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THE ANATOMICAL RECORD 244:155-164 (1996)
Differential Expression of Placental (P)-Cadherin in Sertoli Cells and
Peritubular Myoid Cells During Postnatal Development of
the Mouse Testis
LI-HSIEN LIN AND ROBERT M. DEPHILIP
Department of Cell Biology, Neurobiology, and Anatomy, Ohio State University College of
Medicine, Columbus, Ohio
ABSTRACT
Background: In previous work, RNA transcripts for placental (Pbcadherin, a calcium-dependent cell adhesion molecule, were
identified in the rat testis during the first 4 weeks of postnatal development.
However, the cells in the testis responsible for P-cadherin expression have
not yet been identified.
Methods: We used conventional epifluorescence microscopy to examine
P-cadherin immunoreactivity in cryostat sections of mouse testis and scanning laser confocal microscopy to localize P-cadherin and p-catenin in
wholemount preparations of mouse seminiferous tubules. We used fluorescent phalloidin to identify actin filaments.
Results: Sertoli cells expressed P-cadherin on postnatal days 1,3, and 8,
but not on any day thereafter. In contrast, peritubular cells did not express
P-cadherin during the first week of development, but began to express
P-cadherin on postnatal day 8 and continued expression in adulthood.
p-Catenin was localized near contact areas between peritubular cells on
postnatal days 12 and 15. A mature pattern of actin filament organization in
peritubular cells appeared on day 15 and coincided with the uniform appearance of P-cadherin and p-catenin near areas of contact between adjacent peritubular cells.
Conclusions: During postnatal development of the testis, the earlier expression of P-cadherin by Sertoli cells is replaced by the subsequent expression of P-cadherin by peritubular cells. The expression of P-cadherin
in peritubular cells is correlated temporally with the expression of P-catenin and the development of a mature network of actin filaments and is
consistent with a role in intercellular adhesion and junction formation.
0 1996 Wiley-Liss, Inc.
Key words: Actin cytoskeleton, Catenin, Cell Adhesion, Seminiferous tubules
P-cadherin belongs to a family of cell surface adhesion molecules that are characterized by their dependency on calcium for adhesive function (Takeichi,
1988). Like other “classical” cadherin molecules such
as epithelial (El- and neural (N)-cadherin, P-cadherin
was identified in functional cell adhesion assays and
consists of a large extracellular domain that specifies
adhesion, a transmembrane portion that fixes the molecule in the plasma membrane, and a cytoplasmic domain that interacts with actin microfilaments via linking molecules called catenins (Kemler, 1992). It has
been suggested that the formation of a cadherin-catenin complex is necessary for full adhesion of cadherins
(Ozawa et al., 1990).
Cadherins play a “morpho-regulator’, function during development and contribute to the formation of
body structure (Edelman, 1988). In this capacity, cadherins allow cells to recognize each other, aggregate,
0
1996 WILEY-LISS. INC.
and form definitive structures. One such example of
this function is seen during kidney development when
mesenchymal cells of the lateral plate mesoderm condense, initiate E-cadherin expression, and participate
with E-cadherin-expressing cells of the ureteric bud in
nephron formation (Vestweber et al., 1985). Termination of cadherin expression also can have an effect as
seen in the development of the neural tube, when certain ectodermal cells cease expression of E-cadherin
and define the neural plate (Hatta and Takeichi, 1986).
These cells subsequently express N-cadherin and form
Received June 19, 1995; accepted September 27, 1995.
Address reprint requests to Robert M. DePhilip, Department of Cell
Biology, Neurobiology, and Anatomy, 4072 Graves Hall, Ohio State
University, Columbus, OH 43210.
156
LIN AND DEPHILIP
the neural tube. Cadherins also play a “mechanostatic”
role in maintenance of adult structure (Ozawa et al.,
1989). This function is seen most easily in epithelial
cells, where cadherins are responsible for an initial adhesive event that results in the formation and maintenance of specific intercellular junctions (Gumbiner et
al., 1988).
We are studying the expression of cadherins in the
testis to determine their role in organogenesis and in
the establishment of the various cell-cell interactions
and junctions that are responsible for normal testicular
function. A picture of cadherin expression in the testis
began to emerge when cDNA probes and specific antibodies were used to determine the presence of different
cadherins during testicular development. Transcripts
for N-cadherin have been identified in RNA isolated
from rat (Cyr et al., 1992; Wu et al., 1993; Byers et al.,
1994) and mouse testis (MacCalman et al., 1993). An
antiserum against the putative cell adhesion recognition sequence of N-cadherin was shown to be reactive
toward the surfaces of isolated spermatogenic cells and
Sertoli cells and was able to reduce germ cell binding to
Sertoli cells in vitro by 3 0 4 0 % (Newton et al., 19931,
suggesting a role for N-cadherin in germ cell adhesion
to Sertoli cells. Transcripts for E-cadherin have also
been demonstrated in rat testis (Wu et al., 1993)) and
E-cadherin protein has been localized immunohistochemically to germ cells in 8-day-old mouse testis
(Wu et al., 1993) and to interstitial cells in fetal and
newborn rat testis (Byers et al., 1994). Finally, transcripts for P-cadherin have been identified in rat testis
RNA with highest expression occurring during the first
2 weeks of postnatal development and lower levels observed a t weeks 3 and 4 (Cyr et al., 1992; Wu et al.,
1993).Although P-cadherin protein has been identified
in homogenates of rat testis on immunoblots, P-cadherin expression has not yet been demonstrated in the
testis using immunohistochemistry.
The purpose of this work was to determine which
cells in the developing mouse testis express P-cadherin.
For these studies, we examined P-cadherin expression
in the mouse rather than in the rat as we did earlier
(Wu et al., 1993), in order to take advantage of a wellcharacterized, commercially available monoclonal antibody, PCD-1, that is specific for mouse P-cadherin
(Nose and Takeichi, 1986). Conventional epifluorescence microscopy and scanning laser confocal microscopy were used to examine P-cadherin expression in
cryostat sections of mouse testis and in wholemount
preparations of mouse seminiferous tubules. We show
that Sertoli cells and peritubular cells both expressed
P-cadherin, but in nonoverlapping patterns. Sertoli
cells expressed P-cadherin during the first week of
postnatal development, but not thereafter. Peritubular
cells did not express P-cadherin during postnatal week
1,but did so during week 2, and later. The appearance
of P-cadherin in peritubular cells paralleled the appearance of p-catenin, one of the molecules that links
cadherins to the actin cytoskeleton, and occurred near
the time that actin microfilaments organized into the
pattern characteristic of mature peritubular cells. This
work raises interesting questions regarding the function of P-cadherin in Sertoli cells and peritubular cells
and about the differential regulation of P-cadherin in
these two cell types.
MATERIALS AND METHODS
Animals
ND4 Swiss Webster mice were purchased from Harlan Sprague-Dawley (Indianapolis, IN) and were maintained on a cycle of 12 h of light and 12 h of darkness.
Pups were housed with their mothers, who had free
access to food and water. Pups were killed by cervical
dislocation. Maintenance and experimental use of all
animals complied with the NIH Guide for the Care and
Use of Laboratory Animals.
lmmunoblot Analysis
Freshly dissected tissues were homogenized in
buffer “0” (O’Farrell, 1975) containing protease
inhibitors as described (Wu et al., 1993). Samples were
sonicated for 30 sec on ice, clarified by centrifugation
at 15,850 xg for 15 min, and boiled for 5 min before
electrophoresis on 7.5% polyacrylamide reducing slab
gels containing sodium dodecyl sulfate (Laemmli,
1970). Rainbow’” prestained protein markers (Amersham Co., Arlington Heights, IL) and p-galactosidase
(molecular mass = 116 kDa) were included in each
gel. Proteins were then electrophoretically transferred
to BA83 0.2 pm2 pore size nitrocellulose (Schleicher &
Schuell, Keene, NH) using a two-step procedure (Wu
et al., 1993). Nitrocellulose blots were incubated for 30
min a t room temperature in blocking buffer consisting
of Dulbecco’s phosphate-buffered saline (pH 7.2)
containing 1.5 mM CaC1, and 1.0 mM MgC1, (DPBS),
5% nonfat dry milk, and 0.1% Tween-20. The rat
monoclonal antibody against mouse P-cadherin
(PCD-1) (Nose and Takeichi, 1986) was purchased
from Zymed Laboratories (South San Francisco, CA).
Blots were incubated overnight at 4°C with 5 pglml
PCD-1 antibody in blocking buffer. After three washes
with DPBS-0.1% Tween-20, blots were incubated for
1.5 h at room temperature in a 1:2500 dilution of
alkaline phosphatase-conjugated, goat antirat IgG
antiserum (Promega, Madison, WI) in 50 mM TrisHC1-150 mM NaC1, pH 8.2 (TBS) containing 5%
nonfat dry milk and 0.1% Tween-20. Blots were then
washed three times with TBS containing 0.1%
Tween-20 and bound antibodies were detected using a
substrate mixture of nitro blue tetrazolium and
5-bromo-4-chloro-3-indoyl phosphate (Sigma Chemical
Co., St. Louis, MO). P-Galactosidase in the marker
lane was stained with Fount India drawing ink
(Pelikan, F.R.A.). A control for nonspecific binding of
the secondary antibody was carried out on duplicate
blots that were not exposed to primary antibody, but
were treated with the secondary antibody and the
chromogen.
lmmunofluorescenceMicroscopy
Tissues were embedded in O.C.T. Compound (Miles,
Elkhart, IN) and frozen in liquid nitrogen. Cryostat
sections (7-10 pm) were cut, thawmounted on 0.6%
gelatin-coated slides, and stored a t -20°C. Sections
were rehydrated in DPBS and fixed for 10 min with
3% paraformaldehyde in DPBS. After three washes
with DPBS, sections were extracted with 0.5% (v/v)
Triton X-100 in DPBS for 5 min at room temperature
using gentle agitation. Extraction with Triton X-100
was found empirically to reduce nonspecific immuno-
157
P-CADHERIN IN MOUSE TESTIS
reactivity. DPBS containing 5% normal goat serum
(Jackson ImmunoResearch Lab., West Grove, PA) was
then applied t o sections for 1 h at room temperature.
Sections were incubated with 10 pg/ml PCD-1 in
DPBS-5% normal goat serum for 1 h at 37°C. After
three washes with DPBS, sections were incubated in a
1:30 dilution of rhodamine-conjugated, goat antirat
IgG antiserum (HyClone Laboratories, Logan, UT) in
DPBS-5% normal goat serum for 30 rnin at room
temperature. After three washes with DPBS, some
sections were incubated with 5 pg/ml Hoechst 33258
for 5 min to visualize nuclei (Haneji and Koide, 1988).
All sections were mounted using nine parts glycerol
and one part DPBS before examining with conventional epif luorescence microscopy. Control sections in
which the primary antibody was omitted were examined in each experiment.
Wholemount Preparation of Seminiferous Tubules
for lmmunohistochemistry
The tunica albuginea of the testis was removed and
seminiferous tubules were gently rinsed in a plastic
culture dish containing 2 ml of ice cold DPBS. Seminiferous tubules were fixed in two steps, first by gently
adding an equal volume of ice cold DPBS containing
3%paraformaldehyde into the dish (final concentration
of paraformaldehyde = 1.5%)and incubating on ice for
30 min, followed by replacement of this solution with
3%paraformaldehyde and incubation for an additional
30 min. Continuous, gentle agitation was used in all
steps described below. After washing three times with
DPBS, each testis was cut into smaller pieces (-1-2
mm3) and extracted with 0.05% (dv) Triton-X 100 in
DPBS for 5 min at room temperature. Tubule fragments were rinsed and 2-3 tubules, each 1-2 mm in
length, were transferred to one well of a 96-well tissue
culture plate. Tubule fragments were incubated in
DPBS-5% normal goat serum for 1 h at room temperature, followed by incubation with primary antibodies
in DPBS a t 37°C for 1 h.
The PCD-1 antibody was used a t a concentration of 10
pg/ml. Mouse monoclonal antibody against chicken
p-catenin (Johnson et al., 1993) was applied as an undiluted supernatant of a hybridoma culture. After incubation, tubule fragments were rinsed and incubated
with rhodamine-conjugated, goat antirat IgG (1:lOO) or
goat antimouse IgG (1:lOOO)antiserum, as appropriate,
at room temperature for 30 min. Tubule fragments were
washed three times with DPBS prior to mounting in
glycerol-DPBS medium. The preparations were examined using conventional epifluorescence microscopy as
well as the MRC-600 Confocal Laser Scanning Imaging
System (Bio-Rad Laboratories, Life Science Group,
Melville, NY). Confocal images were captured and photographed using a digital film recorder (GCC Technologies, Bedford, MA). Control wholemount tubules were
prepared without incubation with primary antibodies
and were examined in each experiment, using the same
gain and black level settings as used for samples incubated with primary antibodies. Actin filaments were
visualized after incubating fixed and detergent-extracted tubules for 5 min with 0.1 pg/ml fluoresceinlabeled phalloidin (#P-5282, Sigma Chemical Co.).
RESULTS
lmmunodetection of P-cadherin in Mouse Testis
and Epididymis
The specific reactivity of PCD-1 toward P-cadherin
in mouse skin has been demonstrated (Nose and Takeichi, 1986).To establish the reactivity of PCD-1 toward
mouse testis, we examined homogenates of mouse testis on immunoblots. We included homogenates of
mouse epididymis in our analysis because high expression of P-cadherin RNA transcripts in the rat epididymis has been reported (Cyr and Robaire, 1991). PCD-1
identified an immunoreactive band with a molecular
mass of 118 kDa in homogenates of skin, testis, and
epididymis prepared from 7-day-old mice (Fig. 1A).
PCD-1 did not react with homogenates of mouse liver.
The expression of P-cadherin in skin and its absence in
liver is consistent with previous immunoblot results
using this antibody (Nose and Takeichi, 1986). PCD-1
reactivity in cryostat sections of mouse epididymis was
concentrated at contact sites between adjacent epithelial cells (Fig. 1B) and suggested that epithelial cells
are responsible for the expression of P-cadherin RNA
transcripts that have been detected in RNA isolated
from the intact organ (Cyr and Robaire, 1991). The
P-cadherin reactivity in mouse skin was associated
with cells in the outer root sheath and hair matrix of
follicles (Fig. 1C) and was consistent with previous localization of P-cadherin in human skin (Hirai et al.,
1989). No specific reactivity of antibody PCD-1 was
found in cryostat sections of mouse liver (Fig. 1D).
Next, we explored the spatial distribution of P-cadherin immunoreactivity in mouse testis during the
first 15 days of postnatal development in cryostat sections. Germ cells, Sertoli cells, and peritubular cells
were identified after counterstaining nuclei with
Hoechst 33258, a fluorescent dye for DNA (Haneji and
Koide, 1988). Germ cell nuclei were large and contained evenly fluorescent chromatin, whereas Sertoli
cell nuclei were small and contained clusters of
brightly fluorescent heterochromatin. Peritubular cell
nuclei were flattened and separated from the seminiferous epithelium by a space occupied by the basement
membrane between Sertoli cells andperitubular cells.
On postnatal day 1, P-cadherin immunoreactivity
appeared in both the basal and central regions of the
developing seminiferous tubules (Fig. 2A). P-cadherin
in the basal region of day 1 tubules was assigned to
Sertoli cells because a direct correlation could be made
between P-cadherin reactivity and Hoechst-stained
Sertoli cell nuclei (Fig. 2A,B). P-cadherin in the central
region of day 1 tubules was also attributed to Sertoli
cells, specifically to Sertoli cell processes that extend
between gonocytes to reach the center of the cords. It is
unlikely that P-cadherin reactivity in the center of the
cords is associated with the gonocyte surface because
the surface contour of gonocytes is smooth, whereas the
P-cadherin reactivity in the center of the tubules is
irregular. We never observed P-cadherin reactivity associated with the smooth contours of germ cells, despite
the numerous opportunities to view such profiles in
sectioned material. The absence of P-cadherin reactivity within the cytoplasm of gonocytes resulted in large
areas within the cords that exhibited no fluorescent
signal. On postnatal day 3 P-cadherin reactivity was
158
LIN AND DEPHILIP
Fig. 1 , Immunoblot detection and immunofluorescence localization
of P-cadherin in mouse tissues. Immunoblot in A contains protein (30
pg) isolated from testis (t), epididymis (el, skin ( s ) ,liver (1) of a postnatal day 7 mouse. Arrow on the right indicates P-cadherin immunoreactivity at 118 kDa. Positions of molecular mass markers are indicated on the left in kDa. Bands around 50 kDa seen in all lanes at the
asterisk represent nonspecific reactivity of the secondary antibody.
Immunofluorescence of P-cadherin was localized on cryostat sections
of day 5 epididymis (B) and day 7 skin (C),but not day 7 liver (D).
Arrowheads in B indicate three of many examples of P-cadherin reactivity at areas of lateral contact between epididymal cells. In C, a
cross section through the basal region of five hair follicles is shown
and P-cadherin reactivity associated with cells in the outer root
sheath (0s)and the hair matrix (hm) is indicated. B, x 250; C, x 400;
D, x 325.
concentrated in the basal region of the cords and was
associated again with Sertoli cells (Fig. 2C). Central
regions of the tubules on day 3 often contained clusters
of gonocytes that excluded P-cadherin reactivity, demonstrating clearly that P-cadherin was not present a t
sites of contact between gonocytes. By postnatal day 8,
the majority of gonocytes had migrated from the central regions of the tubules to the basement membrane.
P-cadherin reactivity re-appeared in the central region
of the tubules from which gonocytes had departed and
again occupied both the basal and central regions, suggesting that Sertoli cell processes had regained access
to the center of the tubule (compare A and E, Fig. 2).
An additional feature of P-cadherin reactivity on day 8
was a thin, discontinuous line of fluorescent signal between Sertoli cell nuclei and peritubular myoid cell
nuclei (Fig. 2E) that was not seen on day 1, nor on day
3. During subsequent development of the seminiferous
tubules, this linear reactivity of P-cadherin a t the margins of the tubules intensified, whereas the intratubular reactivity diminished, so that by day 12, P-cadherin
in the testis was seen exclusively at the tubular margins and was absent within the tubules (Fig. 3A).
The association of P-cadherin with peritubular cells
was suggested when tangential sections of day 15 tubules were examined (Fig. 3B), and P-cadherin reactivity could be seen outlining a network of large cells a t
the tubule margin. The identification of these cells as
peritubular cells was confirmed in wholemount preparations of tubules, processed for immunohistochemistry and examined with conventional fluorescence microscopy (Fig. 3C). The large size of these cells and
their location on the surface of seminiferous tubules
identified them as peritubular cells and distinguished
them from Sertoli cells that are smaller and are located
below the tubule surface. Furthermore, sinusoidal endothelial cells were removed during isolation of seminiferous tubules for wholemount examination and did
not contribute to P-cadherin reactivity on the tubule
surface. P-cadherin continued to be associated with
peritubular cells on postnatal day 60, the latest age of
development studied here (not shown). No P-cadherin
P-CADHERIN IN MOUSE TESTIS
159
Fig. 2. Immunofluorescence localization of P-cadherin during early
postnatal development of the mouse testis. P-cadherin reactivity in
cryostat sections from 1-,3-, and 8-day-old mouse testes is presented
in A, C, and E,respectively. DNA was visualized in the same sections
by counterstaining with Hoechst dye 33258 and the corresponding
micrographs are shown in B, D, and F. Arrows in A and B indicate the
same five Sertoli cells and demonstrate that P-cadherin is associated
with Sertoli cells. Arrows in E indicate the discontinuous line of
P-cadherin reactivity between Sertoli cell nuclei and pertibular cell
nuclei and should be compared to the corresponding area indicated by
arrows in F. In E and F, note the presence of P-cadherin reactivity and
the absence of cell nuclei in the center of the tubules. Sc, P-cadherin
reactivity associated with Sertoli cells in the basal region of a seminiferous tubule sectioned tangentially. g, germ cell nucleus. A, B, E,
F, x 500; C and D, x 450.
immunoreactivity was ever detected in the interstitial
region of the testis at anv
" aae- studied.
postnatal development, wholemount preparations of
day 7 and dav 15 seminiferous tubules were compared
using confocd microscopy. A focal plane at the sirface
of day tubules revealed that only a few of the large,
flattened peritubular cells were outlined by P-cadherin
reactivity-(Fig. 4A). The P-cadherin reactivity associated with Sertoli cells within day 7 tubules was dem-
u
Demonstration of P-Cadherin in Seminiferous Tubules
using Confocal Microscopy
To visualize P-cadherin both on the surface and
through the thickness of seminiferous tubules during
160
LIN AND DEPHILIP
8 (Fig. 2E). By day 15, all peritubular cells were outlined by P-cadherin, creating an impressive network of
reactivity a t contact sites between adjacent cells (Fig.
4C). P-cadherin reactivity associated with peritubular
cells also could be demonstrated when the focal plane
passed through the diameter of day 15 tubules (Fig.
4D). Here, a single layer of peritubular cells was immunoreactive at the lateral margins of the tubule,
whereas the multiple layers of cells within the tubules
were not reactive. Thus P-cadherin reactivity on peritubular cells and the lack of reactivity on Sertoli cells
and germ cells within day 15 tubules were demonstrated both in cryostat sections (Fig. 3B) and wholemount preparations (Fig. 4D).
Actin Filament Organization and p-Catenin Expression in
Peritubular Cells during Postnatal Development
Fig. 3. Immunofluorescence localization of P-cadherin during later
postnatal development of mouse testis. P-cadherin reactivity in cryostat sections from 12- and 15-day-old mouse testis is presented in A
and B, respectively. Arrows in A and B indicate the discontinuous line
of P-cadherin reactivity surrounding seminiferous tubules (t) cut in
cross section. In B, P-cadherin reactivity is observed outlining peritubular cells (p) in tubules cut tangentially. There is no P-cadherin
reactivity within the seminiferous tubules at these ages. In C, the
continuous network of P-cadherin reactivity outlining peritubular
cells in tubules from 15-day-old mouse testis is demonstrated in
wholemount preparations examined with conventional microscopy.
The plane of focus is at the surface of the tubules. A, x 325; B and C ,
x 500.
onstrated again when the focal plane passed through
the tubule diameter (Fig. 4B) and should be compared
to the P-cadherin reactivity on cryostat sections on day
The adhesive function of cadherins is dependent on
an association with actin filaments that is mediated by
catenins (Kemler, 1993). Having demonstrated that
P-cadherin is expressed at contact sites between all
peritubular cells on day 15, it was appropriate to examine the organization of actin filaments in peritubular cells and, in addition, to ask whether any catenin
molecule could be localized at the peritubular cell surface. Actin filament bundles in mature peritubular
cells are arranged in a characteristic orthogonal pattern and have been studied in adults of several species
using fluorescent phallotoxins (Vogl and Soucy, 1985;
Vogl et al., 1985; Maekawa et al., 1994). The postnatal
development of peritubular cell actin bundles has been
studied in rats, where the adult pattern appears on, or
slightly before postnatal day 22 in the Sprague-Dawley
strain (Russell et al., 1989), or on day 30 in the Wistar
strain (Maekawa et al., 1995). To correlate the development of the actin cytoskeleton in peritubular cells
with P-cadherin expression, we incubated wholemount
preparations of seminiferous tubules with fluorescent
phalloidin and examined the preparations using confocal microscopy. Actin bundles perpendicular to the long
axis of seminiferous tubules appeared first and were
detected in the majority of peritubular cells on postnatal day 7 (Fig. 5A). Perpendicular actin bundles were
still the predominant type in peritubular cells on day
12 (Fig. 5B). On day 15, actin bundles parallel to the
long axis of tubules were found in the majority of peritubular cells and joined the perpendicular bundles to
create the mature orthogonal network (Fig. 5C). At all
ages, strong fluorescent signal corresponding to cortical actin defined the perimeter of peritubular cells.
p-Catenin immunoreactivity outlined several peritubular cells on day 12 (not shown) and the majority of
peritubular cells on day 15 (Fig. 5D), the same day that
P-cadherin appeared uniformly at peritubular cell contact sites (Fig. 4C) and that the mature actin network
was established (Fig. 5C).
DISCUSSION
P-cadherin has been localized to two cell types in the
developing mouse testis-Sertoli cells within the seminiferous tubules and peritubular cells in the tubule
boundary tissue. However, the temporal expression of
P-cadherin in these two cells was quite different. P-cadherin expression in Sertoli cells was observed on post-
P-CADHERIN IN MOUSE TESTIS
161
Fig. 4. Immunofluorescence localization of P-cadherin in wholemount preparations of seminiferous tubules using confocal microscopy. P-cadherin reactivity is demonstrated in tubules from 7- (A and
B) and 15- (Cand D) day-old mouse testis. The plane of focus is either
at (A and C) or below (B and D) the surface of the tubules. In A, an
example of P-cadherin reactivity outlining a large peritubular cell is
indicated (arrow). In B, P-cadherin reactivity associated with cells
within the seminiferous tubules is shown. In C, P-cadherin reactivity
outlines all peritubular cells on the surface of day 15 tubules. In D,
the plane of focus is through the diameter of a day 15 tubule and
demonstrates P-cadherin reactivity associated with peritubular cells
(example at arrow) at the lateral margins of the tubule. There is no
P-cadherin reactivity associated with cells within seminiferous tubules from the 15-day-oldmouse. The width of the tubule in C and D
is exaggerated because the specimen was compressed under the coverslip to better demonstrate the P-cadherin network. A-D, x 500.
natal days 1 , 3 , and 8, but not on day 12 or thereafter.
P-cadherin expression in peritubular cells was first observed on day 8, became progressively stronger, and
was seen as late as postnatal day 60. The detection of
P-cadherin protein in the testis seen here immunohistochemically should be compared with previous identification of P-cadherin RNA transcripts (Cyr et al.,
1992; Wu et al., 1993). The level of P-cadherin transcripts is highest during the first 2 weeks of postnatal
development and lower at weeks 3 and 4. It is likely
that P-cadherin mRNA identified during week 1is produced by Sertoli cells, whereas transcripts expressed a t
lower levels during weeks 3 and 4 are synthesized by
peritubular cells.
Functions for P-cadherin in the early postnatal development of Sertoli cells can be suggested by considering three features of the seminiferous tubules during
this period. First, Sertoli cells and germ cells undergo
an important change in relative position as gonocytes
move from the center of the cords to their permanent
position adjacent to the basement membrane (McGuinness and Orth, 1992). This migration of gonocytes is
thought to be crucial for providing the proper number
of germ cells to sustain spermatogenesis. P-cadherin
may help re-establish contact between Sertoli cells as
gonocytes migrate between them and thus maintain
tubule structure during this period of cell migration.
Second, Sertoli cells in the newborn mouse demonstrate gap junctions and rudimentary occluding junctions (Nagano and Suzuki, 1976). The gap junctions
decrease in number and size between birth and the end
of week 1,whereas the occludingjunctions continue to
form and eventually divide the seminiferous tubule
into apical and basal compartments. P-cadherin expressed during postnatal week 1 may facilitate gap
junction formation between Sertoli cells, a role proposed for N-cadherin in mouse sarcoma cells (Matsuzaki et al., 1990) and for E-cadherin in mouse epidermal cells (Jongen et al., 1991). Since P-cadherin
expression within seminiferous tubules declines to an
undetectable level between day 8 and day 12, before
the Sertoli cell occluding junctions mature around day
16 (Nagano and Suzuki, 1976), any involvement of
P-cadherin in occluding junction formation must be
early, rather than late in this process. Finally, the loss
of P-cadherin expression by Sertoli cells between day 8
162
LIN AND DEPHILIP
Fig. 5. Distribution of actin filament bundles and p-catenin immunoreactivity in peritubular cells.
Wholemount preparations of seminiferous tubules were incubated with fluorescein-conjugated phalloidin and examined using confocal microscopy. Actin filament distribution was observed in peritubular
cells from 7- (A), 12- (B),and 15- (C) day-old tubules. Arrows in A and B indicate actin bundles that are
perpendicular to the long axis of the seminiferous tubule. In C, actin filaments parallel to the long axis
of tubules joined the perpendicular fibers to create the mature lattice network in the majority of peritubular cells. Black arrowheads in A, B, and C indicate cortical actin at the perimeter of peritubular
cells. In D, p-catenin immunoreactivity outlined the majority of peritubular cells on the surface of
15-day-old tubules. A-D, x 500.
and day 12 coincides with their decreased potential for
proliferation. The labelin index of mouse Sertoli cells
after incorporation of [BHI-thymidine declines from
11%on postnatal day 6 t o 0.1%on day 12 (Kluin et al.,
1984).Since P-cadherin expression has been associated
with cells having a high rate of proliferation (Hirai et
al., 1989), the loss of P-cadherin by Sertoli cells may
signal a more mature, postmitotic stage of differentiation. Whatever its role in Sertoli cells, P-cadherin may
function independently of known catenins, since we
were unable to detect p-catenin within mouse seminiferous tubules (not shown), and only weak staining with
antibodies against a-and p-catenin was reported in rat
seminiferous tubules during fetal development and on
postnatal day 10 (Byers et al., 1994).
The localization of P-cadherin and 6-catenin at areas
of intercellular contact suggests an adhesive function
for P-cadherin in peritubular cells. Furthermore, the
appearance of a mature lattice network of actin bundles
at about the same time that P-cadherin and p-catenin
completely outline the peritubular cells is consistent
with the idea that a P-cadherin-p-catenin-actin
fil-
ament complex is being formed to achieve intercellular
adhesion and junction formation. The identification of
discrete junctions between peritubular cells using electron microscopy is challenging because of the limited
profiles that are available in sectioned material. Nevertheless, cytoplasmic dense bodies, responsible for
cellular adhesion and similar to those seen in smooth
muscle cells, have been described between mouse peritubular cells (Ross, 1967). It is reasonable to predict
that strong junctions between peritubular cells are necessary to maintain the integrity of the peritubular cell
sheath as it undergoes the contractions responsible for
the movement of testicular fluid and spermatozoa. Our
demonstration of 6-catenin in peritubular cells of the
mouse is consistent with previous work reporting p- and
a-,but not y-catenin in developing and mature peritubular cells of the rat (Byers et al., 1994).
The question of whether germ cells also express
P-cadherin remains unanswered. In sections containing germ cell aggregates that excluded Sertoli cells, it
was clear that P-cadherin was not localized at contact
sites between adjacent germ cells. Therefore, it is un-
P-CADHERIN IN MOUSE TESTIS
likely that the observed variations in immunostaining
during the first 15 postnatal days can be attributed to
changes in the germ cell population that occur during
this period (BellvB et al., 1977). However, we cannot
rule out the possibility that P-cadherin is expressed at
contact sites between germ cells and Sertoli cells. We
have shown that germ cells in the 8-day-old mouse testis express E-cadherin (Wu et al., 1993) and have observed E-cadherin on germ cells throughout the first
week of postnatal development (not shown). Because
adhesion between cells expressing different types of
cadherin has been observed (Volk et al., 1987), an interaction between E-cadherin-expressing germ cells
and P-cadherin-expressing Sertoli cells during the first
week of postnatal development must be considered. In
addition, cadherins have been shown t o bind to integrins (Cepek et al., 1994), although this binding is not
a general feature of cadherin activity. Integrin subunits have been identified in the developing testis (Palombi et al., 1992; Frojdman and Pelliniemi, 19941, and
it is possible that P- or E-cadherin interact with integrins during gonadogenesis.
Finally, aside from discussion of P-cadherin function
in Sertoli and peritubular cells, these results raise interesting questions regarding the differential regulation of P-cadherin in two cell types that are separated
from each other only by a basement membrane. Although their microenvironments may differ, Sertoli
cells and peritubular cells are subject to the same endocrine environment, yet Sertoli cells express P-cadherin when peritubular cells do not and vice versa. The
loss of P-cadherin expression by Sertoli cells coincides
with a decrease in the amount of bioactive Mullerian
inhibiting substance to an undetectable level (Taketo
et al., 1993). Mullerian inhibiting substance is produced by immature Sertoli cells and is responsible for
regression of the female reproductive ducts in male embryos, but has an inductive effect on Sertoli cells. It will
be important to determine whether Sertoli cells autoregulate their expression of P-cadherin via Mullerian
inhibiting substance. When considering the regulation
of P-cadherin in peritubular cells, it may be significant
that the expression of P-cadherin parallels the maturation of peritubular cells, a process that is androgendependent (Hovatta, 1972).The androgen regulation of
P-cadherin in peritubular cells can be tested in vivo
and in vitro. The developing testis may prove to be a
valuable model system to study the differential regulation of the P-cadherin gene. A unique feature of this
system is that Sertoli cells and peritubular cells can act
as positive and negative controls of P-cadherin expression for each other at specific stages of testis development.
ACKNOWLEDGMENTS
We thank Margaret J. Wheelock and Keith R.
Johnson (University of Toledo) for their gift of antibody
against p-catenin. We thank Kenneth H. Jones (Ohio
State University) for his review of the manuscript before submission. This work was supported by NIH
Grant HD-19735.
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