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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online September 20, 2005 as doi:10.1096/fj.05-3962fje. |
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Department of Histology and Medical Embryology,
* Department of Medical Physiopathology, and Centro di Eccellenza Biologia e Medicina Molecolare, University of Rome "La Sapienza," Italy
1Correspondence: Department of Histology and Medical Embryology University of Rome "La Sapienza" Via Antonio Scarpa, 14 00161 Rome, Italy. E-mail: mario.stefanini{at}uniroma1.it
SPECIFIC AIMS
The recent demonstration of the presence of "regulated upon activation of normal T cells expressed and secreted" (RANTES) in the follicular fluid and that this molecule is an in vitro chemoattractant for human spermatozoa, suggested that ß-chemokines could be the signaling molecules responsible for sperm chemotaxis in the human. To test this hypothesis, we have verified the presence of ß-chemokine receptors CCR5 and CCR3 on the surface of human ejaculated sperm cells.
PRINCIPAL FINDINGS
1. CCR5 and CCR3 are expressed on sperm head of human ejaculated spermatozoa
Cytometric analysis, performed on fresh nonpermeabilized cells, demonstrated that both chemokine receptors were strongly expressed on the sperm surface of healthy normozoospermic subjects (Fig. 1)
. Immunofluorescence experiments located CCR5 on the periacrosomal region of the plasma membrane whereas CCR3 had a diffused distribution on the sperm head (Fig. 2)
. Western blot analysis confirmed the presence of both receptor proteins of the expected molecular weights. Our data agree with the recent demonstration by RT-PCR that CCR5 mRNAs are present in ejaculated human spermatozoa but represent the first evidence for the presence of CCR3 on the surface of human spermatozoa.
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2. Percentage of spermatozoa expressing CCR5 and CCR3 varied in different subjects
Cytometric analysis of CCR5 demonstrated that CCR5 expression in the positive sperm ranged between 5- and 50-fold above its isotype control, suggesting that CCR5 expression on the sperm membrane is finely regulated during spermiogenesis. In addition, the percentage of chemokine receptor positive sperm varied in the cohort studied: in the majority of the subjects (28 of 33) 25% to 75% of the sperm was CCR5 positive, whereas in the remainder the sperm was either all positive (3 cases) or all negative (2 cases) (Fig 1A-E
). These different cytometric profiles were confirmed by immunofluorescence.
Data on the presence of CCR3 refer to 8 of the 33 normozoospermic subjects studied for CCR5. CCR3 was observed in the sperm head with a localization more diffuse than that observed for CCR5. Five subjects had all spermatozoa strongly expressing CCR3; and in three, half of the sperm population was positive (Fig 1F-H
). In all eight subjects studied, CCR3 expression was higher than that observed for CCR5, both as percentage of cells expressing the receptor and as amount of receptor present in each cell.
3. Absence of CCR5 expression in the sperm plasma membrane of a subpopulation of spermatozoa may be related to sperm haplotypes mutated for the CCR5 gene
Segregation of the alleles that occurs in haploid germ cells may produce, in individuals who are heterozygous for a specific gene, two genetically different sperm cell populations that can be revealed as two different expression profiles by cytometric analysis. Product sharing via intercellular bridges conjoining differentiating germ cells is thought to guarantee gametic equality despite haploid gene activity in mammalian spermiogenesis. However, it has been reported that haploid-expressed membrane proteins and sperm antigens cannot diffuse through the cytoplasmic bridges, resulting in functionally different sperm cells in each male. Since CCR5 and 3 are membrane proteins, they belong to a subset of molecules not necessarily redistributed among germ cells connected by intercellular bridges. We investigated the possible presence of a 32 nucleotide deletion (
32) in the CCR5 gene, which is responsible for the lack of CCR5 receptor expression on the cell membrane. The genetic 32 deletion was detected, together with the wild-type allele (32+/–), in 2 of the 13 subjects displaying sperm that were half negative and half positive for CCR5, whereas it was not observed in two subjects with all sperm cells positive for CCR5 who was used as a control (32+/+). These genotyping data demonstrate that expression of CCR5 on the membrane of half sperm population may be due to heterozygotic mutation in the relative gene. The two different CCR3 expression patterns need to be further investigated to verify their possible genetic origin.
CONCLUSIONS AND SIGNIFICANCE
Sperm chemotaxis consists of the regulation of sperm movement direction which occurs at the time of fertilization, once the capacitated sperm is in close proximity to the egg. The induction of chemotaxis in human sperm by FF in vitro appears to be well established and a series of molecules present in the FF of several mammalian species, including humans, have been considered as possible sperm chemoattractants. It was reported recently that chemokines are involved in several human reproductive events and that sperm cells are capable of migrating by following a chemoattractant gradient of soluble ß-chemokine factors such as RANTES, which has been found in a variety of genital tract fluids and in the FF. Based on these observations and on the presence of a stable CCR5 mRNA isoform in human spermatozoa, it has been proposed that the signaling system RANTES and its receptors play a role in sperm chemotaxis. Here we demonstrate the presence of the CCR5 and CCR3 on the human sperm plasma membrane, thus confirming the specificity of this molecular mechanism. Preliminary cytometric evidence, obtained in same subjects studied for both CCR3 and CCR5, demonstrated that CCR1 is present on the human sperm, although in a smaller percentage of cells (data not shown). It is conceivable that more than one chemokine receptor is involved in the chemotactic sperm recruitment induced by molecule(s) present in the follicular fluid; on the other hand, ß-chemokines such as RANTES are able to bind to more than one receptor. The functional role of the chemokine-chemokine receptor system in sperm chemotaxis remains to be evaluated by inhibition and competition experiments for each ligand present in FF and of the receptors identified in human sperm.
Chemokine receptor 5 is considered the main coreceptor for cell entry used by macrophage-tropic HIV-1 viral strains, those most commonly involved in sexual transmission of HIV-1 infection; chemokine receptor 3 is described to facilitate infection by a subset of primary HIV-1 isolates as well as to promote efficient infection of microglia and CNS by the virus. Individuals bearing the 32–/– defective CCR5 gene are resistant to HIV infection despite multiple sexual viral exposures. Heterozygous individuals for this mutation appear to exhibit a slower progression of the disease and a lower blood viral load. Although it is known that semen may contain both cell-free virions HIV-1 infected leukocytes and that viruses adhering to leukocyte and sperm cell surfaces are carried to the sexual partner, the mechanisms of viral adhesion to sperm cells remain poorly understood. We recently proposed that the presence of CCR5 on the sperm membrane, alone or in association with other molecules, may allow HIV-1 interaction with spermatozoa. The demonstration of CCR3 expression on the sperm head surface represents, in our opinion, additional evidence in favor of the possibility that ß-chemokine receptors represent the molecular machinery responsible for HIV-1 adhesion to the sperm surface, thereby shedding light on how spermatozoa act as cellular carriers of virions during sexual transmission of HIV-1 infection.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-3962fje;
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