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Endocrinology Vol. 144, No. 6 2360-2367
Copyright © 2003 by The Endocrine Society

Follicle-Stimulating Hormone Suppresses Cytosolic 3,5,3'-Triiodothyronine-Binding Protein Messenger Ribonucleic Acid Expression in Rat Granulosa Cells

CheMyong Ko, Nicole A. Grieshaber, Inhae Ji and Tae H. Ji

Department of Chemistry (N.A.G., I.J., T.H.J.) and Department of Clinical Sciences (C.K.), University of Kentucky, Lexington, Kentucky 40506

Address all correspondence and requests for reprints to: Dr. Tae H. Ji, Department of Chemistry, University of Kentucky, Lexington, Kentucky 40506-0055. E-mail: tji{at}uky.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
FSH plays crucial roles in differentiation of granulosa cells and development of follicles. Considering the broad scope of FSH effects, a large number of genes are likely responsive to the hormone. However, only a limited number of genes have been identified as FSH-regulated genes, particularly during the preantral stage. In an attempt to better define genes involved in follicular development, we examined primary granulosa cell cultures, an undifferentiated rat ovarian granulosa cell line and rat ovaries, using differential display, quantitative RT-PCR, Northern blot analysis, and in situ hybridization. We report, for the first time, that nicotinamide adenine dinucleotide phosphate-dependent cytosolic T3-binding protein mRNA is expressed in the ovary, particularly in the granulosa cell layer of preantral and early antral follicles, but not in large preovulatory follicles. Its expression markedly declines in response to FSH, which is dependent on the period of the exposure. This FSH-responsive down-regulation is dependent on granulosa cell differentiation and follicular development. FSH down-regulates the mRNA via the adenylyl cyclase/cAMP pathway, and the down-regulation requires de novo synthesis of a regulatory protein(s). The cytosolic T3-binding protein may play a significant role in the regulation of steroidogenesis and follicular development in the mammalian ovary.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
FSH STIMULATES GRANULOSA cell differentiation and follicular development. It is responsible for inducing estrogen production and preventing the apoptosis of early antral follicle cells in rodents (1). In growing follicles, FSH mediates continued mitotic activity of granulosa cells, and decreased FSH responsiveness is associated with follicular atresia (2). These FSH activities are initiated when FSH binds to and activates the FSH receptor. FSH receptor mRNA is expressed in granulosa cells as early as the primary stage of follicular development (3). The importance of FSH and its receptor is clear, as female mice homozygous for a defective FSHß are infertile due to the arrest of follicular development at the preantral stage (4). The ovarian phenotype of FSH receptor-knockout mice is similar to that observed in FSH-knockout mice (5). It has been shown that FSH elicits peptide and steroid hormone production in granulosa cells by inducing the expression of its target genes (2). Due to the broad scope of the FSH effects, a large number of genes are likely responsive to the hormone. However, only a limited number of FSH-regulated genes have been identified to date, such as inhibin/activin subunits and steroidogenic enzymes. In particular, little is known about the FSH-responsive genes at the preantral stage. This is, in part, due to the lack of a suitable experimental system.

The rat ovarian granulosa (ROG) cell line is a useful system. It was established from immature granulosa cells of the rat ovary (6) and grows in a defined serum-free medium containing activin A but not FSH. ROG cells show many characteristics of undifferentiated immature cells, lacking steroidogenesis and the LH receptor. Upon exposure to FSH, the cells become postmitotic and highly steroidogenic, similar to mature granulosa cells of a dominant follicle. FSH-stimulated ROG cells also become dependent on the continued presence of FSH and will undergo apoptosis upon its removal (6). In addition, ROG cells form a structure resembling a follicle when cultured in the presence of an oocyte/cumulus cell complex (7). We have shown that the actin cytoskeleton in ROG cells quickly rearranges within 3 h of exposure to FSH, leading to changes in cell-cell interactions (8). To identify the FSH-responsive genes, we examined differences in gene expression caused by exposure of rat granulosa cells to FSH using mRNA differential display. Here, we present the evidence that FSH down-regulates expression of nicotinamide adenine dinucleotide phosphate, reduced (NADPH)-dependent cytosolic T3-binding protein (CTBP) in rat granulosa cells. CTBP may play a significant role in the regulation of steroidogenesis and follicular development in the mammalian ovary.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
DMEM, Ham’s-F12, and antibiotics for tissue culture were obtained from Life Technologies, Inc. (Gaithersburg, MD). Restriction enzymes, reverse transcriptase, T7 and SP6 RNA polymerases, and Taq DNA polymerase were obtained from New England Biolabs, Inc. (Beverly, MA). [{alpha}-32S]Uridine triphosphate and [{alpha}-32P]dCTP were obtained from Amersham Pharmacia Biotech (Piscataway, NJ). Oligonucleotides were synthesized by Sigma (Coralville, IA). FSH, human chorionic gonadotropin (hCG), and activin A were purchased from the National Hormone and Peptide Program (Torrance, CA). Pregnant mare’s serum gonadotropin (PMSG) was purchased from Sigma.

Animals, hormone treatment, granulosa cell isolation, and culture
ROG cells were cultured as previously described (6). Briefly, ROG cells were maintained in suspension in a defined serum-free medium consisting of F12-DMEM supplemented with activin A (25 ng/ml), insulin (10 µg/ml), transferrin (5 µg/ml), {alpha}-tocopherol (0.1 µg/ml), progesterone (10 nM), BSA (0.1%), and aprotinin (25 µg/ml) in the absence of antibiotics. Activin A (25 ng/ml) was replenished every 24 h. The cells were provided with fresh media once a week, pooled every 2 wk by centrifugation at 1000 rpm for 5 min, and replated at 1:2.

All animals were handled according to the guidelines for care and use of animals set by the National Institutes of Health and the University of Kentucky Institutional Animal Care and Use Committee. Eighteen- to 21-d-old Sprague Dawley female pups with nursing mothers were purchased from Harlan Breeding Company (Indianapolis, IN) and housed in a photoperiod of 14-h light/10-h darkness with lights on at 0500 h. For in situ hybridization analysis, rats were injected sc with 15 IU PMSG in 0.1 ml PBS at 22 or 23 d of age. Some of the rats primed with PMSG for 48 h were additionally injected ip with 10 IU hCG.

For granulosa cell culture, immature rats were sc injected daily with 1.5 mg of 17ß-estradiol at 21, 22, and 23 d of age. Ovaries were isolated from the rats on d 24, and granulosa cells exhibiting a small antral phenotype (9) were collected in cold serum-free 4F medium consisting of 15 mM HEPES (pH 7.4), 50% DMEM, and 50% Ham’s F12 with bovine transferrin (5 µg/ml), human insulin (2 mg/ml), hydrocortisone (40 ng/ml), and antibiotics. After cells were washed three times in 4F medium, they were plated on serum-coated (10) six-well plates at a density of approximately 1 x 106 cells per well and incubated in the humidified atmosphere of 5% CO2 at 37 C. After 16 h, FSH (30 ng/ml) or forskolin (10 µM) were added to the cultures. For the inhibition of protein synthesis or transcription, cycloheximide (10 µg/ml) or {alpha}-amanitin (30 µg/ml) were added, respectively, 1 h before hormone treatment.

Differential display
ROG cells were incubated in the absence of FSH (0 h) or in the presence of FSH (30 ng/ml) for 6 h in triplicate, and total RNA was extracted. Pooled total RNA was used as a template for differential display of mRNA analyses using the Delta Differential Display Kit (CLONTECH Laboratories, Inc., Palo Alto, CA) according to the manufacturer’s instruction. cDNA fragments were reamplified by PCR, cloned into PCR 2.1 TA cloning vector (Invitrogen, San Diego, CA) and sequenced on a Beckman Coulter (Fullerton, CA) CEQ 2000 capillary sequencer.

Northern blot
For Northern analysis, 4–20 µg of total RNA per sample was resolved on 1.2% agarose gels containing 2.2 M formaldehyde and blotted to nylon membranes (Nytran SuPerCharge, Schleicher \|[amp ]\| Schuell, Keene, NH). [{alpha}-32P]Deoxy-CTP-labeled cDNA probes were prepared from the CTBP clone using random primers. Blots were hybridized overnight at 42 C in 50% (vol/vol) formamide, 5x SSPE, 5x Denhardt’s reagent, 0.1% (wt/vol) sodium dodecyl sulfate (SDS), and 200 mg/ml denatured, fragmented herring testis DNA. Filters were washed once at low stringency [5x standard saline phosphate/EDTA (0.15 M NaCl/10 mM phosphate (pH 7.4)/1 mM EDTA (SSPE), 0.1% SDS; 25 C)] and twice at high stringency (0.1x SSPE, 1% SDS; 62 C) for 45 min and visualized on phosphor imager (FLA-2000; Fuji, Stamford, CT).

RT-PCR
RT-PCR was performed as previously described (11). Total RNA (1–2 µg) was reverse-transcribed at 37 C in 20 µl using random hexamer (500 ng) and Moloney murine leukemia virus reverse transcriptase (10 U; New England Biolabs, Inc.). cDNA in 2 µl was added for a total 25-µl reaction mixture containing the primers (200 ng each), 0.4 mM deoxynucleotide 5'-triphosphate mixture, and Taq DNA polymerase (2.5 U) in 1x PCR buffer (10 mM Tris, pH 8.3; 50 mM KCl; 1.5 mM MgCl2; and 0.01% gelatin). All PCR amplifications were carried out for 20, 25, and 30 cycles on an MJ Research, Inc. (Cambridge, MA) Minicycler. PCR products were separated by 2% agarose gel electrophoresis, stained with SYBR Green I (Molecular Probes, Inc., Eugene, OR), and visualized on a phosphor imager. The primers were: 5'-ctg act ggc gag aac tgg atg-3' and 5'-aca gta tgc agg ctt cgc tcc-3' for 160-bp CTBP; 5'-gct ttc cct ctg ttg acc cac-3' and 5'-aga tgt tga ggg cag ctc gat-3' for 255-bp inhibin {alpha}; and 5'-ctg aag gtc aaa ggg aat gtg-3' and 5'-gga cag agt ctt gat gat ctc-3' for 194-bp L-19 as an internal control (12).

In situ hybridization
Frozen ovaries were cut in 20-µm sections using a MICROM HM 505 E cryostat (Richard-Allen Scientific, Kalamazoo, MI) and mounted onto Superfrost/Plus Microscope slides (Fisher Scientific, Pittsburgh, PA). Sections were fixed, pretreated, and hybridized with antisense and sense RNA probes as previously described (11). Using T7 or SP6 polymerase, [{alpha}-35S]uridine triphosphate-labeled RNA probes were synthesized from clones in pBluescript II vector (Stratagene, La Jolla, CA). RNA probes (107 cpm/ml) in hybridization buffer consisting of 50% formamide, 5x SSPE, 2x Denhardt’s reagent, 10% dextran sulfate, 0.1% SDS, and 100 µg/ml yeast tRNA were applied to sections, which were incubated in a humidity chamber at 47 C for 16–18 h. After hybridization, the sections were treated with ribonuclease (RNase) A (20 µg/ml) at 37 C for 30 min, washed repeatedly in increasingly lower concentrations of SSC down to 0.1x SSC at 58 C, and dehydrated through an ethanol series. The slides were exposed to Kodak (Rochester, NY) BIOMAX MR film for 2 d and processed for liquid emulsion autoradiography using NTB-2 emulsion (Kodak) for 3–6 wk. Developed sections were stained with Gill’s formulation no. 2 hematoxylin solution (Fisher Scientific). Tissues were examined on a Nikon (Melville, NY) Microphot-SA microscope under bright- and dark-field optics. Sense riboprobes were used as a control for nonspecific binding.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Differential display of mRNA shows decrease of CTBP mRNA expression in response to FSH
To identify FSH-responsive genes in granulosa cells, ROG cells were cultured in the absence or presence of 30 ng/ml FSH for 6 h, total RNA was isolated, and mRNA was differentially displayed on sequencing gels. Bands, which showed intensity difference between control and FSH-treated groups, were excised, PCR-amplified, cloned, and sequenced. One of the clones corresponded to nucleotides 768-1210 of the full-length cDNA of CTBP (GenBank accession no. Y17328), with one base mismatch (Fig. 1Go). This clone is also referred as µ-crystallin, because it was first isolated from lens crystals (13). We used the clone to isolate a cDNA encompassing the full-length open reading frame from 22-d-old immature Sprague Dawley rat ovaries (Fig. 1AGo). The sequence of our clone showed five nucleotide mismatches as compared with the nucleotide sequence of the reported Norway rat cDNA clone. These mismatches do not alter the open reading frame and amino acid sequence. The deduced protein comprises 313 amino acids and shares 97% and 87% sequence identity with the mouse (GenBank accession no. AF039391) and human (GenBank accession no. U85772) clones, respectively (Fig. 1BGo). These variant amino acids may impact the structure and functions of the proteins, because they are considered interchangeable (14). Considering these interchangeable residues, the homologies between the rat with the mouse and human sequences are 100% and 93.4%, respectively. These results suggest that they are virtually identical proteins.



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  		Figure 1. Nucleotide sequence of clone JK32 and rat CTBP cDNA and protein sequence comparison. A, The nucleotide sequences of Sprague Dawley rat CTBP (upper rows) and Norway (lower rows) rat are compared. The given nucleotide sequence (upper rows) is from Sparague-Dawley rat cDNA. Note the 5five nucleotide mismatches between the two sequences. The start and stop codons are indicated by underlined bold letters. Broken lines represent identical nucleotides. B, Deduced CTBP amino acid sequence was aligned with mouse µ-crystallin (NP057878) and human CTBP (AAB81564) sequences. Dashed lines and asterisks represent identical residues and gaps, respectively.

 
CTBP mRNA expression in granulosa cells of small follicles
To localize the in vivo CTBP mRNA expression, an adult rat ovary was subjected to in situ hybridization. The CTBP mRNA signal was detected only in small, growing follicles but not in atretic follicles and corpus lutea (Fig. 2AGo). To verify the differentiation- and development-dependent expression of CTBP, we compared the CTBP expression with that of inhibin {alpha}, a known FSH-responsive gene whose expression is dependent on granulosa cell differentiation and follicular development. Tandem ovarian sections were hybridized with an inhibin {alpha} antisense probe. All of the follicles that showed the CTBP mRNA signal expressed inhibin {alpha} mRNA, and furthermore, no other follicles showed the signal (Fig. 2Go, A and B). The granulosa cell layers of the small follicles were conspicuously labeled with the CTBP probe, and the theca cells were also labeled (Fig. 2CGo). No signal was detected from the sections hybridized with CTBP sense probe (Fig. 2DGo).



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  		Figure 2. Localization of inhibin {alpha} and CTBP mRNAs in adult rat ovary. Tandem ovarian sections of an adult rat ovary were hybridized with antisense probes for inhibin {alpha} (A) and CTBP (B and C) and subjected to liquid emulsion autoradiography (left panels) followed by hematoxylin staining (right panels). Both inhibin {alpha} and CTBP mRNA signals are seen in granulosa cells of preantral follicles (arrow) and early antral follicle (arrowhead), but not in atretic follicles (AtF) and corpus lutea (CL). GC, Granulosa cell; T, theca cell; InT, interstitial cell. Photographs are taken at x20 magnification for A and B and x200 for C.

 
FSH suppresses CTBP mRNA expression in primary granulosa cell culture
The differential display showed that the CTBP mRNA level in ROG cells diminished upon 6-h exposure to FSH (data not shown). Although ROG cells show some of the characteristics of undifferentiated granulosa cells (Fig. 2Go) as previously reported (6, 8), we decided to test the FSH responsiveness of CTBP mRNA in a more physiologically relevant system. Granulosa cells were isolated from immature rats treated with 17ß-estradiol and cultured in the presence or absence of FSH for 1–48 h. The RNA expression level was measured by semiquantitative RT-PCR. The RT-PCR analyses revealed an intriguing picture of the time- and hormone-dependent expression of CTBP mRNA (Fig. 3Go). Compared with the abundant expression in untreated granulosa cells, CTBP mRNA was barely detectable within 6 h of exposure to FSH. This down-regulation lasted until 12 h, at which time CTBP mRNA levels began to rise. However, even by 48 h of exposure, the mRNA levels were below the original level before FSH treatment. In contrast to the time- and FSH-dependent fluctuation in CTBP mRNA, an internal control, L-19 mRNA, maintained a stable level with marginal variation. The normalized levels of CTBP mRNA support our conclusion of the down- and up-expression of CTBP mRNA in the primary granulosa cell culture during FSH exposure.



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  		Figure 3. CTBP mRNA expression in primary granulosa cell culture. Granulosa cells were isolated from the ovaries of 24-d-old immature rat primed with 17ß-estradiol. The cells were cultured in the presence of 30 ng/ml FSH for up to 48 h, and RNA was extracted and amplified for CTBP using quantitative RT-PCR. In addition, L-19 mRNA was probed as an internal control. CTBP cDNA and L-19 cDNA were amplified in the same reaction. The CTBP values were normalized with the corresponding values of L-19, and two independent experimental values were averaged. + FSH, Cultured in the presence of FSH; - FSH, cultured in the absence of FSH.

 
Transient expression of CTBP mRNA primarily at early stage of follicular development
The analysis of CTBP mRNA extracted from the cultured primary granulosa cells, as shown in Fig. 2Go, reflects the FSH- and time-dependent changes in the cells. However, it is unclear whether those same changes indeed occur in vivo, and if so, whether in the same time-dependent manner and sequence of down- and up-expression. In addition, there is the outstanding question of whether the down- and up-regulation is also dependent on in vivo cell differentiation and follicle development. In a step to address these questions, in situ hybridization was performed on ovarian sections of immature rats that were primed with PMSG for 0–48 h. In the ovaries of nonprimed rats, CTBP mRNA was detected primarily in preantral and early antral follicles (Fig. 4AGo). Furthermore, it was found exclusively in the granulosa cell layers, consistent with the results shown in Fig. 2Go. As expected, PMSG stimulated follicular growth (Fig. 4Go, B–F). During this follicular development, strong CTBP mRNA signals were observed consistently in small follicles, mostly at preantral and early antral stages.



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  		Figure 4. Follicular stage-dependent expression of CTBP mRNA. Immature 22- to 23-d-old rats were primed with a single injection of PMSG for 0 (A), 3 (B), 6 (C), 24 (D), and 48 (E) h, followed by priming with an hCG injection for 1 (F) and 6 h (G and H). The ovaries were excised, sectioned, and hybridized with the CTBP antisense probe (A–G) or inhibin {alpha} antisense probe (H). In addition to the dark-field autoradiographs in the left panel, the corresponding bright-field images of hematoxylin staining are aligned in the right panels. Arrows and arrowheads indicate preantral and early antral follicles, respectively. PO, Preovulatory follicle; AtF, atretic follicle. Magnification, x40.

 
By 48 h of exposure to PMSG, many follicles reached the large antral stage. However, it was difficult to precisely define PMSG-dependent changes in the CTBP mRNA expression levels (Fig. 4FGo). Therefore, we became interested in the hCG effect. The PMSG-treated rats were additionally primed with hCG for 6 h (Fig. 4GGo). This hCG treatment completely abolished the CTBP signal in large antral follicles, particularly in preovulatory follicles, whereas a high level of CTBP mRNA expression persisted in small follicles. These results indicate that CTBP mRNA is expressed in preantral and early antral follicles, but not in the follicles further developed beyond the early antral stage, particularly in preovulatory follicles. This expression pattern raised the unlikely, yet fundamental, question whether the transcription machinery was shut down altogether, but not specifically for CTBP transcription, in the large follicles. To test this possibility, a tandem ovarian section was probed for inhibin {alpha} transcript. Figure 4HGo shows strong signals of inhibin {alpha} mRNA in the large antral and preovulatory follicles. This result clearly demonstrates not only that active transcription is ongoing in those follicles but also the selective gene transcription of inhibin {alpha} and not CTBP. Taken together, our results suggest that CTBP mRNA expression in small follicles declines as the follicles develop and grow in response to PMSG and hCG, suggesting a complex regulatory mechanism.

De novo protein synthesis is required for the FSH-induced suppression of CTBP mRNA expression
The FSH-dependent CTBP mRNA decrease takes time: it was noticeable by 6 h of exposure to FSH but not 3 h of exposure (Figs. 3Go and 4Go). This is in contrast to the quick response of the cytoskeletal gene expression within 3 h of exposure to FSH (15). This and the putative complex regulatory mechanism of the CTBR mRNA regulation raise the logical question whether or not FSH is directly involved in the decline. To address this question, the effects of cycloheximide (CHX), a translation inhibitor, and {alpha}-amanitin, a transcription inhibitor, were examined. Granulosa cells isolated from the ovaries of immature rats primed with 17ß-estradiol were cultured for 6 h with or without FSH and in the presence or absence of antibiotics. RNA was extracted, and CTBP mRNA was measured by RT-PCR with the internal control, L-19. In the presence of CHX, the FSH-dependent decrease in CTBP mRNA was less than that in the absence of CHX (P < 0.05; Fig. 5Go). The result suggests that de novo synthesis of a protein(s) is involved in the FSH-dependent decline of CTBP mRNA. In contrast, CHX treatment increased the CTBP mRNA level in the absence of FSH, suggesting the possibility that de novo protein synthesis is also involved in maintaining the steady state of CTBP mRNA in granulosa cells of the small follicles. Because the protein factor is associated with the decrease in the CTBP mRNA level, it would be interesting to see whether the protein is an RNase(s).



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  		Figure 5. Effects of cycloheximide and {alpha}-amanitin treatment on the FSH/forskolin-induced expression of CTBP mRNA. Granulosa cells were isolated from 17ß-estradiol primed 24-d-old immature rat ovaries and cultured with FSH (30 ng/ml) or forskolin (FSK, 10 µM) in the presence or absence of CHX (10 µg/ml), or {alpha}-amanitin (AMA, 30 µg/ml) for 6 h. CHX, a translation inhibitor, and AMA, a transcription inhibitor, were pretreated to the cell cultures 1 h before hormone treatment. Total RNA was isolated and analyzed for CTBP and L-19 mRNA by semiquantitative RT-PCR assay using 23 cycles for CTBP and 20 cycles for L-19, which was used as an internal control. CTBP cDNA and L-19 cDNA were amplified in the same reaction. Values shown are the range of two independent experiments along with the mean, indicated by bars. *, P < 0.05 vs. lane 5.

 
The FSH-dependent down-regulation of CTBP mRNA involves de novo synthesis of a protein. Therefore, it is logical to raise the question whether the protein synthesis involves transcriptional regulation. To this end, we tested the effect of {alpha}-amanitin, a transcription inhibitor. Cotreatment with FSH and {alpha}-amanitin completely abolished CTBP mRNA expression, whereas {alpha}-amanitin alone did not significantly impact the expression level compared with the control (Fig. 5Go). A simple explanation of these results is that the de novo synthesis of the protein factor does not require transcription. It is unclear, however, whether the FSH-dependent down-regulation of CTBP mRNA involves suppression of gene transcription, although the mRNA level of a gene is likely transcriptionally regulated.

FSH is capable of activating two distinct signal pathways, the adenylyl cyclase/cAMP pathway and the phospholipase Cß/inositol phosphate and diacyl glycerol pathway. We have previously demonstrated that FSH activates the adenylyl cyclase/cAMP pathway to quickly induce the massive reorganization of the cytoskeletons with dramatic morphological changes (8). To determine whether FSH down-regulates CTBP mRNA via the adenylyl cyclase/cAMP pathway, the cells were treated with forskolin, instead of FSH, which activates adenylyl cyclase and induces cAMP production. Forskolin mimicked the effect of FSH (Fig. 5Go): the drug treatment reduced the CTBP mRNA level, which was partially prevented by cycloheximide, thus confirming the FSH action.

Tissue-specific expression of CTBP mRNA
Because this is the first study on CTBP mRNA expression in the rat, we were prompted to determine tissue distribution of the CTBP transcript. Total RNA was isolated from the liver, stomach, pancreas, lung, bladder, kidney, intestine, brain, and cerebellum of an adult female rat, the testis of an adult male rat, and ovaries of immature rats primed with PMSG with or without hCG. The mRNA appeared in a band of 1.3 kb (Fig. 6Go), suggesting a single transcript. Its expression could not be detected in the stomach, pancreas, lung, bladder, intestine, and testis. The brain showed the highest level of the mRNA, but the cerebellum of the brain did not, suggesting site-specific expression in the brain. Expression was abundant also in the liver and kidney. In addition to these tissues, the CTBP mRNA level in ovaries was examined to directly verify the RT-PCR and in situ hybridization results shown in Figs. 2–5GoGoGoGo. The Northern blot results are consistent with our observations described so far. For example, the adult rat ovary showed a relatively low level of CTBP mRNA. However, the mRNA level was significant in the immature rat ovary primed with PMSG for 12 h but gradually declined as the rats were primed with PMSG for longer periods and additionally with hCG. These results verify that CTBP mRNA is responsive to FSH, and the hormone down-regulates the gene transcripts. In addition, the tissue-specific expression suggests a role of CTBP in tissues other than the eye lens.



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  		Figure 6. Tissue-specific expression of CTBP mRNA in adult rats. The relative mRNA expression level of the CTBP was compared among different tissues by Northern blotting. Twenty micrograms of total RNA from adult tissues and 4 µg of total RNA from immature rat ovaries were separated on 1.2% agarose gel, hybridized with CTBP probe. Lane 1, Liver; 2, stomach; 3, pancreas; 4, lung; 5, bladder; 6, kidney; 7, intestine; 8, brain; 9, cerebellum; 10, testis; 11, adult ovary; 12, ovary from PMSG 12-h-treated immature rat; 13, ovary from PMSG 24-h-treated immature rat; 14, ovary from PMSG 48-h-treated immature rat; 15, ovary from PMSG 48-h- and hCG 6-h-treated immature rat. The blot was stripped and rehybridized with L-19 probe. Note strong intensities of CTBP transcript in liver, kidney, brain, and immature ovaries and no signal from stomach, pancreas, lung, and bladder. Locations of size marker are indicated.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have made a novel observation that CTBP mRNA is expressed in the ovary, particularly in the granulosa cell layer of preantral and early antral follicles, but not in large preovulatory follicles. Its expression is responsive to FSH, which is dependent on the granulosa cell differentiation and follicular development. FSH down-regulates the mRNA via the adenylyl cyclase/cAMP pathway, and mainly by a posttranscriptional mechanism. The down-regulation requires de novo synthesis of a regulatory protein(s), and the CTBP mRNA level is likely regulated by mRNA degradation.

In agreement with our Northern blot result (Fig. 6Go), a similar tissue-specific expression pattern of CTBP mRNA (Fig. 6Go) was observed in the mouse (16). However, specific expression of CTBP transcript in the ovaries has never been described. In this study, expression of CTBP mRNA in rat granulosa cells is demonstrated by several lines of evidence: differential display, semiquantitative RT-PCR, in situ hybridization, and Northern blot. To obtain unequivocal evidence, we examined several different targets: ROG cells, primary granulosa cell culture, and entire ovaries at various stages displaying follicles in a wide range of development. These rigorous examinations led to a number of interesting and potentially significant observations. The conclusion that FSH impacts expression of CTBP mRNA is based on the following observations. FSH treatment for 6 h consistently resulted in the noticeable decline of the CTBP mRNA level in ROG cells, primary granulosa cell cultures, and in growing follicles in the PMSG-primed rat ovaries. This FSH-responsive and preferential expression in preantral and early antral follicles suggests that the expression is dependent on granulosa cell differentiation and follicular development. Consistently, primordial follicles also expressed CTBP mRNA, but large antral follicles showed no or only marginal levels of the mRNA. The down-regulation of CTBP mRNA by FSH in granulosa cells was mediated, at least in part, by the adenylyl cyclase/cAMP signal pathway, because forskolin simulated the FSH action. The CTBP mRNA level appears to decline by mRNA degradation as well as transcription inhibition, but it is not clear how much of the transcriptional inhibition is responsible for the FSH-induced down-regulation. On the other hand, the down-regulation clearly requires de novo synthesis of protein(s), likely from the existing mRNA. It will be interesting to see whether the protein factor is an RNase(s).

Crystalline proteins were initially isolated from the transparent eye lens, and therefore their distribution has been thought to be restricted to the lens and to have only refractive functions (17). However, different crystalline proteins share no significant nucleotide and amino acid sequence homology and are found in tissues other than the eye lens, suggesting other functions. In fact, based on the amino acid sequence homology with enzymes, several nonlens functions have been suggested for µ-crystallin, such as lysine and ornithine cyclodeaminase (13) and a reductase possibly involved in amino acid metabolism (16, 18). The only demonstrated nonlens function for µ-crystallin is nicotinamide adenine dinucleotide phosphate (NADPH)-regulated thyroid hormone binding (19). In search of thyroid hormone binding protein, a protein was isolated that showed high specific binding affinity to thyroid hormone (T3) in a NADP-dependent manner. Subsequent amino acid sequencing and cDNA cloning revealed that the protein was identical with µ-crystallin. Recently, another group (20) has clearly demonstrated that the protein binds T3 and transfers the hormone into the nucleus, where it interacts with its nuclear receptor, the thyroid hormone receptor. Consistent with this, CTBP was also found in thyroid hormone target tissues such as brain, retina, muscle, skin, kidney, and liver (13, 16). Our Northern blot analysis showed similar tissue-specific expression.

Are thyroid hormone and its binding protein involved in the ovulation cycle? Recently, it has been shown that adequate levels of circulating T3 are important for normal female reproductive functions. Changes in T3 levels result in menstrual disturbances, impaired fertility, and altered pituitary gonadotropin secretion in humans and animals (21, 22, 23). T3 modulates FSH and LH action on steroidogenesis in porcine (24, 25) and human (26) granulosa cells in vitro. Consistent with these observations, T3 binding protein and T3 receptor mRNA have been found in mammalian granulosa cells (27, 28).

Some actions of T3 are exerted by its direct contact to target molecules. However, T3 is widely recognized for binding to nuclear receptors and regulating transcription. These T3 receptors belong to the super family of ligand-dependent transcription factors that include the receptors for steroids, retinoids, and vitamin D (19). The steroid receptors have four general functions: binding steroids, shuttling between the cytosol and nucleus, transporting the steroid, and interacting with genes in the nucleus to regulate transcription. In contrast, the T3 receptors do not shuttle between the cytosol and nucleus and therefore cannot transport the ligand, T3, from the cytosol to the nucleus (29). Instead, T3 receptors remain bound to their target genes, regardless of ligand binding (30). Therefore, the thyroid receptors need a cytosolic ligand carrier to transport thyroid hormones from the cytosol to the nucleus. It will be interesting to see whether CTBP fulfills the role of the carrier (20).

How would such a putative thyroid hormone carrier fit into the FSH-induced estrogen surge? It has been shown that T3 inhibits the aromatase activity (31) and down-regulates the aromatase mRNA expression. Therefore, an intriguing possibility is for FSH to lower the level of the thyroid hormone carrier and, thus, the T3 supply to the sites of the aromatase activity. This would be consistent with our observation but needs to be rigorously tested.

In conclusion, CTBP was identified as a FSH-responsive gene in granulosa cells. mRNA encoding this protein is abundantly expressed in immature follicles, but upon exposure to FSH, the transcript level sharply decreased to an undetectable level. This down-regulation is accomplished via the adenylyl cyclase/cAMP pathway, by de novo synthesis of a regulatory protein(s). This down-regulation of CTBP may be an integral part of the FSH-induced surge of estrogen production in granulosa cells.


   Footnotes
 
This work is supported by NIH Grants DK-51469 and HD-18702.

Abbreviations: CHX, Cycloheximide; CTBP, cytosolic T3-binding protein; hCG, human chorionic gonadotropin; NADPH, nicotinamide adenine dinucleotide phosphate (reduced); PMSG, pregnant mare’s serum gonadotropin; RNase, ribonuclease; ROG, rat ovarian granulosa.

Received November 8, 2002.

Accepted for publication February 5, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Hsueh AJ, McGee EA, Hayashi M, Hsu SY 2000 Hormonal regulation of early follicle development in the rat ovary. Mol Cell Endocrinol 163:95–100[CrossRef][Medline]
  2. Richards J 1994 Hormonal control of gene expression in the ovary. Endocr Rev 15:725–751[Abstract/Free Full Text]
  3. Oktay K, Briggs D, Gosden RG 1997 Ontogeny of follicle-stimulating hormone receptor gene expression in isolated human ovarian follicles. J Clin Endocrinol Metab 82:3748–3751[Abstract/Free Full Text]
  4. Kumar TR, Wang Y, Lu N, Matzuk MM 1997 Follicle stimulating hormone is required for ovarian follicle maturation but not male fertility. Nat Genet 15:201–204[CrossRef][Medline]
  5. Dierich A, Sairam MR, Monaco L, Fimia GM, Gansmuller A, LeMeur M, Sassone-Corsi P 1998 Impairing follicle-stimulating hormone (FSH) signaling in vivo: targeted disruption of the FSH receptor leads to aberrant gametogenesis and hormonal imbalance. Proc Natl Acad Sci USA 95:13612–13617[Abstract/Free Full Text]
  6. Li R, Phillips DM, Moore A, Mather JP 1997 Follicle-stimulating hormone induces terminal differentiation in a predifferentiated rat granulosa cell line (ROG). Endocrinology 138:2648–2657[Abstract/Free Full Text]
  7. Li R, Mather JP 1997 Lindane, an inhibitor of gap junction formation, abolishes oocyte directed follicle organizing activity in vitro. Endocrinology 138:4477–4480[Abstract/Free Full Text]
  8. Grieshaber NA, Boitano S, Ji I, Mather JP, Ji TH 2000 Differentiation of granulosa cell line: follicle-stimulating hormone induces formation of lamellipodia and filopodia via the adenylyl cyclase/cyclic adenosine monophosphate signal. Endocrinology 141:3461–3470[Abstract/Free Full Text]
  9. Alliston TN MA, Buse P, Firestone GL, Richards JS 1997 Follicle stimulating hormone-regulated expression of serum/glucocorticoid-inducible kinase in rat ovarian granulosa cells: a functional role for the Sp1 family in promoter activity. Mol Endocrinol 11:1934–1949[Abstract/Free Full Text]
  10. Fitzpatrick SL, Richards JS 1991 Regulation of cytochrome P450 aromatase messenger ribonucleic acid and activity by steroids and gonadotropins in rat granulosa cells. Endocrinology 129:1452–1462[Abstract/Free Full Text]
  11. Ko C, In YH, Park-Sarge OK 1999 Role of progesterone receptor activation in pituitary adenylate cyclase activating polypeptide gene expression in rat ovary. Endocrinology 140:5185–5194[Abstract/Free Full Text]
  12. Chan YL LA, McNally J, Peleg D, Meyuhas O, Wool IG 1987 The primary structure of rat ribosomal protein L19. A determination from the sequence of nucleotides in a cDNA and from the sequence of amino acids in the protein. J Biol Chem 262:1111–1115[Abstract/Free Full Text]
  13. Kim RY, Gasser R, Wistow GJ 1992 µ-Crystallin is a mammalian homologue of Agrobacterium ornithine cyclodeaminase and is expressed in human retina. Proc Natl Acad Sci USA 89:9292–9296[Abstract/Free Full Text]
  14. Schultz Sr G 1979 Principle of protein structure. New York: Springer-Verlag; 14–16
  15. Grieshaber NA, Ko C, Grieshaber SS, Ji I, Ji TH 2003 Follicle-stimulating hormone-responsive cytoskeletal genes in rat granulosa cells: class I ß-tubulin, tropomyosin-4, and kinesin heavy chain. Endocrinology 144:29–39[Abstract/Free Full Text]
  16. Aoki N, Ito K, Ito M 2000 µ-Crystallin, thyroid hormone-binding protein, is expressed abundantly in the murine inner root sheath cells. J Invest Dermatol 115:402–405[CrossRef][Medline]
  17. Magabo KS, Horwitz J, Piatigorsky J, Kantorow M 2000 Expression of ßB(2)-crystallin mRNA and protein in retina, brain, and testis. Invest Ophthalmol Vis Sci 41:3056–3060[Abstract/Free Full Text]
  18. Segovia L, Horwitz J, Gasser R, Wistow G 1997 Two roles for µ-crystallin: a lens structural protein in diurnal marsupials and a possible enzyme in mammalian retinas. Mol Vis 3:9[Medline]
  19. Vie MP, Evrard C, Osty J, Breton-Gilet A, Blanchet P, Pomerance M, Rouget P, Francon J, Blondeau JP 1997 Purification, molecular cloning, and functional expression of the human nicotinamide-adenine dinucleotide phosphate-regulated thyroid hormone-binding protein. Mol Endocrinol 11:1728–1736[Abstract/Free Full Text]
  20. Mori J, Suzuki S, Kobayashi M, Inagaki T, Komatsu A, Takeda T, Miyamoto T, Ichikawa K, Hashizume K 2002 Nicotinamide adenine dinucleotide phosphate-dependent cytosolic T3 binding protein as a regulator for T3-mediated transactivation. Endocrinology 143:1538–1544[Abstract/Free Full Text]
  21. Erfurth EM, Hedner P 1986 Thyroid hormone metabolism in thyroid disease as reflected by the ratio of serum triiodothyronine to thyroxine. J Endocrinol Invest 9:407–412[Medline]
  22. Webster JR, Moenter SM, Barrell GK, Lehman MN, Karsch FJ 1991 Role of the thyroid gland in seasonal reproduction. III. Thyroidectomy blocks seasonal suppression of gonadotropin-releasing hormone secretion in sheep. Endocrinology 129:1635–1643[Abstract/Free Full Text]
  23. Shi ZD, Barrell GK 1992 Requirement of thyroid function for the expression of seasonal reproductive and related changes in red deer (Cervus elaphus) stags. J Reprod Fertil 94:251–259[Abstract/Free Full Text]
  24. Chan WK, Tan CH 1986 Inhibition of follicle-stimulating hormone induction of aromatase activity in porcine granulosa cells by thyroxine and triiodothyronine. Endocrinology 119:2353–2359[Abstract/Free Full Text]
  25. Maruo T, Hayashi M, Matsuo H, Yamamoto T, Okada H, Mochizuki M 1987 The role of thyroid hormone as a biological amplifier of the actions of follicle-stimulating hormone in the functional differentiation of cultured porcine granulosa cells. Endocrinology 121:1233–1241[Abstract/Free Full Text]
  26. Goldman S, Dirnfeld M, Abramovici H, Kraiem Z 1993 Triiodothyronine (T3) modulates hCG-regulated progesterone secretion, cAMP accumulation and DNA content in cultured human luteinized granulosa cells. Mol Cell Endocrinol 96:125–131[CrossRef][Medline]
  27. Bandyopadhyay A, Roy P, Bhattacharya S 1996 Thyroid hormone induces the synthesis of a putative protein in the rat granulosa cell which stimulates progesterone release. J Endocrinol 150:309–318[Abstract/Free Full Text]
  28. Wakim AN, Polizotto SL, Buffo MJ, Marrero MA, Burholt DR 1993 Thyroid hormones in human follicular fluid and thyroid hormone receptors in human granulosa cells. Fertil Steril 59:1187–1190[Medline]
  29. Oppenheimer JH 1985 Thyroid hormone action at the nuclear level. Ann Intern Med 102:374–384
  30. Lazar MA 1993 Thyroid hormone receptors: multiple forms, multiple possibilities. Endocr Rev 14:184–193[Abstract/Free Full Text]
  31. Gregoraszczuk EL, Slomczynska M, Wilk R 1998 Thyroid hormone inhibits aromatase activity in porcine thecal cells cultured alone and in coculture with granulosa cells. Thyroid 8:1157–1163[Medline]



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