Gregoraszczuk, Ewa

Rak, Agnieszka

Jagiellonian Univeristy Repository

INTRODUCTIONGhrelin, an endogenous ligand of GHSR, is a 28-amino acidpeptide recognized to have a wide range of physiological roles inmany species including regulation of food intake and stimulationof growth hormone secretion (1). It is synthesized predominantlyin the stomach but has also been identified in a variety of otherorgans such as the bowels, kidney, thyroid, lung, lymphatictissue, placenta, hypothalamus, and pituitary (2).The expression of ghrelin and its receptor have beendetected in reproductive tissues in the human, rat, pig, sheep andchicken (3-7). Ghrelin mRNA expression was consistentlydetected in the rat ovary throughout the estrous cycle andpregnancy, with higher levels present in the corpus luteum andduring the first days of gestation (5). Zhang et al. (6) showed thatthe highest ghrelin mRNA expression levels in the pig ovaryoccur in the diestrous phase and the lowest in the proestrousphase. Our previously published data demonstrated that ghrelinwas secreted by porcine ovarian follicles collected from ovariesof prepubertal animals and by modification of aromatase activitystimulated estradiol secretion (8, 9). Moreover, the ERK 1/2 andPI-3 kinase pathways may be potential signals of ghrelin mediatethe cell proliferation and apoptosis of porcine ovary cells (10).There are no data showing changes of ghrelin and itsreceptor in porcine ovarian follicles collected from animalsduring prepubertal and regular estrous cycle. In this study, weexamined the gene and protein expression of ghrelin, and itsreceptor, and the concentration of both forms of ghrelin, Ac andUnAc, in different size of ovarian follicles collected fromovaries of prepubertal and cycling animals.MATERIALS AND METHODSSample collectionCrossbed gilts (Large White×Polish Landrace) ofprepubertal (5-6 months age) and cycling (7-8 months age)animals were used in the experiment. Small (3-4 mm; SF, n=5),medium (4-5 mm; MF, n=6) and large (6-7 mm; LF, n=6)follicles were obtained from porcine prepubertal ovaries. Fromcycling animals, follicles were obtained at days 4-6 (2-4 mm; SF,n=6), 10-12 (4-6 mm; MF, n=6) and 16-18 (8-12 mm; LF, n=5)of the estrous cycle. Follicular fluids were aspirated from theovarian follicles.Experimental procedureTo assay ghrelin and GHSR gene expression, ovarianfollicles were collected, immediately frozen in liquid nitrogenand stored at -70°C until RNA extraction. To determinateconcentrations of Ac and UnAc forms of ghrelin and proteinexpression of ghrelin and GHSR-1a, whole ovarian follicles werehomogenized in lysis buffer, centrifugation and the whole proteinJOURNAL OF PHYSIOLOGY AND PHARMACOLOGY 2012, 63, 2, 195-199www.jpp.krakow.plShort communicationA. RAK-MARDYLA, E.L. GREGORASZCZUKEXPRESSION OF GHRELIN AND ITS RECEPTOR IN PORCINE OVARIAN FOLLICLES COLLECTED FROM PREPUBERTAL AND ESTROUS CYCLE ANIMALSDepartment of Physiology and Toxicology of Reproduction, Chair of Animal Physiology, Institute of Zoology, Jagiellonian University, Cracow, PolandGhrelin, a hormone predominantly found in the stomach, was described as a factor that controls female reproductivefunction. Using real time PCR and western blot, we measured gene and protein expression of ghrelin and its receptor.Enzyme-like immunoassay (ELISA) was used to measure the concentration of acylated (Ac) and unacylated (UnAc)forms of ghrelin as well as levels of estradiol (E2) in follicular fluid. For all analyses, we compared small, medium andlarge ovarian follicles collected from ovaries of prepubertal and estrous cycling pigs. We demonstrated that the geneexpression levels of ghrelin significantly increased in ovarian follicles from cycling animals, with the maximumexpression in large follicles, without any change in prepubertal. However, the protein expression of ghrelin and itsconcentration was increased with increasing follicle size both in prepubertal and cycling animals and it was positivelycorrelated with E2 levels in follicular fluid. In addition, both receptor growth hormone secretagogue receptor (GHSR)and GHSR type GHSR-1a expression were significantly higher in ovarian follicles from cycling animals thanprepubertal. Results of this study suggest the possibility of local synthesis of ghrelin in the ovarian follicles and point toimportant modulatory functions for ghrelin before puberty and during the estrous cycle.K e y  w o r d s : estradiol, estrous cycle, ghrelin, growth hormone secretagogue receptor-1a, ovary, prepubertal, protein expressioncontent in lysates was measured with Bradford method. Afterhomogenization, supernatants and follicular fluid were collectedand stored at -70°C.Real-time PCR analysisWe examined ghrelin and GHSR gene expression by reversetranscription real-time PCR method as describe in our previousstudy (11). Briefly, total RNA was isolated using the High PureRNA Tissue Kit (Roche, Germany) and 1 µg of total RNA wastreated with deoxyribonuclease I and reverse transcribed usingthe transcriptor first strand cDNA Synthesis Kit (Roche,Germany). Real-time PCR analyses were performed using theStepOne real-time PCR system (Applied Biosystems). ThemRNA expression of the ghrelin and GHSR was quantifiedusing TaqMan Gene Expression Assays (Ss03392360_m1,RefSeq: NM_213807.1; and Ss03383123_s1, RefSeq:NM_214180.1 respectively, Applied Biosystems). 18S rRNAlevels were determined as an endogenous control assay(Hs99999901_s1, RefSeq: X03205.1, Applied Biosystems).Quantitative PCR was performed with 100 ng of cDNA, 1 µLgene expression assay, and 10 µL TaqMan PCR master mix(Applied Biosystems). Expression of ghrelin and GHSR mRNAwere normalized to the levels of 18S rRNA.Western blot methodSamples were subjected to SDS-page and immunoblottingusing specific antibodies against ghrelin and GHSR-1a (both1:200, Santa Cruz Biotechnology) followed by peroxidase-conjugated secondary antibodies and chemiluminescencedetection. The blots were visualised using the ChemiDocTM andquantified using the Image LabTM 2.0 Software (BioRadLaboratories).Hormone determinationThe levels of Ac, UnAc isoforms of ghrelin and E2 weredetermined using commercially available ELISA kits(Biovendor). The inter- and intra-run precision of Ac ghrelin: of8.3% and 8.1%, and of UnAc ghrelin: 3.80% and 3.20%, and ofE2: 6.72% and 2.71%, respectively.Statistical analysesStatistical analysis was performed using Statistical 6.0(StatSoft, Poland). Data were analyzed by one-way analysis ofvariance (ANOVA) followed by the Tukey honestly significantdifference (HSD) multiple range test. All data are expressed asthe mean ±S.E.M.RESULTSExpression of ghrelin receptor and ghrelin in porcine ovarianfollicles from prepubertal and estrous cycle animalsBoth mRNA and protein of ghrelin receptor weresignificantly increased in ovarian porcine follicles from cyclinganimals compared to prepubertal (p<0.05, Fig. 1).196Fig. 1. Expression of ghrelin receptor in small (SF), medium(MF) and large (LF) ovarian follicles collected from ovaries ofprepubertal and cycling animals; (A) mRNA of GHSR, (B)protein and semi-quantitative densitometry of Western blotanalysis of GHSR-1a. All data are expressed as the mean±S.E.M. Different letters indicate statistically significantdifferences among groups (p<0.05).Ghrelin mRNA are expressed in the ovarian folliclescollected from prepubertal and cycling animals (Fig. 2). Theexpression levels were consistent across all sizes of follicles inprepubertal, but significantly increase ghrelin mRNAexpression was observed in cycling animals, with the maximumexpression in large follicles (p<0.05, Fig. 2A). In bothprepubertal and cycling ovarian follicles, the protein expressionof ghrelin was significantly increased with increasing folliclesize (p<0.05, Fig. 2B).Concentrations of ghrelin in ovarian follicles from prepubertaland estrous cycling animalsIn prepubertal animals the total concentration of ghrelin washigher in ovarian follicles (68.77, 71.01 and 95.19 pg/ml for SF,MF and LF, respectively) than it was in the follicular fluid(35.48, 42.14 and 46.02 pg/ml, Fig. 3A). The concentration ofghrelin in prepubertal ovarian follicles was correlated withincreasing levels of estradiol (3.87, 7.22 and 9.58 pg/ml in SF,MF and LF, respectively; p<0.05; Fig. 3B).The total concentration of ghrelin (both forms) in ovaries ofcycling animals was higher in ovarian follicles than follicularfluid (ovarian follicles: 109.19, 130.8 and 148.36 pg/ml andfollicular fluid: 46.47, 52.34 and 53.32 pg/ml in SF, MF, and LF,respectively; Fig. 3C). As was the case for prepubertal animals,ghrelin was correlated with levels of estradiol (4.73, 7.35 and14.49 pg/ml, respectively in SF, MF and LF; Fig. 3D, p<0.05)in follicular fluid.DISCUSSIONIn the present study, we demonstrated that ovarian ghrelinmRNA expression significantly increased in cycling animals,without any change in prepubertal. However, protein expressionof ghrelin and its concentration was increased with increasingfollicle size both in ovaries of prepubertal and cycling animals.We demonstrated that both GHSR and GHSR-1a expression wassignificant higher in cycling animals than prepubertal. Thepresented study was agreement with observation by Barreiro etal. (12) who showed that the expression of the GHSR-1areceptor increases after puberty and reaches peak values inadulthood; this suggests that during pubertal development a shiftin the pattern of splicing of the GHSR gene takes place in rattestis, thereby favoring the expression of the biologically activeGHSR-1a, which might imply considerable changes in netghrelin sensitivity with sexual maturation.Puberty is characterized by increasing concentrations ofestradiol, which are driven by increasing concentrations ofpituitary gonadotrophins. Ghrelin by inhibiting GnRH and LHsecretion in the prepubertal period (13, 14), could be involved inpuberty. Our previously published data demonstrated a stimulatoryaction of ghrelin on estradiol secretion and aromatase proteinexpression with parallel antiapoptotic actions in prepubertalovarian follicles, supported a possible local action of ghrelin justbefore puberty (9). Results of the present study show that levels ofghrelin in prepubertal ovarian follicles are correlated with estradiolconcentration. Lebenthal et al. (15) demonstrated that increased197Fig. 2. Expression of ghrelin in small (SF), medium (MF)and large (LF) ovarian follicles collected from ovaries ofprepubertal and cycling animals; (A) mRNA, (B) protein andsemi-quantitative densitometry of Western blot analysis. Alldata are expressed as the mean ±S.E.M. Different lettersindicate statistically significant differences among groups(p<0.05).steroid hormone levels in prepubertal children led to a markeddecline in circulating plasma levels of ghrelin in boys, but not girls.They suggested that estradiol can be positively correlated withghrelin levels, which is in accordance with our suggestion.In cycling animals, expression of ghrelin and concentrations ofboth, Ac and UnAc, isoforms of ghrelin increased with the size ofovarian follicles. UnAc ghrelin is the major circulating form andconstitutes 80-90% of circulating total ghrelin. We suggest, as wedid for the large follicles of prepubertal animals, that the increasinglevels of ghrelin are a result of local production of ghrelin in thefollicles. However, high concentrations of UnAc ghrelin could bealso the result of the filtrate from plasma not only local synthesis.To our knowledge there are no data showing effect of UnAc ghrelinin the ovarian follicles function. The last paper by Romani et al.(16) showed that UnAc ghrelin significantly reduced progesteroneand vascular endothelial growth factor (VEGF) release in culturedhuman luteal cells. Zhang et al. (6) showed that ghrelin mRNAexpression depended on the stage of the porcine estrous cycle, withlowest expression in proestrous and maximum expression inestrous and diestrous. Similarly, ghrelin mRNA expression in themouse ovary was dependent on the estrous cycle, with the lowestexpression in proestrous and the maximum value in the diestrousphase (5). Expression of ghrelin and its receptor also increased inhypothalamus and pituitary during pregnancy (17). Moreover,ghrelin mediated circadian timing in the gastrointestinal tract (18).In summary, this study provides evidence that expressionand concentration of ghrelin increases in ovarian follicles fromprepubertal pig and follicular development in cycling animals.Results of this study suggest of local synthesis of ghrelin in theovarian follicles and points to important auto/paracrinemodulation of ghrelin in puberty and during estrous cycle.Acknowledgements: This work was supported by theDS/MND/WBiNoZ/IZ/6/2011.Conflict of interests: None declared.REFERENCES1. Horvath TL, Diano S, Sotonyi P, Heiman M, Tschop M.Minireview: ghrelin and the regulation of energy balance - a198Fig. 3. Concentrations of (A) both Ac and UnAc of ghrelin in follicular fluid and in small (SF), medium (MF) and large (LF) ovarianfollicles collected from prepubertal animals; (B) estradiol in follicular fluid from prepubertal animals; (C) both Ac and UnAc of ghrelinin follicular fluid and ovarian follicles collected from cycling animals; (D) estradiol in follicular fluid from cycling animals. All dataare expressed as the mean ±S.E.M. Different letters indicate statistically significant differences among groups (p<0.05).hypothalamic perspective. Endocrinology 2001; 142: 4163-4169.2. van der Lely AJ, Tschop M, Heiman ML, Ghigo E.Biological, physiological, pathophysiological, andpharmacological aspects of ghrelin. Endocr Rev 2004; 25:426-457.3. Gaytan F, Barreiro ML, Chopin LK, et al.Immunolocalization of ghrelin and its functional receptor, thetype 1a growth hormone secretagogue receptor, in the cyclichuman ovary. J Clin Endocrinol Metab 2003; 88: 879-887.4. Du C, Xilingaowa CG, Wang C, Li H, Zhao Y, SiqingaowaCJ. Expression of the orexigenic peptide ghrelin in the sheepovary. Domest Anim Endocrinol 2009; 36: 89-98.5. Caminos JE, Tena-Sempere M, Gaytan F, et al. Expressionof ghrelin in the cyclic and pregnant rat ovary.Endocrinology 2003; 144: 1594-15602.6. Zhang W, Lei Z, Su J, Chen S. Expression of ghrelin in theporcine hypothalamo-pituitary-ovary axis during the estrouscycle. Anim Reprod Sci 2008; 109: 356-367.7. Rak A, Szczepankiewicz D, Gregoraszczuk EL. Expressionof ghrelin receptor, GHSR-1a, and its functional role in theporcine ovarian follicles. Growth Horm IGF Res 2009; 19:68-76.8. Rak A, Gregoraszczuk EL. Ghrelin levels in prepubertal pigovarian follicles. Acta Vet Hung 2009; 57: 109-113.9. Rak A, Gregoraszczuk EL. Modulatory effect of ghrelin inprepubertal porcine ovarian follicles. J Physiol Pharmacol2008; 59: 781-793.10. Rak-Mardyla A, Gregoraszczuk E. ERK 1/2 and PI-3 kinasepathways as a potential mechanism of ghrelin action on cellproliferation and apoptosis in the porcine ovarian follicularcells. J Physiol Pharmacol 2010; 61: 451-458.11. Rak-Mardyla A, Gregoraszczuk E, Karpeta A, Duda M.Expression of ghrelin and its receptor in porcine corpusluteum development and the inhibitory effects of ghrelin onprogesterone secretion, 3-beta-hydroxysteroid dehydrogenase(3βHSD) activity and protein expression. Theriogenology2012; 77: 1505-1512.12. Barreiro ML, Suominen JS, Gaytan F, et al. Developmental,stage-specific, and hormonally regulated expression ofgrowth hormone secretagogue receptor messenger RNA inrat testis. Biol Reprod 2003; 68: 1631-1640.13. Lebrethon MC, Aganina A, Fournier M, Gerard A, ParentAS, Bourguignon JP. Effects of in vivo and in vitroadministration of ghrelin, leptin and neuropeptide mediatorson pulsatile gonadotrophin-releasing hormone secretionfrom male rat hypothalamus before and after puberty. 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Gut clock:implication of circadian rhythms in the gastrointestinal tract.J Physiol Pharmacol 2011; 62: 139-150.R e c e i v e d : February 20, 2012A c c e p t e d : April 6, 2012Autor's address: Dr. Agnieszka Rak-Mardyla, Department ofPhysiology and Toxicology of Reproduction, Chair of AnimalPhysiology, Institute of Zoology, Jagiellonian University, 9Gronostajowa Street, 30-387 Cracow, Poland; E-mail: agnieszka.rak@uj.edu.pl199

Expression of ghrelin and its receptor in porcine ovarian follicles collected from prepubertal and estrous cycle animals

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