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  • Levofloxacin mg In a previous paper we demonstrated immunohi


    In a previous paper, we demonstrated immunohistochemically the Levofloxacin mg of GHSR-1a in acrosomes and peripheral region of spermatid heads as well as in acrosomes of the spermatids undergoing spermiation [19]. The present study was undertaken to demonstrate the GHSR-1a expression in rat epididymal spermatozoa using immunohistochemistry, immunofluorescence and Western blotting techniques. Moreover, we examined the effects of ghrelin on sperm motility, intracellular free calcium ion concentration and induction of apoptosis.
    Materials and methods
    Discussion The results of the present study demonstrated the expression of GHSR-1a at the proacrosomal and acrosomal sites of rat spermatids and epididymal spermatozoa and provided evidence for its functional meaning. GHSR-1a expression was revealed by immunohistochemistry and immunofluorescence and was confirmed by Western blotting with three different anti-GHSR-1a antibodies. The characteristic localization of immunostaining in spermatids and epididymal spermatozoa was consistent with our previous results [19] and may represent the expression of at least one of the GHSR-1a isoforms. It is of interest that the localization of the receptor proteins within the acrosomal region has also been shown for leptin in boar sperm [23], [24] and for inositol 1,4,5-triphosphate in dog, rat, hamster and mouse sperm [27]. Moreover, in all of the cases the binding of ligands to the receptors induced a response related to sperm motility or acrosome reaction. The possible functional role of GHSR-1a within the rat seminiferous epithelium has been discussed previously [19]. However, spermatozoa, especially in rodents, are susceptible to laboratory procedures and respond to procedural manipulations with cell damage or death [25], [28]. In comparison to bulls, boars and rams, repeated pipetting and extended centrifugation times significantly decreased rat sperm motility as well as membrane and acrosomal integrity [25]. Thus, to protect sperm against apoptosis and to minimize the impact of procedural manipulations, a special approach had to be applied to record the effects of the activation of the ghrelin receptor signaling pathway in epididymal spermatozoa. The results of our experiments indicated that ghrelin, at concentrations 10−9 and 10−6mol/L, affected rat epididymal spermatozoa. At these concentrations, especially at a concentration of 10−6mol/L, ghrelin significantly elevated the intracellular Ca2+ level and raised the percentage of spermatozoa with progressive motility but did not influence the apoptotic processes of spermatozoa. Under these conditions, ghrelin neither changed the proportion of phosphatidylserine translocation evaluated with annexin V conjugated to FITC, nor altered the percentage of the spermatozoa with active caspase-3 within the incubated spermatozoa sample. This suggests that the apoptosis of spermatozoa was unaffected by the employed concentrations of ghrelin. No any proapoptotic effects of ghrelin (at concentration of 100–1000pg/mL) were observed also by other authors in an in vitro experiment with a chorioncarcinoma cell line used [29]. Moreover, our results confirmed a phenomenon observed by other authors [30], which is that spermatozoa in early apoptosis are motile. Ghrelin at a concentration of 10−6mol/L significantly increased the percentage of spermatozoa showing progressive movement. Furthermore, rat spermatozoa responded to ghrelin with an increasing intracellular concentration of Ca2+ which may be related to sperm motility. Interestingly, after the addition of ghrelin to the medium, rat spermatozoa reacted with an elevated intracellular Ca2+ level within a few seconds, suggesting that the action of grehlin is mediated through the ghrelin receptor. The elevated intracellular Ca2+ level could then be registered by flow cytometry for 20min, showing the functional excitation of spermatozoa for a longer time. Opposing effects of ghrelin as a result of GHSR-1a activation were recently reported in mouse germinal cells in vitro[31]. Applying the patch clamp technique, the authors found that ghrelin at 0.1μmol/L reversibly inhibited T-type Ca2+ channel currents, and this inhibitory effect could be blocked by a selective antagonist of GHSR-1a. However, they used a heterogeneous sample of spermatogenic cells isolated from mouse testes.