Molecular Human Reproduction, Vol. 5, No. 8, 732-736,
August 1999
© 1999 European Society of Human Reproduction and Embryology
Detection of mRNA transcripts of cyclic nucleotide phosphodiesterase subtypes in ejaculated human spermatozoa
1 Department of Biochemistry, and 2 Department of Dermatology, Andrological Unit, University of Leipzig, Liebigstrasse 21, D-04103 Leipzig, Germany
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Multiple types and subtypes, including splice variants, of cyclic nucleotide phosphodiesterases (PDE) have been shown to be expressed in various tissues and organs. They control the intracellular level of cyclic nucleotides and are involved in hormonal signalling. In human spermatozoa, PDE play an important role in the regulation of motility, capacitation and acrosome reaction. The aim of this study was to investigate which transcripts of the different PDE types and subtypes could be found in human spermatozoa using reverse transcription–polymerase chain reaction (RT–PCR). Ejaculated spermatozoa from 10 single semen samples as well as another three semen sample pools were separated by swim-up and were investigated by RT–PCR. We obtained PCR products of the PDE types/subtypes 1A/B/C, 2, 3A/B, 4A/B/C, 5, and 8 with different intensities. Control PCR for leukocyte contamination were negative and contamination by other somatic cells was excluded by the spermatozoa preparation protocol, immunohistochemistry and visual examination. These results demonstrated for the first time that human ejaculated spermatozoa contain an extended pattern of PDE mRNA transcripts.
cyclic nucleotide phosphodiesterase-subtypes/human spermatozoa/polymerase chain reaction
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Introduction |
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Various cellular functions are regulated by cyclic adenosine monophosphate (cAMP) or cyclic guanine monophosphate (cGMP) concentrations. The intracellular value of these cyclic nucleotides is a dynamic equilibration between synthesis and degradation (Rossi et al., 1985
). Cyclic nucleotide phosphodiesterases (PDE), 3,5-cyclic-5-nucleotidylhydrolases (EC 3.1.4.17), degrade the cyclic nucleotides to the respective nucleotide monophosphates by hydrolysis of the phosphodiester bond. Multiple PDE types/subtypes exist (Beavo, 1995), which differ with respect to amino acid composition, substrate specifities and affinities, their sensitivity to activators, subcellular distribution and expression in various cells, tissues and organs (Conti et al., 1995). Specific inhibitors are useful tools in characterizing the different types (Fisch et al., 1998). PDE are classified in eight types (1–8), each with different subtypes and further with various splice variants (Beavo and Reifsnyder, 1990; Conti et al., 1995; Manganiello et al., 1995; Fisher et al., 1998a). Recently, a further type PDE 9 (Fisher et al., 1998b) has been described.
PDE have also been found in male gametes (Gray et al., 1971). PDE 1 and PDE 4 may play a special role in mammalian spermatozoa (Naro et al., 1996). PDE type 1 preferentially cleaves cGMP and was found to be activated by calcium and calmodulin (Beavo, 1995) and is likely to be involved in the acrosome reaction, that is initiated by an influx of calcium ions into the spermatozoon (Gearon et al., 1994). Several studies have suggested that sperm motility (McKinney et al., 1994; Jaiswal et al., 1996; Nassar et al., 1999), capacitation (Visconti et al., 1995; Galantino-Homer et al., 1997; Lamirande et al., 1997) and the acrosome reaction (Tesarik et al., 1992) are regulated by PDE. Thus, PDE 4, which has a specific affinity for cAMP and could also be controlled by phosphorylation (Conti et al., 1995), seems to affect sperm motility. The influence of PDE types 1 and 4 on sperm motility and the acrosome reaction has been confirmed using specific PDE inhibitors (Fisch et al., 1998).
Little is known about the synthesis of the PDE types/subtypes in human ejaculated spermatozoa. A better insight into the expression of PDE types/subtypes is provided by the investigation of mRNA content and gene encoding human PDE (Miki et al., 1996). A highly sensitive method of detecting small quantities of mRNA is reverse transcription–polymerase chain reaction (RT–PCR). We used this method in the following study to examine the existence of mRNA transcripts of PDE types and subtypes in human spermatozoa.
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Semen samples
Investigations were performed in 10 single semen samples from 10 patients and 3 pooled semen samples from 11 patients consulting the Department of Andrology, University of Leipzig, for infertility problems. The semen samples were examined in accordance with the World Health Organization guidelines (WHO, 1992). The leukocytes were identified by their CD45 antigen in the immunocytochemistry technique (WHO, 1992). We accepted the ejaculates for the experiments only if leukocytes were <1.0x106/ml, the sperm concentration was >20x106/ml, at least 30% of the spermatozoa had a normal morphological appearance and >50% of the spermatozoa showed an intact acrosome, determined by binding of fluorescein isothiocyanate (FITC)-conjugated Pisum sativum agglutinin (Cross et al., 1986
). Highly viscous ejaculates and semen samples with positive mixed antiglobulin reaction (MAR) test, i.e. >10% spermatozoa with adherent particles were excluded.
Preparation of spermatozoa
Spermatozoa were prepared by density gradient centrifugation and swim up-technique. The isotonic gradient was composed of minimal essential medium (MEM, ICN Biomedicals, Eschwege, Germany) and Percoll 40% (v/v) to avoid selection of spermatozoa and to remove adherent particles from the spermatozoal surface. The liquefied semen samples were diluted with an equal volume of MEM and layered on top of a 2 ml gradient. After centrifugation (20 min at 400 g) the pellets were washed twice (300 g for 10 min) with 10 ml MEM containing 1 mg/ml bovine serum albumin. The pellets were gently overlaid with human tubal fluid (HTF; Quinn et al., 1985) modified by additional 20 mM HEPES (Sigma, Deisenhofer, Germany). The tubes were placed in an incubator for 60 min. The top 1.0 ml of the HTF was removed and the separated spermatozoa were further investigated by RT–PCR.
Extraction of mRNA
Polyadenylated (polyA+) mRNA was isolated from swim-up prepared spermatozoa (1.0–15.0x106 cells) using the Dynabead mRNA Isolation Kit (Dynal, Hamburg, Germany) following the manufacturer's instructions. Dynabeads are paramagnetic polystyrene latex particles with covalently linked dT25 oligonucleotides, that allow preparation of highly purified, DNA-free polyA+ mRNA through magnetic separation.
RT–PCR amplification
The detection of PDE transcripts followed the previously described method (Rohwedder et al., 1996) for mRNA transcripts in human ejaculated spermatozoa. Briefly, the cDNA reactions were performed at 42°C for 60 min in a 80 µl reaction mixture containing 20 µl of the isolated mRNA, 250 pmol oligo dT12–18 primer (Pharmacia, Freiburg, Germany), 4 µl 10 mM dNTPs (Boehringer Mannheim, Mannheim, Germany), 16 µl 5x AMV reverse transcriptase buffer (Promega, Mannheim, Germany) and 25 IU AMV reverse transcriptase (Promega). The PCR amplifications were carried out in a 50 µl reaction mix containing 4 µl of prepared cDNA or control template, 20 pmol of each primer, 0.2 mM dNTPs (Boehringer Mannheim) and 5 µl 10x PCR buffer as well as 5 IU polymerase-mix from ExpandTM High Fidelity PCR System (Boehringer Mannheim). The applied PCR primers are shown in Table I. For positive control reactions cDNA prepared from human blood leukocytes and human fat tissue in our laboratory as well as cDNA libraries from human lung and brain (Stratagene, Heidelberg, Germany) were used as PCR input (Figure 1). To allow a semi-quantitative comparison of the amount of PDE mRNA between semen samples and positive control tissues ~1x106 cells of human blood leukocytes (in agreement with the minimum number of spermatozoa in single semen samples) were used for mRNA isolation. The yield of cDNA of all other positive control tissues was adapted by the strength of PCR amplification products for the ß-actin gene with the blood leukocyte cDNA sample (data not shown). Samples were amplified in a thermal cycler (Gene Amp PCR System 2400, Perkin Elmer, Langen, Germany) programmed for 40 cycles. The cycle profiles and the expected lengths of the resulting PCR products are listed in Table II. The PCR products (10 µl of each) were analysed on 1.2% agarose gels stained with ethidium bromide and molecular sizes were determined with a 2000 bp ladder (BioRad, München, Germany). For all PCR amplifications negative controls (water only, as well as mRNA without RT instead of cDNA) and positive controls were included.
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Results |
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RT–PCR was used to detect the mRNA transcripts of most PDE subtypes (Figure 2
). To verify the identity of the PCR products, an amplification product of each PDE type was cloned into pCRTM-2.1 by TA-cloning (Invitrogen, DeSchelp, Netherlands). Positive plasmids were characterized by restriction digestion and partly sequenced using a T7 promoter primer derived from the vector. Alignment of all resulting sequences with published sequence information identify all PCR amplification products are PDE-specific.
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A set of 10 swim-up-prepared single semen samples (1.0–6.0x106 cells) and another three semen samples pooled from 11 different patients (8.0–15.0x106 cells) were analysed by RT–PCR for phosphodiesterase types/subtypes. Specific bands of the molecular weight were observed in the PCR for PDE types/subtypes 1A/B/C; 2; 3A/B; 4A/B/C; 5 and 8 (Figure 1
). All semen samples were positive for PDE 4A and 4B and nine out of 10 investigated probes were positive for PDE 3B and PDE 8. Their PCR signals were very strong compared with the positive control. Weak signals were detected for PDE types/subtypes 1A/B/C, 2, 3A, 4C and 5. PCR products were found in five (PDE 1A), six (PDE 1C, 4C and 5), seven (PDE 2 and 3A) and eight (PDE 1B) of the 10 single ejaculates and in all of the three investigated semen sample pools (Figure 1). Transcripts for the PDE types/subtypes 4D and 7 were only detectable in one semen sample as a very weak signal.
All semen RNA samples were tested by PCR without prior reverse transcription to be sure that the PCR signals were not caused by contaminating DNA. In these control PCR reactions no products for any PDE were detected in all investigated semen samples. To exclude the possibility that the positive signals were a result of leukocyte contamination we used a primer pair for the CD4 gene, which is expressed at high levels by these cells (Littman, 1987) but not by spermatozoa (Chiang et al., 1994). There was no detectable contamination with the CD4 gene. Contamination of semen samples with leukocytes and immature spermatogenetic cells were excluded by the preparation-technique of spermatozoa (density gradient centrifugation, washing and swim up-technique), immunocytochemistry (WHO, 1992) and visual examination of the purified spermatozoa as well as confirmed by PCR controls. From these results we conclude that ejaculated human spermatozoa contain mRNA of PDE subtypes, and that a remnant of mRNA carried-over from contamination with somatic cells is improbable.
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Discussion |
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In the present study, we demonstrated the presence of mRNA for several PDE types/subtypes in ejaculated human spermatozoa for the first time. The results show that spermatozoa after density gradient centrifugation and swim up-preparation contain mRNA for the PDE types/subtypes 1A/B/C, 2, 3A/B, 4A/B/C, 5, and 8 with different intensities (Figure 1
). PDE are of growing interest as targets for therapeutic intervention. Therefore, in context with the complexity of the PDE gene family, it will be of increasing significance to establish precisely which of the many different PDE types/subtypes are expressed in a given cell type (Sonnenburg et al., 1998). Single semen samples showed specific bands of the expected molecular weight for several PDE subtypes but a definite signal was not observed in the PCR for PDE 4D and PDE 7 in all samples investigated. A strongly specific band was found in most of the single samples for PDE 1B, 3B, 4A, 4B and PDE 8. Amplification products of PDE types 1A/C, 2, 3A, 4C, and 5 were only observed in a part of the samples as weak signals. The comparison of the PDE mRNA pattern between all investigated semen samples showed no correlation between the several detectable PDE mRNA. Also, there was no relationship between the number of spermatozoa in the samples (1–6x106 cells) and the pattern of PDE types was found. Pooled semen samples represented a summary of PDE transcripts of single samples. The strength of the PCR signals in the pooled spermatozoa correlated with the frequency of PCR amplification products detectable in single samples and the lack of PDE 4D and PDE 7 was confirmed.
The presence of mRNA molecules in ejaculated spermatozoa has previously been demonstrated for the protamine-2 gene (Miller et al., 1994), for the c-myc gene (Kumar et al., 1993), and for the HLA class I genes G and B (Chiang et al., 1994). These results led to the assumption that the `sleeping' genome in spermatozoa may not to be so inactive after all (Kramer and Krawetz, 1997). Up to now it has not been clear whether the mRNA transcripts are products of an active synthesis in ejaculated spermatozoa or whether they are synthesized at an earlier stage of spermatogenesis and then stored in ribonucleoprotein particles (RNPs).
The PCR results agree with protein investigations, which found PDE 1 and PDE 4 by using selective inhibitors (Fisch et al., 1998), as well as with our former radiochemical determination of PDE activity in human spermatozoa from normal semen samples (Glander and Dettmer, 1992).
The existence of PDE in male germ cells is well documented. Gray et al. first demonstrated the presence of PDE in human spermatozoa (Gray et al., 1971). PDE represent a superfamily of structurally and functionally-related enzymes (Conti et al., 1991). More than 30 different forms have so far been identified and grouped into several broad gene families (Beavo, 1995). Some of the forms appear to be tissue-specific in their expression and differentially regulated, but all of the forms of PDE have the potential to regulate concentrations of the second messenger, cAMP or cGMP (Epstein, 1998). The concentrations of these cyclic nucleotides are controlled by two enzymatic processes: the synthesis by cyclases and the breakdown by cyclic nucleotide phosphodiesterases (Rossi et al., 1985). The hydrolysis of cyclic nucleotides is performed at a rate ~9–600-fold faster than the rate of cyclic nucleotides formation suggesting that the PDE play the dominant role in the control of cyclic nucleotides level in spermatozoa (Cheng and Boettcher, 1982).
The function of the cyclic nucleotides in spermatozoa seems to be involved in maturation, energy metabolism, and the acrosome reaction (Gearon et al., 1994) besides multiple roles in the regulation of testicular function (Rossi et al., 1985). Investigations of PDE functions in male germ cells have been performed indirectly via influence of PDE inhibitors on sperm functions such as sperm motility (Rees et al., 1990; Calogero et al., 1998; Fisch et al., 1998) or acrosome reaction (Tesarik et al., 1992; Fisch et al., 1998) or detection of enzyme activities (Glander and Dettmer, 1992). The question of which PDE-isoenzyme influences which sperm function in which way is unknown in detail. However, there is no doubt that PDE types/subtypes are also regulated by phosphorylation. This common mechanism results in signal transduction (Conti et al., 1995), especially via PDE 1, 3, 4 and 5; we detected the transcripts of these types/subtypes in human ejaculated spermatozoa.
In summary, we have shown that human ejaculated spermatozoa contain an extended pattern of mRNA transcripts of PDE. The functions of the resulting PDE types/subtypes in spermatozoa require further investigation.
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3 To whom correspondence should be addressed
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References |
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Submitted on February 3, 1999; accepted on April 29, 1999.
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