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Regular Article |
a Department of Animal Sciences, The Ohio State University, Columbus, Ohio 43210
b Department of Obstetrics and Gynecology, Indiana University School of Medicine, Indianapolis, Indiana 46202
c Medical Sciences, Indiana University, Bloomington, Indiana 47405
ABSTRACT
Objectives were to sequence and examine the expression of the estrogen receptor ß (ERß) in the sheep ovary. The sequence of the ovine ERß (oERß) was determined using reverse-transcription polymerase chain reaction (RT-PCR) and cloning techniques. The reading frame of oERß contained 527 amino acids and exhibited high overall homology with cow (98%), rat (88%), and human (88%) ERß. In addition, an oERß isoform having a 139-base pair deletion (oERß1) was identified. The predicted amino acid sequence of this isoform is lacking the ligand-binding and carboxyl-terminal transactivation domains. The oERß protein and mRNA were determined in ovaries obtained from ewes on Days 0 (first day of estrus), 2, 6, and 10 of the estrous cycle and Day 30 of gestation. Immunohistochemistry showed that oERß protein was located in granulosa cells, the ovarian surface epithelium, endothelium, and Day 2 corpus luteum (CL). Weak immunostaining for ERß was detected in the theca interna. Relative steady-state amounts of oERß mRNA in the CL were determined using semiquantitative RT-PCR. Amounts of oERß mRNA were greater (P < 0.05) during CL formation (Day 2) than at later stages. The oERß to oERß1 mRNA ratio was lower (P < 0.05) on Day 2 than on Day 10 or Day 30 due to a decrease in amounts of oERß1. Results indicate that the oERß is a 527-amino acid protein expressed in specific cells of the ovary. Changes in relative amounts of full-length oERB and a deletion isoform in CL occurred during the estrous cycle, suggesting that these two types of ERß might regulate estrogen actions during early CL development in sheep.
corpus luteum, estradiol receptor, follicle, ovary
INTRODUCTION
Estrogens are important regulators of reproductive processes in female mammals. Estrogen receptor 
(ER) and ERß mediate the actions of estrogens by regulating transcription of target genes. Most tissues of female reproductive organs express both ER and ERß [1] and the relative expression levels of these receptors may play a major role in mediating estrogen actions in a particular tissue. It is not clear whether ER and ERß have specific functions in cells that coexpress them or whether they interact by forming ER/ß heterodimers, to regulate the expression of common target genes [2, 3].
The ERß has been consistently detected in the membrana granulosa of follicles of different species including rats, mice, humans, marmosets, and cattle [4–11]. Some of these studies demonstrated that ERß was the predominant, or the only, estrogen receptor expressed in granulosa cells [7, 8, 11]. Although ER mRNA and protein have been shown to be present in ovine granulosa cells [12], expression of ERß in the sheep ovary has not been examined. Low amounts of ERß mRNA have been detected in the corpus luteum (CL) [1, 6, 13–17] and theca cells [1, 6] of several species. In addition to being highly expressed in granulosa cells, functional studies have shown that ERß can stimulate transcriptional activation in these cells [9, 18]. The importance of ERß in ovarian function has been demonstrated in ERß knockout mice that exhibited a high incidence of ovulation failure and decreased number of offspring [19].
Although multiple isoforms of the ERß mRNA have been detected and characterized [17, 20–27], little is known about the regulation of ERß isoform expression and functional significance. Differential tissue expression and ability of C-terminal ERß isoforms to form dimers with wild-type ER or ERß that bind DNA have been reported in humans [24]. A rat ERß isoform containing an 18-amino acid insertion in the DNA-binding domain (DBD) inhibited estrogen-induced transcription and might function as a dominant negative regulator of estrogen actions in vivo [23]. The ERß seems to be important in ovarian function of several species so far examined; however, studies on ERß in sheep are lacking. The objectives of the present experiments were to sequence and determine the expression of ERß in the sheep ovary during the estrous cycle and early pregnancy.
MATERIALS AND METHODS
Animals and Tissue Collection
Ovaries were obtained from ewes (Targhee x Hampshire cross) on Days 0 (first day of estrus), 2, 6, or 10 of the estrous cycle and Day 30 of pregnancy (n = 2–4 ewes per group). A portion of the CL was dissected from luteal phase ovaries, frozen in liquid nitrogen, and then stored at -80°C. Residual tissue from ovaries containing CL, and ovaries collected at estrus, were fixed for histological determinations. Procedures for handling the experimental animals were approved by our Institutional Animal Use and Care Committee.
Cloning and Sequencing
Total RNA was isolated from ovarian tissue using the RNeasy procedure (Qiagen, Valencia, CA). Integrity of RNA was verified by visually estimating the 28s to 18s rRNA ratio on pictures of formaldehyde-agarose gels stained with ethidium bromide. Purity of RNA was determined by calculating the 260–280 nm absorbance ratio. Reverse transcription-polymerase chain reaction (RT-PCR) was performed using a commercial kit (Perkin Elmer, Roche Molecular Systems, Inc., Branchburg, NJ). Reactions for first-strand cDNA synthesis included 5 mM MgCl2, 1x reaction buffer, 1 mM dNTPs, 1 U/µl RNase inhibitor, 2.5 U/µl murine leukemia virus reverse transcriptase, 2.5 µM random hexamers, and 0.5 µg of total RNA per 10 µl of reaction volume. Tubes were incubated for 10 min at room temperature, 30 min at 42°C, 5 min at 99°C, and then chilled to 5°C.
Primers for amplification of overlapping and nested fragments of oERß were designed based on the human [28], bovine [10], and ovine (partial sequence, GenBank AF110817) ERß sequences. The primers used for the amplification of oERß fragments are shown in Table 1. Polymerase chain reactions were performed for 35 cycles in 50-µl mixtures containing 10 µl of RT reaction, 1.5 or 2 mM MgCl2, 0.3 µM primers, 200 µM dNTPs, and 1.25 U of Taq DNA polymerase. Conditions for thermal cycling were: denaturation for 1 min at 95°C, annealing for 45 sec at 55°C, and extension for 2 min at 72°C, with the exception of fragments 1 and 6 for which annealing temperature started at 65°C and decreased 0.5°C per cycle to 55°C (touchdown PCR). The last cycle included 7 min additional of extension at 72°C that was followed by cooling to 5°C. Products were analyzed by agarose-gel electrophoresis, visualized by ethidium bromide staining and UV illumination, and then photographed.
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The PCR products were cloned into pCR2.1 vector using the Original TA cloning kit (Invitrogen, Carlsbad, CA). Primers h584 and h1479 generated two bands. Both bands remained following PCR optimization and were cloned simultaneously. Sequence analysis demonstrated that one of the products was an exon 5-deleted isoform of oERß (named oERß1). Both strands of two to three clones for each product representing possible ERß fragments were sequenced. The reading frame of ERß was determined by multiple sequence alignment using MACAW [29], a program available at the National Center for Biotechnology Information (NCBI) ftp server. The cDNA and predicted amino acid sequences of oERß and oERß1 were then compared to reported sequences using the Basic Local Alignment Search Tool at the NCBI Internet page (http://www.ncbi.nlm.nih.gov/). Homology analysis among protein sequences of the ovine, bovine, and rat ERß was performed using ClustalW (BioEdit; North Carolina State University, Raleigh, NC).
Immunohistochemistry
Tissue localization of full-length oERß was determined using a polyclonal rabbit antibody directed against a C-terminal synthetic peptide of the rat ERß (PA1-310; Affinity Bioreagents, Golden, CO). This 19-amino acid peptide (CSSTEDSKNKESSQNLQSQ) has 9 residues (47%) identical to the equivalent ovine sequence. An average of 6 amino acids has been reported to constitute antigenic determinants in peptides [30]. Ovaries were incubated overnight at 4°C in 4% paraformaldehyde in PBS (pH 7.4). The tissues were then rinsed in PBS and cryoprotected by infiltration of 30% sucrose in PBS at 4°C. The tissues were sectioned into blocks, embedded in Tissue Fixing Medium (Triangle Biomedical Sciences, Durham, NC), frozen in liquid nitrogen, and stored at -80°C. The blocks were cut on a cryostat into 8-µm sections and the sections were mounted onto slides (Superfrost/Plus; Fisher Scientific, Pittsburgh, PA), and dried at room temperature for 1–2 h. Sections were then postfixed in 4% paraformaldehyde in PBS for 10 min, rinsed, dehydrated in ascending ethanol solutions, and stored at -80°C.
Antigen retrieval was performed by heating the slides to 95°C in citrate buffer (pH 6.0; DAKO Corp., Carpinteria, CA) for 25 min. Slides were cooled to room temperature in the same buffer and then washed in PBS. Endogenous peroxide was blocked by incubation with 0.3% hydrogen peroxide in 40% methanol for 30 min. Slides were washed three times (5 min per wash) using 0.5% Tween 20 in PBS (PBS-Tween). All subsequent washings were performed in the same manner unless otherwise indicated. Sections were treated with normal goat serum (#G9023; Sigma, St. Louis, MO) for 20 min, washed, and then incubated overnight at 4°C in anti-ERß primary antibody diluted to 8 µg/ml in PBS-Tween containing 1% BSA. Negative control sections were incubated in antibody diluent alone or in antibody preabsorbed with a 10-fold excess of peptide (PEP 007; Affinity Bioreagents). Sections were washed and incubated for 1 h with secondary antibody (biotinylated goat anti-rabbit, 1/400; Vector Laboratories, Burlingame, CA) and then 1 h with ABC reagent (Vector Laboratories). After washing, the slides were treated with nickel-intensified diaminobenzidine (DAB substrate kit; Vector Laboratories) for 5–8 min, rinsed in water, and then in PBS, dehydrated, and coverslipped. Sections were viewed using a microscope, photographed with a SPOT RT CCD camera and SPOT software (Diagnostic Instruments, Inc., Sterling Heights, MI), and processed for publication using Adobe Photoshop 5.5 (Adobe Systems, Mountain View, CA).
Reverse Transcription-PCR of oERß in CL
Total RNA was isolated from CL tissue using TRI Reagent (MRC, Inc., Cincinnati, OH) and reverse transcribed as described. Amplifications of oERß, ovine ribosomal protein L19 (oL19), and the simultaneous amplification of oERß and oERß1, were performed using 5-µl aliquots (equivalent to 250 ng of total RNA) of the same reverse transcription reaction. Primers for oERß were designed based on the sequence described in the present study. A 493-base pair (bp) fragment of the full-length oERß was amplified using the primers 482U (sense, 5'-GGT CAT GTG AAG GAT GTA AG-3') and 974D (antisense, 5'-ACT TGG TCG TAC AGG CTG AG). The primer 974D anneals within exon 5 of the oERß transcript and specifically amplifies the full-length oERß and not the oERß1 isoform. The full-length oERß (895-bp fragment) and oERß1 (756-bp fragment) were amplified simultaneously using primers 482U and 1376D (antisense, 5'-AGG AGC ATC AGC AGG TTA GC-3').
Initially, glyceraldehyde-3 phosphate dehydrogenase (GAPDH) was chosen as an internal control. Expression of GAPDH decreased with day of CL development (data not shown), making it an unsuitable control. Ribosomal protein L19 (L19) was then evaluated using primers for rat L19 [31]. A single PCR product of predicted size was obtained and sequenced. This product of 497 bp exhibited high homology with L19 of other species including the human (92%, GenBank X63527), mouse (90%, GenBank NM-009078), and rat (89%, GenBank J02650). New primers (sense: 5'-AGC TGT GAC TGT CCA TTC-3', and antisense: 5'-CTT GCG AGC CTT GTC TGC-5') were synthesized based on this sequence and used to amplify a 299-bp fragment of oL19.
The same touchdown (35 cycles) and standard PCR protocols described above were used for the simultaneous amplification of oERß and oERß1, and for the amplification of oERß (30 cycles) and oL19 (22 cycles), respectively. The number of cycles for each PCR protocol was set within the exponential portion of the amplification curve that was determined by sampling PCR reactions every other cycle in preliminary assays. Reverse transcriptase was replaced by water in a set of reactions run as controls to detect bands not generated by RNA. Resulting products were analyzed by agarose-gel electrophoresis and stained with ethidium bromide. Pictures of gels were taken using instant film (Polaroid T57), and cDNA bands were subjected to densitometric analysis using the Image-J program from the National Institutes of Health (available on the Internet at http://rsb.info.nih.gov/ij/). Relative mRNA abundance was estimated by calculating the ratio of intensities of oERß to oL19 bands.
Statistical Analyses
Analyses were performed using SYSTAT [32]. The effects of day on the relative amounts of oERß mRNA and on the oERß to oERß1 transcript ratio were determined by ANOVA. Means were compared using protected least significant difference tests.
RESULTS
Ovine ERß Sequence
The oERß reading frame has 1581 nucleotides, and its predicted translation generated a protein composed of 527 amino acids (Fig. 1). This protein exhibited high overall homology with the bovine (98%, GenBank AF110403), rat (89%, GenBank CAA05631), and human (88%, GenBank Q92731) ERß (Fig. 2A). Comparisons within the different regions of the receptor showed the highest mean homology for the C (or DBD), followed by the E/F (or ligand-binding domain, LBD), D, and A/B regions (Fig. 2B).
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An isoform lacking 139 nucleotides in the E/F region (nucleotide 945–1083, bolded and underlined in Fig. 1) was detected during sequence analysis. The deleted isoform appeared as a clearly visible band when primers spanning the deleted region were used for cDNA amplification. As expected, when one of the primers was designed to bind within the deleted region, only a single product (full-length oERß) was detected (Fig. 3). Translation of the deleted isoform showed a shift in the reading frame and a stop codon in the E/F region, indicating that this isoform would generate a truncated protein that lacks most of the LBD and the putative C-terminal activation domain (Fig. 4). The 139-bp deletion begins at the 3'-end of exon 4 (C at position 945, Fig. 1) and ends at the 3'-end of exon 5 (G at position 1083, Fig. 1). Exon 5 of human ERß has 138 bp [5], and homology analysis demonstrated the same number of base pairs for exon 5 of the oERß. Therefore, this oERß isoform probably originated by elimination of exon 5 through alternative splicing. Results indicated that the oERß1 mRNA is relatively abundant in the ovary (Fig. 3).
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Immunohistochemistry of oERß in the Ovary
Immunostaining for oERß was observed in nuclei of granulosa cells of preantral and antral follicles, at all days examined (Fig. 5, A–C). Immunostaining for oERß was also observed in the ovarian surface epithelium (OSE; Fig. 5D), endothelium (Fig. 5, A and G), and in a proportion of cells of Day 2 CL (Fig. 5H). Granulosa cells of follicles having structural signs of atresia (e.g., dispersed granulosa cells) also showed specific oERß staining (Fig. 5E). Theca interna of antral follicles exhibited weak immunostaining for oERß (Fig. 5, B and C). No immunostaining was observed in negative controls that included sections incubated with immunoabsorbed antibody or in sections for which the antibody was omitted.
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Ovine ERß mRNA in CL
The relative amounts of oERß mRNA was influenced by day of CL development (P = 0.02). Amounts of oERß mRNA decreased (P < 0.05) after Day 2 and remained low up to Day 30 (Fig. 6, A and B). The oERß to oERß1 mRNA ratio was lower (P < 0.02) on Day 2 than on Days 10 or 30. The ratio was lower (P = 0.04) on Day 6 than on Day 10, and there was a tendency (P = 0.06) for a lower ratio on Day 6 than on Day 30 (Fig. 6C).
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DISCUSSION
The predicted oERß protein is composed of 527 amino acids, and its sequence is very similar to the ERß of cattle [10], a closely related species. The DBD of oERß was 100% homologous with a variety of species including the cow, human, and other species of bird and fish, which is consistent with the notion that the DBD is a highly conserved region of steroid receptors. The translation start site of ERß is not well defined. Initially, methionine at position 54 (Fig. 2A) was considered a possible start site in the human [33] and rat [4]. The ERß sequences of human and rat subsequently described have start sites equivalent or upstream to the sheep (Fig. 2A). Similar to the cow, the oERß sequence has a valine at position 46 instead of a methionine, which was considered a possible initiation codon in the rat [4] and mouse [34].
Though multiple isoforms of the ERß gene have been reported in different species, only the isoform lacking exon 5 was detected in the sheep ovary in the present study. The exon 5-deleted isoform of ERß has been found in reproductive organs and mammary glands of humans [22, 27], mice [22], and granulosa cells of cattle [17]. Transient transfection studies demonstrated that the cow exon 5-deleted isoform was unable to transactivate an estrogen-response element (ERE) construct, probably due to the lack of LBD [17]. It is not known whether this transcriptionally inactive isoform competes with full-length ERß for binding to ERE.
The abundance of oERß1 mRNA was approximately half the full-length oERß at the beginning of CL development (Day 2) and then gradually decreased, becoming very low by Day 10 and remaining low by Day 30. Full-length oERß became the predominant isoform (about six times higher than oERß1) in Day 10 and Day 30 CL. This is the first study that demonstrates changes in expression patterns of ERß isoforms during CL development. The biological significance of these changes in ERß isoform expression are not known at present, but it may be that loss of the truncated isoform allows estrogenic stimulation of CL through decreased competition for ERE on target genes.
Production of the truncated oERß isoform might also impinge upon synthesis of the full-length receptor. Because the oERß1 isoform seems to originate by alternative splicing, changes in its rate of synthesis would probably alter the amounts of full-length oERß mRNA. Therefore, the factors that regulate alternative splicing could indirectly influence the synthesis of full-length oERß mRNA. Alternative splicing occurs by complex interactions between RNA and proteins, among them some members of the small nuclear ribonucleoproteins and serine-arginine splicing factors [35]. The changes in expression patterns of ERß isoforms during development in particular cell types might be a modulatory mechanism of estrogen actions in the ovary [9].
Localization of oERß in granulosa cells of preantral and antral follicles is consistent with previous reports in other species [4–12]. It is worth noting that the immunostaining observed in the present study would include only the full-length oERß, not the truncated protein lacking the LBD generated by the oERß1 transcript. Staining intensity of oERß protein in granulosa cells at different days of the estrous cycle was similar, suggesting that major changes in oERß expression in granulosa cells are absent in sheep and that oERß might be important for regulation of most stages of follicular development. Immunoreactive ERß was present in granulosa cells of antral follicles undergoing atresia. Whether this is only a residual or functional protein remains to be determined. Little is known about control of ERß gene expression in granulosa cells. A regulatory event that has been documented in rats and rhesus monkeys is a decrease in ERß mRNA [13] or protein [7, 36] after the administration of ovulatory doses of hCG. Somewhat related is the finding that the preovulatory LH surge also inhibits aromatase (CYP19) transcription and increases degradation of aromatase mRNA [37]. This would result in decreased synthesis of 17ß-estradiol, the predominant ligand of estrogen receptors. Therefore, the ovulatory LH surge could attenuate estrogenic effects in the preovulatory follicle.
Cells of the membrana granulosa (which usually express high levels of ERß) and theca interna differentiate into large and small luteal cells, respectively [38]. Although it is not known whether ERß is downregulated in granulosa cells of sheep by the preovulatory LH surge, our results indicate that ERß mRNA and protein were present during early CL development (Day 2) and then decreased in more developed CL. In humans, amounts of ERß mRNA were high during the early luteal phase and then decreased [16], similar to what we observed in the sheep. In the rhesus monkey, however, the opposite pattern of ERß gene expression has been reported [39].
Zieba et al. [40] determined that ER immunostaining in sheep CL was weak on Day 6 and increased by Day 9 of the estrous cycle. These results indicate that ER has a different pattern of expression than ERß, suggesting that the roles of these receptors in the sheep CL might differ. The functions of estrogens in the ovine CL are not known. Estrogens are considered to be luteotropic in the rat, rabbit, and pig [41]. Proliferation of small luteal cells, fibroblasts, and endothelial cells during CL formation is very intense and seems to be influenced by growth factors, LH, and growth hormone [41]. In this study, ERß was detected in endothelial cells and might have a role in the intense vascularization occurring during CL formation. Estrogens regulate transcription of vascular endothelial growth factor (VEGF), a potent inducer of angiogenesis [42], and both VEGF and its type 2 receptor are highly expressed in the bovine CL during the early luteal phase [43].
We determined that oERß protein is present in the sheep OSE, which agrees with results in humans [44, 45] and cynomolgus monkeys [15]. Likewise, cells of sheep OSE contain ER [12]. Enzymatic activity originating from the OSE has been associated with follicular rupture during ovulation in sheep [46]. Cultured bovine OSE cells synthesize growth factors and growth factor receptors that are regulated by gonadotropins [47, 48]. FSH stimulates multiplication of OSE cells, and this effect has been related to ovarian cancer cell proliferation [49]. It is not known whether ERß mediates regulation of these or other unknown processes in the OSE.
In summary, the oERß reading frame encodes a 527-amino acid protein that is expressed in multiple cell types of the ovary, particularly in granulosa cells of follicles where it might be an important mediator of autocrine actions of estradiol. We are reporting, for the first time, changes in ERß isoform expression during CL development, the significance of which remains to be determined.
ACKNOWLEDGMENTS
The support of Amrita Ahluwalia of Indiana University and the personnel at the Sheep Unit of the Ohio Agricultural Research and Development Center, Wooster, are greatly appreciated.
FOOTNOTES
First decision: 9 January 2001.
1 Salaries and research support provided by State and Federal funds appropriated to The Ohio Agricultural Research and Development Center and The Ohio State University. This research is manuscript no. 42-00AS and was supported by funds from the National Institutes of Health, PHS CA74748 to K.P.N. and CA74748 postdoctoral award to H.C. 
2 Correspondence: H. Cárdenas, Department of Animal Sciences, The Ohio State University, 2029 Coffey Rd., Columbus, OH 43210. FAX: 614 292 7116; cardenas-seijas.2{at}osu.edu
Accepted: February 15, 2001.
Received: November 28, 2000.
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