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Department of Cell Biology and Physiology, University of Pittsburgh (M.J.S., J.P.S., S.C.W., A.J.Z., W.H.W.), and Division of Immunogenetics, Children’s Hospital of Pittsburgh, (S.B.), Pittsburgh, Pennsylvania 15261
Address all correspondence and requests for reprints to: Dr. William H. Walker, 820 Scaife Hall, Department of Cell Biology and Physiology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261. E-mail: walkerw+{at}pitt.edu
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Abstract |
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Introduction |
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Numerous genes in Sertoli cells important for quantitatively normal spermatogenesis are induced by FSH and cAMP; however, the extent to which these genes require CREB as a trans-activator in vivo is not fully understood. Potential CREB-regulated Sertoli cell products include factors required to maintain germ cell metabolism as well as those needed to support germ cell growth and differentiation (4, 5, 6, 7, 8, 9). Also, CREB may induce transcription factors required to activate other genes supportive of spermatogenesis (10, 11, 12, 13, 14, 15), hormones that regulate spermatogenesis (16, 17), and antiapoptotic factors (18, 19). Interestingly, Sertoli cells exhibit striking stage-specific increases in CREB messenger RNA levels in vivo (20) during the 12.9-day, 14-stage (I–XIV) cyclical process of spermatogenesis in rats (21). Together, these data support the idea that stage-specific up-regulation of CREB activity is related to the induction of Sertoli cell genes required for spermatogenesis.
Gene knockout studies have provided limited information related to the function of CREB in spermatogenesis. Although partial knockout of the CREB gene interferes with germ cell development at the spermatocyte stage (22), complete knockout of the CREB gene results in perinatal death (23). To better focus on the actions of CREB in Sertoli cells and to determine whether CREB is required for spermatogenesis, we attempted to inhibit CREB activity by employing a nonreplicating adenovirus directing the expression of a CREB mutant that cannot be phosphorylated on serine 133 (AdCREBm1) (24). In this study we show that transfection of primary Sertoli cells with AdCREBm1 represses CREB-mediated transcriptional activity in vitro. In addition, we demonstrate that injection of adenovirus into seminiferous tubules in vivo results in the expression of adenovirus-derived gene expression in Sertoli cells, but not in germ cells. Finally, we report that AdCREBm1 infection of seminiferous tubules does not affect Sertoli cell viability, but results in spermatocyte apoptosis and the subsequent elimination of later germ cells in vivo.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Adenoviral and plasmid transfection of primary Sertoli
cells
Primary Sertoli cell cultures in 60-mm2
plates were transfected with AdCREBm1 or Adß-gal (24)
(1 x 1010 adenovirus particles/ml) in DMEM
containing 1% FBS. For toxicity studies, cells were trypsinized
48 h postinfection, and the percentage of live cells was
calculated after addition of trypan blue. Plasmid transfections of
primary Sertoli cells were performed using a calcium phosphate protocol
as previously described (15). The DNA precipitate was
added 3 h after the addition of adenovirus. The cells were washed
with PBS, and serum-free medium containing supplements used for
culturing Sertoli cells was added 4 h after transfection.
Forskolin and isobutylmethylxanthine (IBMX) or vehicles were added
12 h before cell recovery to induce the phosphorylation and
activation of endogenous CREB.
Detection of ß-galactosidase activity
Primary Sertoli cells transfected with Adß-gal and frozen
tissue sections from Adß-gal-injected testes (described below) were
washed with PBS and then fixed for 1 h in 2% paraformaldehyde,
followed by three PBS washes. X-Gal staining solution, consisting of
0.02 M
K3Fe(CN)6, 0.02
M
K4Fe(CN)6·3H2O,
1 mM MgSO4, and 0.1% X-gal in
HEPES-buffered saline, was added to cells in a humidified chamber and
incubated at 37 C for 24 h. Cells and tissue sections were
examined microscopically for the presence of blue stain in their
nuclei. In some cases tissue sections were poststained using cresyl
violet. For Adß-gal-infected tissue sections, the percentage of
tubules displaying ß-galactosidase activity was determined by
counting the total number of stained and unstained seminiferous tubules
(stained tubules were defined as those containing at least three
stained nuclei) in a tubule cross-section. At least three sections each
from three animals were assayed to derive the percentage of
seminiferous tubules displaying ß-galactosidase activity.
In vivo adenovirus injections, terminal
deoxynucleotidyltransferase-mediated deoxy-UTP nick end labeling
(TUNEL) assays, analysis of spermatogenesis disruption, and
testosterone RIA
Adult rats were anesthetized, the testes were surgically
exposed, and a small hole was made through the tunica albuginea using a
26-gauge needle. The adenovirus, suspended in sterile PBS and green
food coloring, was delivered into either the region of the rete testis
or the lumen of seminiferous tubules from a 1-cc syringe fitted with an
18-gauge needle attached to a drawn-out glass pipette by Tygon tubing.
Into the left testis, a solution of 2 x
1010 particles/ml Adß-gal virus was delivered.
Into the right testis a mixture of 1 x 1010
particles/ml Adß-gal virus and 1 x 1010
particles/ml AdCREBm1 virus was injected. Usually one to three
injections, totaling 50 µl, were required to deliver the viral
solution (as visualized by the green dye) to an area encompassing 30%
of the visible tubules of the testis. The testes were replaced, and the
incision was closed. Rats were killed either 4 or 14 days
postinjection.
To evaluate adenoviral effects on spermatogenesis, testes were removed 14 days postinfection, fixed in Bouin’s solution, and paraffin embedded. Tissue sections (5 µm) were stained using a periodic acid-Schiff staining kit (Sigma, St. Louis, MO) to allow morphological evaluation of the tubules. The volume fractions (a measure of relative number) of round spermatids and pachytene spermatocytes present in stage VII and VIII seminiferous tubules were compared for noninfected, Adß-gal-infected, and Adß-gal- plus AdCREBm1-infected testes using a point-counting method (26). Briefly, for each section, a grid of 400 intersecting points with a known area was superimposed over individual seminiferous tubules, and the number of round spermatids or pachytene spermatocytes contacting grid intersection points (Pn) was determined relative to the total number of grid intersection points (Pt) covering the tubule. The derived ratio (Pn ÷ Pt) was defined as the volume fraction of germ cells present. One tissue section from each of 3 rats infected with either Adß-gal plus AdCREBm1 (a total of 115 stage VII and VIII tubules) or Adß-gal alone (a total of 138 stage VII and VIII tubules) was used to determine mean volume fraction. Mean volume fractions of round spermatids and pachytene spermatocytes in 12 stage VII and VIII seminiferous tubules were also determined in a noninfected rat for comparison purposes. For analysis of apoptosis, 10-µm frozen sections from testes 4 days postinfection were evaluated using a TUNEL assay kit (Roche Molecular Biochemicals). Apoptotic cells were identified by fluorescence microscopy.
Plasma testosterone was assayed in duplicate using a previously described RIA (27) employing antiserum T3–125 (Endocrine Sciences, Inc., Tarzana, CA). The mean sensitivity of the assay was 0.05 ng/ml.
Immunocytochemistry
Frozen sections (10 µm) of adenovirus-infected testes were
fixed in 4% paraformaldehyde for 5 min, permeabilized for 1 min in
ice-cold 100% MeOH, and dried completely, followed by blocking for
16 h with normal goat serum, 0.5% BSA, and 0.15% glycine. The
testis tissue or cultured cells were then incubated for 12–24 h with
nonimmune serum or the ED1 monoclonal antibody directed against a
myeloid cell-specific lysosomal membrane protein (Serotec,
Oxford, UK; catalogue no. MCA341). For colorimetric staining, antimouse
biotinylated secondary antibody (Vectastain Elite ABC Kit,
Vector Laboratories, Inc., Burlingame, CA) was added, and
bound antibodies were detected as described by the manufacturer using
0.02% 3-amino-9-ethylcarbazole, 5%
N,N-dimethylformamide, 0.015%
H2O2, and 0.1
M sodium acetate, pH 5.0, as the colorimetric
reagent. Slides were washed in H2O and
counterstained with hematoxylin.
Statistical analysis
Comparisons of spermatid and spermatocyte volume fractions were
performed for Adß-gal-infected and AdCREBm1- plus Adß-gal-infected
testes using an unpaired t test and StatView version 4.5
software (Abacus Concepts, Inc., Berkley, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). To determine the potential cellular
toxicity resulting from virus infection, primary Sertoli cells were
infected with 1 x 1010 particles/ml of
Adß-gal or an adenovirus-expressing CREBm1 (AdCREBm1). Three
days after adenoviral infection, the cells were collected, and the
percentage of live cells was determined by trypan blue exclusion.
There was no significant toxicity associated with infection of
either virus (Fig. 1B).
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, C and D). Addition of
Adß-gal or AdCREBm1 did not affect basal levels of
c-fosCAT (data not shown). Adß-gal also did not alter the
induction of c-fosCAT by forskolin and IBMX. However,
infection of the cells with AdCREBm1 resulted in the complete
inhibition of c-fos promoter induction by forskolin and
IBMX. These studies demonstrated that AdCREBm1 efficiently infects
Sertoli cells in vitro without exerting any apparent toxic
effect on the cell and that AdCREBm1 is able to repress CRE-mediated
transcription.
Adenovirus-derived products are expressed in Sertoli cells, but not
germ cells, after infection of seminiferous tubules
Although the testis is an immune-privileged site (28, 29), adenovirus injection into the rete testis or intratubular
space can result in immune reactions (30). Therefore,
adenoviral vectors were injected directly into the seminiferous tubule
lumen that excludes immune cells by virtue of the blood-testis barrier
formed by specialized tight junctions between Sertoli cells
(31). To determine the efficiency of adenoviral uptake and
gene expression by this method, Adß-gal (50 µl of 1 x
1010 particles/ml) was injected into rat
seminiferous tubules, and 4 days later, the testes were removed, and
expression of the adenoviral-derived ß-galactosidase gene was
assessed by colorimetric staining of testis tissue sections. Adenoviral
infection occurred in clusters of seminiferous tubule cross-sections,
and the efficiency of adenovirus uptake varied from 5–22% of the
total tubules depending on the individual injected testis and the
positions of the cross-sections observed within the testis (Fig. 2A). This measurement may understate
infection efficiency, as the arbitrary cut-off defining successful
infection was set at three or more ß-galactosidase positive nuclei
per cross-section, but numerous cross-sections contained one or two
ß-galactosidase-positive nuclei. Furthermore, other cells may not
produce the threshold of ß-galactosidase activity required to observe
staining. Although it is difficult to totally rule out some infection
of spermatogonia, the positioning and shape of the
ß-galactosidase-positive nuclei at the periphery of the seminiferous
tubule (Fig. 2, B and C) suggest that predominately Sertoli cells are
infected by adenovirus. Similar results were reported in an earlier
study in which adenovirus was delivered via intratesticular or rete
testis injection (30).
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). Therefore, stage VII and VIII
tubules were used to quantify AdCREBm1-induced disruption of
spermatogenesis. The mean volume fractions of spermatids (a measure of
the relative number; see Materials and Methods) in stage VII
and VIII seminiferous tubules were determined for Adß-gal- plus
AdCREBm1-infected, Adß-gal-infected, and uninfected testes. Infection
of testes with Adß-gal alone did not markedly alter the volume
fraction of spermatids compared with that of uninfected testes
(8.3 ± 1.3 x
10-2 vs.
9.7 x 10-2,
respectively; Fig. 3B). However, the mean volume fraction of spermatids
in Adß-gal- plus AdCREBm1-infected testes (3.9 ± 0.8 x
10-2) was significantly
reduced (P < 0.05) compared with the mean volume
fraction of spermatids in testes infected with Adß-gal alone. In
contrast, the mean volume fraction of spermatocytes in Adß-gal- plus
AdCREBm1-infected testes (3.9 ± 0.3 x
10-2) was not
significantly different (P > 0.05) from the mean
volume fraction of testes infected with Adß-gal alone (4.3 ±
0.6 x 10-2).
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). In contrast to the Adß-gal- plus
AdCREBm1-infected testes, none of the seminiferous tubules from
Adß-gal-infected testes or uninfected testes were devoid of
spermatids or met the criteria for disrupted spermatogenesis.
The disruption of spermatogenesis did not appear to be caused by
localized immune cell destruction of tissue, as an antibody (ED1)
recognizing a lysosomal membrane glycoprotein specific to myeloid cells
showed little or no infiltration into the interstitial space by immune
cells in three experiments employing the tubule lumen microinjection
technique (Fig. 4A). In contrast,
delivery of the adenovirus via the rete testis resulted in localized
regions of immune cell infiltration in each of three experiments (Fig. 4B). Disruption of spermatogenesis also did not appear to result from
damage to Leydig cells or decreased testosterone production, as the
mean testosterone level of AdCREBm1-injected rats (2.48 ± 0.85
ng/ml; n = 6) was comparable to that in noninjected controls
(2.19 ± 0.30 ng/ml; n = 2).
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). In contrast, clusters of
seminiferous tubules containing numerous apoptotic germ cells were
evident in AdCREBm1-infected testes (Fig. 4D). The cells undergoing
apoptosis in AdCREBm1-infected testes were predominately pachytene
spermatocytes, although some apoptotic spermatogonia, preleptotene and
zygotene spermatocytes as well as spermatids were observed (Fig. 4E).
No apoptotic activity was observed in Sertoli cells. Together, these
data suggest that expression of CREBm1 in Sertoli cells causes
apoptosis in early germ cells and the subsequent absence of more mature
germ cells 14 days after infection. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Fourteen days after adenoviral injection, testes infected with AdCREBm1 had a significant increase in tubules with disrupted spermatogenesis, including an absence of round spermatids. In contrast, the less mature pachytene spermatocytes and the more mature elongated spermatid germ cells were not affected. Due to the well characterized kinetics of germ cell development in the rat (21), the lack of round spermatids 14 days after the injection of AdCREBm1 suggests that pachytene spermatocytes are the major cell type adversely affected by AdCREBm1 infection.
AdCREBm1-induced germ cell death did not appear to be the result of nonspecific responses to adenovirus, as testes injected with Adß-gal showed no increased apoptosis or loss of germ cells. Immune responses that were previously observed within 10 days of injection into the rete testis or the intertubular space (30) also did not appear to be responsible for cell death, because the immune response, as measured by the lymphoid-specific ED-1 antibody, was limited by the use of the intratubular injection protocol. Furthermore, although some spermatogonia and spermatids were TUNEL positive, predominately spermatocytes underwent apoptosis in response to AdCREBm1 addition to seminiferous tubules. Interestingly, Sertoli cells do not appear to undergo apoptosis or necrosis when CREBm1 is overexpressed, implying that CREB is not required for Sertoli cell survival. Instead, the major effects of blocking Sertoli cell CREB action appears to be directed to the spermatocyte stage of germ cell development.
Earlier studies performed by Blanchard and Boekelheide suggest that Sertoli cells are more susceptible to adenovirus infection during stages II–VI (30). Interestingly, Sertoli cell CREB messenger RNA levels peak in stages II–VI (20); therefore, Sertoli cell gene expression may be particularly sensitive to changes in AdCREBm1-induced CREB activity during these stages. It is possible that the AdCREBm1-directed expression of CREBm1 during stages II–VI may disrupt the cyclical induction of CREB-regulated genes that are required to produce factors needed by spermatocytes for survival.
Because of the blood-testis barrier, the Sertoli cell must provide many
factors required for the maintenance and development of germ cells
(32). In Sertoli cells, CREB is known to directly regulate
or can be linked to the regulation of a number of genes that contribute
to germ cell survival (see Table 1).
These potential CREB-regulated survival factors include growth factors
for spermatogonia and spermatocytes as well as transcription factors
that may induce the production of other products required for
spermatogenesis. CREB also induces genes required for the production of
germ cell nutrients and iron transport to germ cells. Particularly
relevant for germ cell survival is stem cell factor (SCF), also known
as Kit ligand or Steel factor. FSH and cAMP induce SCF expression in
Sertoli cells, and there are three potential CREB-binding sites in the
SCF promoter (7, 8). In the absence of SCF stimulation,
spermatogonia and spermatocytes undergo apoptosis (33, 34).
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In summary, this report demonstrates that the introduction of a nonphosphorylatable CREB mutant into Sertoli cells in vitro can effectively disrupt CREB-mediated transcription without adverse toxic effects on the cell. In addition, although adenoviral infection of seminiferous tubules is limited to Sertoli cells, the AdCREBm1 adenovirus causes spermatocyte apoptosis. Although we cannot exclude the possibility of some occult effect of the AdCREBm1 vector, based on the data available we hypothesize that CREBm1 interferes with Sertoli cell production of one or more critical survival factor(s) required by germ cells, and that in the absence of CREB-inducible survival factors, these cells are eliminated by apoptosis.
Studies are underway to confirm that CREBm1 alters the expression of potential germ cell survival factors or apoptotis-promoting genes in Sertoli cells. The AdCREBm1 adenovirus will be instrumental for these studies, as, in contrast to plasmid-based transient transfection reporter gene assays, infection of cells with AdCREBm1 allows for studies of the regulation of endogenous CREB-regulated genes in their normal chromatin context in cultured cells and in vivo.
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Acknowledgments |
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Footnotes |
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Received August 4, 2000.
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80% confluent) isolated
from 16-day-old rats were transfected with Adß-gal and stained
72 h later. Blue-stained cells (represented here by various
intensities of dark nuclei) indicative of Adß-gal infection represent
over 90% of the cell population. Bar, 100 µm. B,
Primary Sertoli cells transfected with no virus (control), Adß-gal,
or AdCREBm1 (1 x 1010 adenovirus particles/ml) were
assayed for cell viability 72 h after transfection by trypan blue
dye exclusion. Results shown are the mean of two observations each from
two experiments ± SE. C, AdCREBm1 represses
CREB-mediated transcription. Primary Sertoli cells (2 x
105) infected with either Adß-gal or AdCREBm1 were
transfected with 2 µg of a vector containing the CREB-responsive
c-fos promoter linked to the CAT gene (c-fosCAT).
Forskolin induced promoter activity in the absence of adenovirus (lanes
2 and 3 vs. lane 1). Infection of cells with Adß-gal
had no effect on promoter induction by forskolin (lanes 4 and 5).
Infection of cells with AdCREBm1 decreased forskolin induction of the
c-fos promoter to basal levels (lanes 6 and 7). D, The
summary of three experiments, as shown in C, is presented. The activity
of the c-fosCAT reporter plasmid under the various
conditions is expressed as the fold induction ± SE
relative to c-fosCAT basal activity.
