chorionic gonadotropin receptor mRNA in rat ovarian cells during follicular development and luteinization.

Hormonal Regulation of Luteinizing Hormone/Chorionic Gonadotropin Receptor mRNA in Rat Ovarian Cells during Follicular Development and Luteinization

Deborah L. Segaloff*, Haiyun Wang*, and JoAnne S. Richards The Population Council (D.L.S., H.W.) New York, New York 10021 Department of Physiology and Biophysics (D.L.S., H.W.) University of Iowa College of Medicine Iowa City, Iowa 52242 Department of Cell Biology (J.S.R.) Baylor College of Medicine Houston, Texas 77030

To determine whether hormone-dependent changes in the levels of LH/CG receptor in the rat ovary are associated with changes in expression of LH/CG receptor mRNAs, total RNA from rat follicles and corpora lutea at various stages of development was prepared and analyzed by Northern blots and/or solution hybridization. Whereas small antral follicles contained low amounts of LH/CG receptor mRNAs, the growth of preovulatory (PO) follicles was associated with an increase in all LH/CG receptor mRNA transcripts. Induction of LH/CG receptor mRNAs in granulosa cells of hypophysectomized rats was dependent on the synergistic effects of estradiol and FSH. An LH/CG surge in vivo or LH treatment of PO follicles in vitro caused a rapid decline of all LH/CG receptor mRNAs in PO follicles, which was prevented by cycloheximide. Newly formed corpora lutea (days 1-4 postovulation) contained low amounts of LH/CG receptor mRNAs unless the rats were pregnant or treated exogenously with PRL. During pregnancy, corpora lutea isolated on days 4-19 of gestation contained high levels of LH/CG receptor mRNAs, which decreased markedly on days 21 and 24, the time of functional luteolysis and decreasing LH/CG receptor levels.

and 3) induction and maintenance by PRL in corpora lutea of gestation. (Molecular Endocrinology 4: 1856-1865, 1990)

INTRODUCTION

The LH/CG receptor is obligatory for initiating gonadal responses to circulating LH or hCG. The binding of LH (in the male and the nonpregnant female) or hCG (in the pregnant female) to cell surface LH/CG receptors present on gonadal target cells results in the stimulation of cAMP accumulation (1, 2), which causes an increase in steroid biosynthesis (3, 4). It has been well documented that the levels of LH/ CG receptors (as measured by hormone binding) in the ovary are under complex hormonal control. The hormonal modulation of LH/CG receptors and related changes in cellular responsiveness to LH/CG have been especially well characterized in the rat ovary (5, 6). Thus, the differentiation of granulosa cells that occurs during the growth of small antral (SA) follicles to preovulatory (PO) follicles is known to be accompanied by an estradiol- and FSH-dependent induction in the numbers of LH/CG receptors per cell (7-9) and an increase in LH/CG-responsive adenylyl cyclase activity (9-11). These effects of FSH are due to its ability to increase intracellular cAMP levels (12-15). The effect of estradiol appears to be mediated by estradiol receptors present in rat granulosa cells (16, 17). The acquisition of increased numbers of LH/CG receptors is required for LH-induced ovulation and the initiation of luteinization. In concert with these events, the in vivo midcycle surge of LH results in the desensitization of the LH/CGresponsive adenylyl cyclase (18, 19) and the down-

These studies demonstrate that hormonal regulation of LH/CG receptor mRNA in rat ovarian cells parallels changes in LH/CG receptor levels and involves diverse molecular mechanisms, including 1) induction by the synergistic actions of estradiol and low concentrations of cAMP (elicited by FSH) in developing follicles, 2) inhibition by high concentrations of cAMP (elicited by LH/CG) in PO follicles, 0888-8809/90/1856-1865S02.00/0 Molecular Endocrinology Copyright © 1990 by The Endocrine Society

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The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 13:12 For personal use only. No other uses without permission. . All rights reserved.

LH/CG Receptor mRNA Regulation

regulation of the LH/CG receptor in PO follicles (7, 20, 21). If pregnancy ensues, however, there is a recovery of LH/CG receptors in the corpora lutea. The recovery and maintenance of LH/CG receptors in rat corpora lutea have been shown to be PRL/placental lactogen dependent (22, 23). Until recently, it has not been possible to examine the molecular mechanisms underlying the hormonal regulation of the LH/CG receptor. The cloning of the cDNAs for rat luteal and porcine testicular LH/CG receptors (24, 25) now allows one to begin to address these questions directly. In the studies described herein we examined whether hormone-induced changes in the numbers of LH/CG receptors in the rat ovary were associated with changes in the amounts or relative abundance of the different LH/CG receptor mRNA transcripts.

RESULTS

Within the rat ovary, LH/CG receptors are known to be expressed in granulosa and thecal cells of PO follicles and in corpora lutea. To determine the relative abun"*» and sizes of LH/CG receptor mRNA transcripts lese different cell types a Northern blot was . As shown in Fig. 1, several LH/CG receptor .nscripts are present in the different cell types; i abundant ones are 6.7, 4.3, 2.6, and 1.2 kb

6.7 —

4.3 —

kb 2.6 — 1.2 —

PO PO CL GC TC Fig. 1. LH/CG Receptor mRNAs in Rat Ovarian Cells Known to Express LH/CG Receptors Granulosa and thecal cells of PO follicles were isolated from 30-day-old immature female rats that had been injected with a low dose of hCG (0.15 IU twice daily for 2 days). Corpora lutea were isolated from the ovaries of rats on day 13 of gestation. The data shown are from a Northern blot prepared from 20 ng total RNA of each sample, which was probed with a 32P-labeled rat luteal LH/CG receptor cDNA (see Materials and Methods for details).

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in size.1 Corpora lutea from day 13 of pregnancy contained more of each of the LH/CG receptor mRNA transcripts than did granulosa or thecal cells, indicating that the transcripts were expressed in a coordinated manner in the different tissues. In the experiments that follow, the hormonal regulation of LH/CG receptor expression has been examined during follicular development and luteinization. LH/CG Receptor mRNA Induction during Follicular Development One of the hallmarks of the differentiation of granulosa cells during follicluar development is the acquisition of cell surface LH/CG receptors (26, 27). Previous studies have suggested that the induction of the LH/CG receptor in granulosa cells is due to de novo synthesis of the receptor (13, 28). To determine whether this increase in LH/CG receptor numbers was related to changes in LH/CG receptor mRNAs, we compared the number and abundance of LH/CG receptor transcripts present in granulosa cells from SA follicles, which have little or no LH/CG-binding activity, with those in granulosa cells from PO follicles, which exhibit elevated amounts of LH/CG-binding activity. As shown in Fig. 2, PO granulosa cells contain markedly higher amounts of LH/CG receptor mRNAs than do granulosa cells of SA follicles. Furthermore, the multiple LH/CG receptor mRNAs appear to be coordinately induced. It has been well documented that the induction of LH/CG receptors in rat granulosa cells in vivo (5, 7) and in vitro (13, 29, 30) is dependent upon both estradiol and FSH (acting through cAMP). To determine which of these hormones regulates LH/CG receptor mRNAs in granulosa cells, granulosa cells were isolated from intact immature rats left untreated or primed in vivo with estradiol. The cells were then cultured for 48 h in serum-free medium containing different hormone additions, after which [125l]hCG-binding activity and LH/CG receptor mRNAs were determined. As shown in Fig. 3, granulosa cells isolated from estradiol-primed rats and cultured with no additions (or with estradiol alone; data not shown) displayed no detectable LH/CG receptor mRNA or [125l]hCG-binding activity. The addition of FSH alone stimulated a marked increase in both LH/CG receptor mRNA levels and [125l]hCG binding/The further addition of estradiol (in the presence of FSH) had no effect. In granulosa cells from untreated female rats cultured under similar conditions, the addition of FSH alone caused only a modest increase in the amount of LH/CG receptor mRNA and [125l]hCG-binding activity, both of which were augmented significantly if estradiol was present with FSH during the 48-h culture period. Although one might initially conclude from these data that FSH alone can induce the expression of LH/CG 1 The apparent discrepancy between the sizes of the different mRNA transcripts reported here and those previously reported (24) is due to the fact that we now use RNA markers to calibrate the gels instead of the DNA markers used in the previous experiments.


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E-Primed Rats

Untreated Rats

6.7 -

6.7

4.3-

4.3

kb

kb

2.6-

2.6 1.2-

1.2 NA F SA GC

EF

NA F

EF

PO GC

Fig. 2. Induction of LH/CG Receptor mRNA during Rat Granulosa Cell Differentiation Granulosa cells were isolated from SA follicles of immature female rats 26 days of age or from PO follicles from immature female rats injected with a low dose of hCG (0.15 IU twice daily for 2 days). The data shown are from a Northern blot prepared from 20 ng total RNA of each sample, which was probed with a 32P-labeled rat luteal LH/CG receptor cDNA, as described in Materials and Methods.

20-

o CO

receptor mRNA in granulosa cells and that estradiol augments this response, one cannot exclude the possibility that low levels of estrogen were present in the untreated intact immature rat ovary and that these were sufficient to permit a subsequent induction of LH/CG receptor mRNA by FSH alone in the cultured cells. To address this issue, we examined the content of LH/CG receptor mRNA in granulosa cells of immature female rats that had been hypophysectomized and treated in vivo with estradiol alone, FSH alone, or a combination of estradiol and FSH. The results, shown in Fig. 4, clearly show that granulosa cells isolated from hypophysectomized rats have no detectable LH/CG mRNA and that neither estradiol alone nor FSH alone can induce LH/CG receptor mRNAs. However, when estradiol-primed hypophysectomized rats were injected with the same dose of FSH, LH/CG receptor mRNA was induced, indicating that the synergistic actions of estradiol and FSH are required for the induction of LH/CG receptor message in granulosa cells. Importantly, the content of LH/CG receptor mRNA induced by FSH and estradiol in these cells is comparable to that observed in granulosa cells isolated from a PO follicle of an intact rat. LH/CG Receptor mRNA in Response to the Ovulatory LH/CG Surge The initiation of luteinization by an ovulatory dose of LH/CG has been associated with a loss of LH/CGbinding activity (7, 19-21). To determine whether this

O U

IO-

1 NA F

EF

1""""" NA F

EF

Fig. 3. Induction of LH/CG Receptor mRNA in Cultures of Granulosa Cells Derived from Immature Rats Granulosa cells were isolated from ovaries of intact immature female rats that had been estradiol (E) primed (left panels) or were untreated (right panels). The cells were cultured for 48 h with no additions (NA) or with 50 ng/ml FSH (F) or 10 nM estradiol plus 50 ng/ml FSH (EF). At the end of the incubation, some cells were used for the isolation of total RNA, which was analyzed by Northern blots for LH/CG receptor mRNAs (top panels), and some cells were used to measure [125l]hCGbinding activity (lower panels), as described in Materials and Methods.

phenomenon is associated with decreased levels of LH/ CG receptor mRNAs, hormonally primed immature female rats containing PO follicles were injected with an ovulatory concentration of hCG. Two, 4, and 6 h later the content of LH/CG receptor mRNA in the PO follicles was quantitated. As shown in Fig. 5, there was a rapid loss in the content of LH/CG receptor mRNA, declining to 75% and 25% of control values by 2 and 4 h, respectively. The down-regulation of LH/CG-R mRNA by LH/CG in vivo (Fig. 5) could be reproduced in vitro. As shown in Fig. 6, intact PO follicles isolated and then incubated with a high concentration of LH exhibit markedly lower amounts of LH/CG receptor mRNAs than control PO follicles incubated without LH. Importantly,


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6.7 —

6.7 4.3 —

4.3

kb

kb

2.6 —

2.6 1.2 —

1.2 Treatment: LH/CG-R mRNA:

H HE HF HEF 0.6 0.4 0.3 6.4

+ LH

-LH

10.6

Fig. 4. Induction of LH/CG Receptor mRNA in Granulosa Cells of Hypophysectomized Rats Injected with Estradiol and/or FSH Immature female rats were hypophysectomized at 26 days of age. They then were untreated (H) or were injected with estradiol (HE) only, FSH only (HE), or a sequential combination of E then FSH (HEF; see Materials and Methods for details). The rats were killed after the given treatment, the ovaries were removed, and the granulosa cells were isolated. Total RNA was prepared from the granulosa cells, some of which were used for Northern analysis of LH/CG receptor mRNAs and some of which was used to quantitate the levels of LH/CG receptor mRNA by solution hybridization. The latter are expressed as picograms of LH/CG receptor mRNA per ng total RNA and are shown below the Northern blot.

Fig. 6. Down-Regulation of LH/CG Receptor mRNAs in Isolated PO Follicles Incubated with LH PO follicles were isolated from ovaries of immature 30-dayold rats after treatment with a low dose of hCG (0.15 IU twice daily for 2 days) and were incubated for 6 h in DMEM-F-12 (1:1) at 37 C with 95% oxygen-5% CO2 in the absence (left) or presence (right) of ovine LH (500 ng/ml). At the end of the incubation, total RNA was prepared, which was used for a Northern analysis of LH/CG receptor mRNAs.

LH/CG-R mRNA (pg/ng total RNA) 0

1

2

3

4

5

6

7

NA LH

Fo

LH/CG-R mRNA (pgAig RNA) 5

10

15

20

Fo + CHX Control

+ hCG, 2h

+hCG, 4h

+hCG, 6h

Fig. 5. Down-Regulation of LH/CG Receptor mRNA in PO Follicles of Rats Injected with hCG Intact immature female rats were injected with a low dose of hCG (0.15 IU twice daily for 2 days) to stimulate the growth of PO follicles. They were then injected with an ovulatory dose of hCG (10 IU, iv) and killed 2,4, or 6 h later. The ovaries were removed and PO follicles isolated. Total RNA was prepared and assayed for LH/CG-R mRNA by solution hybridization (see Materials and Methods for details). The results are expressed as picograms of LH/CG receptor mRNA per ^g total RNA (mean ± SEM of duplicate samples). The errors in each point, however, are too small to be detected in this figure.

Fig. 7. Levels of LH/CG Receptor mRNA in PO Follicles Incubated with LH or Forskolin PO follicles were isolated from ovaries of immature 29-dayold rats after treatment with a low dose of hCG (0.05 IU twice daily for 2 days) and were incubated for 6 h in DMEM-F-12 (1:1) at 37 C with 95% oxygen-5% CO2 containing no additions, LH (500 ng/ml), forskolin (Fo; 10 fiM), or forskolin (Fo; 10 >IM) plus cycloheximide (CHX; 10 ^g/m\). At the end of the incubation, total RNA was prepared and assayed for LH/CGR mRNA by solution hybridization (see Materials and Methods for details). The results are expressed as picograms of LH/CG receptor mRNA per ng total RNA (mean ± SEM of duplicate samples).

the different LH/CG receptor transcripts appear to be coordinately decreased in this 6-h period examined. Because LH and hCG stimulate the production of cAMP, we next determined whether the LH/CG-induced decrease in LH/CG receptor mRNAs was mediated by cAMP. For this, PO follicles were isolated and incubated for 6 h with forskolin (10 HM). AS shown in Fig. 7, forskolin (10 ^M) caused the amounts of LH/CG


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receptor mRNA to decrease to levels comparable to those in follicles incubated for the same length of time with LH (500 ng/ml). These data suggest that the rapid LH/CG-induced decrease in LH/CG receptor message in the rat PO follicle is mediated by cAMP. To test whether cAMP was affecting LH/CG receptor mRNA levels directly or if it was inducing the synthesis of a protein that, in turn, affects LH/CG receptor mRNA levels, we examined the effect of cycloheximide on forskolin-induced down-regulation of the LH/CG receptor message. As also shown in Fig. 7, cycloheximide inhibited the ability of forskolin to decrease LH/CG receptor mRNAs in the PO follicles. Similar results were observed when the PO follicles were incubated with LH in the presence of cycloheximide (data not shown). LH/CG Receptor mRNA Levels in Rat Corpora Lutea during Pregnancy Although the LH/CG receptor and its mRNA are reduced dramatically by the ovulatory surge of LH, if pregnancy ensues the expression of LH/CG receptors is recovered (6). To determine whether the changes in LH/CG receptor mRNA parallel those in LH/CG-binding activity in rat corpora lutea during pregancy, LH/CG receptor mRNA content was examined on different days of gestation. As shown in Fig. 8, LH/CG receptor mRNA was undectable on the first day of gestation. By day 4 of pregnancy, however, the levels of LH/CG-R mRNA were elevated, with maximal steady state amounts occurring between days 7-15. The highest content of LH/CG receptor mRNA in the corpora lutea was comparable to the level observed in PO thecal cells and was about 2-fold higher than that observed in PO

granulosa cells (see Fig. 2). During the time of functional luteolysis (days 19-23 of gestation) LH/CG receptors decreased (6), and this was clearly associated with a concomitant decrease in the content of LH/CG receptor mRNA in the corpora lutea. It has been well documented that the appearance of LH/CG receptors in rat corpora lutea by day 4 of gestation is dependent upon pituitary PRL (22, 23). To analyze whether the observed increase in LH/CG receptor mRNA content in rat corpora lutea on day 4 of gestation (see Fig. 8) was dependent on PRL, the following experiment was performed. Rats were given an ovulatory dose of hCG and were either left untreated or injected 24 h later with PRL, estradiol (which stimulates pituitary PRL production), or PRL plus estradiol (see Materials and Methods for protocols). Ninety-six hours after the injection of hCG, the content of LH/CG receptor mRNA in the corpora lutea of these nonpregnant animals was measured. The results, shown in Table 1, clearly show that the appearance of LH/CG receptor mRNA in rat corpora lutea is dependent upon PRL. Indeed, the levels of LH/CG receptor mRNA in the corpora lutea of these nonpregnant rats treated with PRL, estradiol, or PRL plus estradiol were comparable to those in the corpora lutea of a rat 4 days into pregnancy (see Table 1).

DISCUSSION

Expression of the LH/CG receptor in the rat ovary is under diverse hormonal control. As a first step toward determining the transcriptional and translational controls governing these changes in receptor expression, we have identified several mRNA transcripts present in

LH/CG-R mRNA (pg/ng total RNA) 10 SA Follicle

GC TC

PO Follicle

GC TC

20

Day 1

Table 1. Effects of PRL on the Induction and Maintenance of LH/CG Receptor mRNA Levels in the Corpora Lutea of Nonpregnant Rats 96 h after Ovulation Treatment

Day 4 Day 7

Corpora Lutea (days gestaslion)

Day 11 Day 12 Day IS Day 19 Day 21 Day 23

Fig. 8. LH/CG Receptor Levels in Rat SA Follicles, PO Follicles, and Corpora Lutea Granulosa or thecal cells were isolated from PO follicles of 30-day-old intact immature female rats that had been injected with a low dose of hCG (0.15 IU twice daily for 2 days) or from SA follicles of untreated immature female rats. Corpora lutea were isolated from rats on the indicated day of gestation. Total RNA was prepared and assayed for LH/CG receptor mRNA by solution hybridization (see Materials and Methods for details). The results are expressed as picograms of LH/CG receptor mRNA per ^g total RNA (mean ± SEM of duplicate samples).

LH/CG Receptor mRNA (pg/^g total RNA)

0 Control 96 h, No Additions 96 h, + PRL 96 h, + E 96 h, + PRL + E

23.5 5.3 26.1 22.8 25.5

± 0.5 ±0.1 ±0.1 ± 0.3 ± 0.8

Day 4 of Pregnancy

22.0 ± 0.1

The growth of PO follicles of immature female rats was stimulated by injections of low concentrations of hCG, after which ovulation and luteinization were induced with an ovulatory dose of hCG. The rats were then treated with PRL alone, estradiol alone, or PRL and estradiol. Corpora lutea were isolated from the rat ovaries 96 h after the ovulatory dose of hCG. Corpora lutea were also isolated from the ovaries of rats on day 4 of pregnancy. LH/CG receptor mRNA contents in the corpora lutea were assayed by solution hybridization. Results are expressed as picograms of LH/CG receptor mRNA per ng total RNA (mean ± SEM of duplicate determinations).



the rat ovary and have determined some aspects of their regulation. Specifically, four LH/CG receptor transcripts have been resolved by Northern blot analyses, which appeared to be coordinately regulated during various hormone-induced stages of ovarian cell differentiation. The nucleotide sequence of each of the rat ovarian LH/CG receptor mRNAs remains to be determined and, therefore, which transcript(s) encodes for the 93-kDa cell surface LH/CG receptor is presently not known. In addition to the full-length cDNA encoding a functional 93-kDa LH/CG receptor, several other rat luteal LH/CG receptor cDNAs were originally isolated. These encoded for variant forms of the receptor containing truncations and/or deletions as well as some containing intronic sequences (31). Thus, some of the RNA transcripts observed on the Northern blots may encode for these variant receptor species. It is unclear, however, whether mRNAs coding for LH/CG receptor variants are actually translated and, if they are, whether they are translated into stable proteins. Western blots of wheat germ agglutinin-purified rat luteal extracts that are incubated with either [125l]hCG or one of several site-specific antibodies that encompass different domains of the receptor consistently reveal only one 93kDa LH/CG receptor protein (32) (our unpublished data). These data suggest that mRNAs encoding for variants of the LH/CG receptor that would be of significantly different mass than the 93-kDa form are not expressed on the plasma membrane. Furthermore, although cDNAs have been identified in both rat luteal and porcine testicular libraries which encode for truncated LH/CG receptors containing only the extracellular domain (25, 31), the secretion of a LH/CG-binding protein has not been observed. Although the details regulating the sizes of the LH/ CG receptor mRNAs remain to be determined, it is clear that changes in the total amount of LH/CG receptor mRNA parallel changes in LH/CG binding activity. These results suggest that the presence (and possible expression) of altered forms of the mRNA do not interfere with the induction and expression of mRNA encoding the 93 kDa functionally active LH/CG receptor. Because all transcripts appear to be coordinately regulated, no selective advantage (or disadvantage) appears to be conveyed by any one species of mRNA at a given stage of granulosa/luteal cell differentiation. During the development of PO follicles in vivo, LH/ CG receptor mRNA was dramatically increased, indicating that the appearance of LH/CG-binding activity and LH/CG-responsive adenylyl cyclase activity in granulosa cells of PO follicles is dependent on an increase in receptor mRNA. In vivo and in vitro studies further documented that the increase in LH/CG receptor mRNA and [125l]hCG-binding activity in PO granulosa cells is dependent on the synergistic actions of estradiol and FSH. The synergistic effects of estradiol and FSH not only regulate expression of mRNA for the LH/CG receptor, but also induce mRNA for RII/3, the regulatory subunit of type II cAMP-dependent protein kinase (A-

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kinase) (33, 34), cholesterol side-chain cleavage cytochrome P450 (P450scc) (35), and inhibin a-subunit (36). The effects of estradiol and FSH appear to involve interactions of the estradiol receptor and A-kinase pathways present in rat granulosa cells and known to be involved in granulosa cell differentiation (5,37,38). However, the factors in each pathway that are required for this synergistic induction of LH/CG receptor mRNA remain to be determined. One might speculate that FSH, acting through the A-kinase, phosphorylates the estradiol receptor (39), allowing estradiol receptor transactivation of the LH/CG receptor gene. Alternatively, estradiol may induce a protein (frans-acting factor) that is phosphorylated by A-kinase. These are not mutually exclusive nor all encompassing hypotheses and will only be resolved by characterization of the regulatory domains in the LH/CG receptor gene and other genes specifically expressed in granulosa cells of developing follicles. The rapid loss of LH/CG receptor mRNA in PO follicles that occurs in response to an ovulatory dose of LH/CG in vivo (40) or in vitro demonstrates that high levels of LH/CG have inhibitory effects on transcription of the LH/CG receptor gene and/or on LH/CG receptor mRNA stability. Because forskolin could mimic the effects of LH on LH/CG receptor mRNA in PO follicles incubated in vitro, the LH/CG-induced decrease in receptor mRNA appears to be mediated at least in part by high concentrations of cAMP. Therefore, the inhibitory effects of high concentrations of cAMP contrast with the stimulatory effects of low concentrations of cAMP in inducing LH/CG receptor mRNA (see above). These opposing effects of different concentrations of cAMP on LH/CG receptor mRNA levels may explain why the induction of LH/CG receptors in granulosa cells by FSH, cholera toxin, or cAMP analogs is submaximal at high concentrations of these stimuli (13, 25, 41). Because the effects of forskolin or LH/CG on decreasing LH/CG receptor mRNA can be reversed by cycloheximide, it appears that the synthesis of a labile protein is required for the rapid loss of mRNA for this receptor. Whether this involves the synthesis of a negative regulatory factor acting on the LH/CG receptor gene and/ or a factor altering mRNA stability remains to be resolved. This rapid LH/CG-induced decrease appears to be specific for LH/CG receptor mRNA, because mRNA for P450scc in PO follicles incubated under similar conditions is induced with ovulatory doses of LH or high concentrations of forskolin (35, 42). The LH/CG receptor and its cognate mRNA are also regulated by cAMP in MA-10 Leydig tumor cells (43). LH/CG or 8-bromo-cAMP (8-Br-cAMP) added to MA10 cells transiently increases the content of LH/CG receptor mRNA and then causes a subsequent decline in message levels. The time courses and magnitude of the effects of LH/CG and 8-Br-cAMP on LH/CG receptor mRNA are comparable. These and other data support the conclusion that the LH/CG-induced loss of LH/ CG receptor mRNA in these cells is mediated by cAMP (43). Whereas the levels of LH/CG receptor parallel the


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changes in LH/CG receptor mRNA in 8-Br-cAMPtreated cells, however, the down-regulation of LH/CG receptors by LH/CG in MA-10 cells precedes the LH/ CG (cAMP)-induced loss of LH/CG receptor mRNA. Thus, quantitatively, the primary cause of LH/CG-induced down-regulation of LH/CG receptors in MA-10 cells is the increase in the rate of receptor degradation occurring during the receptor-mediated endocytosis of the hormone-receptor complex (44, 45); a secondary contribution is the loss of LH/CG receptor mRNA. This is in apparent contrast to the regulation of the LH/CG receptor in the rat ovary, where the LH/CG-induced loss of LH/CG receptor mRNA occurs relatively quickly (c.f. Fig. 5). It is important to note that cAMP also regulates steady state mRNA levels of the /3-adrenergic receptor (46-49), a membrane protein that is structurally related to the LH/CG receptor (24, 25, 50) and also couples to a Gs protein. It has been shown that cAMP transiently increases /3-adrenergic receptor mRNA during short term incubations, but then decreases them during long term incubations (46-49). Thus, the regulation of the levels of mRNA for Gs protein-coupled receptors by cAMP may be a generally important mechanism for negative feedback control. Loss of LH/CG receptor mRNA and LH/CG-binding activity during LH/CG-induced luteinization in the rat ovary is not irreversible (6, 7, 21-23, 40). If PRL is present during the early stages of luteal cell differentiation (days 1-14) or if placental lactogen is present during the second half of pregnancy, LH/CG mRNA is induced and maintained at elevated levels until functional luteolysis occurs between days 19-23 of gestation. Once again, the induction and maintenence of LH/ CG receptor mRNA by PRL/placental lactogen during pregnancy are associated with functional LH/CG-binding activity (6). Although LH/CG and LH/CG-responsive adenylyl cyclase activity appear to be required for maintenance of luteal cell function in vivo in the rat through day 12-13 of gestation (51), the functional role of LH/ CG and its receptor during the second half of pregnancy in the rat is less clear. The regulation of LH/CG receptor mRNA by PRL in luteal cells is similar to the pattern observed for the expression of a2-macroglobulin in the rat corpora lutea of gestation (52). However, the regulation of these two gene products differs markedly from the regulation of mRNA for two steroidogenic genes, P450scc (35, 42) and aromatase (P450 aromatase) (53). As mentioned above, P450scc is induced by the LH/CG surge in vivo (35) and in vitro (42). Once induced, P450scc is constitutively maintained in luteinized granulosa cells (42) and is modulated, not induced, by PRL (42). In contrast, P450 aromatase mRNA is rapidly reduced in PO granulosa cells by LH/CG, but is then constitutively maintained at a low level in luteinized cells. In vivo, PRL induces aromatase mRNA after, but not before, day 10-12 of gestation (54), and in vitro is inhibitory (55). Thus, regulation of LH/CG receptor mRNA by PRL shows some, but not all, of the features by which other

gene products in the ovary are regulated by this pituitary hormone. The mechanisms by which PRL regulates these different genes may involve different pathways. In summary, hormonal regulation of LH/CG receptor mRNA in ovarian cells involves diverse molecular mechanisms, including 1) induction by the synergistic actions of estradiol and low concentrations of cAMP (elicited by FSH) in developing follicles, 2) inhibition by high concentrations of cAMP (elicited by LH/CG) in PO follicles, and 3) induction and maintenance by PRL in corpora lutea of gestation. The changes in LH/CG receptor mRNA parallel those in LH/CG-binding activity during ovarian cell differentiation, indicating that regulation of LH/CG receptor mRNA is a major determinant in the expression of LH/CG receptor numbers in these cells.

MATERIALS AND METHODS Hormones and Supplies hCG (CR-123), ovine LH (NIH oLH-23), and ovine FSH (NIH oFSH-16) were obtained from the National Hormone and Pituitary Progam (Baltimore, MD). Estradiol was obtained from Sigma (St. Louis, MO). Electrophoretic supplies were obtained from Bio-Rad (Richmond, CA), and tissue culture reagents from Gibco (Grand Island, NY). Animals Intact immature female rats were obtained from Holtzman Co. (Madison, Wl). Immature hypophysectomized female rats were obtained from Johnson Laboratories (Chicago, IL) 1 day after surgery (day 26). Animal Models Pregnant Rat Corpora lutea were isolated from timed pregnant rats on days 1 (sperm positive) through 23 (parturition). Ovulating Rat SA follicles were obtained from intact immature rats (25-28 days of age). PO follicles were obtained from intact 30-day-old rats that had been treated with a low dose of hCG (0.15 IU/0.2 ml, sc) twice daily for 2 days (56). Hypophysectomized Rat Immature female rats were hypophysectomized at 26 days of age. They were either left untreated or injected with estradiol only (once daily for 3 days with 1.5 mg), with FSH alone (twice daily for 2 days with 1.0 mg), or with estradiol then FSH (6, 7, 35). Nonpregnant Rat with PRL-lnduced Corpora Lutea Maturation of PO follicles was stimulated by treatment of immature rats with a low dose of hCG as described above. Rats were then injected iv with an ovulatory dose of hCG (10 IU) to cause ovulation and luteinization of PO follicles (56). To maintain functional corpora lutea, animals were injected 24 h after the ovulatory dose of hCG with 250 mg ovine PRL twice daily (42, 52), with 75 ng estradiol (57), or with PRL and estradiol. In Vitro Models Incubation of Isolated PO Follicles PO follicles from hormonally primed intact rats (see above) were incubated in Dulbecco's Modified Eagle's Medium (DMEM)-Ham's F-12 (1:1) at 37 C and with 95% oxygen-5% CO2 for 6 h in the absence or presence of the indicated compounds. After the



incubation, follicles in each treatment group (~60/group) were pooled, collected by centrifugation, and extracted to prepare total RNA as described below. Primary Cultures of Granulosa Cells Immature female rats (day 26) were untreated or were primed with estradiol (1.5 mg once daily for 3 days). The ovaries were removed, and the granulosa cells were harvested and cultured (30,33) in serumfree DMEM-F-12 (1:1) for 48 h at 37 C with the indicated additions. At the end of the incubation, cells were either extracted to prepare total RNA or assayed for [125l]hCGbinding activity (see below).

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[125l]hCG Binding Highly purified hCG was iodinated as described previously (63). Granulosa cells, in 24-well plates, were washed four times with 1-ml portions of PBS, pH 7.4. They were then incubated for 5 h at 37 C with [125l]hCG (100 ng/ml) in the absence (three or four wells) or presence (one or two wells) of 50 IU crude hCG. At the end of the incubation, the cells were set on ice and washed five times with 1 -ml portions of ice-cold PBS, pH 7.4. After solubilizing the cells in 100 ml 0.5 N NaOH, the contents of each well were transferred with cotton swabs to a plastic tube and counted in a 7-counter. Values shown represent specific counts bound.

LH/CG Receptor mRNA Detection by Northern Analyses and Solution Hybridization

Acknowledgments Corpora lutea dissected from intact ovaries were homogenized in 5 M guanidine isothiocyantate, and total RNA was prepared by cesium chloride centrifugation (58). Total cytoplasmic RNA was isolated from cultured granulosa cells or from isolated PO follicles or theca or granulosa cells (59) using Nonidet P-40 as described by Pelham (59). For Northern analysis 20 ^g total RNA were denatured and electrophoresed in a 1 % formaldehyde-agarose gel and then transferred to a nylon (Biodyne, ICN, Irvine, CA) membrane. A prehybridization preceded hybridization overnight at 42 C with a nick-translated 32P-labeled pGEM-4Z vector (Promega, Madison, Wl) containing nucleotides 1-622 of the rat luteal LH/CG receptor cDNA (24). This portion of the LH/CG receptor cDNA encodes for a large portion of the extracellular domain of the receptor. The blot was washed four times in 2 x SSC (saline sodium citrate) and 0.1% sodium dodecyl sulfate (SDS) at room temperature (15 min/washing) and then twice in 0.1 x SSC and 0.1% SDS at 60 C (30 min/washing). The resulting blots were exposed to x-ray film at - 7 0 C with intensifying screens. The sizes of mRNA transcripts were determined using RNA markers (Boehringer Mannheim Biochemicals, Indianapolis, IN) of 7.4, 5.3,2.8,1.9,1.6,1.0, 0.6, 0.4, and 0.3 kb. For solution hybridizations, a pGEM-4Z vector (Promega) containing nucleotides 1-622 of the rat luteal LH/CG receptor cDNA (24) was used to prepare unlabeled sense and 35Slabeled antisense RNA probes (60). Specific mRNA levels were measured by incubating 1-3 ^g total RNA with 3 x 104 cpm 35 S-labeled antisense RNA in a total volume of 20 n\ buffer containing 0.6 M NaCI, 10 mM Tris-CI, 4 mM EDTA, and 0.05% SDS, pH 7.4, for 16-20 h at 68 C. The single stranded RNA was then digested, and the double stranded RNA was precipitated (61, 62). The concentration of LH/CG-R mRNA was calculated by comparison with a standard curve generated using the unlabeled sense strand and expressed as picograms per fig total RNA. The quantitation was performed under conditions in which the assay gave a linear response with increasing concentrations of total RNA. The data shown in a given figure are from one representative experiment. Although the absolute values of LH/CG receptor mRNA of a given sample varied somewhat between experiments (and thus between different figures), the relative changes shown are representative. Whenever possible, the LH/CG receptor mRNA in a given experiment was analyzed by both Northern blotting and solution hybridization. The Northern blots allow determination of whether all or some of the LH/CG receptor mRNA transcripts are hormonally regulated, whereas the solution hybridization assay provides a more accurate quantitation of the changes in the total amount of LH/CG receptor mRNA. Due to low yields of mRNA in some experiments, however, it was not always possible to perform both assays. In those situations, the assay yielding the information most pertinent to the particular experiment was performed.

We wish to thank Dr. M. Ascoli for helpful suggestions and for critically reading the manuscript.

Received August 6, 1990. Revision received September 4, 1990. Accepted September 21, 1990. Address requests for reprints to: Dr. Deborah L. Segaloff, Department of Physiology and Biophysics, The University of Iowa College of Medicine, Iowa City, Iowa 52242. This work was supported by NIH Grants HD-22196 (to D.L.S.) and HD-16272 (to J.S.R.). * Current address: Department of Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, Iowa 52242.

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Hormonal regulation of gonadotropin receptor mRNA in rat ovary during follicular growth and luteinization.

Gonadotropin-induced up- and down-regulation of rat ovarian LH receptor message levels during follicular growth, ovulation and luteinization.

Induction of follicular luteinization by equine chorionic gonadotropin in cyclic guinea pigs.

Ovarian stimulation with human chorionic gonadotropin and equine chorionic gonadotropin affects prostacyclin and its receptor expression in the porcine oviduct.

Hyperinsulinemia and human chorionic gonadotropin synergistically promote the growth of ovarian follicular cysts in rats.

Human chorionic gonadotropin induces c-myc mRNA expression via TSH receptor in FRTL-5 rat thyroid cells.

CG receptor.

Estradiol receptor content in rat granulosa cells during follicular development: modification by estradiol and gonadotropins.

Effect of follicular fluid and oestradiol on the luteinization of rat granulosa cells in vitro.

Human chorionic gonadotropin and ovarian and placental steroidogenesis.

Cellular localization of ovarian proopiomelanocortin messenger RNA during follicular and luteal development in the rat.

Ovarian clear-cell carcinoma producing estradiol and human chorionic gonadotropin.

Ovarian superstimulation using FSH combined with equine chorionic gonadotropin (eCG) upregulates mRNA-encoding proteins involved with LH receptor intracellular signaling in granulosa cells from Nelore cows.

The porcine ovarian follicle: III. Development of chorionic gonadotropin receptors associated with increase in adenyl cyclase activity during follicle maturation.

Activated ovarian endothelial cells promote early follicular development and survival.

Human chorionic gonadotropin causes an estrogen-mediated induction of rat ovarian carbonyl reductase.

Properties of human chorionic gonadotropin produced in vitro by ovarian carcinoma cells.

Changes in fibroblast growth factor 9 mRNA in granulosa and theca cells during ovarian follicular growth in dairy cattle.

Immunolocalization of GnRHRI, gonadotropin receptors, PGR, and PGRMCI during follicular development in the rabbit ovary.

Preovulatory follicular fluid steroid levels in stimulated and unstimulated cycles triggered with human chorionic gonadotropin.

Human chorionic gonadotropin and rat mammary cancer prevention.

Gonadotropin-releasing hormone and chorionic gonadotropin gene expression in human placental development.

choriogonadotropin receptor in rat ovary during luteinization.

chorionic gonadotropin (CG) receptor and human CG for receptor-hormone interaction.