Delivery of ferric ion to mouse spermatozoa is mediated by lipocalin internalization

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Abstract

The aim of this study was to illustrate the further process of 24p3 protein after association with epididymal spermatozoa. We have previously identified a caput-initiated 24p3 protein, which interacts with the spermatozoa surface in vitro. In the present study, we investigate another role of the 24p3 protein with spermatozoa. Mouse epididymal spermatozoa exhibit the ability to bind spontaneously with exogenous 24p3 protein, a part of which is further internalized into the spermatozoa in epididymal caput. We have now focused on this issue using freshly prepared spermatozoa from caudal region of epididymis. First, the cytosolic fractionation of spermatozoa has revealed that biotinylated 24p3 protein signal could be detected by supplying biotinylated protein under 37 °C incubation after 30 min at this experiment. Further, flow cytometric analysis of FITC-protein containing spermatozoa has revealed two distinct types of fluorescent spermatozoa, and microscopical experimentation with fluorescent FITC-24p3 protein has shown that the 24p3 protein did accumulate in the cytosolic portion of spermatozoa. All of these events, which showed protein uptake into the cell, demonstrated time- and temperature-dependence of endocytotic characteristics, these constituting the critical points in the process of endocytosis for spermatozoa as for other cells. Using a fluorometric method, the binding affinities of ferrous ion and ferric ion to 24p3 protein were shown to be (1.5 ± 0.2) × 106 and (3.0 ± 0.4) × 107 M−1, respectively. We have also determined the internalization of this protein in the transition of iron into spermatozoa. We report here that spermatozoa, from the caudal epididymis, demonstrate the ability to bind with 24p3 protein and further internalize it and deliver the ferric ion to the spermatozoa via protein internalization. We suggest that the 24p3 protein plays a physiological role in spermatozoa in the context of protein–ligand complex internalization.

Section snippets

Materials and methods

Chemicals, biochemicals, and bio-assay kits. An Enhanced Chemiluminescence plus kit (ECL) was purchased from Amersham–Pharmacia Biotech (Buckinghamshire, UK). Anti-rabbit IgG-horseradish peroxidase conjugate (HRP-conjugated anti-rabbit IgG) prepared from goats and fluorescein isothiocyanate (FITC) were obtained from Sigma (St. Louis, MO, USA). Desferrioxamine mestylate (DFO) and diethylene triamine pentaacetic acid (DTPA) were purchased from Sigma (St. Louis, MO, USA). A permeable ester form of

24p3 protein accumulation in the spermatozoa cytosol

As described previously, the epididymal caput was found to produce a large quantity of 24p3 protein in luminal fluid and was also found to be associated with caudal spermatozoa, for which the 24p3 protein had virtually vanished [14]. To test whether there was any further processing of the spermatozoa in the epididymal caput after protein association, we extracted the proteins from the different portions of the caput spermatozoa. Using Western blotting, we detected the location of 24p3 protein

Discussion

A prerequisite for understanding the physiological function of lipocalins is some knowledge pertaining to the mode of delivery of the bound ligands into the cells. Evidence suggests that lipocalin binds to certain cells and internalizes the protein into these cells [16], whereas others suggest that the lipocalin present creates a direct signal that induces various physiological processes to commence [26]. The interaction of 24p3 protein with murine spermatozoa resulting in an increase in the

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