0013-7227/91/1281-0388$02.00/0 Endocrinology Copyright © 1991 by The Endocrine Society

Vol. 128, No. 1 Printed in U.S.A.

Evidence that Human Chorionic Gonadotropin/ Luteinizing Hormone Receptor Down-Regulation Involves Decreased Levels of Receptor Messenger Ribonucleic Acid* YETTA M. HOFFMAN, HELLE PEEGEL, MARJA J. E. SPROCK, QING-YU ZHANG, AND K. M. J. MENON Endocrine Laboratory, Departments of Obstetrics/Gynecology and Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109

ABSTRACT. Injection of pseudopregnant rats with pharmacological doses of hCG leads to a characteristic decrease in LH/ hCG binding by the isolated luteal cells. The steady state levels of LH/hCG receptor mRNA were determined in rat ovaries during hCG-induced down-regulation of the receptor. Northern blots were performed using a 20-mer probe corresponding to a guanine cytosine-rich carboxyl-terminal untranslated region of the LH/hCG receptor cDNA. The hybridization of the probe to LH/hCG receptor mRNA was highly specific, since the probe hybridized only to rat luteal cell RNA fraction, with no signal detected in nontarget tissues. The LH/hCG receptor level was quantitated by [125I]hCG binding to the isolated membrane fractions from the corresponding treatment and control groups.

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T IS NOW well established that the gonadotropins hCG and LH regulate ovarian function by binding to specific receptors on the luteal cell surface (1, 2). In the pseudopregnant rat, the binding of [125I]hCG to luteal cell receptors is related to the physiological state of the ovary (3, 4), and the binding activity parallels the ability of tissue to secrete progesterone (5). In the pseudopregnant rat, injection of a supraphysiological dose of hCG abolishes the capacity of isolated luteal cells to bind [125I] hCG (6, 7). This loss of hCG-binding sites could be attributed to a number of causes, including receptor internalization, receptor degradation, modification of the receptor protein leading to a loss of hormone-binding activity, or a decrease in receptor mRNA resulting from diminished transcriptional activity or increased message degradation. Since the sequence for the LH/hCG receptor cDNA

Examination of mRNA levels of the receptor during hCG-induced down-regulation showed a steady decrease from 0-24 h, followed by a gradual increase to control levels from 24-72 h corresponding to days 8-9 of pseudopregnancy. The [1?5I]hCGbinding activity during down-regulation paralleled the mRNA profile in both the experimental and control groups. Examination of the levels of mRNA for a-actin showed no change during this period, suggesting that the loss of LH/hCG receptor mRNA at 24 h was not due to a general loss of mRNA in luteal cells. These results suggest that hCG-induced down-regulation of the LH/hCG receptor in luteal cells involves regulation of the receptors at the message level. {Endocrinology 128: 388-393, 1991)

corresponding to the 92K dalton receptor from rat luteal cells is now available (8), we have investigated the fate of LH/hCG receptor mRNA in rat ovary during the gonadotropin-induced refractory state. Our data show that during hCG-induced down-regulation of LH/hCG receptors in rat luteal cells, the loss of binding activity closely parallels a decrease in receptor mRNA.

Materials and Methods Materials

Received August 2, 1990. Address all correspondence and requests for reprints to: Dr. K. M. J. Menon, Department of Obstetrics and Gynecology, University of Michigan, Ann Arbor, Michigan 48109-0278. * This work was supported by NIH Grant HD-06656. Presented in part in preliminary form at the 72nd Annual Meeting of The Endocrine Society, Atlanta, GA, June 1990.

hCG was a gift from the Center for Population Research, NICHHD (Bethesda, MD). The sodium salt of 125I, [a-32P] dCTP (>3000 Ci/mmol) and [7-32P]ATP (>4500 Ci/mmol) were obtained from ICN (Irvine, CA). Salmon sperm DNA and hCG for animal treatment were obtained from Sigma (St. Louis, MO). PMSG was purchased from Calbiochem (La Jolla, CA). Klenow fragment of DNA polymerase was obtained from BRL (Gaithersburg, MD). The a-actin cDNA was a gift from Dr. Bob Lyons of this University. BCA reagents for protein assay were obtained from Pierce (Rockford, IL). Nitrocellulose was obtained from Millipore (Bedford, MA), and X-Omat film for autoradiography was obtained from Kodak (Rochester, NY).

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REGULATION OF GONADOTROPIN RECEPTOR mRNA All other chemicals used were ultrapure reagents designed for use in molecular biology. Animals and tissues Pseudopregnancy was induced using the procedure described by Parlow (9). Immature female rats were injected with 50 IU PMSG, followed by 25 IU hCG 56 h later, which represented day 0. When indicated, down-regulation was induced in pseudopregnant rats by injecting 50 IU hCG on day 5 of pseudopregnancy; control rats received saline. Ovaries were collected and either processed immediately or stored in liquid nitrogen until needed. Membrane preparation A partially purified membrane fraction was prepared as described previously from our laboratory (10). Briefly, ovaries were homogenized in 5 vol PBS (0.15 M NaCl-0.05 M phosphate pH 7.4) and centrifuged at 400 x g for 4 min. The pellet was washed once, and the combined supernatants were centrifuged at 700 X g for 10 min. The pellet was again washed once, and the combined supernatants were centrifuged at 9000 X g for 15 min. The resulting pellet was suspended in 4 vol PBS based on tissue weight and stored at —70 C until used. Protein was determined by the method of Smith et al. (11) using BCA reagents. hCG binding assays Specific binding of [125I]hCG to membrane preparations was performed using a procedure previously described by this laboratory (10). hCG was radioiodinated using the chloramine-T method (12) to give a specific activity of 40-60 /iCi/jug. Nonspecific binding was determined by the addition of a 1000-fold excess of unlabeled hCG. Bound receptor-hCG complexes were precipitated with polyethylene glycol and separated from free [125I]hCG by centrifugation. RNA extraction RNA was extracted from tissues using the procedure of Chomczynski and Sacchi (13). Briefly, tissues were homogenized in a solution of guanidine isothiocyanate acidified with 2 M sodium acetate, pH 4.0, and extracted with water-saturated phenol and chloroform-isoamyl alcohol (49:1). RNA remaining in the aqueous phase was precipitated overnight at —20 C using 3 vols ethanol. RNA was quantified spectrophotometrically, and the purity was determined by the ratio of A26o/A28oNorthern blot Procedures were essentially the same as those described by Davis et al. (14). Total RNA was separated by electrophoresis in 1.2% agarose gel containing 3% formaldehyde. RNA was blotted to nitrocellulose using a 10 X saline-sodium citrate buffer, pH7 (SCC). The blot was heated at 80 C under vacuum for 2 h and then hybridized. Deoxyoligonucleotide probe construction A LH/hCG receptor mRNA probe complimentary to bases 2677-2697 of the COOH-terminal untranslated region of the

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cDNA sequence published by McFarland et al. (8) was synthesized. The sequence of the probe was as follows: 5'GCTGAGATGGGCTCCTGGCC 3'. Uniqueness of this probe sequence was checked by comparing it to other known rodent sequences found in the Genebank (release 48) and National Biomedical Research Foundation (release 11) databases by using Beckman's Microgenie program (version 5.0, Palo Alto, CA). Labeling of probes

LH/hCG receptor probe was radiolabeled by the procedure of Davis et al. using [7-32P]ATP and T4 polynucleotide kinase (14). a-Actin cDNA probe was labeled using [«-32P]dCTP and the Klenow fragment of DNA polymerase (15). Unincorporated radioactivity was removed from labeled probes using a Sephadex G-50 column. Hybridization of blots Procedure was essentially the same as that described by Sambrook et al. (16). Blots were prehybridized at 42 C for 2 h in a solution containing 10 mg salmon sperm DNA and 2 X hybridization buffer [1.5 M NaCl-0.1 M TES (pH 7.1)-0.1 M EDTA-2 X Denhardt's] diluted 1:1 with deionized formamide. The probe (2 x 107 cpm) was hybridized to blots overnight at 42 C in fresh buffer. Hybridized blots were washed twice with 2 X SSC containing 0.1% sodium dodecyl sulfate (SDS) for 20 min at room temperature and once at 60 C for 30 min. The washed blots were exposed overnight at —70 C in a Kodak XOmatic cassette containing intensifying screens. Stripping hybridized blots LH/hCG receptor probe was stripped from hybridized Northern blots by washing once with a boiling solution of 0.05 X SSC containing 0.01 M EDTA and 0.1% SDS for 20 min. Blots were then washed twice with boiling 0.1 x SSC containing 0.1% SDS for 20 min. Blots were briefly rinsed with 0.1 X SSC containing 0.1% SDS at room temperature, blotted dry on Whatman 3MM filter paper (Clifton, NJ), and exposed to x-ray film overnight at -70 C.

Results To determine that the constructed 20mer probe was capable of hybridizing specifically with LH/hCG receptor mRNA present in rat luteal tissue, total RNA was extracted from pseudopregnant rat ovary and from the nontarget tissues, such as adrenal gland, heart, kidney, liver, and lung, and subjected to Northern blot. There was no hybridization of probe to nontarget tissues, but the rat ovarian preparation showed hybridization of the probe to one major 4.4-kilobase (kb) band and to a minor 3.3-kb band (Fig. 1). The 4.4-kb region corresponds to the size of LH/hCG receptor mRNA reported previously (8). Since McFarland et al. (8) used DNA markers for size estimation of the receptor mRNA, similar standards were used in the present study to make a comparison to

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REGULATION OF GONADOTROPIN RECEPTOR mRNA

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a b

-28S

-18S

FIG. 1. Hybridization of LH/hCG receptor probe to RNA from different tissues. Each lane contained 30 /ug total RNA. Numbers on the left indicate DNA size markers in kilobases. The positions of 28S and 18S ribosomal RNA are shown on the right. Samples shown are ovary (lane a), fully down-regulated ovary (lane b), heart (lane c), lung (lane d), kidney (lane e), adrenal (lane f), and liver (lane g).

their results. The 4.4-kb band, when run against RNA standards rather than DNA standards, corresponds to 6.7 kb in size. The presence of the 4.4-kb band in ovary and its absence in nontarget tissues shows that the 20mer probe was hybridizing specifically to LH/hCG receptor mRNA. The amount of hybridizable mRNA in ovaries during different stages of pseudopregnancy was then examined. The changes in the 4.4-kb LH/hCG receptor mRNA on different days of pseudopregnancy are shown in Fig. 2. As can be seen in the autoradiogram, the 4.4-kb receptor

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mRNA was maximally expressed on day 8 and began to diminish toward the end of pseudopregnancy. Then [125I] hCG binding studies were performed using partially purified membrane fractions isolated from ovaries obtained at the same time points as those used for RNA extraction to determine whether the appearance of the 4.4-kb band showed a correlation to the hormone-binding activity of the receptor. As seen in Fig. 3, the [125I]hCG-binding activity paralleled the appearance of the 4.4-kb band representing LH/hCG receptor mRNA. Studies were then designed to determine whether the down-regulation of LH/hCG receptors seen after hCG injection was due to a decrease in the receptor mRNA levels. In initial experiments, 50 IU hCG were administered on day 5 of pseudopregnancy, and ovaries were isolated 24 h later. The RNA extracted from ovaries that had been treated in this manner did not show the presence of the 4.4-kb receptor mRNA (Fig. 1, lane b). The time course of LH/hCG receptor mRNA disappearance after hormone injection was then examined. Pseudopregnant rats were injected with 50 IU hCG on day 5 of pseudopregnancy, and ovaries were collected 1.5, 3, 6, 9, 12, 24, 48, and 72 h later. Control rats received an equal volume of saline. One group of ovaries was used for RNA analysis, and the other for determination of [125I]hCGbinding activity. The autoradiogram (Fig. 4A) of Northern blots shows that hCG injection caused a decrease in hybridizable mRNA as early as 6 h after treatment. The receptor mRNA was undetectable at 24 h and started to reappear at 48 h. The corresponding controls (Fig. 4B) showed no decrease during these time periods, but, rather, demonstrated an increase in the 4.4-kb band as pseudopregnancy progressed. The [125I]hCG binding data were parallel to the hybridized RNA levels. Binding activity in down-regulated ovaries decreased by 6 h, reached a minimum at 24 h, and reappeared at 48 h (Fig.

FIG. 2. Hybridization of receptor probe to RNA during a time course of pseudopregnancy. Each lane contained 30 fig total RNA. Numbers at the left indicate DNA size markers in kilobases. The positions of 28S and 18S ribosomal RNA are shown on the right. Samples shown were run in duplicate. Lanes 4, 6, 8, 10, 12, and 14 represent days of pseudopregnancy.

-28S

-18S 1.0-

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REGULATION OF GONADOTROPIN RECEPTOR mRNA A

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9 12 24 48 72

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10B FlG. 3. [12SI]hCG-binding activity during pseudopregnancy. Rats were killed on the indicated day after hCG injection, and a membrane fraction was prepared from isolated ovaries. Aliquots of membranes were incubated with 300,000 cpm [125I]hCG in the presence or absence of unlabeled hCG. Binding is expressed as picomoles of [125I]hCG bound per mg protein. Each data point represents the mean ± SEM of triplicate determinations.

5A). Binding activity in control ovaries showed a steady increase (Fig. 5B). To rule out the possibility that hCG injection caused an overall decrease in ovarian RNA and not just the receptor mRNA, blots previously hybridized with receptor probe were stripped and rehybridized with 32P-labeled «-actin cDNA. The autoradiogram (Fig. 6) showed no significant changes in actin mRNA at any time points after hCG injection. Additionally, cytochrome P450scc mRNA levels assayed using the same RNA preparations showed an increase at the same time points.1 These results suggest that the loss of hybridizable LH/hCG receptor mRNA after hCG injection is not due to a general decrease in transcription. Discussion In the present study we used a 20mer probe to monitor LH/hCG receptor mRNA from rat luteal cells. The specificity of the probe to detect the receptor mRNA was confirmed by demonstrating the absence of the receptor mRNA in nontarget cells, such as liver, heart, lung, adrenal gland, and kidney, and by the close correlation between the [125I]hCG-binding activity and the receptor mRNA abundance. The probe selected was unique as it did not show similarity to any known rodent sequences. Additionally, the size of the major labeled band in our Northern blots corresponded to that reported by McFarland et al (8). 1

Peegel, H., and K. M. J. Menon, unpublished results.

1.5 12

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48 72

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1.0FIG. 4. Hybridization of receptor probe to RNA during a time course of down-regulation. Numbers on the left indicate DNA size markers in kilobases. The positions of 28S and 18S rRNA are shown on the right. Each lane contained 30 ^g total RNA. Rats were treated as described in Materials and Methods. A, Hybridization of receptor probe to downregulated RNA 1.5, 3, 6, 9, 12, 24, 48, and 72 h after hCG injection. B, Hybridization of receptor probe to control RNA 1.5, 12, 24, 48, and 72 h after saline injection.

Although a loss of hormone-binding activity after hCG treatment was demonstrated several years ago, the underlying molecular events leading to loss of receptor sites are not yet understood. With the availability of the probe to accurately measure the changes in LH/hCG receptor mRNA, we wished to examine the phenomenon of hCGinduced down-regulation of the receptors in luteal cells. Our data clearly show that during the fully down-regulated state, there is a total disappearance of LH/hCG receptor mRNA. This was most striking 24 h after hCG treatment. The close correlation between hCG-binding activity and receptor message levels during hCG-induced down-regulation clearly shows that the loss of binding activity after hormone injection is due to the loss of receptor mRNA. This loss of receptor mRNA could result from a decreased synthesis or increased breakdown of preexisting mRNA. The present studies cannot conclusively delineate between these possibilities.

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REGULATION OF GONADOTROPIN RECEPTOR mRNA

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FIG. 6. Hybridization of actin cDNA to RNA during a time course of down-regulation (as shown in Fig. 4). LH/hCG receptor probe was stripped from a Northern blot of a time course of receptor downregulation by washing twice with a boiling solution of 0.1 x SSC-0.1% SDS, and the blot was rehybridized with labeled a-actin cDNA. A, Probe hybridization to down-regulated RNA. Lanes represent 1.5, 3, 6, 9, 12, 24, 48, and 72 h after hCG injection. B, Hybridization of probe to control groups. Lanes represent 1.5, 12, 24, 48, and 72 h after saline injection. 12

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48

60

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FIG. 5. [125I]hCG-binding activity during the time course of downregulation. Rats were treated, and membrane fractions were prepared from isolated ovaries, as described in Materials and Methods. Aliquots of membranes were incubated with 300,000 cpm [125I]hCG in the presence and absence of unlabeled hCG. Binding is expressed as picomoles of [125I]hCG specifically bound per mg protein. Each data point represents the mean ± SEM of triplicate determinations. A, Binding activity for the time course of down-regulation; B, binding activity for corresponding controls.

The regulation of hCG receptor mRNA levels plays an important role in controlling the responsiveness of the luteal cells to gonadotropins. Regulatory elements responsive to cAMP might play a crucial role in this process, since luteal cells show a response to hCG, resulting in an increase in cAMP levels. Furthermore, mRNA for cytochrome P450scc, which is known to be induced in response to cAMP (17), was increased during the gonadotropin-induced desensitized state. Thus,

cAMP may regulate ovarian cell function, resulting in an increase or decrease in specific mRNA levels. After our study was completed, LaPolt et al, (18) in a communication reported LH/hCG receptor mRNA levels during follicular growth, ovulation, and luteinization and showed correlation between receptor mRNA levels and the ligand-binding activity. The close correlation between the receptor mRNA levels and ligand-binding activity observed in our studies agrees in general with that reported by LaPolt et al. (18). One minor difference, however, was that in the present study, the LH/hCG receptor mRNA content returned to the control level 72 h after hCG injection, while in the studies reported by LaPolt et al. (18), the receptor mRNA content did not show a corresponding increase with the [125I]hCG-binding activity after ovulation induction. Possibly, this suggests an interesting difference in physiological regulation of the receptor between the two different models. In summary, we have shown that LH/hCG receptor mRNA expression in the pseudopregnant rat ovary parallels gonadotropin binding, and we conclude that the loss of hCG binding seen during hCG-induced downregulation is, at least in part, due to the loss of the receptor mRNA. From these studies, it is also clear that the LH/hCG receptor is a protein with a rapid turnover, since a significant decrease in both the [125I]hCG-binding activity and receptor mRNA was seen as early as 6 h after hCG injection.

References 1. Menon KMJ, Gunaga K 1974 Role of cyclic AMP in reproductive processes. Fertil Steril 25:732 2. Niswender GS, Sawyer HR, Chen TT, Endres DB 1980 Action of luteinizing hormone at the luteal cell level. In: Thomas JA, Singhal RL (eds) Advances in Sex Hormone Research. Urban and Schwarzenberg, Baltimore, vol 4:153-86 3. Ryan RJ, Birnbaumer L, Lee CY, Hunzicker-Dunn M 1977 Gonadotropin interactions with the gonad as assessed by receptor binding and adenylyl cyclase activity. In: Greep BO (ed) International Review of Physiology. University Park Press, Baltimore, vol 13:85-152

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REGULATION OF GONADOTROPIN RECEPTOR mRNA 4. Hwang J, Menon KMJ 1980 Evidence that the subunit structure of gonadotropin receptor is preserved during regression of rat corpus luteum. Biochem Biophys Res Commun 137:546-51 5. Lee CY, Tetesishi K, Ryan RJ, Jiang NS 1975 Binding of human chorionic gonadotropin by rat ovarian slices: dependence on the functional state of the ovary. Proc Soc Exp Biol Med 148:505-7 6. Conti M, Harwood JP, Hseuh AJW, Dufau ML, Catt KJ 1976 Gonadotropin-induced loss of hormone receptors and desensitization of adenylate cyclase in the ovary. J Biol Chem 251:7729-31 7. Sen K, Azhar S, Menon KMJ 1979 Desensitization of gonadotropin binding sites, activation of adenosine 3'5'-cyclic monophosphatedependent proteins kinase(s), and regulation of steroidogenesis in rat ovary. J Biol Chem 254:664-71 8. McFarland KC, Sprengel R, Phillips HS, Kohler M, Rosemblit N, Nikolics K, Segaloff DL, Seeburg PH 1989 Lutropin-choriogonadotropin receptor: an unusual member of the G protein-coupled receptor family. Science 245:494-9 9. Parlow AF 1958 A rapid bioassay method for LH and factors stimulating LH secretion. Fed Proc 17:402 10. Azhar S, Menon KMJ 1976 Effect of phospholipases on the binding of 125I-choriogonadotropin by membrane associated and solubilized receptors. J Biol Chem 251:7398-404 11. Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BS, Klenk DC

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1985 Measurement of protein using bioinchorinic acid Anal Biochem 150:76-85 Catt KJ, Dufau ML, Tsuruhara TJ 1972 Radioligand-receptor assay of luteinizing hormone and chorionic gonadotropin. J Clin Endocrinol Metab 34:123-32 Chomczynski P, Sacchi N 1987 Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156-9 Davis LG, Dibner MD, Battey JF 1986 Methods in Molecular Biology. Elsevier, New York Feinberg AP, Vogelstein B 1983 A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132:6-13 Sambrook J, Fritsch EF, Maniatis T 1989 Molecular Cloning—A Laboratory Manual, Ed 2. Cold Spring Harbor Laboratory, Cold Spring Harbor John ME, Simpson ER, Waterman MR, Mason JI1986 Regulation of cholesterol side-chain cleavage cytochrome P-450 gene expression in adrenal cells in monolayer culture. Mol Cell Endocrinol 45:197-204 LaPolt PS, Oikawa M, Xiao-Chi J, Dargan K, Hsueh JW 1990 Gonadotropin-induced up- and down-regulation of rat ovarian LH receptor message levels during follicular growth, ovulation and luteinization. Endocrinology 125:3277

Erratum The title of the article by J. P. Schroder-Van der Elst and D. Van der Heide (Endocrinology 127: 16561664, 1990) should read, "Thyroxine, 3,5,3'-triiodothyronine, and 3,3',5'-triiodothyronine concentrations in several tissues of the rat: effects of amiodarone and desethylamiodarone on thyroid hormone metabolism." The printer regrets the error.

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luteinizing hormone receptor down-regulation involves decreased levels of receptor messenger ribonucleic acid.

Injection of pseudopregnant rats with pharmacological doses of hCG leads to a characteristic decrease in LH/hCG binding by the isolated luteal cells. ...
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