Silencing of the Gene for the {alpha}-Subunit of Human Chorionic Gonadotropin by the Embryonic Transcription Factor Oct-3/4

Limin Liu1, Douglas Leaman2, Michel Villalta3 and R. Michael Roberts

Departments of Biological Sciences (L.L.) and Animal Sciences and Biochemistry (D.L., M.V., R.M.R.), University of Missouri, Columbia, Missouri 65211


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
CG is required for maintenance of the corpus luteum during pregnancy in higher primates. As CG is a heterodimeric molecule, some form of coordinated control must be maintained over the transcription of its two subunit genes. We recently found that expression of human CG ß-subunit (hCGß) in JAr human choriocarcinoma cells was almost completely silenced by the embryonic transcription factor Oct-3/4, which bound to a unique ACAATAATCA octameric sequence in the hCGß gene promoter. Here we report that Oct-3/4 is also a potent inhibitor of hCG {alpha}-subunit (hCG{alpha}) expression in JAr cells. Oct-3/4 reduced human GH reporter expression from the -170 hCG{alpha} promoter in either the presence or absence of cAMP by about 70% in transient cotransfection assays, but had no effect on expression from either the -148 hCG{alpha} or the -99 hCG{alpha} promoter. Unexpectedly, no Oct-3/4-binding site was identified within the -170 to -148 region of the hCG{alpha} promoter, although one was found around position -115 by both methylation interference footprinting and electrophoretic mobility shift assays. Site-directed mutagenesis of this binding site destroyed the affinity of the promoter for Oct-3/4, but did not affect repression of the promoter. Therefore, inhibition of hCG{alpha} gene transcription by Oct-3/4 appears not to involve direct binding of this factor to the site responsible for silencing. When stably transfected into JAr cells, Oct-3/4 reduced the amounts of both endogenous hCG{alpha} mRNA and protein by 70–80%. Oct-3/4 is therefore capable of silencing both hCG{alpha} and hCGß gene expression. We suggest that as the trophoblast begins to form, reduction of Oct-3/4 expression permits the coordinated onset of transcription from the hCG{alpha} and hCGß genes.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
CG is a luteotropic factor and the primary signal for maternal recognition in higher primates, including the human (1, 2). It consists of an {alpha}-subunit (hCG{alpha}), common to all the glycoprotein hormones, and a distinct ß-subunit (hCGß) responsible for biological specificity. To produce biologically active hCG, the two subunit molecules must be produced in a coordinated manner. Not unexpectedly, therefore, synthesis of hCG{alpha}- and ß-subunits is reasonably well balanced in the first trimester of human pregnancy (3, 4), although little is understood about how this coordinated synthesis is achieved.

Transcriptional regulation appears to be paramount in the control of hCG production (1, 2). The hCG{alpha} subunit is encoded by only a single gene (5, 6), whereas there is a cluster of six hCGß subunit genes (1, 7). Multiple regulatory elements in the upstream promoter region of the hCG{alpha} gene have been found to be important for transcriptional activation of the gene in choriocarcinoma cells (8, 9, 10, 11, 12, 13, 14, 15). They include two tandem repeats of a cAMP response element (CRE), a complex upstream regulatory element (URE), the {alpha}-activator ({alpha}-ACT) element, the junctional regulatory element (JRE), and the CCAAT region. The transacting factors that bind to these elements, with the exception of the CRE-binding protein (CREB), remain to be identified and cloned. Although much less studied, the hCGß gene also contains multiple regulatory regions that contribute toward expression in choriocarcinoma cells (16, 17, 18, 19). Curiously, the promoter regions of hCG{alpha}- and ß-subunit genes share little similarity, and no transcription factor important for regulation of both genes in developing trophectoderm has been identified.

Oct-3/4, characterized by its conserved POU DNA-binding domain, is expressed in totipotent/pluripotent embryonic cells and is a strong candidate for a regulator of early embryogenesis (20, 21, 22, 23, 24, 25). In mouse, Oct-3/4 mRNA and the protein itself have been detected in early stage trophectoderm but not in trophoblast cells after the blastocyst has hatched from the zona pellucida (20, 23, 24). By analogy with the mouse, down-regulation of Oct-3/4 in the human embryo probably coincides with the first expression of hCG in trophectoderm.

Recently, we found that hCGß expression in JAr human choriocarcinoma cells was almost completely silenced by Oct-3/4 (25). Reporter expression from an hCGß gene promoter was reduced about 90% by Oct-3/4 in transient transfection assays. Oct-3/4 specifically bound to a sequence (CAATAATCA; -276/-268) in the hCGß gene promoter as measured in electrophoretic mobility shift assays (EMSAs) and methylation interference footprinting analyses. Mutation of this binding site abolished Oct-3/4 repression (25). Here we report that Oct-3/4 is also a potent inhibitor of hCG{alpha} expression in JAr cells and suggest that a loss of Oct-3/4 expression in developing trophectoderm may permit the coordinated production of both CG subunits in the early human embryo.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Inhibition of hCG{alpha}-GH Expression by Oct-3/4
A -170 hCG{alpha} promoter is sufficient to provide full expression and cAMP responsiveness to a reporter gene in human choriocarcinoma cells (26, 27, 28). Initial experiments with the -170 hCG{alpha}-CAT construct showed that it was inhibited in JAr cells in a dose-dependent manner by pcDNA3-Oct-3/4 cotransfection and that 8-bromo-cAMP (1 mM), despite enhancing promoter activity approximately 10-fold in controls, did not reverse the Oct-3/4 inhibition (data not shown).

To define where Oct-3/4 effects in the promoter were manifested, various truncated promoter constructs were tested in the cotransfection assay (Fig. 1Go). Whereas human GH (hGH) expression from -170 hCG{alpha}-GH was inhibited approximately 70% by pcDNA3-Oct-3/4 transfection, there was no inhibition of the -99 construct. Similarly, Oct-3/4 failed to inhibit expression from the -148 hCG{alpha} promoter, which lacks the URE (11, 26) but contains intact CRE elements (8, 9, 10). Therefore, a site within the -170 to -149 URE region is likely responsible for the Oct-3/4 effects. When -170 hCG{alpha}-GH was cotransfected with an Oct-1 expression plasmid, hGH expression was not affected (112% ± 17 of the control value).



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Figure 1. Inhibition of hCG{alpha}-hGH by Oct-3/4 CoTransfection in JAr Cells

The hCG{alpha}-hGH constructs were cotransfected with either pcDNA3-Oct-3/4 or pcDNA3 in the controls. The Oct-3/4-binding site in the hCG{alpha} promoter (Oct, marked by the bold underline here and in Fig. 3Go) was destroyed in the µ-170 hCG{alpha}-GH by site-direct mutagenesis (*). URE and the two identical CREs in the hCG{alpha} promoter are shown. The results were the means (±SEM) of at least five independent experiments; the SEM are interexperimental values. Values marked with different letters are statistically different (P < 0.001). Because truncations and other mutations affect control values in the absence of Oct3/4 transfection, all results are calculated as percent of controls, i.e. nontransfected cultures. Human GH expression values in the reporter assay in the absence of Oct-3/4 were as follows: -170 hCG{alpha}-hGH, 263,320 ± 3,320; -148 hCG{alpha}-hGH 7,259 ± 509; -99 hCG{alpha}-hGH 1,728 ± 18; µ-170 hCG{alpha}-hGH, 95,493 ± 7,600. The background values in the absence of the transfected constructs was 210 ± 20.

 
Inhibition of -170/-lOO hCG{alpha} Enhancer Activity by Oct-3/4
The -170/-100 region of the hCG{alpha} promoter transcriptionally activated a variety of heterologous promoters in human trophoblast cell lines (8, 10, 29). Whether Oct-3/4 could inhibit these enhancer effects was studied by cotransfection of pcDNA3-Oct-3/4 with a series of hCG{alpha}-TKGH constructs in which hCG{alpha} fragments (-170/-53, -172/-100, and -153/-129) had been fused to a TKGH hybrid gene (Fig. 2Go). Human GH reporter expression from the thymidine kinase (TK) promoter was increased 9- and 6-fold by the -170/-53 and -170/-100 hCG{alpha} fragments, respectively, in the absence of Oct-3/4 (Fig. 2Go). When pcDNA3-Oct-3/4 was cotransfected, hGH expression from -170/-53{alpha}-TKGH and -170/-100{alpha}-TKGH was in each case reduced to about 30% of control values. Expression from the -153/-129 construct, which lacked the URE but contained an intact CRE, was not affected.



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Figure 2. Inhibition of hCG{alpha}-TKGH by Oct-3/4 Cotransfection in JAr Cells

The hCG{alpha}-TKGH constructs were cotransfected with either pcDNA3-Oct-3/4 or pcDNA3. Human GH expression is shown as a percentage of that from TKGH in the absence of Oct-3/4 cotransfection. The results are the means (+SEM) of at least four independent experiments.

 
There was concern that some of the Oct-3/4 effects shown in Fig. 2Go might be through the TK promoter, which has a canonical ATTTGCAT octamer motif (30). This sequence, therefore, was removed. Expression from the modified construct (-170/-53{alpha}-2.3TKGH) was inhibited by Oct-3/4 about as effectively as the original -170/-53{alpha}-TKGH construct (Fig. 2Go).

Binding of Oct-3/4 to the hCG{alpha} Promoter in Vitro
Oct-3/4 synthesized by coupled in vitro transcription and translation formed a complex with a 32P-labeled -170/-53 hCG{alpha} promoter fragment (Fig. 3AGo, lane 2). Such a labeled complex was absent when the reticulocyte lysate alone was used (lane 1). It was not detected when the OCT consensus oligonucleotide was added as competitor (lane 3).



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Figure 3. Binding of Oct-3/4 to the hCG{alpha} Promoter In Vitro

A, EMSA with 32P-labeled -170/-53 hCG{alpha} as a probe. Oct was used as a competitor (100-fold molar excess; lane 3). B, Methylation interference footprint of the hCG{alpha} promoter in the presence of Oct-3/4. The -170/-53 hCG{alpha} fragment was radiolabeled at the 5'-end of its sense strand. The free (F) probes and the Oct-3/4-bound (B) probes were cleaved at the methylated sites and resolved on an 8% sequencing gel. The sites where methylation apparently interfered with Oct-3/4 binding are indicated with arrows. Lanes 1 and 3 are identical. C, EMSA with [32P]-170/-100 hCG{alpha} as a probe. The Oct-3/4-hCG{alpha} probe complex is indicated by an arrow. D, EMSA with 32P-labeled µ-170/-100 hCG{alpha}. The nucleotides A (-111) and T (-110) within the Oct-3/4-binding region of the hCG{alpha} promoter (see Fig. 3EGo) were altered to C and G, respectively, in µ-170/-100 hCG{alpha} by site-directed mutagenesis. Equal amounts of probe were used in panels C and D. In panel D, most of the free probe had run from the gel. E, The hCG{alpha} promoter sequence around the methylation interference sites. The boxed region (Oct) represents a motif with some similarity (six nucleotides of eight) for the optimal binding sequence of the POU domain established for Oct-1 (4).

 
Methylation interference analysis with a probe that had been methylated in the sense strand at positions -115 and -114 clearly bound Oct-3/4 less well than unmethylated probe (Fig. 3BGo). The -117 to -110 sequence within the hCG{alpha} gene promoter is identical at six positions to the consensus octamer motif (Fig. 3EGo). Methylation at other positions in the sense strand of -170/-53 hCG{alpha} (Fig. 3BGo) or at positions in the antisense strand of -170/-100 hCG{alpha} (data not shown) did not appear to interfere with Oct-3/4 binding.

Effect of Mutating the 0ct-3/4 Binding Site in the hCG{alpha} Promoter
The -170/-100 hCG{alpha} fragment formed a single complex with Oct-3/4 in the EMSA (Fig. 3CGo), but after the nucleotides A (-111) and T (-110) within the putative Oct-3/4 binding region (Fig. 3EGo) were changed to C and G, respectively, the resulting mutant µ-170/-100 hCG{alpha} fragment was unable to bind to Oct-3/4 (Fig. 3DGo). Whether this Oct-3/4 site defined by mobility shift assays and by methylation interference was necessary for Oct-3/4 inhibition of the hCG{alpha} promoter was studied by fusing the µ-170/-100 hCG{alpha} fragment to the -99 hCG{alpha} promoter to form the µ-170 hCG{alpha}-GH and -chloramphenicol acetyl transferase (CAT) constructs. As expected (13), reporter expression from the mutant promoter was only about 30% (±10%) of that from the wild type -170 hCG{alpha} promoter in the absence of Oct-3/4 cotransfection. However, both hGH and CAT expression from the mutant promoter were repressed as strongly as from the wild type promoter by Oct-3/4 (Fig. 1Go; data not shown for CAT constructs).

It would appear that even though there is a well defined Oct-3/4-binding site on the hCG{alpha} promoter, it is not required for Oct-3/4 inhibitory effects.

Inhibition of Endogenous hCG{alpha}-Subunit Production in JAr Cells by 0ct-3/4 Stable Transfection
To study the effect of Oct-3/4 on endogenous hCG{alpha} gene expression, JAr cells were stably transfected with pcDNA3-Oct-3/4 (25). Total RNA was isolated from both stable Oct-3/4 clones and stable control clones and subjected to a ribonuclease protection assay in the presence of an antisense hCG{alpha} RNA probe expected to hybridize to a 440-bp fragment of the hCG{alpha} transcript. As shown in Fig. 4AGo, the quantity of hCG{alpha} mRNA in both of the stable Oct-3/4 clones tested (S1 and S4) was clearly much lower than in the two stable control clones (C1 and C2) and in the normal JAr cells (J). In contrast, the content of ß-actin mRNA was comparable among all clones, whether they expressed Oct-3/4 or not. When quantitated by densitometry and normalized to ß-actin mRNA, the hCG{alpha} mRNA content of the clones expressing Oct-3/4 was about 23% of that in the controls (Fig. 4BGo).



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Figure 4. Decrease of Endogenous hCG{alpha} mRNA Levels in JAr Cells after Oct-3/4 Stable Transfection

A, Ribonuclease protection assays were carried out as described in Materials and Methods to determine relative amounts of hCG{alpha} mRNA in stable JAr clones. Clones S1 and S4 had been stably transfected with pcDNA3-Oct-3/4, C1, and C2 with pcDNA3. Lane J represents RNA from normal JAr cells. The protected hCG{alpha} fragment and the internal ß-actin control are indicated by arrows. No protection was observed when yeast RNA was run as a negative control. B, The signals were then quantitated by densitometry. The exposure time used to obtain appropriate optical densities in x-ray film was 30 min for ß-actin and 20 min for hCG{alpha}. All hCG{alpha} values were then normalized by comparison with ß-actin. hCG{alpha} mRNA levels in the clones expressing Oct-3/4 are significantly lower than in controls (P < 0.03).

 
Production of the hCG{alpha} subunit measured by a RIA was significantly reduced (P < 0.001) in the clones expressing Oct-3/4 (Fig. 5Go). For clones S1 and S4, the amount of hCG{alpha} was 33% and 19%, respectively, of the average production of two control lines C1 and C2.



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Figure 5. Reduced Production of the Endogenous hCG{alpha} Subunit in JAr Cells Stably Transfected with pcDNA3-Oct-3/4

Stable clones C1, C2, S1, and S4 are the same as in Fig. 4Go. Amounts of hCG{alpha} secreted by the stable clones were measured by a RIA that used a rabbit antiserum specific to the hCG{alpha} subunit. The results are the means (±SEM) of four independent experiments. hCG{alpha} production in the clones expressing Oct-3/4 is significantly lower than in controls (P < 0.001).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In this study, we have found that the transcription factor Oct-3/4 is a potent repressor of hCG{alpha} gene expression in JAr choriocarcinoma cells. Stable expression of Oct-3/4 reduced the amounts of both endogenous hCG{alpha} mRNA and hCG{alpha} protein by 70 to 80% in these cells. Oct-3/4 also strongly inhibited reporter expression from the hCG{alpha} gene promoter in transient transfection assays. Nevertheless, the mechanism whereby hCG{alpha} transcription is repressed by Oct-3/4 appears distinct from the manner in which hCGß transcription is silenced. Whereas the Oct-3/4-binding site in the hCGß promoter is critical for its repression, the binding site in the hCG{alpha} promoter appears dispensable.

Transient transfection experiments with the hCG{alpha} promoter-reporter gene constructs revealed selective inhibition of the promoter by Oct-3/4 relative to controls (Figs. 1Go and 2Go). The responsive region was located between -170 and -149 (Fig. 1Go), but no direct binding of Oct-3/4 to this region could be demonstrated (Fig. 3Go, B and D), although the existence of a cryptic site available in vivo cannot be ruled out. Expression from constructs lacking the -170/-149 region were not affected by Oct-3/4 expression. The inhibitory effects, therefore, were not due to sequence-independent squelching of some general transcription factor as has been observed for the transcriptional repressor Dr1/Dc2 (31, 32). Preliminary experiments, less complete than those shown in Figs. 1Go and 2Go and performed with a CAT rather than a GH reporter gene, have shown that a deletion of the -160/-155 region on the promoter (the µVIII hCG-CAT construct of 26 abolished the responsiveness of the promoter to Oct-3/4. Curiously, however, as assessed by EMSA and by methylation interference assays (Fig. 3Go, A–C), Oct-3/4 was found to associate with the promoter not within the URE, but at another site (-117/-110). However, mutation of this sequence had no influence on the ability of Oct-3/4 to repress the hCG{alpha} promoter (Fig. 1Go). The significance, if any, of Oct-3/4 binding remains unclear.

Oct-3/4 probably exerts its effects within the URE region (-177/-141) of the hCG{alpha} promoter (Fig. 1Go). The narrower -170/-149 region responsive to Oct-3/4 is believed to bind at least three different nuclear proteins from choriocarcinoma cells in vitro (11, 15, 18, 26). The URE2/TSE binding factor that requires the sequence from -177 to -156 (12, 15) is probably unable to bind the shorter -170 hCG{alpha} promoter used primarily in the present study and is unlikely, therefore, to be the target of Oct-3/4. The UREl-binding protein (12, 26) and the {alpha}ACT-binding protein (15), on the other hand, are known to bind the -170 promoter directly. A possible mechanism of Oct-3/4 inhibition is that it interacts directly with one or more of these URE-associated transcriptional activators and influences their abilities to associate with either the promoter or CREB, the CRE-binding transcription activator (33), in the downstream CRE elements upon which the URE depends exclusively for its enhancing activity (10, 11, 12). An alternative hypothesis is that Oct-3/4 sequesters (squelches), or represses by some other means, transcriptional coactivators necessary for proper functioning of the URE-associated factors. Some POU-containing proteins, including Oct-1, regulate transcription through their cooperative interactions with other transcription cofactors (34, 35, 36, 37).

In summary, the gene for hCG{alpha}-subunit, like those for the ß-subunit, has been shown to be a target of the embryonic transcription factor Oct-3/4. Both subunit genes are coordinately repressed in the presence of Oct-3/4, a phenomenon that may explain why these genes are silent in the inner cell mass of the human embryo but expressed in trophectoderm. Surprisingly, the mechanisms by which silencing of the two genes is mediated appear to differ.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Construction of Plasmids
The -170/+44 hCG{alpha} (SanI-BamHI), -99/+44 hCG{alpha} (RsaI-BamHI) and -148/+29 hCG{alpha} (isolated by SacI and HindIII digestion of pµIXhCG{alpha}-CAT; 26 promoter fragments (8) were fused to the hGH gene of the p0GH plasmid (Nichols Institute Diagnostics, San Juan Capistrano, CA) to form p-170 hCG{alpha}-GH, p-99 hCG{alpha}-GH, and p-148 hCG{alpha}-GH, respectively. The mutant µ-170/-100 hCG{alpha} fragment was produced by PCR with oligonucleotide primer µ{alpha}OctR (see Table 1Go) and was cloned upstream of the -99 hCG{alpha}-GH fusion gene to form pµ-170 hCG{alpha}-GH, in which the µ-170/-100 and -99/+44 hCG{alpha} fragments were in the same orientation (confirmed by DNA sequencing). The hCG{alpha}-CAT plasmids, p-170 hCG{alpha}-CAT, p-99 hCG{alpha}-CAT, and pµ-170 hCG{alpha}-CAT, were constructed in a way similar to that described for hCG{alpha}-GH construction.


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Table 1. Synthetic Oligonucleotides used in PCR, EMSA, Methylation Interference Assay, and Mutagenesis

 
The -170/-53 (DdeI-DdeI) and the -170/-100 (DdeI-RsaI) hCG{alpha} fragments were cloned upstream of the TKGH fusion gene through the HindIII site in the pTKGH plasmid (Nichols Institute Diagnostics) to form the plasmid p-170/-53{alpha}-TKGH and p-170/-100{alpha}-TKGH, respectively. The p-170/-53{alpha}-2.3TKGH plasmid was constructed by fusing the -170/-53 hCG{alpha} fragment to a 2.3-kb EcoRI fragment of the TKGH fusion gene, which was devoid of the -198/-79 TK promoter fragment that contains a consensus octamer motif (ATGCAAAT at -131/-138; 30 . Oligonucleotide {alpha}CREf and {alpha}CREr (Table 1Go) were annealed to each other to form the double-stranded {alpha}CRE fragment. Two tandem repeats of the {alpha}CRE fragment were then cloned into the HindIII site of pTKGH to form p-153/-129{alpha}-TKGH. The hCG{alpha}-GH, hCG{alpha}-CAT, and hCG{alpha}-TKGH constructs were confirmed by DNA sequencing.

A 440-bp PstI hCG{alpha} cDNA fragment that covers exon 1, 2, and part of exon 3 of the hCG{alpha} gene (5) was isolated and cloned into the PstI site of the pGEM-4Z vector (Promega, Madison, WI). The orientation of the hCG{alpha} fragment relative to the T7 promoter in the pGEM-hCG{alpha} plasmid was determined by DNA sequencing.

Construction of pcDNA3-Oct4 from pcMV-Oct4 (22) was described previously (25). Expression plasmid pCGOct-1 was provided by Dr. W. Herr (38). Plasmids p0GH, pTKGH, and pXGH5 were purchased from Nichols Institute Diagnostics.

Transient Transfection, hGH RIA, and CAT Assay
The phCG{alpha}-GH plasmid (2 µg) was cotransfected with either pcDNA3-Oct-3/4 (2 µg) or pcDNA3 (2 µg) into JAr cells in six-well tissue culture plates (25). The amount of hGH secreted was measured by a specific RIA (25). Expression from the phCG{alpha}-TKGH construct was analyzed in a similar manner. Either pcDNA3-Oct-3/4 (2 µg) or pcDNA3 (2 µg) was cotransfected with phCG{alpha}- CAT (2 µg) and a pTKGH internal control plasmid (0.2 µg) into JAr cells. CAT activity was then normalized in relation to hGH production (25). ANOVA and a standard Student’s t test were used for statistical analyses (39).

EMSA and Methylation Interference Analysis
Oct-3/4 protein, produced by coupled in vitro transcription and translation in a reticulocyte lysate (25), was used with the 32P-labeled -170/-53 hCG{alpha}, -170/-100 hCG{alpha}, and µ-170/-100 hCG{alpha} fragments in electrophoretic mobility shift assays (25). The double-stranded OCT oligonucleotide, which had been produced by annealing oligonucleotide OCTf and OCTr (Table 1Go), was included as a competitor in some reactions. Single-ended labeled -170/-53 hCG{alpha} fragments were prepared and subjected to methylation interference analyses (25).

Stable Transfection of Oct-3/4, Ribonuclease Protection Assay, and RIA of hCG
JAr cells were stably transfected with pcDNA3-Oct-3/4 or pcDNA3 (25). Ribonuclease protection assays of the total RNA (10 µg) isolated from several stable clones (-2 x 108 cells) were performed by standard procedures (25). Antisense hCG{alpha} probe was synthesized from pGEM-hCG{alpha} by using T7 bacteriophage RNA polymerase in the presence of {alpha}-[32P]CTP (800 Ci/mmol, DuPont NEN, Weymouth, MA). Antisense human ß-actin RNA probe was synthesized to an 80-fold lower specific activity (by diluting the original 800 Ci/mmol {alpha}-[32P]CTP with a concentrated solution of unlabeled CTP) from the pTRI-ß-actin-125-human antisense control template (Ambion Inc, Austin, TX). The human ß-actin probe (10,000 cpm) was used together with the hCG{alpha} probe (100,000 cpm) for each protection assay. The relative amounts of radiolabel in the protected ß-actin and hCG{alpha} fragments were measured by densitometry. The radiolabel in hCG{alpha} mRNA was then normalized relative to the label in ß-actin mRNA.

The amount of hCG{alpha} (either as the free subunit or as the subunit of intact hCG heterodimer) secreted by cultured JAr cells (~2 x 106 cells per culture) over a 24-h period was measured by a double-antibody RIA (40). The procedure employed rabbit anti-hCG{alpha} antiserum (AFP-310784) and hCG{alpha} subunit standard (CR-119). Both AFP-310784 and CR-119 were supplied by the National Hormone and Pituitary Program (NIDDK, Rockville, MD). Mean intra- and interassay coefficients of variation were 3.5% and 6.4%, respectively.


    ACKNOWLEDGMENTS
 
We thank Dr. H. R. Schöler for pCMV-Oct4, Dr. W. Herr for pCGOct-1, Dr. I. Boime for hCG{alpha} cDNA clone, Dr. J. H. Nilson for pµIXhCG{alpha}-CAT, Dr. D. H. Keisler for reagents and help in the RIA of hCG{alpha}, and the National Hormone and Pituitary Program, NIH, for anti-hCG{alpha} antiserum and hCG{alpha} subunit standard. We thank Ellen Swanson for help in preparing the manuscript.


    FOOTNOTES
 
Address requests for reprints to: R. Michael Roberts, 158 Animal Sciences Research Center, University of Missouri-Columbia, Columbia, Missouri 65211.

This work was supported by NIH Grants HD-21896 and HD-29843. This paper is a contribution from the Missouri Agricultural Experiment Station, Journal Series Number 12,488.

1 Present address: Duke University Medical Center, Department of Medicine, Durham, NC 27710. Back

2 Present address: Department of Molecular Biology, Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195-5210. Back

3 Present address: Servicio de Investigaci 92 n Agraria Diputaci 92 n General de Aragon, Apartado 727, 50.080-Zaragoza, Spain. Back

Received for publication February 21, 1997. Revision received July 3, 1997. Accepted for publication July 17, 1997.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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