry
Serial sections, 5 pm thick, were cut from the paraffin-embedded embryos. Sections from buffered HgC1,
glutaraldehyde-fixed embryos were treated with
Lugol’s solution to remove mercuric salts prior to histochemical staining.
The battery of lectin-HRP conjugates used in the
previous study was employed here (Fazel et al., 1987).
This included conjugates for visualizing glycoconjugate
with terminal a- and @-galactose, N-acetylgalactosamine, N-acetylglucosamine, sialic acid, and a-fucose
as well a s a conjugate with affinity for the core region
of N-linked oligosaccharides. Agglutinins from Ulex
europaeus (UEA I) and Lotus tetragonolobus (LTA)
MATERIALS AND METHODS
Embryos from two strains Of mice (C57BLi6J) and
C3HiHeJ) were used’
mice were mated Overnight with males of the f%3me strain, and day 0 of gestation was assigned a t the appearance of the vaginal
c
1990 WILEY-LISS, INC
Received January 15, 1990; accepted March 16, 1990.
Address reprint requests to Dr. S.S. Spicer, Department of Pathology and Laboratory Medicine, Medical Universlty of South Carolina.
171 Ashley Avenue, Charleston, S.C. 294‘25.
178
A.R. FAZEL ET AL.
Fig. 1. Large migrating PGCs lying along the epithelium of the
dorsal mesentery (arrowheads) stain selectively in a transverse section of an 11 day mouse embryo. PGCs appear separated from the
epithelium of the dorsal mesentery where they reach the genital
ridge. A, aorta; M, mesonephric tube; UG, urogenital ridge; DM, dorsal mesentery; MV, mesenteric vessel. Stained with LTA-HRP conjugate. x 200.
Flg. 2. Enlargement of the area outlined in Figure 1. LTA binding
is localized to the cell surface and cytoplasmic foci possibly representing Golgi cisternae. x 1000.
were purchased from Sigma Chemical Co., St. Louis,
MO. Agglutinin from Aleuria aurantia (orange fungus
agglutinin, OFA) was kindly supplied by Dr. M.
Kochibe, Department of Biology, Faculty of Education,
Gunma University, Gunma 371, Japan.
The procedures for conjugating lectins and staining
sections with the lectin-HRP conjugate were similar to
those described previously (Faze1 et al., 1987).In brief,
the deparaffnized and demercurialized sections were
immersed 30 min in pH 7.2, 0.1 M phosphate buffer
containing 0.1 M CaCl,, MgCl,, and MnC1,. Sections
were then incubated 3 h in a similar buffer containing
10-20 pgiml of lectin conjugated to horseradish peroxidase (Schulte and Spicer, 1983). Sections were then
incubated in the diaminobenzidine-H,O,
substrate
medium for peroxidase to visualize sites of lectin binding. Cytochemical controls included substitution of unconjugated lectin for the lectin-HRP conjugate, exposure to unconjugated HRP and substrate medium, and
exposure of sections to 10-20 pgiml of each lectin HRP
conjugate containing 0.1 M concentration of the sugar
for which the lectin possessed specific affinity.
RESULTS
On day 9 of gestation, part of the posterior cell surface and a cytoplasmic focus interpreted a s the Golgi
zone (not shown) in the epithelium of the dorsal section
of the hindgut and adjacent cells reacted with both
LTA and OFA. No other sites in the developing embryo
bound the LTA-conjugate. However, several structures
such as regions of the developing nervous system
shared affinity for OFA.
From day 10 through day 13 of gestation, the period
when the PGCs start then end their migration, LTA
and OFA conjugates stained the surface of the germ
cells and a cytoplasmic area mainly located beside the
nucleus (Figs. 1-5; Table 1).Among the various HRPlectin conjugates tested, only the two fucose-binding
lectins, LTA and OFA, reacted with the mouse PGCs
during their migration.
PRIMORDIAL GERM CELL MARKERS
179
Fig. 3. In a sagittal section of a day 12 mouse embryo, only PGCs
stain. The germ cells underlie the epithelium in the urogenital ridge.
H, heart; L, liver. LTA-HRP conjugate. x 150.
Fig. 4. Enlargement of cells in Figure 3. The cell surface, a large
cytoplasmic region in some cells located beside the nucleus, and small
cytoplasmic foci react strongly with LTA. x 1000.
LTA staining was confined to PGCs from day 10
through day 13 during their migration, except for the
epithelia of facial placodes, which stained from day 11
to day 14. By gestational day 14, structures other than
PGCs, as, for example, mesonephric and renal tubules,
became LTA positive (Figs. 6 and 7). OFA, however,
differed from LTA in reacting with other sites, such a s
gut epithelium, renal tubules, and neural tubes, a t earlier gestational stages (Fig. 5).
LTA reactivity was diminished on the surface of
PGCs a t day 14 (Figs. 6 and 7). On day 15, cells distributed like the PGCs seen a day earlier showed discrete foci of LTA binding in the cytoplasm, but no lectin
reactivity on the cell surface (Fig. 8) . No LTA binding
persisted after day 15 (Fig. 9). PGCs stained with OFA
until day 15.
The course of PGC’s migration was the same a s that
described in rat embryos (Faze1 et al., 1987) except that
180
A.R. FAZEL ET AL.
Fig. 5. In a transverse section of day 13 embryo, PGCs are localized within the developing gonads
(arrows).Other structures such as developing neural tube (N), collecting tubules of the kidneys (K), part
of the gut epithelium (lower right), and scattered cells in the liver (L)also bind this lectin. Stained with
OFA-HRP conjugate. x 150.
TABLE 1. Staining by peroxidase conjugated
fucose-binding lectins in developing mouse embryos
9
LTA
PGCs
Other sites
OFA
PGCs
Other sites
UEAI
PGCs
Other sites
+
10
11
+
+
-
-
+
+
+
+
Day of gestation
12
13
14
+
+
+
:i
~.
- :i
+
+
+
+
t
t
+
+
+
-
~
3
-
-
-
-
-
-
-
-
-
-
+
15
2
?
+
2
+
-
+
z
'
16
-
+
-
+
-
+
'All other lectin-HRP conjugates tested failed to react with mouse
PGCs at any time of development (see Faze1 et al., 1987, Table I).
'Staining was mainly located in cytoplasmic foci, possibly lysosomes,
in contrast to surface reactivity seen a t earlier stages.
.'Except for staining of the epithelia of the facial placodes.
all observed migrating PGCs were in contact with the
epithelium of the dorsal mesentery and positive cells
were not detected in the stroma of the dorsal mesentery. Briefly, the germ cells appeared around the posterior section of the hindgut and, a s dorsal mesentery
formed, they migrated through it to the urogenital
ridge and reached their final destination in the developing gonads. Mouse PGCs did not react with any
tested lectin after day 15 of gestation when migration
had terminated.
None of the staining reported for PGCs or other sites
was seen in any of the cytochemical controls.
DISCUSSION
The migration of the PGCs in mouse embryo via
hindgut and dorsal mesentery toward the gonadal
ridges in the dorsal body wall has been well established
(Clark and Eddy, 1975; Tam and Snow, 1981;McLaren,
PRIMORDIAL GERM CELL MARKERS
Figs. 6 and 7. Transverse and frontal sections, respectively, of mouse
embryo a t day 14 of gestation. PGCs (small arrows) in the developing
gonads ( * ) appear to be losing their reactivity, retaining cytoplasmic
but little surface staining. For the first time in the fetal development,
181
other histologic sites reveal affinity for the lectin as, for example,
mesonephric tubules (M) and developing collecting tubules (large arrows) in kidney (K). LTA HRP conjugate. x 250.
182
A.K. FAZEL ET AL.
Fig. 8. Transverse section through the gonad on day 15 of gestation
shows some PGCs (arrows)that retain staining in cytoplasmic bodies.
Cell surface reactivity has disappeared. Mesonephric tube (M)
remains positive. LTA-HRP conjugate. x 300.
Fig. 9. Transverse section through a day 16 embryo showing that
PGCs are no longer lectin positive in the developing gonad (*). Collecting tubules of the kidney ( K ) and a mesonephric tubule (M)
stain,
however. L, Liver. LTA-HRP conjugate. x 220.
183
PRIMORDIAL GERM CELL MARKERS
1983; Fujimoto et al., 1985). The present selective
staining of mouse primordial germ cells affirms the
previous perception showing a similar migratory route.
Observations in this study testify to the presence of a
distinctive glycoconjugate on the surface of mouse
PGCs a s on the PGCs in the rat (Fazel e t al., 1987)
during their migration. However, the glycoconjugate
on PGCs of mouse embryos differs from that on the rat
PGCs in containing a-fucose instead of terminal a-Nacetylgalactosamine.
The mouse PGCs differed from rat PGCs in another
respect. After cessation of migration, PGCS in both
species lost affinity for the lectin that stained them
selectively. However, PGCs failed thereafter to stain
with any lectin conjugate in mice, whereas they exhibited affinity for another GalNAc-binding lectin, Wisteria floribunda agglutinin (WFA), in the rat embryo.
In contrast to rats, it was accordingly impossible to
delineate the PGC’s participation in the development
of the mouse gonad. Male and female gonads in the
mouse embryo could not be distinguished a s they were
in rats (Fazel et al., 1987)where organization of lectinreactive PGCs into seminiferous tubules identified the
male gonad during its development.
Lectin binding to migrating PGCs on days 9 to 12
appeared localized to the cell surface and a cytoplasmic
area. Although quite diffuse in some cell profiles, presumably because of diffusion of reaction product, the
cytoplasmic staining commonly appeared discrete and
located beside the nucleus. The cytoplasmic staining
was interpreted as revealing nascent glycoprotein during biosynthesis in Golgi cisternae and prior to transport to the glycocalyx. This view is supported by numerous observations at the light (Stoward et al., 1980)
and electron (Sato and Spicer, 1982) microscopic levels
showing lectin reactivity with glycoconjugate in Golgi
cisternae and appears consistent with biochemical
knowledge that glycosylation of protein invariably depends on Golgi-associated transferases.
The disappearance of LTA receptors from the surface
of PGCs after they settled in the gonad coincided with
the appearance of reactivity for the lectin in cytoplasmic foci. Such concurrent changes could be explained
on the basis of internalization of lectin reactive glycocalyx to lysosomes for degradation. Down-regulation
by such a mechanism of a surface constituent essential
to migration would be expected after the cell had
reached its destination.
Although LTA, OFA, and UEA I have nominal binding specificity for a-fucose, their preferences as to linkage and underlying sugar chain structure differ. OFA
binds to fucose residues of carbohydrate side chains but
does not require a particular linkage to the penultimate sugar (Kochibe and Furukawa, 1980). OFA has
been found to bind to a considerable fraction of the
rapidly transported fucose-labeled glycoproteins in
rabbit retinal ganglionic cells (Ohlson et al., 1985) a s
well a s to the major portion of the fucose in rat brain
which is linked (a1+3) to N-acetylglucosamine
(GlcNAc) (Krusius and Finne, 1977,1978; Gustavsson
et al., 1982; Ohlson and Karlsson, 1983).
UEA I and LTA, on the other hand, are thought to
show the highest affinity for fucose-linked (al-2) to
galactose or other more complex difucosyl structures
(Pereira and Kabat, 1974; Allen e t al., 1977; Pereira et
al., 1978). It has been shown that LTA does not bind to
fucose-linked (011-6) to GlcNAc and has a very poor
affinity for the fucose (al+3)-GlcNAc linkage (Susz
and Dawson, 1979). Histochemical studies from this
laboratory comparing human salivary glands from
secretors and nonsecretors provided evidence for affinity of LTA but not UEA for (al+4)-linked fucose
(Laden et al., 1984). The failure of mouse PGCs to bind
UEA and the affinity of these cells for LTA indicate
that the PGC glycocalyx contains glycoconjugate possessing fucose bound a1-4 to the neighboring residue.
The staining with LTA restricted to PGCS and the
widespread binding of OFA in several tissues and cell
types of the mouse embryo are consistent with the narrow specificity of LTA and broader specificity requirement for OFA.
Demonstrating in murine PGCs a glycoconjugate
unique to the embryo supports the concept, held from
observations of r a t embryo, that the migratory mechanism also unique to this cell type entails reaction between an interstitial constituent and a complex carbohydrate exclusively present on this cell’s glycocalyx. If
surface glycoconjugate participates in such a mechanism, its structure is not limited to content of a single
type of terminal residue on the basis of physicochemical properties of the particular sugar but can
vary a t least in the nature of the terminal constituent.
Unlike in the rat embryo, the present findings do not
support an epiblastic origin of the PGCs in mouse embryos. It was not possible to detect positive cells migrating from epiblast to the adjacent area of the mouse
hindgut with the lectin methods. If PGCs originate in
this species from epiblast and then migrate toward the
hindgut, they presumably possess different macromolecules which direct them to their early destination.
Based on the staining observed with conjugated LTA
and OFA, it appears that PGCs form from mesenchyme
around hindgut epithelium of possible mesodermal origin. If this is the case, and since mesodermal cells form
from epiblast layers, we can say that PGCs are a second
generation of the ectodermal layer.
ACKNOWLEDGMENTS
The authors wish to thank Ms. Charlene Kerr for her
excellent technical assistance. The expert editorial assistance of Mrs. Leslie Harrelson is gratefully acknowledged.
LITERATURE CITED
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A.R. FAZEL ET AL.
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