Metabolic Status and Ghrelin Regulate Plasma Levels and Release of Ovarian Hormones in Layer Chicks

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1 Physiol. Res. 66: 85-92, 2017 Metolic Sttus nd Regulte Plsm Levels nd Relese of Ovrin Hormones in Lyer Chicks A. V. SIROTKIN 1,2,3, A. H. HARRATH 3, R. GROSSMANN 4 1 Deprtment Zoology nd Anthropology, Constntine the Philosopher University, Nitr, Slovki, 2 Deprtment Genetics nd Reproduction, Reserch Institute of Animl Production, Lužinky, Slovki, 3 Zoology Deprtment, College of Science, King Sud University, Riydh, Sudi Ari, 4 Deprtment Functionl Genomics & Bioregultion, Friedrich Loeffler Institute, Mriensee, Neustdt, Germny Received Jnury 26, 2016 Accepted My 20, 2016 On-line Octoer 26, 2016 Summry The im of the present study ws to exmine the role of nutritionl sttus, the metolic hormone ghrelin nd their interreltionships in the control of chicken hormones involved in the regultion of reproduction. For this purpose, we identified the effect of food deprivtion, dministrtion of ghrelin 1-18 nd their comintion on plsm levels of testosterone (T), estrdiol (E), rginine-vsotocin (AVT) nd growth hormone (GH) s well s the relese of these hormones y isolted nd cultured ovrin frgments. It ws oserved tht food deprivtion reduces plsm T nd E nd increses plsm AVT nd GH levels. Food restriction lso reduced the mount of E produced y isolted ovries, ut it did not ffect the ovrin secretion of T nd AVT. No ovrin GH secretion ws detected. dministered to d liitum fed chickens did not ffect plsm T nd E levels, ut it did increse plsm GH nd AVT concentrtions. Moreover, it prtilly prevented the effect of food deprivtion on plsm E nd AVT levels, ut not on T or GH levels. dministrtion to control irds promoted ovrin T, ut not E or AVT relese nd reduced T nd no other hormonl outputs in irds sujected to food restriction. Our results (1) confirmed the ovrin origin of the min plsm T nd E nd the extr-ovrin origin of the min lood AVT nd GH; (2) showed tht food deprivtion-induced suppression of reproduction my e cused y suppression of T nd E nd the promotion of AVT nd GH relese; (3) suggest the involvement of ghrelin in control chicken E, AVT nd GH output; nd (4) indictes tht ghrelin cn either mimic or modify the effect of the intke of low clories on chicken plsm nd ovrin hormones, i.e. it cn medite the effect of metolic stte on hormones involved in the control of reproduction. Key words Ovry Testosterone Estrdiol GH Argininevsotocin Corresponding uthor A. V. Sirotkin, Deprtment Zoology nd Anthropology, Constntine the Philosopher University, Nitr, Slovki. Fx: E-mil: sirotkin@ukf.sk Introduction Reproduction is n energy-demnding process; therefore, it should e synchronized with optiml nutritionl conditions. It hs een postulted tht the hypotheticl meditor of the nutritionl effect on reproduction (1) should e ffected y nutrition nd (2) controls reproduction, nd (3) its chnges should mimic nd modify the effect of nutrition on reproduction. On the sis of these criteri, it is proposed tht the nutritionl sttus ffects mmmlin reproductive processes vi the metolic hormones, leptin, ghrelin nd oesttin (Ten- Sempere 2008, Nvrro nd Kiser 2013, Ro nd Ten- Sempere 2014, Sirotkin 2014). In irds, mlnutrition inhiits ovrin folliculogenesis, ovultion/egg lying nd ovrin hormones relese (Hocking 2004, Sirotkin nd Grossmnn 2015) most likely vi the induction of PHYSIOLOGICAL RESEARCH ISSN (print) ISSN (online) 2017 Institute of Physiology of the Czech Acdemy of Sciences, Prgue, Czech Repulic Fx , e-mil: physres@gfu.cs.cz,

2 86 Sirotkin et l. Vol. 66 ovrin folliculr cells poptosis (Pczosk-Elisiewicz et l. 2003). Mlnutrition is ssocited with chnges in plsm nd rin ghrelin s well s its receptors (Kiy et l. 2007, 2013, Sirotkin et l. 2013). i.m. dministrtion in chicken is le to reduce plsm progesterone (P) levels (Sirotkin et l. 2015), which directly lters prolifertion, poptosis, steroidogenesis nd protein kinses in cultured ovrin cells (Sirotkin et l. 2006, Sirotkin nd Grossmnn, 2007, 2008), nd prevents the food restriction-induced decrese in ovrin testosterone (T), estrdiol (E) nd rginine-vsotocin (AVT) relese (Sirotkin et l. 2015). Such dt hve demonstrted the importnce of ghrelin in integrting nutrition nd reproduction nd its potentil pplicility for the improvement of frm vin reproduction. Nevertheless, the meditory role of ghrelin in the metolic control of ovrin functions hs only een previously demonstrted in one study (Sirotkin et l. 2015). Moreover, in the descried study, ghrelin nd food restriction effects only on hormonl relese y ovrin tissue in vitro, ut not on plsm hormones under in vivo conditions, were exmined. The generl im of the present study ws to exmine the role of the nutritionl sttus, metolic hormone ghrelin nd their interreltionships in the control of chicken steroid nd peptide hormones involved in the regultion of reproduction. For this purpose, we used oth in-vivo nd in-vitro pproches to identify the effect of food deprivtion, dministrtion of ghrelin 1-18 nd their comintion on plsm level of hormones (T, E, AVT nd growth hormone, GH) whose re known utocrine/prcrine nd endocrine regultors of oth mmmlin nd vin ovrin functions (Sirotkin 2005, 2014, Lun et l. 2014, Hri 2015) s well s the relese of these hormones y isolted nd cultured ovrin frgments. Mterils nd Methods Animl experiments, tissue collection nd culture Young (pproximtely 8 months of ge) White Leghorn hens (LSL), weight kg with n egg-lying rte of more thn 95 %, were housed in individul cges under stndrd conditions t the Experimentl Sttion of the Institute of Animl Science on photoperiod 12L:12D (illumintion ). The conditions of their cre, mnipultions nd use corresponded the instructions of the EC no. 178/2002 nd relted EC documents, nd the protocols were pproved y the locl ethics commission. After two-month dpttion period to the conditions of the experimentl frms, the hens were divided into four experimentl groups: (1) the control group ws fed d liitum, no hormone tretment; (2) the group fed d liitum nd treted with i.m. injection of humn recominnt reserch grde ghrelin 1-18 (Peptides Interntionl Inc., Louisville, Kentucky, USA) (this truncted ghrelin nlogue ghrelin 1-18 mimicked the effect of full-length ghrelin 1-28 on chicken ovrin cells (Sirotkin nd Grossmnn 2008); (3) the group sujected to food deprivtion, no hormone tretment; nd (4) the group sujected to food deprivtion nd treted with i.m. injection of humn recominnt ghrelin The nimls of the food-deprivtion groups hd no ccess to food during the entire experiment for 72 h, wheres ll of the nimls hd permnent ccess to drinking wter. Hormonl tretments comined with food deprivtion egn together with food restriction. ws dissolved in sterile 0.7 % NCl immeditely prior to the strt of the experiments nd injected i.m. t doses 30 µg/niml in 1 ml of 0.7 % NCl. This consecutive injection ws done for 3 dys, every h, in the dytime (t 8.00 nd 18.00). This dose, injection nd smpling time (detils re elow) were comprle to the mount of hormones in the chicken orgnism nd those treted with previously reported experiments (Kiy et l. 2007, Sirotkin et l. 2013, Sirotkin nd Grossmnn 2015). Next, 1.5 h fter the lst injection (etween 9.00 nd m.), the nimls were killed y decpittion. Their lood ws collected in heprinized tues, nd the plsm ws seprted y 10 min centrifugtion t 500 g nd frozen t -70 C until rdioimmunossy (RIA) or enzyme immunossy (EIA). The lrgest (F1) follicles were isolted from the ovry. The stge of folliculogenesis ws determined y recording the time of the lst oviposition nd y the size nd position of the next ovrin follicle. Frgments of the folliculr wll (5 mm in dimeter, weight 24±8 mg) were isolted s previously descried (Sirotkin nd Grossmnn 2003, 2006). After wshing three times in sterile culture medium (DMEM/F-12 1:1 mixture supplemented with 10 % ovine fetl serum nd 1 % ntiiotic-ntimycotic solution (ll from Sigm, St. Louis, USA), these frgments were cultured without tretment for 2 dys in 2 ml culture medium in Flcon 24-well pltes (Becton Dickinson, Lincoln Prk, USA) t 38.5 C under 5 % CO 2 in humidified ir. This

3 2017, Feeding nd Hormones in Chicken 87 protocol yields the mximl ccumultion of ovrin hormones in the culture medium, which is the most relile chrcteristic of ovrin secretory ctivity (Sirotkin nd Grossmnn 2003). Immunossy Concentrtions of testosterone (T), estrdiol (E), rginine-vsotocin (AVT) nd growth hormone (GH) were determined in 25 µl liquots of plsm or incution medium y EIA nd RIA, whose were previously vlidted for use in culture medium (Sirotkin et l. 2006, Sirotkin nd Grossmnn 2007). These hormones were considered s the indices of ovrin secretory ctivity, stress, response to hormonl stimuli nd the key regultors of oth mmmlin (Sirotkin 2014) nd chicken (Sirotkin nd Grossmnn 2006, 2007, 2008, Lun et l. 2014, Sirotkin 2014, Hri 2015) ovrin functions. T ws ssyed ccording to Münster (1989) using ntiser ginst steroids (produced in the Institute of Animl Science, Neustdt, Germny). The sensitivity of the ssy ws 10 pg/ml. The cross-rectivity of the T ntiserum ws 96 % to dihydrotestosterone, 3 % to ndrostenedione, 0.01 % to P 4 nd E 2, 0.02 % to cortisol nd <0.001 % to corticosterone. The inter- nd intrssy coefficients of vrition were 12.3 % nd 6.8 %, respectively. E concentrtions were evluted ccording to Münster (1989) using ntiser ginst steroids (produced y the Institute of Animl Science, Neustdt, Germny) with n ssy sensitivity of 5 pg/ml. The cross-rectivity of the E 2 ntiserum ws <2 % to estrone, 0.3 % to estriol, % to T nd < % to P 4 nd cortisol. The inter- nd intrssy coefficients of vrition did not exceed 16.6 % nd 11.7 %, respectively. AVT ws determined using RIA ccording to Gry nd Simon (1983). The nti-avt ntiserum ws kindly provided y Dr. D.A. Gry (Mx-Plnk Institute for Physiologicl nd Clinicl Reserch, Bd Nuheim, Germny), which cross-rected 1.0 % with mesotocin nd ngiotensin II. The sensitivity of the RIA ws 0.3 pg/ml. The inter- nd intrssy coefficients of vrition did not exceed 8.8 % nd 7.2 %, respectively. GH ws mesured using the EIA sed on the EIA used for porcine GH (Serpek et l. 1993) nd dpted for determintion of chicken GH (Zheng et l. 2007). Chicken GH for stndrds (AFP-9020C), iodintion (AFP 7678B) nd ntiserum ginst chicken GH (AFP , dilution 1:720,000) were kindly provided y Dr. A.P.F. Prlow (Ntionl Hormones nd Pituitry Progrm, Bethesd, USA). This ntiserum hs 0.7 % cross-rectivity with chicken prolctin nd <0.001 % cross-rectivity with P 4, T, E 2 nd AVT. The sensitivity of the ssy ws 0.2 ng/ml. The inter- nd intrssy coefficients of vrition did not exceed 12.9 % or 10.8 %, respectively. Sttistics The dt shown re the men of the vlues otined in three seprte experiments performed on seprte dys using independent nimls (8 nimls per group) nd their ovries. In ech in vitro experiment, ech experimentl group consisted of six culture wells with ovrin frgments. Assys of the hormone levels in the incution medi were performed in duplicte. The vlues of the lnk control were sutrcted from the vlue determined using RIA/EIA in the cell-conditioned medium to exclude ny non-specific ckground (less thn 15 % of the totl vlues). The rtes of sustnce secretion were clculted per mg tissue/dy. Significnt differences etween the experiments were evluted using two-wys ANOVA. When effects of the tretments were reveled, dt otined from the experimentl nd control groups were compred using the Wilcoxon- Mnn-Whitney multiple rnge test with Sigm Plot 11.0 sttisticl softwre (Systt Softwre, GmH., Erkrth, Germny). Differences compred to control were considered significnt if P<0.05. Results In chicken lood plsm T, E, AVT nd GH were detected (Fig. 1). The culture medium conditioned y cultured ovrin frgments contined norml mounts of T, E nd AVT, ut not of mesurle GH (Fig. 2). These prmeters were ffected y food restriction, dministrtion of ghrelin nd the comintion of the ove fctors. Food deprivtion significntly reduced the concentrtions of oth T (Fig. 1A), E (Fig. 1B) nd incresed the level of oth AVT (Fig. 1C) nd GH (Fig. 1D) in plsm. dministered to d liitum fed chickens did not ffect plsm T (Fig. 1A) or E (Fig. 1B) levels, ut it did increse plsm AVT (Fig. 1C) nd GH (Fig. 1D) concentrtions. Moreover, ghrelin dministrtion could prtilly prevent the effect of food restriction on plsm E (Fig. 1B) nd AVT (Fig. 1C), ut not on T (Fig. 1A) or GH (Fig. 1D) levels.

4 88 Sirotkin et l. Vol. 66 Anlysis of hormones produced y ovrin tissue in vitro demonstrted tht food deprivtion reduced the mount of E produced y the ovry (Fig. 2B), ut it did not ffect the ovrin secretion of T (Fig. 1A) or AVT (Fig. 2C). dministrtion to control irds promoted ovrin T (Fig. 2A), ut not E (Fig. 2B) or AVT (Fig. 2C) relese. Furthermore, ghrelin reduced T (Fig. 2A), ut not E (Fig. 2B) or AVT (Fig. 2C) output y the ovries of irds sujected to food deprivtion. Discussion Our oservtions showed the vilility of steroid hormones, AVT nd GH in chicken lood plsm nd the relese of steroid hormones nd AVT y cultured chicken ovrin tissue, ut our ssy filed to detect mesurle GH production y chicken ovrin cells reported y other investigtors (Lun et l. 2014, Hri 2015) which cn e explined y low GH production y chicken ovrin cells in our experiments. 700 A 350 B Testosteron concentrtion (pg/ml) Estrdiol concentrtion (pg/ml) Norml feeding Norml feeding C D AVT concentrtion (ng/ml) GH concentrtion (ng/ml) Norml feeding Norml feeding Fig. 1. Effect of food restriction nd dministrtion of ghrelin 1-18 (in vivo) on the levels of testosterone (A), estrdiol (B), rgininevsotocin (C) nd growth hormone (D) in chicken lood plsm. Dt re the men ± S.E.M. Differences etween the groups t P<0.05 were considered significnt: ) effect of hormones dministrtion (differences etween control nd hormone-treted chicken); ) effect of food restriction (differences etween corresponding groups of chickens sujected nd not sujected to food restriction).

5 2017, Feeding nd Hormones in Chicken 89 Testosteron relese (pg/mg tissue/dy) A Estrdiol relese (pg/mg tissue/dy) B Norml feeding Norml feeding 12 C AVT relese (pg/mg tissue/dy) Norml feeding Fig. 2. Effect of food restriction nd dministrtion of ghrelin in vivo on the relese of testosterone (A), estrdiol (B) nd rgininevsotocin (C) y isolted chicken ovrin frgments. Legends re similr to those presented in Fig. 1. A comprison of the hormones tht re ville in generl circultion nd those tht re produced y isolted ovrin tissues nd its chnges under the effect of extr-ovrin fctors demonstrtes tht some hormones found in lood (E, T, AVT) re minly ovrin, ut other fctors (GH) re minly extr-ovrin in origin. Furthermore, differences in the mechnisms controlling the relese of these hormones were oserved. For exmple, food deprivtion reduced E in oth plsm nd ovrin frgment-conditioned medium. This finding suggests tht the negtive metolic stte directly ffects ovrin E output. In contrst, the effect of food restriction on plsm T nd AVT, ut not on their relese y isolted ovrin tissue, suggest tht the metolic stte controls T nd AVT relese vi upstrem extr-ovrin regultory mechnisms (proly vi hypothlmo-hypophysil system involved in metolic control of gondl functions, Ro nd Ten-Sempere 2014). One such extrovrin regultor could e ghrelin. could influence chicken ovrin steroid hormones in our previous (Sirotkin et l. 2006, 2015, Sirotkin nd Grossmnn 2007, 2008) nd present studies. In some previous (Sirotkin et l. 2006, Sirotkin nd Grossmnn 2008), ut not in our present experiments, ghrelin promoted chicken ovrin AVT output, which cn e explined y vritions in initil stte of AVT producing cells etween the experiments. Furthermore, our present oservtions of ghrelin-induced increse in plsm GH

6 90 Sirotkin et l. Vol. 66 confirmed previous findings (Budet nd Hrvey 2003) tht ghrelin is physiologicl GH secretgogue not only in the mmmlin, ut lso in the vin pituitry. The oserved ghrelin-induced chnges could e due to influence of ghrelin on hormonl regultors of reproduction t the level of the ovry (steroids), the upstrem hypothlmo-hypophysil system (GH) or on the differentition of CNS nd ovrin tissue. The site nd fine mechnisms of ghrelin influence on orgnisms require further studies, ut the present oservtions suggest the ction of ghrelin t oth CNS nd gondl level. In our experiments, the pronounced effect of ghrelin on testosterone relese y ovrin tissue (Fig. 2A) ws not ssocited with the corresponding chnges in plsm testosterone (Fig. 1A). It suggests tht the direct ction of ghrelin on the ovry could e msked y dditionl fctors in CNS or generl circultion ffecting steroid trnsport, inging or degrdtion. Our oservtions confirmed previous reports tht food deprivtion reduces ovrin steroid hormones (Pczosk-Elisiewicz et l. 2003, Sirotkin nd Grossmnn 2015) nd promotes GH (Buyse et l. 2000, 2002) levels in chicken plsm. The food deprivtioninduced increse in lood AVT levels oserved in our experiments nd the fsting-induced reduction in ovrin AVT relese oserved in our previous (Sirotkin nd Grossmnn 2015) ut not our present studies indicted tht the metolic stte cn ffect lood nd mye ovrin AVT. Vritions in initil stte of ovrin AVT producing cells etween the experiments could influence ovrin AVT response not only to ghrelin (detils re ove) ut lso to food restriction. The steroid hormones GH nd AVT re known regultors of oth mmmlin nd vin reproductive processes including ovrin steroidogenesis (Sirotkin 2005, 2014, Lun et l. 2014, Hri 2015). Thus, it is possile tht food deprivtion cn ffect reproductive processes vi chnges in the relese of these peptide nd steroid hormones. In ddition, the metolic stte cn ffect these hormonl regultors of reproduction vi ghrelin. Food consumption ffects the production of chicken ghrelin, ghrelin cyltion nd ghrelin receptor (Kiy et l. 2007, 2013, Sirotkin et l. 2013, Sintuin et l. 2014). Furthermore, this is the first evidence tht ghrelin cn mimic the effect of food deprivtion on plsm GH nd AVT, nd tht ghrelin cn modify the effect of food deprivtion on chicken plsm hormones. Tken together, comined with the dt on the importnce of ghrelin in the control of sic ovrin functions (detils re ove), these evidence suggest tht ghrelin cn e the key hormone mediting the effect of the metolic stte on downstrem hormonl regultors of vin ovrin functions. Conflict of Interest There is no conflict of interest. Acknowledgements The uthors express their deep grtitude to G. Neuhus for the skillful technicl help in the rrngement of the experiments nd AVT RIA, I. Stelter for EIA of the steroid hormones, K. Tothov nd Z. Kuklov for ssistnce in the sttisticl processing of the dt, s well s to Dr. D. A. Gry (Mx-Plnk Institute for Physiologicl nd Clinicl Reserch, Bd Nuheim, Germny) for the kind gift of ntiserum ginst AVT. We thnk Dr. A. P. F. Prlow (Ntionl Hormones nd Pituitry Progrm, Bethesd, MD, USA) for kindly providing regents for the chicken GH ssy. This work ws finncilly supported y the Germn nd Slovk Ministries of Agriculture nd y the Denship of Scientific Reserch t King Sud University Reserch group No. RG#164. References BAUDET ML, HARVEY S: -induced GH secretion in domestic fowl in vivo nd in vitro. J Endocrinol 179: , BUYSE J, DECUYPERE E, DARRAS VM, VLEURICK LM, KÜHN ER, VELDHUIS JD: Food deprivtion nd feeding of roiler chickens is ssocited with rpid nd interdependent chnges in the somtotrophic nd thyrotrophic xes. Br Poult Sci 41: , BUYSE J, JANSSENS K, VAN DER GREYTEN S, VAN AS P, DUCUYPERE E, DARRAS VM: Pre-nd postprndil chnges in plsm hormone nd metolite levels nd heptic deiodinse ctivities in mel-fed roiler chickens. Br J Nutr 88: , GRAY DA, SIMON E: Mmmlin nd vin ntidiuretic hormone: studies relted to possile species vrition in osmoregultory systems. J Comp Physiol 151: , 1983.

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