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Regular Article |
a Center for Reproductive Biology, School of Molecular Biosciences, Washington State University, Pullman, Washington 99164-4660
b Department of Cell Biology, Georgetown University Medical Center, Washington, District of Columbia 20007
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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kinases, spermatogenesis, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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More than 70 AKAPs have been identified to date, and several are expressed exclusively in male germ cells and are critical in sperm viability. They include S-AKAP84 [6], AKAP82, and AKAP110 [4]. Sperm motility is largely regulated by PKA-mediated phosphorylation events [4, 7]. The addition of anchoring inhibitor peptides (AIPs), which compete with the PKA RII/AKAP binding domain, inhibits sperm motility [8]. However, mutant mice lacking the RII alpha subunit have motile sperm and are fertile, suggesting that AIPs may have an effect that is independent of AKAP-anchored PKA RII signaling [9].
PKA interacts with AKAPs through a conserved amino acid sequence that is located at the N-terminus of the PKA regulatory subunit [4]. The sequence forms a hydrophobic pocket that binds the amphipathic helix of the PKA binding domain located on AKAP [4]. Ropporin also contains this conserved N-terminus amino acid sequence and interacts with AKAPs in the fibrous sheath of sperm flagella [5]. AIPs inhibit PKA-AKAP interactions but they do not interact with AKAPs; instead, AIPs contain the amphipathic helix found in AKAPs, and they interact with PKA regulatory subunits [8].
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Male W/Wv mutant mice (WCB6F1) and male wild type litter mates at 6 wk of age were purchased from Jackson Laboratory (Bar Harbor, ME). Balb/c mice were housed at the Eastlick Vivarium at Washington State University or at Georgetown University. Male Balb/c mice at 4–6 wk of age were injected (i.p.) with 40 mg/kg of busulfan (Sigma, St. Louis, MO) as previously described [10, 11]. Testes were harvested 30–35 days following injection with busulfan, and total RNA was extracted as described below.
Cell Isolation and RNA Isolation
Sertoli cells and interstitial cells were isolated from 15- to 30-day-old male Balb/c mice as previously described [12] and maintained in serum-free Dulbecco modified Eagle medium for 4 days at 35°C. Type A spermatogonia were isolated from 8-day-old Balb/c mice, and pachytene spermatocytes and round spermatids were isolated from adult mice as described by Bellve and colleagues [13] with minor modifications [14]. Total RNA was extracted from various tissues isolated from 40-day-old mice following the acid guanidinium-phenol-chloroform method [15]. Total RNA was purified from cultured Sertoli cells, cultured interstitial cells, and isolated germ cell populations using TRIzol (Gibco BRL, Gaithersburg, MD) according to manufacturer's specifications. Poly(A)+-enriched RNA was isolated by a single pass over oligo(dT) cellulose column following the method in Current Protocols in Molecular Biology [16].
Differential Display Reverse Transcriptase-Polymerase Chain Reaction
Differential display (DD) was performed using the Hieroglyph Kit (Genomyx, Foster City, CA). Reverse transcriptase (RT) reactions were carried out using 200 ng of total RNA isolated from the testes of W/Wv and wild-type mice, and from isolated type A spermatogonial cells. RT reactions (20 µl) were performed in duplicate at 42°C for 60 min using two different RT enzymes, avian myeloblastosis virus (AMV) RT (Gibco BRL) and Superscript II (Gibco BRL) with an oligo(dT) primer (200 nM) of 5'-
(dT12)MN-3' (M = A, G, or C; N = A, T, G, or C) in 1x buffer according to manufacturer's specifications for each RT enzyme. The underlined sequence of the oligo(dT) primer is half of the T7 promoter sequence. Complementary DNA samples (2 µl) were amplified by polymerase chain reaction (PCR) in duplicate 20-µl reactions containing 1x buffer, 1.5 mM MgCl2, 20 µM dNTPs, the 3' oligo(dT) primer (0.2 µm), one arbitrary 5' primer (0.2 µM; listed below, ARP1-4), 0.125 µCi (
-33P)dATP, and 1 unit of AmpliTaq (Perkin Elmer, Gaithersburg, MD). Reactions were carried out at 95°C for 2 min, then for 4 cycles at 94°C for 30 sec, 46°C for 30 sec, and 72°C for 2 min; followed by 26 cycles at 94°C for 30 sec, 60°C for 30 sec, and 72°C for 2 min; and a final extension at 72°C for 7 min. Negative controls were included in which RNA, RT, or cDNA was replaced with deionized water. Samples were analyzed by denaturing polyacrylamide (4.5% LR-Optimized HR-1000TM; Genomyx, Foster City, CA) gel electrophoresis using a genomyxLR GX100 DNA Sequencer (Genomyx). Complementary DNA fragments were visualized by autoradiography using Biomax film (Kodak, Rochester, NY). Putative germ cell-specific cDNA fragments were excised from the gel and incubated in 100 µl of deionized water overnight at room temperature. Complementary DNA fragments were reamplified by PCR in 100-µl reactions containing 1x buffer, 20 µM dNTPs, 1.5 mM MgCl2, 0.2 µM full-length T7 primer, 0.2 µM full-length M13 reverse (M13r), 0.5 units Taq DNA polymerase (Gibco BRL), and 40 µl of cDNA for 30 cycles at 94°C for 30 sec, 60°C for 30 sec, 72°C for 2 min, and a final extension at 72°C for 7 min. PCR products were then cloned into p-GemT Easy Vector (Promega, Madison, WI) using standard protocols. The arbitrary primers used for the DDRT-PCR were as follows: ARP1, 5'-
CGACTCCAAG-3'; ARP2, 5'-GCTAGCATGG-3'; ARP3, 5'-GACCATTGCA-3'; and ARP4, 5'-GCTAGCAGAC-3'. The underlined sequence in the arbitrary primer is half of the M13r (-48) sequence.
DNA Sequencing and Synthesis
Oligonucleotide primers SP6END (5'-AGCTATGCATCGAACGCGTT-3') and T7END (5'-TTGGACCCGACGTCGCA-3') were used to sequence the DDRT-PCR clones. The amplified DD fragments contain the T7 and M13r sequences; therefore, primers SP6END and T7END were designed to bind to the polylinker region between the SP6 and T7 promoters of the p-GemT Easy Vector, respectively. DNA sequencing analysis was done by the Laboratory of Biotechnology and Bioanalysis at Washington State University. Sequences of cloned DDRT-PCR fragments were analyzed by the GenBank/European Molecular Biology Laboratory database using the basic local alignment search tool (BLAST) for homology identification. Full-length cDNA was submitted to GenBank using the Bankit Internet program at the National Center for Biotechnology Information.
Northern Blot Analysis
Poly(A)+-enriched RNA (6–8 µg) from specific organs, cultured Sertoli cells, and cultured interstitial cells samples were fractioned in a 1% agarose/formaldehyde gel and transferred to a nylon membrane (Hybond-N, Amersham Pharmacia, Piscataway, NJ) and UV cross-linked (UV Stratagene 1800, La Jolla, CA). The DDRT-PCR cDNA fragment was radiolabeled with (-32P)dATP using the Rad Prime DNA Labeling Kit (Gibco BRL). Northern blots were hybridized overnight at 42°C with labeled cDNA probes in 50% formamide, 50 mM NaH2PO4, 5x Denhardt, 5x SSC, 0.1% SDS, 2% dextran, and 1 mM EDTA. Following hybridization, blots were washed in 2x SSC/0.1% SDS for 10 min at room temperature, 1x SSC/0.1% SDS for 30 min at 65°C, and 0.1% SSC/0.1% SDS for 30 min at 65°C. After blots were washed, they were placed in a phosphor screen cassette (Molecular Dynamics, Sunnyvale, CA) and allowed to expose a phosphor screen for 8–12 h. The signals were detected using a Molecular Dynamics PhosphorImager 445 SI and ImageQuant software (Molecular Dynamics, Sunnyvale, CA). The ribosomal protein S2 (ChoB) cDNA probe was used as a control for loading RNA [17].
5' Rapid Amplification of cDNA Ends
5' Rapid amplification of cDNA ends (RACE) were done by using the 5' RACE system version 2.0 (18374-058; Gibco BRL). Briefly, first-strand cDNA reaction was done in 25-µl reactions with 20 mM Tris (pH 8.4), 50 mM KCl, 2.5 mM MgCl2, 10 mM dithiothreitol (DTT), 100 nM gene-specific primer, 400 µM dNTPs, 3 µg of total RNA from testes of 40-day-old mice, and 200 units of Superscript II RT at 50°C for 60 min. Following the 60-min incubation, the cDNA reactions were heated to 70°C for 10 min, then placed at 37°C, and 1 µl of RNase mix (Gibco BRL) was added and incubated for 30 min. The cDNA was then purified on a GlassMax spin column (Gibco BRL) by adding 120 µl of binding buffer to the cDNA reaction mixture, which was transferred to the GlassMax spin column and centrifuged at 13 000 x g for 20 sec. The cartridge was washed four times with 0.4 ml of cold wash buffer and twice with cold 70% ethanol. Complementary DNA was eluted from the cartridge with 50 µl of distilled water preheated to 65°C. Purified cDNA was then tailed with terminal deoxynucleotidyl transferase (TdT) in a reaction containing 10 mM Tris (pH 8.4), 25 mM KCl, 1.5 mM MgCl2, 200 µM dCTP, 10 µl of purified cDNA, and 1 µl of TdT for 10 min at 37°C. The cDNA fragment was amplified by PCR using 20 mM Tris (pH 8.4), 50 mM KCl, 1.5 mM MgCl2, 200 µM dNTP, 400 nM nested gene-specific primer, 400 nM abridged anchored primer, 5 µl of tailed cDNA, and 0.5 units of Taq for 35 cycles at 94°C for 30 sec, 55°C for 1 min, 72°C for 4 min, and a final extension at 72°C for 10 min. PCR products were visualized and gel-purified on a 1% agarose gel, cloned into p-GemT Easy Vector, and sequenced. The procedure was repeated with new primers until the full-length gene was sequenced. Also, reverse primers were generated to verify the sequence following the protocol outlined for the RT-PCR. Antisense gene-specific primers were as follows: 104B31, 5'-AAGCTGTATTTGGCACCAAGT-3'; 104BR1, 5'-GATCGTTCTGAGCTTTTCTACTGGCA-3'; 104BR2, 5'-TCGGGCAGCGGCATCGTTCT-3'; 104BR3, 5'-GCGGCGGCCTGAGACGTTGT-3'; 104BR4, 5'-GCAGCGGGCGGCGAGGA-3'; 104R5, 5'-GGACCCGCCGCCACCTCA-3'; and 104BR6, 5'-ACTGTCCGCCACCTCAGGAAA-3'. Sense gene-specific primers were 104B51, 5'-AGTAGAAAAGCTCAGAACGATC-3'; 104B52, 5'-CCGCCGCGGTCTCCCCAGAA-3'; and 104B53, 5'-TTTCCTGAGGAGGCGGACAGT-3'.
In Situ Hybridization
The ASP cDNA fragment corresponding to the PCR product using primers 104B31 and 104B51 (290 base pairs [bp]) was cloned into p-GemT Easy Vector. The plasmid (2 µg) containing the ASP cDNA was linearized with either restriction enzymes BamHI (antisense) or HindIII (sense) to produce templates for in vitro transcription according to the manufacturer's specifications (Gibco BRL). The linearized plasmids were treated with 1 µl of 20 mg/ml proteinase K for 10 min at room temperature, extracted with 1 volume of buffered phenol, then precipitated in 0.1 volume of 3 M sodium acetate and 2.5 volumes of ethanol. Antisense and sense probes were labeled with 33P in reactions consisting of 10 µl of (-33P)UTP; 1 µl (20 units) RNasin (Promega); 50 units of T7 or Sp6 RNA polymerase (Gibco BRL); 1x transcription buffer; 10 mM DTT; 2.5 mM rATP, rGTP, and rCTP; and 25 ng of template according to the manufacturer's specifications. In situ hybridization analyses were done on testes from 40-day-old mice as described by Lok et al. [18]. Slides were hybridized overnight at 50°C, dipped in Kodak emulsion NTB-3 (VWR), and exposed for 4 days at room temperature.
RT-PCR and Southern Blot Analyses
For RT-PCR, 1 µg of total RNA from isolated from type A spermatogonia, pachytene spermatocytes, round spermatids, W/Wv mutant testes, spleen, skeletal muscle, brain, kidney, heart, liver, lung, ovary, and testes from 40-day-old mice and cultured Sertoli cells isolated from 15- to 30-day-old mice were reverse transcribed in a 20-µl reaction at 42°C for 60 min using 0.2 units of Superscript II (Gibco BRL), 50 ng of oligo(dT) primer in 1x cDNA buffer according to the manufacturer's specifications. PCR was done in a reaction volume of 50 µl with 1 µl of the RT reaction, 1x buffer, 20 µM dNTPs, 1.5 mM MgCl2, 400 nM gene-specific primer 104B31, 400 nM gene-specific primer 104B53, and 0.5 units of Taq DNA polymerase (Gibco BRL) for 30 cycles at 94°C for 30 sec, 60°C for 30 sec, 72°C for 1 min, and a final extension at 72°C for 7 min. PCR products were heat-denatured, separated on a 1.0% agarose gel, visualized with ethidium bromide, transferred to a nylon membrane (Hybond-N, Amersham Pharmacia) under alkaline conditions, and UV cross-linked (UV Stratagene 1800). An internal oligonucleotide specific for the predicted PCR product was end-radiolabeled and used as a probe for Southern blot analyses. The internal oligonucleotide was 104B51 (see above). End labeling reactions were done in 20-µl reactions with 1 unit of T4 kinase (Gibco BRL), 1x forward reaction buffer, 12 pmol of oligonucleotide, and 25 µCi (
-32P)ATP at 37°C for 10 min. Blots were hybridized overnight as described above. Following hybridization, blots were washed in 1x SSC/0.1% SDS for 15 min at room temperature, then in 0.1x SSC/0.1% SDS at 65°C for 15 min. Signals were detected using the PhosphorImager as described above. RT-PCR and Southern blot analyses were done in triplicate. Ubiquitin was used as a control for the RT-PCR analyses. Ubiquitin primers were as follows: UB-1, 5'-AGTCCACCCTGCACCTGGTTCTCCG-3' and UB-2 5'-CCTCAAGCGCAGGACCAAGTGCAGAG-3' for the PCR; and UB-3, 5'-AGACCCTGACTGGTAAGACCATTACCCTCG-3' as the internal probe. DNA was replaced with deionized water for a negative control, and 10 ng of p-GemT Easy Vector containing the ASP cDNA generated from the RT-PCR using 104B31 and 104B53 primers.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In this study we used the differential display technique to identify a gene specifically expressed in developing germ cells. We have identified a novel murine cDNA that has homology to a recently reported AKAP-associated protein (ASP) found in human sperm using DDRT-PCR technology. DDRT-PCR is an effective method for separating and visualizing a subpopulation of cDNA to identify differentially expressed genes [19–21]. By comparing a cDNA subpopulation from isolated type A spermatogonia with those from W/Wv mouse testis, we identified mRNAs that are expressed in type A spermatogonia but not in somatic cells of the testis. The W/Wv mouse testis has been characterized as germ cell-deficient [22, 23], thereby providing an excellent source of testicular somatic cells. Further analysis of cDNA fragments was done only if they were absent in the W/Wv testis samples and present in both type A spermatogonia and wild-type testis samples, analyzed by both AMV and Superscript II RT reactions, and duplicate PCRs, on multiple samples. Using primer set 10-4, AP 10 (MN = AG) and ARP-4, we identified a cDNA fragment of a novel ASP. The autoradiogram of the germ cell-specific cDNA candidate is illustrated in Figure 1. The 420-bp cDNA fragment was excised from the spermatogonia and wild-type sample lanes, reamplified by PCR, cloned, and sequenced. The sequence of the DDRT-PCR cDNA insert was used to search the GenBank database using BLAST and did not match any known mouse sequences in the database; however, it did have homology to a human ASP (GenBank accession number AF239723).
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5' Rapid Amplification of cDNA Ends
To determine the full-length cDNA sequence of the ASP cDNA, gene-specific primers were designed from the DDRT-PCR cDNA fragment and 5' RACE-PCR was used to amplify the upstream sequence from adult mouse testis. The full-length cDNA sequence was 864 bp, excluding the poly(A)+ tail (GenBank accession number AF305427). The mRNA sequence contains a putative open reading frame of 230 amino acids. ASP has 90% amino acid similarity with a previously described ASP characterized in human sperm (Fig. 2A) [24]. The N-terminal (amino acids 6–47) of ASP has 46% identity and 65% similarity with the PKA RII dimerization/AKAP binding domain of the PKAR type II subunits found in fungus, mouse, rat, bovine, and human peptide sequences (Fig. 2B).
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Northern Blots
To verify that the cDNA fragment isolated from the differential display was expressed only in germ cells, Northern blot analyses were done on poly(A)+-enriched RNA isolated from the testes of 5-, 15-, 25-, and 40-day-old wild type mice and testis samples from 60-day-old mice that had been treated with busulfan 30 days previously. The DDRT-PCR cDNA insert was used to generate random primed probes for Northern blot analyses as described in Materials and Methods.
Testis samples collected at these ages of development correspond to specific events during mouse spermatogenesis. Total testis RNA from 5-day-old mice were used as a source of poly(A)+-enriched RNA to verify the expression of the DDRT-PCR clones in spermatogonia. The testis samples from 15-day-old mice coincide with the first meiotic division during spermatogenesis. Testis samples from 25-day-old mice coincided with the formation of haploid spermatids, and the 40-day-old testis samples represented sexually mature adult mice [25]. Likewise, to verify that the ASP mRNA was not expressed in testicular somatic cells, total testis RNA from mice that were treated with busulfan 30 days prior to collection were used as a germ cell-deficient model. The ASP mRNA was not detected in testis samples from busulfan-treated mice, but it was detected in testis samples from 5-, 15-, 25-, and 40-day-old mice. Furthermore, the expression of the ASP gene increased with testis sample age (Fig. 3A). Although the intensity of the ASP signal appears to be constant in 5-, 15-, 25-, and 40-day old samples, much less mRNA was present in the 25- and 40-day-old samples, compared with the 5- and 15-day-old samples as shown by the ChoB intensity (Fig. 3B). ChoB is not regulated by age in the testis [17, 18].
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The expression of ASP mRNA was analyzed in isolated testis cell types and other reproductive tissues. Northern blot analyses were done on poly(A)+-enriched RNA from cultured Sertoli cells and cultured interstitial cells isolated from 15- and 30-day-old mice and from ovaries, uterus, epididymis, and testes from 40-day-old mice (Fig. 4). The ASP mRNA was not detected in ovary, uterus, epididymis, cultured Sertoli cells, or interstitial cells. The testis from 40-day-old mice was the only sample that showed the presence of the ASP mRNA.
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Finally, we examined the expression of ASP mRNA in other mouse tissues. Northern blot analyses were done on poly(A)+-enriched RNA isolated from spleen, skeletal muscle, brain, kidney, heart, liver, lung, and testes from 40-day-old mice (Fig. 5). ASP was detected only in testis samples from 40-day-old mice. Expression of this gene was specific to the testicular germ cells and was not detectable by Northern blot analysis in other mouse tissues examined here.
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In Situ Hybridization
In situ hybridization analyses were done on cross-sections of 40-day-old mice testes to localize the expression of the ASP gene (Fig. 6). Expression of the ASP gene was limited to cells within the seminiferous tubules. Specifically, the cells in the adluminal compartment had the highest expression of ASP mRNA, whereas cells in the basal compartment had much lower levels of expression. Furthermore, expression of the ASP gene did not appear to vary with the different stages of the seminiferous epithelium. Consistent with Northern blot analysis, expression of the ASP gene was not detected in interstitial cell populations, suggesting that the expression is either very low or absent in these cells types.
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Reverse Transcriptase-Polymerase Chain Reaction
To determine whether gene expression of the ASP gene was restricted to the testis, RT-PCR analyses were done on spleen, skeletal muscle, brain, kidney, heart, liver, lung, ovary, and testes from 40-day-old mice and cultured Sertoli cells isolated from 15- to 30-day-old mice (Fig. 7). Gene-specific primers were used to amplify ASP cDNA from the specific mouse tissues, and an internal oligonucleotide was end-labeled and used as a probe for Southern blot analyses of the resulting PCR products to authenticate the PCR products. The ASP PCR product was detected only in testis samples from 40-day-old mice by RT-PCR (Fig. 7).
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To determine whether ASP mRNA was present in specific germ cell populations, total RNA was isolated from these populations and analyzed by RT-PCR. Gene-specific primers for ASP were used to amplify ASP cDNA from isolated type A spermatogonia, pachytene spermatocytes, and round spermatids. In addition, RT-PCR was done on total testis samples from 40-day-old wild-type mice and W/Wv mutant mice. Southern blot analyses were used to authenticate the resulting RT-PCR products by probing with an end-labeled gene-specific internal oligonucleotide (Fig. 8). Each isolated germ cell population expressed the ASP gene. A faintly detectable PCR product was produced from W/Wv mutant mouse cDNA sample.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Carr and coworkers characterized the binding function and identified the human homolog of the ASP in sperm [24]. They screened a human testis library using a yeast two-hybrid system designed to identify proteins that interact with AKAP110 and identified several proteins, including PKA RII, ropporin, and a novel protein, designated ASP. It was further demonstrated that the ASP peptide interacts with AKAP110 in a method similar to that of PKA RII and ropporin during the yeast two-hybrid screen [24]. Although the authors did not show a translated peptide in the testis or spermatozoa, they did show that the expression of the ASP gene was limited to the testes. This finding is supported by the data from our studies.
Several proteins, including ropporin and AKAP110, have been shown to be expressed specifically in germ cells and have protein products in spermatozoa [5, 26]. Ropporin protein is localized in the principal piece segment and the cytoplasmic droplet at the distal end of the midpiece [5, 24]. AKAP110 protein is localized in the principal piece and the dorsal margin of the acrosomal segment [24]. If ASP interacts with ropporin and AKAP110 in spermatozoa, then a reasonable deduction is that ASP would be localized in the principal piece of the spermatozoa and may be involved in motility. This possible role for ASP in sperm motility is supported by Vijayaraghavan and coworkers [8] who showed that anchoring inhibitor peptides (AIPs) can inhibit sperm motility by competing with the N terminal AKAP/PKA RII interaction domain. ASP contains an AKAP/PKA RII interaction domain and could be sensitive to AIPs, thereby inhibiting ASP interactions with AKAPs and PKA RII subunits. In RII knockout mice it was shown that sperm were motile and fertility was not inhibited, which suggests that other proteins besides RII are sensitive to AIPs [9]. Identification of novel peptides such as ASP, which interact with AKAPs, expands the possibilities of how cell signaling is being regulated during sperm motility.
Although we can speculate on a possible function of ASP in sperm by using the findings of Carr and coworkers, we cannot rule out the possibility that ASP interacts with other AKAPs and mediates cytoskeletal events other than sperm motility. We demonstrated that this mRNA is expressed in several premeiotic and postmeiotic germ cell populations. AKAPs such as AKAP84 have a specific role in condensing spermatids [6]. It is possible that ASP is facilitating cytoskeletal reorganization during spermiogensis.
In summary, we have cloned and localized to germ cells a novel murine cDNA that encodes a peptide that has homology to the AKAP-associated protein in human testis. We have shown that this novel murine transcript is expressed solely in the developing germ cells. Further characterization of this ASP protein and identification of more novel mRNAs and their resulting protein products will lead to a better understanding of how spermatogenesis is controlled.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by National Institutes of Health grants R37 HD10808 to M.D.G. and R01 HD33728 to M.D. 
2 Correspondence. FAX: 509 335 9688; griswold{at}mail.wsu.edu
Accepted: January 8, 2002.
Received: July 19, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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