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

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

reproductive biology 12 (2012) 293–300 Available online at www.sciencedirect.com journal homepage: http://www.elsevier.com/locate/repbio Original R...

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reproductive biology 12 (2012) 293–300

Available online at www.sciencedirect.com

journal homepage: http://www.elsevier.com/locate/repbio

Original Research Article

Expression of ghrelin receptor (GHSR-1a) in rat epididymal spermatozoa and the effects of its activation Andrzej Łukaszyk a,*, Małgorzata Kotwicka b, Anna Jankowska b, Aldona Kasprzak a, Marcin Rucin´ski a, Karolina Sterzyn´ska a, Agnieszka Zio´łkowska a, Piotr Sawin´ski a, Marek Ruchala c a

Department of Histology and Embryology, Poznan´ University of Medical Sciences, Poznan´, Poland Department of Cell Biology, Poznan´ University of Medical Sciences, Poznan´, Poland c Department of Endocrinology, Metabolism and Internal Medicine, Poznan´ University of Medical Sciences, Poznan´, Poland b

article info

abstract

Article history:

In this study we demonstrated the expression of the ghrelin receptor GHSR-1a in rat sperma-

Received 6 November 2011

tids and epididymal spermatozoa, as well as some effects of ghrelin on the spermatozoa

Accepted 15 March 2012

in vitro. For the demonstration of GHSR-1a the immunocytochemical, immunofluorescence and Western blotting techniques were applied using three different types of antibodies. The

Keywords:

response of spermatozoa to ghrelin was tested in a series of in vitro experiments and their

Rats

effects were evaluated using confocal microscopy and flow cytometry. GHSR-1a protein was

Epididymal spermatozoa

found as expressed in the Golgi and acrosomes of spermatids and acrosome regions or the

GHSR-1a

head cell membrane of epididymal spermatozoa. The GHSR-1a expression in spermatozoa

Calcium ions

was also confirmed by Western blot. No differences were found in percentage of spermatozoa

Sperm motility

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 ghrelin increased, while 10

4

6

mol/L

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. # 2012 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

1.

Introduction

Ghrelin and the functionally antagonistic leptin have been recognized as signaling molecules that are involved in the

mechanisms controlling both energy balance and reproduction by acting on various levels of neuroendocrine axis and within the testis [1–3]. Testicular expression of the peptides, their functional receptors and their transcripts were demonstrated in rodents and humans, and some essential data

* Corresponding author at: Department of Histology and Embryology, University of Medical Sciences, S´wie˛cickiego Street 6, 60-781 Poznan´, Poland. E-mail address: [email protected] (A. Łukaszyk). 1642-431X/$ – see front matter # 2012 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved. http://dx.doi.org/10.1016/j.repbio.2012.09.002

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reproductive biology 12 (2012) 293–300

concerning their role in regulatory mechanisms in testis was evaluated [4–12]. When leptin was found to be indispensable for testicular physiology and male fertility, the role of ghrelin in this respect appeared explicitly hard to elucidate. Mice lacking leptin or having mutations within the leptin receptor are infertile and exhibit impaired spermatogenesis, while a lack of ghrelin does not affect male fertility [13–16]. However, some effects of increased ghrelin level have been observed in the course of experimental hyperghrelinemia in rats, or as a consequence of ghrelin gene overexpression in Prader–Willi syndrome in humans [17,18]. An impairment of LH and testosterone secretion, delayed pubertal timing and local defects in spermatogenic cell line maturation were observed in both these cases. Both ghrelin and leptin, as well as their receptors were demonstrated locally in Leydig, Sertoli and spermatogenic cells. The expression of ghrelin was ascribed to Leydig and Sertoli cells [2,3,10]. In human, ovine and rat testes, ghrelin was also demonstrated immunohistochemically in gametogenic cells [8,19,20], and expression of the growth hormone secretagogue receptor type 1a (GHSR-1a) in rat gametogenic cells was documented up to the step of spermiation [19]. The knowledge concerning the expression of the ghrelin receptor in spermatozoa is obscure but expression of the leptin receptor was confirmed in the ejaculated spermatozoa of humans and boars. In humans, the immunoreactivity of the leptin receptor was localized in the sperm tail [21,22]. No correlation was found between concentration of leptin in seminal plasma and sperm motility. In vitro recombinant human leptin failed to alter the parameters of sperm motility as well as the percentage of capacitated and acrosome-reacted spermatozoa [22]. Interestingly, leptin receptor expression in boar spermatozoa was localized in the acrosomal region [23,24] and its activation by leptin resulted in an enhancement of sperm capacitation and motility, stimulation of acrosin and phosphorylation of the BCL2 protein [24]. The presence and role of the ghrelin receptor in spermatozoa as well as its species specificity have been poorly recognized. If ghrelin exerts an action concurrent to leptin, then this justifies searching for the expression and possible functional role of GHSR-1a in rat spermatozoa. In a previous paper, we demonstrated immunohistochemically the expression 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.

were approved by the Local Ethical Commission for Experimentation on Animals. The male rats were sacrificed by decapitation. At autopsy the testes were fixed in Bouin’s fluid or 4% (v/v) paraformaldehyde and embedded in paraffin. The epididymides were secured for isolation of spermatozoa. The paraffin sections of testes were used for immunocytochemical demonstration of GHSR-1a receptor in seminiferous epithelium. The epididymal spermatozoa were isolated and treated carefully to minimize stress effect on the integrity of plasma membranes [25]. Samples of epididymal sperm were washed twice in PBS and centrifuged at 600  g for 10 min. The pellet was then resuspended and used for: 1/preparation of smears on SuperFrost/Plus microscope slides for demonstration of GHSR-1a expression by immunohistochemistry and immunofluorescence, 2/Western blotting, and 3/in vitro experiments exploring possible effects of the receptor activation by ghrelin.

2.

Materials and methods

2.3.

2.1.

Animals and collection of tissues

The resuspended pellet was homogenized in RIPA buffer and centrifuged at 600  g for 30 min at 4 8C to remove debris. Protein concentrations were determined by Lowry method. Rat hypothalamus was used as positive control. Samples of protein (25 mg) were loaded into each lane, separated on a 16% (w/v) SDS-PAGE gel, and transferred onto PVDF membrane

The animals used in this study were adult, 90-day-old, male Wistar rats kept under standard conditions of temperature (21  2 8C) and lighting (14L/10D) with free access to food pellets and water. The protocol of the study and procedures

2.2.

Immunohistochemistry/immunofluorescence

The 5 mm thick testis sections mounted on SuperFrost/Plus microscope slides were deparaffinized. The sections were preincubated with 1% (v/v) H2O2 to inhibit endogenous peroxidase, microwaved and treated with primary antibody overnight at 4 8C. Two different rabbit anti-GHSR-1a polyclonal antibodies (Phoenix Pharmaceuticals, CA, USA, and Millipore Co., Bedford, MA, USA) were used. After washing in phosphate-buffered saline (PBS; 3  3 min) the sections were incubated with the secondary biotinylated anti-rabbit IgG (20 min) and with the streptavidin-biotin-peroxidase complex (LSAB2; 20 min), and subjected to classical ABC technique [26]. The color reaction was evoked with 0.05% (w/v) diaminobenzidine dissolved in 0.05 mol/L Tris-HCl buffer, pH 7.6, supplemented with 0.001% (v/v) H2O2. Finally, the slides were counterstained with hematoxylin. Positive reaction manifested, in at least three sequential sections, as a dark brown or black precipitate in the cells. For negative control the IgG or complete rabbit serum were used instead of antibodies. The smears of epididymal spermatozoa were fixed in 4% (w/v) formaldehyde and frozen. After thawing, the smears were washed 3 times in PBS, microwaved (15 min), washed again in PBS, blocked with H2O2 and serum, and then incubated with anti-GHSR-1a antibodies (Phoenix Pharmaceuticals, CA, USA, or Millipore Co., Bedford, MA, USA) overnight at 4 8C. The antigen–antibody complexes were revealed according to the procedure described above for testis sections. Smears used for immunofluorescence were incubated overnight with the primary antibody, followed by treatment with the fluorescein isothiocyanate (FITC) labeled secondary antibody (for 1 h in darkness at room temperature) and mounted in diamidino-2-phenylindole (DAPI) containing mounting medium.

Western blotting

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295

(Millipore Co., MA, USA). Membranes were cut to obtain three parts, each containing two protein samples from sperm cells and hypothalamus. Non-specific binding was blocked by immersing the membranes in 5% (w/v) BSA. The membranes were then treated separately: one was incubated with antiGHSR-1a goat polyclonal antibody (Santa Cruz Biotechnology, Inc., CA, USA) at 1:500 dilution, the second was incubated with Anti-Ghrelin Receptor Type 1a (Millipore Co., MA, USA) at 1:500 dilution, and the third was incubated with anti-GHSR-1a antibody (Phoenix Pharmaceuticals, CA, USA) at 1:50 dilution. After washing with TBST (10 mmol/L Tris, pH 8.0, 150 mmol/L NaCl, 0.05% (v/v) Tween-20) for 3  10 min, the first membrane was further incubated with horseradish peroxidase-conjugated anti-goat IgG (Sigma, St. Louis, MO, USA) at a 1:10000 dilution. The second and third pieces of membrane were treated with horseradish peroxidase-conjugated anti-rabbit IgG (Sigma, St. Louis, MO, USA) at a 1:5000 dilution for 1 h at room temperature. The membranes were washed for 3  10 min with TBST and signals were detected by ECL (Amersham, Life Science, Little Chalfont, Bucks, UK).

spermatozoa were first immobilized in 1% (w/v) agarose and then treated with 10 6 or 10 9 mol/L of ghrelin. Forty-five images were collected (each 10 s) and used to study the kinetics of calcium ion changes. Moreover, the sperm loaded with Fluo-3 (as described above) and treated for 20 min with 10 6 or 10 9 mol/L of ghrelin were analyzed using flow cytometry. To distinguish dead from intact cells, the sperm were stained with propidium iodide [50 mg/mL]. Spermatozoa not treated with ghrelin were used as a control.

2.4.

A sperm suspension (1  106 cells/mL) was incubated with 10 6 or 10 9 mol/L ghrelin for 2 h at 37 8C, and phosphatidylserine translocation (PST) was evaluated using Annexin V (An-V) conjugated with fluorescein isothiocyanate (AnV-V-FITC; Ex/Em = 494/518 nm; Roche, Germany) according to the manufacturer’s protocol. The cells were additionally stained with propidium iodide (PI; 50 mg/mL; Ex/Em = 535/617 nm; Sigma–Aldrich, MO, USA) to assess the viability of the spermatozoa under study. The analysis was performed with a confocal microscope (LSM 510, Zeiss, Germany) and flow cytometry (FACSCalibur, Becton-Dickinson, NJ, USA).

The fluorescence signals of labeled spermatozoa were analyzed by flow cytometry (FACSCalibur, Becton-Dickinson, NJ, USA). For each experiment 10,000 spermatozoa were examined. The sperm population was gated with 908 and forwardangle light scatter to exclude debris and aggregates. The green fluorescence of both Annexin-V-FITC, FITC-DEVD-FMK and Fluo-3 was indicated by an argon laser (488 nm) and measured in the FL1 channel (515–545 nm). The red fluorescence of propidium iodide was excited by the same argon laser and was detected in FL3 channel (>650 nm). All data were collected and examined using CellQuest Pro software, version 5.2.1 (BectonDickinson, NJ, USA). For each experiment the density dot plots were drawn.

2.5.

2.9.

Effect of ghrelin on phosphatidylserine translocation

Effect of ghrelin on caspase-3 activity

The spermatozoa (1  106 cells/mL) were incubated with 10 6 or 10 9 mol/L ghrelin for 2 h at 37 8C and then the percentage of the spermatozoa with active caspase-3 was calculated according to the fluorescence of FITC-conjugated caspase-3 inhibitor (FITC-DEVD-FMK; Ex/Em = 485/535 nm; Calbiochem, Germany). The procedure was performed according to the manufacturer’s protocol. To estimate the number of dead cells, after 35 min of incubation with FITC-DEVD-FMK, propidium iodide [50 mg/mL] was added and incubation was continued for the next 10 min. Spermatozoa were analyzed by confocal microscopy (LSM 510, Zeiss, Germany) and by flow cytometry (FACS Calibur, Becton-Dickinson, NJ, USA).

2.6. Effect of ghrelin on intracellular free calcium ion concentration Fluo-3 (Molecular Probes, OR, USA; Ex/Em = 488/526 nm) was used to study changes in the free calcium ion level in sperm cells. Fluo-3 fluorescence intensity is directly calcium dependent, so it expresses intracellular calcium ions concentration. The cells (1  105 sperm cells/mL) were incubated with 4 mmol/L Fluo-3 for 45 min at 37 8C according to the manufacturer’s protocol. For confocal microscope analysis the

2.7.

Effects of ghrelin on sperm motility

Spermatozoa (1  105 sperm cells/mL) suspended in Hams F-10 medium were treated with ghrelin (10 9, 10 6 or 10 4 mol/ L) for 2 h at 37 8C and then the percentage of cells displaying progressive movement was calculated for each treatment. The ghrelin data were compared to that of untreated cells. The minimum number of analyzed cells was 200.

2.8.

Flow cytometry

Statistical analysis

The numerical data are presented as mean  SD. All the results were analyzed statistically using the Kruskal–Wallis test with the Dunn post hoc test. The level of significance was set at p < 0.05.

3.

Results

3.1. Expression of GHSR-1a in the rat seminiferous epithelium and epididymal spermatozoa Immunohistochemical expression of ghrelin receptor in the rat seminiferous epithelium was demonstrated with Millipore polyclonal antibodies (Fig. 1). With this type of antibody the immunostaining marks probably an isoform of GHSR-1a selectively present in Golgi region and acrosomes of spermatids of all steps, including the step of spermiation. In stage VIII of the germinal epithelium cycle, the staining labels the acrosomes of step 8 round spermatids and step 19 spermatids released to the tubule’s lumen (Fig. 1A). In stage VIII/IX of the cycle only acrosomes of step 8 spermatids are labeled (Fig. 1A and B). In stage XII/XIII of the cycle, the staining labels the heads of maturing spermatids, and in stage IV the heads of

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Fig. 1 – Ghrelin receptor (GHSR-1a) expression in rat testis demonstrated immunohistochemically using anti-GHSR-1a polyclonal antibodies (Millipore) and the classic ABC technique. The positive staining marks succeeding steps of acrosome formation in spermatids and heads of spermatids undergoing spermiation: (A) spermatids of 7, 8 and 19 steps of spermiogenesis in stage VIII of the germinal epithelium cycle (right upper corner), stage VIII/IX (left tubule) and stage VII (right lower corner), (B) acrosomes of step 8 spermatids in stage VIII/IX of cycle, (C) proacrosomes of step 4 spermatids and heads of step 17 spermatids in stage IV of the cycle (right upper corner) and heads of maturing spermatids in stage XII/XIII (left tubule). Counterstained with hematoxylin.

maturing spermatids and proacrosomes of step 4 spermatids are labeled (Fig. 1C). Using immunohistochemistry (Fig. 2A–C) and immunofluorescence (Fig. 2D–F), GHSR-1a expression in acrosomal and postacrosomal regions was demonstrated in the rat spermatozoa isolated from epididymis. The positive immunohistochemical reaction at the level of spermatozoa was documented with both antibody types (Fig. 2A and B). The immunofluorescence staining emphasized the membrane profile at both the convex and concave surface of the heads (Fig. 2E and F). Western blot analysis, irrespective of the anti-GHSR-1a protein antibodies used revealed a 41 kDa band corresponding

to GHSR-1a in the protein extract from rat epididymal spermatozoa. Fig. 3A–C demonstrates the sperm GHSR-1a (lane 2) in comparison to the control, i.e. the GHSR-1a extracted from rat hypothalamus (lane 1).

3.2. Effects of ghrelin on viability and apoptosis of epididymal sperm Flow cytometry and confocal microscopy in control sperm (not exposed to ghrelin) identified the following four sperm subpopulations: (1) viable cells without phosphatidylserine translocation (PST) – An-V /PI , (2) viable cells

Fig. 2 – Expression of GHSR-1a in the heads of rat epididymal spermatozoa demonstrated with immunohistochemical and immunofluorescence techniques. Phoenix Pharmaceuticals (A) and Millipore (B) antibodies or complete rabbit serum as negative control (C) was applied in the classic ABC technique. DAPI (D) and Millipore antibodies conjugated with fluorescein-labeled secondary antibody (E) were used in the immunofluorescence experiment; (F) overlapping images of DAPI and fluorescein.

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297

3.3. Effect of ghrelin on intracellular free calcium ion concentration Fig. 3 – The ghrelin receptor (GHSR-1a) protein expression determined by Western blot in epididymal spermatozoa of adult male rats. Different anti-GHSR-1a goat polyclonal antibodies from Santa Cruz Biotechnology (A), Millipore (B) and Phoenix Pharmaceuticals (C) were applied in this experiment. Lane 1: hypothalamus (positive controls), lane 2: sperm cells.

with PST – An-V+/PI , (3) dead cells without PST – An-V /PI+, and (4) dead cells with PST – An-V+/PI+. Phosphatidylserine translocation in viable spermatozoa was observed mostly within the middle piece. In dead cells, both the middle piece and the acrosome region showed the presence of phosphatidylserine in the membrane outer layer. Ghrelin did not influence phosphatidylserine translocation. None of the analyzed ghrelin concentrations altered ( p > 0.05) the number of spermatozoa expressing PST (data not shown). The presence of the active form of caspase-3 was observed in the spermatozoa and a number of spermatozoa with active caspase-3 showed a progressive movement. The highest expression of the enzyme was detected within the middle piece. Ghrelin did not change the number of spermatozoa exhibiting caspase-3 activity (data not shown).

In most intact spermatozoa, the highest concentration of intracellular free calcium ions was observed within the middle piece (arrow at the Fig. 4A; analysis time 0 s). For these spermatozoa, 10 6 mol/L ghrelin caused a rapid increase (observed for a few seconds after the stimulation) of calcium ions (Fig. 4B), initiated within the head (arrow at the Fig. 4A; analysis time 55 s) and then moved into the distal part of the sperm tail (arrow at the Fig. 4A; analysis time 418 s). In cells showing an initially high level of intracellular free calcium ion concentration within the head (arrowhead at the Fig. 4A; analysis time 0 s), ghrelin increased the accumulation of calcium ions within the middle piece and distal part of tail (arrowhead at the Fig. 4A; analysis time 55–418 s). Lower dose of ghrelin induced similar changes; however, the kinetics of the process was slower (Fig. 4C) than that of higher ghrelin dose (Fig. 4B). Spermatozoa subjected to 20 min incubation with 10 6 mol/L ghrelin manifested higher calcium ion levels compared to controls (Fig. 4D). Spermatozoa incubated with 10 9 mol/L ghrelin for the same period of time showed no significant differences in calcium level compared to controls (data not shown).

3.4.

Effect of ghrelin on sperm motility

The initial percentage of sperm cells in progressive movement was 20.2  3.9%. After 2 h of incubation, the percentage of cells

Fig. 4 – The effects of ghrelin on free calcium ion concentration in rat spermatozoa. (A) Topography of calcium ion concentration changes in single spermatozoa in a given time period after stimulation with ghrelin (10S6 mol/L). Arrows and arrowheads indicate the changes in the respective spermatozoa over time. (B) Free calcium ion concentration kinetics assessed for 2 selected spermatozoa stimulated with ghrelin (G; 10S6 mol/L). (C) Free calcium ion concentration kinetics assessed for 4 selected spermatozoa stimulated with ghrelin (G; 10S9 mol/L). (D) Flow cytometry analysis of calcium ion concentration in sperm cells after 20 min incubation with ghrelin (10S6 mol/L). The OX axis presents the values of fluorescence intensity of Fluo-3 which expressed the calcium ion concentration; FL1-H: spectrum of fluorescent channel, 515–545 nm.

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Percentage of spermatozoa with progresive movement

[%] 50

b Mean Mean ±SD Mean ±1.96*SD

45

ab 40

ab

35 30

a

25

c 20 15 10 5 0

C

C2h

G10 -9

G10 -6

G10 -4

Fig. 5 – The effects of ghrelin on the progressive movement of spermatozoa. Bars with different superscripts are significantly different ( p < 0.01). C: control, C 2h: control after 2 h, G10S9: after 2 h of incubation with ghrelin (10S9 mol/L), G10S6: after 2 h of incubation with ghrelin (10S6 mol/L), G10S4: after 2 h of incubation with ghrelin (10S4 mol/L), SD: standard deviation.

in progressive movement was insignificantly higher ( p > 0.05; 25.9  6.6%). Ghrelin at dose 10 9 mol/L did not affect ( p > 0.5) the examined parameter (28.1  6.6%), but at dose 10 6 mol/L significantly ( p < 0.01) increased the percentage of spermatozoa exhibiting progressive movement (20.2  3.9% vs. 32.1  7.9%; Fig. 5). In contrast, ghrelin at dose 10 4 mol/L significantly ( p < 0.01) decreased the percentage of sperm cells in progressive movement in comparison to 2-h control and all other ghrelin concentrations.

4.

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 GHSR1a 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 1,4,5triphosphate 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 6 mol/L, affected rat epididymal spermatozoa. At these concentrations, especially at a concentration of 10 6 mol/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 100–1000 pg/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 6 mol/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 20 min, 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 0.1 mmol/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. The presented results speak for the direct effects of ghrelin on rat spermatozoa, which are accomplished by the activation of the GHSR-1a receptor. Thus, the results seem to be of physiological significance in vivo since ghrelin, the natural ligand of GHSR-1a, was found to be present in the seminal plasma, at least in humans [32]. However, the indirect effects of ghrelin on sperm function should also be mentioned since a report was published on the improvement of the viability of rat spermatozoa in vitro as a result of ghrelin administration to rats in vivo [33]. The authors treated 45-day-old rats with 1 nmol of ghrelin daily for 10 days and found both increased spermatozoa progressive movement values and increased membrane integrity which could be observed up to 40 days after the last injection. However, because ghrelin possesses

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multiple functions, these results may be a consequence of its action at different levels of the neuro-endocrine axis which controls reproductive functions in rats. In discussing the dose-dependence of ghrelin on rat spermatozoa in vitro, the problem of cell response to the increasing concentration of the peptide present in the incubation medium should be taken into account. In this paper we report a reduction in the percentage of the spermatozoa progressive movement when the concentration of ghrelin in the medium was increased to 10 4 mol/L. This effect may be caused by the down-regulation of the ghrelin receptor in spermatozoa. The in vivo effects of experimental hyperghrelinemia have been demonstrated in prepubertal male rats, showing a decrease in serum LH and testosterone levels and delayed timing of puberty [18]. Hyperghrelinemia associated with impairment of the neuroendocrine axis and gonadal function has also been observed in humans with Prader–Willi syndrome [17,34]. In conclusion, in this study we reported the expression of the ghrelin receptor in rat spermatozoa and in vitro response of the spermatozoa to ghrelin. The receptor function was dependent on ligand concentration, and included protection of sperm viability and increase in sperm motility. These phenomena occurred optimally at 10 6 mol/L of ghrelin concentrations. Higher concentration of ghrelin (10 4 mol/L) had an opposite effect on sperm motility. Thus, our report includes spermatozoa as a site of ghrelin signal reception in the male reproductive system, at least in rats, and raises the question of whether this system also exists in the male gametes of other species, including humans.

Acknowledgment This research was supported by grant number 3951/P01/ 2006/31 from the Ministry of Science and Higher Education, Poland.

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