Expression of ghrelin receptor (GHSR-1a) in rat epididymal spermatozoa and the effects of its activation.

In this study we demonstrated the expression of the ghrelin receptor GHSR-1a in rat spermatids and epididymal spermatozoa, as well as some effects of ghrelin on the spermatozoa in vitro. For the demonstration of GHSR-1a the immunocytochemical, immunofluorescence and Western blotting techniques were applied using three different types of antibodies. The response of spermatozoa to ghrelin was tested in a series of in vitro experiments and their effects were evaluated using confocal microscopy and flow cytometry. GHSR-1a protein was found as expressed in the Golgi and acrosomes of spermatids and acrosome regions or the head cell membrane of epididymal spermatozoa. The GHSR-1a expression in spermatozoa was also confirmed by Western blot. No differences were found in percentage of spermatozoa showing annexin-V binding and expression of active form caspase-3 between control and ghrelin-treated spermatozoa. This result may indicate no pro-apoptotic effects of ghrelin neither at 10(-9) nor 10(-6)mol/L concentration. Ghrelin (10(-6)mol/L) increased free intracellular calcium ion concentration in the rat spermatozoa. Moreover, stimulation with 10(-6)mol/L ghrelin increased, while 10(-4)mol/L ghrelin decreased the number of spermatozoa showing progressive motility. In conclusion, the expression of the GHSR-1a receptor in spermatozoa, as well as ghrelin influences on sperm motility and intracellular calcium ion concentration suggest that such biological effects of ghrelin may be produced under in vivo conditions.

The analysis of exogenous ghrelin plasma activity and tissue distribution.

OBJECTIVES: Ghrelin presents a multiplicity of biological functions, what is consistent with widespread expression of this peptide and its receptors. Ghrelin may act locally, but it may also influence distant cells. The aim of the study was to assess plasma activity of exogenous ghrelin and its distribution in rats. DESIGN: Plasma radioactivity of (125)I-ghrelin (cpm) was analyzed in blood specimens collected after (125)I-ghrelin administration. Tissue uptake of (125)I-ghrelin (cpm/mg) was evaluated in 27 tissues obtained during an autopsy performed 1, 2 and four hours after (125)I-ghrelin administration. The radioactivity of the tissue specimen (cpm) was divided by the weight of the specimen (mg). RESULTS: Plasma (125)I-ghrelin radioactivity decreased rapidly after peptide administration. The half-life time of (125)I-ghrelin was 15-18 minutes. The analysis of (125)I-ghrelin distribution revealed three profiles of its tissue uptake. The first profile was characterized by decreasing radioactivity (e.g. brain, kidney, liver). Increasing tissue radioactivity followed by a gradual decrease (second profile) was observed for example in stomach, intestine and thyroid. The third profile was described as a relatively stable radioactivity (e.g. lung, myocardium). Despite of Lugol's solution administration, thyroid uptake of (125)I-ghrelin was notably higher than in other tissues (second and third profile). CONCLUSIONS: Exogenous ghrelin uptake in tissues that produce this peptide suggests, that ghrelin influences the biology and function of these cells also in endocrine way. Similarly, the accumulation of peptide observed in the third profile (e.g. thyroid) may reflect a potential role of ghrelin in these organs.

Immunohistochemical and hybridocytochemical study on ghrelin signalling in the rat seminiferous epithelium.

The results of presented study demonstrate expression of ghrelin, its functional receptor GHSR-1a and their genes in spermatogenic cells of rat testis suggesting their functioning within seminiferous epithelium. The immunohistochemical and hybrydocytochemical expression, of proteins and transcripts, was estimated taking into account the cycle of seminiferous epithelium and phases of spermatogenesis. Both transcripts and ghrelin was found to show nuclear expression and scarcely cytoplasmic. Expression of genes for ghrelin and GHSR-1a was shown in early spermatocytes and round spermatids representing transcriptional phases of meiosis and spermiogenesis. Ghrelin was evidenced to show nuclear expression in two stage-specific windows, in late spermatogonia, in spermatocytes up to early pachytenes, and again in spermatids of acrosome and early maturation phase of spermiogenesis. In late pachytenes, secondary spermatocytes, round spermatids, maturing spermatids and spermatozoa the reaction is lacking. With two types of antibodies against the GHSR-1a used the two different patterns of immunostaining was evidenced suggesting two isoforms of GHSR-1a. The first evidenced GHSR-1a in cytoplasm of spermatocytes, cell membrane and acrosomes of spermatids, Sertoli cell processes and heads of spermatozoa. With second type of antibodies the immunostaining marks all steps of evolution of acrosome in spermatids. It is believed that site of ghrelin expression in seminiferous epithelium may indicate its role in local regulations, not excepting the intracellular signalling. Immunostaining pattern for GHSR-1a seems to suggest both its participation in the cross-talk among the cells and also process of furnishing gametes with GHSR-1a for its response to ghrelin in seminal plasma or female reproductive tract.