Regulation of luteinizing hormone receptor messenger ribonucleic acid levels by gonadotropins, growth factors, and gonadotropin-releasing hormone in cultured rat granulosa cells.

0013-7227/91/1285-2449$03.00/0 Endocrinology Copyright© 1991 by The Endocrine Society

Vol. 128, No. 5 Printed in U.S.A.

Regulation of Luteinizing Hormone Receptor Messenger Ribonucleic Acid Levels by Gonadotropins, Growth Factors, and Gonadotropin-Releasing Hormone in Cultured Rat Granulosa Cells* GARY N. PIQUETTEf, PHILIP S. L A P O L T $ , MAMORU OIKAWA, AND AARON J. W. HSUEH§ Department of Reproductive Medicine, M-025, University of California-San Diego, La Jolla, California 92093

PRL. In contrast, treatment of cells with basic fibroblast growth factor or epidermal growth factor suppressed FSH induction of LH receptor mRNA in a dose-dependent manner, whereas treatment with insulin-like growth factor-I had no effect. In addition, GnRH suppressed FSH-stimulated LH receptor mRNA levels in a dose-dependent manner; the effects of GnRH could be counteracted by coincubation with a GnRH antagonist, suggesting mediation by specific GnRH-binding sites. These studies demonstrated that the observed stimulatory effects of gonadotropins (FSH, LH, and PRL) and the inhibitory effects of growth factors (epidermal growth factor and basic fibroblast growth factor) and GnRH on LH receptor content are correlated to their regulation of LH receptor mRNA levels. The granulosa cell culture system should provide a useful model for studying LH receptor gene regulation. (Endocrinology 128: 2449-2456, 1991)

ABSTRACT. The induction of LH receptors in granulosa cells is prerequisite for ovarian follicles to ovulate and form corpora lutea. Earlier studies have demonstrated the modulatory role of gonadotropins, growth factors, and GnRH on ovarian LH receptor content. We have now analyzed the influences of gonadotropins (FSH, LH, and PRL), several growth factors, and GnRH on LH receptor mRNA levels in cultured granulosa cells. Cells were obtained from immature estrogen-treated rats and cultured in medium containing FSH with or without growth factors or GnRH for 48 h. Some cells were also treated with FSH for 48 h, followed by treatment with FSH, LH, or PRL for another 2 days. Cellular total RNA was extracted, and blot hybridization with 32P-labeled LH receptor cRNA or 28S ribosomal RNA cDNA probes was performed. Treatment of granulosa cells with FSH increased the levels of five species of LH receptor mRNAs in a dose- and time-dependent manner. In FSH-primed cells, LH receptor mRNA levels were maintained by FSH, LH, and

T

HE INDUCTION of LH receptors in granulosa cells is an obligatory step for ovarian follicles to ovulate and develop into corpora lutea. Several studies have shown that FSH increases LH receptor numbers in rat granulosa cells through a protein synthesis-dependent event (1-4). After granulosa cells are preincubated with FSH to induce LH receptors, LH receptors are maintained by FSH, LH, and PRL (4-6). Insulin-like growth factor-I (IGF-I) enhances (7, 8), whereas basic fibroblast growth factor (bFGF) (9-11), epidermal growth factor (EGF) (9, 12), and GnRH (12-14) inhibit the induction Received December 5,1990. * This work was supported by NIH Grant HD-23273. t Postdoctoral Fellow supported by National Research Service Award HD-07410. X Postdoctoral Fellow supported by National Research Service Award HD-07252. § To whom all correspondence and requests for reprints should be sent. Present address: Division of Reproductive Biology, Department of Gynecology/Obstetrics, Stanford University Medical Center, Stanford, California 94305.

of granulosa cell LH receptors by FSH. Recent cloning of rat and porcine LH receptor cDNAs (15, 16) indicated that LH receptors belong to the Gprotein-coupled receptor family, with seven transmembrane domains, and are closely related to the FSH and TSH receptors (17, 18). All of these glycoprotein receptors have large extracellular domains with leucine-rich repeats, which presumably confer hormone binding specificity. Using a LH receptor cDNA fragment isolated from rat testes corresponding to a portion of the extracellular domain, we have demonstrated that ovarian LH receptor message levels are regulated during follicular growth, ovulation, and luteinization (19). To ascertain whether gonadotropins, growth factors, and GnRH directly modulate LH receptor mRNA in ovarian cells, we cultured rat granulosa cells with FSH with or without increasing doses of bFGF, EGF, and GnRH or primed these cells with FSH followed by treatment of mature granulosa cells with FSH, LH, or PRL. We then analyzed steady state LH receptor mRNA levels

2449

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 November 2015. at 10:29 For personal use only. No other uses without permission. . All rights reserved.

REGULATION OF LH RECEPTOR mRNAs

2450

to detect possible modulation of LH receptor gene regulation. Materials and Methods Hormones and reagents Ovine FSH (NIH FSH S17; 20 x NIH FSH Si U/mg), ovine LH (NIH LH S25; 2.3 x NIH LH Si U/mg), and ovine PRL (NIH PRL S15; 30.5 IU/mg) were obtained from the National Hormone and Pituitary Distribution Program, NIDDK, NIH. EGF (purity, >98%) and IGF-I (purity, >95%) were purchased from Collaborative Research (Waltham, MA). Recombinant bFGF was a generous gift from Dr. Andreas Sommer (Synergen, Boulder, CO). GnRH and the GnRH antagonist ([Ac-D-Phe\DpCl-Phe2,D-Trp36]GnRH) were the generous gifts of Dr. N. C. Ling (Whittier Institute, La Jolla, CA). Androstenedione, herring sperm DNA, and yeast tRNA were obtained from Sigma Chemical Co. (St. Louis, MO), and McCoy's 5A medium (modified, serum-free), streptomycin sulfate, penicillin, and L-glutamine were purchased from Grand Island Biochemical Co. (Santa Clara, CA). Enzymes for RNA analyses were obtained from Bethesda Research Laboratories (Gaithersburg, MD), and [a-32P]CTP from Amersham (Arlington Heights, IL). The plasmid containing the human 28S ribosomal RNA (rRNA) gene (pA) was a generous gift from Dr. Iris L. Gonzalez (Hahnemann University, Philadelphia, PA). All other chemicals were reagent grade obtained from Fisher Scientific (Pittsburgh, PA). Granulosa cell culture Granulosa cells were obtained from immature 28-day-old Sprague-Dawley female rats pretreated with a diethylstilbestrol capsule implant 5 days earlier (Johnson Laboratories, Bridgeview, IL), as previously described (20). Cells were cultured in 12 x 75-mm polypropylene culture tubes (Falcon, Cockeysville, MD) at a concentration of 500,000 viable cells (as determined by trypan blue exclusion) in 0.5 ml McCoy's 5A medium at 37 C in a humidified 95% air-5% CO2 atmosphere. McCoy's 5A medium was supplemented with antibiotics (penicillin, 100 U/ml; streptomycin sulfate, 100 Mg/ml) and 10~7 M androstenedione, with or without added hormones. Preparation of LH receptor cRNA probe and 28S rRNA cDNA probe The 32P-labeled LH receptor cRNA probe was derived from a rat LH receptor cDNA fragment obtained by reverse transcription/polymerase chain reaction of rat testicular RNA, followed by subcloning the PCR product into the pGEM4Z vector (Promega, Madison, WI), as previously described (19). The 408-basepair (bp) cRNA probe (SA, 1 X 108 cpm//ig) was synthesized with the Riboprobe II system (Promega) (21) using the LH receptor cDNA fragment which corresponds to extracellular domain bases 441-849 of the rat LH receptor cDNA reported by McFarland et al. (15). The 28S rRNA 32P-labeled cDNA probe was prepared using the random primer method (22). A 1600-bp £coRI-£amHI fragment (pABE) of the exon region of the human 28S rRNA gene was used as the template for probe synthesis (23, 24).

Endo • 1991 Vol 128 • No 5

RNA analysis After hormone treatment, cultured cells were washed once in ice-cold saline and then snap-frozen in a dry ice-ethanol bath and stored at —70 C. Total RNA from each sample was prepared using the Nonidet-P40 method (25). For Northern blot analysis, samples were fractionated on denaturing agarose gels and then transferred to nitrocellulose or nylon membranes (Schleicher and Schuell, Keene, NH) (25). No difference in RNA recovery or hybridization sensitivity was observed between nylon or nitrocellulose membranes. Nylon membranes were washed for 5 min with 5-fold concentrated sodium chloride-sodium citrate buffer (5 X SSC; 1 X SSC = 150 mM sodium chloride and 15 mM sodium citrate, pH 7.0) after transfer to remove residual agarose. For slot blot analysis, RNA was dissolved in 10 mM Tris and 1 mM EDTA (pH 8.0) containing 0.1% sodium dodecyl sulfate (SDS) and denatured for 15 min at 60 C in 4 M formaldehyde. Samples were then adjusted to 6 X SSC and applied to nitrocellulose filters using a slot blot apparatus (Schleicher and Schuell). Replicate filters were prepared for hybridization to LH receptor cRNA as well as 28S rRNA cDNA probes. Nitrocellulose and nylon membranes were baked for 2 h at 80 C under vacuum to immobilize RNA. Nylon membranes were washed for 1 h before prehybridization in 0.1 x SSC-0.5% SDS at 65 C. Hybridization of the rat LH receptor probe to membranes was carried out under highly stringent conditions, as previously described (19). Briefly, baked membranes were prehybridized for at least 2 h (64 C) in a solution containing 5 X SSC, 50% formamide, 1.6-fold concentrated Denhardt's solution (25), 1 mM EDTA, 250 /ig/ml heat-denatured and sonicated herring sperm DNA, and 500 ^g/vaX yeast tRNA. Hybridization was carried out at 64 C overnight in the same solution containing 2 x 106 cpm/ml 32P-labeled probe. Sequence analysis of the 28S rRNA gene fragment predicts an 89% nucleotide homology to a corresponding sequence in the rat 28S rRNA gene (26, 27). For hybridization with the 28S cDNA probe, conditions were as described above, except prehybridization and hybridization temperatures were 42 C. A single specific hybridization signal corresponding to the ethidium bromide staining of 28S rRNA was observed under these stringency conditions. After hybridization, membranes were washed in 2 x SSC and 0.1% SDS for 5 min at room temperature, followed by two washes (64 C; 20 min each) in 0.1 x SSC and 0.1% SDS. Filters were exposed to Kodak XAR-5 film (Eastman Kodak, Rochester, NY) at -70 C for 1-3 days. Data analysis The relative intensity of slot blot hybridization signals was determined using a transmittance scanning densitometer and computer software program (Hoefer Scientific Instruments, San Francisco, CA). LH receptor mRNA levels were normalized based on 28S rRNA content in duplicate samples, as determined by blot hybridization, and were expressed as the relative ratio between treatment groups. The hybridization signal of 28S rRNA per ng total RNA did not change significantly (P > 0.05) with hormone treatments. Statistical significance of differences among groups was determined by analysis of variance, followed by Scheffe's post-hoc test, and the Mann-Whitney U


REGULATION OF LH RECEPTOR mRNAs test. For Northern blot analyses, all experiments were repeated two or more times, with one representative experiment presented. The RNA content of pooled samples was determined by absorbance at 260 nm. The same amount of total RNA was loaded for each group, and equal loading was confirmed after staining of the 28S rRNA by ethidium bromide. For slot blot analyses, the data represent the mean ± SE of three to six cultures.

Results Time-dependent induction of LH receptor mRNA by FSH The number of LH receptors in granulosa cells from immature rats is increased by FSH in a time- and dosedependent manner (1-4). To determine whether the increase in LH receptor numbers is correlated with increases in LH receptor mRNA content, granulosa cells were incubated for 0, 12, 24, 36, 42, and 48 h with or without 30 ng/ml FSH. LH receptor mRNA levels were quantitated using slot blot methods and normalized with 28S rRNA levels. As shown in Fig. 1A, the LH receptor mRNA in untreated (control) cells decreased over 48 h, whereas FSH significantly (P < 0.05) increased LH receptor mRNA in a time-dependent manner, with peak induction (>5-fold) between 36-48 h after incubation. In Northern blot analysis of the LH receptor mRNA (Fig. IB), FSH treatment induced one strong hybridization signal at 7.0 kilobases (kb), with weaker signals at 4.2, 2.5, and 1.8 kb. Very weak signals at 1.2 kb were also present with longer exposure times.

A

2451

Dose-dependent stimulation of LH receptor mRNA by FSH Granulosa cells were incubated for 48 h with no hormone or with increasing doses (1-100 ng/ml) of FSH. LH receptor mRNA levels were quantitated using slot blots and normalized with 28S rRNA levels. FSH significantly (P < 0.05) increased LH receptor mRNA in a dose-dependent manner (Fig. 2), with maximal (3-fold) stimulation between 30 and 100 ng/ml FSH (P < 0.05). The LH receptor mRNA content of cells treated with 1 or 3 ng/ml FSH was not significantly different from the control value (P > 0.05). Effects of FSH, LH, and PRL treatment on LH receptor mRNA in FSH-primed cells Granulosa cells were primed with 30 ng/ml FSH for 2 days to induce LH and PRL receptors (3, 5, 6), then incubated with 30 ng/ml FSH or increasing doses of LH or PRL for an additional 48 h. Slot blot analysis was performed, followed by hybridization with LH receptor cRNA and 28S rRNA cDNA probes. After 48-h incubation, levels of LH receptor mRNAs in cells treated with maximal doses of LH or PRL were not significantly different from those in the FSH-treated cells (P > 0.05), but were greater than those in the control cells (P < 0.05; Fig. 3). There was a dose-dependent effect of LH or PRL on LH receptor mRNA content; 10 or 100 ng/ ml LH were more potent than 1 ng/ml LH, and 1001000 ng/ml PRL were more potent than 10 and 30 ng/ ml PRL.

B 1.0-

O—O Control • — • FSH

C

xn

FSH kb

28S-

-7.0 -4.2

18S-

-1.8 - 12

-2.5

0

12

24

36

48 h

|

0.5-

o J5 o

INCUBATION TIME

FIG. 1. Time course of induction of LH receptor mRNA by FSH. A, Total RNA (~1.5 /ig) from granulosa cells cultured in medium alone (control) or 30 ng/ml FSH for various times was applied to nitrocellulose membranes in duplicate using a slot blot apparatus, followed by hybridization to the LH receptor probe or the 28S rRNA probe. Each time point represents the mean ± SEM of four separate cultures. Values are normalized for 28S rRNA levels and expressed relative to the FSH group at 48 h (1.0). B, Total RNA (-1.5 /ig) from cells treated for 48 h with medium alone (C) or FSH (30 ng/ml) was fractionated on a denaturing agarose gel and transferred to nitrocellulose membranes for blot hybridization using the LH receptor probe. The migrating positions of the 28S and 18S rRNAs are indicated. The Northern blot is representative of two experiments.

0 1

10 30 FSH, ng/ml

100

FIG. 2. Dose-dependent stimulation of granulosa cell LH receptor mRNA levels by FSH. Cells were cultured for 48 h in medium alone [control (C)] or with increasing doses of FSH. Total RNA (~1.5 ng) was extracted from cells and applied to nitrocellulose membranes in duplicate using a slot blot apparatus, followed by hybridization to the LH receptor probe or 28S rRNA probe. Slot blot data are the mean ± SEM of four separate cultures, each normalized for 28S rRNA levels, as determined by hybridization. Values are expressed relative to the 30 ng/ml FSH dose (1.0).



2452

B

Prolactin u xn oo ^

1.5-

Endo • 1991 Voll28^No5

•

i

b F E F S GG C H F F

SB I 1.0 kb

o ^5

28 S *

o 18S-

0

CF S H

1

10

100

1000

ng/ml

FIG. 3. Effects of treatment with FSH, LH, or PRL on LH receptor mRNA levels in mature granulosa cells. Granulosa cells were pretreated for 48 h with 30 ng/ml FSH to induce functional LH and PRL receptors. Cells were then washed and reincubated for 48 h with medium alone (C), FSH (30 ng/ml), or increasing doses of LH or PRL. Total RNA (~1.5 ng) was applied to nitrocellulose membranes in duplicate using a slot blot apparatus, followed by hybridization to the LH receptor probe or the 28S rRNA probe. Each point represents the mean ± SEM of four separate cultures, each normalized for 28S rRNA content and expressed relative to the FSH group (1.0).

Effects of treatment with bFGF, EGF, and IGF-I on LH receptor mRNA levels Earlier studies have demonstrated that treatment of granulosa cells in vitro with IGF-I enhances (7, 8), whereas treatment with bFGF or EGF suppresses (9-12) FSH induction of [125I]hCG binding in granulosa cells. In this study granulosa cells were incubated for 48 h with FSH, bFGF, EGF, or medium alone. Some granulosa cells were treated with FSH plus IGF-I (10 ng/ml) or FSH plus increasing doses of EGF or bFGF. The content of LH receptor mRNAs in cultured granulosa cells was then analyzed. As shown in Fig. 4A, treatment of cells with FSH alone (30 ng/ml) induced five species of LH receptor mRNA, but treatment with bFGF alone (10 ng/ ml) or EGF alone (10 ng/ml) did not induce LH receptor mRNA levels. However, as shown in Fig. 4B, cotreatment with FSH (30 ng/ml) and increasing doses of bFGF inhibited the levels of all five species of the FSH-induced LH receptor mRNA in a dose-dependent manner; the 30 ng/ml dose of bFGF completely inhibited LH receptor mRNA levels. Cotreatment with 30 ng/ml FSH and 10 ng/ml EGF also inhibited FSH-induced LH receptor mRNA levels, with a decrease in all five species of LH receptor mRNA (Fig. 4B). In contrast, a similar dose of IGF-I (10 ng/ml) was ineffective in altering LH receptor mRNA in FSH-treated cells (Fig. 4B). Also, IGF-I alone did not induce LH receptor mRNA (data not shown). To determine whether the suppressive effect of EGF

28S

28S-

FIG. 4. Effect of treatment with bFGF, EGF, or IGF-I on basal and FSH-induced LH receptor mRNA levels. A, Cells were incubated for 48 h with control medium (C), FSH (30 ng/ml), EGF (10 ng/ml), or bFGF (10 ng/ml). B, Cells were incubated for 48 h with FSH (30 ng/ ml) with increasing doses of bFGF, 10 ng/ml IGF-I (FI), or 10 ng/ml EGF (FE). Total RNA (1 ng) was fractionated on denaturing agarose gels and transferred to nylon membranes for hybridization to the LH receptor probe. The migrating positions of the 28S and 18S rRNAs are indicated. The lower panel depicts ethidium bromide staining of 28S rRNA of the transferred samples.

on FSH-induced LH receptor mRNA was dose dependent, granulosa cells were cotreated with FSH (30 ng/ml) and increasing doses of EGF. As shown in Fig. 5, levels of all five species of the FSH-induced LH receptor mRNA were suppressed by EGF in a dose-dependent manner; the 3 ng/ml dose completely inhibited LH receptor mRNA levels, suggesting that EGF has a more potent inhibitory effect than bFGF. Dose-dependent inhibition of FSH-stimulated LH receptor mRNA by GnRH Granulosa cells were incubated for 48 h with 30 ng/ml FSH alone or FSH plus increasing doses (0.01-10 nM) of GnRH. Cotreatment with FSH and increasing doses of GnRH inhibited the levels of all five species of FSHinduced LH receptor mRNAs in a dose-dependent manner (Fig. 6). Although without effect by itself on FSH induction of LH receptor mRNA levels, the GnRH antagonist (300 nM) was able to counteract the inhibitory effect of 10 nM GnRH on FSH action (Fig. 7). Furthermore, GnRH alone did not induce LH receptor mRNA (data not shown). Discussion This study demonstrated that FSH treatment induced five different transcripts of LH receptor mRNAs in



2453

FSH

+

GnRH 0 .01.1 1 10 (nMl

0 .1 .3 1 3 10 Ing/ml]

kb

28S-

18S-

28 S FIG. 5. Dose-dependent suppression of FSH-induced LH receptor mRNA levels by EGF. Cells were incubated for 48 h with FSH (30 ng/ ml) with or without increasing doses of EGF (0.1-10 ng/ml). Total RNA (5 ng) was fractionated on denaturing agarose gels and transferred to nitrocellulose membranes for hybridization using the LH receptor probe. The migrating positions of the 28S and 18S rRNAs are indicated. The lower panel depicts ethidium bromide staining of 28S rRNA of the transferred samples.

cultured granulosa cells in a dose- and time-dependent manner. After FSH priming, mature granulosa cell LH receptor mRNA levels were further maintained by FSH, LH, or PRL. Although treatment of cells with bFGF or EGF alone was without effect, cotreatment of cells with FSH plus bFGF or EGF inhibited FSH induction of LH receptor mRNA levels. In contrast, IGF-I alone or in combination with FSH had no effect on LH receptor mRNA levels. Although GnRH also suppressed the FSH action in a dose-dependent manner, a GnRH antagonist counteracted the inhibitory effect of GnRH, suggesting that the observed effect of GnRH is mediated by specific receptors. Granulosa cells from preantral follicles have a negligible number of LH-binding sites, whereas priming with FSH induces functional LH and PRL receptors (1-4). Once induced, LH receptors are maintained by FSH (46), LH (4), or PRL (5, 6). The maintenance of rat LH receptors by LH and PRL is consistant with the luteotropic action of LH and PRL in rats and implicates the

28S-

18S-

28S FIG. 6. Dose-dependent inhibition of FSH-induced LH receptor mRNA levels by GnRH. Cells were incubated for 48 h with FSH (30 ng/ml) with or without increasing doses of GnRH (0.01-10 nM). Total RNA (5 ng) was fractionated on denaturing agarose gels and transferred to nitrocellulose membranes for Northern blot hybridization using the LH receptor probe. The migrating positions of the 28S and 18S rRNAs are indicated. The ethidium bromide staining of 28S rRNA of the samples is shown.

important role of LH receptors in corpora lutea development. The present observations of stimulatory effects of the gonadotropins FSH, LH, and PRL on granulosa cell LH receptor mRNA levels in vitro suggests that the previously observed (1-4) increases in LH binding induced by these hormones are closely related to the regulation of LH receptor mRNA levels. The observed maintenance of message levels by these gonadotropins may be a result of increased LH receptor gene transcription and/or message stability (i.e. resistance to degradation). Recent in vivo studies by LaPolt et al. (19) showed that rat ovarian LH receptor and its mRNA levels are up- and down-regulated by gonadotropins. LH receptor numbers were increased during PMSG stimulation of follicle growth. After an ovulatory dose of hCG, these levels declined sharply, then gradually increased during luteinization. The observed changes in LH receptor number during follicle growth, ovulation, and luteinization were associated with similar changes in LH receptor message levels. The stimulatory effect of FSH, LH, and



2454

FSH

+

F S G A G A H

28S-

18S-

28S FlG. 7. Counteractive effect of a GnRH antagonist on GnRH inhibition of FSH-induced LH receptor mRNA levels. Cells were incubated for 48 h with FSH alone (30 ng/ml) or with FSH plus GnRH (G; 10 nM) and/or the GnRH antagonist (A; 300 nM). Total RNA (1 Mg) was fractionated on denaturing agarose gels and transferred to nylon membranes for hybridization to the LH receptor probe. The migrating positions of the 28S and 18S rRNAs are indicated. The ethidium bromide staining of 28S rRNA of the samples is shown.

PRL on LH receptor mRNA levels in granulosa cells in vitro is consistent with the stimulatory effect of exogenous PMSG during follicular growth and endogenous PRL during luteinization in vivo. Because of the difficulties involved in mimicking the in vivo LH surge in cultured granulosa cells, down-regulation of LH receptor mRNA in vitro was not observed. Since the actions of LH and FSH on LH receptor numbers are mediated through the protein kinase-A pathway (6, 12), the present observation of induction and maintenance of LH receptor message levels by these hormones presumably results from activation of the same second messenger system. The second messenger system in the mediation of PRL action, however, remains unknown at this time. In contrast to the stimulatory effects of gonadotropins on LH receptors, both bFGF (9-12) and EGF (9, 12) suppressed LH receptor induction by FSH. Our study demonstrates that the suppression of LH-binding sites

Endo • 1991 Voll28-No5

by these growth factors correlates with the decreases in receptor mRNA levels. Because bFGF is an angiogenic factor originally extracted from corpora lutea (28, 29), the ability of bFGF to inhibit the induction of LH receptors and LH receptor mRNA levels suggests that bFGF may be involved in the modulation of LH receptors during corpora lutea development. EGF has also been localized in the ovary (30) and inhibits granulosa cell LH receptor and its mRNA, indicating a role for EGF in modulating ovarian LH receptors. Although both bFGF (11) and EGF (12) impair cAMP-dependent LH receptor induction, several studies (29, 31, 32) suggest that these growth factors may act distally to cAMP formation via an alternative second messenger pathway. Previous studies have shown that IGF-I (7,8) enhances hCG binding to granulosa cells cultured in the presence of FSH. Nonetheless, in this study, treatment with IGFI (10 ng/ml) alone or in combination with 30 ng/ml FSH had no effect on granulosa cell LH receptor mRNA levels, suggesting that IGF-I increases LH binding through a mechanism other than regulation of the receptor message level. However, further studies considering time- and dose-responses may be required to fully understand the mechanisms of IGF-I action in LH receptor regulation. GnRH and its agonists inhibit multiple FSH-dependent functions in granulosa cells (9,13,14), acting through specific receptors (33, 34). Our study further demonstrates that GnRH inhibits LH receptor mRNA levels and that the action of GnRH is mediated through these GnRH receptors, because a GnRH antagonist counteracted the inhibitory effect of GnRH on LH receptors mRNA levels. The inhibition of LH receptor induction may be mediated by GnRH of ovarian origin, because GnRH mRNA has recently been localized in rat ovaries (35). Although GnRH may inhibit LH receptor formation by suppressing cAMP levels (12), several studies (36-38) have shown that GnRH action may be mediated through the protein kinase-C pathway. The observed suppression of LH receptor mRNA by GnRH may also be mediated through this pathway. Future analysis of the promoter region of the LH receptor gene may provide an interesting model to analyze the mechanisms involved in the inhibitory actions of GnRH, bFGF, and EGF. The present study demonstrated at least five species of LH receptor mRNAs in cultured rat granulosa cells after hybridization under high stringency with a probe specific for the coding region of the LH receptor extracellular domain. The presence of multiple species of LH receptor mRNAs has been previously reported in rat corpora lutea (15), whole rat ovaries during the follicular and luteal phases (19), rat testes (39), and porcine ovaries and testes (16). Interestingly, the larger species of rat testicular LH receptor mRNAs are up- and down-regulated by gonadotropins, whereas the 1.8-kb species is


REGULATION OF LH RECEPTOR mRNAs unaffected by gonadotropin treatment (39). In contrast, all LH receptor mRNA species, including the 1.8-kb species, are up- and down-regulated in the rat ovary (19). Furthermore, a probe corresponding to the transmembrane domain of the LH receptor hybridizes to all but the 1.8-kb species of the rat testes, suggesting that the smaller transcript codes for a truncated form of the LH receptor (39). Truncated forms of LH receptor cDNA have been isolated and may represent transcripts that code for soluble receptors (16, 40, 41). In this regard, the various species of LH receptor mRNAs observed in this study may be a result of alternate splicing and/or transcription start sites that may code for different forms of the receptor. This study analyzed steady state LH receptor mRNA levels in cultured granulosa cells. Further studies will be needed to elucidate whether the observed effects of various hormones on LH receptor mRNAs are a result of changes in LH receptor transcription, message stability, or both. Because our findings implicate diverse pathways for regulation of the LH receptor gene, the present cultured rat granulosa cells will serve as a useful model to study the mechanisms involved in LH receptor gene regulation.

Acknowledgments We thank Ms. C. Sincich for technical assistance, Dr. Andreas Sommer for the bFGF, Dr. N. C. Ling for the GnRH and GnRH antagonist, Dr. Iris L. Gonzalez for the human 28S gene plasmid, and the National Hormone and Pituitary Program, NIDDK, for the gonadotropins.

References 1. Zeleznik AJ, Midgley Jr AR, Reichert Jr LE 1974 Granulosa cell maturation in the rat: increased binding of human chorionic gonadotropin following treatment with follicle-stimulating hormone in vivo. Endocrinology 95:818-825 2. Richards JS, Ireland JJ, Rao MC, Bernath GA, Midgley Jr AR, Reichert Jr LE 1976 Ovarian follicular development in the rat: hormone receptor regulation by estradiol, follicle stimulating hormone and luteinizing hormone. Endocrinology 99:1562-1570 3. Erickson GF, Wang C, Hsueh AJW 1979 FSH induction of functional LH receptors in granulosa cells cultured in a chemically defined medium. Nature 279:336-337 4. Jia XC, Hsueh AJW 1984 Homologous regulation of hormone receptors: luteinizing hormone increases its own receptors in cultured rat granulosa cells. Endocrinology 115:2433-2439 5. Casper RF, Erickson GF 1981 In vitro heteroregulation of LH receptors by prolactin and FSH in rat granulosa cells. Mol Cell Endocrinol 23:161-171 6. Hsueh AJW, Adashi EY, Jones PBC, Welsh TH 1984 Hormonal regulation of the differentiation of cultured ovarian granulosa cells. Endocr Rev 5:76-127 7. Davoren JB, Kasson BG, Li CH, Hsueh AJW 1986 Specific insulinlike growth factor (IGF) I- and II-binding sites on rat granulosa cells: relation to IGF action. Endocrinology 119:2155-2162 8. Adashi EY, Resnick CE, Svoboda ME, Van Wyk JJ 1985 Somatomedin-C enhances induction of luteinizing hormone receptors by follicle-stimulating hormone in cultured rat granulosa cells. Endocrinology 116:2369-2375

2455

9. Mondschein JS, Schomberg DM 1981 Growth factors modulate gonadotropin receptor induction in granulosa cell cultures. Science 211:1179-1180 10. Baird A, Hsueh AJW 1986 Fibroblast growth factor as an intraovarian hormone: differential regulation of steroidogenesis by an angiogenic factor. Regul Peptide 16:243-250 11. Oury F, Darbon JM 1988 Fibroblast growth factor regulates the expression of luteinizing hormone receptors in cultured rat granulosa cells. Biochem Biophys Res Commun 156:634-643 12. Knecht M, Catt KJ 1983 Epidermal growth factor and gonadotropin-releasing hormone inhibit cyclic AMP-dependent luteinizing hormone receptor formation in ovarian granulosa cells. J Cell Biochem 21:209-217 13. Hsueh AJW, Ling NC 1979 Effect of an antagonist analog of gonadotropin releasing hormone upon ovarian granulosa cell function. Life Sci 25:1223-1230 14. Hsueh AJW, Wang C, Erickson GF 1980 Direct inhibitory effect of gonadotropin-releasing hormone upon follicle-stimulating hormone induction of luteinizing hormone receptor and aromatase activity in rat granulosa cell. Endocrinology 106:1697-1705 15. McFarland KC, Spengel 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 245:495-499 16. Loosfelt H, Misrahi M, Atger M, Salesse R, Thi MTVH-L, Jolivet A, Guiochon-Mantel A, Sar S, Jallal B, Gamier J, Milgrom E 1989 Cloning and sequencing of porcine LH-hCG receptor cDNA: variants lacking transmembrane domain. Science 245:525-528 17. Sprengel R, Braun T, Nikolics K, Segaloff DL, Seeburg PH 1990 The testicular receptor for follicle stimulating hormone: structure and functional expression of cloned cDNA. Mol Endocrinol 4:525530 18. Parmentier M, Libert F, Maenhaut C, LeFort A, Gerard C, Perret J, VanSande J, Dumont JE, Vassart G 1989 Molecular cloning of the thyrotropin receptor. Science 246:1620-1622 19. LaPolt PS, Oikawa M, Jia XC, Dargan C, Hsueh AJW 1990 Gonadotropin-induced up- and down-regulation of rat ovarian LH receptor message levels during follicular growth, ovulation and luteinization. Endocrinology 126:3277-3279 20. Bicsak TA, Tucker EM, Cappel S, Vaughan J, Rivier J, Vale W, Hsueh AJW 1986 Hormonal regulation of granulosa cell inhibin biosynthesis. Endocrinology 119:2711-2719 21. Melton DA, Krieg PA, Rebagliati MR, Maniatis T, Zinn K, Green MR 1984 Efficient in vitro synthesis of bioactive RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acid Res 12:7035-7056 22. Feinberg AP, Vogelstein B 1983 A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132:6-13 23. Erickson JM, Rushford CL, Dorney DJ, Wilson GN, Schmickel RD 1981 Structure and variation of human ribosomal DNA: molecular analysis of cloned fragments. Gene 16:1-9 24. Gonzalez IL, Gorski JL, Campen TJ, Dorney DJ, Erickson JM, Sylvester JE, Schmickel RD 1985 Variation among human 28S ribosomal RNA genes. Proc Natl Acad Sci, USA 82:7666-7670 25. Maniatis T, Fritsch EF, Sambrook J 1982 Molecular Cloning-A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor 26. Chan Y-L, Olvera J, Wool IG 1983 The structure of rat 28S ribosomal ribonucleic acid inferred from the sequence of nucleotides in a gene. Nucleic Acids Res 11:7819-7831 27. Hadjiolov AA, Georgiev OI, Nosikov VV, Yavachev LP 1984 Primary and secondary structure of rat 28S ribosomal RNA. Nucleic Acids Res 12:3677-3693 28. Gospodarowicz D, Cheng J, Lui G-M, Bohlen P1985 Corpus luteum angiogenic factor is related to fibroblast growth factor. Endocrinology 117:210-213 29. Adashi EY, Resnick CE, Croft CS, May JV, Gospodarowicz D 1988 Basic fibroblast growth factor as a regulator of ovarian granulosa cell differentiation: a novel non-mitogenic role. Mol Cell Endocrinol 55:7-14 30. Hsu C-J, Holmes SD, Hammond JM 1987 Ovarian epidermal


2456

31.

32.

33. 34. 35.


growth factor-like activity. Concentrations in porcine follicular fluid during follicular enlargement. Biochem Biophys Res Commun 147:242-247 Galway AB, Oikawa M, Ny T, Hsueh AJW 1989 Epidermal growth factor stimulates tissue plasminogen activator activity and messenger ribonucleic acid levels in cultured rat granulosa cells: mediation by pathways independent of protein kinases-A and -C. Endocrinology 125:126-135 LaPolt PS, Yamoto M, Veljkovic M, Sincich C, Ny T, Tsafriri A, Hsueh AJW 1990 Basic fibroblast growth factor induction of granulosa cell tissue-type plasminogen activator expression and oocyte maturation: potential role as a paracrine ovarian hormone. Endocrinology 127:2357-2363 Clayton RN, Harwood JP, Catt KJ 1979 Gonadotropin-releasing hormone analogue binds to luteal cells and inhibits progesterone production. Nature 282:90-92 Jones PBC, Conn PM, Marian J, Hsueh AJW 1980 Binding of gonadotropin releasing hormone agonist to rat ovarian granulosa cells. Life Sci 27:2125-2132 Oikawa M, Dargan C, Ny T, Hsueh AJW 1990 Expression of gonadotropin-releasing hormone and prothymosin alpha messenger ribonucleic acid in the ovary. Endocrinology 127:2350-2356

Endo • 1991 Voll28«No5

36. Knecht M, Ranta T, Feng P, Shinohara O, Catt KJ 1985 Gonadotropin-releasing hormone as a modulator of ovarian function. J Steroid Biochem 23:771-778 37. Wang J, Leung PCK 1987 Role of protein kinase C in luteinizing hormone-releasing hormone (LHRH) stimulated progesterone production in rat granulosa cells. Biochem Biophys Res Commun 146:939-944 38. Huckle WR, McArdle CA, Conn PM 1988 Differential sensitivity of agonist- and antagonist-occupied gonadotropin-releasing hormone receptors to protein kinase C activators. J Biol Chem 263:3296-3302 39. LaPolt PS, Jia XC, Sincich C, Hsueh AJW 1991 Ligand-induced down-regulation of testes and ovary luteinizing hormone receptors is preceded by tissue-specific inhibition of alternatively processed LH receptor transcripts. Mol Endocrinol 5:397-403 40. Tsai-Morris CH, Beuzko E, Wang W, Dufau ML 1990 Ihtronic nature of the rat luteinizing hormone receptor gene defines a soluble receptor subspecies with hormone binding activity. J Biol Chem 32:19385-19388 41. Bernard NP, Meyers RV, Moyle WR 1990 Cloning of rat lutropin (LH) receptor analogs lacking the soybean lectin domain. Mol Cell Endocrinol 71:R19-R23


Hormonal regulation of follicle-stimulating hormone receptor messenger ribonucleic acid levels in cultured rat granulosa cells.

Regulation of inhibin subunit messenger ribonucleic acid levels by gonadotropins, growth factors, and gonadotropin-releasing hormone in cultured rat granulosa cells.

Differential regulation of thyroid hormone receptor messenger ribonucleic acid levels by thyrotropin-releasing hormone.

chorionic gonadotropin receptor messenger ribonucleic acid transcripts.

Regulation of cytochrome P450 aromatase messenger ribonucleic acid and activity by steroids and gonadotropins in rat granulosa cells.

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

Transcriptional regulation of ferritin messenger ribonucleic acid levels by insulin in cultured rat glioma cells.

Ontogeny and pituitary regulation of testicular growth hormone-releasing hormone-like messenger ribonucleic acid.

Tissue distribution, characterization, and regulation of messenger ribonucleic acid for growth hormone receptor and serum binding protein in the rat.

Single cell levels of hypothalamic messenger ribonucleic acid encoding luteinizing hormone-releasing hormone in intact, castrated, and hyperprolactinemic male rats.

Gonadotropin regulation of c-fos and c-jun messenger ribonucleic acids in cultured rat granulosa cells.

Expression and regulation of growth hormone (GH) receptor messenger ribonucleic acid (mRNA) in rat adipose tissue, adipocytes, and adipocyte precursor cells: GH regulation of GH receptor mRNA.

Discordant effects of aging on prolactin and luteinizing hormone-beta messenger ribonucleic acid levels in the female rat.

Dynamic regulation of follicle-stimulating hormone-beta messenger ribonucleic acid levels by activin and gonadotropin-releasing hormone in perifused rat pituitary cells.

Mechanism of regulation of thyrotropin-releasing hormone receptor messenger ribonucleic acid in stably transfected rat pituitary cells.

Regulation of rat hypothalamic preprogrowth hormone-releasing factor messenger ribonucleic acid by dietary protein.

Parathyroid hormone messenger ribonucleic acid in the rat hypothalamus.

Activin stabilizes follicle-stimulating hormone-beta messenger ribonucleic acid levels.

Regulation of insulin-like growth factor I messenger ribonucleic acid levels by serum in cultured rat fibroblasts.

Growth hormone-releasing hormone messenger ribonucleic acid in the hypothalamus of the adult male rat is increased by testosterone.

Biphasic effects of exogenous ganglioside GM3 on follicle-stimulating hormone-dependent expression of luteinizing hormone receptor in cultured granulosa cells.

Luteinizing hormone induces progesterone receptor gene expression in cultured porcine granulosa cells.

Cellular localization and hormonal regulation of follicle-stimulating hormone and luteinizing hormone receptor messenger RNAs in the rat ovary.

neurophysin-I messenger ribonucleic acid in bovine granulosa cells increases after the luteinizing hormone (LH) surge and is stimulated by LH in vitro.