DOMESTIC ANIMAL ENDOCRINOLOGY
Vol. 8(1):1-13, 1991
PROTEIN KINASE C IN PREOVULATORY FOLLICLES FROM THE HEN OVARY J.L. Tilly and A.L. Johnson
Department of Animal Sciences Rutgers, the State University of New Jersey New Brunswick, NJ 08903-0231 Received September 20, 1990
INTRODUCTION
Recent evidence indicates that liberation of 1,2-diacylglycerol (DAG) following ligand-receptor activation of phospholipase C and the ensuing metabolism of specific membrane phospholipids initiates a cascade of several second messenger pathways, including the activation of protein kinase C (PK-C) (for review, see 1-4). For instance, hydrolysis of phosphatidylinositol 4,5-bisphosphate by phospholipase C transiently generates free DAG, which remains intercalated within the cellular membrane, and inositol 1,4,5-trisphosphate (IP3), which is released to the cytosolic compartment. Diacylglycerol, in the presence of calcium and phospholipid, is believed to activate PK-C by decreasing the calcium requirement of the kinase from the milli- to the micromolar range, thus rendering the enzyme fully active without a net change in cytosolic calcium concentrations (5, 6). However, the active lifespan of DAG is very short since the compound is rapidly phosphorylated via diacylglycerol kinase to phosphatidic acid, which can be recycled back into the membrane p h o s p h o l i p i d pool, a n d / o r h y d r o l y z e d by d i a c y l g l y c e r o l lipase to arachidonic acid (AA), which can serve directly as a second messenger or be used for the synthesis of eicosanoids (1, 2). Inositol 1,4,5-trisphosphate, a second metabolite generated following phosphoinositide hydrolysis, is thought to mobilize calcium from cytoplasmic stores via specific binding sites located within the endoplasmic reticular membrane (7, 8). The increase in cytosolic calcium concentrations initiated by IP 3 is proposed to be involved in the activation of calcium/calmodulin dependent protein kinases and/or PK-C. Similar to DAG, IP 3 is only transiently available and is rapidly metabolized to biologically inactive inositol phosphate by-products which can be used for the resynthesis of membrane phosholipids (9). Protein kinase C was originally characterized as a calcium-, diacylglycerol- and phospholipid-dependent serine-threonine protein kinase (5, 10-12). Since its initial characterization, it has been determined that PK-C exists as a ubiquitous family of closely related isoenzymes which are the products of different genes and/or result from alternative splicing of a single gene, and have distinct biochemical properties and tissue distribution (13-21). Protein kinase C exists as a single-chain, 77-80 kD polypeptide containing two major structural domains which can be generated following proteolytic cleavage with calpain. One fragment, of approximately 51 kD, is hydrophilic and contains the catalytic domain which is active in the absence of essential cofactors. The hydrophobic, 26 kD regulatory domain contains the calcium and DAG binding sites, and is therefore believed to be involved in binding of the kinase to the cellular membrane upon activation (2, 6, 22, 24-29). In its inactive state, PK-C is thought to reside primarily in the cytosolic fraction; however, activation of the enzyme is frequently correlated with its translocation to the membrane microenvironment by, as yet, an unidentified mechanism (6, 28). Copyright © 1991 by Domendo, Inc.
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In the mammalian ()vary, the presence of PK-C activity has been clearly demonstrated (30-34), and several hormones have been implicated in the regulation of PK-C expression and/or activation (35-40). Furthermore, a role for PK-C in modulating ovarian function (e.g. cAMP production, steroidogenesis and plasminogen activator mRNA levels/activity) has been established by several independent researchers (41-51), collectively indicating that this second messenger pathway ,nay play a significant role in the regulation of ovarian function similar to that described for the well characterized adenylyl cyclase/cAMP system. Studies directed at understanding the possible involvement of DAG and PK-C in the regulation of ovarian function have been greatly aided by the discovery that tumor-promoting phorbol esters (e.g. phorbol 12-myristate 13-acetate, PMA; phorbol 12,13-dibutyrate, PDB) or synthetic diacylglycerol analogs (e.g. 1-oleoyl-2acetylglycerol, OAG) can be substituted for endogenous DAG as specific activators of PK-C (2, 52, 53). Additionally, PK-C is believed to be the major phorbol ester receptor within intact cells (53-57). Keeping these aspects in mind, this review describes our current knowledge concerning the presence of PK-C in the avian ()vary and its possible role(s) in the regulation of ovarian steroidogenesis and plasminogen activator (PA) activity. Although it is recognized that additional pathways activated following phosphoinositide hydrolysis (e.g. IP~/calcium/calmodulin and AA/eicosanoids) likely play important roles in the regulation of ovarian function of the hen, these topics will not be addressed in detail here. For information concerning these pathways and their roles in the hen ovary, the reader is referred elsewhere (IP~/calcium/calmodulin: 58-61; AA/eicosanoids: 62-67). EVIDENCE FOR THE EXISTENCE OF PK-C IN THE AVIAN OVARY
Initial studies conducted with hen granulosa cells and phorbol esters demonstrated that binding of 3H-PDB to granulosa cell cytosolic protein is saturable and can be displaced by increasing concentrations of PMA but not phorbol 13-monoacetate (an inactive analog of PMA which does not bind to nor activate PK-C) (68). Furthermore, binding of radiolabelled-PDB to cytosolic protein is associated with its subsequent redistribution to the membrane fraction (68), suggesting the specific translocation of PDB and its receptor (presumably PK-C) to the membrane, a process generally associated with the activation of PK-C. More recently, we have shown that both granulosa and theca cells of the most mature preovulatory (Fj) follicle contain immunoreactive PK-C, and that prior exposure of granulosa cells to PMA results in the translocation of immunoreactive PK-C from the cytosolic to the membrane fraction (granulosa: 66; theca: unpublished data). Although the antibody used in these studies is reported to recognize the ot (Type lid and 13 (Type II), but not y (Type I), subspecies of PK-C, further studies in our lab using monoclonal antibodies specific for each subtype suggest that the 13 form is the predominant immunoreactive subspecies in both brain cerebral cortex and ovarian granulosa tissue, with negligible amounts of eL and y (Figure 1). The lack of detectable 0¢ and y subspecies is apparently due to either very low/nonexistent levels, or the inability of the mammalian monoclonal antibodies to recognize the chicken moieties. Furthermore, preliminary evidence from our laboratory and others have identified the presence of a calcium- and phospholipid-dependent protein kinase within granulosa tissue of hen preovulatory follicles (Table 1) (69). However, the levels of kinase activity in granulosa cell protein are relatively low compared to those levels found in protein preparations of hen cerebral cortex (Table 1). The reason for the substantially lower levels of PK-C activity within ovarian tissue is unclear, but it
PROTEIN KINASE C IN THE HEN OVARY
3
A 1
2
3
4 -.r
s
5
6
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8
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2
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Fig. 1. Western blot of protein kinase C from brain and granulosa tissues. Panel A. Immunoreactivity following electrophoresis of 100 p.g crude cytosolic protein from rat (lanes 1,4,7), hen (lanes 2,5,8) and bovine (lanes 3,6,9) cerebral cortex using specific monoclonal antibodies (Seikagaku Kogyo Co., Tokyo, Japan) directed against rat ~ (lanes 1-3), 13 (lanes 4-6) and ~/(lanes 7-9) protein kinase C subspecies. Panel B. Immunoreactivity from 150 p.g crude cytosolic protein from hen granulosa tissue: lane 1, c~; lane 2, 13; lane 3, "/. Note that the cx and ~/subspecies from hen brain and granulosa tissues are too low to detect, or alternatively, are not immunoreactive with the mammalian monoclonal antibodies. Details of the Western blot analysis have been previously described (66).
m a y b e the result o f high levels o f an e n d o g e n o u s PK-C inhibitor as has b e e n rec e n t l y d e s c r i b e d for h u m a n p l a c e n t a l a n d rat o v a r i a n tissues (70). In a n y case, t h e s e d a t a t a k e n collectively clearly indicate the p r e s e n c e o f PK-C w i t h i n o v a r i a n tissue o f the d o m e s t i c hen, a n d its characteristic r e s p o n s i v e n e s s to t u m o r - p r o m o t ing p h o r b o l esters.
EFFECTS OF PIK-C ON STEROIDOGENESIS G r a n u l o s a T i s s u e . T h e first p u b l i s h e d r e p o r t attributing a specific role for PK-C in t h e a v i a n o v a r y a r o s e f r o m s t u d i e s w h i c h d e m o n s t r a t e d that PMA a n d O A G m o d u l a t e luteinizing h o r m o n e ( L H ) - i n d u c e d s t e r o i d o g e n e s i s in h e n g r a n u l o s a cells c o l l e c t e d from the F1 follicle (71). T h e s e c o m p o u n d s d o n o t affect b a s a l p r o g e s t e r o n e o r a n d r o g e n p r o d u c t i o n , b u t a t t e n u a t e LH-stimulated s t e r o i d o g e n e s i s in a d o s e - d e p e n d e n t fashion. A s u b s e q u e n t r e p o r t f r o m A s e m a n d T s a n g (72) c o n f i r m e d t h e s e initial f i n d i n g s o f a n i n h i b i t o r y a c t i o n o f PMA o n L H - p r o m o t e d s t e r o i d o g e n e s i s in h e n g r a n u l o s a cells. T h e actions o f PMA are p r e s u m e d specific for PK-C activation since c o m p a r a b l e effects are o b t a i n e d w i t h a d i a c y l g l y c e r o l a n a l o g b u t n o t w i t h the inactive PMA analog, p h o r b o l 1 3 - m o n o a c e t a t e (71). In fact,
TABLE 1. PROTEIN KINASE C ACTIVITYIN HEN BRMN CORTEX, AND GRANULOSATISSUE COLLECTEDFROM THE LARGEST PREOVULATORY(Fi) FOLLICLE)
Tissue N Specific Activity~ Cerebral Cortex 4 22.0 -+ 3.9 F, Granulosa 3 3.4 ~ 2.0 *Activity (pmol 32p transferred/min/mg protein) was measured from cytosolic protein which was prepared as previously described (66). The assay was conducted with (~/-~aP)-ATPin the absence and presence of CaCI2 (15 mM) plus phosphatidylserine (50 Izg)/diolein (6 I~g) and calf thymus histone H1 (100 ~g), as described by Kikkawa et al. (95). 2Mean ± SE (N=number of replicate experiments).
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TILLY AND JOHNSON
the inhibitory effects of PMA are not limited to LH-induced steroid producti(m since steroidogenesis stimulated by other hormonal ligands which act via adcnylyl cyclase/cAMP in hen granuk)sa cells (i.e. vasoactive intestinal peptide; 73) is similarly suppressed (71). A subsequent study clarified that the actions of PK-C on LH-promoted steroid output are directed at sites both prior and distal to cAMP formation (adenylyl cyclase, and cytochrome P,~,,~ function/cholesterol transport, respectively) (74). Again, the effects of PMA are believed specific tk)r PK-C activation since the inhibitory actions of PMA on LH-stimulated cAMP formation are reversed in a dosedependent fashion by the putative PK-C inhibitor, staurosporine (74). Ahhough staurosporine is known to inhibit not only PK-C, but also PK-A activity, in both dispersed hen granulosa cells (75; unpublished data) and in cell-free preparations of PK-A (75), the results from the aforementioned studies cannot be attributed to inhibitory effects of staurosporine on PK-A activity. More recently, in an effort to clearly elucidate the site of action of PK-C distal to cAMP formation, we have evaluated the effects of PMA on levels of immunoreactive P,s0,~ protein and the kinetic parameters of P,~.~. activity in crude mitochondrial protein prepared from F~ granulosa tissue. Results from these studies indicate that pretreatment of cells with PMA and LH does not affect the levels of immunoreactive P~s0~, protein compared to vehicle or LH-pretreated cells, but decreases the ability of P,~,~ isolated from mitochondria to convert 25-hydroxycholesterol to pregnenolone (unpublished data). From these studies, we propose that PK-C may directly phosphorylate P,~,,~ in avian granulosa tissue, thereby reducing the ability of the enzyme to effectively convert cholesterol precursor to pregnenolone. Phosphorylation studies using radiolabelled-ATP as a phosphate donor are currently underway to substantiate this proposal. Finally, granulosa cells isolated from less mature, growing tkJlicles (6-8 mm in diameter; approximately 3 weeks prior to ovulation) in the hen ovary have been shown to be steroidogenically incompetent, failing to produce progesterone in response to LH, follicle-stimulating hormone (FSH), 8-bromo-cAMP or 25-hydroxycholesterol (76, 77). However, we have found that preincubation of these granulosa cells with forskolin for 24 hr renders the cells c o m p e t e n t to metabolize 25-hydroxycholesterol to progesterone, suggesting the induction of P~s0~,~ activity (Figure 2; Tilly JL, Kowalski KI and Johnson AL, unpublished data). Interestingly, coincubation of 6-8 mm fkJlicle granulosa cells with forskolin in the presence of PMA abolishes the stimulatory actions of the adenylyl cyclase activator on the induction of P~s0~ activity, but does not affect the ability of these granulosa cells to metabolize pregnenolone to progesterone (Figure 2). From a physiological standpoint, we have proposed that the activation of PK-C within granulosa cells of the hen serves at least two specific functions associated with steroidogenesis. First, it has previously been reported that a dramatic increase in adenylyl cyclase activity occurs in F~ granulosa cells during the hours preceding the preovulatory LH and progesterone surges (78), which is followed by a shortterm period of steroidogenic "desensitization" (79-81). During this period of "desensitization", g o n a d o t r o p i n - i n d u c e d adenylyl cyclase activity is markedly decreased, leading to the termination of the p r e o v u l a t o r y p r o g e s t e r o n e surge. However, the decrease in progesterone production by F~ granulosa cells cannot be attributed solely to a decrease in LH-stimulated adenylyl cyclase activity, since granulosa cells collected during this densensitization period also fail to produce progesterone in response to cAMP analogs (82). Therefore, the short-term suppression of LH-promoted steroidogenesis and the subsequent termination of the pre-
PROTEIN KINASE C IN THE HEN OVARY
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o
100
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i! CON
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Fig. 2. Progesterone production from 6-8 mm follicle granulosa cells incubated for 3 hr in the absence and presence of 25-hydroxycholesterol (CHOL; 2500 ng) or pregnenolone (P4 20 ng) following a 24-hr preincuhation with vehicle (CON; control), PMA (100 riM), forskolin (FOR; 10 I.LM)or PMA + forskolin. Data are expressed as the mean _+ SE of results from 3 replicate incubations. These data would indicate that activation of the protein kinase C system negatively modulates the induction of functional P,s,,,,~ stimulated by cAMP/protein kinase A in granulosa cells of developing follicles, but does not significantly alter 313-hydroxysteroiddehydrogenase activity.
ovulatory progesterone surge, which o c c u r as a result of a decrease in responsiveness to LH both prior and distal to cAMP formation, m a y be initiated via PK-C activation. Secondly, the induction of steroidogenic c o m p e t e n c e in avian granulosa cells of developing follicles 2-3 w e e k s prior to ovulation by the cAMP/PK-A pathw a y m a y be tightly regulated by a tonic, s u p p r e s s i v e effect o f PK-C activity. Although the cellular site of action of PK-C on the induction of steroidogenic c o m p e t e n c y in maturing avian granulosa cells is unclear at present, w e are currently evaluating the effects of PK-C on P.,~0~c~ messenger RNA expression, levels of immunoreactive protein and e n z y m e activity in these cells. T h e c a T i s s u e . In theca cells collected from the s e c o n d largest preovulatory (F,) follicle, PMA (but not p h o r b o l 13-monoacetate) and OAG have b e e n s h o w n to suppress LH-stimulated a n d r o s t e n e d i o n e production in a c o n c e n t r a t i o n - d e p e n d e n t manner, but d o not alter c o r r e s p o n d i n g cAMP levels or progesterone output (83). Similar effects of these c o m p o u n d s have also b e e n reported for steroid production by theca cells of less mature, developing follicles (6-8 m m in diameter) in the hen ovary (84), suggestive of a functional PK-C system within less mature ovarian follicles. These findings contrast those reported for avian granulosa cells, since there appears to be solely a post-cAMP inhibitory action of PK-C in theca tissue, apparently at C17,20-1yase. In addition, basal levels of progesterone, a n d r o s t e n e d i o n e and estradiol are increased following incubation of F e or 6-8 m m follicle theca cells with PMA, further indicating that distinct differences exist in the actions of PK-C within granulosa and theca cells. In contrast, it appears the role of PK-C in m o d u lating theca cell steroidogenesis is similar regardless of the stage of follicular development. Marrone and Asem (85) have recently reported that a dramatic decrease in the ability of theca cells to p r o d u c e a n d r o s t e n e d i o n e (but not cAMP) in r e p o n s e to LH a c c o m p a n i e s the progression of follicles from the F2 to the F, stage of develop-
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TILLY AND JOHNSON
ment. Since the effects of PMA and OAG on LH-promoted steroidogenesis m F, theca are quite similar to those reported to occur during this transitional period, m vivo, we have p r o p o s e d that activation of PK-C may indeed mediate the loss (~f androgenic capacity in F~ theca (83). EFFECTS OF PK-C ON PLASMINOGEN ACTIVATOR (PA) ACTIVITY
We have previously reported that both granulosa and theca cells of preovulato~" follicles contain significant levels of the serine protease, plasminogen activator (63, 64, 66, 73, 86-88). However, the second messenger regulation of PA activity is markedly different in granulosa versus theca cells. For instance, we have shown that activation of the cAMP/PK-A pathway attenuates PA activity in granulosa cells whereas this system serves a stimulatory role in the adjacent theca layer (63, 64, 86). In contrast, the role of PK-C in regulating granulosa and theca PA activity would a p p e a r to be similar in that incubation of either cell type with PMA (but not phorbol 13-monoacetate) causes a dose-dependent increase in PA activity (64, 86). The actions of PK-C apparently require both messenger RNA and protein synthesis since inclusion of the RNA synthesis inhibitor, actinomycin-I), or the protein synthesis blocker, cycloheximide, abolish the stimulatory actions of PMA on PA activity (86). The physiological roles of increased PA activity stimulated by PK-C in granulosa and theca tissue of the hen have yet to be firmly established; however, considering the differential regulation of PA activity by second messenger pathways in granulosa versus theca cells, and the fact that dramatic changes in levels of PA activity occur during the course of follicular d e v e l o p m e n t and ovulation (89), we have p r o p o s e d several roles fbr PK-C relative to PA activity. In granulosa tissue, it appears PK-C may mediate the induction PA activity by h o r m o n e s which are believed to be involved in such processes as cellular differentiation and proliferation (e.g. growth factors), whereas in the adjacent theca layer, PK-C-rnediated induction of PA activity may be involved in the rupture of the follicular stigma at ovulation. HORMONES WHICH MAY ACT VIA PK-C IN THE HEN OVARY
L u t e i n | z | n g H o r m o n e . Hertelendy et al. (61) have reported that LH initiates the metabolism of m e m b r a n e phosphoinositides in hen granulosa cells via a direct activation of phospholipase C (PLC). Concurrent with the decrease in phosphatidylinositol 4,5-bisphosphate following exposure to LH, IP~ is generated and intracellular calcium concentrations increase. Furthermore, the same study indicated that PLC activity apparently increases during the last two days of follicular maturation (as follicles progress from the F~ to the F, and finally to the F l stage of development), clearly indicating the existence of an LH-sensitive, PLC-IPscalcium-signalling pathway in avian granulosa cells. In contrast, a recent report from Johnson et al. (67) demonstrated that both PLC and PEA2, but not LH, stimulate the release of 3H-AA from prelabelled F2 theca cells. These data would indicate the existence of a PLC/PLA2-sensitive m e m b r a n e signalling p a t h w a y within hen theca tissue which is not measurably responsive to LH stimulation. In any case, the extent to which PK-C is or is not activated in avian granulosa or theca cells fk~llowing exposure to LH is not clear at present. G r o w t h F a c t o r s . We have provided evidence that the actions of growth factors, specifically those of epidermal growth factor (EGF) and transff)rming growth factor alpha (TGF0~), on hen granulosa cell steroidogenesis (88) and plasminogen activator activity (86-88) may be mediated via the PK-C system. These proposals are derived from studies which show that the actions of these growth factors are mira-
PROTEIN KINASE C IN THE HEN OVARY
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icked by the phorbol ester, PMA, and can be blocked following cotreatment of cells with either of two putative protein kinase C inhibitors [H-7 and staurosporine: (86, 87)]. The physiological function of growth factors and their relationship to PKC activation in the avian ovary have yet to be unequivocally established; however, in light of a recent report that EGF directly promotes the in vitro proliferation of chicken granulosa cells (90), we have proposed that this and other actions of EGF are mediated, at least in part, via PK-C activation. P r o s t a g l a n d i n s . Previous studies from our lab have indicated that, similar to the actions of PMA, prostaglandins (PGs) E1 and E2 stimulate PA activity in both granulosa and theca cells of mature preovulatory follicles (63,64). Additionally, the stimulatory actions of PGs on PA activity in both cell types are reversed in a dosedependent fashion following coincubation with staurosporine (unpublished data). In hen granulosa cells, the cellular regulation of PA activity is tightly controlled by the interactions of two opposing second messenger pathways: DAG/PK-C (stimulatory) and cAMP/PK-A (inhibitory) (86). Although direct evidence for the involvement of PK-C in mediating the stimulatory effects of PGs on granulosa PA activity are lacking, we have proposed this to be the mechanism of action since PMA mimics (and s t a u r o s p o r i n e reverses) the effects of PGs, and the inhibitory cAMP/PK-A system is clearly not involved. In avian theca cells from the most mature (F 1) preovulatory follicle, PGs also increase PA activity; however, the mechanism of action is not clear since both the DAG/PK-C and cAMP/PK-A pathways increase PA activity (64). Since results from previous reports have shown that membrane receptors for PGEs can be coupled to more than one intracelluar signalling pathway [e.g. both cAMP/PK-A and phosphoinositide metabolism/PK-C; (91)], it is proposed that PGs act via both systems in theca cells for maximal induction of PA activity. This may be of greatest consequence as the time of impending ovulation approaches, since prostaglandin levels are known to be higher in the two largest preovulatory follicles (as compared to less mature follicles in the hierarchy) and substantial increases in PG levels occur in the largest preovulatory (F1) follicle during the periovulatory period (92, 93). Thus, increased proteolytic activity initiated by PGs, combined with increased contractions of follicular smooth muscle bundles within the F 1 follicle in response to prostaglandins (94), may aid in rupture of the weakened stigma and expulsion of the ovum. POSSIBLE ROLES FOR DAG ASIDE FROM PK-C ACTIVATION
One additional point worthy of note concerns the fate of DAG once produced from membrane phospholipids. Since DAG is rapidly acted upon by a specific lipase (forming AA) and/or kinase (forming phosphatidic acid), it is evident that the production of DAG may not only result in the activation of PK-C, but subsequently lead to the formation of additional bioactive second messengers such as AA. We have recently provided evidence that AA is not only released in avian ovarian cells following stimulation with physiologic (e.g. PLC, PLA2) or pharmacologic (e.g. the ionophore A23187) agents, but that AA itself (either produced endogenously via PLA2 or added exogenously) directly influences steroidogenesis and plasminogen activator activity in granulosa and theca cells, in vitro (65-67). Furthermore, the actions of AA are apparently specific and not mediated via lipoxygenase or cyclooxygenase metabolites, nor via the activation of PK-C. Thus, the initial generation of one second messenger (e.g. DAG) is sufficient to cause to activation of several discrete pathways which may act separately or in concert to modify specific cellular responses.
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TILLY AND JOHNSON
G R A N U L O S A CELL
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Fig. 3. Schematic diagram depicting the generation of phosphoinositide metabolites and their actions on the steroidogenic pathway within granulosa and theca cells of preovulatory hen follicles. Note that prorein kinase C has both a stimulatory (+) and inhibito W (-) eflE'ct on steroidogenesis in theca cells depending upon the hormonal environment (see text for further discussion). Abbreviations used are: AA, arachidonic acid; cAMP, cyclic adenosine 3',5'-monophosphate; 1,2-DAG, 1,2-diacylglycerol; EGF. epidermal growth factor; 36-HSD, 36-hydroxysteroid dehydrogenase; LH, luteinizing hormone: PK-A, protein kinase A: PK-C, protein kinase C; PLA_,, phospholipase A=,; PLC, phospholipase C; P,~ ..... cytochrome P.s
Vol. 8(1):1-13, 1991
PROTEIN KINASE C IN PREOVULATORY FOLLICLES FROM THE HEN OVARY J.L. Tilly and A.L. Johnson
Department of Animal Sciences Rutgers, the State University of New Jersey New Brunswick, NJ 08903-0231 Received September 20, 1990
INTRODUCTION
Recent evidence indicates that liberation of 1,2-diacylglycerol (DAG) following ligand-receptor activation of phospholipase C and the ensuing metabolism of specific membrane phospholipids initiates a cascade of several second messenger pathways, including the activation of protein kinase C (PK-C) (for review, see 1-4). For instance, hydrolysis of phosphatidylinositol 4,5-bisphosphate by phospholipase C transiently generates free DAG, which remains intercalated within the cellular membrane, and inositol 1,4,5-trisphosphate (IP3), which is released to the cytosolic compartment. Diacylglycerol, in the presence of calcium and phospholipid, is believed to activate PK-C by decreasing the calcium requirement of the kinase from the milli- to the micromolar range, thus rendering the enzyme fully active without a net change in cytosolic calcium concentrations (5, 6). However, the active lifespan of DAG is very short since the compound is rapidly phosphorylated via diacylglycerol kinase to phosphatidic acid, which can be recycled back into the membrane p h o s p h o l i p i d pool, a n d / o r h y d r o l y z e d by d i a c y l g l y c e r o l lipase to arachidonic acid (AA), which can serve directly as a second messenger or be used for the synthesis of eicosanoids (1, 2). Inositol 1,4,5-trisphosphate, a second metabolite generated following phosphoinositide hydrolysis, is thought to mobilize calcium from cytoplasmic stores via specific binding sites located within the endoplasmic reticular membrane (7, 8). The increase in cytosolic calcium concentrations initiated by IP 3 is proposed to be involved in the activation of calcium/calmodulin dependent protein kinases and/or PK-C. Similar to DAG, IP 3 is only transiently available and is rapidly metabolized to biologically inactive inositol phosphate by-products which can be used for the resynthesis of membrane phosholipids (9). Protein kinase C was originally characterized as a calcium-, diacylglycerol- and phospholipid-dependent serine-threonine protein kinase (5, 10-12). Since its initial characterization, it has been determined that PK-C exists as a ubiquitous family of closely related isoenzymes which are the products of different genes and/or result from alternative splicing of a single gene, and have distinct biochemical properties and tissue distribution (13-21). Protein kinase C exists as a single-chain, 77-80 kD polypeptide containing two major structural domains which can be generated following proteolytic cleavage with calpain. One fragment, of approximately 51 kD, is hydrophilic and contains the catalytic domain which is active in the absence of essential cofactors. The hydrophobic, 26 kD regulatory domain contains the calcium and DAG binding sites, and is therefore believed to be involved in binding of the kinase to the cellular membrane upon activation (2, 6, 22, 24-29). In its inactive state, PK-C is thought to reside primarily in the cytosolic fraction; however, activation of the enzyme is frequently correlated with its translocation to the membrane microenvironment by, as yet, an unidentified mechanism (6, 28). Copyright © 1991 by Domendo, Inc.
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TILLY AND JOHNSON
In the mammalian ()vary, the presence of PK-C activity has been clearly demonstrated (30-34), and several hormones have been implicated in the regulation of PK-C expression and/or activation (35-40). Furthermore, a role for PK-C in modulating ovarian function (e.g. cAMP production, steroidogenesis and plasminogen activator mRNA levels/activity) has been established by several independent researchers (41-51), collectively indicating that this second messenger pathway ,nay play a significant role in the regulation of ovarian function similar to that described for the well characterized adenylyl cyclase/cAMP system. Studies directed at understanding the possible involvement of DAG and PK-C in the regulation of ovarian function have been greatly aided by the discovery that tumor-promoting phorbol esters (e.g. phorbol 12-myristate 13-acetate, PMA; phorbol 12,13-dibutyrate, PDB) or synthetic diacylglycerol analogs (e.g. 1-oleoyl-2acetylglycerol, OAG) can be substituted for endogenous DAG as specific activators of PK-C (2, 52, 53). Additionally, PK-C is believed to be the major phorbol ester receptor within intact cells (53-57). Keeping these aspects in mind, this review describes our current knowledge concerning the presence of PK-C in the avian ()vary and its possible role(s) in the regulation of ovarian steroidogenesis and plasminogen activator (PA) activity. Although it is recognized that additional pathways activated following phosphoinositide hydrolysis (e.g. IP~/calcium/calmodulin and AA/eicosanoids) likely play important roles in the regulation of ovarian function of the hen, these topics will not be addressed in detail here. For information concerning these pathways and their roles in the hen ovary, the reader is referred elsewhere (IP~/calcium/calmodulin: 58-61; AA/eicosanoids: 62-67). EVIDENCE FOR THE EXISTENCE OF PK-C IN THE AVIAN OVARY
Initial studies conducted with hen granulosa cells and phorbol esters demonstrated that binding of 3H-PDB to granulosa cell cytosolic protein is saturable and can be displaced by increasing concentrations of PMA but not phorbol 13-monoacetate (an inactive analog of PMA which does not bind to nor activate PK-C) (68). Furthermore, binding of radiolabelled-PDB to cytosolic protein is associated with its subsequent redistribution to the membrane fraction (68), suggesting the specific translocation of PDB and its receptor (presumably PK-C) to the membrane, a process generally associated with the activation of PK-C. More recently, we have shown that both granulosa and theca cells of the most mature preovulatory (Fj) follicle contain immunoreactive PK-C, and that prior exposure of granulosa cells to PMA results in the translocation of immunoreactive PK-C from the cytosolic to the membrane fraction (granulosa: 66; theca: unpublished data). Although the antibody used in these studies is reported to recognize the ot (Type lid and 13 (Type II), but not y (Type I), subspecies of PK-C, further studies in our lab using monoclonal antibodies specific for each subtype suggest that the 13 form is the predominant immunoreactive subspecies in both brain cerebral cortex and ovarian granulosa tissue, with negligible amounts of eL and y (Figure 1). The lack of detectable 0¢ and y subspecies is apparently due to either very low/nonexistent levels, or the inability of the mammalian monoclonal antibodies to recognize the chicken moieties. Furthermore, preliminary evidence from our laboratory and others have identified the presence of a calcium- and phospholipid-dependent protein kinase within granulosa tissue of hen preovulatory follicles (Table 1) (69). However, the levels of kinase activity in granulosa cell protein are relatively low compared to those levels found in protein preparations of hen cerebral cortex (Table 1). The reason for the substantially lower levels of PK-C activity within ovarian tissue is unclear, but it
PROTEIN KINASE C IN THE HEN OVARY
3
A 1
2
3
4 -.r
s
5
6
?
8
9
:~
2
3
Fig. 1. Western blot of protein kinase C from brain and granulosa tissues. Panel A. Immunoreactivity following electrophoresis of 100 p.g crude cytosolic protein from rat (lanes 1,4,7), hen (lanes 2,5,8) and bovine (lanes 3,6,9) cerebral cortex using specific monoclonal antibodies (Seikagaku Kogyo Co., Tokyo, Japan) directed against rat ~ (lanes 1-3), 13 (lanes 4-6) and ~/(lanes 7-9) protein kinase C subspecies. Panel B. Immunoreactivity from 150 p.g crude cytosolic protein from hen granulosa tissue: lane 1, c~; lane 2, 13; lane 3, "/. Note that the cx and ~/subspecies from hen brain and granulosa tissues are too low to detect, or alternatively, are not immunoreactive with the mammalian monoclonal antibodies. Details of the Western blot analysis have been previously described (66).
m a y b e the result o f high levels o f an e n d o g e n o u s PK-C inhibitor as has b e e n rec e n t l y d e s c r i b e d for h u m a n p l a c e n t a l a n d rat o v a r i a n tissues (70). In a n y case, t h e s e d a t a t a k e n collectively clearly indicate the p r e s e n c e o f PK-C w i t h i n o v a r i a n tissue o f the d o m e s t i c hen, a n d its characteristic r e s p o n s i v e n e s s to t u m o r - p r o m o t ing p h o r b o l esters.
EFFECTS OF PIK-C ON STEROIDOGENESIS G r a n u l o s a T i s s u e . T h e first p u b l i s h e d r e p o r t attributing a specific role for PK-C in t h e a v i a n o v a r y a r o s e f r o m s t u d i e s w h i c h d e m o n s t r a t e d that PMA a n d O A G m o d u l a t e luteinizing h o r m o n e ( L H ) - i n d u c e d s t e r o i d o g e n e s i s in h e n g r a n u l o s a cells c o l l e c t e d from the F1 follicle (71). T h e s e c o m p o u n d s d o n o t affect b a s a l p r o g e s t e r o n e o r a n d r o g e n p r o d u c t i o n , b u t a t t e n u a t e LH-stimulated s t e r o i d o g e n e s i s in a d o s e - d e p e n d e n t fashion. A s u b s e q u e n t r e p o r t f r o m A s e m a n d T s a n g (72) c o n f i r m e d t h e s e initial f i n d i n g s o f a n i n h i b i t o r y a c t i o n o f PMA o n L H - p r o m o t e d s t e r o i d o g e n e s i s in h e n g r a n u l o s a cells. T h e actions o f PMA are p r e s u m e d specific for PK-C activation since c o m p a r a b l e effects are o b t a i n e d w i t h a d i a c y l g l y c e r o l a n a l o g b u t n o t w i t h the inactive PMA analog, p h o r b o l 1 3 - m o n o a c e t a t e (71). In fact,
TABLE 1. PROTEIN KINASE C ACTIVITYIN HEN BRMN CORTEX, AND GRANULOSATISSUE COLLECTEDFROM THE LARGEST PREOVULATORY(Fi) FOLLICLE)
Tissue N Specific Activity~ Cerebral Cortex 4 22.0 -+ 3.9 F, Granulosa 3 3.4 ~ 2.0 *Activity (pmol 32p transferred/min/mg protein) was measured from cytosolic protein which was prepared as previously described (66). The assay was conducted with (~/-~aP)-ATPin the absence and presence of CaCI2 (15 mM) plus phosphatidylserine (50 Izg)/diolein (6 I~g) and calf thymus histone H1 (100 ~g), as described by Kikkawa et al. (95). 2Mean ± SE (N=number of replicate experiments).
4
TILLY AND JOHNSON
the inhibitory effects of PMA are not limited to LH-induced steroid producti(m since steroidogenesis stimulated by other hormonal ligands which act via adcnylyl cyclase/cAMP in hen granuk)sa cells (i.e. vasoactive intestinal peptide; 73) is similarly suppressed (71). A subsequent study clarified that the actions of PK-C on LH-promoted steroid output are directed at sites both prior and distal to cAMP formation (adenylyl cyclase, and cytochrome P,~,,~ function/cholesterol transport, respectively) (74). Again, the effects of PMA are believed specific tk)r PK-C activation since the inhibitory actions of PMA on LH-stimulated cAMP formation are reversed in a dosedependent fashion by the putative PK-C inhibitor, staurosporine (74). Ahhough staurosporine is known to inhibit not only PK-C, but also PK-A activity, in both dispersed hen granulosa cells (75; unpublished data) and in cell-free preparations of PK-A (75), the results from the aforementioned studies cannot be attributed to inhibitory effects of staurosporine on PK-A activity. More recently, in an effort to clearly elucidate the site of action of PK-C distal to cAMP formation, we have evaluated the effects of PMA on levels of immunoreactive P,s0,~ protein and the kinetic parameters of P,~.~. activity in crude mitochondrial protein prepared from F~ granulosa tissue. Results from these studies indicate that pretreatment of cells with PMA and LH does not affect the levels of immunoreactive P~s0~, protein compared to vehicle or LH-pretreated cells, but decreases the ability of P,~,~ isolated from mitochondria to convert 25-hydroxycholesterol to pregnenolone (unpublished data). From these studies, we propose that PK-C may directly phosphorylate P,~,,~ in avian granulosa tissue, thereby reducing the ability of the enzyme to effectively convert cholesterol precursor to pregnenolone. Phosphorylation studies using radiolabelled-ATP as a phosphate donor are currently underway to substantiate this proposal. Finally, granulosa cells isolated from less mature, growing tkJlicles (6-8 mm in diameter; approximately 3 weeks prior to ovulation) in the hen ovary have been shown to be steroidogenically incompetent, failing to produce progesterone in response to LH, follicle-stimulating hormone (FSH), 8-bromo-cAMP or 25-hydroxycholesterol (76, 77). However, we have found that preincubation of these granulosa cells with forskolin for 24 hr renders the cells c o m p e t e n t to metabolize 25-hydroxycholesterol to progesterone, suggesting the induction of P~s0~,~ activity (Figure 2; Tilly JL, Kowalski KI and Johnson AL, unpublished data). Interestingly, coincubation of 6-8 mm fkJlicle granulosa cells with forskolin in the presence of PMA abolishes the stimulatory actions of the adenylyl cyclase activator on the induction of P~s0~ activity, but does not affect the ability of these granulosa cells to metabolize pregnenolone to progesterone (Figure 2). From a physiological standpoint, we have proposed that the activation of PK-C within granulosa cells of the hen serves at least two specific functions associated with steroidogenesis. First, it has previously been reported that a dramatic increase in adenylyl cyclase activity occurs in F~ granulosa cells during the hours preceding the preovulatory LH and progesterone surges (78), which is followed by a shortterm period of steroidogenic "desensitization" (79-81). During this period of "desensitization", g o n a d o t r o p i n - i n d u c e d adenylyl cyclase activity is markedly decreased, leading to the termination of the p r e o v u l a t o r y p r o g e s t e r o n e surge. However, the decrease in progesterone production by F~ granulosa cells cannot be attributed solely to a decrease in LH-stimulated adenylyl cyclase activity, since granulosa cells collected during this densensitization period also fail to produce progesterone in response to cAMP analogs (82). Therefore, the short-term suppression of LH-promoted steroidogenesis and the subsequent termination of the pre-
PROTEIN KINASE C IN THE HEN OVARY
5
10000 ~" O
[] [] [] \1~
1000
o
o
100
&
10
CON FOR FOR+PMA PMA /
I
24 h prelncubation
i! CON
CHOL
P5
i
3 h Incubation
Fig. 2. Progesterone production from 6-8 mm follicle granulosa cells incubated for 3 hr in the absence and presence of 25-hydroxycholesterol (CHOL; 2500 ng) or pregnenolone (P4 20 ng) following a 24-hr preincuhation with vehicle (CON; control), PMA (100 riM), forskolin (FOR; 10 I.LM)or PMA + forskolin. Data are expressed as the mean _+ SE of results from 3 replicate incubations. These data would indicate that activation of the protein kinase C system negatively modulates the induction of functional P,s,,,,~ stimulated by cAMP/protein kinase A in granulosa cells of developing follicles, but does not significantly alter 313-hydroxysteroiddehydrogenase activity.
ovulatory progesterone surge, which o c c u r as a result of a decrease in responsiveness to LH both prior and distal to cAMP formation, m a y be initiated via PK-C activation. Secondly, the induction of steroidogenic c o m p e t e n c e in avian granulosa cells of developing follicles 2-3 w e e k s prior to ovulation by the cAMP/PK-A pathw a y m a y be tightly regulated by a tonic, s u p p r e s s i v e effect o f PK-C activity. Although the cellular site of action of PK-C on the induction of steroidogenic c o m p e t e n c y in maturing avian granulosa cells is unclear at present, w e are currently evaluating the effects of PK-C on P.,~0~c~ messenger RNA expression, levels of immunoreactive protein and e n z y m e activity in these cells. T h e c a T i s s u e . In theca cells collected from the s e c o n d largest preovulatory (F,) follicle, PMA (but not p h o r b o l 13-monoacetate) and OAG have b e e n s h o w n to suppress LH-stimulated a n d r o s t e n e d i o n e production in a c o n c e n t r a t i o n - d e p e n d e n t manner, but d o not alter c o r r e s p o n d i n g cAMP levels or progesterone output (83). Similar effects of these c o m p o u n d s have also b e e n reported for steroid production by theca cells of less mature, developing follicles (6-8 m m in diameter) in the hen ovary (84), suggestive of a functional PK-C system within less mature ovarian follicles. These findings contrast those reported for avian granulosa cells, since there appears to be solely a post-cAMP inhibitory action of PK-C in theca tissue, apparently at C17,20-1yase. In addition, basal levels of progesterone, a n d r o s t e n e d i o n e and estradiol are increased following incubation of F e or 6-8 m m follicle theca cells with PMA, further indicating that distinct differences exist in the actions of PK-C within granulosa and theca cells. In contrast, it appears the role of PK-C in m o d u lating theca cell steroidogenesis is similar regardless of the stage of follicular development. Marrone and Asem (85) have recently reported that a dramatic decrease in the ability of theca cells to p r o d u c e a n d r o s t e n e d i o n e (but not cAMP) in r e p o n s e to LH a c c o m p a n i e s the progression of follicles from the F2 to the F, stage of develop-
6
TILLY AND JOHNSON
ment. Since the effects of PMA and OAG on LH-promoted steroidogenesis m F, theca are quite similar to those reported to occur during this transitional period, m vivo, we have p r o p o s e d that activation of PK-C may indeed mediate the loss (~f androgenic capacity in F~ theca (83). EFFECTS OF PK-C ON PLASMINOGEN ACTIVATOR (PA) ACTIVITY
We have previously reported that both granulosa and theca cells of preovulato~" follicles contain significant levels of the serine protease, plasminogen activator (63, 64, 66, 73, 86-88). However, the second messenger regulation of PA activity is markedly different in granulosa versus theca cells. For instance, we have shown that activation of the cAMP/PK-A pathway attenuates PA activity in granulosa cells whereas this system serves a stimulatory role in the adjacent theca layer (63, 64, 86). In contrast, the role of PK-C in regulating granulosa and theca PA activity would a p p e a r to be similar in that incubation of either cell type with PMA (but not phorbol 13-monoacetate) causes a dose-dependent increase in PA activity (64, 86). The actions of PK-C apparently require both messenger RNA and protein synthesis since inclusion of the RNA synthesis inhibitor, actinomycin-I), or the protein synthesis blocker, cycloheximide, abolish the stimulatory actions of PMA on PA activity (86). The physiological roles of increased PA activity stimulated by PK-C in granulosa and theca tissue of the hen have yet to be firmly established; however, considering the differential regulation of PA activity by second messenger pathways in granulosa versus theca cells, and the fact that dramatic changes in levels of PA activity occur during the course of follicular d e v e l o p m e n t and ovulation (89), we have p r o p o s e d several roles fbr PK-C relative to PA activity. In granulosa tissue, it appears PK-C may mediate the induction PA activity by h o r m o n e s which are believed to be involved in such processes as cellular differentiation and proliferation (e.g. growth factors), whereas in the adjacent theca layer, PK-C-rnediated induction of PA activity may be involved in the rupture of the follicular stigma at ovulation. HORMONES WHICH MAY ACT VIA PK-C IN THE HEN OVARY
L u t e i n | z | n g H o r m o n e . Hertelendy et al. (61) have reported that LH initiates the metabolism of m e m b r a n e phosphoinositides in hen granulosa cells via a direct activation of phospholipase C (PLC). Concurrent with the decrease in phosphatidylinositol 4,5-bisphosphate following exposure to LH, IP~ is generated and intracellular calcium concentrations increase. Furthermore, the same study indicated that PLC activity apparently increases during the last two days of follicular maturation (as follicles progress from the F~ to the F, and finally to the F l stage of development), clearly indicating the existence of an LH-sensitive, PLC-IPscalcium-signalling pathway in avian granulosa cells. In contrast, a recent report from Johnson et al. (67) demonstrated that both PLC and PEA2, but not LH, stimulate the release of 3H-AA from prelabelled F2 theca cells. These data would indicate the existence of a PLC/PLA2-sensitive m e m b r a n e signalling p a t h w a y within hen theca tissue which is not measurably responsive to LH stimulation. In any case, the extent to which PK-C is or is not activated in avian granulosa or theca cells fk~llowing exposure to LH is not clear at present. G r o w t h F a c t o r s . We have provided evidence that the actions of growth factors, specifically those of epidermal growth factor (EGF) and transff)rming growth factor alpha (TGF0~), on hen granulosa cell steroidogenesis (88) and plasminogen activator activity (86-88) may be mediated via the PK-C system. These proposals are derived from studies which show that the actions of these growth factors are mira-
PROTEIN KINASE C IN THE HEN OVARY
7
icked by the phorbol ester, PMA, and can be blocked following cotreatment of cells with either of two putative protein kinase C inhibitors [H-7 and staurosporine: (86, 87)]. The physiological function of growth factors and their relationship to PKC activation in the avian ovary have yet to be unequivocally established; however, in light of a recent report that EGF directly promotes the in vitro proliferation of chicken granulosa cells (90), we have proposed that this and other actions of EGF are mediated, at least in part, via PK-C activation. P r o s t a g l a n d i n s . Previous studies from our lab have indicated that, similar to the actions of PMA, prostaglandins (PGs) E1 and E2 stimulate PA activity in both granulosa and theca cells of mature preovulatory follicles (63,64). Additionally, the stimulatory actions of PGs on PA activity in both cell types are reversed in a dosedependent fashion following coincubation with staurosporine (unpublished data). In hen granulosa cells, the cellular regulation of PA activity is tightly controlled by the interactions of two opposing second messenger pathways: DAG/PK-C (stimulatory) and cAMP/PK-A (inhibitory) (86). Although direct evidence for the involvement of PK-C in mediating the stimulatory effects of PGs on granulosa PA activity are lacking, we have proposed this to be the mechanism of action since PMA mimics (and s t a u r o s p o r i n e reverses) the effects of PGs, and the inhibitory cAMP/PK-A system is clearly not involved. In avian theca cells from the most mature (F 1) preovulatory follicle, PGs also increase PA activity; however, the mechanism of action is not clear since both the DAG/PK-C and cAMP/PK-A pathways increase PA activity (64). Since results from previous reports have shown that membrane receptors for PGEs can be coupled to more than one intracelluar signalling pathway [e.g. both cAMP/PK-A and phosphoinositide metabolism/PK-C; (91)], it is proposed that PGs act via both systems in theca cells for maximal induction of PA activity. This may be of greatest consequence as the time of impending ovulation approaches, since prostaglandin levels are known to be higher in the two largest preovulatory follicles (as compared to less mature follicles in the hierarchy) and substantial increases in PG levels occur in the largest preovulatory (F1) follicle during the periovulatory period (92, 93). Thus, increased proteolytic activity initiated by PGs, combined with increased contractions of follicular smooth muscle bundles within the F 1 follicle in response to prostaglandins (94), may aid in rupture of the weakened stigma and expulsion of the ovum. POSSIBLE ROLES FOR DAG ASIDE FROM PK-C ACTIVATION
One additional point worthy of note concerns the fate of DAG once produced from membrane phospholipids. Since DAG is rapidly acted upon by a specific lipase (forming AA) and/or kinase (forming phosphatidic acid), it is evident that the production of DAG may not only result in the activation of PK-C, but subsequently lead to the formation of additional bioactive second messengers such as AA. We have recently provided evidence that AA is not only released in avian ovarian cells following stimulation with physiologic (e.g. PLC, PLA2) or pharmacologic (e.g. the ionophore A23187) agents, but that AA itself (either produced endogenously via PLA2 or added exogenously) directly influences steroidogenesis and plasminogen activator activity in granulosa and theca cells, in vitro (65-67). Furthermore, the actions of AA are apparently specific and not mediated via lipoxygenase or cyclooxygenase metabolites, nor via the activation of PK-C. Thus, the initial generation of one second messenger (e.g. DAG) is sufficient to cause to activation of several discrete pathways which may act separately or in concert to modify specific cellular responses.
8
TILLY AND JOHNSON
G R A N U L O S A CELL
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Fig. 3. Schematic diagram depicting the generation of phosphoinositide metabolites and their actions on the steroidogenic pathway within granulosa and theca cells of preovulatory hen follicles. Note that prorein kinase C has both a stimulatory (+) and inhibito W (-) eflE'ct on steroidogenesis in theca cells depending upon the hormonal environment (see text for further discussion). Abbreviations used are: AA, arachidonic acid; cAMP, cyclic adenosine 3',5'-monophosphate; 1,2-DAG, 1,2-diacylglycerol; EGF. epidermal growth factor; 36-HSD, 36-hydroxysteroid dehydrogenase; LH, luteinizing hormone: PK-A, protein kinase A: PK-C, protein kinase C; PLA_,, phospholipase A=,; PLC, phospholipase C; P,~ ..... cytochrome P.s
