Short term effects of genistein on the testes of quail (Coturnix coturnix)

Transcription

1 Reserch Article Turk. J. Vet. Anim. Sci. 2012; 36(3): TÜBİTAK doi: /vet Short term effects of genistein on the testes of quil (Coturnix coturnix) Serp EKİNCİ, Melike ERKAN* Deprtment of Biology, Fculty of Science, İstnul University, 34134, Vezneciler, İstnul - TURKEY Received: Astrct: Although there hve een numer of studies on the effects of phytoestrogens, especilly genistein, on testes, its effects on the testis tissue of irds hve not een studied yet. The present study ims to revel the effects of genistein on quil testes through the use of histologicl nd immunohistochemicl methods. In the study, totl of 64 mle quils (Coturnix coturnix) were used, ech of which ws 50 dys old. Three different doses of genistein were dministered y gvge to the seprte groups t intervls of 15 nd 30 dys. According to the results of the histologicl nlysis nd DNA in situ nick end leling (TUNEL), it ws detected tht high doses of genistein, especilly in 30-dy pplictions, cused more dmge compred to the control group nd the group receiving 15-dy pplictions in terms of histology nd sperm numer. Moreover, it ws lso detected in the 15-dy nd 30-dy ppliction results tht oth low nd high doses of genistein induced poptosis. Key words: Quil, genistein, testis, poptosis Genistein in ıldırcın (Coturnix coturnix) testisi üzerine kıs süreli etkisi Özet: Fitoöstrojenlerin özellikle de genisteinin testis üzerindeki etkisi ile ilgili ir çok çlışm olmsın rğmen kuşlrın testis dokusu üzerindeki etkisini gösteren ir çlışm henüz ulunmmktdır. Çlışmmızd histolojik ve immunohistokimysl yöntemler kullnılrk ıldırcınlrın testis dokusu üzerine genisteinin göstermiş olduğu etkinin çığ çıkrılmsı mçlnmıştır. Çlışmd 64 det 50 günlük erkek ıldırcın (Coturnix coturnix) kullnılmıştır. Genisteinin üç frklı dozu 15 gün ve 30 gün süreyle yrı gruplr mide sondsı yrdımıyl ğızdn verildi. Histolojik inceleme ve in situ DNA uç işretlemesi (TUNEL) sonuçlrın göre genisteinin yüksek dozd ve özellikle 30 günlük uygulmlrınd histolojik ve sperm syısı kımındn kontrole ve 15 günlük uygulmlr göre dh çok hsr neden olduğu, yrıc hem düşük hem de yüksek dozlrının 15 günlük ve 30 günlük uygulm sonuçlrınd poptozu teşvik ettiği elirlenmiştir. Anhtr sözcükler: Bıldırcın, genistein, testis, poptoz * E-mil: merkn@istnul.edu.tr 251

2 Short term effects of genistein on the testes of quil (Coturnix coturnix) Introduction Due to their high protein content, quils re species tht hve een in high demnd ltely, since oth their met nd eggs re cple of meeting food requirements (1,2). Legume grins nd soy products serve n importnt role s niml feeds used in the rising of poultry, including quils (3). Legumes nd soy products contin group of chemicls known s phytoestrogens. Phytoestrogens my hve estrogenic nd ntiestrogenic effects, especilly on the reproductive system, y showing structurl similrity to the hormone estrdiol (4,5). Genistein is one of the most common non-steroid (6) phytoestrogens present in soy products, soy ens, nd other legumes (7,8). Genistein cn show oth estrogenic nd ntiestrogenic effects, competing with estrdiol t the receptor level (9). It is lso known tht genistein stops the cell cycle t the G 2 -M phse (10) nd induces poptosis in testis cells (11). In the present study, the effects of genistein on testis histology nd testiculr cell poptosis in quils were studied using light microscope nd DNA in situ nick end leling (TUNEL). This study ims to revel new dt tht will contriute to our understnding of the reltionship etween nutrition nd the reproductive iology of irds y compring the results of the present study with similr studies of other irds nd mmmls. Mterils nd methods In this study, totl of 64 mle quils (Coturnix coturnix) were used, ech of which hd reched sexul mturity of 50 dys. Ech of the experimentl groups which received genistein (provided y Mikro- Gen Medicine Ind. Inc.) consisted of 8 quils, s did the control group. The genistein ws dministered y gvge. The experimentl groups nd exposure mounts of genistein were s shown in Tle. Histometric nd histologicl methods The testis tissues from the niml groups shown in the Tle were fixed in Bouin s solution for 24 h t 20 C nd emedded in prffin wx. The sections of 5 μm were stined with hemtoxylin nd eosin following the method descried y Humson (12). The dimeters of the seminiferous tuules nd the germinl epithelil height of the control nd experimentl groups were evluted using the stndrd point counting method descried y Attnssov et l. (13). The DNA in situ nick end leling (TUNEL) method After the testis tissues from the groups shown in the Tle were fixed in 10% uffered formlin, preprtes were prepred using histologicl methods. The cells with free DNA ends were mrked using Roche rnd in situ cell deth detection kit, POD, nd stined with Gill s hemtoxylin. Sttisticl nlysis In the nlyses conducted on the experimentl nimls, the differences in testis weights, seminiferous Tle. Control groups nd experimentl groups tht were given genistein. 15 DAYS 30 DAYS Control group I Only corn oil Control group II Only corn oil Experimentl group I 0.5 mg/kg Experimentl group IV 0.5 mg/kg Experimentl group II 25 mg/kg Experimentl group V 25 mg/kg Experimentl group III 50 mg/kg Experimentl group VI 50 mg/kg 252

3 tuule dimeters, germinl epithelil heights, nd poptosis indexes etween the control nd experimentl groups were nlyzed through single direction vrince nlysis (ANOVA). The vlue of P < ws considered to e sttisticlly significnt. In ddition, the differences etween findings of the 15-dy nd 30-dy experimentl groups were evluted sttisticlly using the Scheffe s method of vrince nlysis. A vlue of P < 0.05 ws considered to e sttisticlly significnt. Results As result of the mcroscopic nlysis, the verge testis weight for ech group ws determined nd results re shown in Figure 1. In terms of testis weight, compred to the control group I, experimentl groups II nd III showed significnt increse. When experimentl group V nd experimentl group VI were compred to control group II, significnt increse ws detected in testis weight (P < 0.001). Furthermore, when experimentl group I nd experimentl group IV, experimentl group II nd experimentl group V, nd experimentl group III nd experimentl group VI were compred to ech other, significnt (P < 0.05) increse ws oserved in testis weight. Testis weight (g) dys groups 60 dys groups 0 Control 0.5 mg/kg 25 mg/kg 50 mg/kg Figure 1. Testis weights for control nd experimentl groups (, : P < 0.001). When seminiferous tuule dimeters were evluted, significnt (P < 0.001) decrese ws oserved in experimentl group III s compred to control group I while significnt increse (P < 0.05) ws oserved etween experimentl groups III nd VI. The vlues recorded for the other groups were not found to e significnt. When germinl epithelil heights were evluted in control group I nd in experimentl groups I, II, nd III, the germinl epithelil heights were found to show significnt decrese (P < 0.001) compred to the control group. In experimentl group VI, germinl epithelil height incresed significntly (P < 0.001) compred to control group II. Experimentl group V lso incresed significntly (P < 0.05) in comprison to experimentl group II. In experimentl group VI, significnt increse (P < 0.05) ws oserved in germinl epithelil heights when compred to experimentl group III. The findings for experimentl groups given genistein for 15 dys were exmined nd when results from experimentl groups I, II, nd III were compred with control group I, it ws oserved tht the seminiferous tuules nd germinl epithelil cells mintined their regulr structure. It ws lso seen tht in experimentl groups I nd II, the density nd morphology of the spermtocytes in the seminiferous tuule lumens were similr to those of the control group; in experimentl group III, however, decrese in the density of spermtocytes ws detected (Figure 2). A look t the findings of the experimentl groups given genistein for 30 dys showed tht when experimentl groups IV, V, nd VI were compred with control group II the regulr structure of the seminiferous tuules ws mintined. Intercellulr spces were identified in some prts lthough germinl epithelil cells were in order. In experimentl groups IV, V, nd VI, the morphology of the spermtocytes in the seminiferous tuule lumens ws similr to tht of the control group. However, while the spermtocyte density in experimentl group IV ws similr to tht oserved in the control group, this density ws seen to decrese in experimentl groups V nd VI when compred to the control group (Figure 3). When the groups tht were given genistein for 15 dys were compred with control group I, 253

4 Short term effects of genistein on the testes of quil (Coturnix coturnix) Figure 2. Histologicl sections of quil testes from control groups nd experimentl groups tht were given genistein for 15 dys. (H&E, r = 13 μm). Spermtogonium ( ), primry spermtocyte ( ), spermtid ( ), sperm (S). A. The control group; B. The group given 0.5 mg/kg genistein; C. The group given 25 mg/kg genistein; D. The group given 50 mg/kg genistein. it ws oserved tht the proportion of poptosis incresed significntly in ll groups (P < 0.001). Similrly, in the groups which were given genistein for 30 dys, significnt increse (P < 0.001) in the proportion of poptosis ws oserved in ll groups compred to control group II. Furthermore, when experimentl group I nd experimentl group IV, experimentl group II nd experimentl group V, nd experimentl group III nd experimentl group VI were compred with ech other, it ws detected tht the poptosis proportion in these groups lso incresed significntly (P < 0.05) (Figure 4). Discussion In studies conducted to determine the effects of genistein on testis tissue, testis weight, seminiferous tuule dimeter, the histologicl structure of the testis, nd the mount of poptosis seen in the testes hve ll een considered (13-18). Genistein, which is phytoestrogen, shows vrious effects on the testes y inding estrogen receptors, interfering with sex steroid production, inhiiting protein kinses, preventing cell prolifertion, nd decresing the production of sex steroid inding proteins (19). Mgee (20) hs reported tht the dministrtion of 0.1% genistein nd 0.5% genistein hd no significnt effect on testis weight when compred to control. In study from 2001, however, Ngo et l. (14) showed tht testis weight incresed significntly in rts given 25, 50, or 100 mg/kg of genistein in comprison with the control group. A study undertken y Fritz et l. in 2003 (15) determined tht there ws no increse in the testis weight of rts tht were given 25 mg/kg or 1000 mg/ kg genistein for 35 dys. In nother study in the sme yer (16), 10 mg/kg nd 40 mg/kg genistein were given sucutneously to rts for 3 weeks; it ws found tht in the group tht ws given 10 mg/kg genistein the testis weight did not chnge compred to the control group, lthough in the group which ws given 40 mg/kg, the testis weight decresed compred to the control group. In 2004 study y Adchi et l. (17), 254

5 Figure 3. Histologicl sections of quil testes from control groups nd experimentl groups tht were given genistein for 30 dys. (H&E, r = 13 μm). Spermtogonium ( ), primry spermtocyte ( ), spermtid ( ), sperm (S), expnsion the intercellulr spces ( ). A. The control group; B. The group given 0.5 mg/kg genistein; C. The group given 25 mg/kg genistein; D. The group given 50 mg/kg genistein. Apoptotic index (%) dys groups 60 dys groups 0 Control 0.5 mg/kg 25 mg/kg 50 mg/kg Figure 4. The poptotic index (%) for control nd experimentl groups (, : P < 0.001). 1 mg/kg genistein ws given to neworn rts from the first dy of irth until the fifth dy. When the rts were 12 weeks old, the differences tht occurred in their testes were nlyzed. It ws found tht there ws no difference in the testis weight of the rts in the group given genistein compred to the control group. In study y McClin et l. (18), 7-week-old rts were cute, suchroniclly, nd chroniclly given genistein orlly. They found tht in the rts which were exposed to cute nd suchronic genistein for 4 weeks, the testis weight ws not ffected while in the rts exposed to suchronic genistein for 13 weeks the testis weight incresed in correltion with the genistein dose given compred to the control group. In the chronic experiment, the rts were exposed to 50 mg/kg nd 500 mg/kg genistein for 52 weeks. It ws found tht in these rts the testis weight lso incresed in correltion with the genistein dose 255

6 Short term effects of genistein on the testes of quil (Coturnix coturnix) given compred to the control group. According to the present study, significnt increse ws seen in the groups tht were given 25 mg/kg or 50 mg/kg genistein, wheres there ws no significnt increse in the testis weight in the group tht ws given 0.5 mg/kg genistein when compred to the control group. In study y Aktş et l. (21), White Leghorn eggs were exposed to 500 μg/egg genistein during emryonic development. They found tht genistein did not hve negtive effect on emryonic testis development. These findings indicte tht, similr to the results otined for other niml groups, low doses of genistein do not cuse ny chnge in the testis weight of irds lthough higher doses increse the testis weight. In 2003 study conducted y Fritz et l. (15) on rts, it ws shown tht genistein did not chnge the seminiferous tuule dimeter significntly. According to the results of the present study, there ws significnt decrese in tuule dimeter in the group tht ws given 50 mg/kg genistein for 15 dys; on the other hnd, there ws sustntil increse in the seminiferous tuule prmeter if genistein ws dministered for 30 dys. It is thought, however, tht this increse ws not relted to the nturl structure of the tissue nd sperm production, eing cused insted y the intercellulr spces formed in the germinl epithelil tissue. No importnt chnge ws oserved in ny of the experimentl groups with regrd to the seminiferous tuule histology when compred to the control group (14,16,17) nd it ws seen tht spermtogenesis continued normlly (17). In the present study, when ll of the experimentl groups were evluted in terms of sperm morphology nd compred to their respective control groups, the significnt decrese in the groups tht were given 50 mg/kg genistein for 15 dys nd 25 mg/kg or 50 mg/ kg genistein for 30 dys suggests tht n overdose of genistein decreses the density of sperm in quils s well s in rts (22). In previously undertken studies, it hs een stted tht genistein hd n poptotic effect on spermtogenic cells found in the testes nd tht this effect ws relted to the dose dministered (11,13,23). In the present study, in ll of the groups tht were given genistein for 15 or 30 dys, the poptosis index incresed significntly in correltion with the dose when compred to the control group. In conclusion, this study showed tht genistein, one of the most dominnt phytoestrogens found in soy, soy products, nd other legume species used in the feeding of poultry, induces poptosis in correltion with the dose in the testes of quil, one of the est representtives of poultry. Acknowledgment This study ws supported y the Reserch Fund of İstnul University (Project numer T-2639). References 1. Özcn, M., Ekiz, B., Güneş, H.: Jpon ıldırcınlrınd (Coturnix coturnix jponic) gruplndırılmış yumurt ğırlığı ve çıkım ğırlığının üyüme performnsı üzerine etkileri. İ. Ü. Vet. Fk. Derg., 2001; 27: Shin, N., Shin, K., Onderci, M., Srkr, F.H., Doerge, D., Prsd, A., Kucuk, O.: Effects of dietry genistein on nutrient use nd minerl sttus in het-stressed quils. Exp. Anim., 2006; 55: Ylçın, S., Sehu, A.: Bıldırcın rsyonlrın ktıln di fiğin (Vici stiv L.) üyüme, krks rndımnı ve zı kn prmetreleri üzerine etkisi. Tr. J. of Veterinry nd Animl Sciences, 1998; 22: (In Turkish with English strct) 4. Setchell, K.D.R., Cssidy, A.: Dietry isoflvones: iologicl effects nd relevnce to humn helth. The Journl of Nutrition, 1999; 129: Mskrinec, S.: The effect of phytoestrogens on hot flshes. Nutrition Bytes, 2003; 9: Oplk, M., Kmińsk, B., Ciereszko, R., Dusz, L.: Genistein ffects testosterone secretion y Leydig cells in roosters (Gllus gllus domesticus). Reproductive Biology, 2004; 4: Ski, T., Furoku, S., Nkmoto, M., Shuto, E., Hosk, T., Nishiok, Y., Sone, S.: The soy isoflvone equol enhnces ntigen-specific IgE production in ovlumin-immunized BALB/c mice. J. Nutr. Sci. Vitminol., 2010; 56: Mrtínez-Montemyor, M.M., Otero-Frnqui, E., Mrtinez, J., De L Mot-Peyndo, A., Cuno, L.A., Dhrmwrdhne, S.: Individul nd comined soy isoflvones exert differentil effects on metsttic cncer prgression. Clin. Exp. Metstsis, 2010; 27:

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