Differential signalling for enhanced hexose uptake by interleukin (IL)-3 and IL-5 in male germ cells

Abstract

We studied the expression and function of the IL (interleukin)-3 and IL-5 family of receptors in male germ cells. RT (reverse transcription)-PCR showed expression of mRNAs encoding the alpha and beta subunits of the IL-3 and IL-5 receptors in human testis, and the presence of IL-3 and IL-5 receptors alpha and beta proteins was confirmed by immunoblotting with anti-alpha and anti-beta antibodies. The immunolocalization studies showed expression of these receptors in the germ line in the human testis and in human and bovine ejaculated spermatozoa. Functional studies with bull spermatozoa indicated that IL-3 signalled for increased uptake of hexoses in these cells at picomolar concentrations compatible with expression of functional high-affinity IL-3 receptors in these cells. In contrast, IL-5 failed to induce increased hexose uptake in bull spermatozoa. Experiments using HL-60 eosinophils that express functional IL-3 and IL-5 receptors confirmed that IL-3, but not IL-5, signalled for increased hexose uptake. Our findings suggest that differential signalling for increased hexose uptake by heteromeric high-affinity IL-3 and IL-5 receptors in mammalian spermatozoa is a property that depends on the identity of the alpha-subunit forming part of the alphabeta-complex and is not a property specific to the germ cells.

Figure 1. Expression of IL-3 and IL-5 receptors in human germ cells and bovine spermatozoa

(A) RT-PCR of human IL-3 and IL-5 mRNAs. Total human testis RNA was subjected to RT-PCR using primers specific for the IL-3 and IL-5 α subunits or the βc subunit. PCR products corresponding to the IL-3 α (510 bp), IL-5 α (450 bp) and the βc (570 bp) subunits are shown in lanes 2, 3 and 4 respectively. The migration in the agarose gel of a series of DNA 100-mer size standards is shown in lane 1. (B, C) Identification of IL-3 and IL-5 receptors in spermatozoa. Membrane proteins isolated from human (B) and bovine (C) spermatozoa were fractionated by SDS/PAGE, transferred on to Immobilon membranes, and probed with anti-(IL-3 α-subunit), anti-(IL-5 α-subunit) and anti-(βc subunit) antibodies, followed by incubation with a secondary antibody coupled to horseradish peroxidase. Pre-adsorbed antibodies incubated with the peptides used to elicit them were used as negative controls (lanes labelled +). Sizes on the left in kDa indicate the migration of molecular-mass standards.

Figure 2. Localization of IL-3 and IL-5 receptors in human testis

Testis sections were incubated with anti-IL-3 α-subunit (a, b), anti-IL-5 α-subunit (c, d) and anti-βc subunit (e, f) antibodies followed by incubation with a secondary antibody conjugated to horseradish peroxidase. Testis section incubated with anti-(IL-3 α-subunit) antibody in the presence of the peptide used to elicit it did not show a positive reaction (g, h). Arrows show positive staining in male germ cells. L, lumen of the seminiferous tubules. Scale bars, 20 μm.

Figure 3. Immunolocalization of IL-3 and IL-5 receptors in human and bovine spermatozoa

Human (A) and bovine (B) spermatozoa were spread on to coated slides and probed with the anti-(IL-3 α-subunit) (a), anti-(IL-5 α-subunit) (c) and anti-βc-subunit (e) antibodies, followed by incubation with a secondary antibody conjugated to horseradish peroxidase. No reactivity was observed with pre-adsorbed antibodies (b, d and f).

Figure 4. Effect of IL-3 and IL-5 on DOG uptake in bovine spermatozoa and in HL-60 eosinophils

(A) The DOG uptake by bovine spermatozoa in response to concentrations of IL-3 (•) and IL-5 (▪) ranging from 0 to 1 nM at 30 min treatment time is shown. The results are displayed as nmol/min for DOG uptake and in cells treated with IL-3 and IL-5 relative to cells that were not treated. GM-CSF (○) and TNFα (▴) were used as positive and negative controls respectively. Results are the means±S.D. of four samples. (B) Time-course of the effect of GM-CSF (○), IL-3 (•) and IL-5 (▪) on DOG uptake by HL-60 eosinophils. Cells were treated with 1 nM GM-CSF, IL-3 and IL-5 for the time periods indicated, before measuring the uptake of DOG. Results are the mean of two experiments with three replicates each. (C) Dose-dependence of the effect of GM-CSF (○), IL-3 (•) and IL-5 (▪) on DOG uptake by HL-60 eosinophils. Cells were incubated for 30 min in the presence of different concentrations of GM-CSF, IL-3 and IL-5, and uptake of DOG was measured afterwards. Results are the means±S.D. of four samples, and represent one of three similar experiments.