J. Embryol. exp. Morph. 81, 93-104 (1984)
Printed in Great Britain © The Company of Biologists Limited 1984
93
Characterization of a cell-surface differentiation
antigen of mouse spermatogenesis: timing and
localization of expression by immunohistochemistry
using a monoclonal antibody
By KATHLEEN B. BECHTOL
The Wistar Institute of Anatomy and Biology, 36th Street at Spruce,
Philadelphia, Pennsylvania 19104, U.S.A.
SUMMARY
The XT-1 antigen, bound by monoclonal antibody XT-I, is a differentiation antigen of germ
cells in the mouse testis. As seen in immunoperoxidase-stained tissue sections from several
juvenile ages and adult, the antigen becomes detectable on early (leptotene/zygotene) spermatocytes and increases in staining during spermatocyte development. During spermatid
development the distribution of the determinant shifts from its relatively uniform surface
distribution on spermatocytes to a more restricted localization on the base of the head, tail and
cytoplasmic lobe of the elongating spermatid. The antigen is not detectable on juvenile or
adult Sertoli cells. Detection of the antigen is dependent on the presence of germ cells of
appropriate developmental stage. It is, thus, a marker for spermatocytes and later germ cells,
for a cell-surface molecule related to spermatogenesis and for redistribution and/or modification of the molecule during spermatid elongation.
INTRODUCTION
Spermatogenesis is an ideal system for the study of development because of
several useful and well-documented characteristics. First, the system is relatively
simple involving only two types of cells, the developing germ cells and the
epithelial Sertoli cells. Second, there are clear morphological markers of the
progress of cells in spermatogenic development. Third, because of the regular
timing of entry of new generations of germ cells into spermatogenesis and the
consistent progression of the germ cells through spermatogenesis, recurrent
associations of germ cells are formed within each tubule section. These associations allow identification of the precise state of development of each germ cell
to within plus or minus 22 h within the 34-5-day (mouse) differentiation sequence
(Oakberg, 1956a,b). Lastly, relatively large amounts of material are available.
Spermatogenesis is a developmental system which proceeds from stem cell to
product in the adult, and moreover, does so repeatedly.
The cell surface (integral membrane components, junctions, peripherally
associated components) is the means through which the cell interacts with its
EMB81
94
K. B. BECHTOL
environment. It is thought to play important roles in regulating development and
in producing differentiated function (Subtelny & Wessels, 1980). One approach to
study the role of the cell surface in differentiation is analysis of differentiation antigens. Several populations of cell-surface differentiation antigens of testicular
germ cells have been described using polyclonal immune sera, and recently
monoclonal antibodies have been used to identify individual antigens or sets of
antigens crossreactive at a single antigenic determinant. A set of antigens (defined
by a rabbit anti-mouse spermatogonia serum exhaustively absorbed with somatic
cells) is present on all of the germ cells while they reside within the epithelium of
the testicular tubule (Millette, 1979; Millette & Beilve, 1980). Presumably during
elongation of the spermatids these antigens (determinants) become differentially
distributed to the cytoplasmic lobe of the spermatid with little or none of these
antigens remaining on the head or tail of the spermatid. At release of the spermatozoa from the epithelium, the physical budding off of the residual cytoplasmic
body from the sperm separates most or all of this set of antigens from the sperm
proper (Millette, 1979; Millette & Bellve, 1980). Antigens which are not detected
on spermatogonia, but are found on spermatocytes have been detected by several
different immune sera (O'Rand & Romrell, 1977; Millette & Bellve, 1977; Tung &
Fritz, 1978; O'Rand & Romrell, 1981) and monoclonal antibodies (Bechtol,
Brown & Kennett, 1979; Gaunt, 1982). Some of these antigens continue to be expressed on the sperm surface, while others become undetectable by the elongatedspermatid stage. Still other surface antigens are not detectable on either spermatogonia or spermatocytes and first appear on late spermatids (Tung, Bebe-han &
Evan, 1979; O'Rand & Romrell, 1980). Quantitative and qualitative shifts in cell
surface composition of germ cells as they progress through spermatogenesis have
also been observed by comparing electrophoretic gel patterns of proteins and
glycoproteins solubilized from germ cells separated according to size (developmental stage) (Boitani, Geremia, Rossi & Monesi, 1980; Millette & Moulding,
1981; Romrell, O'Rand, Sandow & Porter, 1982; Kramer & Erickson, 1982).
The XT-1 antigen, bound by monoclonal antibody XT-I (IgG2a), is a testisspecific differentiation antigen of mouse germ cells. By quantitative-absorption
radioimmunoassay using cell suspensions from whole testis of several juvenile
ages and adult, the average concentration of XT-1 in the testis is seen to increase
from undetectable in the 8-day juvenile mouse to a peak around 22 days after
birth, followed by a decline to about 50 % of that concentration in the adult
testis. Antigen is detectable on epididymal sperm in concentrations per mg of
protein about 4 % of that in the adult testis (Bechtol et al. 1979; Bechtol, Jonak
& Kennett, 1980). The antigen is found in the 13-day juvenile mouse, and this
is consistent with its first appearance on either spermatogonia or early spermatocytes (Nebel, Amaroso & Hackett, 1961). The present study was undertaken to determine more precisely the developmental expression of XT-1 and to
describe its distribution on spermatozoa. As seen in immunoperoxidase-stained
tissue sections, the antigen becomes detectable on early (leptotene/zygotene)
Germ-cell differentiation antigen
95
spermatocytes and increases in staining during the long pachytene spermatocyte
phase. During haploid cell development the distribution of the determinant
shifts from its relatively uniform surface distribution on spermatocytes to a more
restricted localization on the base of the head, tail and cytoplasmic lobe of the
elongating spermatid. The XT-1 determinant is thus a marker of spermatocytes
and later germ cells and of a surface molecule which is related to their differentiation. It also provides a paradigm for alteration of the cell surface during spermatid elongation.
MATERIALS AND METHODS
Animals
129/SV and BALBcJ mice were maintained as inbred colonies at The
Wistar Institute. Results obtained using the two strains were indistinguishable.
W7+(C3H/HeJ)-(Russell, Lowson & Schabtach, 1957) and W V /+(C57B1/6J)
were obtained from Jackson Laboratory, Bar Harbor, Maine, and were bred to
produce W x /W v black-eyed-white and W/+ white-spotted heterozygotes.
Antibodies and staining procedures
The XT-I (IgG2a) monoclonal anti-testis antibody was produced by a subclone
of the original XT-I hybridoma (Bechtol et al. 1979) which has lost the yi heavy
chain of the P3-X63Ag8 myeloma parent. Antibody was concentrated from
culture supernatant (HY medium, Kennett, 1980) by precipitation with 50 %
saturated (NH4)2SO4 or was purified from serum-free culture supernatant
(Murakami et al. 1982) by pH4-5 elution from a protein A affinity column (Ey,
Prowse & Jenkin, 1978; Protein A Sepharose, Pharmacia). Negative controls
were similar preparations of an IgG2a monoclonal antibody, UK8, which does
not bind testis, and of supernatants of the SP2/0 myeloma and 0-5 % bovine
serum albumin. All three gave similar results.
Immunoperoxidase staining was performed on 5 /mi sections of tissue fixed for
1 to 2 h in Bouin's fixative or 20 % formalin, followed by 75 % ethanol overnight
and standard embedding in wax. Paraffin was removed from the sections by the
method of Sainte-Marie (1962), and immunoperoxidase staining was performed
using affinity-purified, horseradish peroxidase-coupled rabbit anti-mouse Ig
(Kirkegaard and Perry Laboratories, Inc.), diaminobenzidine (Aldrich Chemical Company, Inc.), and OsO4 (Electron Microscopy Sciences), as described by
Jonak (1980).
The XT-1 antigenic activity was not detectable after embedding in several
water-permeable plastics tested. Therefore, to obtain 1 /zm immunoperoxidasestained sections, sections were processed as described above except that they
were placed on plastic coverslips, and, following immunoperoxidase staining,
they were embedded in epon and 1 fim sections were cut parallel to the plane of
the coverslip.
96
K. B. BECHTOL
The 'stage' of spermatogenesis present in each tubule cross-section was
estimated from haematoxylin (H)- and eosin (E)-stained and periodic acidSchiffs base (PAS)- and fast-green-stained sections serially adjacent to the
immunoperoxidase-stained section using the criteria (e.g. morphology of spermatids), described in Oakberg (1956a). Photomicrographs of immunoperoxidase
staining were taken with Tri-X Pan film, ASA 400, using an Olympus BH
microscope with green filter.
Immunofluorescent staining was performed on cells derived from adult testis
by mincing the tubules. Cells were incubated with XT-I or UK8 for 1 h at 0°C,
washed three times, and resuspended in a 1/120 dilution of biotinylated-rabbit
anti-mouse Ig (Vector Labs) for l h at 0°C. The cells were then washed and
incubated with FITC-avidin DCS (Vector Labs) for 1 h at O °C. The unfixed cells
were observed with a Leitz Orthoplan microscope equipped with phase-contrast
and a Phoem epi-illumination system. Photomicrographs were taken with Tri-X
Pan film, ASA 800.
RESULTS
XT-1 in the testis cycle
Immunoperoxidase-stained tubule sections from juvenile and adult testes
demonstrate the developmental expression of XT-1 antigen in a segment of testis
tubule (Fig. 1). The cell associations (12 stages, Oakberg, 1956a) of mouse
spermatogenesis are numbered around the circle, and stained tubules in most of
these stages are shown in the outer circle. This outer circle reconstructs the
events in continuously cycling epithelium of the adult testis. As is well documented, new generations of germ cells enter spermatogenesis near the rim of the
tubule and progress from mitotic spermatogonia through meiotic spermatocytes
to haploid spermatids as time proceeds. In effect, the whole tubule section with
time passes repeatedly around this cycle with the individual germ cell progressing
farther in development and nearer the centre of the tubule until its release as a
sperm in stage VII. Each turn of the circle requires 8-6 days of the 34-5-day
development of the germ cell (Oakberg, 1956&).
The spiral part of Fig. 1 shows XT-I-stained sections of tubules from juvenile
mice. Each cross section in a pie-shaped segment of the figure, for example in the
Fig. 1. Developmental expression of XT-1 is reconstructed using XT-I
immunoperoxidase-stained tubule cross sections. The least developed tubule is at the
centre of the figure. Tubules with progressively older associations of cells are shown
proceeding spirally outward. Tubules are placed on a particular radius of the figure
according to their stage of spermatogenesis, as determined from adjacent serial
sections stained with H and E and with PAS and fast green. Lower left inset is tubule
section of adult W*/Wv testis immunoperoxidase-stained with XT-I. The format of
this figure is based on the cycles of cell associations in testis tubules which has been
described in many species. Staging for the mouse is based on Oakberg, 1956a,b.
Bars = 100 jum. 5jum sections.
Germ-cell differentiation antigen
97
pie segment including roman numeral IV, is in the same stage of spermatogenesis, though the more mature generations of germ cells are missing from some
tubules, depending on the number of generations that have been initiated in that
tubule. Some of the earlier cells are more easily seen in the juvenile tubules
98
K. B. BECHTOL
where there are fewer cells, the diameter of the tubule is less and the younger
cells form layers several cells thick.
The complete development of expression of XT-1 on a single generation of
germ cells can be seen by following that generation as it develops in the adult
tubules of the outer circle of the diagram or by following the lumenal layer of
germ cells in each tubule of the spiral proceeding from the centre tubule to the
outer circle, as described below.
Spermatogonia and immature Sertoli cells
The central tubule of Fig. 1 (tubule A) and the three subsequent sections
(B,C,D) contain only spermatogonia and immature Sertoli (somatic epithelial)
cells, neither of which stain with XT-I.
Spermatocytes
The central cell layer of the next two tubules (E,F) contain antigen-positive
leptotene/zygotene spermatocytes. Early-pachytene spermatocytes are
developing in the following two tubules (G,H; stages I to V), and these show
increased XT-1 expression. These spermatocytes are surrounded by a more
peripheral layer of unstained spermatogonia. Intense XT-I staining is seen on the
late-pachytene spermatocytes of the next two tubules (I,J; stages VIII through
X). In those tubules the peripheral layers contain both lightly stained early
spermatocytes and unstained spermatogonia.
Diplotene primary spermatocytes and spermatocytes in meiosis are found in
the centre of the next two tubules (K,L; stages XI and XII). These cells are past
the peak of XT-I staining (compare with the previous two tubules), though they
still stain more intensely than the more peripheral zygotene spermatocytes in the
same tubules. Two cells in this stage XII tubule are in the process of division.
Such cells are intensely stained by the reagents in the absence of XT-I, and their
antigen expression, therefore, cannot be determined in these experiments.
Spermatids
Antigen-positive, round spermatids are found in tubules of stages I through
VIII following meiosis (i.e. M-P, the next four sections in Fig. 1). At stage IV
(N), the round spermatids are approximately similar in staining to the more
peripheral pachytene spermatocytes. As the tubule section progresses through
stages VI and VII the round spermatids remain apparently unchanged in staining
intensity, while the more peripheral pachytene spermatocytes increase in staining intensity as seen in the previous turn around the spiral.
At stage VIII, elongation of the spermatid's nucleus begins. Along with this,
the bulk of the spermatid cytoplasm begins to move caudally, away from the
nucleus to form the cytoplasmic lobe which protrudes into the lumen of the
tubule (Oakberg, 1956a). All tubules in the outer circle of Fig. 1 (Q-Y) contain
Germ-cell differentiation antigen
X
99
et
pc
9
et
Fig. 2. Partial cross sections of tubules from adult mouse testis immunoperoxidasestained with XT-I (A, B, D) or an IgG2a monoclonal antibody which does not bind
testis (C), then embedded in plastic and recut to 1/^m thickness. Lumen of each
tubule is toward the top. Basement membrane is toward the bottom and is marked
by a short dash, g = spermatogonia, Ic = leptotene spermatocytes, pc = pachytene
spermatocytes, rt = round spermatids, et = elongating spermatids. Bar = 20jum.
elongating spermatids lining the lumen. Because the elongating cells are less
than 5 //m in diameter, they are best observed in the thinner, 1 jum sections of Fig.
2. The antigen-positive spermatids in Fig. 2A are very near the beginning of
elongation (as determined by adjacent serial sections stained with H and E, and
with PAS and fast green). By Fig. 2B the spermatids have clearly begun to
elongate and the cytoplasmic lobe of the spermatid is clearly visible by antigen
staining (arrow). In Fig. 2D the elongating spermatids are near release and the
antigen-positive midpiece of the tail, housfng the mitochondria, is clearly visible
(arrow).
100
K. B. BECHTOL
Fig. 3. Testicular sperm (A and B) and elongating spermatids (C and D) were
mechanically released from the tubule of adult mouse testis and stained by indirect
immunofluorescence using XT-I antibody (A and C). Panels B and D are phasecontrast photographs of the samefield.Indirect staining with UK8 (IgG2a) produced
no detectable fluorescence (not shown). The smaller arrowhead marks the intersection of the midpiece (above) and the principal piece (below) of the tail. The
cytoplasmic lobe (large arrowhead in D) of the elongating spermatid lies along the
midpiece near the head (arrow). The residual droplet of cytoplasm (large arrowhead
in B) left on the sperm after its release from the epithelium remains on the midpiece
near the head of the testicular sperm. Bar = 10 jum.
A representative spermatid (C & D) and a released testicular sperm (A & B)
stained in suspension using indirect immunofluorescence are shown in Fig. 3.
The lobe of cytoplasm (large arrowhead, 3D) and the midpiece of the tail (from
sperm head to small arrowhead, 3D and total length shown below head, 3B)
show surface staining. The principal piece of the tail (from small arrowhead
downward, 3D) also shows detectable uniform staining, though it appears much
less intense, at least in part for geometric reasons since the distance looked
through in that part of the tail is shorter than in the midpiece. There appears to
be a small area of staining at the base of the head at the point of attachment of
the tail. The remainder of the head is negative.
Sertoli cells
Immature Sertoli cells are not stained by XT-I (Fig. 1, centre tubule). To test
for antigen expression on mature Sertoli cells, the genetically germ-cell-depleted
testes of adult W x /W v mice were immunoperoxidase stained with XT-I. These
mice are deficient in germ cells because in the embryo the primordial germ cells
do not increase normally in number and few migrate to the genital ridge (Mintz
& Russell, 1957). Nevertheless, the Sertoli cells mature, showing the characteristic changes in mitochondrial and nuclear morphology, appearance of specialized
junctions and pumping of fluid (Handel & Eppig, 1979). XT-1 is not detectable
in the testis tubules of adult W x /W v mice (Fig. 1, lower left insert), though it is
expressed normally in their W/+ sibs (not shown).
In normal adult testes there are occasionally found sections of tubule in which
one or more generations of germ cells is missing from the normal stage association
Germ-cell differentiation antigen
101
or where no apparent spermatogenesis is occurring. When such sections are
stained for XT-1, germ cells stain as expected based on their morphology,
irrespective of absence of younger or older cells and the Sertoli cells remain
unstained.
DISCUSSION
The XT-1 differentiation antigen of mouse spermatogenesis is expressed on
the surface of spermatocytes and later germ cells. There is an increase in the
intensity of surface immunostaining as germ cells proceed through spermatocyte
differentiation. This presumably is due to an increase in concentration of the XT1 determinant on the cell surface, and not to an optical artifact based on the
increasing size of the cell. All of the spermatocytes have diameters greater than
the 5/im thickness of the sections. Moreover, at high magnification (xlOOO),
examination of individual cells over their surface in many different focal planes
confirms the staining difference between cells in different stages observed at
lower magnification. The XT-1 determinant continues to be expressed after
meiosis, on spermatids, and at elongation of the spermatid, detection of the
determinant becomes restricted to the base of the head, cytoplasmic lobe and
tail. The determinant(s) detected by XT-I binding on spermatocytes and sperm
are probably identical or nearly identical since by Scatchard plot the antibody
binds to both cells with the same Ko (unpublished observation).
Two other individual antigens of spermatogenesis which are also expressed on
sperm have been identified, RSA-1 (rabbit) and 1B3 (mouse). Both of these stain
the whole sperm surface with some variation in intensity and patchiness of the
distribution (O'Rand & Romrell, 1981; Gaunt, 1982). Surface antigens that arise
during spermatogenesis and are then found on the spermatozoon can either be
distributed over the entire sperm surface (O'Rand & Romrell, 1981; Gaunt,
1982) or restricted to one or more areas of the sperm head and tail (Millette &
Bellve, 1977; O'Rand & Romrell, 1980; Tung, Yanagimachi & Yanagimachi,
1982). Several domains of sperm surface antigens (intrinsic membrane components and molecules adhering from fluids of the reproductive tract) have been
described, including the entire sperm head, the acrosomal and post-acrosomal
regions, the whole tail, the midpiece and the principal piece of the tail (e.g.
Myles, Primakoff & Bellve, 1981; Feuchter, Vernon & Eddy, 1981; Schmell,
Gulyas, Yuan & August, 1982a,6). The restricted distribution of XT-1 staining
on sperm may reflect 1) a redistribution of the existing surface molecule bearing
the XT-1 determinant or restricted localization of incorporation of new antigen
molecules during spermatid elongation, 2) chemical modification of the antigenic
molecule on the developing head which alters the XT-1 determinant so that it no
longer binds the XT-I antibody, and/or 3) masking of the XT-1 determinant by
other molecules newly associated with it.
The XT-1 antigen is detected on the surface of germ cells in situ (Figs 1-3) and
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K. B. BECHTOL
in cell suspension (Bechtol, Jonak & Kennett, 1980), provided the cells have not
previously been exposed to proteolytic enzymes (Bechtol et al. 1979). The antigen is not detected on juvenile Sertoli cells in normal testis, on Sertoli cells in
aspermatogenic segments of normal adult testis, or on Sertoli cells in genetically
germ-cell-depleted adult mice. The XT-1 determinant thus appears to be a
marker for spermatocytes and later germ cells, though it cannot be ruled out
from the above data that Sertoli cells express the antigen where they contact or
lie near the antigen-positive germ-cell stages. Detection of the antigen is in any
case dependent on the presence of germ cells of the appropriate developmental
stage.
The monoclonal antibody (XT-I) and the antigenic moiety it binds can serve
1) as useful markers for spermatocytes and later germ cells, 2) as markers for at
least one form of a surface molecule which is quantitatively related to male germcell differentiation, and 3) as markers of a molecular redistribution or modification which occurs at the surface of the germ cell during spermatid elongation.
The description presented herein serves as a foundation on which to build
molecular and functional studies of the antigen.
This research was supported by grants GM-23892 and CA-10815 from the National Institutes of Health. The author wishes to thank Ms Marian Butts and Ms Elsa Agio for their
excellent technical assistance, Mr Joe Weibel for embedding and cutting the 1 /im plastic
sections, and Drs Barry Zirkin and Gerd Maul for their interested discussion.
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{Accepted 5 December 1983)