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A Novel Isoform of Human Cyclic 3',5'-Adenosine Monophosphate-Dependent Protein Kinase, C-s, Localizes to Sperm Midpiece¹

^a Institute of Medical Biochemistry, University of Oslo, N-0317 Oslo, Norway ^b Andrology Laboratory, Department of Gynecology and Obstetrics, The National Hospital, N-0027 Oslo, Norway ^c

ABSTRACT

Using rapid amplification of cDNA ends, a cDNA encoding a novel splice variant of the human C catalytic subunit of cAMP-dependent protein kinase (PKA) was identified. The novel isoform differed only in the N-terminal part of the deduced amino acid sequence, corresponding to the part encoded by exon 1 in the previously characterized murine C gene. Sequence comparison revealed similarity to an ovine C variant characterized by protein purification and micropeptide sequencing, C-s, identifying the cloned human cDNA as the C-s isoform. The C-s mRNA was expressed exclusively in human testis and expression in isolated human pachytene spermatocytes was demonstrated. The C-s protein was present in ejaculated human sperm, and immunofluorescent labeling with a C-s-specific antibody indicated that C-s was localized in the midpiece region of the spermatozoon. The majority of C-s was particulate and could not be released from the sperm midpiece by cAMP treatment alone. Furthermore, detergent extraction solubilized approximately two-thirds of the C-s pool, indicating interaction both with detergent-resistant cytoskeletal and membrane structures. In addition, we recently identified the regulatory subunit isoforms RI, RII, and an A-kinase anchoring protein, hAKAP220 in this region in sperm that could target C-s. This novel C-s splice variant appeared to have an independent anchor in the human sperm midpiece as it could not be completely solubilized even in the presence of both detergent and cAMP.

cAMP, sperm, sperm motility and transport, spermatid, spermatogenesis, testes

INTRODUCTION

Activation of cAMP-dependent protein kinase (PKA) by cAMP elicits initiation and maintenance of flagellar movement in mature spermatozoa [1, 2]. The PKA tetrameric holoenzyme, composed of two catalytic subunits (C) and a regulatory subunit (R) dimer is activated upon binding of cAMP by release of catalytically active monomeric C subunit [3]. Reactivation of motility of demembranated sperm can be achieved either by cAMP or by active C subunit [4]. Although the underlying mechanisms are still unknown, sperm have been shown to contain a distinct adenylyl cyclase [5, 6], PKA type I and II isozymes [7] and phosphodiesterases [8] as well as distinct PKA C isoforms [9–11], implying that sperm have the machinery to generate cAMP and mediate its effects. Furthermore, anchoring of PKA through A-kinase anchoring proteins (AKAPs) to distinct intracellular sites in sperm is believed to be important for regulating sperm motility since disruption of the AKAP–PKA interaction results in motility arrest [12]. Thus, PKA may be essential for sperm function and consequently for fertility.

There is a substantial complexity of PKA isozymes due to heterogeneity in catalytic (C, Cß, C, PrKX [13]) and regulatory (RI, RIß, RII, RIIß) subunits. The various R and C subunits are encoded by distinct genes localized on different chromosomes [14]. Furthermore, several splice variants of the catalytic subunit have previously been reported: human C-2 [15], Aplysia C-N2 [16], ovine C-s [11], bovine Cß2 [17], mouse Cß1, Cß2, and Cß3 [18], as well as a pseudogene for murine C, Cx [19]. In addition, the PKA subunit isoforms show cell-specific expression and differential regulation in several cells and tissues. They also reveal different subcellular localization, mainly due to binding to AKAPs [20, 21].

In this report we describe the cloning of a novel human C isoform, identified as the human C-s protein on the basis of its similarity to a recently characterized C isoform that was identified by purification and peptide sequencing of C from ovine sperm [11]. The human C-s protein was present in testis as well as ejaculated sperm and C-s localized to the midpiece of human sperm.

MATERIALS AND METHODS

Isolation of cDNA Clones by 5'-Rapid Amplification of cDNA Ends

Human testis 5'-rapid amplification of cDNA ends (RACE)-ready cDNA was purchased from Clontech Laboratories Inc., Palo Alto, CA (cat. no. 7414–1). A primary polymerase chain reaction (PCR) with primer C-2137 (5'-GGACTTGGCCTCTCCTGTTCCCTTTTG-3', nucleotides 2137 to 2163 in the human C cDNA sequence) or primer C-1363 (5'-TGTGGGGAAAGAGGAAGGGAAAAGT-3', nucleotides 1363 to 1387 in the human C cDNA sequence) and anchor-specific primer (AP1, Clontech) was performed. A subsequent secondary PCR was performed using primer C-188 (5'-AAACTGATCCAAGTGGGCTGTGTTC-3', nucleotides 188 to 212 in the human C cDNA sequence) or primer C-624 (5'-GCGAAACCGAAGTCTGTCACCTG-3', nucleotides 624 to 646 in the human C cDNA sequence), and anchor-specific primer (AP2, Clontech). The Clontech Advantage PCR kit (Clontech cat. no. K1905-1) was used in both PCR reactions with 25 cycles of denaturation at 94°C for 30 sec and with annealing and extension at 68°C for 4 min. The 848- and 560-base pair (bp) PCR products were subcloned and sequenced.

Sequencing of cDNA Clones

Plasmids were sequenced on both strands by the dideoxy chain termination method [22] using cycle sequencing protocols (ThermoSequenase kit cat. no. US79760; Amersham Life Science Inc., Cleveland, OH), ³³P-labeled dideoxynucleotides (cat. no. AH9539, Amersham) and a combination of vector- and insert-specific primers. Nucleotide and amino acid sequence data were analyzed using the GCG program package (program manual for the Wisconsin package, version 8, September 1994; Genetics Computer Group, Madison, WI).

Fractionation of Germ Cells from Human Testis

Germ cells were isolated by combined treatment with trypsin and DNase [23], followed by separation using a centrifugal elutriation method essentially as described elsewhere [24]. Purity of the cell fractions was evaluated by analysis of DNA content by flow cytometry and phase contrast microscopy.

Sperm Fractionation

Mature motile sperm from human donors were purified using the swim-up procedure [25]. Removal of sperm heads was performed by sonicating sperm for 3 min and subsequent sedimentation of heads at 1500 x g for 5 min. For Triton X-100 extraction, sperm or sperm tails were extracted 30 min with 1% Triton X-100 in the presence or absence of 20 µM cAMP essentially as described elsewhere [11].

Oligonucleotide Probes

Specific probes were made using oligo 5'-AAGAAGGGCAGCGAGCAGGAGAGCGTGAAAGAATTCTTAG-3' corresponding to positions 102 to 141 in the human C cDNA sequence (C-1) and oligo 5'-CTGAGAACAGGACTGAGTGATGGCTTCCAACTCCAGCGAT-3' corresponding to positions 1 to 40 in the human C-s cDNA sequence (C-s), in an oligomerization protocol as described elsewhere [26].

Northern Analysis

Total RNA from human germ cell fractions was extracted by the guanidine isothiocyanate/CsCl method as previously described [27]. Northern analysis was performed using 10–20 µg RNA/lane. Ethidium bromide staining of the gel verified the loading in each lane. Filters with human germ cell fractions or multiple human tissues (Clontech, cat. no. 7759–1) were probed with ³²P-labeled C-1 or C-s probes.

Antibodies

Anti-C-s antiserum was made by immunizing rabbits with hemocyanine-coupled synthetic peptides (Antigen EP 990209, NH₂-ASNSSDVKE-CONH₂; Eurogentec, Seraing, Belgium) corresponding to amino acids 1 to 9 of C-s and used at a dilution of 1/100 for immunofluorescence. Anti-C-s antiserum was enriched for IgG on protein A sepharose columns (Pharmacia, Stockholm, Sweden) and subsequently affinity-purified on columns with peptide coupled to CNBr-activated Sepharose 4B (Pharmacia) and used at 2 µg/ml for immunoblotting. C antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA; cat. no. sc-905) was used at 2 µg/ml to detect all isoforms of PKA C subunits.

Immunological Procedures

Immunoblot analysis was performed as described elsewhere [28]. Immunoreactive proteins were detected by horseradish peroxidase-labeled protein A (Amersham) in the second layer and developed using ECL (Amersham cat. no. RPN2106). Immunofluorescence was performed on detergent-extracted mature motile sperm from human donors (see above) fixed on poly-L-lysine-coated glass slides with 3% paraformaldehyde and permeabilized with 0.5% Triton X-100 for 15 min. Immunofluorescence detection was performed as described previously [29]. The DNA was labeled with 0.1 µg/ml Hoechst 33342. Cells were examined on an Olympus AX70 epifluorescence microscope using a 100x objective. Photographs were taken with a Phototonic Science charge-coupled device camera and the OpenLab software (Improvision Co., Coventry, UK).

RESULTS

Identification of Human C-s

In order to look for the possible presence of differentially spliced C transcripts, 5'-RACE was performed using a human testis cDNA library and primers situated downstream of exon 1. This yielded one clone of 560 bp where the 3' region (nucleotides 40 to 560) shared identity with the previously characterized human C-cDNA [30] from nucleotide 126 to 646. Using the BLASTN program (basic local alignment and search tool [31]), similarity to the murine Cx pseudogene [19] was found from position 14 to 560. An ATG start codon was identified (positions 19 to 21), giving rise to 180 amino acids of reading frame that did not contain any stop codons. Whereas the 173 C-terminal amino acid residues of this partial reading frame was identical to positions 16 to 188 in the human C protein, the seven amino acids not present in the previously reported C sequence constituted a novel N-terminus. Figure 1A shows the amino acid sequences corresponding to exon 2 in human C (boxed) and the N-terminal amino acids that differ between the two peptide sequences (bold). The N-terminal seven amino acids were homologous (four identical amino acids out of six, in addition to the methionine) to a distinct C protein, C-s, purified and peptide sequenced from ovine sperm [11] (Fig. 1B), demonstrating that the human C splice variant most probably is the C-s isoform.

View larger version (14K): [in this window] [in a new window]   FIG. 1. A) Sequence alignment of human C isoforms. The cloned C-s cDNA sequence was translated into its corresponding amino acid sequence. The peptide sequences of C (top) and C-s (bottom) that correspond to exons 1 (bold) and the first 28 amino acids of exon 2 (boxed) from the C gene are shown. B) Sequence comparison of human (top) and ovine (bottom) C-s peptide sequences. Vertical lines denote homology

Tissue- and Cell-Specific Expression of Human C-s mRNA

To determine which human tissues expressed C and C-s mRNAs, specific probes for C (now designated C-1) and C-s were radioactively labeled and used to probe a Northern blot with human tissues. As shown in the top panel of Figure 2A, C-s mRNA was expressed exclusively in the testis. The C-1 probe detected expression in all tissues and cell types examined (Fig. 2A, lower panel). To explore further C-s mRNA expression in human testis, germ cells were examined for the expression of the C-s mRNA (Fig. 2B). Male human germ cells were isolated and fractionated as described in Materials and Methods. C-s mRNA was expressed in fractions 4 and 5, enriched in pachytene spermatocytes (PS) (lanes 4 and 5), but was not detected in fractions 1–3 enriched in round spermatids (RS) (lanes 1–3).

View larger version (37K): [in this window] [in a new window]   FIG. 2. A) Tissue distribution of human C mRNAs. A human multiple tissue blot (Clontech Inc., 2 µg poly(A)+ RNA per lane) was probed with 32P-labeled C-s-specific (top panel) or a C-1-specific (bottom panel) probes (see Materials and Methods). Lines indicate migration of RNA molecular weight markers (kb). B) Germ cell suspensions from human testis were fractionated by centrifugal elutriation, and total RNA was extracted and subjected to Northern blot analysis with a 32P-labeled C-s-specific probe. Fractions were analyzed by flow cytometry for DNA content and separated into haploid cells (round spermatids [RS]) and tetraploid cells (pachytene spermatocytes [PS]). Diploid cells were considered to be spermatogonia, secondary spermatocytes, or contamination by somatic cells and leukocytes. Lane 1, 57% RS, 7% PS; lane 2, 41% RS; lane 3, 16% RS, 7% PS; lane 4, 21% PS, 3% RS; lane 5: 51% PS, 4% RS. Twenty micrograms of total RNA was loaded with the exception of lane 2 (10 µg). The probe detected the C-s mRNA of approximately 2.8 kb (top arrow) as compared to migration of 18S rRNA (bottom arrow)

The C-s Protein Is Present in Human Testis and Sperm

An antiserum directed toward a peptide corresponding to amino acids 1 to 9 of C-s was affinity purified and used for immunoblotting analysis. Immunoblotting of human tissue and cell homogenates revealed a 39-kDa immunoreactive band present in testis and sperm (Fig. 3A, top panel, lanes 2 and 3, respectively) but not in any other cells or tissues examined (Fig. 3A, top panel, lanes 4 to 7). As expected, heterologously expressed murine C and human C proteins did not react with the antiserum (Fig. 3A, lanes 1 and 8, respectively). The immunospecificity of the 39-kDa band in human testis and sperm was demonstrated by competition with the antigen (Fig. 3A, lower panel, lanes 2 and 3, respectively). The presence of other C isoforms in these tissues as well as immunoreactivity of the standards was demonstrated by probing a parallel blot using an anti-C antibody that recognizes all known C isoforms (Fig. 3B).

View larger version (36K): [in this window] [in a new window]   FIG. 3. Human C-s immunoreactive protein in sperm. Nitrocellulose-immobilized proteins from human cell and tissue lysates were subjected to immunoblot analysis using either the C-s antiserum (A) in the absence (upper panel) or presence (lower panel) of 2 mM peptide antigen, or an anti-C antibody reactive to all C-subunit isoforms (B). Lane 1, recombinant murine C standard (500 ng); lane 2, human testis (20 µg protein); lane 3, human sperm lysate (106 sperm); lane 4, human T-cells (20 µg); lane 5, human B cells (20 µg); lane 6, human brain (20 µg); lane 7, HeLa cells (20 µg); lane 8, recombinant human C (100 ng)

The C-s Protein Localizes to Detergent-Resistant Structures in Sperm Midpiece

The subcellular localization of human C-s was examined in sperm by immunofluorescence using the C-s antiserum (Fig. 4A, red). In ejaculated sperm preparations extracted with Triton X-100 (Fig. 4A, left panel) or left untreated (results not shown), C-s labeling was restricted to the midpiece region. The staining was shown to be specific because preimmune serum or an unrelated antibody did not produce any signal by immunofluorescence in sperm (results not shown), and the staining was competed with the corresponding peptide (Fig. 4A, right panel). To examine further the solubility of C-s in human sperm midpiece, we extracted sperm tails with detergent both in the absence and presence of cAMP. Figure 4B shows immunoblotting of insoluble (P) and soluble (S) fractions from human sperm tails extracted with Triton X-100 (lanes 1 and 2) or Triton X-100 in combination with 20 µM cAMP (lanes 3 and 4), using the anti C-s antibody. In both cases approximately two-thirds could be solubilized from the particulate fraction. Addition of cAMP to the extraction buffer did not significantly alter the C-s signal in either fraction as compared to treatment with Triton X-100 alone, indicating that a pool of C-s was insoluble also in the presence of cAMP.

View larger version (21K): [in this window] [in a new window]   FIG. 4. A) Subcellular localization of human C-s in sperm cells. Human motile sperm were extracted with 1% Triton X-100, fixed with ethanol on glass slides, and subjected to immunofluorescense analysis using the C-s antiserum (red staining) in the absence (left panel) or presence of 2 mM peptide antigen (middle panel). Cells were concomitantly DNA stained with Hoechst (blue). B) Detergent extraction of human sperm tails. Sperm tails were extracted with 1% Triton X-100 (lanes 1 and 2) or 1% Triton X-100 and 20 µM cAMP (lanes 3 and 4), fractionated into soluble (S) and unsoluble (P) fractions, and subjected to immunoblotting using the anti-C-s antibody

DISCUSSION

In the present study, we report the cloning and characterization of a partial cDNA-sequence encoding C-s, an alternatively spliced PKA C subunit that contains a novel N-terminus most probably encoded by a yet uncharacterized alternative exon 1 of the C gene. We demonstrate that the C-s mRNA is expressed in human male germ cells and that the C-s protein is present in ejaculated sperm. Furthermore, our results demonstrate C-s localization to detergent-soluble and detergent-resistant structures in the sperm midpiece.

The length of the N-terminal sequence derived from exon 1 (seven amino acids in both species) and sequence similarity between ovine and human C-s indicate that they have the same origin. In addition, the murine Cx pseudogene [19] translates into a predicted N-terminal region (MPSSSND) with the same length that shares homology (four identical residues out of six) to both ovine and human C-s. Thus, it is possible that the murine Cx pseudogene originated by retroposition of a C-s mRNA. This indicates the expression of C-s in three different species, which would mean that C-s has been conserved during evolution.

We have previously described another testis-specific C subunit, the C isoform [9] that is present only in primates and is derived from an intronless retroposon [10]. Sequence alignment demonstrated that C-s is clearly distinct from the C isoform, in agreement with a previous report on ovine C-s [11]. However, the expression pattern of C-s seen in human male germ cells resembles that of C, and it is possible that these two isoforms utilize similar germ cell-specific promoter elements. Although the exact role of these proteins in human spermatogenesis and sperm function remains to be elucidated, the presence of two distinct C isoforms in the human testis and sperm suggest either a need for substantial redundancy in order to preserve function or support a notion that different isoforms and alternatively spliced gene products serve distinct and specific sperm functions.

The solubility properties of human C-s are reflected by the observation that the presence of both Triton X-100 and cAMP was inadequate to extract this protein fully from the sperm midpiece. This is in contrast to the previous report on ovine C-s [11] where ovine C-s could be extracted only by Triton X-100 when added in combination with cAMP. However, the authors did not account for the unsoluble material after this extraction. Thus, it is possible that a pool of C-s is insoluble in the presence of cAMP also in ovine sperm. Nevertheless our data support the notion that the C-s subunit has unusual solubility properties. An intriguing possibility is that in addition to its anchoring to an R subunit, the shortened C-s amino terminus (as compared to the normal length C) exposes hydrophobic regions that interact with membranous or cytoskeletal structures [32]. This would render the free monomeric C-s insoluble both in the absence and presence of cAMP. Such a hypothesis is supported by the fact that C-s lacks the N-terminal phosphorylation site at Ser10, present in other isoforms of the catalytic subunit [33]. Mutagenesis at Ser10 renders the catalytic subunit insoluble [34] possibly as a result of exposing hydrophobic regions. Independent anchoring of C-s may also provide an explanation of the fact that RII knockout mice are fertile [35]. We have recently shown that the RI and RII subunits of PKA are anchored by the A-kinase anchoring protein, AKAP220, to detergent-resistant structures in the sperm midpiece, whereas S-AKAP84 associates with membrane structures in the same region [36]. Thus, AKAP220/S-AKAP84, RI/RII, and C-s may constitute signaling complexes present in the midpiece region of human sperm specialized for regulating motility and/or other sperm functions.

ACKNOWLEDGMENTS

We thank Sissel Eikvar, Marianne Nordahl, Liv Paltiel, and Åse Strutz for excellent technical assistance.

FOOTNOTES

First decision: 8 March 2000.

¹ This work was supported by the Norwegian Cancer Society, the Norwegian Research Council, Anders Jahres Foundation for the Promotion of Science, and NOVO Nordic Research Foundation Committee.

² Correspondence: Bjørn S. Skålhegg, Institute of Medical Biochemistry, University of Oslo, P.O. Box 1112 Blindern, N-0317 Oslo, Norway. FAX: 47 22 85 14 97; bjorn.skaalhegg{at}basalmed.uio.no

Accepted: April 3, 2000.

Received: February 2, 2000.

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