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Endocrinology Vol. 140, No. 5 2297-2306
Copyright © 1999 by The Endocrine Society

ARTICLES

Type 4 Cyclic Adenosine Monophosphate-Specific Phosphodiesterases Are Expressed in Discrete Subcellular Compartments during Rat Spermiogenesis1

Michele Salanova2, Sang-Young Chun3, Saveria Iona, Claudia Puri, Mario Stefanini and Marco Conti

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The type 4 cAMP-specific phosphodiesterases (PDE4) are a family of closely related enzymes with similar catalytic domains and divergent amino- and carboxyl-terminus domains. Multiple PDE proteins with heterogeneous amino termini are derived from each gene. To understand the significance of this heterogeneity, the expression and localization of variants derived from PDE4A and PDE4D genes was investigated during spermatogenesis in the rat. RNase protection analysis with mRNA for testes at different ages of development showed that two transcripts (PDE4D1 and PDE4D2) are expressed at day 10 and 15 of age and become undetectable thereafter. An additional PDE4D transcript appears at day 30 and increased during testid maturation. This latter transcript codes for a long variant of the PDE4D gene and is expressed in germ cells as demonstrated by RNase protection with RNA from isolated pachytene spermatocytes and round spermatids. The presence of a corresponding PDE4D protein with a molecular mass of 98 kDa was established by immunoprecipitation and Western blot analysis with antibodies specific for PDE4D and by immunoaffinity chromatography purification of the 98 kDa variant from isolated germ cells. PDE4A transcripts were also expressed in pachytene spermatocytes and round spermatids. Two polypeptides encoded by these PDE4A transcripts were expressed in pachytene spermatocytes, reached a maximum in round spermatids, and declined thereafter. Immunofluorescence analysis demonstrated a localization of the PDE4D protein in the manchette and in a periacrosomal region of the developing spermatid, a localization confirmed by immunogold electron microscopy. Conversely, the PDE4A was mostly soluble in the cytoplasm of round spermatids. These data demonstrate that PDE4D and PDE4A variants are expressed at different stages and localized in distinct subcellular structures of developing spermatids. Different properties of the mRNAs derived from the two genes and localization signals are responsible for the temporal and spatial expression of the different PDE4 isoenzymes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE CYCLIC nucleotide-dependent signal transduction pathway plays a crucial role in cell replication, differentiation, and in the function of a terminally differentiated cell (1, 2). Following translation of external cues, cAMP is synthesized by adenylyl cyclases and regulates the cell function by activating PKA (3). In addition to phosphorylation of proteins in the cytoplasm, the activated catalytic subunit of PKA translocates to the nucleus to phosphorylate several transcription factors including CREB or CREM (4). Phosphorylation of CREB and CREM are associated with activation of transcription. Recently, the concept has emerged that compartmentalization of the cAMP signaling may dictate the specificity of responses. Controlled diffusion of cAMP to physically separate compartments may regulate discrete pools of PKA, producing phosphorylation of proteins involved in distinct cell functions (5, 6). cAMP degradation and inactivation is catalyzed by cyclic nucleotide phosphodiesterases (PDEs) (7). These enzymes are thought to play an important role in cAMP compartmentalization by controlling cAMP diffusion and its access to different PKAs (8, 9).

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.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Culture medium
MEM and DMEM-F12 used were from Gibco BRL (Grand Island, NY). Restriction enzymes used were from Boehringer Mannheim (Indianapolis, IN) or from Gibco BRL. [32P]-UTP (400–800 Ci/mmol) and 32P-dCTP (3000 ci/mmol) were from DuPont NEN (Boston, MA). Unless otherwise indicated, all chemicals were the purest grade available from Sigma Chemical Co. (St. Louis, MO).

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.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characterization of the PDE4D variants expressed during testis development
We have previously shown that a PDE4D protein is expressed in Sertoli cells of the seminiferous tubule. The expression of this protein is dependent on FSH stimulation of the Sertoli cell (31, 32). With the elucidation of the structure of the PDE4D gene, it has emerged that five different splicing variants are derived from this gene (12, 27, 33). The short PDE4D1 and PDE4D2 variants are expressed in the immature Sertoli cell and regulated by cAMP via regulation of transcription or mRNA stabilization (31, 34). The long variants (PDE4D3, PDE4D4 and PDE4D5) are regulated by PKA-dependent phosphorylation and may be targeted to different subcellular structures (35, 36).

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. 1Go). When transcript-specific probes were used, different patterns of mRNA expression for the short and long PDE forms were observed (Fig. 1Go). 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. 1Go). 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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  		Figure 1. Expression of different PDE4D transcripts during testis development. RNA was extracted from testes according to the procedure described in Materials and Methods. A probe corresponding to the 3'-end of the PDE4D mRNA common to all variants was used for the RNase protection reported in panel A, whereas a probe specific for PDE4D1 and PDE4D2 together with a probe corresponding to a region common to PDE4D3, PDE4D4, and PDE4D5 variants was used for the RNase protection reported in panel B. A GAPDH probe was used as a control of the amount of message used. A representative experiment of the three performed is reported.

 
To further determine which cells of the seminiferous tubules were expressing the short and long forms of PDE4D, RPA was performed with mRNA extracted from enriched pachytene spermatocyte and round spermatid fractions. RNA extracted from immature Sertoli cells cultured in the absence or presence of FSH was used as a control. While the mRNA for the short forms was most abundant in the Sertoli cells and inducible by FSH, mRNA for the long forms was predominantly expressed in the two germ cell fractions (Fig. 2Go).



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  		Figure 2. Expression of different PDE4D transcripts in somatic and germ cells of the testis. Germ cells at the stage of pachytene spermatocytes and round spermatids were prepared by sedimentation at unit gravity. Immature Sertoli cells were isolated and cultured in the presence or absence of FSH for 24 h. RNA was extracted from these enriched cell populations according to the procedure detailed in Materials and Methods. A probe corresponding to the 3'-end of the PDE4D transcripts common to all variants was used for the RNase protection reported in panel A, whereas a probe specific for PDE4D1 and PDE4D2 together with a probe corresponding to a region common to PDE4D3, PDE4D4, and PDE4D5 variants was used for the RNase protection reported in panel B. A GAPDH probe was used as a control of the amount of message used.

 
These findings indicate that different PDE4D variants are expressed in somatic and germ cells of the seminiferous tubules and that a long form of PDE4D is expressed during meiosis and spermiogenesis. The expression of an additional PDE4 gene in spermatids (PDE4A) has been previously reported (20, 25, 37).

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. 3AGo). 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. 3CGo). An additional polypeptide of 72 kDa was observed only with the K116 antibody (Fig. 3AGo). Because this polypeptide is recognized only by the PDE4 nonselective antibody (Fig. 3Go, 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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  		Figure 3. PDE4D and PDE4A protein expression in the testis and germ cells. A, Western blot of extracts from adult testis. The soluble fraction from rat testis was immunoprecipitated with PDE4D-selective (M3S1) and nonselective (K116) antibodies. As a control, immunoprecipitation was performed in the absence of IgG (-Ab). The immunoprecipitated samples were separated on SDS-PAGE, transferred on Immobilon membrane, and probed with the nonselective polyclonal antibody (K116). B, Western blot of the PDE immunopurified from germ cells. Extracts from total germ cell populations were immunoaffinity-purified on a column with immobilized anti-PDE4D monoclonal antibody. The fractions containing the highest amount of PDE activity were concentrated and separated by SDS-PAGE. After transfer to Immobilon membrane, the blot was probed with the nonselective polyclonal antibody K116. The recombinant PDE4D3 was loaded on a separate lane as a control (4D3). C, Expression of PDE4A in the adult testis. Extracts from adult rat testis and isolated epidydimal spermatozoa were immunoprecipitated with PDE4A-selective (AC55 antiserum) and nonselective (K116) antibodies or preimmune serum (PS). The immunoprecipitated samples were then separated on SDS-PAGE, transferred on Immobilon membranes, and probed with an affinity purified PDE4A-specific antibody (AC55).

 
Triton X-100 extracts from total germ cells were further purified on an immunoaffinity column with PDE4D-selective antibodies immobilized to Sepharose (38). This purification yielded a peak of PDE activity that coeluted with a immunoreactive polypeptide of 98 kDa (Fig. 3BGo). 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. 3BGo).

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. 4Go). 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. 4Go). 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. 5Go).



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  		Figure 4. Immunolocalization of PDE4D in different stages of the seminiferous epithelium cycle. Immunofluorescence was performed as described in Materials and Methods using the PDE4D-selective antibody M3S1. A representative photomicrograph of the pattern of immunofluorescence observed at stage XIII (a), stage I (c), stage V (e), and stages VII–VIII (g) of the seminiferous epithelium cycle is reported together with the corresponding phase contrast images (b,d,f,h) (x378). The monoclonal antibody M3S1 recognized PDE antigens in the seminiferous epithelium in a stage-specific pattern.

 


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  		Figure 5. Immunostaining of adult rat testis with a nonselective PDE4 antibody. a, Fluorescence at stages VII–VIII of the seminiferous epithelium cycle. b, Phase contrast (x650). c, K116 preabsorbed with the immunogen peptide. d, Phase contrast (x430). K116 recognizes PDE antigens on elongating and maturing spermatids.

 
A puncated staining with the PDE4D-specific antibody was observed in pachytene spermatocytes at stage XIII of the seminiferous tubule cycle (Fig. 4AGo). 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. 6Go). 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. 6aGo).



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  		Figure 6. PDE4D immunostaining on isolated germ cells and spermatozoa. a, Fluorescence on isolated germ cells from adult rat testis. b, Phase contrast (x650). c, Fluorescence on mature spermatozoa isolated from cauda epidydimous. d, Phase contrast (x650). e, Elongating spermatids, fraction F2 from Staput, at steps 11–12 of spermiogenesis. f, Phase contrast (x650). M3S1 antibody recognized PDE antigens on the dorsal region of the head of maturing spermatids and in a transitory structure of elongating spermatids, the manchette. M3S1 recognizes PDE antigens only in elongating and maturing germ cells (spermatids). Epidydimal spermatozoa were negative when stained with M3S1.

 
This localization in the manchette was verified at the ultrastructural level. Immunogold particles were mostly localized on the microtubules of the manchette as well as in a region corresponding to the basal body of the spermatid. The localization in the periacrosomal region and not in the acrosome (Fig. 7bGo) confirmed the observation made on cryosections and on isolated cells.



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  		Figure 7. Immunoelectromicroscopy localization of PDE4D in elongating spermatids. Spermatids at acrosomal and maturation phases of spermiogenesis. Protein A gold labeled M3S1 antibodies decorate manchette (M) and connecting piece (CP) at step 13 (a) and subacrosomal space (S) and connecting piece at step 15 (b). Control experiment performed by omitting M3S1 antibody (c). a, HM23 embedded section, x20,000; b, HM20 embedded section x27,500; c. HM20 embedded section, x27,500.

 
To compare the expression and localization of the PDE4D with the previously identified PDE4A forms, a double staining experiment was performed on adult rat testis (Fig. 8Go). The PDE4A-specific antibody recognized antigens that were present mostly in the cytoplasm of round and elongating spermatids (Fig. 8bGo), where the PDE4D-specific signal was not detectable (Fig. 8aGo), suggesting that two different PDE gene products are expressed at different times during spermiogenesis.



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  		Figure 8. Comparison of the expression and localization of PDE4A and PDE4D in spermatids. M3S1 (specific for PDE4D) and AC55 (specific for PDE4A) staining on adult rat testis. a, M3S1 detected with fluoresceine-conjugated secondary antibodies, in green (x260); b, AC55 detected with rhodamine-conjugated secondary antibodies, in red (x260).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Deletion and site-directed mutagenesis studies have established that in a PDE4 protein the catalytic domain is flanked by regulatory and subcellular targeting domains (31, 41, 42). Here we report evidence that a variant derived from PDE4D is transiently expressed during spermatid development and is associated with cytoskeletal structures of elongating spermatids. Conversely, PDE4A variants are expressed mostly in round spermatids and are localized in the cytosol of these cells.

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{alpha} and seven-transmembrane receptors coupled to this Gs{alpha} 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).


   Acknowledgments
 
We are thankful to Caren Spencer and Kathleen Horner for the editorial review of the manuscript.


   Footnotes
 
1 The work described was supported by NIH Grant RO1-HD-31544 (to M.C.), by "MURST (40% and 60%) and by Consiglio Nazionale delle Ricerche Grant No. 95.02941.CT14 (to M.S.). Back

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. Back

3 Current affiliation: Hormone Research Center, Chonnam National University, Kwangju 500–757, Korea. Back

Received August 20, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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