THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1987 by The American Society of Biological Chemists, Ine

Vol. 262, No. 5, Issue of Februery 15, pp. 2339-2344 1987 Printed in f.f.S.A.

Hormonal Regulation of Tissue Plasminogen Activator Secretion and mRNA Levels in Rat Granulosa Cells*

(Received for publication, May 23,1986)

Marcia L. O’Connell$, Rita Caniparis, and Sidney StricklandST From the $Department of Pharmacological Sciences, State University of New York, Stony Brook, New York 11794-8651 and the Ilstituto di I s ~ o ~ ~ ~ ed ~ m b r i o l o g ~ Generule, University of Rome, Via A. Scarpa 14, 0016i Rome, Italy

Granulosa cells from immature rats produce tissue plasminogen activator (tPA) in response to follicle stimulating hormone (FSH) or luteinizing hormone (LH) both in vitro and in vivo. We have used the in vitro system to investigate the level at which the hor- monal induction of tPA is regulated. Within 12 h fol- lowing FSH addition, a dramatic but transient increase in tPA secretion occurs for by 24 h secretion returns to basal levels. This pattern of enzyme induction is similar with LH, but the onset of the increase is de- layed. When steady-state tPA mRNA levels are exam- ined after hormone treatment, the results mirror those obtained if one measures enzyme activity; a large in- crease in tPA mRNA followed by a decrease to basal levels is observed with both hormones, and the lag in induction by LH is also apparent. These results dem- onstrate that the regulation of tPA activity by gonad- otropins occurs at the level of the steady-state concen- tration of the mRNA. In the presence of cycloheximide, the induction of tPA mRNA by FSH or LH is not greatly affected, indicating that this phase of the response to gonadotropins does not require the synthesis of new protein. However, the decrease in tPA mRNA levels observed 24 h after FSH treatment is affected by cy- cloheximide, in that the drug delays the reduction in mRNA levels seen with hormone alone.

Plasminogen activator is produced in the immature rat ovary in response to injection of either follicle stimulating hormone (FSH)‘ or luteinizing hormone (LH) and in the mature cycling rat ovary soon after the endogenous gonado- tropin surge that precedes ovulation (Beers et al., 1975; Strick- land and Beers, 1976). Two distinct ovarian cell types, gran- ulosa cells and thecal cells, are responsible for the majority of the plasminogen activator production. Each of these cell types secretes a different form of the enzyme in response to hor- mone treatment, with granulosa cells producing mainly tissue- type plasminogen activator (tPA) and thecal cells producing the urokinase type (Canipari and Strickland, 1985; Ny et al., 1985). _ . _ _ ~

* This work was supported by American Cancer Society Grant BC- 525, National Institutes of Health Grant HD17875, NATO Collabo- rative Research Grant RG.85/0456, and American Heart Association Grant 84 1242. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore he hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

11 Established Investigator of the American Heart Association. ’ The abhreviations used are: FSH, follic~e-stimulating hormone;

LH, luteinizing hormone; PMSG, pregnant mare Serum gonadotropin; PA, plasminogen activator; tPA, tissue-type plasminogen activator; DME medium, Dulhecco’s modified Eagle’s medium; SDS, sodium dodecyl sulfate.

When granulosa cells are cultured in vitro in the presence of either FSH or LH, a dramatic increase in tPA is detected in the medium (Strickland and Beers, 1976; Liu et al., 1981; Martinat and Combarnous, 1983; Wang and Leung, 1983; Too et al., 1984; Canipari and Strickland, 1985; Reich et al., 1985). Little is known about the molecular mechanism for this induction. One possibility was that FSH and LH were reduc- ing the activity in the medium of a tPA inhibitor, either by decreasing its production or by attenuating its activity. In this regard, Protein C has been shown to increase the fibrinolytic activity in endothelial cell cultures via reduction of inhibitor activity (Sakata et aL, 1985), and it has been reported that FSH-primed granulosa cells do secrete a fibrinolytic inhibitor (Ny et aL, 1985). A second possibility was that the hormones were actually stimulating tPA production by modulating the levels of its mRNA, as is seen in human melanoma cells upon the addition of the tumor promoter phorbol 12-myristate 13- acetate (Opdenakker et al., 1983). Similarly, phorbol 12-my- ristate 13-acetate and calcitonin increase the levels of the urokinase-type mRNA in mouse MSV-3T3 cells and porcine kidney cells, respectively (Belin et al., 1984; Nagamine et ai., 1983).

The regulation of granulosa tPA secretion is of additional interest since after hormone treatment i n vitro, activity in the medium increases linearly for about 12 h but then is main- tained at a constant level with no further increase. Negative feedback inhibition of tPA expression has been reported to occur in fibroblasts (Kadouri and Bohak, 1983) and could explain the above observation if high extracellular levels could somehow suppress secretion. It is also possible that in addition to stimulating enzyme production, FSH and LH induce an inhibitor of the enzyme whose effects are not seen for 12-16 h. The inhibition of tPA activity in HTC hepatoma cells by glucoco~icoids has been shown to occur via induction of inhibitor secretion, which diminishes the PA activity in the medium (Cwikel et aE., 1984). Finally, the maintenance of a constant level of enzyme activity could be the result of a decrease in tPA production due to phenomena such as cell differentiation or desensitization to hormone (Degen et at., 1985).

Although both FSH and LH induce tPA production, details of their modes of action appear to be different. In uitro a lag is detected in the response of granulosa cells to LH, but not in the response to FSH (Canipari and Strickland, 1986). Recent evidence suggests that since essentially all of the PMSG-primed granulosa cells possess FSH receptors, FSH is capable of inducing the entire population directly. With LH, only a subset of the cells has sufficient LH receptors to be stimulated directly. It is the prostaglandins produced by these directly stimulated cells that appear to stimulate the remain- ing majority of the cells (Canipari and Strickland, 1986).

This paper demonstrates that the gonadotropins induce a

2339

2340 Hormonal Regulation of tPA mRNA in Granulosa Cells

rapid and dramatic increase in the steady-state levels of tPA mRNA but that this increase is transient. Thus, the secretion of tPA into the medium parallels the levels of its mRNA.

EXPERIMENTAL PROCEDURES

Materials

Rats of the Sprague-Dawley strain were obtained from Charles River Breeding Laboratories, Inc. Rat FSH (rFSH-1-6) and LH (rLH- 1-6) were obtained from the National Hormone and Pituitary Program of the National Institutes of Health. PMSG was purchased from Organon Diagnostics. Nitrocellulose was purchased from Schleicher and Schuell. The rat tissue-type plasminogen activator cDNA clone was a generous gift of Drs. E. Waller (The Rockefeller University, New York) and E. Reich (State University of New York at Stony Brook).

Methods

Preparation of Granulosa Cell Cultures-Female rats, 26 days old, were injected subcutaneously with 5 IU of PMSG in 0.02 ml of 0.9% NaCl, killed by CO, asphyxiation 48 h later, and the ovaries were then removed.

Granulosa cell cultures were prepared by a modification of the method of Crisp and Denys (1975) as described in detail (Strickland and Beers, 1976). Briefly, the contents of individual follicles were expressed into medium, and the granulosa cells were collected and cultured at a cell density of 1.2 X 107/100-mm dish in 10 ml of DME medium with 8% fetal bovine serum at 37 "C in a 7% COz atmosphere. Using this procedure the attached cells are 90% viable, and electron microscopic examination has previously revealed that approximately 90% have an ultrastructural morphology characteristic of granulosa cells (Beers et al., 1975).

For studies based on enzyme activity, the cells were cultured for the indicated period of time in the presence of 100 ng/ml FSH or LH. Harvest fluid was then collected and assayed for P A by incubating the samples with plasminogen and assaying the plasmin generated using a chromogenic substrate assay (Verheijen et al., 1982; Andrade- Gordon and Strickland, 1986).

Isolation of Total RNA from Granulosa Cells-RNA from granulosa cells grown in culture was isolated by combining a modified version of the guanidinium thiocyanate procedure developed by Chirgwin et al. (1979) and the CsCl method developed by Glisin et al. (1974). Briefly, after the indicated number of hours in culture the cells were washed two times with 0.9% NaCl. Cells were harvested by solubili- zation in 4 M guanidinium thiocyanate, 1 M P-mercaptoethanol (ap- proximately 1 ml of guanidinium mixture/3.0 X lo6 cells). This mixture was layered over a 2.2-ml cushion of 5.7 M CsCl, 0.1 M EDTA in SW 41 tubes. After centrifugation at 33,000 rpm for 24 h at 17 "C in an SW 41 fixed angle rotor, the guanidinium layer, the viscous DNA interface, and the CsCl layer were removed with suction to within 1 cm of the bottom of the tube. The remaining CsCl was decanted, and the bottom 2 cm of the tube cut off and placed on ice. The RNA pellet was dissolved in 0.5 ml of H,O for 20 min and transferred to a polypropylene tube. The bottom of the centrifuge tube was washed with an additional 0.5 ml of H20, which was transferred to the polypropylene tube. The salt concentration was adjusted to 0.3 M using 3.0 M sodium acetate, and the RNA was precipitated with 2 volumes of EtOH overnight at -20 'C or for 1 h at -70 "C. The final RNA pellet was dissolved in 1 ml of H,O.

Northern Analysis-Hybridization of immobilized RNA to radio- labeled DNA probes was performed according to Thomas (1980). Briefly, 15 pg of total RNA was separated by electrophoresis on a 0.8% agarose gel containing 6% formaldehyde. The gel was then washed twice in 10 X SSC (0.15 M NaCI, 0.015 M sodium citrate, pH 7.0) for 10 min at room temperature and the RNA transferred to nitrocellulose by capillary blotting with 20 X SSC for 20 h. Following transfer, the nitrocellulose filter was soaked in 10 x SSC for 10 min, baked in uacm for 2 h at 80 "C, and placed in a plastic bag with 1 ml/cm prehybridization buffer (6 X SSPE (0.18 M NaCl, 0.010 M NaP04, pH 7.7,O.OOl M EDTA), 5 X Denhardt's solution (0.2 mg/ml Ficoll, 0.2 mg/ml polyvinylpyrrolidone, 0.2 mg/ml bovine serum al- bumin), 0.1% SDS, 0.1 mg/ml salmon sperm DNA, 50% formamide) for 4-24 hours at 42 "C. The filters were hybridized for 24 h at 42 "C in prehybridization buffer containing a rat tPA cDNA clone radiola- beled using the random priming method of Feinberg and Vogelstein (1983). The filters were washed at 65 "C for 30 min in approximately

300 ml of 2 X SSC, 0.1% SDS (twice), 1 X SSC, 0.1% SDS (once), 0.2 X SSC, 0.1% SDS (once), and exposed to XAR film. The filters were probed a second time with a random primed mouse 18 S ribosomal RNA probe (Arnheim and Kuehn, 1979) after having been stripped with 50 mM NaOH at room temperature for 30 min.

Determination of Protein Synthesis in Granulosa Cells-For exper- iments involving the use of cycloheximide, the level of protein syn- thesis in the presence and absence of the drug was determined by culturing small aliquots of the granulosa cells in the presence of [35S] methionine. Granulosa cells (2.5 X lo5) were cultured in 24-well dishes with or without 10 pg/ml cycloheximide in 300 p1 of methio- nine-free DME medium with 0.1% bovine serum albumin. After 30 min, 15 pCi/ml [3SS]methionine and 100 ng/ml FSH or LH were added to the cultures. After 3 h labeled proteins were trichloroacetic acid precipitated onto Whatman No. 3" paper and counted in scintillation fluid.

RESULTS

It has previously been shown that when rat granulosa cells are cultured in vitro in the presence of either FSH or LH, there is a dramatic increase in the extracellular levels of tissue plasminogen activator activity. After a lag period, the length of which depends on the hormone used for stimulation (Can- ipari and Strickland, 1986), this increase in extracellular tPA activity is linear and is maintained for 8-10 h. However, between 10 and 16 h after addition of hormone, the level of activity reaches a plateau and little further increase is ob- served (see Fig. 1). Both the increase in tPA activity and the constant level observed at longer culture times could have several explanations. For example, modulation of PA inhibitor levels could influence tPA activity in the medium. Further- more, it has been reported that secretion of tPA is subject to negative feedback inhibition (Kadouri and Bohak, 1983), and this mechanism could explain an initial increase followed by a plateau.

To gain more insight into the pattern of the induction mechanism, granulosa cells from PMSG-primed rats were plated at different densities and cultured for 24 h in the presence of 100 ng/ml FSH. Samples of conditioned medium were collected at various times after hormone addition and assayed for plasminogen activator. As shown in Fig. 1, the time profiles for accumulated enzyme activity are similar at all cell concentrations with respect to the time when the plateau is reached, but not with respect to the level of enzyme activity at the plateau. This latter phase is dependent on cell concentration, showing an increase with an increase in cell density. These results suggest that the concentration of the extracellular enzyme is not responsible for limiting further secretion of tPA.

The above observation implies that negative feedback in- hibition is not occurring. However, it does not answer the question of whether enzyme secretion was decreasing as a consequence of desensitization and/or cell differentiation or proceeding at the same rate with the extracellular activity reaching a steady-state due to degradation of the enzyme or an increase in inhibitor activity. PMSG-primed granulosa cells are known to produce an inhibitor; however, this activity is actually suppressed after addition of FSH or LH (Ny et al., 1985). Therefore, since secretion of an inhibitor cannot be responsible for these observations, we investigated changes in the rate of secretion of tPA at different culture times. Gran- ulosa cells were cultured in the presence of hormone, and at specific times during the 24-h period the medium was changed, the cells cultured for an additional 2 h, and the medium conditioned during these 2 h collected and assayed for tPA activity. Fig. 2 shows that after approximately 2 h with FSH and 4-6 h with LH there is a pronounced increase in the amount of tPA being secreted. The rate of production peaks at 8 h for FSH and 10 h for LH, after which there is a

Hormonal Regulation of tPA mRNA in Granulosa Cells 2341

0.7

CELL N ~ ~ B E ~ x 105

1.5 3c

1.0 t 0.5 o

0.5 E c m

0.4 l- a W

z u 4 m 0.3 a

m 0 ffl U

0.2

0.1

0 . o Y I I I I I 1 I I , I I I I I I I I I I I

2 4 6 8 10 12 14 16 18 20 22 24 HOURS

FIG. 1. Time course of accumulated tPA activity induced by FSH at different granulosa cell coneen- trations. Granulosa ceiis were plated at a density of 1.5,1.0, or 0.5 X 106 viable ceIls/7-mm well in 0.2 ml of DME medium supplemented with 8% FBS. Cells were cultured in the presence of 100 ng/ml FSH added at time 0. A t the indicated times, harvest fluid was collected and stored at -20 "C until assayed for plasminogen activator activity. The activity in the medium from cells cultured in the absence of hormone was the same at all cell concentrations and remained at basal levels throughout the 24-h period. The y axis values represent the amount of p-nitroaniline release by plasmin during a 4-h assay.

significant decrease in secretion for both hormones. The above results indicate that although FSH and LH induce secretion of tPA in granulosa cells, the induction is transient, and the decrease in production is most likely the result of desensitization and/or differentiation.

To investigate the level at which this transient induction is regulated, the influence of the gonadotropins on the steady- state levels of tPA mRNA was explored. Granulosa cells were cultured in the presence of either FSH or LH, and at various times after hormone addition, total RNA was extracted and examined for tPA mRNA accumulation. Fig. 3 illustrates that stimulation of rat granulosa cells in vitro with either FSH (100 ng/ml) or LH (100 ng/ml) results in the appearance of a 2.8-kilobase RNA species that hybridizes to a rat tPA probe, a size in agreement with that of the human tPA mRNA (Pennica et al., 1983). The steady-state level of the tPA mRNA reaches a maximum after 8-12 h in the presence of either FSH or LH (Fig. 3). However, the initial time course of induction by the two hormones differs; FSH induces a detectable increase by 2 h, while there is a lag in the response of the cells to LH of approximately 4 h. At later times the profiles for hormone responsiveness are more similar. Be- tween 12 and 16 h after either hormone treatment the levels

of tPA mRNA have decreased to approximately 30-50% of the maximal level, and 24 h after treatment with either hormone the mRNA levels decrease almost to basal levels (Fig. 3). There is no increase in the mRNA in the absence of hormone (data not shown). To determine if equivalent amounts of total RNA were present in each lane, the blots were hybridized a second time with a probe specific for 18 S rRNA. The two autoradiograms were scanned on a densitom- eter, and the values for the tPA mRNA were normalized to those of the rRNA species. These values are indicated as percent maximum stimulation and demonstrate an induction of tPA mRNA which is greater than 50-fold.'

To determine if protein synthesis were required for the maximal induction of tPA mRNA by these hormones, the protein synthesis inhibitor cycloheximide was used. The drug was added to the granulosa cell cultures 30 min before FSH or LH addition, and RNA was collected 8 h later. When cycloheximide was added to cultures which were then treated with FSH, the steady-state level of the tPA mRNA (Fig. 4) was approximately 70% the level seen with hormone alone,

Exact quantitation of the degree of induction is difficult to deter- mine due to the extremely low level of tPA mRNA in the unstimulat~ cells.

__

2342 Hormonal Regulation of tPA mRNA in Granulosa Cells

0.7

4 FSH

0.6

0.5

E c LD

0.4 l- a W u z $ 0.3 m a

0 cn

0.2

0 . 1

0.0 2-4 4-6 6-8 6-10 10-12 12-14 22-24

HOURS FIG. 2. Plasminogen activator activity measured at 2-h intervals for 24 h in vitro in the presence

of FSH or LH. Granulosa cells were plated at a density of 1.5 X 10’ viable cells/’l-mm well in 0.2 ml of DME medium supplemented with 8% FBS. Cells were cultured in the presence of 100 ng/ml FSH or 100 ng/ml LH added at time 0. At the indicated times the medium was changed and the conditioned medium collected 2 h later. The medium conditioned for 2 h was then assayed for plasminogen activator activity.

indicating that the induction is not greatly affected by the later and RNA extracted. As expected, the addition of cyclo- presence of the drug. The results were similar when cyclohex- heximide 4 h after hormone addition had little effect on the imide was added to cells cultured in the presence of LH (Fig. increase in the steady-state levels of tPA mRNA seen after 8 5 ) . In this case tPA mRNA was 61% of the fully induced level, h (Fig. 6). However, examination of the level of the mRNA indicating that under these conditions the drug does not have a dramatic effect on the ability of LH to stimulate the increase in tPA mRNA. Cycloheximide alone did not increase tPA mRNA levels (Fig. 4). To confirm the fact that protein syn- thesis was inhibited in each experiment, the incorporation of [35S]methionine into trichloroacetic acid-precipitable mate- rial was measured. In each case, protein synthesis after cyclo- heximide treatment was <2% of controls (data not shown). It should also be noted that based on cell morphology and the yield of RNA from the cultures, cells exposed to cycloheximide for 8 h were viable.

Sixteen hours after stimulation with FSH the level of tPA mRNA is less than 20% of the level of mRNA seen after 8 h (Fig. 3). To investigate if this decrease is affected by cyclo- heximide, the drug was added to the granulosa cell cultures 4 h3 after FSH addition. Cells were then collected 4 and 12 h

We initially attempted adding cycloheximide 30 min prior to FSH addition and collecting RNA 24 h later, but no RNA could be isolat.ed. An observation of the cells indicated that most had died. This necessitated adding the drug 4 h after FSH addition and collecting the cells 12 h later. Under these conditions the cells remained viable.

16 h after hormone addition shows that the expected 90% decrease in the levels of the message does not occur; rather the level of the mRNA is still approximately 90% of the fully induced level observed in the presence of cycloheximide (Fig. 6).

DISCUSSION

The experiments in this paper demonstrate that the regu- latory mechanism for control of tPA expression in granulosa cells is via modulation of the steady-state level of its mRNA. Two common mechanisms for the regulation of messenger RNA levels are consistent with the data. The hormones could be either stimulating transcription of the tPA gene or stabi- lizing tPA mRNA in the cell. Since stimulation of tPA secre- tion by both LH (Strickland and Beers, 1976) and FSH (Knecht, 1986) is inhibited by actinomycin D, which inhibits transcription, the former explanation seems most likely. Fur- ther experiments, with which one can measure transcription rates, will more clearly define how the message levels are being influenced.

The transient nature of the hormonal induction of tPA and

Hormonal Regulation of tPA mRNA in Granulosa Cells FSH LH

1.5 2 4 8 12 24 4 8 12 24 HRS

LH - - +

2343

c YC

0 8 49 100 51 7 14 62 100 28 O/o

FIG. 3. Time course of tPA mRNA levels following induc- tion by FSH or LH. Granulosa cells at a concentration of 1.2 X lo7 cells/lOO-mm dish in 10 ml of DME medium with 8% FBS were plated for the indicated number of hours with either FSH or LH at a concentration of 100 ng/ml. Control cultures were incubated for 1.5 h in the absence of hormone in order to recover viable cells that adhere to the plate. Cells were then collected and RNA extracted. Total cellular RNA (15 pg) was electrophoresed on an agarose gel, transferred to nitrocellulose, and the filter was probed with a random primed tPA cDNA clone, as described under “Experimental Proce- dures.” The percent stimulation was determined by first scanning the autoradiogram obtained from the above experiment on a microden- sitometer to determine relative amounts of tPA mRNA (the method, according to dilution experiments performed in our laboratory, gives an estimate of relative tPA mRNA levels (Laskey, 1980)). Second, the blot was stripped, hybridized with a probe for 18 S rRNA, and scanned a second time to estimate the amount of total RNA in each lane. The values obtained for the tPA peak were divided by the corresponding 18 S rRNA peak to normalize for variability in RNA yield, transfer efficiency, etc. The normalized values were then ex- pressed as percent of the maximal stimulation (%), with the maxi- mally stimulated sample arbitrarily assigned a value of 100%. Dis- crepancies between visual estimation and the normalized values (e.g. LH 8 and LH 12) can be accounted for by the rRNA values used for normalization.

- + + 1.5 8 8

CYC - - FSH 8 H R S

0 100 70 0 %

FIG. 4. Effect of cycloheximide on induction of tPA mRNA by FSH. Granulosa cells plated as in Fig. 3 were treated with or without cycloheximide (CYC) at a concentration of 100 pg/ml at time 0 for 30 min. FSH was then added to the cultures indicated at a concentration of 100 ng/ml. Eight hours after FSH addition cells were collected and RNA extracted. Total cellular RNA (15 pg) was electrophoresed on a denaturing agarose gel, transferred to nitrocel- lulose, and the filters were probed with a random primed tPA cDNA clone. (The apparent discrepancy in molecular weights in the FSH lanes appears to be due to the fact that the two RNA samples migrated slightly differently in the gel as evidenced by the ribosomal species, and not to a true difference in molecular weight.) The percent stimulation (%) normalized to an rRNA signal was determined by densitometer scanning (see the legend for Fig. 3).

its mRNA in granulosa cells in vitro is also characteristic of the production of the enzyme in vivo (Beers and Strickland, 1978); both display an initial increase followed by an equally dramatic decrease after approximately 12 h. One interpreta-

100 61

FIG. 5. Effect of cycloheximide on induction of tPA mRNA by LH. The experimental protocol was the same as in Fig. 4 but with 100 ng/ml LH and a cell density of 2.5 X lo7 cells/lOO-mrn dish. The high density was used to facilitate a rapid response to LH (Canipari and Strickland, 1986). The percent stimulation (%) normalized to an rRNA signal was determined by densitometer scanning (see the legend for Fig. 3).

8 8 16 16 H R S

+ C Y C - + -

100 61 18 55 O/o

FIG. 6. Effect of cycloheximide on tPA rRNA steady-state levels between 4 and 16 h after FSH addition. Granulosa cells were treated with 100 ng/ml FSH at time 0. Four hours after FSH addition, cycloheximide was or was not added as indicated at a concentration of 100 pg/ml. Eight and 16 h after FSH addition, cells were collected and RNA extracted, electrophoresed, transferred to nitrocellulose, and probed with the random primed tPA cDNA clone. The percent stimulation (%) normalized to an rRNA signal was determined by densitometer scanning (see the legend for Fig. 3).

tion for the decrease in tPA activity is that the cells have become refractory to FSH. This idea is consistent with the fact that soon after the gonadotropin surge in vivo, granulosa cells in ovulated follicles begin to differentiate into luteal cells, which involves events such as the initiation of steroid biosynthesis, desensitization to hormone (for a review, see Hsueh et al., 1984), and subsequent internalization of receptor hormone complexes (Lee and Takahashi, 1977; Schwa11 and Erickson, 1983). Furthermore, these events are also observed after in vitro gonadotropin treatment of preovulatory granu- losa cells (Amsterdam et al., 1979; Nimrod and Lamprecht, 1980). This desensitization to hormone would ensure that tPA enzyme activity was present only at the time of ovulation and that once the oocyte had escaped, secretion of the enzyme would cease.

Concerning the effects of cycloheximide on the induction of PA mRNA, examples of both dependence on (Nagamine et al., 1983) and independence of (Waller and Schleuning, 1985) protein synthesis have been previously observed. In the case of granulosa cells, most of the FSH- and LH-stimulated increase in tPA mRNA levels that occurs within the first 8 h does not require protein synthesis. However, in several exper-

2344 Hormonal Regulation of tPA mRNA in Granulosa Cells

iments the stimulation by LH was diminished by the inhibi- tion of protein synthesis when cells were incubated with the druc for 12 h, and cycloheximide reproducibly affected LH induction more than FSH induction (data not shown). Pre- vious work has suggested that these gonadotropins induce tPA secretion in two ways (Canipari and Strickland, 1986): directly via hormone receptors, and indirectly via prostaglan- dins, which are produced by the directly stimulated cells. Since many of the cells possess fewer LH receptors than FSH receptors, the indirect pathway is more evident with LH. Therefore, it is perhaps the extent of direct versus indirect stimulation that accounts for the effect we have observed of cycloheximide on induction by LH at later times. This inter- pretation would suggest that the direct induction by hormones does not require protein synthesis, but the indirect stimula- tion via prostaglandins does, perhaps to allow for the synthe- sis of enzymes required for prostaglandin production/secre- tion (Clark et al., 1978).

Cycloheximide also alters the pattern seen at later times after FSH induction such that the cells maintain a relatively high level of the tPA message. This observation indicates that either protein synthesis is required for the disappearance of the message or that some secondary effect of the drug is changing the normal response of the cells to the hormone. In particular, it is possible that cycloheximide is actually stabi- lizing the tPA mRNA, since cycloheximide inhibits protein synthesis by freezing ribosomes on mRNAs (Baliga et al., 1969), such that an mRNA being actively translated at the time when the drug is added can be “stabilized” in a complex with ribosomes (see Sive et al., 1984). Experiments with other protein synthesis inhibitors such as puromycin, which inhibits translation by a different mechanism, will have to be per- formed in order to distinguish between the possibilities. In addition, a determination of the stability of the mRNA in the presence and absence of hormone could provide insight into the normal mechanism for the decrease in the tPA mRNA levels.

The magnitude of the response of rat ovarian cells to the gonadotropins FSH and LH has now facilitated the investi- gation of not only the cell biology of plasminogen activator production, such as which cell types produce which enzymes, but also the molecular biology of its regulation. Our initial investigation of the molecular events that occur in granulosa cells has led to the realization that regulation of enzyme production occurs at the level of the steady-state concentra- tion of the tPA mRNA. Additional experiments should lead to a further clarification of the mechanism by which these hormones affect mRNA levels and ultimately to a better understanding of the physiological significance of these mo- lecular events.

REFERENCES Amsterdam, A., Nimrod, A., Lamprecht, S. A., Burstein, Y., and

Andrade-Gordon, P., and Strickland, S. (1986) Biochemistry 25, Lindner, H. R. (1979) Am. J. Physiol. 236, E129-El38

4033-4040

Arnheim, N., and Kuehn, M. (1979) J. Mol. Biol. 134,743-765 Baliga, B. S., Pronczuk, A. W., and Munro, H. N. (1969) J. Biol.

Chem. 244,4480-4489 Beers, W. H., and Strickland, S. (1978) in Novel Aspects of Reproduc-

tiue Physiology (Spilman, C. H., and Wilks, J. W., eds) pp. 13-35, Spectrum Publications, Inc., Jamaica, NY

Beers, W. H., Strickland, S., and Reich, E. (1975) Cell 6,387-394 Belin, D., Godeau, F., and Vassalli, J.-D. (1984) EMBO J. 3, 1901-

Canipari, R., and Strickland, S. (1985) J. Biol. Chem. 260, 5121-

Canipari, R., and Strickland, S. (1986) Endocrinology 118, 1652-

Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J., and Rutter, W.

Clark, M. R., Marsh, J. M., and LeMaire, W. J. (1978) J. Biol. Chem.

Crisp, T. M., and Denys, F. R. (1975) in Electron Microscopic Concepts of Secretion (Hess, M., ed) pp. 3-33, John Wiley and Sons, New York

Cwikel, B. J., Barouski-Miller, P. A., Coleman, P. L., and Gelehrter, T. D. (1984) J. Biol. Chem. 259 , 6847-6851

Degen, J. L., Estensen, R. D., Nagamine, Y., and Reich, E. (1985) J.

Feinberg, A. P., and Vogelstein, B. (1983) Anal. Biochem. 132,6-13 Gliiin, V., Crkvenjakov, R., and B p s , C. (1974) Biochemistry 13,

Hsueh, A. J. W., Adashi, E. Y., Jones, P. B. C., and Welsh, T. H., Jr.

Kadouri, A., and Bohak, Z. (1983) BiolTechnology 1,354-358 Knecht, M. (1986) Endocrinology 118, 348-353 Laskey, R. A. (1980) Methods Enzymol. 65, 363-371 Lee, C. Y., and Takahashi, H. (1977) Endocrinology 101,869-875 Liu, W.-K., Burleigh, D. B., and Ward, D. N. (1981) Mol. Cell. Endocr.

Martinat, N., and Cambarnous, Y. (1983) Endocrinology 113 , 433-

Nagamine, Y., Sudol, M., and Reich, E. (1983) Cell 3 2 , 1181-1190 Nimrod, A., and Lamprecht, S. A. (1980) Biochem. Biophys. Res.

Ny, T., Bjersing, L., Hsueh, A. J. W., and Loskutoff, D. J. (1985) Endocrinology 116 , 1666-1668

Opdenakker, G., Ashino-Fuse, H., Van Damme, J., Billiau, A., and De Somer, P. (1983) Eur. J. Biochem. 131,481-487

Pennica, D., Holmes, W. E., Kohr, W. J., Harkins, R. N., Vehar, G. A., Ward, C. A., Bennett, W. F., Yelverton, E., Seeburg, P. H., Heyneker, H. L., Goeddel, D. V., and Collen, D. (1983) Nature 301,214-221

Reich, R., Miskin, R., and Tsafriri, A. (1985) Endocrinology 116,

Sakata, Y., Curriden, S., Lawrence, D., Griffin, J. H., and Loskutoff,

Schwall, R. H., and Erickson, G. F. (1983) J. Biol. Chem. 258,3442-

Sive, H. L., Heintz, N., and Roeder, R. G. (1984) Mol. Cell. Biol. 4,

Strickland, S., and Beers, W. H. (1976) J. Biol. Chem. 2 5 1 , 5694-

Thomas, P. S. (1980) Proc. Natl. Acad. Sci. U. S. A. 77,5201-5205 Too, C. K. L., Bryant-Greenwood, G. D., and Greenwood, F. C. (1984)

Verheijen, J . H., Mullaart, E., Chang, G. T. G., Kluft, C., and

Waller, E. K., and Schleuning, W.-D. (1985) J. Biol. Chem. 260,

Wang, C., and h u n g , A. (1983) Endocrinology 112 , 1201-1207

1906

5125

1659

J. (1979) Biochemistry 18, 5294-5299

253,7757-7761

Biol. Chem. 260,12426-12433

2633-2637

(1984) Endocr. Reu. 5, 76-127

2 1,63-73

435

Commun. 92,905-911

516-521

D. J . (1985) Proc. Nutl. Acad. Sci. U. S. A. 82, 1121-1125

3445

2723-2734

5702

Endocrinology 115 , 1043-1050

Wijngaards, G. (1982) Thromb. Huemostasis 48, 266-269

6354-6360