ARTICLE. E. Pavlova 1, N. Atanassova 1, C. McKinnell 2, R.M. Sharpe 2 1 Institute of Experimental Morphology, Pathology and Anthropology with Museum,

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1 DOI:.554/5YRTIMB..3 OPPOSITE MODELS OF EXPRESSION OF ANDROGEN RECEPTOR (AR) AND RETINOIC ACID RECEPTOR-α (RAR-α) IN THE ONSET OF MALE GERM CELL DEVELOPMENT IN HORMONALLY MANIPULATED RATS E. Pvlov, N. Atnssov, C. McKinnell, R.M. Shrpe Institute of Experimentl Morphology, Pthology nd Anthropology with Museum, Bulgrin Acdemy of Sciences, Sofi, Bulgri MRC, Humn Reproductive Sciences Unit, The Queen s Medicl Reserch Institute, Edinburgh, UK Correspondence to: Ekterin Pvlov E-mil: e_bnkov@yhoo.com ABSTRACT Vitmin A together with testosterone (T) nd FSH ply n essentil role in regulting spermtogenesis. Androgens nd Vitmin A ct through specific nucler receptors: AR, retinoic cid receptors (RAR-α, β, γ). Present study imed to investigte reltionship between expression of RAR-α nd AR in the initition of spermtogenesis during puberty in conditions of neontl hormonl mnipultion. Experimentl model of neontl tretment with diethylstilbestrol (DES), GnRH-ntgonist, T ws used. Immunostining for RAR-α nd AR; -point counting were pplied. Absolute nucler volume (ANV) ws estimted for Sertoli (SC), germ cells (GC) nd their subtypes. At onset of puberty (d8) over expression of RAR-α ws evident in SCs fter DES- but not GnRH tretment. Co-dministrtion of T+DES restored the norml pttern of wek immunorectivity. An opposite model of expression ws seen for AR loss of expression fter DES but not GnRH ppliction nd recovery by T-therpy. Similr reduction ws found for spermtogonil ANV in both groups, wheres ANV of spermtocytes (Sc) is reduced in greter extent by DES (x) thn GnRH (6x). Preleptotene (Pl), leptotene (L) nd zygotene (Z) stges ws lso more ffected by DES thn GnRH. Sertoli cell support towrd spermtocytes (Sc/SC rtio) ws more ffected by DES (4x decrese) thn GnRH (x) nd the sme tendency is found for Pl/SC nd L-Z/SC. In conclusion, dt suggest possible interply between retinoid nd ndrogen signling in Sertoli cells in the differentition of mle germ cells. The nti-ndrogenic effect of estrogens on meiotic germ cells is probbly medited by ugmenttion of RAR- α expression. Keywords: RAR-α, AR, ndrogens, estrogens, spermtogenesis Introduction Vitmin A together with testosterone (T) nd FSH ply n essentil role in regulting spermtogenesis. Androgens (testosterone nd 5α-dihydrotestosterone) control the development, differentition, nd function of mle reproductive system. Androgen ction is medited through specific nucler receptor ndrogen receptor (AR) nd in the testis AR is loclized in Sertoli, peritubulr nd Lydig cells, but not in germ cells (5). In ddition to plying fundmentl role in very diverse processes such s vision nd the growth nd differentition of numerous types of cell, vitmin A (retinol) nd its principl biologiclly ctive derivtive, retinoic cid, re clerly involved in the regultion of testiculr functions. An excess of vitmin A leds to testiculr lesions nd spermtogenetic disorders, wheres deficiency induces erly cesstion of spermtogenesis nd dversely ffects testosterone secretion (7). Retinoids pper to exert their ction on the three min testiculr cell types (Sertoli, germinl nd Leydig cells), s they ct on the BIOTECHNOL. & BIOTECHNOL. EQ. 6//SE 7 signling pthwys. Vitmin A cts through two fmilies of retinoid receptors, retinoic cid receptors (RAR) nd retinoid X receptors (RXR), ech with three subtypes α, β nd γ, loclized in the testis (3, 4). Mice mutnt for retinoic cid α receptors nd retinoid X β receptors re sterile. Hving in mind the importnt role of both ndrogens nd vitmin A, the im of the present study is to investigte reltionship between expression of RAR-α nd AR in the initition of spermtogenesis during puberty in conditions of neontl hormonl mnipultion. Mterils nd Methods We used experimentl model for mnipultion of neontl hormonl environment by tretment with: DES- µg in µl corn oil on dys, 4, 6, 8, nd ; ntgonist of gondotrophin-relesing hormone (GnRH) -mg/kg in µl 5% mnnitol on dys nd 5; co-dministrtion of µg DES nd µg testosterone-propionte (TP) nd µl corn oil (vehicle) s control. Rts from ll tretment groups were subsequently smpled on dy 8 (erly puberty). Immunohistochemistry for RAR-α nd AR ws pplied. In situ detection of germ cell poptosis by TUNEL method () nd subsequent 5 YEARS ROUMEN TSANEV INSTITUTE OF 6-7 OCTOBER, SOFIA

2 -point counting using clock-fce smpling of 5 fields (35 points) were used (). We estimted bsolute nucler volume (ANV) for Sertoli cells (SC), germ cells (GC) - spermtogoni (A-Sg, In nd B-Sg) nd spermtocytes (Sc) including preleptotene (pl), leptotene nd zygotene (L+Z) nd pchytene (Ph), s well s the rtios of GC/SC. Comprison of the different prmeters of the vrious tretment groups ws mde using Student s t- test. Results nd Discussion Exposure to estrogens during neontl life is reported to cuse delyed development of the testis nd permnent impirment of spermtogenesis in dulthood tht dversely ffect totl germ cell (GC) nd Sertoli cell (SC) popultion (). Neontl dministrtion of estrogens suppressed FSH production t the time when this hormone is essentil for initition of spermtogenesis t puberty. For tht reson the negtive effect of estrogens were ttributed to suppression of gondotropin secretion during the tretment tht result in inhibition of testosterone (T) production by Leydig cells, s well (9). On dy 8 in the control group GC development proceeds to the lte pchitene stge of meiotic prophse-i. The lumen ws observed in most seminiferous tubules. There were seen single poptotic cells. DOI:.554/5YRTIMB..3 Very wek immunorectivity of RAR-α ws found in Sertoli cells, some peritubulr nd Leydig cells (Fig. ). Over expression of RAR- α ws prominent in perinucler region of Sertoli cells fter tretment with DES- but not GnRH. Codministrtion of testosteron with DES restored the norml pttern of wek immunorectivity. An opposite model of expression ws seen for AR loss of expression fter DES but not GnRH ppliction nd recovery by therpy with testosteron (Fig. ) tht corresponds to results of other collectives (6). Estrogen dministrtion might interfere with ndrogen production or ction in ddition to ctivting estrogen receptor (ER) -medited pthwys. This both possibilities would fundmentlly lter the ndrogen/estrogen blnce by lowering ndrogen ction nd elevting estrogen ction. Suppression of ndrogen production nd ction (expression of AR) is n integrl prt of the mechnism vi which estrogen effect mle reproduction (). DES- induced chnges in AR expression were prlleled by chnges in Leydig cell volume per testis (9% reduction thn control) (8). Germ cell poptotic index ws evluted t similr extent in DES nd GnRH treted rts corresponding to equl reduction of plsm T nd FSH levels (dt not shown). Fig.. Effect of neontl ndrogen suppression on the immunoexpression of RAR-α in Sertoli cells of rt testis on dy 8 fter tretment with DES- μg, GnRH or therpy with TP. x 63 BIOTECHNOL. & BIOTECHNOL. EQ. 6//SE 73 5 YEARS ROUMEN TSANEV INSTITUTE OF 6-7 OCTOBER, SOFIA

3 DOI:.554/5YRTIMB..3 Fig.. Immunoexpression of AR in the testis of 8-dy old rts treted with DES- µg, GnRH or DES-+T. x Quntittive nlysis on dy 8 reveled tht both tretments (DES nd GnRH) cused similr reduction (3 times) by spermtogonil bsolute nucler volume, wheres nucler volume of spermtocytes is reduced in greter extent by DES ( times) thn GnRH (6 times) (Fig. 3A). Differences between men vlues of were significnt tht implied the direct estrogen ction on meiotic germ cells. The similrities in the ction of high levels of estrogen nd those induced by GnRH (both tretments inhibit T-production by Leydig cells) indicte tht gondotrophin suppression is involved in indirect mechnism of ction of DES. Both tretments cused more pronounced reduction in dvnced types of spermtogoni, In nd B (8%) thn in A-Sg (35%) (Fig. 4А). Among the spermtocytes, leptotene-zygotene stges were most sensitive to different hormonl tretments nd the effect of DES (3-fold decrese in ANV) ws significntly more severe thn those of GnRH (8-fold decrese) (Fig. 5-A). Preleptotene stge ws lso more ffected by DES (8 fold) thn GnRH (3.5-fold). The Rtio spermtocytes/spermtogoni decresed in lrger extent in DES (3.4 times) thn GnRH (twice) compring to control. GnRH DES- DES-+T. GnRH DES- DES-+T Absolute Nucler Volume (mm 3 ) Totl Germ Cells Totl Spermtogoni Totl Spermtocytes Fig. 3. ANV (mm 3 ) of testiculr germ cell types (A) nd germ cell types per unit Sertoli cell nucler volume (B) on dy 8 of control nd neontlly treted rts with GnRH, DES- μg or co-dministrtion of DES- μg nd µg TP Dt represent men vlue ± SE (p<.5; p<.; p<.); - sttisticly significnt compred to GnRH Germ/Sertoli Cell Rtio TGC/Sertoli TotlSg/Sertoli TotlSc/Sertoli BIOTECHNOL. & BIOTECHNOL. EQ. 6//SE 74 5 YEARS ROUMEN TSANEV INSTITUTE OF 6-7 OCTOBER, SOFIA

4 DOI:.554/5YRTIMB GnRH DES- DES-+T.45.4 GnRH DES- DES-+T Absolute Nucler Volume (mm 3 ) Spermtogoni/Sertoli Cell Rtio A-Spermtogoni In+B Spermtogoni A-Sg/Sertoli In+B Sg/Sertoli Fig. 4. ANV (mm 3 ) of different types Spermtogoni (A) nd ANV of spermtogonil cell types per unit Sertoli cell nucler volume (B) on dy 8 of control nd neontlly treted rts with GnRH, DES- μg or co-dministrtion of DES- μg nd µg TP Dt represent men vlue ± SE (p<.5; p<.; p<.). In+B Sg-Intermedite nd B- Spermtogoni Absolute Nucler Volume (mm3) GnRH DES- DES-+T pl-s c L-Z S c Ph-Sc Fig. 5. ANV (mm 3 ) of different types Spermtocytes (A) nd ANV of spermtocytic cell types per unit Sertoli cell nucler volume (B) on dy 8 of control nd neontlly treted rts with GnRH, DES- μg or co-dministrtion of DES- μg nd µg TP Dt represent men vlue ± SE (p<.5; p<.; p<.). pl-sc- Preleptotene Spermtocytes; L-Z Sc- Leptotene nd Zygotene Sc; Ph-Sc- Pchitene Sc Spermtocytes/Sertoli Cell Rtio pl-sc/sertoli L-Z Sc/Sertoli Ph-Sc/Sertoli GnRH DES- DES-+T The function of Sertoli cells to support germ cells, known s efficiency of spermtogenesis, ws evluted by estimtion of ANV of germ cells per unit Sertoli cell ANV. Supporting function of Sertoli cells towrd totl germ cell (TGC) popultion ws significntly suppressed by both tretments nd in DES nimls this reduction ws more pronounced ( times lower thn control) thn GnRH (.5times) (Fig. 3B). The rtio between spermtogoni nd Sertoli cells were eqully reduced in both experimentl groups wheres Sertoli cell support towrd spermtocytes ws more ffected by DES (4-fold decrese) thn GnRH (twice) (Fig 3B) nd the sme tendency is found for pl/sc nd L-Z/SC rtios (Fig. 5B). Our dt suggest direct ction of estrogens on GC (Sg nd Sc) rther thn indirect mechnism vi SC nd their supporting function. Sertoli nd germ cells were reported to express ER-β nd the direct dverse effect of high estrogen levels on functionl mturtion of SC ws demonstrted by Shrpe et l. (). Conclusions Our dt suggest possible interply between retinoid nd ndrogen signling in Sertoli cells in the differentition of germ cells, prticulrly spermtocyte development. The nti-ndrogenic effect of estrogens on meiotic germ cells is probbly medited by ugmenttion of RAR-α expression. Acknowledgements This work ws supported by Grnt DO /3/9 of Ntionl Science Fund of the Ministry of Eduction nd Science of Bulgri nd by the Europen Socil Fund nd Republic of Bulgri, Opertionl Progrmme Development of Humn Resources 7-3, Grnt BG5PO /46 from BIOTECHNOL. & BIOTECHNOL. EQ. 6//SE 75 5 YEARS ROUMEN TSANEV INSTITUTE OF 6-7 OCTOBER, SOFIA

5 REFERENCES. Atnssov N. (7) D. Sci. Thesis, Sofi, p Atnssov N., McKinnell C., Wlker M., Turner K.J., Fisher J.S., Morley M., Millr M.R., Groome N.P., Shrpe R.M. (999) Endocrinology, 4, Doyle T., Brun K.W., McLen D.J., Wright R., Griswold M.D., Kim K. (7) Ann. N. Y. Acd. Sci.,, Dufour J. nd Kim K.H. (999) Biol. Reprod., 6, Gelmn E.P. () J. Clin. Oncol., (3), Kushik M.C., Misro M.M., Sehgl N., Nndn D. () Andrologi, 4(3), DOI:.554/5YRTIMB Liver G., Rouiller-Fbre V., Pirult C., Levcher C., Hbert R. () Reproduction, 4, McKinnell C., Atnssov N., Willims K., Fisher J.S., Wlker M., Turner K.J., Sunders T.K., Shrpe R.M. () J. Androl., (), Pvlov E., Atnssov N., Shrpe R. (8) Act Morphol. et Antropolo., 3, 7-.. Shrpe R.M., Atnssov N., McKinnell C., Prte P., Turner K.J., Fisher J.S., Kerr J.B., Groome N.P., Mcpherson S., Millr M.R., Sunders P.T.K. (998) Biol. Reprod., 59, BIOTECHNOL. & BIOTECHNOL. EQ. 6//SE 76 5 YEARS ROUMEN TSANEV INSTITUTE OF 6-7 OCTOBER, SOFIA

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