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Division of Reproductive Biology (M.Sa., M.C.), Department of Gynecology and Obstetrics, Stanford University School of Medicine, Stanford, California 94305; Department of Histology and Medical Embryology (S.-Y.C., S.I., C.P., M.St.), University of Rome "La Sapienza", Rome, Italy 00162
Address all correspondence and requests for reprints to: Marco Conti, M.D., Division of Reproductive Biology, Department of Gynecology and Obstetrics, Stanford University School of Medicine, Stanford, California 94305-5317. E-mail: marco.conti{at}forsythe.stanford.edu
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Type 4 phosphodiesterases (PDE4) hydrolyze cAMP with high affinity and are regulated by several intracellular signaling pathways (10, 11). Four genes encoding PDE4 are present in the mammalian genome and each gene encodes several different transcriptional units originating in at least 14 different PDE4 proteins (10, 11). Differences in regulation and subcellular localization of the variants may explain this heterogeneity (10). Phosphorylation of the PDE4D3 variant, but not of the PDE4D1 and PDE4D2 variants, was demonstrated in FRTL-5 cells (12, 13); similarly, a PDE4A5 form is activated by GH via a SP6 kinase-mediated phosphorylation (14).
In addition, it has been proposed that the heterogeneity at the amino terminus of the different PDE4s reflects differences in subcellular localization of the variants (9). The PDE4A1 variant is mostly membrane bound in the cortex and cerebellum and is possibly localized in synaptic membranes (15). Similarly, the recombinant PDE4A1 protein expressed in COS cells is particulate, whereas a 25-amino acid truncated form in the amino terminus is recovered in the soluble fraction of the cell (16, 17). These observations have led to the proposal that the amino terminus of different PDE4s contains signals for compartmentalization. In support of this hypothesis, an additional PDE4A variant, PDE4A4, contains a polyproline region that interacts with SH3 domains in a reconstitution system (18). The human counterpart is recovered in both the soluble and particulate fraction of COS cells (19).
Although the above data are suggestive of differential targeting of different PDE4s, most of the conclusions are based on overexpression of recombinant proteins, and little information is available on the subcellular localization of the native PDE4. Using germ cells as a model for cell differentiation, we have compared the expression and compartmentalization of variants derived from two PDE4 genes during spermatid development in situ. In previous studies, we determined that a PDE4A splicing variant accumulates in round spermatids (20). With the present report, we have identified a previously undetected PDE4D variant that is expressed in pachytene spermatocytes and during the spermatid elongation phase. Furthermore, we provided evidence that this variant interacts with cytoskeletal structures and that its localization is different from the localization of the PDE4A variants expressed in the same cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell isolation and cultures
Male Sprague Dawley rats of different ages were used in all
experiments. Sertoli cell-enriched primary cultures were prepared from
small explants of seminiferous epithelium, using rat testes of
20-day-old animals according to established procedures (21) and
cultured at 32 C in defined MEM. After dilution, the
collagenase-dispersed cell suspension was centrifuged at 1200 rpm for
10 min at room temperature and, after several washings, the cells thus
obtained were cultured for 3 days. Contaminating germ cells were
removed by a 2–3 min incubation in 20 mM Tris-HCl, pH 7.4,
hypotonic medium (22). Cells were harvested at least 24 h after
hypotonic shock. In those experiments where cells were stimulated with
hormones, ovine FSH was added to a final concentration of 500 ng/ml for
8 h.
Total germ cells were isolated from adult rat testis by two subsequent collagenase digestions (0.33% Collagenase type I 220 U/mg, Worthington Biochemical Corp., Freehold, NJ). After the first digestion, the collagenase was diluted with PBS, and the supernatant containing interstitial cells was discarded. Following the second enzymatic digestion, the pellet was washed several times with PBS to remove the residual collagenase and the tubules devoid of the peritubular wall were disrupted mechanically. After 5 min of sedimentation, the sedimented fraction containing somatic cells was discarded while the germ cells were recovered from the supernatant by centrifugation at 1200 rpm in a tabletop clinical centrifuge. After several washings of the pellet with PBS, the total germ cell suspension was used for mRNA extraction or immunocytochemistry experiments as described below.
Germ cell fractions enriched in round spermatid (F3) or pachytene spermatocyte (F5) were prepared by the above described total germ cell preparation by sedimentation at unit gravity in albumin gradient (STAPUT) (23). The composition of these enriched cell populations has been previously reported (24).
Epidydimal spermatozoa were isolated from cauda epidydimis according the methods described (20). After several washings with PBS, the cell suspension was used for Western blot or for immunocytochemistry experiments as described below.
RNA preparation
Total RNA was extracted from rat testes at different ages,
Sertoli cell cultures, enriched cell fractions of round spermatids (F3)
and pachytene spermatocytes (F5), using Tri Reagent (Molecular Research Center, Inc., Cincinnati, OH) following the
manufacturer’s protocol and then precipitated with cold ethanol. The
dried pellets for each preparation were dissolved in ribonuclease-free
H2O and stored at -80 C for further analysis.
RNase protection assay
The 276-bp probe A common to all PDE4D transcripts and
corresponding to the nucleotide 1634–1910 of rat PDE4D1 sequence was
generated by PCR as previously described (25). Probe B was obtained by
restriction digestion of the pGEM-SC8 plasmid (26) with
EcoRI and StuI, and the excised fragment
was subcloned into pBlueScript. The predicted protected fragment for
PDE4D1 was 291 bp, whereas the predicted protected fragment for PDE4D2
was 200 bp. Probe C was obtained from the
EcoRI-StuI digestion of the pCMV5-PDE4D3 plasmid
(12) and was subcloned in pBlueScript (Stratagene, La
Jolla, CA). The probe encompassed the first 400 bp of rat PDE4D3 open
reading frame. The presence of PDE4D4 or PDE4D5 transcripts yielded a
protected fragment of 350 bp. After a linearization with
SalI and purification, the plasmids were used as a template
to generate the RNA probe. GAPDH construct from Ambion, Inc. (Austin, TX) was linearized by StyI to generate
a probe of 190 bp and a protected probe of 135 bp, and transcription
was performed using T7 RNA polymerase. The RNA Century Marker Template
Set (Ambion, Inc.), which is comprised of five linearized
plasmids for use as templates in an in vitro transcription
reaction, was used for synthesis of RNA size standards. In
vitro transcription was performed on each linearized template (1
µg) using a Transcription In Vitro System II
(Promega Corp., Madison, WI) following the manufacturer’s
instructions and using either T3 or T7 polymerase (20 U/µg of probe)
and 50 µCi of 32P-UTP. At the end of the reaction,
digestion with 20 U of RNase free DNase I per reaction was performed at
37 C for 15 min. The probes were then precipitated with ethanol and
ammonium acetate to remove unincorporated radionucleotides. The pellets
were dissolved in loading buffer and loaded on a 8% urea/acrylamide to
separate full-length probes.
RNase protection assays were then performed using the RPA II kit (Ambion, Inc.) following the manufacturer’s instructions. Forty micrograms of total RNA were hybridized overnight at 45 C with 106 cpm of [32P] labeled probe. Free probe was further digested with RNase, and protected fragments were fractionated on a 8% urea/acrylamide gel.
Antibodies
Four antibodies specific for PDE4 were used for Western blot and
immunocytochemistry experiments. The monoclonal antibody (M3S1) is
selective for PDE4D and was raised against the carboxyl terminus of
PDE4D fused to glutathione-S-transferase (GST-PDE4D) (27). The
PDE4A-selective polyclonal antibody affinity purified (AC55) was raised
against the carboxyl terminus region of PDE4A fused to GST (GST-PDE4A)
(20). The PDE4 nonselective polyclonal antibody (K116) that recognizes
PDE4A, PDE4B, PDE4C, and PDE4D, was raised against a synthetic peptide
corresponding to a conserved region in the regulatory domain present in
all PDE4 (27). The above antibodies were previously tested for
selectivity and specificity in immunoprecipitation and Western blot
analyzes (27). A fourth antibody raised against the carboxyl terminus
of human PDE4D was a generous gift from Dr. Florian Gantner (Byk
Goulden, Germany). Western blot analysis with recombinant proteins
indicated that this latter antibody is specific for PDE4D (data not
shown).
Immunoprecipitation
After removal of the capsula albuginea, adult rat testis was
homogenized in isotonic buffer (250 mM sucrose, 20
mM Tris HCl pH 7.8, 1 mM EDTA, 10
mM ß-mercaptoethanol) including a mixture of protease
inhibitors (50 mM benzamidine, 0.5 µg/ml leupeptine, 0.7
µg/ml pepstatin, 4 µg/ml aprotinin, 10 µg/ml soybean trypsin
inhibitor, and 2 mM phenymethysulforide). The homogenate
was centrifuged for 30 min at 20.000 x g to obtain a
soluble fraction. The pellet was extracted in RIPA buffer and clarified
by centrifugation at 100,000 x g 30 min at 4 C to
obtain the particulate fraction. Soluble and particulate fractions were
incubated with M3S1 (diluted 1:100 vol/vol) or K116 (diluted 1:100
vol/vol) antibodies for 1 h at 4 C in continuous mixing.
Antigen-antibody complexes were precipitated with protein A-Sepharose
(polyclonal) or Protein G-Sepharose (monoclonal) beads.
Immunoprecipitated samples were then washed with the same
homogenization buffer, and the adsorbed proteins were eluted with 1%
SDS in PBS and analyzed by SDS-PAGE and Western blot.
Western blot analysis
Samples were diluted in sample buffer (62.5 mM Tris
HCl (pH 6.8), 10% glycerol, 2% (wt/vol) SDS, 0.7 M
2ß-mercaptoethanol, 0.0025% (wt/vol) bromophenolblue), separated on
8% SDS-PAGE gel and transferred to Immobilon membranes
(Millipore Corp., Bedford, MA). The membranes were
incubated overnight at 4 C in 5% BSA (wt/vol) dissolved in TBS-T
solution (0.1% Tween-20, 20 mM Tris HCl and 14
mM NaCl, pH 7.6) to reduce nonspecific background and
then incubated with selective or nonselective PDE4 antibodies for
1 h at room temperature. All the antibodies were diluted 1:100
(vol/vol) in TBS-T containing 0.1% BSA and 1% normal goat serum
(Vector Laboratories, Inc., Burlingame, CA). After
extensive washing, the blots were incubated for 1 h with
peroxidase-conjugated secondary antibodies (ECL Amersham Corp., Arlington Heights, IL) diluted 1/5000 (vol/vol) in TBS-T.
The secondary antibodies were detected using a luminescence method (ECL
Amersham Corp.) and recorded by exposure to x-ray
film.
Immunofluorescence analysis
Adult rat testes were cut in to small pieces and quickly frozen
in liquid nitrogen in OCT compound (Miles, Diagnostics Division,
Elkhart, IN). Approximately 5 µm cryosections were cut in a
Leitz cryostat (Leitz, Wetzelar, Germany),
mounted on poly-L-lysine slides and processed for
immunofluorescence. Total germ cells were isolated from adult testis
according to the Staput method described above and were then spread on
poly-L-lysine-coated slides, at a concentration of 500,000
cells/ml, by using a citospin (Shandon Inc., Pittsburgh, PA, citospin
3).
The slides were subsequently fixed in ethanol/acetone (1:1 vol/vol) for 10 min at -20 C and processed for immunocytochemistry. The specimens were incubated 30 min with 1% normal goat serum (Vector Laboratories, Inc., Burlingame, CA) to reduce nonspecific staining and then incubated for 1 h at room temperature in a humidified chamber with PDE4-specific antibodies M3S1 (25 ng/ml) or K116 (diluted 1:100 vol/vol). Primary antibodies were visualized by fluorescein-conjugated secondary antibodies (FITC-conjugates antibodies, Vector Laboratories, Inc.). For double staining, M3S1 (25 ng/ml) and AC55 (20 ng/ml) antibodies were mixed together and used as described above; M3S1 was detected with antimouse fluorescein-conjugated (FITC) secondary antibodies, whereas AC55 was detected with antirabbit rhodamine-conjugated (TRITC) secondary antibodies (Vector Laboratories, Inc.). After extensive washing with PBS, the slides were mounted with Vectorshield mounting medium (Vector Laboratories, Inc.) and analyzed with a fluorescence equipped light microscope (Axoplan, Carl Zeiss, Oberkochem, Germany). The specificity of the primary antibodies staining was monitored by preabsorbing the primary antibodies with the peptides or fusion proteins used as immunogens (1 µg/ml). The specificity of the secondary antibodies was also evaluated by staining after omission of the primary antibody.
Immunoelectromicroscopy
Small fragments of adult rat testis were fixed in
8% buffered paraformaldehyde and infused in 1.8% sucrose. Samples
were then dehydrated in a cryosubstitution apparatus (Leica Microsystems Inc., Deerfield, IL) and embedded in Lowicryl HM23
or HM20 (28, 29). Ultrathin sections were first incubated at room
temperature with M3S1 antibody (25 ng/ml) and, after extensive washing
in PBS, were incubated with protein A gold (15 nm) (30). Control
experiments were performed by preabsorbing M3S1 antibody with the
fusion protein used as immunogen (1 µg/ml) and or by incubating the
sections with unrelated antibodies. Sections were then stained with
uranyl acetate and lead citrate and observed with a Hitachi Scientific Instruments, Inc. (Mountain View, CA) H7000
electron microscope.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine which of the variants derived from the PDE4D gene is
expressed in seminiferous tubule cells during testis development, RNase
protection was performed with RNA from testes of animals of different
ages. A probe corresponding to the 3'-end of the PDE4D mRNA common to
all PDE4D transcripts was used together with two probes specific for
the transcripts encoding the short (PDE4D1 and PDE4D2) and long
(PDE4D3, PDE4D4, and PDE4D5) variants. With the probe corresponding to
the common 3'-end of the PDE4D mRNA, a protected fragment of 275 bp was
present at all ages (data not shown), even though an overall increase
in mRNA levels was observed between 10 and 90 days of age (Fig. 1
). When transcript-specific probes were
used, different patterns of mRNA expression for the short and long PDE
forms were observed (Fig. 1). The levels of mRNAs coding for the short
forms and corresponding to protected fragments of 280 and 170 bp were
maximal at 15 days of age and declined thereafter. Conversely, the mRNA
for the long PDE4D forms (protected fragments of 350 bp) was barely
detectable at 10–15 days and increased dramatically at days 30, 40,
and 90 (Fig. 1). Overexposure of the autoradiogram indicated the
presence of an additional 400 bp protected fragment corresponding to
the PDE4D3 transcript. The levels of this transcript did not change
significantly during testis development (data not shown).
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A PDE4D protein with distinct electrophoretic properties is
expressed in germ cells
To determine whether the PDE4D RNA detected in germ cells is
translated into a protein, immunoprecipitation and Western blot
analysis were performed with total testis or isolated germ cell
preparations. Soluble fractions from adult testis were
immunoprecipitated, and Western blot analysis on the immunoprecipitated
fractions was performed with both PDE4 nonselective and PDE4D-selective
antibodies. Both antibodies identified an immunoreactive polypeptide of
98 kDa in the soluble fraction of the testis homogenate (Fig. 3A). Approximately 50% of the 98 kDa
protein was recovered in the particulate fraction (data not shown). The
PDE4A-specific antibody recognized a doublet of 86, and 93 kDa (Fig. 3C). An additional polypeptide of 72 kDa was observed only with the
K116 antibody (Fig. 3A). Because this polypeptide is recognized only by
the PDE4 nonselective antibody (Fig. 3, A and B), it must represent
either a PDE4B or PDE4C variant; the identity of this protein was not
further investigated.
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). The
migration of the polypeptide derived from germ cells was clearly
distinct from that of the recombinant PDE4D3 which migrates with an
apparent molecular weight of 93K (Fig. 3B). Attempts were made to determine the exact stage of expression of this PDE4D variant during spermatogenesis. While the PDE4 nonselective antibody immunoprecipitated 80% of the PDE activity present in the round spermatid fraction, the PDE4D-selective antibody immunoprecipitated only 10% of the activity (data not shown). Upon Western blot with the antibodies specific for PDE4D, no immunoreactive polypeptide could be associated with the small amount of activity recovered in the immunoprecipitation (data not shown), suggesting that the PDE4D protein is either expressed at low levels only in a subpopulation of germ cells or it is recovered mostly in the particulate fraction of the homogenate.
Immunofluorescence localization indicates late expression during
spermiogenesis
The data reported above indicate that a variant derived from the
PDE4D gene is expressed during rat spermiogenesis together with
previously characterized PDE4A variants (20). The site of expression
and localization of these proteins was further studied by
immunofluorescence in adult rat testis. A PDE4D-selective monoclonal
antibody and a polyclonal antibody specific for the PDE4A forms were
used for the localization, whereas a polyclonal antibody that
recognizes all the PDE4 proteins was used to confirm the data obtained
with the other antibodies.
When cryosections of adult rat testis were stained with the
PDE4D-selective antibody (M3S1), staining was present in a region
surrounding the acrosome of elongating and maturing spermatids (Fig. 4). Specifically, the staining was first
detected in elongating spermatids at step 13 of spermiogenesis (stage
XIII of the seminiferous epithelium cycle) and increased in the
successive steps reaching a maximal intensity at steps 18–19 (stage
VIII of the cycle), before the release of spermatozoa from the
seminiferous epithelium (spermiation) (Fig. 4). Released spermatozoa in
the lumen of the tubules were negative (see below). The specificity of
the immunolocalization was confirmed by blocking the antibody with the
fusion protein used as immunogen (data not shown). An additional
polyclonal antibody specific for PDE4D gave a pattern of staining
identical to that obtained with the PDE4D-specific monoclonal antibody
(data not shown). Finally, the PDE4 nonselective polyclonal antibody
also recognized the periacrosomal region of elongating spermatids (Fig. 5).
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). This staining was blocked by preadsorption of the antibody
and was confirmed by a second PDE4D-specific antibody (data not shown).
The identity of these structures was not further investigated.
Immunocytochemistry experiments on isolated germ cells were performed
to confirm the staining in elongating spermatids. Cytospin preparations
of total germ cells and enriched fractions of pachytene spermatocytes,
round spermatids, and epididymal spermatozoa were used for this
analysis (Fig. 6). The PDE4D-selective
antibody staining on isolated germ cells confirmed the pattern of
staining observed with the testis sections. Additional stainining was
observed in a region corresponding to the manchette, a transitory
cytoskeletal structure of elongating spermatids present at step 11 of
spermiogenesis (39, 40). This structure rich in microtubules bundles
would not be resolved in 5-µm thin cryosections. Isolated round
spermatids showed some additional staining in a region corresponding to
the centrosome or the Golgi apparatus (Fig. 6a).
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) confirmed the
observation made on cryosections and on isolated cells.
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The PDE4A-specific antibody recognized antigens that were present
mostly in the cytoplasm of round and elongating spermatids (Fig. 8b),
where the PDE4D-specific signal was not detectable (Fig. 8a),
suggesting that two different PDE gene products are expressed at
different times during spermiogenesis.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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While the mRNAs for PDE4A and PDE4D are expressed at roughly the same time during meiotic prophase in pachytene spermatocytes and in round spermatids, the accumulation of the encoded proteins follows a different pattern. The PDE4A is expressed early during spermiogenesis and accumulates in the soluble fraction of the cell. While the PDE4D mRNA is clearly detectable in round spermatids, no corresponding protein could be detected either by Western blot analysis or immunofluorescence localization. This indicates that the PDE4D mRNA is not efficiently translated at the round spermatid stages but is translated only later at steps 11–18 of spermiogenesis. There are several precedents for a translational control during spermiogenesis. The protamime mRNA (43, 44) contains elements at the 3'-end that controls the timing of translation during spermiogenesis; it is transcribed at steps 5–6 of spermiogenesis and translated later at steps 9–10. Whether similar regulatory elements controlling translation are present in the PDE4D mRNA is not known. An alternative possibility is that the PDE4D protein is uniformly expressed at low levels during spermiogenesis and is detectable by immunofluorescence only when it is concentrated in the manchette or in the periacrosomal space of elongating spermatids.
The PDE4D protein that we have identified migrates with an apparent MW 98K on SDS-PAGE, a mobility clearly different from the rat PDE4D1 (72K), PDE4D2 (68K), PDE4D3 (93K) or PDE4D4 (105K) variants (12, 27, 33). The migration of this immunoreactive protein is instead similar or identical to the migration of the PDE4D5 variant recently cloned from human libraries (33). The finding that the mRNA expressed in germ cells contains the exons included in the long forms is also suggestive that the PDE4D retrieved from spermatids is either identical to PDE4D5 or differs from the other long variants only in the amino terminus leader. The PDE4D5 variant has a unique amino terminus of 89 amino acids that is homologous to the amino terminus of the PDE4B3 and PDE4C2 variants (33). No recognizable signatures for subcellular localization are present in this domain.
The immunolocalization of the PDE4D in developing spermatids demonstrated an interaction of this protein with two structures. The immunofluorescence and immunogold electron microscopy showed that the PDE4D variant is in close proximity to microtubules present in the transitory structure of the manchette. This localization is specific because staining could be blocked by using the fusion protein that has been used as immunogen. In addition, two additional polyclonal antibodies against different epitopes of PDE4D also stained the same region of the elongating spermatids. Thirdly, several other microtubule structures present in the Sertoli cells were not stained by either antibody, again suggesting that the antibodies used do not directly recognize the microtubules. Also consistent with the targeting of this PDE with insoluble structure, is the finding that considerable amounts of the 98-kDa immunoreactive protein were recovered in the insoluble fraction of testis extracts. That other PDE4D variants may interact with cytoskeletal structures has been demonstrated by immunofluorescence localization in cultured thyroid cells (36).
While studies on the transcription factor CREM have pointed to an
important role for the cAMP-dependent pathway in spermatid
differentiation, the mode of regulation of cAMP in these cells is
unknown (45, 46). All the components of the cAMP-dependent pathway are
expressed in spermatids, with the possible exception of
Gs
and seven-transmembrane receptors coupled to this
Gs protein. The coordinate expression of two PDE4 genes
suggests that cAMP degradation is regulated during spermiogenesis in a
temporal and spatial manner. One PDE4A protein is expressed in round
spermatids in the soluble fraction of the cell. This is followed by
expression of a predominantly particulate PDE4D in elongating
spermatids. The PDE4D is probably degraded before spermiation, and the
only PDE4 form detectable in spermatozoa is the PDE4A form. Since it
has been suggested that PDE may play an important role in signal
compartmentalization, our data are compatible with this view. Recently,
anchoring proteins tethering the PKA regulatory subunits to different
spermatid structures have been described (47, 48, 49). The expression of
these scaffold proteins during spermiogenesis may play an important
role in the morphogenic restructuring that occurs during spermatid
elongation. As suggested by the disruption of PKA targeting (50), they
may also play an important role in targeting PKAs to flagellar
structures involved in the control sperm motility. Whether targeting of
PDEs to similar structures plays a role in flagellar motility remains
to be determined. This possibility however, is supported by our recent
observation that a PDE4 controls a distinct pool of cAMP involved in
the regulation of motility in human spermatozoa (51).
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Acknowledgments |
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Footnotes |
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2 Supported by University of Rome "La Sapienza", a fellowship from
"Fondazione Cenci Bolognetti", Rome, Italy, and from the Dean’s
Fellowship at Stanford University, Stanford, CA.
3 Current affiliation: Hormone Research Center, Chonnam National
University, Kwangju 500–757, Korea.
Received August 20, 1998.
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