HIV-1 chemokine co-receptor CCR5 is expressed on the surface of human spermatozoa

Viruses adhering to the sperm surface are described in the semen of HIV-1-infected individuals, although viral adhesion mechanisms have yet to be fully understood. We demonstrate, by cytometric analysis and immunofluorescence microscopy, the presence of β-chemokine receptor 5 (CCR5) on the periacrosomal region of ejaculated spermatozoa. CCR5 expressed on the sperm cell surface may allow sperm to act as virion cellular carriers during the sexual transmission of HIV-1 infection.

Chemokines belong to the cytokine superfamily and represent a group of small proteins that, through their specific receptors, mediate leukocyte traffic in various tissues and inflammation, thus playing an important role in many physiological and pathological processes ^[1]. The two main subfamilies of chemokines include the α-chemokines and the β-chemokines ^[1], whose specific receptors are classified, respectively, as α and β chemokine receptors ^[2].

It has been reported that chemokines are involved in several human reproductive events ^[1], including sperm chemotaxis, ovulation, implantation and menstruation ^[3]. More recently, it has been demonstrated that human spermatozoa contain messenger RNA coding for the β-chemokine receptors 1 and 5 (CCR1 and CCR5) that bind the β chemokine 'regulated upon activation of normal T cells expressed and secreted' (RANTES), and that RANTES has a chemotactic effect on human sperm ^[4].

HIV-1 uses chemokine co-receptors for cell entry, and CCR5 is the main co-receptor used by macrophage-tropic viral strains, which are those most commonly involved in the sexual transmission of HIV-1 infection ^[2].

During HIV-1 infection, the virus may be present in the semen as cell-free virions in the seminal fluid, within infected leukocytes or carried by sperm cells ^[5]. Molecular mechanisms involved in the viral adhesion to the sperm surface remain to be fully understood, because the presence of CD4 cells on the membrane of spermatozoa remains a matter of debate ^[6]. No chemokine co-receptor has yet been reported to be expressed on the surface of ejaculated human spermatozoa.

We report data demonstrating the presence of a high level of CCR5 protein on the periacrosomal region of ejaculated spermatozoa.

By cytometric analysis, using a specific primary anti-CCR5 monoclonal antibody (R&D Systems, Minneapolis, MN, USA) and phycoerythrin-conjugated secondary monoclonal antibody, we demonstrated the presence of CCR5 on the surface of unfixed freshly isolated sperm cells. CCR5 protein was detected in 20 of the 22 healthy subjects studied (91%).

Different CCR5 expression patterns were observed, with all sperm cells expressing CCR5 in four subjects (Fig. 1a) and two sperm populations, either negative or positive for CCR5, in the remaining 16 subjects. The purity of the sperm fractions analysed was demonstrated by their haploid DNA content, measured using the DNA binding dye propidium iodine ^[7].

Fig. 1

The fluorescence signal for the CCR5 antigen was higher in sperm cells than in freshly isolated peripheral blood mononuclear cells from healthy donors, used as positive controls (data not shown), and ranged from 5 to 50 times above its isotype control, thus demonstrating that CCR5 is strongly expressed on the plasma membrane of ejaculated spermatozoa and finely regulated during spermiogenesis.

The cellular location of the CCR5 protein, evaluated by immunofluorescence, showed a strong fluorescent signal on the surface of the sperm head corresponding to the periacrosomal region of the cells (Fig. 1b,c). As regards the amount of positive cells, immunofluorescence microscopy observations confirmed the results obtained by flow cytometric analysis. These data provide a solid ground for the possible involvement of the β-chemokine receptor system in human sperm physiology.

Preliminary genotyping data suggest that the lack of CCR5 expression in the sperm plasma membrane could be related to sperm haplotypes mutated for the CCR5 gene. In individuals who are heterozygous for a specific gene, the segregation of the alleles that occur in haploid germ cells may entail the production of two genetically different sperm cell populations that can be revealed as two different expression profiles by cytometric analysis ^[8].

The presence of CCR5 on the sperm membrane suggests that, in the male genital tract, HIV-1 might interact with spermatozoa through this receptor, either alone or in association with other molecules ^[9]. The presence of a surface galactoglycolipid, galactosyl-alkyl-acylglycerol, structurally related to galactosylceramide and capable of specifically binding the gp120 has been reported on the sperm membrane ^[10]. In addition, modes of HIV adhesion to spermatozoa using receptors other than CD4 cells have been suggested ^[11]. CCR5 expressed on the sperm cell surface may represent the molecular machinery of sperm-virus interaction, thereby shedding light on how spermatozoa act as cellular carriers of virions during the sexual transmission of HIV-1 infection ^[12].

Binding inhibition experiments with anti-human CCR5 antibodies may shed further light on the physiological role of CCR5 in sperm chemotaxis as well as on the role of sperm as an HIV-1 cellular carrier.

Sponsorship: This work was supported by grants (30C.71 and 30D.74) from the Programma Nazionale Ricerca sull'AIDS (Istituto Superiore di Sanità).