Induction of prostaglandin H synthase in rat preovulatory follicles by gonadotropin-releasing hormone.

0013-7’227/92/1306-3512$03.00/O Endocrinology Copyright 0 1992 by The Endocrine

Vol. 130, No. 6 Society

Printed

in U.S.A.

Induction of Prostaglandin H Synthase in Rat Preovulatory Follicles by Gonadotropin-Releasing Hormone* WINONA

Y. L. WONG

Department

of

AND

JOANNE

S. RICHARDS

Cell Biology, Baylor College of Medicine,

Houston,

Texas 77030

ABSTRACT. Two distinct isoforms of prostaglandin (PG) endoperoxide synthase (PGS) have been identified in rat ovarian tissues: rPGSi (mol wt. 70,000-72,000) is induced by FSH and LH in preovulatory follicles, whereas the other isoform (mol wt, 69.000) is not. Induction of rPGSi is associated with LH-stimulated increases in PG biosynthesis obligatory for ovulation. Because GnRH, like LH, can also stimulate the synthesis of PGs and ovulation in the rat, this study was undertaken to determine which isoform of PGS might be induced by GnRH, in what cell type, and by what intracellular pathways. Results show that GnRH at relatively low concentrations (1O-8-1O-7 M) induced the same isoform of PGS (rPGSi) in the same cell type (preovulatory granulosa cells) and within the same 5- to 7-h time course as did LH. Unlike LH and FSH, GnRH did not cause a major increase in CAMP, nor did GnRH induce lutein-

ization. The effects of GnRH on rPGS, in preovulatory follicles were not mimicked by known activators of protein kinase-C (phorbol myristate acetate, bryostatin, diacyglycerol, and (+/bonomycin). Epidermal growth factor (but not basic fibroblast growth factor or platelet-derived growth factor), which activates a receptor-associated tyrosine kinase, caused a small increase in rPGSi. Genistein, a selective inhibitor of tyrosine kinases, blocked GnRH and LH induction of rPGSi. Taken together these results suggest that the mechanisms by which GnRH and LH selectively induce rPGS, in granulosa cells of preovulatory follicles before ovulation may converge at some step within a cellular tyrosine kinase cascade. Furthermore, the mechanisms responsible for inducing rPGS, are distinct from those required for cellular luteinization. (Endocrinology 130: 3512-3521, 1992)

T

dins (PGs) after GnRH stimulation of preovulatory follicles (13), the ability of a PG synthesis inhibitor (indomethacin) to block GnRH stimulation of ovulation (13), and the ability of GnRH to stimulate PG production by granulosa cells in vitro (16), PGs are presumed to mediate GnRH as well as LH stimulation of ovulation (17-21). The synthesis of PGs is dependent on the availability of arachadonic acid, the metabolic precursor for prostanoid biosynthesis, and the presence/activity of PG endoperoxide synthase (PGS), the rate-limiting enzyme converting arachadonic acid to the PGs, thromboxane and prostacyclin. Based on the isolation of highly homologous cDNA clones encoding ovine (22, 23), mouse (24), and human (25) PGS enzymes, it has been postulated that only one gene encodes one PGS enzyme (26). However, we have recently shown that the rat ovary contains two immunodistinct mol wt (M,) variants of PGS, each of which is expressed in specific cell types and differentially regulated by gonadotropic hormones (27). The PGS isoform (M,, 70,000-72,000; rPGSi), which is induced in granulosa cells of preovulatory follicles by FSH/LH/ forskolin (27-29), exhibits biochemical and structural features (30) distinct from those of the ovine, mouse, and human genes previously cloned (22-25), but is similar to PGS-related proteins for which cDNAs have recently

HE HYPOTHALAMIC decapeptide GnRH plays a primary role in regulating the synthesis and release of the pituitary gonadotropins FSH and LH (l-3). Extrahypothalamic sites for the synthesis (4-7) and action of this peptide have also been reported. In thymocytes, GnRH can regulate the appearance of the cell surface interleukin-2 (IL-2) receptor (4) involved in mitogenic activity. In the rat ovary both inhibitory and stimulatory actions have been reported and appear to be related in part to the stage of follicular development (8). For example, GnRH and GnRH agonists (GnRHa) have been shown to inhibit FSH stimulation of follicular development in uiuo (9-11) and prevent FSH-mediated differentiation of granulosa cells in. vitro (8, 11). In contrast, GnRH and GnRHa mimic certain physiological effects of LH on preovulatory follicles, including stimulation of ovulation in hormonally primed hypophysectomized rats (12, 13) and perfused proestrous ovaries (14) as well as induction of oocyte maturation of follicle-enclosed oocytes (15). Based on the observed increase in prostaglanReceived October 23,199l. Address all correspondence and requests for reprints to: JoAnne S. Richards, Department of Cell Biology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030. * This work was supported by NIH Grant HD-16229. 3512

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GnRH

INDUCTION

been cloned from Rous sarcoma virus-transformed chicken embryo fibroblasts (31) and mitogen-stimulated 3T3 cells (32). The other isoform (M, = 69,000; rPGS,) is expressed constitutively in rat ovarian tissues (thecal cells, corpus luteum, and residual tissue), kidney, and uterus, but is not induced by FSH or LH and, thus, appears to be similar to the more ubiquitous form of the enzyme originally cloned. The evidence that there are at least two distinct isoforms of PGS encoded by two different genes and that FSH/LH and GnRH are presumed to act via distinct intracellular pathways raised the possibility that each hormone might selectively regulate the expression of a specific PGS gene. Therefore, the following study was undertaken to determine which isoform(s) of the enzyme was induced by GnRH, in which follicular cell type induction occurred, at what stage(s) of follicular development a response to GnRH was observed, and by what intracellular signalling pathway(s) the effects of GnRH were mediated.

Materials

and Methods

Animals

Intact immature female rats were obtained from Holtzman Co. (Madison, WI). Tissues

Small antral (SA) follicles (-12-15 follicles/rat) were dissected manually from ovaries of immature 29-day-old rats. Preovulatory (PO) follicles (-10 follicles/rat) were isolated from ovaries of immature 29-day-old rats that had beentreated for 2 days with a low doseof hCG (0.15 IU, twice daily) (33). Follicle incubations

Follicles (80 follicles/treatment group) were incubated in Dulbecco’sMinimum Essential Medium and Ham’s F-12 nutrients (DMEM:FlB; 1:l) at 37 C and with 95% oxygen-5% CO, for l-7 h in the presence of hormones, inhibitors, and other compounds,as indicated for each experiment. After the incubations, follicles in each treatment were pooled, collected by centrifugation, frozen, and stored at -70 C until appropriate cell extracts were prepared. In selectedexperiments, granulosa and thecal cells of PO follicles were isolated before or after follicle incubations by manual puncture and gentle scraping of the follicles under a dissecting microscope. Granulosa and thecal cells were also frozen and stored before the preparation of cell extracts. Medium sampleswere savedfor RIAs. Phorbol myristate acetate (PMA), genistein, and dexamethasonewere eachdissolvedin ethanol, cycloheximide, and epidermalgrowth factor (EGF) in medium, ol-amanitin, and ionomycin in dimethylsulfoxide. Diacylglycerol (DAG) was dissolved in chloroform, dried before use, and resuspendedin medium by sonication. Compoundswere addedin volumes lessthan 1% of the total incubation volume.

OF PGS

Preparation

of

3513 cellular

extracts

All tissueswere homogenized,as describedpreviously (28, 29), in PE buffer (10 mM potassiumphosphate,pH 6.8, and 1 mM EDTA) and centrifuged at 30,000x g to obtain a particulate (membrane) pellet and a soluble (cytosol) cell fraction. The pellet was resuspendedin PE buffer containing 10 mM 3-[3cholamidopropyl)dimethyl-ammonio]-l-propansulfanat (CHAPS) and sonicated (three times; 3 set each time). Sonicates were briefly centrifuged (by microfuge; 5 min), and supernatants (solubilized membrane extracts) were saved for further protein and immunoblot analyses. Immunoblot

analyses

Solubilized membrane extract proteins (100-300 Fg) were resolvedby one-dimensionalsodiumdodecyl sulfate-polyacrylamide gel electrophoresis(4.5% stacking gel and 10% separating gel, unless otherwise specified) and electrophoretically transferred to nitrocellulose filters (0.45 pM; Schleicher and Schuell, Keene, NH), as describedpreviously (27-29). Antibodies and iodinated [‘Ylprotein-A (1 x 10 6cpm/ml) were diluted in TBS (10 mM Tris-buffered isotonic saline, pH 7.0) containing 2% Carnation (nonfat) dry milk (Pica Riviera, CA) and 0.1% merthiolate and used sequentially to visualize immunopositive proteins, as described previously (28). Apparent M, were determined using Rainbow M, standards (Amersham, Arlington Heights, IL). Antibodies

Antibodies to PGS were generated in rabbits and affinity purified using ovine PGS asthe antigen and ligand, respectively (27, 28). The antibodies used for this study have been shown to specifically recognize two immunodistinct M, variants of PGS (27, 30). Antibodies 2 (27) and 9181 (30) recognize an isoform of rat PGS (M,, 72,000;PGS72; rPGSi) induced by LH in granulosa cells of PO follicles. Antibody 3 recognizes a constitutive form of PGS present in numerousrat tissues(27, 30). Antibodies to protein kinase-C isoforms (a and PII) were a kind gift of Dr. Alan Fields, (CaseWestern ReserveUniversity, Cleveland, OH) and have been used previously to selectively detect protein kinase-Cisoformsby Western blot analysis (34). RIAs

RIAs for CAMP and progesteronewere performed as previously described,using an antiserum to CAMP kindly provided by Dr. Judith Vaitukaitis (Division of Research Resources, NIH, Bethesda,MD) and antisera to progesteronekindly provided by Dr. Gordon D. Niswender (Department of Physiology, Colorado State University, Fort Collins, CO). PGF,, and PGE, were quantified by RIA of unextracted samplesof incubation medium.RIA for PGF,, wasperformed accordingto the method of Johnson et al. (35), with an intraassaycoefficient of variation of 4.3% and an interassay coefficient of 10.8%. The limit of sensitivity for the PGF,, assaywas 2.5 pg/ml. RIAs for PGE, were performed according to the method of Jacobset al. (36), with intra- and interassay coefficients of variation lessthan 10% and a limit of sensitivity of 8.2 pg/ml. Statistical analyses


3514

GnRH

INDUCTION

OF PGS

Endo. Voll30.

1992 No 6

were based on unpaired t test comparisons between treatment groups. Materials

Ovine LH (NIH oLH-23) and ovine FSH (NIH oFSH-16) were obtained from the National Hormone and Pituitary Program (Baltimore, MD); hCG from Organon Special Chemicals (West Orange, NJ); cy-amanitin, DAG, PMA, LHRH (GnRH), and (D-Ala)LHRH (GnRHa) from Sigma (St. Louis, MO); EGF, platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF) from Upstate Biotechnology, Inc. (Lake Placid, NY); IL-l/3 from Promega (Madison, WI); cycloheximide from Nutritional Biochemicals (Cleveland, OH); ionomycin from Calbiochem (La Jolla, CA); genistein from ICN Biochemicals (Cleveland, OH); CHAPS from Boehringer-Mannheim (Indianapolis, IN); electrophoretic supplies from Bio-Rad (Richmond, CA); tissue culture reagents from Gibco (Grand Island, NY); purified ovine PGS from Oxford Biomedical Research, Inc. (Oxford, MI); ‘251-labeled protein-A from ICN Biomedicals, Inc. (Casa Mesa, CA), and Rainbow M, markers from Amersham. Bryostatin was generously provided by Dr. Alan Fields, (Case Western Reserve University, Cleveland, OH). Results Effects of GnRH and GnRHa on production induction of PGS in PO follicles in vitro

of PGs and

To examine the ability of GnRH and GnRHa (D-AlaGnRH) to regulate PG production in PO follicles, PO follicles were isolated and incubated with various doses of GnRH for 6 h. Concentrations of PGEz and PGF2, were assessed in the incubation medium by RIA. PO follicles produced low amounts of both PGF2, and PGEz in the absence of added hormones (Fig. 1). GnRH stimulated dose-dependent increases in the production of PGFz, and PGE2. At 10e6 M GnRH, PGF1, and PGE2 increased ll- and 46-fold, respectively, to levels comparable to those in LH-stimulated PO follicles (see below; Figs. 7-9 and Table 1). To determine if the GnRH-mediated increases in PGEz and PGF2, were associated with increases in PGS enzyme content, PO follicles were incubated in the presence of LH (500 rig/ml) or GnRH (10e7 M) with or without cY-amanitin (25 pg/ml) for 7 h. Solubilized cell extracts were analyzed for PGS by immunoblotting using specific antibodies, designated 2 and 3 (27). GnRH and LH increased the PGS isoform that is selectively recognized by antibody 2 compared to the constitutive isoform recognized by antibody 3 (Fig. 2). The constitutive form of PGS (M,, 69,000), shown in Fig. 2, was present in all samples, including PO follicles incubated in the absence of LH or GnRH or in the presence of a-amanitin. Although antibody 3 recognizes the induced form of PGS (M,, 70,000-72,000) in the LH and GnRH samples, the cross-reactivity is low and accounts for a 1.5-fold increase

n

PGF2a

IJ

PGE2

1u)o

500 0 PO

10

-a

10 -7

I

10

-6 I

+GnRli 1. GnRH stimulation of PGE, and PGF,, production by PO follicles in uitro. PO follicles (80 follicles/treatment group unless specified otherwise as in Fig. 6) were incubated for 6 h in medium alone (PO) or with GnRH (10-s, lo-‘, and 1Om6 M). After incubation, the PGE, and PGFZ, contents in the incubation media were measured by RIA (see Materials and Methods). In this and subsequent experiments PG values were derived from triplicate measurements of aliquots of a single sample. In this way, PG production can be assocciated with changes in enzyme induction within each experiment.

FIG.

in immunoreactive PGS, rather than the lo- to 40-fold increase observed in these same samples with antibody 2. Furthermore, a-amanitin blocked the induction of the induced form of PGS (rPG&; antibody 2), but did not alter the level of PGS recognized by antibody 3. The production of PGF2, by these follicles correlated with PGS content; control follicles produced low amounts of PGF2, (6.5 pg/ml.follicle), which increased -7-fold in response to GnRH (46 pg/ml.follicle) and LH (60 pg/ ml/follicle), but remained at control levels in the presence of GnRH plus cu-amanitin. Based on these observations, combined with those shown in Table 1 as well as those of previous studies (27, 29), we conclude that GnRH specifically induces rPGSi and not the more ubiquitious form of the enzyme. The temporal induction of rPGSi by GnRH as well as the magnitude of the response (Fig. 3A) were similar to those caused by LH (27-29). Measurement of CAMP in the incubation medium determined that GnRH-treated follicles produced significantly (P < 0.05) more CAMP than PO control follicles, but significantly less CAMP (P < 0.007) than LH-treated follicles by 7 h (Fig. 3B and Table 1). As determined by previous dose-response studies with LH (29), the level of CAMP produced in response to GnRH was not sufficiently high to be the sole mediator of GnRH induction of rPGSi. These results suggest that although capable of mediating an increase in CAMP, GnRH regulates rPGSi content through a CAMP-inde-


GnRH TABLE

1. Production

of CAMP,

progesterone,

and PGF?, CAMP

PO follicle +LH (500 rig/ml) +GnRH (lo-’ M) +LH/genistein +GnRH/genistein +EGF (50 rig/ml) +PMA (200 M)

1.5 280 7.5 380 1.2 2.7 5.2

INDUCTION

by incubated (pmol/ml)

OF PGS

PI follices Progesterone

f 0.5 (3) + 7 (6)",* z!z 0.9 (6)" f 41(5)‘Q f 0.2 (3)b + 0.4 (5)b f 4.0 (SD)

Antibody

#2

Antibody

S3

?? 01 F

(rig/ml)

PGF2,

1.6 + 0.9 (4) 56 f 1 (4)“,* 12.6 f 1.8 (4)” 9.4 1.0 f 0.2 (3)b 3.1 + 0.8 (3)b 4.5 f 5.9 (SD)

The numbers in parentheses refer to values obtained in separate experiments, as described refers to standard deviation; all other values are SEMS. DValues significantly different (P < 0.05 to P < 0.000) from those of PO follicles. * Values significantly different (P < 0.05 to P C 0.001) from those of GnRH follicles.

6 E sE 3 sE 5i

3515

Wml)

46 + 7 (4) 1446 + 172 (5)” 1477 + 267 (5)” 35 95 68 + 9 (4)b in Figs.

1-9, and from

other

data

(not

shown).

SD

Mrx10e3

72 59

FIG. 2. Induction of rPGSi by GnRH and LH. PO follicles were incubated for 7 h in medium alone (control), with LH (500 rig/ml), or with GnRH (10e7 M) with or without a-amanitin (cu-aman; 25 rg/ml). Solubilized membrane extracts were prepared, and 300 pg of each were assessed for PGS content by immunoblot analysis, using antibodies 2 and 3.

pendent mechanism. Progesterone production was also measured for LH- and GnRH-treated follicles. Both GnRH and LH stimulated progesterone production; however, GnRH-treated follicles produced significantly (P < 0.000) less progesterone than LH-treated follicles (Fig. 3B and Table 1). Because GnRH induction of rPGSi appeared to be through a CAMP-independent mechanism, we examined the ability of GnRH to induce PGS in follicles at an earlier stage of development. Specifically, SA follicles from unprimed immature rats were incubated in medium alone, with FSH (1 pg/ml), or with GnRH (10V7 M) for 6 h and assessed for PGS content by Western immunoblotting using antibody 2. Incubation with either FSH or GnRH did not induce rPGSi in SA follicles (Fig. 4A). Correspondingly, PGFzo, production by these SA follicles increased only slightly with FSH and not at all with GnRH treatment (Fig. 4B), indicating that although gonadotropins (LH/FSH/hCG) and GnRH act through different biochemical pathways, their abilities to regulate PGS demonstrate a similar developmental constraint. The means by which FSH stimulates a modest rise in

CAMP pmoleslml

B -Exp. 1

- Exp. 2

GnRH3h GnRH 5h GnRH 7h GnRH + LH 7h GnRHlh GnRH 3h GnRH 5h GnRH 7h LH 7h

06 36 40 216

P nglml 0 94 3 46 349 57 56

0.1

05 33 100 207 0

0 3 7 55

nd 03 44 12 90

FIG. 3. Time-dependent induction of rPGS, in PO follicles treated with GnRH. A, Solubilized membrane extracts were prepared from untreated PO follicles, and PO follicles were incubated with GnRH (10e7 M) for 1,3,5, or 7 h or with LH (500 rig/ml) for 7 h. The extracts (300 pg each) were then assessed for PGS content by immunoblot using antibody 2. ‘Y-Labeled immunopositive bands were visualized by autoradiography, excised from the nitrocellulose blot, and counted on a y-counter. B, CAMP and progesterone (P) contents in the incubation medium of two separate experiments were assessed by RIA.

PGs in the absence of an increase of rPGSi remains to be determined, but may involve increased arachadonic acid release and the low level of the constitutive form of PGS (M,, 69,000; rPGSc) in SA follicles (27). To determine whether PGS induction by GnRH is cell specific, PO follicles were incubated alone, with LH, or with GnRH. After incubation, granulosa cells were separated from thecal cells and assessed separately for rPGSi content. As with LH, induction of PGS by GnRH was specific to granulosa cells of PO follicles (Fig. 5). The faint immunopositive bands observed in the thecal lanes


GnRH

3516

CAMP pmoleslml SA

0.7 60.0

SA + FSH

1.4

GnRH

P nglml

PGF2, w/ml

nd

70.1

3.6

176.3

0.3

INDUCTION

70

FIG. 4. rPGSi content in SA follicles incubated with FSH or GnRHa. A, SA follicles were incubated for 6 h alone, with FSH (1 pg/ml), or with GnRH (lo-’ M), and solubilized membrane extracts (300 pg) were assessed for PGS content by immunoblot analysis using antibody 2. Purified sheep seminal vesicle PGS (oPGS; 50 ng; Oxford Biomedical) was included as a standard. B, Media from the incubated follicles were assessed for CAMP, progesterone (P), and PGF*, content by RIA. Granulosa

OF PGS

Endo. Voll30.

1992 No 6

6A). To insure that PMA was not used at toxic doses, lower doses of PMA (25, 50, and 100 nM) were also tested. These lower PMA doses had no effect on follicular rPGSi content (not shown) and failed to stimulate PGE, or PGS, production to levels comparable to those in LH-treated follicles (Fig. 6B and Table l), although a modest (2-fold) increase in PGEz production was observed with 100 nM PMA in this study. Furthermore, when a time course was conducted using 200 nM PMA, neither rPGSi (antibody 2) nor the more ubiquitous form of PGS recognized by antibody 3 (data not shown) was markedly increased. Bryostatin, a pharmacological agent capable of activating specific members of the PKC family and structurally different from PMA (34), was also included in follicle incubations. Neither bryostatin (10,50, and 100 nM) nor DAG (20 pug/ml) induced rPGSi in preovulatory follicles (data not shown). The inability of PMA, bryostatin, and DAG to induce PGS was not due A

Theta

!flr~lO'~

CPM

503 110

3667

4411

134

165

80

B

PGF2a PGE2

FIG. 5. Content of rPGSi in granulosa and thecal cells from PO follicles treated for 6 h with LH or GnRH. PO follicles were incubated alone (PO), with LH (500 rig/ml), or with GnRH (10e7 M) for 6 h. Granulosa and thecal cells were isolated and assessed separately for PGS content by immunoblot analysis using antibody 2. oPGS (50 ng) was included as a standard. Immunopositive protein bands were quantitated as described in Fig. 2.

are believed to be derived from granulosa contaminants of the thecal cell preparations. These results indicate that both the CAMP-dependent and GnRH-stimulated pathways that induce rPGSi are present in granulosa, but not thecal, cells of PO follicles. Effects of protein kinase-C activators on PGS content

To examine the possible role of the PKC pathway (3, 37, 38) in GnRH induction of rPGSi, PO follicles were incubated with pharmacological or physiological activators of PKC enzymes. PMA (200 nM) neither stimulated rPGSi nor altered the induction of rPGSi by LH (Fig.

26rlM

PO

6onM

1

1oonM

LH I

PMA

FIG. 6. Effects of PMA on rPGSi content and PG synthesis by PO follicles. A, PO follicles were incubated with LH (500 rig/ml) with or without PMA (200 nM) for 7 h or with PMA alone for 3,5, or 7 h. The PGS content was assessed by immunoblot analysis (antibody 2) and compared to that in follicles incubated alone for 7 h. B, PO follicles were incubated alone, with LH (500 rig/ml), or with various doses of PMA (10,50, and 100 nM) for 6 h. PGE? and PGF,, were measured in the incubation media by RIA and corrected for the number of incubated follicles (picograms per ml/follicle).


GnRH INDUCTION to the absence of responsive C-kinase isoforms, as both PKCa and PKCpII (two proteins shown to be selectively translocated in response to PMA and bryostatin) (34) were detected in PO follicles by Western immunoblot (data not shown). Bryostatin also failed to mimic the effects of GnRH on progesterone, CAMP, and PGF2, synthesis (data not shown). Effects of tyrosine phosphorylation and growth factors on PGS induction in vitro

Because none of the PKC activators used was sufficient to reproduce the GnRH-mediated induction of rPGSi in PO follicles, studies were undertaken to examine the possible participation of other known signalling pathways. Employing the strategy of calcium depletion (39, 40), PO follicles were preincubated with EGTA and ionomycin (EGTA/ionomycin) for 1 h and then treated with GnRH or LH or incubated alone for 6 h. The EGTA/ionomycin treatment regimen completely blocked the induction of PGS protein by GnRH and LH (Fig. 7). EGTA/ ionomycin treatment also blocked the stimulation of PGFz, synthesis by GnRH. However, this regimen did not completely abrogate the LH stimulation of PGF2, production. Genistein, an inhibitor of tyrosine kinases (41), was used to determine whether tyrosine kinase-mediated phosphorylation was involved in the regulation of PGS. Follicles were incubated with LH or GnRH in the presence or absence of genistein. The effects of both LH and GnRH on rPGSi were completely abolished by this com-

OF PGS

pound (Fig. 8A). Correspondingly, stimulation of PGFz, production was also blocked by genistein (Fig. 8B). Curiously, genistein also significantly reduced LH- and GnRH-stimulated steroidogenesis (Fig. 8B and Table 1). The ability of genistein or EGTA/ionomycin to block the effects of LH were not due to inhibition of adenylyl cyclase, because CAMP production remained high in the incubations with these agents (PO and LH, 618 pmol/ ml vs. PO, LH, and genistein, 412 pmol/ml; PO and LH, 200 pmol/ml vs. PO, LH, and EGTA/ionomycin, 166.7 pmol/ml; see Table 1). These results suggest that calcium and tyrosine phosphorylation contribute to the CAMPdependent and -independent pathways of PGS regulation. To further examine the role of tyrosine phosphorylation in the regulation of PGS, PO follicles were incubated with various growth factors (PDGF, bFGF, and EGF) that activate receptor-associated tyrosine kinase activity and with cytokines (IL-la! and IL-l@), all of which have been shown to alter granulosa cell function (42-47) and in some cells, including granulosa cells, increase PGs (43, 48, 49). Of the peptides tested (data not shown), only EGF increased rPGSi content in the incubated follicles. The induction of rPGSi by EGF was similar to that observed with low concentrations of FSH, but was much less than that observed in follicles treated with high FSH (Fig. 9B). EGF did not cause increases in CAMP that were significantly different from values in PO control A 0 ;i

PGF2a PGS

EGTMono, EGTAnono,

44

LH

1505

a5

Hsri

PmJl

PO

3517

B

CAMP pmoles/ml

80

LH

37

GnRH

PO+LH

616.0

PO+LH+Gen

412.0

PO + GnRH

or x lo-3

P rig/ml

72

59

FIG. 7. Effects of calcium depletion on rPGSi induction by LH or GnRHa. A, PO follicles were incubated with EGTA (1 mM) and ionomycin (4 pM) for 1 h, and then LH (500 rig/ml) or GnRHa (10m7 M) was added to the medium, and the incubations were continued for 6 h more. Solubilized membrane extracts were prepared, and PGS content was assessed by immunoblot (300 rg protein; antibody 2) and compared to that in PO follicles incubated for 6 h with or without LH. B, PGF,, in the incubation medium was measured by RIA.

PO + GnRH Gen

+

526 9.4

7.2

13.3

0.6

0.6

PGPZcl pg/ml 1230 35

1142 nd

FIG. 8. Effects of genistein (Gen) on rPGS, induction by LH and GnRH. A, PO follicles (-SO/group) were incubated for 6 h alone or with LH (500 rig/ml) with or without genistein (30 pg/ml). In a separate experiment PO follicles were incubated for for 7 h with GnRH (10e7 M) in the presence or absence of genistein. Soluble membrane extracts (300 pg protein) were assessed for PGS content by immunoblot analysis using antibody 2. B, CAMP, progesterone (P), and PGF2, concentrations in medium samples were determined by RIA.


3518

GnRH

INDUCTION

Endo. Vol130.

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1992 No 6

TABLE 2. Progesterone production by granulosa cells isolated from PO follicles treated 6 h with either LH or GnRH

A

Progesterone (rig/ml) Whole follicles

B CAMP pmoles/ml

PGF2a

P y/ml

w/ml

FSH

500

150

FSH

50

7.8

10.0

84

FSH

50 + lono

1.2

3.7

89

EGF

1.5

15.2

95

GnRH

7.2

13.3

58.2

1031

Granulosa cells cultured 7 days

GnRH, 3 h 0.45 GnRH, 5 h 2.53 GnRH, 7 h 6.5 + 1.8 f 0.14 43.8 + LH,7h 47.8 f 12 PO follicles were incubated for 6 h with LH (500 rig/ml) or GnRH (lo-’ M) and at the end of the incubations, the granulosa cells were isolated, plated out (DMEMF12, 1:l; 1% FCS; 250,000 cells/ml) in 24multiwell dishes and cultured for 7 days. Medium samples were collected from follicles incubated with GnRH at 3, 5, and 7 h, from LHincubated follicles at 7 h, and from granulosa cell cultures (accumulation for days 5-7). Progesterone content was measured by RIA. Values for progesterone represent the mean f SEM of 6 separate culture wells derived from the pool of granulosa cells isolated from each group of PO follicles at 7 h.

1142

FIG. 9. rPGS, content in PO follicles treated with low and high doses of FSH, with low doses of FSH plus ionomycin, or with EGF. A, PO follicles were incubated with a high dose of FSH (500 rig/ml), with a low dose of FSH (50 rig/ml) with or without ionomycin, with EGF (50 rig/ml), or with GnRH (10-r M) for 7 h. The PGS content of prepared solubilized membrane extracts (300 rg) was examined by immunoblotting using antibody 2. B, CAMP, progesterone, and PGF,, in the incubation media were measured by RIA. The GnRH treatment group is the same as that described in Fig. 8. When samples were analyzed using antibody 3, no changes in the ubiquitous form of PGS (rPGSc) were observed. P, Progesterone.

follicles (Table 1). Neither EGF nor the other growth factors/cytokines tested altered the content of the constitutive isoform of PGS (rPGSc) recognized by antibody 3 (data not shown). LH, but not GnRH, stimulates functional luteinization of granulosa cells in vitro

Follicles stimulated to ovulate in vivo by GnRH do not luteinize (9, 10). To verify that GnRH induction of PGS was independent of luteinization in vitro, granulosa cells from PO follicles incubated for 6 h with either LH or GnRHa were isolated, plated (DMEM:FlB, 1:l; 1% FCS), cultured for 7 days, and examined for signs of luteinization, namely production of high levels of progesterone in the absence of further hormonal stimulation (50). As determined previously, both GnRH and LH simulate progesterone production by PO follicles, LH more strongly than GnRH (Table 2). However, when isolated and cultured in the absence of hormones, granulosa cells from LH-treated follicles continued to produce high levels of progesterone, whereas those exposed to GnRH did not. Also observed, but not shown, was the accumulation of lipid droplets in the LH-treated granulosa cells, but not the GnRH-treated cells. Thus, GnRH stimulation of

PG production in PO follicles is not associated with or obligatory for the process of luteinization. Discussion The results of these studies document that GnRH stimulation of PG biosynthesis in rat preovulatory follicles is associated with the rapid, but transient, tissuespecific induction of one isoform of PGS. Based on our previous immunological data (27-29) and recent biochemical/structural studies (30), we conclude that the PGS variant induced by GnRH is localized to granulosa cells and not thecal cells of rat preovulatory follicles and is identical to the enzyme induced by FSH/LH in these same cells. The induced isoform (M,, 70,000-72,000; PGS72), designated rPGSi, is distinct from the ovine (22, 23), mouse (24), and human (25) enzymes for which cDNAs have been cloned, but has N-terminal amino acid sequence that is more homologous to PGS-related gene products of Rous sarcoma virus-transformed chicken embryo fibroblasts (31) and mitogen-activated 3T3 cells (32). The ability of GnRH to induce rPG& was clearly restricted to preovulatory follicles. GnRH did not induce PG biosynthesis in small antral follicles or alter the expression of PGS isoforms in thecal/residual tissue. Based on this information, PGs can be ruled out as effecters of GnRH-mediated inhibition of FSH action at early stages of follicular development (8-11). These results further suggest that the ability of GnRH to block FSH action in granulosa cells of follicles at an early stage of development and to mimic some, but not all, of the actions of FSH/LH at the preovulatory stage of follicular development may be mediated by different intracellular signalling pathways or the interaction of diverse intracellular pathways present at these two stages of granulosa cell differentiation.


GnRH INDUCTION The precise mechanisms by which GnRH mediates these differential effects on granulosa cells at defined stages of follicular development are not entirely clear and would benefit from the cloning and biochemical characterization of pituitary and ovarian GnRH receptor(s) as well as the putative endogenous ovarian ligand(s) of the GnRH receptor. The ability of exogenous GnRH to induce rPGSi and ovulation is not meant to imply that GnRH or GnRH-like peptides are necessarily produced by preovulatory follicles to mediate the effects of LH. Rather, the results of studies conducted herein provide some new insights concerning what mechanisms may be involved in the induction of this novel PGS isoform by these two distinct peptides presumed to act via different intracellular signalling pathways. First, high concentrations of FSH, LH, or forskolin are required to induce rPGSi (27-29). These doses of hormone and forskolin are associated with elevated intracellular concentrations of CAMP, suggesting that high, but not low, levels of this nucleotide induce expression of the rPGSi gene via CAMP-dependent protein kinase (A-kinase). GnRH, however, even at lO-‘j M, caused increases in CAMP that were not of sufficient magnitude to mimic the effects of elevated FSH/LH/forskolin (29). Thus, high intracellular CAMP and subsequent activation of Akinase do not appear to be the only intracellular pathways capable of activating expression of the rPGSi gene in differentiated granulosa cells. Some effects of LH on ovarian cell function have also been associated with increased intracellular Ca2+ and activation of C-kinase (37), presumably via G-protein coupling to phospholipase-C (38). Although the intracellular pathways involved in mediating the actions of GnRH on gonadotropes have been studied extensively (51-53) and also implicate C-kinase as a mediator (3), the results are not conclusive (54). In granulosa cells, GnRH has been shown to bind high affinity specific receptors (9, 55) and stimulate phospholipid metabolism and Ca2+ uptake (37), leading some to postulate the participation of phospholipid-stimulated diacyglycerol accumulation and protein kinase-C pathway activation in GnRH action in these cells as well. Therefore, one explanation of our data would be to implicate the Ckinase family (56) as the common intracellular pathway mediating LH and GnRH induction of rPGSi. However, the results we obtained using known activators of different C-kinase isoforms do not provide a compelling argument for the participation of this enzyme. For example, PMA, a known activator of Ccr-kinase (34) caused a modest (3-fold) increase in PGE2 production by preovulatory follicles. This increase mimics results obtained by others using cultures of granulosa cells (57). However, the increase in PGE2 mediated by PMA was far less than the -46-fold increase in PGE2 production caused by

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either GnRH or LH. More importantly, PMA also failed to induce rPGSi in preovulatory follicles at the various doses and time points tested. Other activators of PKC, bryostatin (PKCPII) (34) and DAG, also failed to mimic the effects of GnRH on rPGSi, adding supportive (albeit indirect) evidence that if activation of a specific isoform of protein kinase-C is involved, it is not the sole mediator of either GnRH or LH action in granulosa cells. The absence of an effect of PMA on rPGSi in preovulatory follicles was unexpected for several reasons. PMA has been shown to increase PGE synthesis in other cell types (58, 59) and has recently been shown to increase the PGS-related TISlO mRNA in 3T3 cells (32). Furthermore, PMA can mimic the ability LH to induce tissue-type plasminogen activator mRNA in granulosa cells (60, 61), although the mechanisms appear different (62). Possible explanations for the absence of an effect of PMA, bryostatin, and DAG in whole follicles compared to granulosa cells is that these agents may rapidly act to affect the function of thecal cells, which, in turn, alters the responses of granulosa cells. Alternatively, these stimulants may be metabolized by the thecal cell layer, rendering them inactive. Evidence that tyrosine phosphorylation may be involved in GnRH and LH induction of rPGSi was provided by two approaches. First, genistein, a potent inhibitor of tyrosine kinases (41), blocked induction of rPGSi by both GnRH and LH in a dose-dependent manner. Second, EGF, which is known to bind and activate EGF receptorassociated tyrosine kinase (63), stimulated a modest increase in the content of rPGSi without affecting expression of the other PGS isoform. That EGF did not induce rPGSi to the same extent as GnRH or LH may indicate that an optimal dose of EGF was not chosen. However, the concentration used was 5-fold greater than that shown to effectively modify other aspects of granulosa cell function (44), to increase de nouo synthesis of PGS in human amnion cells (49), and to increase PGS-related TISlO mRNA in 3T3 cells (32). Alternatively, the differences in PGS induction by EGF and GnRH may be associated with differences in the time course of response to EGF us. GnRH, the numbers of receptors for each ligand, and, possibly, the involvement of C-kinase as well as tyrosine kinase. For example, Kujubu et al. (32) have shown that induction of the PGS-related TISlO mRNA by EGF was more transient than that observed by other mitogenic stimulators, including forskolin, PMA, and serum. Furthermore, differences in the phosphorylation activity of or substrate accessibility to the membraneassociated EGF receptor tyrosine kinase compared to other putative nonreceptor tyrosine kinases may also be involved. This option is supported by the evidence that neither PDGF nor bFGF, two other known activators of cognate receptor tyrosine kinases, were effective in in-


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ducing rPGSi in preovulatory follicles, despite the evidence that these peptides at the doses used can alter granulosa cell function (42,43) and exert small increases in PG production in whole follicles (43) and other tissues (64). IL-lp was also ineffective in stimulating rPGSi, although this cytokine can alter other aspects of granuolosa cell function (46, 47). Multiple pathways have also been implicated in regulating the expression of the chicken CEF-147 gene and the mouse TIS-10 genes, encoding for homologous PGS enzymes. Thus, one way to link the abilities of both GnRH and LH as well as EGF to regulate expression of this novel form of PGS in granulosa cells of preovulatory follicles is to propose that they mediate the phosphorylation of a common substrate that is part of a tyrosine kinase cascade system. One pathway may involve the mitogen-activated protein kinase cascade (65), which is dependent on serinelthreonine as well as tyrosine phophorylation. Althernatively, the protooncogene pp6Oc-src is a nonreceptor tyrosine kinase presumed to be involved in mediating the expression of the chicken homolog of rPGSi in RSV-transformed chicken embryo fibroblasts (31). Importantly, GnRH/LH induction of rPGSi and the increase in the PGS-related genes in chicken embryo fibroblasts (31) and 3T3 cells (32) share two important similarities: rapid induction followed by transient expression. Furthermore, pp60”‘” is known to be a substrate for and is activated by CAMP-dependent protein kinase (66). Conversely, ~~60”‘” can be inactivated by phosphorylation of specific tyrosine residues (66). If LH and GnRH activate a tyrosine kinase cascade, this might account for the transient induction of this gene product by these two different stimuli. Further comparisons of the mechanisms by which GnRH and LH act on granulosa cells should provide an understanding of which intracellular mechanisms are obligatory for those processes that are shared (ovulation and oocyte maturation) and those that are distinct to LH/FSH, namely luteinization. References 1. Andrews W, Maurer R, Conn P 1988 Stimulation of rat luteinizing hormone-beta messenger RNA levels by gonadotropin releasing hormone. J Biol Chem 263:13755-13761 2. Papavasilion S, Zmeici S, Khoury S, Landefeld T, Chin WW, Marshall JC 1986 Gonadotropin-releasing hormone differentially regulates expression of genes- for luteinizing hormone alpha and beta subunits in the male rat. Proc Nat1 Acad Sci USA 83:40264029 3. Conn P, Huckle W, Andrews W, McArdle C 1987 The molecular mechanism of action of gonadotropin releasing hormone (GnRH) in the pituitary. Recent Prog Horm Res 43:29-63 4. Emanuele N, Emanuele M, Tentler L, Kirsteins L, Azad N, Lawrence A 1990 Rat spleen lymphocytes contain immunoactive and bioactive luteinizing hormone-releasing hormone. Endocrinology 126:2482-2486 5. Batticaine N, Morale M, Gallo F, Farinella Z, Marchetti B 1991 Luteinizing hormone-releasing hormone signalling at the lymphocyte involves stimulation of interleukin 2 receptor expression.

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Multifactorial regulation of prostaglandin synthesis in preovulatory goldfish ovarian follicles.

Prostaglandin H synthase.

Xenobiotic metabolism by prostaglandin H synthase.

Steroidogenesis in isolated cells of preovulatory rat follicles.

Sequential changes associated with the degeneration of preovulatory rat follicles.

Prostaglandin H synthase and xenobiotic oxidation.

Effects of tokishakuyakusan and keishibukuryogan on steroidogenesis by rat preovulatory follicles in vivo.

Induction of thromboxane synthase and prostaglandin endoperoxide synthase mRNAs in human erythroleukemia cells by phorbol ester.

Evidence for two antigenically distinct molecular weight variants of prostaglandin H synthase in the rat ovary.

Regulation of prostaglandin H synthase activity in dog urothelial cells.

Inhibition of rat brain prostaglandin D synthase by inorganic selenocompounds.

Lipopolysaccharide induces prostaglandin H synthase-2 in alveolar macrophages.

Chemical modification of prostaglandin H synthase with diethyl pyrocarbonate.

Crystallization of prostaglandin-H synthase for X-ray structure analysis.

Effects of the antiandrogen hydroxyflutamide on progesterone secretion by preovulatory rat follicles in vivo and in vitro.

Preovulatory changes in morphology of rabbit ovarian follicles.

Prostaglandin D2 production and identification of prostaglandin H synthase within canine mast cell granule.

Protein kinase C in preovulatory follicles from the hen ovary.

Prolactin inhibits plasminogen activator activity in the preovulatory follicles.

Arachidonic acid stimulates steroidogenesis in goldfish preovulatory ovarian follicles.

Cooperation of luteinizing hormone signaling pathways in preovulatory avian follicles regulates circadian clock expression in granulosa cell.

Developmental changes in steroidogenesis by equine preovulatory follicles: effects of equine LH, FSH, and CG.

In vitro DNA synthesis by isolated preantral to preovulatory follicles from the cyclic mouse.

H synthase 2 expression in rat endometrial stromal cells.