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BOR - Papers in Press, published online ahead of print June 8, 2005.
Biol Reprod 2005, 10.1095/biolreprod.105.042796
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BIOLOGY OF REPRODUCTION 73, 703–712 (2005)
DOI: 10.1095/biolreprod.105.042796
© 2005 by the Society for the Study of Reproduction, Inc.

Expression of Estrogen Receptor ESR1 and Its 46-kDa Variant in the Gubernaculum Testis1

Christophe Staub 2, 5, Michel Rauch 3, 5, François Ferrière 3, 6, Mélanie Trépos 5, Isabelle Dorval-Coiffec 5, Philippa T. Saunders 7, Gilda Cobellis 4, 5, Gilles Flouriot 6, Christian Saligaut 6, and Bernard Jégou 5

INSERM,5 U625, GERHM, IFR 140, Campus de Beaulieu, Univ Rennes I, Rennes, Bretagne F-35042, France UMR-CNRS 6026,6 IFR 140, Campus de Beaulieu, Univ Rennes I, Rennes, Bretagne F-35042, France MRC Human Reproductive Sciences Unit,7 Centre for Reproductive Biology, Edinburgh EH16 4SB, United Kingdom


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Testicular descent corresponds to migration of the testis from the abdominal cavity to the scrotum and is essential for proper functioning of the testis. Recent advances in the characterization of estrogen receptor (ESR) subtypes and isoforms in various tissues prompted us to study ESRs within the gubernaculum testis, a structure involved in testicular descent. In the rat gubernaculum, we searched for ESR alpha (Esr1) and beta (Esr2) and for the androgen receptor (Ar), androgens being known to regulate testicular descent. Reverse transcription-polymerase chain reaction (RT-PCR) revealed that Esr1, Esr2, and Ar mRNAs were all expressed in the gubernaculum. Using PEETA (Primer extension, Electrophoresis, Elution, Tailing, and Amplification), we established that all Esr1 leader exons, previously identified in other organs, such as the uterus and pituitary, were transcribed in the gubernaculum, with the major form being O/B. The RNA protection assays, RT-PCR, and Western blot experiments revealed that isoform-specific mRNA transcripts generated by alternative splicing of the C-leader sequence on coding exons 1 and 2 of the Esr1 gene gave the 46- and 66-kDa ESR1 proteins. The ESR1 and AR proteins were found to colocalize in the parenchymal cells of the gubernaculum early in development, whereas AR also was strongly expressed in the muscular cells, both during fetal and postnatal life. The ESR2 protein was weakly expressed, principally in the muscular cells, but only once testicular descent had occurred. The levels of the 46-kDa ESR1 variant (ER46) exceeded those of the 66-kDa ESR1 form (ER66) at periods when the gubernaculum developed. Conversely, the 66-kDa form appears to predominate clearly when the gubernaculum growth was low or completed. The possible role of estrogens on the modulation of the androgen-dependent growth of the gubernaculum and, more widely, on testicular descent is discussed.

androgen receptor, developmental biology, estradiol receptor, male reproductive tract


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
During the last decade, concern has been rising over increases in the number of abnormalities associated with the human urogenital tract [1–3]. One of the abnormalities becoming more frequent is malpositioning of the testes, a condition called cryptorchidism [4, 5]. Cryptochidism leads to hypofertility and sterility and is a major risk factor for testicular cancer, the incidence of which also is increasing throughout the world [5, 6]. At present, the cause of cryptorchidism is unclear. A better understanding of the mechanisms controlling the process of gonad descent—a process defined by Scorer [7] as "the movement of the testis from the abdominal cavity to the bottom of a fully developed and fully relaxed scrotum"—is needed before the cause of this condition can be elucidated.

During the initial phase of fetal development, the testes are in the renal position, held by the cranial suspensory ligament (CSL) at the upper pole and the caudal genital ligament (CGL) at the lower pole. The CGL is comprised of parenchymal cells, which form a cord making up part of the gubernaculum organ. This organ also consists of a bulb of mesenchymal cells, which differentiate into the cremaster muscle; and the cremaster muscle is involved in the final positioning of the testes within the scrotum. The initial phase of transabdominal testicular descent, which is controlled by insulin-like factor 3 (INSL3) and androgens, results in the testis being positioned in the lower abdomen during embryo growth [8–10]. The INSL3 controls gubernaculum swelling via its receptor, LGR8 (leucine-rich repeat containing G protein-coupled receptor 8 also known as GREAT or relaxin receptor 2) [11, 12], a process resulting in thickening of the CGL because of increases in water, glycosaminoglycan, and hyaluronic acid content [13]. Androgens mediate the beginning of the regression phase of the CSL [14]. During the second phase of transabdominal descent, androgens trigger the end of the CSL regression phase and stimulate growth of the gubernaculum bulb and, thus, differentiation of the muscular part of the gubernaculum. The traction resulting from this growth facilitates movement in the inguinoscrotal phase of the testicular descent [9, 15–17].

Several lines of evidence indicate that estradiol, diethylstilbestrol (DES), and estrogenic compounds in general also are involved in mediating testicular descent. For example, administration of estradiol [18, 19] or DES [20, 21] to gestating female rats inhibits testicular descent, leading to cryptorchidism in the male offspring. Both gubernaculum swelling and transabdominal testicular migration are blocked by this prenatal exposure to estrogens [19]. The exposure of pregnant women to DES leads to an increase in the incidence of cryptorchidism in male children [22]. There also may be a relationship between increased levels of placental estradiol and the occurrence of cryptorchidism in the exposed babies [23, 24]. Furthermore, transgenic male mice, which overexpress Cyp19a1 (human aromatase) and, thus, exhibit elevated serum estradiol concentrations, display several reproductive tract abnormalities, including cryptorchidism [25]. Finally, evidence recently has accumulated that indicates some xenoestrogens (the so-called endocrine disruptors) are involved in the occurrence of different types of abnormalities of the urogenital tract, including cryptorchidism [3, 26, 27]. The effects of estrogens on testicular descent generally are believed to be indirect, resulting from inhibition of LH production and, consequently, testosterone secretion [28] and/or from inhibition of fetal INSL3 production in the Leydig cells [29–31]. However, the finding that Esr1 knockout male mice have retracted gonads [32] suggests that estrogens play a direct role in the scrotal positioning of the testis.

The present study searched for the presence of the estrogen receptors ESR1 and ESR2 within the rat gubernaculum testis as an indication of the possible direct implication of estrogens in the process that leads to final positioning of the testis. We demonstrate that whereas ESR2 protein was not detected in the fetal gubernaculum, high expression levels of ESR1 were found in the parenchymal cells of the gubernaculum testis, colocalized with the androgen receptor (AR), from 16 days postcoitum (dpc) to 22 days postpartum (dpp). Two isoforms of ESR1, namely ER66 and ER46, were characterized. We hypothesize that changes in their expression during gubernacular development and their possible competitive interaction could represent a mechanism by which estrogens modulate the action of androgens during the different phases of testicular descent.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Sample Collection

Timed pregnant female Sprague-Dawley rats were purchased from Elevage Janvier (Le Genest Saint Isle, Laval, France). They were anesthetized by an i.p. injection of 40 mg/kg of sodium pentobarbital (Sanofi-Synthélabo, Libourne, France). Gubernaculi were microdissected from rat fetuses aged 16, 17, 18, 20, and 21 days dpc and 0.5, 3, 4, 8, 12, and 22 days dpp under a binocular microscope (Olympus B061). After dissection, gubernaculi were immediately frozen in liquid nitrogen and either used to obtain mRNA and protein extracts, fixed in Bouin fluid, and processed into paraffin wax or cryopreserved in Tissue-Teck O.C.T. (Sakura Finetek Europe BV, The Netherlands) for immunohistochemical analysis. All experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (Institute for Laboratory Animal Research [ILAR] of the National Academy of Sciences, Bethesda, MD).

RNA Extraction and Reverse Transcription-Polymerase Chain Reaction Analysis

Total RNA was extracted from each gubernaculum using the method described by Chomczynski and Sacchi [33]. Sequences encoding Esr1 (estrogen receptor {alpha}), Esr2 (estrogen receptor ß), Ar (AR), Cyp19a1 (aromatase), Srd5a1 (5{alpha}-reductase isoform I), and Actb (ß-actin) mRNA were amplified by reverse transcription-polymerase chain reaction (RT-PCR) using the primers listed in Table 1.


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  		TABLE 1. Oligonucleotides used in the RT-PCR experiments

Complementary DNA was prepared from 4 µg of RNA using 40 ng of random hexanucleotides (Boehringer Mannheim, Germany) and 200 U of Moloney murine leukemia virus-reverse transcriptase (M-MLV-RT; Promega, Madison, WI) according to the manufacturer's instructions. The PCR was carried out on 40 ng of cDNA in a final volume of 25 µl containing 0.6 U of Taq polymerase (Qiagen, Courtaboeuf, France) and 0.2 µM of each appropriate 5' and 3' sequence-specific primers (Table 1). Actin amplification was performed as a control for RNA quality, quantity estimation, and RT efficiency. The samples were denatured at 94°C for 5 min. Amplification was carried out using 35 or 40 standard PCR cycles with a 58°C annealing temperature using a thermal cycler. Aliquots of 15 µl of each PCR sample were subjected to electrophoresis on 1.8% agarose gels. Product size was determined by comparison to markers (100-base pair [bp] DNA ladder; New England Biolabs). Bands were visualized after staining with 0.5 mg/ml of ethidium bromide by observation under ultraviolet illumination (Multimage Light Cabinet; Alpha Innotech Corporation, San Leandro, CA). The RT-PCR products were identified by sequencing using the BigDye system (Applied Biosystems, Foster City, CA) and an ABI 310 sequencer (PerkinElmer, Wellesley, MA).

Long Esr1 cDNA Amplifications

Complementary DNA was synthesized from 1 µg of total RNA by RT-PCR using 50 U of expand reverse transcriptase and the conditions recommended by the supplier (Boehringer Mannheim) with the oligonucleotide primer R1 from the 3'-untranslated region (UTR; exon 8) of rEsr1 mRNAs (Table 2; see Fig. 4A). An aliquot (2 µl) of the RT product was then used as a template for two rounds of 35 cycles of PCR amplification. The first round was done in the presence of the C1 and R2 primer pair; the second round was done using the nested primers, C2 and R3 (Table 2; see Fig. 4A). The 5'-primers (C1 and C2) used for the C-rEsr1 cDNA amplification were designed using the published sequence for exon C (GenBank accession no. X98237) [34]. The 3'-primer R2 and the nested primer R3 were designed from the 3' region common to all rEsr1 cDNAs, located immediately upstream from the region used to design the RT primers (see Fig. 4A). Both rounds of amplification were performed using the Expand Long Template PCR System (Boehringer Mannheim) and the conditions recommended by the manufacturer. Ten-microliter aliquots from each reaction were analyzed on a 1% agarose gel stained with ethidium bromide.


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  		TABLE 2. Oligonucleotides used in primer extension, S1 nuclease mapping, and long cDNA amplification of Esr1



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  		FIG. 4. Long template PCR. A) Genomic organization of the rat Esr1 gene showing the position of the oligonucleotides C1, C2, R1, R2, and R3 used. B) Presence of rER66 and rER46 mRNA in the gubernaculum (Gub), MMQ cells (MMQ; positive control), pituitary gland (Pit), uterus (Ut), and liver (Liv). The tRNA was used as a negative control (Control). Both rER66 and rER46 mRNA transcripts were detected in the 20.5-dpc gubernaculum. C) Schematic representation of the structures of the full-length and the amino-terminal truncated rESR1 proteins. The A/B domain is responsible for AF1 transactivation and is missing from the rER46 protein. The 6F11 antibody, which is directed against this amino-terminal domain of the protein, did not recognize the truncated isoform. The exon C domain constitutes the DNA-binding domain (DBD) of the protein. The D domain contains the nuclear localization sequence (NLS), and the E domain constitutes the ligand-binding domain (LBD). The MC20 antibody is directed against the carboxy-terminal F domain and, thus, recognizes both ESR1 isoforms

Modified Primer Extension and S1 Nuclease Mapping of Esr1

The strongly labeled, single-stranded DNA probe needed for S1 nuclease mapping and the long labeled primer used for primer extension were obtained using a specific primer and T7 DNA polymerase in the presence of [{alpha}-32P]dCTP (3000 Ci/mmol) [35, 36]. The probe (0/B) or the long primer ({Sigma}) were then allowed to hybridize to the appropriate RNA samples and subjected to an S1 nuclease digestion or to reverse transcription, respectively.

The {Sigma} long primer was generated by PCR using 1 ng of a vector containing the rEsr1 open reading frame and two specific primers: a biotinylated 5'-primer F1, and a downstream primer R4 located in exon 2 (Table 2; see Fig. 2A).



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  		FIG. 2. Expression of 5'-UTR mRNA isoforms of the rat estrogen receptor-{alpha} (rEsr1) in the liver, uterus, pituitary gland, and gubernaculum. A) Genomic organization of the 5' region of the rat Esr1 gene. The position of all upstream exons described so far is shown. The ATG start codon, the acceptor splice site in exon 1, and the position of the F1, R4, and R5 primers are indicated. Broken lines symbolize splicing sites. The corresponding transcripts are shown as well. B) Total RNA from yeast (negative control), liver (Liv), uterus (Ut), pituitary (Pit), and gubernaculum (Gub) was allowed to hybridize to primer {Sigma} and subjected to RT. The extension products were then separated on a sequencing gel. The extension products corresponding to transcripts I, II, III, IV, and V are indicated. The distribution pattern and intensities of expression of the Esr1 leader exons are similar between the gubernaculum and the pituitary

The 0/B probe was generated by PCR using the rEsr1 cDNA vector and two specific primers: F8 specific to the exon 0/B in the mRNA of the rEsr1 isoform, and primer R3 located in exon 8 (Table 2; see Fig. 3). The RT-PCR product was subcloned into the TA cloning vector pCR3.1 (Invitrogen, Carlsbad, CA) downstream of the T7 promoter. Finally, the 0/B probe was prepared by PCR using the biotinylated T7 and R3 primer pair.



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  		FIG. 3. S1 nuclease protection assays. The principles of the (0/B-1) rEsr1 mRNA detection method are shown on the left. The size and position on the gel of the single-stranded 0/B probe and protected fragments after S1 digestion of the probe/rEsr1 mRNA hybrids also is shown, as are the methionine start codon (AUG) and the position of F8 and R3 primers. Open boxes represent the exons (1–8) encoding rEsr1, and the gray box represents the unique 0/B sequence. Experiments were carried out on total RNA extracts from liver (Liv), uterus (Ut), pituitary (Pit), gubernaculum (Gub), and yeast (negative control). Extracts were allowed to hybridize to the labeled 0/B probe and then treated with S1 nuclease. The undigested (protected) hybrids were then separated on a sequencing gel (right). The undigested probe is shown as well. The leader exon 0/B is spliced on coding exon 1 in the uterus, pituitary, and gubernaculum. Other leader sequences also are spliced at the coding exon 1 site in these tissues. The 0/B leader sequence was not found in the liver, indicating that Esr1 mRNA is produced from a different leader sequence in this tissue

All biotinylated PCR products were isolated on the basis of their binding to streptavidin-coated magnetic beads (Dynal Biotech, Oslo, Norway). This step was carried out as recommended by the manufacturer, and the nonbiotinylated DNA strands were removed using 0.1 M NaOH.

Probe 0/B and {Sigma} primer were then extended by allowing the R4 primer to anneal to the corresponding biotinylated, single-stranded template. After elution of the single-stranded DNA probe and primer by alkaline treatment and magnetic separation, 2 x 105 cpm of the probe or primer were coprecipitated with 30 µg of total RNA and then dissolved in 20 µl of hybridization buffer (80% formamide, 40 mM PIPES [pH 6.4], 400 mM NaCl, and 1 mM EDTA [pH 8]). The probe and primer were then denatured by incubation at 65°C for 10 min. Hybridization reactions were carried out overnight at 55°C.

The S1 digestion and reverse transcription were carried out as previously described [37]. Products were separated by electrophoresis on denaturing polyacrylamide/urea gels.

The most abundant extension products were cut out and eluted from the gel using 500 µl of 0.5 M ammonium acetate, 10 mM magnesium acetate, 1 mM EDTA (pH 8), and 0.1% SDS and then extracted using the Qiaex II gel extraction kit (Qiagen). A poly(C) tail was then added to each of the extension products to allow subcloning into the pCR3.1 plasmid vector (Invitrogen) for sequencing of the product from a PCR using an oligo(dG) adapter primer and a specific primer R5 located in exon 1 (Table 2; see Fig. 2A). All clones were sequenced.

ESR1 Western Immunoblot Analysis

Gubernaculum lysates were prepared using ice-cold lysis buffer (50 mM Tris [pH 6.8], 5 M urea, and 2% SDS; Bio-Rad Laboratories, Hercules, CA) and 1 M dithiothreitol (Quantum-Appligen, Illkirch, France) and then centrifuged at 105 000 x g for 1 h at 4°C. The protein concentration in the supernatants was determined using a red-dot-blot protein-assay technique based on ponceau S staining [38]. Equal amounts of protein from each extract were subjected to electrophoresis on 8% (w/v) SDS-PAGE under reducing conditions. The proteins were then electrotransferred (over 2 h) onto nitrocellulose membranes (Immobilon-PSQ; Millipore, Bedford, MA). Membranes were incubated overnight at 4°C with Tris-buffered saline (TBS [pH 7.6]; 20 mM Tris, 1.4% HCl, and 130 mM NaCl) containing 3% bovine serum albumin, fraction V (BSA; Eurobio, Les Ulis, France) to block nonspecific binding. They were then incubated for 2 h at room temperature in TBS containing 0.1% BSA, 0.2% Tween 20 (Sigma, St Louis, MO), and either the NCL-ER-6F11/2 (final dilution, 1:100; Novocastra, Newcastle upon Tyne, UK) or the MC20 (final dilution, 1:1000; Santa Cruz Biotechnology, Santa Cruz, CA) anti-ESR1 antibodies or an anti-ß-tubulin antibody as a standardization control (final dilution, 1:2000; Pharmingen, BD Biosciences, Franklin Lakes, NJ). After washing with TBS containing BSA (0.1%) and Tween-20 (0.2%), the PSQ membranes were incubated for 1 h at room temperature in TBS-0.1% BSA-0.2% Tween-20 with horseradish peroxidase (HRP)-conjugated anti-rabbit (NA9340V) or anti-mouse (NA931V; final dilution, 1:10 000; Amersham Biosciences, Little Chalfont, Buckinghamshire, UK) antibodies. After washing in TBS-0.1% BSA-0.2% Tween-20, the signal was detected using enhanced chemiluminescence plus Western immunoblot detection system (Enhanced Chemiluminescence System; Amersham Biosciences, Little Chalfont, Buckinghamshire, UK) and Kodak Biomax MR scientific imaging film (Kodak, New Haven, CT). The films were scanned with an Epson Perfection 1200 photo scanning system (Epson France S.A., Levallois Perret, France). The intensity of the bands were then measured using the Alpha Ease FC Stand Alone software (Alpha Innotech, San Leandro, CA).

Immunohistochemistry

Cryosections (thickness, 7 µm) were cut using a cryostat (Cryo-Star HM 560 M; Microm, Walldorf, Germany) at –20°C and then collected on SuperFrost Plus microscope slides (CML, Nemours, France) and air-dried. Sections were then fixed using 3.7% formaldehyde in PBS for 10 min, quickly immersed in cold methanol for 2 min, and stored in PBS/glycerol (v/v) containing 250 mM sucrose and 7 mM MgCl2 at –20°C until further processing. After washing in PBS, sections were incubated for 20 min at room temperature in PBS containing 1% BSA to prevent nonspecific protein binding. Sections were washed with PBS containing 0.1% BSA. Serial sections were incubated overnight at 4°C in PBS containing 0.1% BSA and one of the following primary antibodies: the NCL-ER-6F11/2 anti-ESR1 antibody (final dilution, 1:10; Novocastra), the MC20 anti-ESR1 antibody (final dilution, 1:300; Santa Cruz Biotechnology), or the M22A anti-AR antibody (final dilution, 1:200). This M22A antibody was obtained from a mouse monoclonal hybridoma and was selected by immunoprecipitation of cytosolic tritiated R1881 calf AR complex. It binds to AR on Western blots and has been used on rat prostate as positive control (not shown). Incubation with mouse immunoglobulin (IgG1) (X0931; DAKO, Glostrup, Denmark) was used as a negative control. The sections were then washed with 0.05% Tween 20 in PBS, incubated first with biotin-labeled anti-rabbit (E0432) or anti-mouse (E0464) secondary antibodies (final dilution, 1:500; DAKO) for 60 min at room temperature, then with streptavidin-HRP (P0397; final dilution, 1:500, DAKO) for 30 min at room temperature, and finally with diaminobenzidine (DAB) for 10 min (Sigma Fast 3,3' DAB tablet sets; Sigma). After mounting in Eukitt (Kindler, Freiburg, Germany), the sections were analyzed using a bright-field Olympus AX60TF microscope with monochromatic objectives (Olympus, France) and photographed using a digital macro camera (Kigamo).

For ESR2 immunohistochemistry, we used a sheep polyclonal antibody that previously demonstrated cell-specific patterns of expression in the male urogenital tract of adult mice [39]. Briefly, sections (thickness, 5 µm) were mounted on charged slides (Superfrost; BDH Laboratory Supplies, Poole, UK) and subjected to heat-induced antigen retrieval in 10 mM citrate (pH 6; 5 min at full pressure and then allowed to stand for 20 min). Endogenous peroxidases were blocked by incubating slides in a solution of 3% (v/v) hydrogen peroxide (in methanol) for 30 min on a rocker platform. Sections were washed in water and in TBS (0.05 M Tris [pH 7.4] and 0.85% NaCl) for 5 min. Tissue was then blocked in normal rabbit serum (NRS)/TBS/BSA (Diagnostics Scotland, Carluke, Lanarkshire, UK) diluted 1:4 in TBS plus 5% BSA containing four drops per 1 ml of avidin for 30 min at room temperature (blocking kit SP-2001; Vector Avidin/Biotin, Peterborough, UK). Two 5-min TBS washes were followed by blocking in biotin (four drops/1 ml of TBS) for 20 min. The sections were then washed twice in TBS, then incubated for 16 h at 4°C with sheep anti-estrogen receptor ß diluted 1:500 in TBS. Sections were washed twice for 5 min each time in TBS and incubated with rabbit anti-sheep IgG (Vector, Peterborough, UK) diluted 1:500 in NRS/TBS/BSA for 1 h. Sections were washed in TBS (5 min each wash) incubated in avidin-biotin complex-HRP complex (DAKO) for 30 min, then washed in TBS (two washes for 5 min each wash), and bound antibodies were visualized by incubation with 3,3'-diaminobenzidine tetrahydrochloride (liquid DAB; catalog no. K3468; DAKO). Sections were counterstained with hematoxylin. Images were captured using an Olympus Provis microscope (Olympus Optical Co., London, UK) equipped with a Kodak DCS330 camera (Eastman Kodak, Rochester, NY), stored on a Macintosh PowerPC computer (Apple, Cupertino, CA), and assembled using Photoshop 7 (Adobe, Mountain View, CA).

Statistics

Differences among the p66:p46 ratios between the three characterized phases were analyzed by the nonparametric Mann-Whitney test. Analyses were made using the Statview 5.0 software (SAS Institute, Inc., Cary, NC).


   RESULTS
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
ESR1 Is Expressed Within the Gubernaculum

The expression of Esr1, Esr2, Ar, Cyp19a1, and Srd5a1 in the gubernaculum was investigated by RT-PCR analysis throughout development (Fig. 1). Esr1, Ar, and Srd5a1 were found at all investigated stages when 35 PCR cycles were performed. Esr2 was only detected in the gubernaculi dissected from 18- and 20-dpc rats when 35 PCR cycles were carried out but was detected in the gubernaculi dissected from 18-dpc to 22-dpp rats after 40 PCR cycles. Cyp19a1 was never detected at any stage or in any condition.



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  		FIG. 1. Detection of Esr1, Esr2, Ar, Cyp19a1, Srd5a1, and Actb mRNA by RT-PCR analysis at various stages of gubernacular development. Positive controls were as follows: oviducts (Ovi) from 23-dpp rats for Esr1, ovaries (Ova) from 23-dpp rats for Esr2 and Cyp19a1, and epididymis (Epi) from 15-dpp rats for Ar and Srd5a1. Negative controls were examined by PCR performed with mRNA (–) and water (0) instead of cDNA. Numbers in parentheses are the expected product sizes (see Table 1); the letters a and b correspond to 35 and 40 amplification cycles, respectively. The RT-PCR products were sequenced using the BigDye system (Applied Biosystems, Foster City, CA) and an ABI 310 sequencer (PerkinElmer, Wellesley, MA)

Bearing in mind the complex genomic organization of the Esr1 gene, in which multiple promoters have been identified [40], we first studied what was the major leader exon in the gubernaculum. The most abundant extension products were then sequenced, and analysis revealed the presence of several rEsr1 mRNA isoforms with different 5'-UTRs. rEsr1 mRNA was abundant in the gubernaculum, and all previously described leader exons were present (Fig. 2). The pattern of distribution and cumulative intensity of the rEsr1 mRNA in the gubernaculum were similar to those found in the pituitary and uterus. In contrast, only a few leader sequences were found in the liver (Fig. 2). The 0/B was the leader exon preferentially transcribed in both the pituitary and the gubernaculum.

To confirm the relative predominance of 0/B rEsr1 mRNA isoform over the other isoforms expressed in the gubernaculum, we carried out an S1 nuclease-mapping experiment using a single-stranded DNA probe that was specific for 0/B rEsr1 mRNAs (0/B probe). The S1 nuclease protection assay experiments confirmed our earlier finding that high levels of rEsr1 mRNA were produced in the gubernaculum and that, as in the uterus, the 0/B exon leader sequence predominated in the gubernaculum (O/B mRNAs represent >50% of total protected fragments) (Fig. 3). In addition to the large fragment corresponding to isoforms with the 0/B exon leader sequence, we also isolated a shorter protected fragment, indicating that other 5'-UTR isoforms also were produced in the tissues tested (Fig. 3). The 0/B leader exon was not found in the liver, indicating that Esr1 mRNA is produced from a different leader sequence in this organ.

Two Isoforms of ESR1 Are Present Within the Gubernaculum

Because the Esr1 gene is able to generate by alternative splicing at least two different isoforms of the ESR1 protein, RT-PCR analysis were performed to analyze the overall pattern of rEsr1 expression in the gubernaculum. Full-length rEsr1 cDNA (rER66) was detected in all the tissues and cells tested (Fig. 4B). In addition, a shorter amplicon also was detected in the gubernaculum, MMQ cells, and the liver. This amplicon was approximately 600 bp shorter than the full-length cDNA. Sequencing of the corresponding cDNA sequences revealed that this difference resulted from the deletion of exon 1 (538 bp) and the splicing of the exon C leader sequence on coding exon 2 (rER46).

Western blots of ESR1 were performed with the MC20 and 6F11 antibodies directed against the carboxy- and amino-terminal, respectively, of the ESR1 protein. The MC20 antibody recognized the two ESR1 isoforms, whereas the 6F11 antibody recognized only the full-length protein (Fig. 4C). When the Western blots were performed with the MC20 antibody, strong signals were observed for the 66-kDa isoform in gubernaculum aged from 17 to 20 dpc and for the 46-kDa isoform in gubernaculum aged from 19 dpc to 3 dpp (Fig. 5A). The 66-kDa protein was consistently barely detectable between 4 and 12 dpp but was abundant again by 22 dpp. The same pattern of expression was observed for this full-length ESR1 using the 6F11 antibody (not shown). In contrast, the 46-kDa protein was either very weakly or not detected after 8 dpp. In all the Western blot experiments, a 55-kDa nonspecific band was detected with both ESR1 antibodies. This unspecific staining already has been reported in the literature [41].



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  		FIG. 5. A) ESR1 detection by Western blot analysis. Similar amounts of homogenized lysates from gubernaculi at 16, 17, 18, 19, 20, and 21 dpc as well as at 0, 3, 4, 8, 12, and 22 dpp were separated by 8% SDS-PAGE and immunoblotted with either ESR1 MC20 antibody or tubulin antibody. The oviduct (ovi) from 23-dpp rats was used as positive control. Arrowheads indicate the positions of the ESR1 66- and 46-kDa isoforms. B) Representation of the p66:p46 ratio calculated using the relative intensity of the bands containing the 66- and 46-kDa proteins during gubernacular development. Results are presented as the mean ratio ± SEM calculated from three independent experiments. Three different phases (I–III) were characterized on the basis of the fluctuations in the p66:p46 ratio. Means between phase I and phase II are different (P = 0.003) as well as means between phase II and phase III (P = 0.041). In contrast, no significant difference were observed between phase I and phase III (P = 0.526)

On the basis of fluctuations in the p66:p46 ratio calculated using the relative intensity of the bands detected using the MC20 antibody, we have defined three main phases of ESR1 production (phases I–III) (Fig. 5B). During the first phase (16–21 dpc), the 66-kDa form predominates over the 46-kDa form. During the second phase (birth to 8 dpp), the p66:p46 ratio markedly decreases (P = 0.003) to close to 1, with the two ESR1 forms being detected in near-comparable amounts. During the third phase (12–22 dpp), production of the 66-kDa form increases again, whereas expression of the 46-kDa form is very low. As a result, the p66:p46 ratio in phase III is higher than that in phase II (P = 0.041), but it is not different from that in phase I (P = 0.526).

Localization of Both Isoforms of ESR1 and ESR2 and of AR in Gubernaculum

Immunostaining of ESR1 was observed in the nuclei of parenchymal cells at 21.5 dpc using both the 6F11 antibody and the MC20 antibody (Fig. 6, A and B, respectively), suggesting the absence of a differential location of the two ESR1 isoforms. In contrast, ESR2 immunostaining was never detected in the fetal gubernaculum (data not shown). The AR was detected in the nuclei of the parenchymal cells and in the nuclei of the mesenchymal cells of the gubernaculum at this stage of development (Fig. 6C). The ESR1 was still detectable in the parenchymal cells in the outer ring of the gubernaculum of 22-dpp rats and also in extensions of parenchymal cells within the muscular tissue of this structure (Fig. 6E). In contrast to the strong staining of ESR1 observed in the parenchymal cells, a much weaker staining of ESR2 was detected only postnatally and essentially in the muscular cells (Fig. 6H), with only few parenchymal and vascular cells being labeled. After birth, AR immunostaining was restricted to muscular areas and in vascular cells (Fig. 6F).



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  		FIG. 6. Immunolocalization of ESR1 in 21.5-dpc (A, B, and D) and 22-dpp (E and G) gubernaculi using the 6F11 antibody (A and E) and the MC20 antibody (B) of AR at 21.5 dpc (C) and 22 dpp (F) and of ESR2 in 22-dpp (H and I) gubernaculi. Mouse IgG was used as a control (D, G, and I). The ESR1 immunostaining was strong both in fetal and postnatal gubernaculum and was restricted to the nuclei of the parenchymal cells (arrows in A, B, and E). In the fetal gubernaculum, AR immunostaining was strong and was found both in the nuclei of the parenchymal cells (arrows in C) and in the nuclei of the mesenchymal cells (arrowheads in C). Postnatally, AR labeling was restricted to the muscular cells (arrowheads in F) and in the nuclei of vascular cells (open arrow in F). The ESR2 immunostaining was consistently weak, only observed postnatally, and essentially localized in the nuclei of muscular cells (arrowheads in H) with only a few labeled parenchymal cells (not shown) and in vascular cells (open arrow in H). Bar = 50 µm. Insets in A and C represent a 1.5-fold magnification


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The work of Donaldson et al. [32] revealed that the testes are positioned near the bladder neck rather than within the scrotum in most homozygous Esr1 knockout mice [42, 43]. This finding led us to investigate whether the gubernaculum expresses estrogen receptors and, therefore, likely would be a direct target for estrogens during the process of testicular descent and development in the rat.

Our RT-PCR analysis showed that Esr1 and Esr2 mRNAs were present in the gubernaculum at all stages of development studied. The PEETA (Primer extension, Electrophoresis, Elution, Tailing, and Amplification) technique revealed that Esr1 was expressed within the gubernaculum and that the expression level and the pattern of distribution of the various Esr1 mRNAs were similar to those found in other structures. The presence of all previously identified leader exons—the major form being O/ B—in the gubernaculum, like in other organs, such as the pituitary, uterus, and liver, suggests that several isoforms of this receptor are present in these organs [44, 45]. This idea is consistent with the results obtained from the S1 nuclease mapping, which showed that some of the Esr1 mRNAs in the gubernaculum were generated by splicing of the 0/B leader sequence on coding exon 1 whereas others were generated using a different leader sequence. Both the RT-PCR experiments using primers designed from the C leader sequence and coding exon 8 and the Western blot experiments confirmed these findings. They revealed the presence of mRNA transcripts corresponding to 46- and 66-kDa ESR1 proteins. Thus, we conclude that two major ESR1 isoforms are produced in the gubernaculum and that these proteins are derived from isoform-specific mRNA transcripts generated by alternative splicing of the C leader sequence on coding exon 1 or 2.

The present study establishes that the ESR1 proteins were abundant in the parenchymal cells of the fetal and postnatal gubernaculum testis. It also demonstrated major differences between the expression of ESR1 and that of ESR2. In fact, both Esr2 mRNA and protein were expressed at lower levels than Esr1. Furthermore, ESR2 protein could only be detected postnatally and was localized principally in the muscular cells. Although the role played by ESR2 in the gubernaculum muscular cells remains to be elucidated, it probably is not involved in the process of testicular descent, because the ESR2 protein is absent in the fetal gubernaculum. This idea is consistent with the fact that no abnormality in testicular positioning was noted in Esr2 knockout male mice [46]. In contrast, our results showing that Esr1 is expressed in the gubernaculum at all ages studied is consistent with the results showing malpositioning of the testes occurs in Esr1 knockout mice, caused by a reduction in the volumetric capacity of the cremaster sac (the inner part of the scrotum), leading to overdevelopment of the gubernaculum [32, 47].

As expected, and considering the well-known involvement of androgens in testicular descent, Ar mRNA also was detected in the gubernaculum at all stages of development. We confirm that during fetal life, AR protein is present in the mesenchymal core [48, 49], and we show that it also is present in the parenchymal cell ring of the fetal gubernaculum. After birth, AR localization is restricted to the muscular area, as described previously [49].

Considering the colocalization of ESR1 and AR in the parenchymal cells of the gubernaculum, it is tempting to hypothesize that estrogens and androgens cointervene in development of the gubernaculum. Androgens are known to stimulate gubernacular development [9], whereas estrogens would be expected to inhibit it, which would be consistent with the abundant literature indicating that estradiol, DES, and xenoestrogens are all involved in the occurrence of cryptorchidism [18–27]. We analyzed the promoting sequence of the Ar gene in the rat and found two potential estrogen-responsible elements (ERE mismatched motives), providing a possible molecular pathway via which estrogens could influence androgen action. A role for the estrogen receptor in mediating androgen-driven cell growth and differentiation already has been proposed for AR in the prostate, where estrogens are suspected to limit androgen-induced prostatic growth [50]. Interestingly, the amino-terminal truncated product (ER46) reportedly acts as an inhibitor of ER66 by competing for DNA binding and is thought to play a role in the genomic regulation of breast cancer proliferation [40, 51].

If we draw a parallel between the latter results and the data presented herein, we hypothesize that when the 66-kDa form clearly exceeds that of the 46-kDa form (i.e., during fetal life), the action of estrogens could prevent growth of the gubernaculum. When the p66:p46 ratio is decreasing, competitive inhibition of ER66 by ER46 could reduce estrogen inhibition on androgen action and allow the initiation of the growth of the organ, which actually occurs during the corresponding time frame [47]. Between birth and 8 dpp, when the 46-kDa form is expressed at least as much as the 66-kDa form, inhibition of ESR1 could occur and be required at a time when the gubernaculum develops exponentially [17]. The strong resurgence of expression of the 66-kDa form coincides with the end of gubernacular development [52]. Therefore, it is tempting to hypothesize that the fluctuations in the expression levels of ESR1 and the changes in the ratio of its two isoforms in the parenchymal cells of the gubernaculum are part of the possible regulatory mechanism by which estrogens could modulate androgen action during testicular descent. However, the reality of this interplay between sex hormones awaits further experiments demonstrating direct experimental effects of estrogens on gubernaculum growth.

The lack of Cyp19a1 expression and the presence of Srd5a1 in the gubernaculum are consistent with results obtained in a previous study [16]. These data indicate that the estrogens acting on the gubernaculum most likely originate from another source within the fetus, and even more likely from the mother, because testicular descent and final positioning of gonads within the scrotum are normal in Cyp19a1 knockout male mice [53]. In contrast, our data confirm that conversion of testosterone to 5{alpha}-reduced metabolites can occur locally [16].

To summarize, we demonstrate here that two isoforms of ESR1, ER66 and its truncated variant ER46, are highly expressed in the gubernaculum testis before testicular transabdominal descent occurs. These proteins are translated from isoform-specific mRNA transcripts generated by alternative splicing. The differences in both timing and levels of expression of these two competitive ESR1 proteins may help us to understand, through future studies, how estrogens might regulate the growth of the gubernaculum during testicular descent. We hypothesize that by modulating the androgen-dependent growth of gubernaculum, estrogens (together with INSL3 and androgens) could be involved in coordinating events that control the positioning of the testes within the scrotum. This is important in the context of the search for the cause of cryptorchidism and the xenoestrogen-induced abnormalities of the male urogenital tract, and it is consistent with a recent publication indicating that ESR1 is overexpressed in the cremasteric muscle of cryptorchid boys [54].


   ACKNOWLEDGMENTS
 
The authors wish to thank Julie Lassurguère-Labbé and Sheila MacPherson for their help at various stages of the experiments and Alain Le Nedic for taking care of the animals.


   FOOTNOTES
 
1 Supported by the INSERM, Ministère de la Recherche et Ministère de la Santé, and the EDEN European program, contract number QLK4-CT-2002-00603. Back

2 Correspondence: Bernard Jégou, INSERM, U625, GERHM, Campus de Beaulieu, Univ Rennes I, Rennes, Bretagne F-35042 France. FAX: 33 2 23 23 50 55; bernard.jegou{at}rennes.inserm.fr Back

3 These authors contributed equally to this work Back

4 Current address: Dipartimento di Medicina Sperimentale, II Università di Napoli, Via Costantinopoli, Naples 16-80138, Italy Back

Received: 15 April 2005.

First decision: 12 May 2005.

Accepted: 3 June 2005.


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