Histochem Cell Biol (2004) 122:473–475DOI 10.1007/s00418-004-0706-9

O R I G I N A L P A P E R

Xinggui Tian · Sheng Cui · Jiali Liu · Shaoli Yi

Expression of estrogen receptors in the efferent ductuleof male sheep fetuses during gestation

Accepted: 24 August 2004 / Published online: 5 October 2004� Springer-Verlag 2004

Abstract There is as yet no report about the develop-mental changes of estrogen receptors (ERs) in the malereproductive system of the sheep fetus. In the presentstudy, the testis, efferent ductule, and epididymis of sheepfetuses were collected at days 70, 90, and 120 of gestationand in the newborn lamb. ER alpha (ERa) and ER beta(ERb) were detected by immunohistochemistry. The re-sults showed that ERb staining was negative in all ofthe examined tissues throughout gestation, whereas ERaimmunoreactivity was only located in the nuclei of theefferent ductule epithelium. In addition, both ERa stain-ing intensity and the number of ERa-positive cells werehigher at day 90 of gestation, compared with that atday 70 and at birth. These results suggest that estrogenmay play important roles in efferent ductule developmentin sheep fetuses.

Keywords Estrogen receptor · Testis ·Efferent ductule · Ovine

Introduction

It has been documented that estrogens are involved in thestructural and functional development of the male repro-ductive system (Fisher et al. 1998). The effects of estro-gen are mediated by its receptors (ERs), including ERaand ERb. ER expressions in the testis and epididymis ofhuman (Takeyama et al. 2001), mouse fetuses (Nielsen etal. 2000), and piglets (Nielsen et al. 2001) have been

described. But there is as yet no published informationabout the ontogeny of ER expression in the male repro-ductive system of sheep fetuses. This experiment wastherefore designed to examine the developmental changesof ERa and ERb in the testis, efferent ductule, and epi-didymis of sheep fetuses by immunohistochemistry.

Materials and methods

After the animal use in and experimental design for this study wasapproved by the Chinese Association for Laboratory Animal Sci-ences, pregnant Chinese Xiaowei ewes with known single insemi-nation dates were anesthetized with sodium phenobarbital ondays 70, 90, and 120 of gestation (term=147 days). The fetuseswere exposed by regular caesarean operation. The testis, efferentductule, and the epididymis were then collected by surgical oper-ation from four male fetuses at every stage. In addition, four malelambs were killed with an overdose of sodium phenobarbital on thesecond day after birth and the same tissue specimens as in fetuseswere collected. The collected tissues were fixed in 4% (w/v)paraformaldehyde in phosphate-buffered saline for 24 h and thenprocessed to paraffin wax. Five-micron-thick sections were cut forER immunohistochemical detection.

The procedure for ERa immunohistochemical detection wassimilar to that in our previous reports (Sheng et al. 1998; Cui andGoldstein 2000). Briefly, after the antigen retrieval was performedby microwaving, the tissue sections were treated with 3% H2O2 andnon-specific binding was blocked with 10% normal rabbit serum.Monoclonal anti-ERa antibody (NCL-ER-LH2; Novocastra, UK;1:70) were subsequently added and incubated for 24 h at 4�C. Thesections were then incubated with biotinylated rabbit anti-mouseIgG (Sigma) for 2 h at room temperature. Lastly, ABC complex(Vector Laboratories) was added for 1 h and the peroxidase activitydetected using DAB. ERb was detected with anti-ERb polyclonalantibody (#06-629; Upstate Biotechnology, USA). The immuno-genic peptide corresponding to amino acids 96–113 of ovine ERb(Cardenas et al. 2001), which was used to determine ERb, showsapproximately 93% homology and does not crossreact with ERa.Western blot analysis with #06-629 indicated that the major form ofERb in the sheep adrenal gland extracts was approximately 53 kDa.In addition, immunohistochemistry staining results with #06-629showed that the ERb is expressed in the nuclei of sheep adrenalgland cells (our unpublished data). These results have confirmedthat #06-629 is specific to sheep ERb. The ERb detecting proce-dure was similar to that for ERa except that the secondary antibodywas biotin-conjugated swine anti-rabbit IgG (Dako, Denmark;1:300). The controls included the omission of the primary, sec-

X. Tian · S. Cui ()) · J. Liu · S. YiDepartment of Animal Physiology,College of Biological Sciences, China Agricultural University,100094 Beijing, People’s Republic of Chinae-mail: cuisheng@cau.edu.cnTel.: +86-10-62892668Fax: +86-10-62893443

S. CuiFaculty of Veterinary Medicine,China Agricultural University,100094 Beijing, People’s Republic of China

ondary, and tertiary antibodies, all of which eliminated staining(data not shown).

In order to determine the proportions of the ERa-positive cellnumber accounting for the total epithelial number, 20 round ornearly round efferent ductule cross-sections were chosen randomlyfrom every specimen, and the ERa-positive cell number and totalepithelial number of efferent ductules were counted, as describedby Franca and Godinho (2003). The results were presented as apercentage (mean € SEM) of ERa-positive cells accounting for thetotal epithelial number. Statistical differences were assessed byone-way ANOVA, followed by Student’s t-test, and P<0.05 wasconsidered significant.

Results and discussion

The present study first detected the ontogeny of ERa andERb expressions in the reproductive organs of male sheepfetuses, including testis, epididymis, and efferent ductule.The results demonstrated that ERa was detected only inthe efferent ductule of all examined tissues throughoutgestation (Fig. 1). This is in accordance with the reportsthat ERa expresses in the efferent ductules of fetal mice(Nielsen et al. 2000), piglets (Nielsen et al. 2001), cy-nomolgus macaque monkeys (West and Brenner 1990),and goats (Goyal et al. 1997). ERb immunoreactivity wasnegative in all of the examined tissues at every age ex-amined (data not shown), which corresponds to the resultsfrom human fetal testis and epididymis (Takeyama et al.2001). However, there have been reports about ERa andERb expressions in the testis and epididymis of human(Takeyama et al. 2001) and other mammals, such as rat(Pelletier et al. 2000; Oliveira et al. 2003), most of whichdid not detail ER expressions in the efferent ductule.

These controversial results about ER expressions may bedue to the various species, ages, and the methodologicaldifferences.

Another finding of the present experiment is that theintensity of ERa immunoreactivity is obviously lower atday 70 (Fig. 1A, B) of gestation and at birth (Fig. 1E, F)than at day 90 (Fig. 1C, D) and day 120 of gestation (datanot show). The percentage of ERa-positive cells of ef-ferent ductule epithelia accounting for total efferentductule epithelial cells was 72.3€2.14% at day 70 ofgestation, the earliest age examined. It rose to 91.1€1.27% by day 90 of gestation, significantly higher thanat day 70 (P<0.01), and then did not change untilday 120, after which it decreased to 80.9€3.06% at birth.These results demonstrate that both ERa-immunoreac-tive intensity and ERa-immunopositive cell number arehigher at about the middle stage of gestation than at earlyand late stages of gestation. In addition, the abovechanging pattern of ERa expression is parallel to thechange of plasma estrogen concentrations during gesta-tion (Alexander et al. 1973); these suggest that estrogenhas roles in the development and functions of the efferentductules by combining with ERa. In support of this, theERa knockout mouse shows developmental anomaliesand dysfunctions of efferent ductules, and ER blockagewith ICI 182,780 results in morphological changes ofmouse efferent ductules (Lee et al. 2000).

It has been reported that the epithelium of the effer-ent ductules consists of ciliated and nonciliated cells inmammals (Ilio and Hess 1994). The present results showthat over 80% of the efferent ductule epithelial cells wereERa positive throughout gestation. This seems very con-

Fig. 1A–I Immunohistochemi-cal localization of estrogen re-ceptors (ERs) in the efferentductule, testis, and epididymisof sheep fetuses. ERa is de-tected in the nuclei (brown) ofthe efferent ductule epithelia atday 70 (A, B) and day 90 (C, D)of gestation and of the newbornmale lamb (E, F), but it is im-munonegative in testis (H) andepididymis (G) throughoutgestation. ERb is immunoneg-ative in testis, efferent ductuleepithelia (data not show), andthe epididymis (I). Scale bar100 mm in A, C, E, G–I; 40 mmin B, D, F

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sistent with the report that only nonciliated epithelial cellsexpress ERa (Goyal et al.1997), although we did notidentify the cell types expressing ERa in the efferentductule epithelium.

Acknowledgements This work was supported by grants from theNatural Science Foundation of China (30170693) and the NaturalScience Foundation of China for Outstanding Young Scientists(30325034).

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