0013-7227/91/1296-3200$03.00/0 Endocrinology Copyright © 1991 by The Endocrine Society

Vol. 129, No. 6 Printed in U.S.A.

Localization of Luteinizing Hormone Receptor Messenger Ribonucleic Acid Expression in Ovarian Cell Types during Follicle Development and Ovulation* XIAO-RONG PENG, AARON J. W. HSUEH, PHILIP S. L A P O L T , LARS BJERSING, AND T. NY Departments of Applied Cell and Molecular Biology (X.-R.P., T.N.) and Pathology (L.B.), University of Umea, S-90187 Umea, Sweden; the Division of Reproductive Biology, Department of Obstetrics and Gynecology, Stanford University School of Medicine (A.J.W.H., P.S.L.), Stanford, California 94305-5317; and the Department of Reproductive Medicine, University of California-San Diego (T.N.), La Jolla, California 92093-6025

ABSTRACT. The action of LH is mediated through specific plasma membrane receptors that are both up- and down-regulated in the ovary during the reproductive cycle. Using immature rats treated with PMSG and hCG as a model system, we have studied the regulation and distribution of LH receptor mRNA in different cell types during follicle development, ovulation, and luteinization by Northern blot and in situ hybridization. In untreated rats, LH receptor mRNA was below the detection level in granulosa cells, cumulus cells, and oocytes, while low levels of LH receptor mRNA were found in the thecal cells. After stimulation with PMSG, expression of LH receptor mRNA was enhanced in the thecal-interstitial cells, while a more dramatic increase in receptor mRNA abundance took place in granulosa cells of large tertiary follicles. In these follicles, the abundance of LH receptor mRNA varied among different subpopulations of granulosa cells, with mural granulosa cells close to the basement membrane exhibiting higher levels than gran-

ulosa cells located closer to the antrum, and cumulus cells and the oocyte lacking detectable hybridization signal. The uneven expression of LH receptor mRNA endows different ovarian cells with varying hormonal responsiveness. After an ovulatory dose of hCG, LH receptor mRNA levels were dramatically decreased, particularly in the granulosa cells of preovulatory follicles, to reach the lowest levels just before ovulation. During the transformation of ovulated follicles into corpora lutea, the expression of LH receptor message was again increased. Our results reveal that the previously documented regulation of the LH receptorbinding activity during ovarian development correlates with expression of the LH receptor transcripts, suggesting that the LH receptor gene is regulated in a complex manner during the periovulatory period to achieve cell-specific expression together with gonadotropin induction and suppression of receptor gene activity. (Endocrinology 129: 3200-3207,1991)

T

HE DEVELOPMENT of the ovarian follicle is a highly integrated process. It involves a series of sequential events in which a recruited follicle acquires its structural and functional properties as it matures, ovulates, and becomes luteinized, under the control of a group of endocrine, autocrine, and paracrine factors (13). LH is one of the primary regulators of ovarian follicle development. As with FSH, LH is secreted by the pituitary and is believed to be responsible for the triggering of ovulation and the transformation of the follicle into the corpus luteum (1-3). The action of LH on its target cells is mediated through binding to specific receptors on the cell membrane and Received June 4,1991. Address all correspondence and requests for reprints to: Tor Ny, Ph.D., Department of Applied Cell and Molecular Biology, University of Umea, S-90187 Umea, Sweden. * This work was supported by the Swedish Medical Research Council (Research Grant B92-13X-09709-02B) and NIH Research Grants HD12303 and HD-23273.

subsequent activation of the c AMP -dependent protein kinase-A pathway (4, 5). The LH receptor, therefore, plays a pivotal role in LH-regulated ovarian functions. The responsiveness of target cells to LH has been shown to be regulated by different mechanisms, including changes in the LH receptor content and the functional activity of the receptor itself (5). During granulosa cell differentiation, FSH has been shown to increase the number of LH receptors on granulosa cells by stimulating LH receptor synthesis, while ovulatory doses of LH/hCG decreased the LH receptor level during ovulation (6-9). The responsiveness to LH can also be regulated independent of changes in receptor number, e.g. by receptor desensitization, where the ability of the receptor to activate adenylyl cyclase is attenuated (10). The regulation of LH receptor on different ovarian cell types is complex due to the structural and functional heterogeneity of the ovary. Autoradiographic studies have demonstrated that the number of LH-binding sites

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on granulosa cells in a given follicle is not uniform, but varies among different subpopulations of granulosa cells (11, 12). These findings suggest that the microenviroment of the cell and the interaction of various cell types in the ovary may be crucial for the acquisition of LH receptors on a given cell type. This uneven distribution of LH receptors in different ovarian cell types may endow the cells with distinct hormonal responsiveness. Recent studies demonstrated that gonadotropins regulate LH receptor mRNA levels during ovarian development (13,14). To study the cell type-specific regulation of LH receptor in vivo during follicle differentiation, ovulation, and luteinization, we have examined the gonadotropin-dependent regulation of LH receptor mRNA levels in specific cell types by Northern blot and in situ hybridization using gonadotropin-treated rats as a model system.

Materials and Methods Materials PMSG and Nonidet P-40 were purchased from Sigma Chemical Co. (St. Louis, MO); hCG (CR-121; 13,450 IU/mg) was obtained from Dr. R. E. Canfield through the Centre for Population Research, NICHHD; collagenase was obtained from Worthington Biochemical Corp. (Freehold, NJ); and modified tissue culture medium was purchased from Gibco (Scotland, United Kingdom); the Riboprobe system was purchased from Promega (Madison, WI); [a-32P]UTP (800 Ci/mmol) and [a;tr> S]UTP (1000 Ci/mmol) were obtained from Amersham (Aylesbury, Buckinghamshire, United Kingdom); restriction enzymes were purchased from Boehringer (Mannheim, Germany). Animals Immature (23-day-old) female rats (Sprague-Dawley) were obtained from Alab Laboratory Tjanst AB (Stockholm, Sweden) and fed chow and water ad libitum. A 14-h light, 10-h dark cycle was maintained, with the light cycle initiated at 0600 h. The rats received a single sc injection of 20 IU PMSG and, 48 h later, an ovulatory dose of hCG (10 IU). Ovulation took place between 12-14 h after hCG injection. Animals were killed at different intervals after hormone treatment, and the ovaries or isolated granulosa and thecal-interstitial cells were collected and immediately frozen in liquid nitrogen for further analysis. The ovaries for in situ analysis were frozen in 2-methylbutane at -70 C. Preparation of ovarian cells Granulosa and thecal-interstitial cells were prepared as previously described (15, 16). Briefly, the granulosa cells were released into medium by selectively puncturing the large follicles and collected by centrifugation at 1500 rpm for 5 min. The remaining ovary remnants were further punctured, washed, and digested briefly in 0.08% collagenase for 5 min to remove the remaining granulosa cells. Thecal-interstitial cells were dispersed by further incubating the tissue in 0.4% collagenase for 1 h. The cells were collected by centrifugation, and viability

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was determined with a hemacytometer after staining the cells with trypan blue. RNA preparation and analysis Total RNA from granulosa and thecal-interstitial cells was prepared using the Nonidet P-40 method (17). For Northern blot analysis, samples containing 10 ng (microgram) total RNA were fractionated on 1% agarose gels in the presence of 2.2 M formaldehyde and transferred to nitrocellulose filters, as previously described (18). After baking at 80 C for 2 h under vacuum, filters were prehybridized in 50% formamide, 5 X SSC (1 X SSC = 0.15 M NaCl 0.015 M Na Citrate), 8 x Denhardt's solution (1.6 mg/ml Ficoll, 1.6 mg/ml polyvinypyrolidone, and 1.6 mg/ml of BSA), 0.1% sodium dodecyl sulfate (SDS), 10 mM EDTA, 25 mM Tris-Cl (pH 7.0), 250 Mg/ml heat-denatured herring sperm DNA, and 250 Mg/ml yeast tRNA at 62 C for 2 h. The hybridization was performed in the same solution containing 2 X 106 cpm/ml probe for 16 h at 64 C. The filters were washed once for 15 min in 2 x SSC, 0.1% SDS at room temperature, followed by two washes in 0.1 x SSC containing 0.1% SDS at 66 C for about 40 min. Hybridizations using 0actin probes were performed at 42 C, as previously described (19). The relative abundance of specific transcripts in different preparations was analyzed by densitometric scanning of autoradiographic films and normalized against the corresponding relative amount of /3-actin mRNA. Synthesis of RNA and DNA probes A 408-basepair (bp) DNA fragment of rat LH receptor cDNA corresponding to the region coding for the extracellular domain of LH receptor was subcloned in the pGEM 4Z vector and linearized by Bglll or Apal to generate the templates for antisense or sense RNA probes, respectively (13). To generate a probe complementary to the tissue-type plasminogen activator (tPA) mRNA, a plasmid containing a 400-bp EcoRl fragment of rat tPA cDNA clone X15 subcloned in pGEMl (20) was linearized with Hindlll. The probes were labeled with [32P]- or [35S]UTP for Northern or in situ hybridization using the Riboprobe in vitro transcription system (Promega). T7 RNA polymerase was used for synthesis of both LH receptor and tPA antisense RNA probes, while SP6 RNA polymerase was used for the sense strand probes. The specific activity of the probes varied between 2-5 X 108 cpm//Lig RNA. The single stranded j8-actin DNA probe of 250 bp was prepared by primer extension, as previously described (19). In situ hybridization Frozen ovaries were oriented with respect to the attached oviduct, and 10-/*m sections were cut on a Reichert-Jung cryostat and mounted on microscope slides coated with poly-L-lysine (21). The sections were fixed in 4% paraformaldehyde for 5 min and stored in 70% ethanol until further analysis. The hybridizations were performed in 50% formamide, 2 X SSC, 20% dextran sulfate, 10 mM dithiothreitol, 1 mg/ml yeast tRNA, 1 mg/ml herring sperm DNA, 2 mg/ml RNase-free BSA (22), and approximately 3 X 107 cpm/ml 35S-labeled probe at 50 C overnight. In each experiment, the sections from all animals of all groups were hybridized at the same time with

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the same batch of probes. The slides were washed in 2 X SSC50% formamide for 30 min and then subjected to 1 Mg/ml RNase-A and -Tl digestion at 37 C for 30 min. After RNase treatment, slides were rinsed in 2 x SSC-50% formamide for 30 min at room temperature and dehydrated sequentially in 70%, 80%, and 90% ethanol. The sections were dipped in autoradiographic emulsion (Kodak NTB-2, Eastman Kodak, Rochester, NY) and exposed for 12-18 days at 4 C before they were developed and stained with eosin or eosin and hematoxylin for further analysis by darkfield and light microscopy, respectively. To monitor background levels and the specificity of hybridization, the sense strand of the LH receptor RNA probe was included in each experiment, and some adjacent sections were hybridized with an antisense tPA RNA probe. Photographs were taken with a Zeiss camera on a Zeiss Axiophot photomicroscope (Carl Zeiss, Oberkochen, Germany) or with a Nikon F-301 camera on a Leitz Dialux 20 microscope (Leitz, Rockleigh, NJ) at magnifications of X6.25-32.

A)

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Granulosa cells C

P

3

6

12

Theca-interstitial cells 2

4

C

P

3

6

12

24

28S

18S

Results Northern blot analysis of LH receptor transcripts in granulosa and thecal-interstitial cells during gonadotropin stimulation of ovarian development PMSG increased the [125I]hCG-binding capacity as well as LH receptor mRNA levels in the whole ovary, while an ovulatory dose of hCG decreased these parameters in PMSG-primed rats (6-9, 13). To study the gonadotropin regulation of LH receptor mRNA in different ovarian cell types, we analyzed the LH receptor mRNA levels in granulosa and thecal-interstitial cells during the periovulatory period. As shown in Fig. 1, the level of LH receptor mRNA was undetectable in granulosa cells and very low in thecal-interstitial cells of untreated ovaries (lane C). As reported previously for the whole ovary (13, 22), PMSG treatment for 48 h significantly enhanced the synthesis of all four species of LH receptor mRNA in both cell types (lane P). However, the most dramatic induction took place in granulosa cells, with a smaller increase in thecal-interstitial cells. After hCG treatment, the levels of LH receptor mRNA were down-regulated in both granulosa and thecal-interstitial cells in a time-dependent manner, with the lowest levels reached just before ovulation (12 h post-hCG treatment). After ovulation (24 h after hCG injection), the LH receptor mRNA level was increased in granulosa cells, although the relative level was lower than that in granulosa cells obtained from large maturing follicles after PMSG treatment. Localization of LH receptor mRNA in different ovarian cell types of immature rats and during PMSG-stimulated follicle growth Previous studies have demonstrated that the LH-binding capacity of ovarian cells varies with ovarian cell type

PMSG+hCG

FIG. 1. Northern blot analysis of LH receptor mRNA expressed in granulosa and thecal-interstitial cells during gonadotropin-induced follicle development, ovulation, and luteinization. Immature rats (23-24 days old) were treated with 20 IU PMSG, followed by an ovulatory dose of hCG (10 IU) 48 h later. Granulosa and thecal-interstitial cells were obtained from ovaries, and total RNA was prepared as described in Materials and Methods. Ten micrograms of total RNA were separated on 1% agarose gel containing 2.2 M formaldehyde and transferred to nitrocellulose filters. The filters were hybridized with a 32P-labeled LH receptor antisense RNA probe and /3-actin DNA probe before exposure to x-ray films. The migrations of the 28S and 18S ribosomal RNA are indicated on the left of the gel. A, A representative autoradiography of LH receptor mRNA levels of both granulosa and thecalinterstitial cells collected at different time points during gonadotropininduced follicle differentiation and ovulation. C and P stand for control and PMSG-treated rats, respectively; 3, 6,12, and 24 represent time in hours after hCG treatment. B, Relative amounts of LH receptor mRNA at each time point, estimated by densitometric scanning of autoradiographs and normalized against the relative amounts of /3-actin mRNA in the corresponding samples.

and among different subpopulations of granulosa cells in a given follicle (2, 11, 12). To investigate whether this uneven distribution of LH-binding capacity of ovarian cells correlates with their capacity to express LH receptor mRNA, we used the in situ hybridization technique to examine the distribution of LH receptor mRNA expression in the rat ovary during gonadotropin-induced follicle growth, differentiation, and ovulation. Ovarian sections were taken from untreated rats or from rats at different times after hormone treatment, and hybridized with a 35S-labeled LH receptor RNA probe. As controls for background levels and specificity, adjacent sections were also hybridized with either a RNA

LH mRNA EXPRESSION DURING OVULATION

probe coding for the sense strand of LH receptor or an antisense tPA RNA probe (20). A summary of the results described below is presented in Table 1 and Fig. 2. The ovaries of untreated 23-day-old rats were small and compact, but contained several early tertiary follicles and many primordial, primary, and secondary follicles (Fig. 2A). As indicated in Fig. 2B, LH receptor mRNA in these ovaries was exclusively expressed in interstitial cells, including theca interna, and the level of LH receptor transcript was below the detection limit in granulosa cells, cumulus cells, and oocytes. The intensity of LH receptor mRNA signals was similar in thecal cells of both secondary follicles, which have no antral cavity, and tertiary follicles, which contain a small antrum (Fig. 2B). Forty-eight hours after treatment with PMSG, the ovary had increased considerably in size and contained many large healthy tertiary follicles (Fig. 2D). In these ovaries, the expression of LH receptor mRNA was largely enhanced. The interstitial cells, including thecal cells, showed somewhat higher levels of LH receptor mRNA than before PMSG treatment. In addition, several large tertiary follicles displayed abundant amounts of LH receptor message (Fig. 2, E and F). In the tertiary follicles, the abundance of LH receptor mRNA varied among the subpopulations of granulosa •cells. The mural granulosa cells located next to the basement membrane exhibited the highest level of LH receptor mRNA. The message level gradually decreased in granulosa cells located closer to the antrum. The cumulus cells surrounding the oocyte and the oocyte itself contained undetectable levels of LH receptor mRNA (Fig. 2, E and F). Primordial follicles as well as oocytes and granulosa cells of primary, secondary, and small tertiary follicles in these ovaries also lacked deTABLE 1. Semiquantitative comparison of LH receptor mRNA levels in developing follicles Small

Medium

follicles follicles (150-200 Mm ) (300-400 Mm)

Antral GC Mural GC Thecal cells Interstitial cells Cumulus cells Oocyte

_ —

Preovulatory follicles (>500 Mm)

-hCG

+hCG (12 h)

++

++

+ +++ ++ ++

+++++ +++ +++

+ +

-

-

-

-

-

-

-

++

Twelve to 16 ovarian sections from each animal were hybridized and analyzed under light- and darkfield illumination. The abundance of LH receptor mRNA in different cell types was estimated by counting the number of silver grains on 3-4 sections from each group on a scale of 1-5. The following rating was observed, and the number represents the counts of silver grains on 1 Mm2. —, Same as background; +, 10 silver grains; ++, 15-20 grains; +++, 20-40 grains; +++++, 60-100 grains. GC, Granulosa cells.

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tectable amounts of LH receptor mRNA. The stratified pattern of LH receptor mRNA levels among different populations of granulosa cells of PMSG-treated ovaries was most obvious in medium-sized follicles that were undergoing maturation (Fig. 2F) and was less evident in fully grown follicles, where LH receptor mRNA was more uniformly expressed among the subpopulations of granulosa cells (Fig. 2E). In contrast to large tertiary follicles of PMSG-treated rats, in which LH receptor mRNA was particularly abundant in granulosa cells, the granulosa cells of small follicles lacked detectable transcript levels. In these follicles, LH receptor expression was only observed in thecal-interstitial cells, and the density of the hybridization to both thecal and interstitial cells was rather uniform (Fig. 2E). To exclude the possibility that the observed differences in LH receptor mRNA levels among different subpopulations of granulosa cells and in oocytes was an artifact of the hybridization procedure, an section adjacent to that shown in Fig. 2F was hybridized with a probe complementary to tPA mRNA. As shown in Fig. 2C, tPA mRNA revealed a different distribution than LH receptor mRNA. While tPA mRNA was undetectable in granulosa cells of these PMSG-treated rats, higher levels were detected in thecal-interstitial cells and oocytes. Localization of LH receptor mRNA in ovarian cells during hCG induction of ouulation and early luteinization As shown in Fig. 1, a time-dependent down-regulation of the LH receptor transcripts took place when PMSGprimed rats were injected with an ovulatory dose of hCG. Likewise, in situ hybridization clearly revealed a reduction in LH receptor mRNA levels in both granulosa cells and thecal-interstitial cells of preovulatory follicles. By 6 h after the hCG injection, the ovary had increased further in size, with multiple large nonatretic follicles and obvious edema in the interstitial tissue (Fig. 2G). As shown in Fig. 2, H and I, a significant reduction of LH receptor mRNA can be observed in these ovaries. The LH receptor mRNA signals had distinctly declined in the membrane granulosa of large follicles destined to ovulate, and the signals in the thecal cells were also weaker than before hCG injection. Cumulus cells and oocytes still lacked detectable amounts of LH receptor mRNA. Just before ovulation (12 h after hCG treatment), about 10 preovulatory follicles with a diameter of 1 mm or more were found for each ovary, partly protruding above the ovarian surface (Fig. 2J). The edema was still prominent, particularly in the central part of the ovary. As shown in Fig. 2, K and L, the LH receptor mRNA had decreased to low levels in these ovaries and was mainly localized to thecal and interstitial cells.

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v* «-

i:

R

FIG. 2. Localization of LH receptor mRNA in the rat ovary during gonadotropin-induced follicle development, ovulation, and luteinization. Ovaries from immature rats treated with PMSG and hCG, as described in Fig. 1, were removed at different times after gonadotropin treatment.

LH mRNA EXPRESSION DURING OVULATION

Localization of LH receptor mRNA in corpus luteum and nonouulated follicles Twenty-four hours after hCG injection, when ovulation was completed, young corpora lutea with a small antrum were found (Fig. 2M). As shown in Fig. 2, N and 0, these newly formed corpora lutea expressed low levels of LH receptor mRNA. However, the relative density of the LH receptor mRNA signal in the young developing corpora lutea was lower than that in medium-sized healthy tertiary follicles at the same time point (Fig. 2N) and was much lower compared to that in large tertiary follicles in ovaries taken 48 h after PMSG treatment (Fig. 2E). By 3 days after hCG injection, the corpora lutea were well vascularized and contained large luteal cells surrounding a central cavity infiltrated by fibroblasts (Fig. 2P). As shown in Fig. 2Q, the LH receptor mRNA was abundant in the corpora lutea, with less intense but obvious signals in thecal and interstitial cells. These ovaries contained many atretic tertiary follicles without any LH receptor mRNA signals in the granulosa layer. Adjacent sections hybridized with the sense strand of the LH receptor probe showed no significant signals in the corpora lutea or elsewhere (Fig. 2R).

Discussion Under physiological conditions, the appearance of LHbinding sites on ovarian cells is restricted to different follicles of given stages, as shown by autoradiographic studies (2, 11, 12). To further understand the molecular mechanisms regulating the localization of the LH receptor, we have examined the cell-specific expression of LH receptor mRNA in the ovary in rats. Our results demonstrate that the expression of ovarian LH receptor mRNA is up- and down-regulated by gonadotropins during follicle development, with the most dramatic changes taking place in granulosa cells. PMSG enhanced LH receptor expression, particularly in granulosa cells of large tertiary follicles, while an ovulatory dose of hCG dramatically decreased LH receptor mRNA levels to reach the lowest levels just before ovulation. After the transformation of ovulated follicles into corpora lutea,

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the expression of LH receptor mRNA was increased again. Our studies demonstrate that gonadotropin regulation of LH receptor binding in different ovarian cell types correlates with similar changes in LH receptor mRNA levels, suggesting that changes in LH receptor content during follicle growth, ovulation, and luteinization are due to changes in LH receptor gene transcription. In untreated immature rats, the granulosa cells in both preantral follicles and early tertiary follicles contained negligible levels of LH receptor mRNA. This is consistent with previous studies which showed that granulosa cells from this stage of follicles do not bind [125I]hCG either in vitro or in vivo (6, 7,11). In thecal cells of small follicles, however, LH receptor mRNA was already expressed, which may be essential for initiating the growth of these small follicles by providing them with the potential of LH-mediated androgen biosynthesis (23-26). Estrogens, in turn, interact synergistically with FSH to stimulate the proliferation and differentiation of the granulosa cells (2). After PMSG induction of follicle growth, the expression of LH receptor mRNA was strongly induced in granulosa cells and, to a lesser extent, increased in thecal cells. During follicle maturation, the synthesis of LH receptor mRNA was first detected in mural granulosa cells immediately next to the basement membrane and then gradually extended to the antral granulosa cells close to the antrum. Forty-eight hours after PMSG treatment, LH receptor was dominantly expressed in large healthy tertiary follicles, and the highest level of LH receptor mRNA was found in mural granulosa cells. Much lower expression of LH receptor was found in the granulosa cells close to the antrum, suggesting that mural and antral granulosa cells may respond differently to LH. It is possible that specific cellular microenviroments play an important role in determining the ability of granulosa cells to express LH receptor. Antral granulosa cells may express less LH receptor due to the lack of direct interaction with thecal cells or the influence of factors present in follicular fluid (27, 28), while mural granulosa cells may synergize with thecal cells to induce higher expression of LH receptor. Throughout the entire

A series of 10-jtrn sections was hybridized with 35S-labeled LH receptor antisense or sense RNA probes or with a tPA antisense RNA probe. After being exposed to the emulsion, the sections were developed and stained with eosin and analyzed by darkfield microscopy. The first vertical row shows overview light micrographs of ovaries stained with eosin, and the second and third rows show darkfield micrographs from the same ovaries at higher magnification after hybridization to specific probes. All sections in the second and third rows were hybridized to an antisense LH receptor probe, except C, which was hybridized to an antisense tPA probe, and R, which is the section adjacent to Q hybridized with a LH receptor sense probe. The red and blue horizontal bars represent 500 and 200 ^im, respectively. The hybridization signal appears in white. A and B, Sections obtained from untreated immature rat ovary containing primary and secondary follicles. C, D, E, and F, Sections of ovaries treated with PMSG for 48 h (micrograph C is the section adjacent to F hybridized to an antisense tPA probe); G, H, and I, sections from ovary treated with PMSG, followed by hCG for 6 h; J, K, and L, sections from ovary just before ovulation, 12 h after hCG treatment; M, N, and O, sections from an ovary obtained 24 h after hCG treatment. In micrograph N, note a young corpus luteum (left) and a medium-sized tertiary follicle {right); the expression of LH receptor mRNA is higher in the tertiary follicle. P, Q, and R, Sections obtained 3 days after hCG treatment, showing corpora lutea.

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process of gonadotropin-induced follicle growth and luteinization, the expression of LH receptor mRNA in both oocytes and cumulus cells was at background levels. Although it has been reported that cumulus cells respond to gonadotropins and contain LH- as well as FSH-binding sites (29-31), we were unable to detect LH receptor mRNA in these cells by in situ hybridization. This negative result may due to the sensitivity of the technique, since cumulus cells contain only one ninth as many hCGbinding sites as granulosa cells (30). The difference in LH receptor mRNA expression in different subpopulations of granulosa cells and cumulus cells is also reflected in the appearance of the LH/CG-binding sites observed (2, 11, 12). The present work, therefore, extends to the molecular level earlier autoradiographic and functional studies that revealed different functional properties of these cells, such as the capacity to synthesize steroids and tPA (32) (Peng, X.-R., A. J. W. Hsueh, and T. Ny, in preparation). The mechanism responsible for the initiation of LH receptor mRNA synthesis in thecal cells is not understood, but LH receptor expression was further increased after PMSG treatment. In contrast to that in granulosa cells, the distribution of LH receptor mRNA was more uniform, and the levels of LH receptor mRNA varied only slightly between the thecal cells of small and large follicles. Further development of follicles stimulated by hCG led to the decrease in LH receptor mRNA synthesis. Likewise, in large follicles found in the ovaries 6 h after hCG injection, the signal intensity was much less than that of large tertiary follicles of PMSG-primed ovaries. The LH mRNA levels continued to decrease and were markedly reduced just before ovulation (12 h after hCG injection), particularly in granulosa cells. At this stage, low levels of LH receptor mRNA expression were observed only in thecal cells of large ovulating follicles (J mm in size). This is consistent with previous reports that the number of LH-binding sites in this type of follicle is very low (11, 12). The reduction of LH receptor mRNA after hCG treatment suggests that the marked decline in LH/CG-binding sites is mainly due to a decrease in LH receptor synthesis (13). During the entire process of follicle growth and maturation, LH receptor mRNA levels remained below the level of detection in oocytes, suggesting that the induction of oocyte maturation does not require direct interaction of the ovulatory hormone with the oocyte itself. Rather, the triggering of oocyte maturation may be mediated by permeable factors through gap junctions found between the granulosa and cumulus cells (33, 34). In contrast, significant tPA mRNA levels were found in the oocytes, which is consistent with previous reports showing that tPA activity, antigen, and mRNA are present in

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oocytes (35, 36). Twenty-four hours after hCG treatment, the ovulated follicles underwent luteinization. This was accompanied by the reinitiation of LH receptor gene expression, which gradually increased to very high levels when corpora lutea were formed. The mechanism controlling this process is unknown. It is generally believed that gonadotropins (PMSG and hCG) mediate their effect through cAMP and the protein kinase-A pathway and that most of the effect is achieved by activating gene transcription (2, 37). The difference in the response between thecal and granulosa cells and the fact that gonadotropins can both increase and decrease the level of LH receptor mRNA suggests that complex mechanisms are involved in the regulation of LH receptor gene expression. Other ovarian gene products, such as cytochrome P450i7a aromatase cytochrome P450 and inhibin-a, are also up- and down-regulated by gonadotropins during follicle maturation, ovulation, and luteinization (38-40). Recently, a transcription factor, cAMP-responsive element modulator, that negatively modulates cAMP-induced transcription was isolated (41). It is possible that cAMP-responsive element modulator or similar factors are involved in the regulation of LH receptor and other ovarian proteins that are up- and down-regulated by gonadotropins. In summary, the present results have revealed that LH receptor gene expression in the ovary is regulated by gonadotropins in a cell type- and developmental stagespecific manner. However, the mechanism by which gonadotropin and cAMP can mediate both up- and downregulation of LH receptor gene expression in a tissuespecific manner remains to be investigated.

Acknowledgment We thank Dr. Paschalis Sideras for the kind advice and help in the performance of in situ hybridization.

References 1. Erickson G F 1986 An analysis of follicle development and ovum maturation. Semin Reprod Endocrinol 4:233-254 2. Hsueh AJW, Adashi EY, Jones PBC, Welsh TH 1984 Hormonal regulation of the differentiation of cultured ovarian granulosa cells. Endocr Rev 5:76-127 3. Richards JS, Jahnsen T, Hedin L, Lifka J, Ratoosh S, Durica JM, Goldring NB 1987 Ovarian follicular developments: from physiology to molecular biology. Recent Prog Horm Res 43:231-270 4. Hunzicker-Dunn M, Birnbaumer L 1985 The involvement of adenylyl cyclase and cyclic AMP-dependent protein kinase in luteinizing hormone actions. In: Ascoli M (ed) Luteinizing Hormone Action and Receptors. CRC Press, Boca Raton, pp 57-134 5. Segaloff DL, Sprengel R, Nikolics K, Ascoli M 1990 Structure of the lutropin/choriogonadotropin receptor. Recent Prog Horm Res 46:261-301 6. Nimrod A, Tsafriri A, Lindner HR 1977 In vitro induction of binding sites for hCG in rat granulosa cells by FSH. Nature 267:632-633 7. Erickson GF, Wang C, Hsueh AJW 1979 FSH induction of func-

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8.

9. 10.

11. 12. 13.

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15. 16. 17. 18. 19.

20. 21.

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