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AW JANUCHOWSKI*
BOROWICZ*
P. JAGODZINSKI*
From the * Department of Biochemistry and
Molecular Biology, and Division of Infertility
and Reproductive Endocrinology, Department of Gynecology and Obstetrics,
University of Medical Sciences, Pozna
, Poland.
Correspondence to: Dr Pawe P. Jagodzinski, Department of Biochemistry
and Molecular Biology, University of Medical Sciences, 6
 wicickiego St, 60-781 Pozna, Poland (e-mail:
pjagodzi{at}am.poznan.pl). |
| Received for publication December 2, 2003; accepted for publication March 10, 2004. |
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Abstract |
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Key words: Sperm, mRNA, CCR5, RQ-PCR
Chemokines belong to the cytokine superfamily; they represent a group of
small proteins that stimulate the attraction of leukocytes and mediate
inflammation (Murdoch and Finn,
2000). The two main subfamilies of chemokines include the
-chemokines (CXCs) and the ß-chemokines (CCs)
(Murdoch and Finn, 2000; Kaplansky and Bongrand,
2001).
Recent findings suggest that certain chemokines may be involved in human reproduction and may play profound roles in ovulation, menstruation, implantation, cervical ripening, preterm labor, and sperm chemotaxis (Garcia-Velasco and Arici, 1999).
Follicular fluid causes chemotaxis of sperm to the fertilization site in the female genital tract, but the chemoattractant in follicular fluid has not yet been identified in humans (Eisenbach, 1999; Garcia-Velasco and Arici, 1999).
It has been shown that chemokines such as Regulated upon Activation of Normal T-cells Expressed and Secreted (RANTES) exist in the genital tract fluids (Rohwedder et al, 1996; Hornung et al, 2001). This chemokine affects various cells via binding to CC chemokine receptor 5 (CCR5), which is present on the surface of various cell types (Alkhatib et al, 1996; Filippatos et al, 2003). RAN-TES exhibits a chemotactic effect on human sperm, and this observation may suggest that the CCR5 receptor transcript can be used during spermatogenesis to biosynthesize this receptor (Isobe et al, 2002).
The CCR5 gene is organized into 4 exons and 2 introns; exons 2 and 3 are not interrupted by an intron (Figure 1A). The presence of CCR5 transcript isoforms has been studied only in leukocytes. The CCR5 mRNA isolated from leukocytes exists mainly as 2 isoforms, which consist of exons 1, 2, 3, and 4, or a second isoform without exon 2 (Figure 1B) (Mummidi et al, 1997).
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Materials and Methods |
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TopAbstractMaterials and MethodsResults and DiscussionReferences |
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,
Poland. Spermatozoa were purified by centrifugation through discontinuous
Percoll (Amersham Bioscences, United Kingdom) density gradient (80:40,
vol/vol) and the swim-up technique. Liquefied semen samples were diluted with
an equal volume of minimal essential medium (MEM) and layered on top of a 2-mL
Percoll solution. After centrifugation for 20 minutes at 800 x
g, Percoll pellets were washed twice (300 x g for 10
minutes) with 10 mL MEM containing 1 mg/mL bovine serum albumin and 50 mM
benzamidine chloride. The pellets were gently overlaid with 1.2 mL human tubal
fluid medium (HTFM) modified by the addition of 20 mM HEPES (Sigma,
Deisenhofer, Germany) and 25 µg/mL streptomycin, 15 µg/mL penicillin,
and 10 mg/mL human serum albumin. The tubes were placed in an incubator at
37°C for 60 minutes. The top 1.0 mL of the HTFM was removed and the purity
of the dissolved spermatozoa was examined using an optical microscope equipped
with a 100x oil objective. These separated spermatozoa were used
immediately to isolate RNA, which were reverse transcribed and investigated by
RQ-PCR analysis.
Isolation of Peripheral Blood Mononuclear Cells
Blood samples were obtained from the same individuals whose semen samples
were used to purify spermatozoa. Peripheral blood mononuclear cells (PBMCs)
were isolated by centrifugation over Ficoll-Hypaque (density 1.077
g/cm3) and were used immediately to isolate the RNA, which was
reverse transcribed and investigated by RQ-PCR analysis for the presence of
CCR5 mRNA isoforms.
RQ-PCR Analysis of CCR5 mRNA Isoforms in Spermatozoa and PBMCs
Total RNA was isolated from spermatozoa or PBMCs according to the methods
described by Chomczynski and Sacchi
(1985). RNA was treated with
DNase I (Promega, Madison, Wis) and reverse-transcribed into complementary DNA
(cDNA) using random hexamer priming and Maloney murine leukemia virus (MMLV)
reverse transcriptase (RT-Kit; Sigma Chemical Company, St Louis, Mo).
Quantitative analysis of CCR5 cDNA was performed by RQ-PCR SYBR Green I
analysis (Light Cycler, Roche Diagnostics GmbH, Mannheim, Germany). The CCR5
cDNA was amplified using pairs of primers as shown in the
Table.
For amplification, 2 µL of cDNA was added to 18 µL of PCR mix containing HotStartTaq DNA polymerase, reaction buffer, deoxynucleotide triphosphate mix, SYBR Green I dye, 2.5 mM MgCl2, and primers (QIAGEN Inc, Valencia, Calif). Quantification of copy number was derived from a standard curve of known amount of synthetic DNA template. One RNA sample of each preparation was processed without MMLV RT (RT reaction) to provide a negative control in subsequent PCR reactions. To normalize for the quantity of cellular CCR5 transcripts in each sample, copy numbers were corrected to the amount of cellular glyceraldehyde-3-phosphate dehydrogenase (Table). To ensure that granulocytes did not contaminate the spermatozoa, we excluded them by RT-PCR analysis of myeloperoxidase transcript, which was restricted to granulocytes.
The amount of CCR5 mRNA was expressed in the number of CCR5 transcript copies shared per 106 heterogeneous population of spermatozoa (Table).
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Results and Discussion |
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TopAbstractMaterials and MethodsResults and DiscussionReferences |
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This spermatozoal CCR5 mRNA isoform was shorter than the CCR5 transcript found in human leukocytes (Figure 2; Table). These immune cells contain two major CCR5 mRNA isoforms that comprise exons 1, 3, and 4, and they differ in the presence of exon 2 (Figure 1B) (Mummidi et al, 1997). The differences between the composition of CCR5 transcripts, which were detected in these two types of cells, may result from different requirements for CCR5 mRNA stability in spermatozoa and leukocytes.
It seems that the leukocyte CCR5 mRNAs are use immediately for translation to biosynthesize the mature molecule of the receptor, or they can be degraded by ribonucleases. This relatively short life span of leukocyte CCR5 mRNA allows control of the rate of protein biosynthesis at the level of transcript stability (Schwartzbauer and Menon, 1998). The different composition of CCR5 mRNA in spermatozoa may be attributed to a higher stability of this transcript in the postmeiotic stages of spermatogenesis. This short CCR5 transcript isoform can be used to biosynthesize the CCR5 receptor either during spermatogenesis or during the early stages of embryonic development.
Transcription of many genes in various stages of spermatogenesis may result in biosynthesis of alternatively spliced variants of somatically expressed genes (Hecht, 1990). In general, transcription takes place throughout spermatogenesis from the spermatogonial stage to the haploid, round-elongating spermatid stage. Investigations of the packaging of DNA in human sperm chromatin revealed that in late spermatogenesis, 85% of the DNA is packaged into nucleoprotamine, and 15% into nucleohistone (Gatewood et al, 1990). The biological significance of the two chromatin fractions suggests that they may exhibit different roles. The nucleoprotamine complex is highly condensed and is transcriptionally inactive, whereas the nucleohistone complex could be involved in chromatin decondensation and transcription of genes, which is necessary in the last step of spermatogenesis (Gatewood et al, 1990).
It has been reported that transcription of many genes occurs in the postmeiotic stages of spermiogenesis. These large numbers of mRNAs can be stored for long periods in the nonpolysomal messenger ribonucleoprotein particles prior to their translation in round spermatids (Hecht, 1990). Many transcripts survive the condensation of the spermatid nucleus and are present in human ejaculated spermatozoa (Miller et al, 1994; Wykes et al, 1997). Further in situ investigations are needed to determine the transcriptional activity of CCR5 gene during the different stages of spermatogenesis.
Chemotaxis may serve as a principal process in bringing together human gametes by the transfer of sperm during the fertilization process (Eisenbach and Tur-Kaspa, 1999). The induction of chemotaxis in human spermatozoa by follicular fluid in vitro has been well documented, whereas the chemoattractant in follicular fluid remains unidentified. Follicular fluid contains several types of chemokines, including RANTES, which exhibits a chemotactic effect on human sperm (Machelon et al, 2000). The RANTES effect on target cells is mediated by the binding of this ligand to the CCR5 chemokine receptor. This finding may suggest that the CCR5 mRNA in spermatozoa could be used to biosynthesize CCR5 receptor during spermatogenesis. Further studies should endeavor to find the intracellular localization of CCR5 receptor in sperm and the relationship between the spermatozoal CCR5 transcript and sperm function.
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Acknowledgments |
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acinski, MD, is gratefully
acknowledged. |
Footnotes |
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, Poland. 
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