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Department of Anatomy and Cell Biology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140
Address all correspondence and requests for reprints to: Dr. Andrew Laslett, Department of Anatomy and Cell Biology, Temple University School of Medicine, 3400 North Broad Street, Philadelphia, Pennsylvania 19140. E-mail: alaslett{at}astro.ocis.temple.edu
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Abstract |
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T3 (100 nM) down-regulated NCAM expression in vitro, as assessed by Western blotting and immunofluorescent staining. This contrasted with the continued expression of NCAM in cultures without added T3 but mimicked the disappearance of NCAM from the neonatal rat testis in vivo. In addition, Western analysis confirmed that P-cadherin is highly expressed in the developing rat testes, as it is in those of mice. We found that P-cadherin is strongly expressed in gonocytes and weakly expressed in Sertoli cells. Moreover, unlike NCAM, P-cadherin expression diminishes with time in vitro in the absence of added hormones. In parallel with our observations for NCAM, expression of P-cadherin was also apparently decreased by T3 (100 nM).
Subsequent quantitative analyses of cultures exposed to a range of T3 levels (0.1–100 nM) indicated that T3 causes detachment of many gonocytes in a dose- and time-dependent manner (approximately 80% detached at 100 nM). In addition, Western blotting indicated that lower concentrations of T3 down-regulate NCAM but not P-cadherin. From this we conclude that the apparent decrease in P-cadherin induced by 100 nM T3 and detected on Western blots reflects loss of gonocytes. In contrast, even low levels of T3 appear to down-regulate NCAM production before any significant detachment of gonocytes. Finally, low levels of T3 that did not affect numbers of adherent Sertoli cells nevertheless caused detachment of gonocytes. Thus, our observations identify T3 as a regulator of NCAM expression in neonatal testicular cells and as a modifier of gonocyte/Sertoli cell adhesion in vitro.
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Introduction |
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Extensive study of these cells, both in vivo and in vitro, has yielded some information about regulation of their development. Whereas many potential paracrine mediators can be found in the neonatal testis (3, 4), Sertoli and germ cells also interact via contact mediated mechanisms. They maintain functional gap junctions and are also tightly adherent to each other (5). Moreover, contact between the two cell types is critical for germ cell survival, because our work (5, 6, 7, 8, 9) and that of others (10) indicate that both gonocytes from newborns and maturing germ cells from adults require contact with Sertoli cells to survive for any appreciable length of time in vitro. Thus, maintenance of appropriate germ cell-Sertoli cell interaction is critical for both development and adult function of the testis.
In spite of the recognized importance of Sertoli cell-gonocyte contact, the precise way in which these cells interact and the regulation of their interaction remain largely unexplored. However, we recently demonstrated the importance of neural cell adhesion molecule (NCAM) in attachment between neonatal Sertoli and germ cells in vivo and in vitro (8, 9). Moreover, we found that NCAM is substantially down-regulated after postnatal day 5 in vivo, eventually disappearing from the adult seminiferous epithelium (9). Thus, appropriate expression of NCAM may be particularly critical during neonatal testicular development. In addition, other adhesion molecules, including P-cadherin (11, 12, 13, 14, 15), have been detected in developing testes, although we know little about the function and regulation of these factors. Hence, we have begun to examine more closely the roles played by both NCAM and other adhesive molecules within the developing testis.
Substantial evidence, obtained both in vivo and in vitro, indicates that T3 is an important modulator of testicular development. For example, experimental hypothyroidism leads to a prolonged period of Sertoli cell proliferation and a delay in differentiation of these cells, whereas hyperthyroidism causes a shortened period of Sertoli cell proliferation and accelerated Sertoli cell differentiation (16, 17, 18, 19, 20, 21, 22, 23). In addition, when FSH-stimulated Sertoli cells are exposed to T3 in vitro, their rate of proliferation is reduced, and a number of markers of Sertoli cell differentiation appear (16). Thus, T3 is believed to be an important physiological stimulus that causes Sertoli cells to cease mitotic activity and differentiate. Interestingly, T3 may also regulate germ cell development, because gonocytes persist longer than normal in hypothyroid rat pups (22). This is likely to be mediated via an effect of the hormone on Sertoli cells, which bear receptors for T3 and are the main if not only target within the seminiferous epithelium (19, 24, 25, 26, 27). Thus, T3 may modify signaling between Sertoli cells and gonocytes that is critical for the development of germ cells in neonates. This may involve T3-modified expression of adhesion factors, in particular NCAM, because T3 has been shown to repress NCAM transcription during brain maturation (28). In the present study, we used a well-characterized Sertoli cell-gonocyte coculture system to determine directly whether T3 regulates production of NCAM and/or P-cadherin in neonatal testicular cells, and, if so, how this impacts the fate of gonocytes and/or Sertoli cells. Our findings indicate that T3 induces specific down-regulation of NCAM and that this may play an important role in modifying gonocyte/Sertoli cell adhesion in vitro.
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Materials and Methods |
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Gonocyte-Sertoli cell cocultures
Cocultures of gonocytes and Sertoli cells were prepared as
previously described (5, 8). Briefly, testes from 1- or 5-day-old pups
(day of birth = day 1) were aseptically removed, decapsulated, and
subjected to sequential digestion with collagenase/hyaluronidase and
collagenase. A single cell suspension was obtained by incubating cell
aggregates in cell dissociation buffer (Life Technologies, Inc., Gaithersburg, MD), and cells were plated in either 35-mm
Petri dishes (3.5 x 106 per dish) for
protein analyses or eight chamber culture slides (0.5 x
106 per chamber; Lab-Tek, Nunc, Naperville, IL)
for immunolocalization or quantitation of cell numbers. Surfaces of
both culture dishes and chamber slides were precoated with Matrigel
(Collaborative Research, Waltham, MA), diluted 1:1 with
medium, as previously described (5). Cultures were maintained in
hormone- and serum-free Eagle’s D-Valine MEM (Life Technologies) containing 0.1 mM nonessential
amino acids, 1 mM sodium pyruvate, 3 mM sodium
lactate, 5 µg/ml transferrin, and 50 ng/ml retinol in a
water-saturated environment of 95% air, 5% CO2
at 37 C for up to 15 days. One day after plating, appropriate chambers
and dishes were treated with 0, 0.1, 1, 10, or 100 nM
T3 (Calbiochem, San Diego, CA) in
hormone- and serum-free Eagle’s D-Valine MEM. T3
was prepared fresh and replenished in chambers or dishes every 24
h.
Preparation of protein samples
Tissue samples. Adult rats and rat pups (1, 3, 5, 10, 15, or
23 days old) were killed by CO2 asphyxiation; and
testes were immediately removed, decapsulated, snap-frozen in liquid
nitrogen, and stored at -70 C. Protein samples for Western blotting
were prepared with a modification of the method described by Rougon
et al. (29). Briefly, frozen tissues were thawed;
hand-homogenized on ice in a lysis buffer containing 50
mM Tris (pH 7.4), 1% Triton X-100, 5
mM EDTA, 1.0 mM
phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin and 10 µg/ml
leupeptin; followed by incubation at 4 C for 60 min. Tissue lysates
were then centrifuged at 14,000 x g at 4 C for 30 min,
and supernatants were collected into fresh tubes. Protein
concentrations of the supernatants were determined using a
bicinchoninic protein assay kit (Pierce Chemical Co., Rockford, IL).
Cells. Protein samples were prepared from cocultures after 1, 2, 4, 5, 10, or 15 days in culture. After rinsing gently with fresh medium, adherent cells were harvested with a cell scraper (Fisher Scientific, Pittsburgh, PA), subsequently pelleted, and stored at -70 C until preparation of proteins, as described above for tissues.
Antibodies
Primary antibodies used for Western blotting and
immunofluorescence were: 1) a polyclonal rabbit IgG (RO49) recognizing
the three major isoforms of NCAM (8) (provided as a gift by U.
Rutishauser); and 2) a polyclonal goat IgG recognizing P-cadherin,
which does not cross-react with other members of the cadherin family
(Santa Cruz Biotechnology, Inc., Santa Cruz, CA).
Western Blotting
Proteins were equally loaded on each gel (50–100 µg total
protein/lane) and separated by electrophoresis on a 5 or 7.5%
SDS-polyacrylamide gel, then electrophoretically transferred to a
nitrocellulose membrane. After incubation in 5% dried nonfat milk
powder in Tris buffered saline with 0.1% Tween-20 at 4 C overnight,
membranes were probed with primary antibody for 60 min at room
temperature (RT). After four washes of at least 10 min each in Tris
buffered saline with 0.1% Tween-20, membranes were incubated with the
appropriate horseradish peroxidase-conjugated secondary antibody [goat
antirabbit IgG (Santa Cruz Biotechnology, Inc.)] for RO49
or rabbit antigoat IgG (H+L; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) for anti-P-cadherin for 30 min at RT.
Antibodies specifically bound to the membrane were visualized by
chemiluminescence with a SuperSignal kit (Pierce Chemical Co.), applied according to the manufacturer’s
directions. To verify that lanes had been equally loaded with protein,
some membranes were stripped, using the Chemicon Re-Blot stripping
procedure (Chemicon, Temecula, CA), and reprobed with a mouse
monoclonal IgG specific for ß-tubulin (Roche Molecular Biochemicals, Indianapolis, IN). Analyses of P-cadherin and NCAM
were each performed a minimum of three times, to ensure that results
were reproducible. Densitometric data were obtained from Western blots
using the Bioquant (R & M Biometrics, Inc., Nashville, TN) software
suite (Version 3.00.6). Data were normalized to ß-tubulin and
expressed as a percentage of the untreated control (mean ±
SE).
Northern blotting
Total RNA was obtained from testis tissue or cocultures, using
the QIAGEN RNeasy kit according to the manufacturer’s
instructions(QIAGEN, Valencia, CA). Samples were
collected and stored as for protein samples, and 20 µg RNA/lane was
run on a 1% agarose gel containing formaldehyde, then transferred
overnight, in 20 x SSC, to nylon membrane (Amersham Pharmacia Biotech, Arlington Heights, IL). Membranes were
prehybridized for 30 min in Rapid-Hyb (Amersham Pharmacia Biotech); and boiled 32P-labeled
P-cadherin complementary DNA (cDNA) probe, a gift of M. Takeichi
[clone p28, described by Nose et al. (30) and Cyr et
al. (12)] was then added in Rapid-Hyb. This cDNA probe was
labeled using the Rediprime Kit (Amersham Pharmacia Biotech) according to the manufacturer’s instructions. After
hybridization, membranes were washed in decreasing concentrations of
SSC, 0.1% SDS before being analyzed autoradiographically using Biomax
MS film and intensifying screens (Eastman Kodak Co.,
Rochester, NY).
Immunofluorescence
Gonocyte/Sertoli cell cocultures were fixed in 2%
paraformaldehyde in PBS (pH 7.2) for 20 min at RT, followed by washes
in PBS; and immunolocalization was performed as previously described
(8). Chambers were blocked in 10% normal donkey serum (for P-cadherin)
or 10% normal goat serum (for NCAM) at RT for 60 min and then
incubated with primary antibody (P-cadherin, 1:10 in 10% normal donkey
serum; RO49, 1:100 in 10% normal goat serum) overnight at 4 C. Control
chambers were incubated either in preimmune serum or without primary
antibody. After extensive washing in PBS, chambers were incubated in
the appropriate rhodamine conjugated secondary antibody for 2 h at
RT (P-cadherin: a 1:500 dilution of donkey antigoat IgG; RO49: a 1:1000
dilution of goat antirabbit IgG [both, Jackson ImmunoResearch Laboratories, Inc.]). After rinsing, slides were mounted in
Vectashield (Vector Laboratories, Inc. Burlingame, CA) and
then either viewed and photographed with differential interference
contrast (DIC) and epifluorescence optics on an Orthoplan 2
microscope (Leitz, Rockleigh, NJ) or images were captured
using an Eclipse E800 microscope (Nikon Instrument group,
Melville, NY) attached to a DEI-750 CE digital video camera and
software (Optronics, Boston, MA).
Quantitative analysis of cell number after T3
treatment
To quantify the numbers of cells in
T3-treated and control groups, triplicate
chambers in each group were analyzed as previously described (8, 31),
with modifications as follows. Cultures were viewed at 600x
magnification, using the phase optics of an inverted microscope
(Diaphot-TMD, Nikon Instrument group), with a
1-cm2 grid placed in one eyepiece. The cells
contained within this grid were defined as a field. For comparison of
numbers of gonocytes, a total of 50 fields per chamber were counted,
and 9 fields per chamber for Sertoli cells. Approximately 2000 to 3000
Sertoli cells and at least 100 gonocytes were counted in each control
chamber. To ensure lack of bias in choosing the fields, a routine,
nonrandom pattern of counting was adopted for all chambers. Final data
were expressed as number of cells/mm2 (in each
chamber).
Statistical analysis
For quantitative data obtained as described above, one-way ANOVA
was used to determine whether differences existed among the mean values
for control and treatment groups, and these differences were
subsequently located with a Newman-Keuls test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Moreover, after longer culture, for
either 10 or 15 days, we detected negligible NCAM signal in samples
from T3-treated cultures, whereas NCAM in
vehicle-treated controls remained high at both times. Importantly, even
after prolonged exposure to T3, an essentially
confluent monolayer of Sertoli cells remained in the dishes, though few
gonocytes were observed (Fig. 1B).
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).
Specifically, the localization of NCAM to cell-cell boundaries was
greatly decreased by exposure to T3, although
some diffuse, cytoplasmic fluorescence was still present in the
hormone-treated cells. In addition, it seemed that there were fewer
attached gonocytes and more detached, floating cells in the
T3-treated chambers, compared with
vehicle-treated controls.
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). P-cadherin protein levels were
essentially stable through day 10, dropped slightly by day 15, and then
became low to absent in testes of adults. Expression of P-cadherin mRNA
also tended to decrease during development, although a weak band could
still be seen in samples from adults. Western analysis was also applied
to protein samples obtained from neonatal cells cocultured for 1, 2, or
4 days (Fig. 4A). A single strong band
indicative of P-cadherin was apparent in control cultures after 24 or
48 h. However, essentially no P-cadherin was detected in
protein samples from cocultures maintained for 4 days in
vitro (Fig. 4A).
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). However, germ cells displayed noticeably
stronger fluorescence than did Sertoli cells, and much of this signal
was cytoplasmic, with only a few restricted areas with signal
apparently at cell surfaces.
We also used Western analysis to determine whether 100 nM
T3 affects production of P-cadherin in cocultured
Sertoli cells and gonocytes. We found that it was unchanged after 1 day
of treatment with T3; but, by day 2 in
vitro, there was an obvious decrease in P-cadherin signal in
hormone-treated cultures, compared with controls (Fig. 4A). It was
again noted that there were fewer attached gonocytes and more detached,
floating cells in the T3-treated chambers,
compared with vehicle-treated controls.
Effect of T3 on numbers of cocultured Sertoli cells and
gonocytes
Qualitative changes in the numbers of adherent vs.
floating cells in cocultures were noted when cells were exposed to 100
nM T3. Therefore, we
examined the impact of a range of T3
concentrations (0.1–100 nM) on the numbers of
both gonocytes and Sertoli cells remaining adherent with increasing
time in vitro. Cocultures were prepared from day-1 testes
and incubated with 0, 0.1, 1, 10, or 100 nM
T3, each for 24, 48, or 96 h. Each chamber
was then fixed and quantified to determine the numbers of Sertoli cells
and gonocytes remaining, as outlined in Materials and
Methods. After 24 h, the number of gonocytes remaining in the
monolayers was significantly reduced (P < 0.05) in the
presence of 100 nM T3 but
not at lower doses (Fig. 5A), whereas no
change was found in Sertoli cell numbers in any of the groups after
24 h of T3 treatment. After 48 h, the
numbers of gonocytes were significantly reduced by treatment with 1,
10, or 100 nM T3, whereas
Sertoli cell numbers again remained unchanged in all groups (Fig. 5B).
After 96 h, the numbers of gonocytes remaining in all of the
treated chambers (0.1–100 nM
T3) were lowered substantially, compared with
controls (Fig. 5C). We again detected no change in Sertoli cell numbers
after exposure to 0.1–10 nM
T3. However, by 96 h of treatment with 100
nM T3, there was about a
30% decrease in the numbers of Sertoli cells, compared with control
cultures. Thus, exposure of cocultures to 0.1–10
nM T3, for 24–96 h, causes
loss of gonocytes that is not accompanied by a decrease in Sertoli
cells.
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NCAM. Western blotting was applied to samples obtained from
cocultures incubated for 24–48 h with 0, 0.1, or 10 nMT3. In each of five separate
experiments for which equal loading of lanes was carefully verified, a
decrease in NCAM was seen in T3-treated samples,
compared with controls (see Fig. 6A for
representative Western blot). When densitometry was used to quantify
these Western blots, we noted decreases of approximately 50 and 30% in
samples treated with 10 nM T3 for
24 h or 0.1 nM T3 for 48 h,
respectively, compared with controls (Fig. 6B). We also immunolocalized
NCAM in chambers subjected to the same T3 doses
as for Western blotting and found a marked decrease in NCAM
immunofluorescence at cell-cell boundaries in all hormone-treated
chambers, compared with controls (Fig. 7).
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for
representative Western blot). Similarly, when total RNA obtained from
similarly treated cultures was probed for the presence of P-cadherin
mRNA, we detected no effect of 0.1–10 nM
T3 on its expression (Fig. 8B).
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Discussion |
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Gonocyte survival in vivo, and potentially in vitro, is obviously a result of their ability to avoid apoptosis (32), as well as their expression of appropriate adhesion factors. Thus, it is possible that an apoptotic pathway may be influenced by T3, subsequently affecting gonocyte survival and/or detachment. From our study, this seems unlikely, because we observed no gonocytes displaying morphological evidence of apoptosis, even in cells clearly in the act of rounding up before detachment (not shown). However, this does not rule out the possibility that T3-treatment causes expression of genes, signifying the start of apoptosis. Though not within the scope of the present endeavor, the potential regulation of gonocyte apoptosis by T3 is an important topic and one that we will address in detail in our future work.
Interestingly, even at the highest tested doses of T3, some gonocytes remained adherent to the underlying Sertoli cells, in spite of the virtual absence of NCAM in the cultures. For example, after 4 days of treatment with 100 nM T3, we found that the numbers of gonocytes in treated chambers were about 20% of those seen in controls while an essentially confluent Sertoli cell monolayer was present, in the presence of minimal NCAM expression. This observation raises some interesting questions about the gonocyte population, because those cells that remain adherent after T3 treatment and NCAM down-regulation may represent a subset of germ cells with the potential to express alternative adhesion factors. In our previous work, where we used NCAM antiserum in vitro to demonstrate the function of NCAM as a gonocyte-Sertoli cell adhesive factor, we found that antiserum treatment without addition of T3 resulted in removal of essentially all gonocytes from cultures (8). This is unlike our observations here with T3, where some cells remained adherent in T3-treated chambers, in spite of substantial NCAM down-regulation. Considering the recognized role of T3 in Sertoli cell differentiation, as discussed below, these adherent gonocytes may be adjacent to differentiating Sertoli cells and may receive signals from these cells that are important for their own development. This hypothesis is particularly attractive, in light of the recognized importance of Sertoli cell-gonocyte interaction during development and its presumed role in determining numbers of spermatogonia (32), and is, therefore, one that we will test directly and in some detail in our future studies.
Besides causing failure of gonocyte adhesion, our evidence also indicates that T3 down-regulates NCAM on Sertoli cells. There is a notable decrease in NCAM immunofluorescence at Sertoli-Sertoli cell boundaries after short-term exposure to low levels of T3, and longer-term exposure to higher levels of hormone causes virtual loss of NCAM from these cells. Interestingly, this is not accompanied by detachment of Sertoli cells, an observation in agreement with our earlier finding in studies for which we interfered directly with NCAM function by exposing cultures to specific antisera (8). This may reflect the ability of Sertoli cells to remain attached after loss of NCAM via their adhesion to the underlying matrix. Alternatively, Sertoli cells may express additional, non-NCAM adhesive factors that allow them to remain adherent to each other, in spite of a loss of NCAM. These possibilities will be explored more fully in our future studies.
Our current findings, indicating a role for T3 in regulation of NCAM expression, correlate well with our understanding of the impact of this hormone on testicular development and with available information on NCAM expression during development. We previously immunolocalized NCAM to Sertoli cell-Sertoli cell and Sertoli cell-germ cell interfaces in neonatal testes in vivo and in vitro, and we also showed that NCAM expression in vivo is increasingly down-regulated as postnatal testicular development proceeds (9). In contrast, during extended coculture of gonocytes and Sertoli cells, we found that the pattern of NCAM expression, over time, did not parallel that seen in vivo. Rather, in these cultures, we noted steady production of NCAM, during 15 days of culture, in the absence of any added hormones or growth factors, implying that exogenous factor(s) might be responsible for the down-regulation of NCAM observed in vivo. Based on data presented here, one factor responsible for down-regulation of NCAM in vivo may be thyroid hormone, a suggestion further supported by data indicating a role for T3 in down-regulating NCAM expression in developing brain (28). In addition, the temporal pattern of expression of testicular T3 receptors in vivo also correlates well with diminishing NCAM production, given that the number of available T3 receptors is high early in postnatal development (24, 27), when down-regulation of NCAM occurs, and then decreases later, after NCAM has essentially disappeared from the seminiferous tubule (9). We already recognize T3 as a hormone of major importance for differentiation of the Sertoli cell population and have, until now, attributed this effect largely to its influence on cell proliferation and apparent exit of Sertoli cells from the cell cycle. Our current findings in vitro raise the possibility that T3 may influence other aspects of Sertoli cell and gonocyte development in the neonate, including (but not limited to) expression of adhesive factors important for interaction of these cells.
It is possible that factors other than NCAM may have a role in contact-mediated cell interactions during neonatal testicular development. Messenger RNA (mRNA) for several members of the cadherin family has been detected at relatively high levels in developing mice (11, 14). In addition, P-cadherin has been immunolocalized to the seminiferous epithelium of newborn mice and, after postnatal day 8, found to be down-regulated in the epithelium and restricted to peritubular cells (13). This raises the possibility that P-cadherin may be important for interaction between Sertoli cells and gonocytes during the first postnatal week, and this prompted us to begin exploring its expression in neonatal testicular cells in vitro. After confirming its expression in vivo to be at high levels in testes during the first 2 postnatal weeks, we immunolocalized P-cadherin in cocultures and found it to be preferentially expressed in gonocytes, with only low-to-moderate localization to Sertoli cells. Moreover, its localization is primarily intracellular, with little signal at cell-cell boundaries, implying that P-cadherin may not act as a classical adhesion factor, at least in gonocytes and Sertoli cells under these in vitro conditions. Interestingly, although the qualitative observations made by Lin and DePhilip (13) on P-cadherin localization in testes of newborn mice in vivo were somewhat different from ours, these authors also concluded that P-cadherin does not act as a gonocyte-Sertoli cell adhesion factor in these animals.
When we explored the temporal pattern of P-cadherin production in untreated cocultures, we found that its expression diminishes with increasing time of culture, in direct contrast to the sustained pattern of NCAM production found in similar chambers. Moreover, although inclusion of 100 nM T3 seemed to accelerate the decrease in P-cadherin, as measured by Western analysis, we conclude that this apparent loss of P-cadherin was most likely attributable to detachment of about 80% of the gonocytes after T3-mediated down-regulation of NCAM, because these cells express P-cadherin at extremely high levels, as discussed above. This suggestion is further supported by our finding that P-cadherin protein and mRNA levels remain high after exposure of cocultures to lower doses of T3 that down-regulate NCAM but leave gonocyte numbers unaffected. Thus, T3 does not seem to regulate P-cadherin expression in cultured neonatal testicular cells, at least under the conditions tested thus far.
In summary, our findings provide the first evidence of a role for T3 as a specific regulator of NCAM production by neonatal testicular cells in vitro and suggest that thyroid hormone may play a role in regulation of its expression in vivo. They further suggest that T3, via its action on Sertoli cells, may have a role in determining the ability of some gonocytes to attach to Sertoli cells via non-NCAM mechanisms.
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Footnotes |
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2 Contributed equally to this work.
Received August 31, 1999.
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