General and Comparative Endocrinology 151 (2007) 27–33

www.elsevier.com/locate/ygcen

Estrogen response system in the reproductive tract of the male turtle: An immunocytochemical study

Daniel H. Gist a,¤, Suzanne Bradshaw b, Carla M.K. Morrow c, Justin D. Congdon d, Rex A. Hess c

a Department of Biological Sciences, University of Cincinnati, Cincinnati, OH 45221-0006, USAb Department of Biology, Raymond Walters College, University of Cincinnati, Cincinnati, OH 45236, USA

c Department of Veterinary Biosciences, University of Illinois, Urbana, IL 61802-6199, USAd Savannah River Ecology Laboratory, Aiken, SC 29801, USA

Received 15 August 2005; revised 1 June 2006; accepted 18 June 2006Available online 28 August 2006

Abstract

Portions of the reproductive tract of the male (Trachemys scripta) turtle were examined by immunocytochemistry for evidence of thecapacity to produce and respond to estrogen hormones (via the expression of P450 aromatase and estrogen receptors). Aromatase wasdetected in both the Sertoli and Leydig cells of the testis and was expressed at diVerent levels during the spermatogenic cycle, being high-est in the quiescent testis and lowest during germ cell meiosis. ER� was found in the Leydig cells surrounding the seminiferous tubules aswell as in the epithelial cells of the excurrent canals (rete testis, eVerent ductule, and epididymis). ER� immunoreactivity was found inboth the spermatogonia and Sertoli cells in the testis, and in the epithelial cells of excurrent canals.© 2006 Elsevier Inc. All rights reserved.

Keywords: Testis; Excurrent ducts; Aromatase; ER�; ER�; Turtle

1. Introduction

There is increasing evidence that the vertebrate testismay be both a source and a target for estrogen hormones.In vertebrates ranging from sharks to mammals, testiculartissues have been shown to contain aromatase, the rate-lim-iting enzyme for estrogen formation (Carreau et al., 1999).In mammals, the testicular cell expressing aromatase isuncertain, with Sertoli, Leydig, and even germ cells beingimplicated as sources of this estrogen-producing enzyme. Inamphibians, testicular Leydig cells possess aromatase activ-ity (Callard et al., 1980), but in chondrichthyan Wsh(Callard et al., 1985) and the rooster (Kwon et al., 1995),activity is restricted to developing germ cells; it may beabsent entirely from the teleost testis (Kobayashi et al.,1998).

* Corresponding author. Fax: +1 513 556 5299.E-mail address: daniel.gist@uc.edu (D.H. Gist).

0016-6480/$ - see front matter © 2006 Elsevier Inc. All rights reserved.doi:10.1016/j.ygcen.2006.06.012

Testicular estrogens are capable of entering the bloodand acting distally, but also can remain and act within thetestis itself in a paracrine manner. Receptors for estrogen(ER�,�) have been identiWed in testicular germ cells in vary-ing states of development as well as in Sertoli and Leydigcells (O’Donnell et al., 2001). The role of testicularestrogens is not clear, but both peripheral (hypothalamus,pituitary) as well as local (spermatogenesis) actions havebeen suggested.

Another target for estrogen action in the male is theexcurrent canal system. These canals (rete testis, eVerentductules, epididymis, and vas deferens) are used for thetransport, maturation, and storage of sperm and their pres-ence is restricted to those vertebrates employing internalfertilization. Estrogen receptors are widespread in themammalian excurrent canal system (see Hess et al., 2001)and interference with those receptors or their absence leadsto infertility. Thus the estrogen response system within themammalian male reproductive tract appears to be an

28 D.H. Gist et al. / General and Comparative Endocrinology 151 (2007) 27–33

important factor in the maintenance of fertility (Hess et al.,1997). A similar response system exists in the bird wheregerm cells are reported to produce estrogens (Kwon et al.,1995) and the epididymis contains estrogen receptors(Kwon et al., 1997); the function of this system in birds,however, remains uninvestigated.

Male excurrent ducts make their Wrst appearance in theearly amniotic vertebrates, of which the reptiles are theremaining living group. Whether these ducts are responsiveto estrogen in early amniotes and thus are implicated in thepost-testicular maturation of sperm remains unclear. Thetestes of some reptiles produce estradiol (Callard and Ryan,1977; Ando et al., 1992) and in others epididymal extractspossess putative estrogen receptors (Dufaure et al., 1983;Cardone et al., 1998), but neither the cellular location ofthese receptors nor a physiological role for estrogens inlower vertebrates has been demonstrated. This study wasundertaken to examine the male reproductive system of theturtle for the presence of the enzyme aromatase and estro-gen receptors. In view of the emerging importance of estro-gen hormones in the regulation of fertility, reptilesrepresent the earliest vertebrate capable of postesticularsperm retention and/or modiWcation and thus an earlyexample of how the estrogenic regulation of fertility mayhave evolved.

2. Materials and methods

Adult male turtles (Trachemys scripta) were either purchased fromcommercial suppliers or trapped in the vicinity of Aiken, South Carolina.Upon receipt, they were housed for a short period of time, generally lessthan a week, in a heated room with access to running water under a photo-period of L:D 12:12. Because of Wxation requirements (see below), it wasnot possible to examine ER and aromatase activity in the same turtle. Tur-tles were perfused and Wxed as follows: turtles were anaesthetized with1 mg pentobarbital and the carapace removed. The aortic trunk from theright ventricle (Kent, 1973) was cannulated and perfused with phosphatebuVered saline, pH 7.4 (PBS); the right atrium was lanced to allow fordrainage. As soon as the Xuid emerging from the right atrium ran clear, theperfusate was switched to either 10% neutral buVered formalin (estrogenreceptor) or Bouins (aromatase) and the perfusion continued for an addi-tional 20–30 min. The testes and/or epididymides were then removed andplaced in cold Wxative overnight at 4 °C. Tissues were dehydrated, embed-ded in paraYn, sectioned at 6 �m, and placed on microscope slides (FisherSupurfrost Plus).

2.1. Immunocytochemistry (ICC)

For aromatase immunocytochemistry, turtles were collected weeklyover the period of spermatogenesis (June–October) resulting in a sampleof 25 animals. A polyclonal antibody (rabbit, CYP1A1) developed againsthuman P450 placental aromatase, purchased from Chemicon Interna-tional (Temecula, CA), was used in these experiments. The antibody wasused at a dilution of 1:1000.

Turtles (ND 6) used for the demonstration of ER immunoreactivitywere purchased from Nasco (Ft. Atkins, WI) during the months of Febru-ary through April. Monoclonal anti-ER� antisera (NCL-ER-LH2) werepurchased from Vector Laboratories and used at a dilution of 1:50. Mono-clonal anti-ER� antisera were purchased from Serotec (Oxford, UK) andused at a dilution of 1:25.

We followed the procedure of Kwon et al. (1995) for the ICC. Slideswere incubated in methanol containing 0.6% H2O2 to quench endogenous

peroxidases, then microwaved in 0.01 M citrate buVer (pH 6.0) for 20 minto unmask antigenic sites. Following avidin–biotin blocking and proteinblocking with 10% normal rabbit serum, slides were incubated with theappropriate antibody overnight at 4 °C. Control slides were incubated inPBS. Binding was visualized by using avidin–biotin complex (ABC Kit;Vector Laboratories, Burlingame CA) and the diaminobenzidine (DAB)chromagen. Hematoxylin was used as a counterstain. Images were cap-tured digitally using either a ProgRes C14 (Jenoptik Laser, Germany) or aSpot-2 (Diagnostic Instruments, Inc., Sterling Heights, MI) camera andcompiled using Adobe Photoshop (Adobe Systems, San Jose, CA).

2.2. Western blot analysis

To verify that the antibodies used in the immunocytochemistry reactedwith appropriate proteins isolated from turtle tissues, Western blot analy-ses were performed. Total protein was extracted from pooled turtle testesand epididymides using Tris buVer (0.1 M NaCl, 0.1 M Tris–HCl, pH 7.6,1 mM EDTA) containing 2% SDS and the concentration of extracted pro-tein was determined by Bio-Rad Protein Assay (Bio-Rad, Hercules, CA).Approximately 32 and 64 �g of protein was run on 10% acrylamide ReadyGels (Bio-Rad, Hercules, CA) then transferred to nitrocellulose. The mem-branes were blocked in 3% BSA/TBS (3% BSA, 10 mM Tris–HCl pH 7.5,150 mM NaCl). Each of the primary antibodies used in the immunocyto-chemistry were incubated with the membranes overnight in 0.5% BSA/TBS at room temperature. For aromatase, the CYP1A1 antibody was usedat a dilution of 1:500; for ER�, the NCL-ER-LH2 antibody was used at1:25, and the Serotec ER� antibody at a dilution of 1:25. Followingwashes, the appropriate horseradish peroxidase-conjugated secondaryantibody (Dako, Oxford, UK) was added for 20 min. Reaction productwas detected using the Opti-4CN Substrate and Detection Kit (Bio-Rad,Hercules, CA).

3. Results

3.1. Aromatase

Aromatase immunoreactivity was present in the testesand epididymides of T. scripta (Fig. 1). Western blots ofpooled testicular and epididymal tissue yielded two aroma-tase immunoreactive bands at 48 and 60 kDa (Fig. 1). Thelatter band is close to the molecular weight of human aro-matase (66 kDa). With immunocytochemistry, aromatasewas localized in the cytoplasm of Sertoli cells and in theLeydig cells exterior to the seminiferous tubules (Fig. 2). Inthe Sertoli cells, immunoreactivity was perinuclear in loca-tion (Fig. 2e). Aromatase immunoreactivity was found inthe vicinity of germ cells at all stages of development butwas absent from the cytoplasm of germ cells themselves.Aromatase immunoreactivity was also found in the Leydigcells exterior to the seminiferous tubules (Figs. 2a–d). Boththe size of the Leydig cells and the amount of immunoreac-tivity in the cytoplasm varied throughout the spermato-genic cycle and, at certain times of the year,immunoreactivity obscured the Leydig cell nuclei. Aroma-tase was not detected in eVerent ductules or the epididymisat any time.

To minimize variations in antibody binding and colordevelopment, ICC procedures on testes collected at diVer-ent times of the year were performed at the same time usingthe same batch of reagents. Despite this, there was variationin the intensity of aromatase immunoreactivity in testes col-lected at diVerent times of the year (Fig. 2). The most

D.H. Gist et al. / General and Comparative Endocrinology 151 (2007) 27–33 29

intense reaction in both Leydig cells and Sertoli cells wasobserved in spermatogenically inactive turtles (Figs. 2a andb). In these, spermatogonia were the most abundant germcell type in the seminiferous epithelium. Aromatase immu-noreactivity in Sertoli cell cytoplasm was concentrated inlarge granules overlying the spermatogonia and in betweenthe spermatogonial cells (Figs. 2a and b). In testes undergo-ing meiotic divisions of germ cells, collected in July andAugust (Fig. 2c), there was a reduction in the intensity ofaromatase immunoreactivity in both Sertoli cells and Ley-dig cells. As sperm were released from the seminiferous epi-thelium (Figs. 2d–f), more intense immunoreactivityreappeared in the Sertoli cells but not to the same extent asseen in the quiescent testis.

Immunoreactivity in the Leydig cells followed a similarpattern. In all of the turtles examined, some Leydig cells weredevoid of immunoreactivity while others adjacent to them dis-played considerable immunoreactivity (Fig. 2a). Generally,Leydig cells were hypertrophied and possessed the greatestimmunoreactivity during testicular quiescence and during thematuration phase of the spermatogenic cycle (Fig. 2d).

Fig. 1. Western blot analysis of proteins isolated from pooled testes andepididymides. Proteins transferred to nitrocellulose were reacted withanti-aromatase (Arom), anti-ER� (Era), or anti-ER� (Erb), and signalwas detected using horseradish peroxidase. Arrows indicate the appropri-ate immunoreactive band. Numbers indicate approximate molecularweight.

3.2. ER�

Testes of turtles examined for estrogen receptors were allin the resting or early proliferative stages. Western blots ofprotein extracts demonstrated a single reactive band at58 kDa, close to the weight of ER� (58 kDa) from otherspecies. ER� immunoreactivity was observed in the nucleiof Leydig cells (Fig. 3) unlike aromatase which wasdetected in the cytoplasm of this same cell type. At this timein the spermatogenic cycle, the testes are quiescent butLeydig cells are hypertrophied and are clustered in groupsof 3–4 cells exterior to the seminiferous tubule (Gribbinset al., 2003). Spermatogonia, whether in the resting or earlyproliferative stages, were devoid of devoid of nuclearimmunoreactivity as were Sertoli cells.

ER� immunoreactivity was also present in the nuclei ofepithelial cells lining the eVerent duct system, including therete testis (not shown), eVerent ductules, and epididymalepithelium (Fig. 4). Reactivity was strongest in the eVerentductules and was present in all three regions of the eVerentductule. In contrast, it was absent from cells exterior to theeVerent ductules and from control tissues.

The epididymis of the turtle contains sperm throughoutthe spermatogenic cycle, and did so in the present study.The nuclei of both vesicular cells and basal cells of the epi-didymal epithelium were found to react positively with theER� antisera (Fig. 5); reactivity in both cell types washeterogeneous, being strong in some cells and weaker inadjacent cells. There was no diVerence in immunoreactivityof cells along the length of the epididymis.

3.3. ER�

ER� immunoreactivity was widespread throughout themale reproductive system. Western blots of pooled testicu-lar and epididymal tissue using the monoclonal Serotecanti-ER� preparation yielded four reactive bands at 100,50, 48, and 44 kDa. The 50 kDa band is close to the weightof human ER� (55 kDa). Within the inactive testis, bothspermatogonia and Leydig cells reacted strongly with theER� antisera (Fig. 6), and Sertoli cells reacted to a lesserdegree. In contrast, the myoid cells outside the seminiferoustubules exhibited no immunoreactivity. Epithelial cells ofthe rete testis also reacted strongly. Intense immunoreactiv-ity was seen in epithelial cells of both the eVerent ductulesand epididymis (Fig. 7), in the latter including both vesicu-lar cells and basal cells. Some of the myoid cells exterior tothe ducts also had ER� immunoreactivity, but the numberof reactive cells and their degree of immunoreactivity wasvariable.

4. Discussion

It is apparent from the results that the turtle testispossesses the rate-limiting enzyme necessary for estrogensynthesis and that the testis and excurrent canal system ofthe turtle possess both ER� and ER� proteins. Western

30 D.H. Gist et al. / General and Comparative Endocrinology 151 (2007) 27–33

blot analysis yielded an aromatase immunoreactive band at60 kDa, an ER� immunoreactive band at 50 kDa, and twoER� immunoreactive bands at 48 and 50 kDa. The posi-tions of these immunoreactive bands are suYciently closeto their predicted molecular weights that we are conWdentthat the reaction products described here represent bindingto the appropriate reptilian protein.

In the present study, aromatase immunoreactivity in thetestis was found in both Sertoli cells and Leydig but wasabsent from the germinal cells. Further, immunoreactivityvaried with the testicular cycle. In the turtle, germ cellsundergo spermatogenic events as a unit, and thus at anygiven time most of the germinal cells are at the same step ofthe spermatogenic cycle (Gribbins et al., 2003). The most

intense reaction in both Sertoli and Leydig cells was seen inthe quiescent testis. Sertoli cell immunoreactivity waslocated in the cytoplasm above the layer of spermatogonia.Following spermatogonial proliferation, which displacedthe immunoreactivity towards the lumen, the intensity ofSertoli cell immunoreactivity diminished during the meioticphase of the spermatogenic cycle, only to resume during thespermiogenic phase. A similar pattern of immunoreactivitywas observed in the Leydig cells outside the seminiferoustubules. Leydig cells are hypertrophied during testicularquiescence, and these cells exhibited a greater immunoreac-tivity at that time; a subsequent hypertrophy and increasein immunoreactivity occurred during the maturationalphase of the spermatogenic cycle. To the extent that

Fig. 2. Aromatase immunoreactivity at diVerent points in the spermatogenic cycle. (a) Premeiotic (quiescent). Inset: control testis without primary antisera.(b) Proliferative; (c) meiosis (early); (d) meiosis (late); (e) maturation; (f) postspermiation. Legend: Es, elongating spermatid; L, Leydig cell; m, maturesperm (in lumen); Rs, round spermatid; S, Sertoli cell; Sp, spermatogonium; Sl, leptotene spermatocyte. BarD 25 �m for all panels.

D.H. Gist et al. / General and Comparative Endocrinology 151 (2007) 27–33 31

enzyme presence can predict hormone synthesis and secre-tion, it would appear that estrogen production continuesthroughout the year except during the meiotic phase of thespermatogenic cycle. The timing of aromatase expression inthe turtle testis suggests that testicular estrogens might pos-sibly have a regulatory inXuence on the spermatogenicevents in the testis. Estrogens are increasingly implicated in

Fig. 5. ER� immunoreactivity in the epithelium lining the epididymis ofthe turtle T. scripta. Legend, v, nucleus of vesicular cell; b, nucleus of basalcell; p, pigment in muscular layer; c capillary; s, sperm in epididymislumen. Bar D 10 �m.

the control of spermatogonial proliferation in bothamphibian an reptilian testes, an eVect mediated by theexpression of regulatory genes (ChieY et al., 2000, 2002).

Estrogens secreted by testicular cells can enter the circula-tion, but it has recently been questioned whether the malereptile can secrete detectable quantities of female sex hor-mones (Lance et al., 2003). Alternatively, there is mountingevidence for a paracrine action of testicular estrogens, eitherintratesticular or downstream via the seminal Xuids (seeHess, 2003 for review). Estrogen action is mediated bynuclear receptors that in turn bind to nuclear hormoneresponse elements. Two forms of the estrogen receptor (ER�,ER�) exist in most vertebrates and three forms are found inteleosts (O’Donnell et al., 2001). In the present study, bothalpha and beta ER immunoreactivity were detected duringthe winter when testes were either quiescent or in the earlyproliferative stages of the spermatogenic cycle, and the excur-rent ducts were not receiving sperm. Alpha receptors wererestricted to the Leydig cells and the beta receptors werefound in both spermatogonia and Sertoli cells. In the mam-malian testis, ER� is localized mainly in the Leydig cellswhile ER� is found in most developing germ cells except forelongating spermatids (O’Donnell et al., 2001). Where inves-tigated in other vertebrate groups, ER� is more evenly dis-tributed among germinal and nongerminal (Leydig)elements, whereas ER� expression is higher in the premeiotic

Fig. 3. Photomicrograph of inactive testis showing pattern of ER� immunoreactivity. (a) Control; (b) anti-ER� antisera. Legend, L, Leydig cell; S, Sertolicell; Sp, spermatogonium; U, lumen of seminiferous tubule. Bar D 10 �m.

Fig. 4. ER� immunoreactivity in the epididymides and eVerent ductules of the turtle. (a) Control (no antisera); (b) anti-ER� antiserum. Legend: e, sperm(in lumen), d, eVerent ductules; p, pigment. BarD 100 �m.

32 D.H. Gist et al. / General and Comparative Endocrinology 151 (2007) 27–33

and meiotic germ cells (Arenas et al., 2001; Bouma andNagler, 2001; Wu et al., 2001; Wu et al., 2003; Choi and Hab-ibi, 2003; ChieY and Varriale, 2004). Thus the distribution ofER� and ER� in the turtle testis diVers only slightly fromthat in other vertebrate groups in that ER� is localized in theSertoli cell in addition to spermatogonia. The presence ofER�s in these two cell types in the quiescent testis of the tur-tle, coupled with the intense aromatase immunoreactivity atthe same time in Sertoli cells, supports the suggestion thatestrogen hormones might inXuence premeiotic spermato-genic events. A receptor-mediated role for estrogens in themammalian testis has yet to be deWnitively established, butthere is strong evidence suggesting a regulatory inXuence onspermatogenesis, particularly for ER� because of it’s pre-dominance within the seminiferous tubule (O’Donnell et al.,2001; Hess, 2003). Limited evidence from other vertebrategroups also point to a regulatory role in spermatogenesis,particularly the early events. Estradiol administration stimu-lates proliferation of spermatogonia in otherwise inactive tes-tes in teleosts, amphibians, and reptiles, and in each,tamoxifen or ICI can prevent the process (Minucci et al.,1997; Miura et al., 1999; ChieY et al., 2002); in the eel thisresponse is mediated by ER�.

Aromatase was absent from the excurrent ducts of theturtle reproductive tract, but the epithelium of all regionspossessed both ER� and ER�. This included the rete testis,eVerent ductules, and the epididymal epithelium. The short,aglandular ductus deferens connecting the epididymis to thepenile groove was not investigated. This is the Wrst localiza-tion of estrogen receptors in the excurrent duct system ofmale reptiles. The widespread distribution of both receptortypes in the turtle excurrent duct system is similar to what isobserved in bird (Kwon et al., 1997) and the mammal (seeHess et al., 2001), where both � and � estrogen receptors canbe coexpressed by the same cell in the eVerent ductule (Nieet al., 2002). Actions mediated by ER� in the mammalianexcurrent canals remain unclear, but the absence of absenceof ER� (�ERKO) results in infertility, apparently due todefects at the level of the eVerent ductules (Hess, 2003).

The presence of estrogen receptors in the excurrent ductsof all vertebrate groups examined to date suggests that what-ever eVects estrogens may have upon these ducts are phylo-genetically conserved and are possibly related to the physicalor physiological support of stored sperm. The turtle epididy-mis is not regionally diVerentiated as it is in the mammal(Holmes and Gist, 2004), but both receptor types were seen

Fig. 6. Photomicrograph of inactive testis showing pattern of ER� immunoreactivity. (a) Control; (b) anti-ER� antisera. Legend, L, Lydig cell; S, Sertolicell; Sp, spermatogonium; U, lumen of seminiferous tubule. Bar D 50 �m.

Fig. 7. Photomicrograph of epididymis and eVerent ductules showing pattern of ER� immunoreactivity. (a) Control; (b) anti-ER� antiserum. Legend:v, nucleus of vesicular cell, b, nucleus of basal cell, l, lumen of epididymis, p, pigment in muscular layers of epididymis and eVerent duct, c, blood vessel.e, efferent ductule. Bar D 100 �m.

D.H. Gist et al. / General and Comparative Endocrinology 151 (2007) 27–33 33

in the vesicular and basal cells throughout the length of theepididymis. It was somewhat surprising to observe the strongreceptor (both � and �) immunoreactivity in the excurrentcanals of the turtle, since the testes were quiescent and spermwere stored in the epididymis. However the turtle has anunusual reproductive cycle in that sperm produced in theautumn are stored overwinter in the epididymis to be used inspring matings (Gist et al., 2001). In fact, sperm are retainedin the epididymis throughout the year, and during this timeretain the same motility characteristics (Gist et al., 2000). Ifestrogens contribute to the maintenance of epididymalsperm, then it is to be expected that the epididymal cellswould retain sensitivity for long periods of time.

Note added in proof

A number of commercially available antibodies reactedwith turtle testicular aromatase, including R-10-2, a poly-clonal antibody directed against human placental aroma-tase and CYP1A1, directed against rat hepatic microsomeP450 enzyme. These and the other preparations reactedwith both Sertoli and interstitial cells in the turtle identi-cally to what is reported here. Best results were obtainedwith the CYP1A1 antibody.

Acknowledgment

Portions of this research were supported by Wnancialassistance award DEFC09-96SR18546 from the USDepartment of Energy to the University of GeorgiaResearch Foundation.

References

Ando, S., Ciarcia, G., Panno, M.L., Imbrogno, E., Tarantino, C., BuVone,M., Beraldi, E., Angelini, F., Botte, V., 1992. Sex steroid levels in theplasma and testis during the reproductive cycle of the lizard, Podarciss. sicula Raf. Gen. Comp. Endocrinol. 85, 1–7.

Arenas, M.I., Royuela, M., Lobo, M.V.T., Alfaro, J.M., Fraile, B., Pania-gua, R., 2001. Androgen receptor (AR), estrogen receptor-alpha (ER-�) and estrogen receptor–beta (ER- �) expression in the testis of thenewt, Triturus marmoratus marmoratus during the annual cycle. J.Anat. 199, 465–472.

Bouma, J., Nagler, J.J., 2001. Estrogen receptor-� protein localization inthe testis of the rainbow trout (Oncorhynchus mykiss) during diVerentstages of the reproductive cycle. Biol. Reprod. 65, 60–65.

Callard, G.V., Ryan, K.J., 1977. Gonadotropin action and androgen syn-thesis in enzyme dispersed cells of the turtle (Chrysemys picta). Gen.Comp. Endocrinol. 31, 414–421.

Callard, G.V., Canick, J.A., Pudney, J., 1980. Estrogen synthesis in Leydigcells: structural–functional correlations in Necturus testis. Biol.Reprod. 23, 461–479.

Callard, G.V., Pudney, J.A., Mak, P., Canick, J.A., 1985. Stage-dependentchanges in steriodogenic enzymes and estrogen receptors during sper-matogenesis in the testis of the dogWsh, Squalus acanthias. Endocrinol-ogy 117, 1328–1335.

Cardone, A., Angelini, F., Varriale, B., 1998. Autoregulation of estrogenand androgen receptor mRNA by estrogens and down regulation ofandrogen receptor mRNA by estrogen in primary cultures of lizardtestis cells. Gen. Comp. Endocrinol. 110, 227–236.

Carreau, S., Genissel, C., Belinska, B., Levallet, J., 1999. Sources of oestrogensin the testis and reproductive tract of the male. Int. J. Androl. 22, 211–223.

ChieY, P., D’Amato, G.L.C., Staibano, S., Franco, R., Tramontano, D.,2000. Estradiol-induced mitogen-activated protein kinase (extracellu-lar signal-regulated kinase 1 and 2) activity in the frog (Rana esculenta)testis. J. Endocrinol, 16777–16784.

ChieY, P., D’Amato, L.C., Guarino, F., Salvatore, G., Angelini, F., 2002.17�-estradiol induces spermatogonial proliferation through mitogen-activated protein kinase (extracellular signal-regulated kinase1/2) activityin the lizard (Podarcis s. sicula). Mol. Reprod. Dev. 61, 218–225.

ChieY, P., Varriale, B., 2004. Estrogen receptor � localization in the lizard(Podarcis s. sicula) testis. Zygote 12, 39–42.

Choi, C.Y., Habibi, H.R., 2003. Molecular cloning of estrogen receptoralpha and expression pattern of estrogen receptor subtypes in male andfemale goldWsh. Mol. Cell. Endocrinol. 204, 169–177.

Dufaure, J.P., Mak, P., Callard, I.P., 1983. Estradiol binding activity inepididymal cytosol of the turtle, Chrysemys picta. Gen. Comp.Endocrinol. 81, 61–65.

Gist, D.H., Turner, T.W., Congdon, J.D., 2000. Chemical and thermaleVects on the viability and motility of spermatozoa from the turtleepididymis. J. Reprod. Fertil. 119, 271–277.

Gist, D.H., Dawes, S.M., Turner, T.W., Sheldon, S., Congdon, J.D., 2001.Sperm storage in turtles: a male perspective. J. Exp. Zool. 292, 180–186.

Gribbins, K.M., Gist, D.H., Congdon, J.D., 2003. Cytological evaluation ofspermatogenesis and organization of the germinal epithelium in themale slider turtle, Trachemys scripta. J. Morphol. 255, 337–346.

Hess, R.A., Bunick, D., Lee, K.H., Bahr, J.M., Taylor, J.A., Korach, K.S.,Lubahn, D.B., 1997. A role for oestrogens in the male reproductivesystem. Nature 390, 509–512.

Hess, R.A., Bunick, D., Bahr, J., 2001. Oestrogen, its receptors and function inthe male reproductive tract-a review. Mol. Cell. Endocrinol. 178, 29–38.

Hess, R.A., 2003. Estrogen in the adult male reproductive tract: a review.Reprod. Biol. Endocrinol. 1, 52.

Holmes, H.J., Gist, D.H., 2004. Excurrent duct system of the male turtleChrysemys picta. J. Morphol. 261, 312–322.

Kent, G.C., 1973. Comparative Anatomy of the Vertebrates. Mosby, St.Louis. 378pp.

Kobayashi, T., Nakamura, M., Kajiura-Kobayashi, H., Young, G., Naha-hama, Y., 1998. Immunolocalization of steroidogenic enzymes (P450 scc,P450c17, P450 arom, and 3beta-HSD) in immature and mature testis ofrainbow trout (Oncorhynchus mykiss). Cell Tissue Res. 292, 573–577.

Kwon, S., Hess, R.A., Bunick, D., Kirby, J.D., Bahr, J.M., 1997. Estrogenreceptors are present in the epididymis of the rooster. J. Androl. 18,378–384.

Kwon, S., Hess, R.A., Bunick, D., Nitta, H., Janulis, L., Osawa, Y., 1995.Rooster testicular germ cells and epididymal sperm contain P450 aro-matase. Biol. Reprod. 53, 1259–1264.

Lance, V.A., Conley, A.J., Mapes, S., Steven, C., Place, A.R., 2003. Doesalligator testis produce estradiol? A comparison of ovarian and testicu-lar aromatase. Biol. Reprod. 69, 1201–1207.

Minucci, S., DiMatteo, L., ChieY, P., Pierantoni, R., Fasano, S., 1997. 17�-estradiol eVects on mast cell number and spermatogonial mitotic indexin the testis of the frog, Rana esculenta. J. Exp. Zool. 278, 93–100.

Miura, T., Miura, C., Ohta, T., Nader, M.R., Todo, T., Yamauchi, K., 1999.Estradiol-17� stimulates the renewal of spermatogonial stem cells inmales. Biochem. Biophys. Res. Comm. 264, 230–234.

Nie, R., Zhou, Q., Sassim, E., Saunders, P.K.T., Hess, R.A., 2002. DiVeren-tial expression of estrogen receptors � and � in the reproductive tractsof adult male dogs and cats. Biol. Reprod. 66, 1161–1168.

O’Donnell, L., Robertson, K.M., Jones, M.E., Simpson, E.R., 2001. Estro-gen and spermatogenesis. Endocrine Rev. 22, 289–318.

Wu, C., Ratino, R., Davis, K.B., Chang, X., 2001. Localization of estoregenreceptor � and � RNA in germinal and nongerminal epithelia of thechannel catWsh testis. Gen. Comp. Endocrinol. 124, 12–20.

Wu, K.H., Tobias, M.L., Thornton, J.W., Kelley, D.B., 2003. Estrogenreceptors in Xenopus; duplicate genes, splice variants, and tissue-spe-ciWc expression. Gen. Comp. Endocrinol. 133, 38–49.