Vol. 131, No. 4 Printed in U.S.A.
The Role of Protein Kinase-C in Gonadotropin-Induced Ovulation in the in Vitro Perfused Rabbit Ovary* G. KAUFMAN,
A. M. DHARMARAJAN,
Department of Gynecology and Obstetrics, Baltimore, Maryland 21205
Y. TAKEHARA, Johns Hopkins
C. S. CROPP,
University
AND
E. E. WALLACH
School of Medicine,
ABSTRACT Tumor-promoting phorbol esters are believed to affect ovarian granulosa cell progesterone and prostaglandin (PG) production and possibly ovulation by activating protein kinase-C (PKC). The effects of phorbol esters and PKC inhibitors on ovulation, progesterone, and PG production were examined in an in vitro perfused rabbit ovary. The effect of tranexamic acid, an inhibitor of the conversion of plasminogen activator to plasmin, on phorbol ester-induced ovulation was also examined. Phorbol l&13-dibutyrate (PdBU), a PKC stimulator, induced ovulation in a dose-related manner in the absence of gonadotropins (56%, 200 nM PdBU; O%, 0 nM PdBU; P< 0.05). Perfusate progesterone levels were increased only after 600 nM PdBU treatment, and perfusate PGF2,., PGE,, and 6-keto-PGF],, were increased in a dose-dependent fashion (P < 0.05). Staurosporine, a potent inhibitor of the catalytic domain of PKC, and calphostin-C, a specific inhibitor of the diacyl-
glycerol-binding region, inhibited hCG-induced ovulation in a doserelated manner. Gonadotropin-induced ovulation decreased from 73% without staurosporine to 19% with 1.0 pM staurosporine (P < 0.01). Calphostin-C reduced ovulatory efficiency from 60% to 24% (P < 0.01). However, neither inhibitor decreased progesterone or PGF,, production by ovaries exposed to hCG. hCG-induced oocyte maturation was also unaffected by exposure to either staurosporine or calphostin-C. Tranexamic acid reduced phorbol ester-induced ovulatory efficiency from 67% to 37% (P < 0.05). These findings demonstrate that the calciumdependent PKC pathway is instrumental in gonadotropin-mediated follicular rupture in the rabbit. Although PGs may play an important role in ovulation, they do not appear to be directly responsible for PKC-mediated follicular rupture. (Endocrinology 131: 1804-1809, 1992)
E
Recently, the calcium phospholipid-dependent PKC pathway has been implicated as a second major signal transduction mechanism for hormone-mediated ovarian function (11, 12). The PKC pathway requires the hydrolysis of phosphatidylinositol 4,5-bisphosphate into diacylglycerol (DG) and inositol triphosphate by phospholipase-C. DG activates PKC, which, in turn, phosphorylates specific intracellular proteins. GnRH, LH, and prostaglandin FZn(PGF*,), substancesthat have been implicated in ovulation in the in vitro perfused rabbit ovary, have been shown to increasephosphoinositide metabolism in rat granulosa cells and bovine and rat luteal cells (3, 12-14). Recent evidence also indicates that the murine LH receptor may be capable of activating both PKC and adenylate cyclase, although with different EDsovalues (15). GnRH, PGF2,,,and possibly gonadotropins also mediate steroid hormone production through the PKC pathway (12). A potent phorbol ester, 12-0-tetradecanoyl-phorbol 13-acetate, which activates PKC, can regulate rat granulosa cell progesterone and PG production (11). Phorbol estersare also able to stimulate tissue plasminogen activator (tPA) mRNA production and decreasethe secretion of plasminogen activator inhibitor-l from human endothelial cells, an effect that is potentiated by CAMP (16-18). Plasminogen activator is thought to activate the proteolytic enzyme cascade,eventually resulting in the production of collagenaseand follicular rupture (1). Recently, Guerre, Jr., et al. (19) observed that staurosporine, a PKC inhibitor, inhibits PMSG-induced ovulation in the rat (19). Taken together, these findings implicate a possible role for phospholipid-PKC signal transduction in mediating gonadotropin-induced follicular rupture. The objectives of the study were to investigate: 1) the
of the extracorporeal perfused rabbit ovary to hCG consistently results in ovulation (1). Many substancesappear to be involved locally in the ovulatory process, including proteolytic enzymes (i.e. plasmin and collagenase), arachidonic acid metabolites, histamine, bradykinin, angiotensin-II, oxygen free radicals, and cytokines (1, 2). However, the intracellular signal transduction pathways by which the LH surge or gonadotropin stimulus results in follicular rupture
XPOSURE
remain
unclear.
It is well established that gonadotropins (FSH, LH, and hCG) induce steroidogenesisand cell differentiation through the CAMP-adenylate cyclase pathway (3-5). Oocyte maturation
as well
as gonadotropin-induced
follicular
rupture
may
also be dependent on CAMP (6-9). In contrast, we have observed that (Bu)~cAMP fails to induce ovulation in the absence of gonadotropins (6), and CAMP inhibits hCG-induced ovulation in the in vitro perfused rabbit ovary (10). Although most studies have focused on the CAMP pathway, little attention has been given to protein kinase-C (PKC) signal transduction. Received May 7, 1992. Address requests for reprints to: E. E. Wallach, M.D., Department of Gynecology and Obstetrics, Johns Hopkins Medical Institutions, 600 North Wolfe Street, Houck 264A, Baltimore, Maryland 21205. Address all correspondence to: A. M. Dharmarajan, Ph.D., Department of Gynecology and Obstetrics, Johns Hopkins Medical Institutions, 600 North Wolfe Street, Park 82-202, Baltimore, Maryland 21205. *This work was supported by NIH Grant HD-19430 (to E.E.W.), Population Center Grant HD-06268 (to A.M.D.), the SmithKline Beecham Fellowship (to Y.T.), and the Rockefeller Foundation (to A.M.D.). Presented in part at the 36th Annual Meeting of Society for Gynecologic Investigation, San Diego, CA, March 15-18, 1989.
1804
ROLE
OF PKC
effect of phorbol 12,13-dibutyrate (PdBU), a stimulator of PKC, on ovulation and progesterone and PG production in the absence of gonadotropins; 2) the effect of staurosporine, a potent inhibitor of PKC, on gonadotropin-induced ovulation and progesterone and PG production; 3) the effect of calphostin-C, a specific PKC inhibitor, on gonadotropininduced ovulation and progesterone and PG production; and 4) the effect of tranexamic acid [trans-4-(aminomethyl)cyclohexanecarboxylic acid], an inhibitor of the conversion of plasminogen to plasmin, on PdBU-induced ovulation. To this end, an in vitro perfused rabbit ovary model was used to investigate the effects of these agents on ovarian function in a carefully controlled and monitored environment.
Materials
and Methods
Animals New Zealand White mature female rabbits, weighing 3.0-4.5 kg, were used. All rabbits were caged individually for at least 3 weeks before use. Animals were given water and an unrestricted diet of rabbit chow. Rabbits were anesthetized with iv sodium pentobarbital (32 mg/kg), treated with heparin sulfate (120 U/kg) for anticoagulation, and then subjected to laparotomy. Ovaries were excluded from the study if they were immature or if two or more of the follicles were hemorrhagic at the time of laparotomy. Experimental ovaries were excluded if the paired contralateral control unstimulated ovary ovulated, possibly due to an endogenous LH surge before ovariectomy. All studies were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals.
In vitro perfusion The cannulation procedure and perfusion apparatus have been described in detail previously (20-22). Each ovarian artery was isolated and cannulated in situ after ligation of major anastomotic connections. The ovary with its cannulated vascular pedicle and supportive connective tissue was removed. The number of mature follicles (>1.5 mm in diameter) observed on the surface of each ovary was recorded, and the ovary was immediately placed in the perfusion chamber. The perfusion system consists of a chamber containing the ovary, an oxygenator, a reservoir, and a pulsatile roller pump that maintains perfusate flow at 1.5 ml/min, the approximate blood flow to the rabbit ovary (23). The oxygenator was gassed with 95% 0,.5% CO,. Ovaries were perfused for 10.5 h at 37 C in 150 ml medium 199 (Gibco, Grand Island, NY) supplemented with heparin sulfate (200 U/liter), insulin (20 U/liter), streptomycin sulfate (5b mg/liter), and penicillin G (75 mg/liter). Perfusate samules were collected from the arterial cannula 0, 0.5, 1, 2, 4, 6, 8, and 10 h after hCG or PdBU administration and replaced with an equal volume of fresh medium. Samples were stored at -20 C for later determination of I’G and progesterone concentrations. The ovary was monitored for fresh ovulation points every half-hour. A follicle was considered to be ruptured when the cumulus containing an ovum was observed protruding from the ovarian surface. Ovulatory efficiency was calculated using the following formula: [number of ovulated ova/number of mature follicles] X 100 (20, 21).
Experimental
IN OVULATION
1805
ovaries were treated with 6 PM 4cu-phorbol 12,13-didecanoate, and observations were compared to those of control ovaries. Samples were collected for determination of progesterone and PG concentrations, and ovulatory efficiency was calculated as described previously. In selected experiments mean arterial pressure was determined by a manometer attached to the arterial cannula in both control and treated ovaries. Effects of staurosporine and calphostin-C on hCG-induced ovulation, progesterone and PG production, and oocyte maturation. These experiments were designed to investigate the effect of PKC inhibition on hCGinduced ovulation and progesterone and PGs production. Thirty-six rabbits were used for this experiment. Staurosporine (Sigma; 0.5 mg) and calphostin-C (Kamiya Biomedical, Thousand Oaks, CA; 1.0 mg) were dissolved in 1.0 ml medium 199 or dimethylsulfoxide, respectively. Inhibitor or vehicle alone was dissolved in 150 ml perfusion medium to concentrations of 0.1, 0.5, and 1.0 PM staurosporine or 0.2, 1.0, and 2.0 PM calphostin-C before the onset of the experiment. One hundred international units of hCG were administered via the arterial cannula at 0 h, and samples were collected for determination of progesterone and PG concentrations. Ovulatory efficiency was calculated, and ovulated ova surrounded by their cumulus mass were carefully recovered from the perfusion chamber with a glass pipette. Cumulus cells were removed with gentle agitation after exposure to hyaluronidase (0.34 U/ml; Sigma Chemical Co.). The denuded oocytes were assessed for the presence or absence of germinal vesical breakdown. At the conclusion of the experiment, unruptured oocyte-cumulus complexes were obtained by rupturing the remaining mature intact follicles with a 24-gauge needle. These ova were also assessed cytologically for germinal vesical breakdown. Effect of tranexamic acid on PdBU-induced ovulation. This experiment was designed to investigate the effect of inhibition of the early steps in proteolytic enzyme cascade on phorbol ester-induced ovulation. Six rabbits were used for this experiment. Tranexamic acid (Sigma) was added to the perfusate at a concentration of 10 mM. The contralateral ovary was perfused without tranexamic acid. Thirty minutes after the onset of the perfusion, PdBU (0.2 PM) was added to the perfusate. Ovulatory efficiency was calculated as described above.
Progesterone
and PG RIAs
Progesterone concentrations in perfusate samples were measured using a solid phase kit (Diagnostic Products Corp., Los Angeles, CA). Progesterone antibody is bound covalently to the inner surface of polypropylene assay tubes. All samples and standards were assayed in duplicate. The intra- and interassay variations were 7.5% and 6.6%, respectively. Perfusate concentrations of I’GF>,,, PGE,, and 6-ketoPGFi,,, a metabolite of prostacyclin, were measured by methods previously described (24). PGF2,. and PGEz antibodies each cross-react less than 2% with the other I’G classes tested. The 6-keto-PGF,,, antibody cross-reacts less than 0.1% with the primary I’Gs. For each prostanoid measured, the intraassay coefficient of variation was less than 8%, and samples from each experiment were assayed simultaneously to avoid error due to interassay variation.
Statistical
analysis
Data for PG and progesterone levels were evaluated by analysis of variance, with P < 0.05 considered significant. Comparison of ovulatory efficiencies was performed using x2 analysis, and P < 0.05 was considered significant. Dose-response studies were designed using a balanced block method; the order of the ovaries for each experiment was determined by a random number table.
design
Effect of phorbol ester on ovulation and progesterone and PG production. This experiment was designed to investigate the effect of PKC stimulation on ovulation and progesterone and PG production in the absence of hCG. Twenty-one rabb& were used for this experiment, One millieram of PdBU (Siema Chemical Co., St. Louis, MO) was dissolved in 1 o. v ml 100% ethyl alcohol. Ovaries were placed in the perfusion chamber at -0.5 h, and PdBU (to a final concentration of 0.02, 0.1, 0.2, or 0.6 1~) or vehicle alone was administered via arterial cannula at 0 h. Four
Results Effect of PdBU on ovulation the absence of hCG
and progesterone
and PG levels in
PdBU induced ovulation in the in vitro perfused rabbit ovary in a dose-related manner in the absence of gonadotro-
ROLE
1806
OF PKC
pins (Fig. 1). Ovulatory efficiency varied from 0% for control and 20 nM PdBU-treated ovaries to 56% for 200 nM PdBUtreated ovaries. 4Lu-Phorbol 12,13-didecanoate, an inactive phorbol ester, did not stimulate ovulation in the absenceof hCG even at dosesas high as 6 PM (Fig. 1). Perfusate PGF2,, levels significantly increased from 143.3 f 26.2 pg/ml for 0 nM PdBU-treated ovaries (control) to 893.8 f 175.9 pg/ml after 200 nM PdBU treatment (P < 0.05; Fig. 2). PGE2and 6keto-PGF,,, also increased significantly after 200 nM PdBU administration compared to levels in control ovaries (P < 0.05; Fig. 2). PG concentrations were significantly elevated after 600 nM PdBU administration (P < 0.05; Fig. 2). Perfusate levels of progesterone were unchanged after treatment with 200 nM PdBU compared to control values (Fig. 3). However, perfusate progesterone levels increasedsignificantly after 600
IN OVULATION
Endo. 1992 Vol 131. No 4
nM PdBU treatment (8.2 f 1.4 rig/ml) compared to the control level (3.0 + 0.4 rig/ml; P < 0.05; Fig. 3). Mean arterial pressure was unaffected after exposure of the ovaries to 200 nM PdBU (n = 4; 63 mm mercury) compared with 0 nM PdBU (control; n = 4; 60 mm mercury). Effect of staurosporine on hCG-induced and PG levels, and oocyte maturation
ovulation,
progesterone
hCG-induced ovulation was inhibited by staurosporine compared to that in hCG-treated controls (P < 0.01; see Table 1). Ovulatory efficiency was reduced from 73% for ovaries perfused with hCG alone to 48% for hCG plus 0.5 PM staurosporine-treated ovaries and 19% for hCG plus 1.0 PM staurosporine-treated ovaries. Staurosporine did not affect perfusate progesterone levels (which varied from 13.4 + 3.2 to 26.2 f 13.4 rig/ml) and did not inhibit perfusate PG levels in response to hCG (data not shown). Staurosporine had no effect on hCG-induced oocyte maturation (seeTable 3). Effect of calphostin-C on hCG-induced and PG levels, and oocyte maturation
0
20
100
PdBU
200
600
(nM)
‘6 phorbol 12,13did.canoat,
FIG. 1. Ovulatory efficiency (percent) of PdBU- and 4ol-phorbol12,13didecanoate-treated ovaries compared to control ovaries perfused with medium containing vehicle. Ovaries were treated with the indicated concentrations of phorbol ester, and ovulatory efficiency was calculated as described in Materials and Methods. Each value is expressed as a mean. The values represent 4-12 ovaries/group. *, P < 0.01 us. 0 nM PdBU (control). P < 0.001 for all values taken together.
"
ovulation,
progesterone
hCG-induced ovulation was also inhibited in a dosedependent manner by calphostin-C compared to that in hCG-treated controls (P < 0.05; Table 2). Ovulatory efficiency was reduced from 60% for ovaries perfused with hCG alone to 31% for hCG plus 1.0 PM-calphostin-C-treated ovaries and 24% for hCG plus 2.0 PM calphostin-C-treated ovaries. Calphostin-C did not affect perfusate progesterone levels (which varied from 5.9 + 1.0 to 3.0 + 1.6 rig/ml) and did not inhibit hCG-stimulated PG production (data not shown), nor did calphostin-C influence hCG-induced oocyte maturation (Table 3).
10000-
*
E 2 UJ 5
FIG. 2. Effects of the indicated PdBU concentrations on perfusate PGF,,,, PGE,, and 6-keto-PGF1, levels. Control perfusions (0 nM PdBU) contained vehicle. Measurements of PGs were performed as described in Materials and Methods. The values are expressed as the mean + SE and represent six to nine ovaries per group. *, P < 0.05 VS. 0 nM PdBU (control).
6000
-
n q
ii J .-c
OnM PdBU 200 nM PdBU 600 nM PdBU
6000
-
4000
-
z 2 z 5; 2 n
PGF
2a
PGE
6-keto-PGF 2
la
ROLE
OF PKC
IN OVULATION TABLE 3. Percent germinal vesicle breakdown (GVBD) of ovulated and follicular oocytes perfused with hCG alone or with hCG plus staurosporine or calphostin-C Staurosporine
Ovulated oocytes GVBD (%) Follicular oocytes GVBD (%)
0.1
0.5
1.0
100
100
96
100
100
100
87
100
Calphostin-C 0
Ovulated oocytes GVBD (W) Follicular oocytes GVBD (%) PdBU
(nM)
FIG. 3. Effects of the indicated PdBU concentrations on perfusate progesterone levels. Progesterone values were determined as described in Materials and Methods. The values are expressed as the mean f SE and represent six to nine ovaries per group. *, P < 0.05 us. control.
TABLE in uitro
1. Effect of staurosporine on hCG-induced perfused rabbit ovary
0
10 10 45 33 73
2. Effect of calphostin-C perfused rabbit ovary
0.1
0.5
1.0
8 8
1
7
7
35 19 54
44 21 48
4 31 6 19”
on hCG-induced Calphostin-C
Perfused ovaries (no.) Ovaries ovulated (no.) Mature follicles (no.) Ovulations (no.) Ovulatory efficiency (%)
ovulation in the (PM)
0
0.2
1.0
2.0
11 11
6 6 28 16 57
6 6 51 16 31”
6 5 42 10 24”
57 34 60
Ovaries were perfused with calphostin-C (0.2, 1.0, and 2.0 pM) or vehicle (0 pM) and stimulated to ovulate by hCG (100 IU) 30 min after the onset of perfusion, as described in Materials and Methods. P < 0.001 for all values taken together. ” P < 0.05 compared to control. Effect of tranexamic
acid on PdBU-induced
(MM) 1.0
2.0
90
79
93
70
82
90
95
81
Ovaries were perfused with either staurosporine or calphostin-C, at the indicated concentrations, and hCG (100 IU). Controls (0 PM) received vehicle alone. Ovulated and follicular oocytes were retrieved and denuded, as described in Materials and Methods. The presence or absence of GVBD was assessed.
(PM)
Ovaries were perfused with staurosporine (0, 0.1, 0.5, and 1.0 PM) and stimulated to ovulate by hCG (100 IU) 30 min after the onset of perfusion, as described in Materials and Methods. P < 0.001 for all values taken together. ’ P < 0.01 compared to control. TABLE in vitro
0.2
ovulation in the
%XJrOSpOrine
Perfused ovaries (no.) Ovaries ovulated (no.) Mature follicles (no.) Ovulations (no.) Ovulatory efficiency (%)
(PM)
0
ovulation
Tranexamic acid significantly inhibited phorbol ester-induced ovulation. Ovulatory efficiency was reduced from 67% to 37% (P < 0.05) after treatment of PdBU-exposed ovaries with 10 mM tranexamic acid (see Fig. 4).
PdBU
PdBU
+ 10 mM
tAMCHA
FIG. 4. Effect of 10 mM tranexamic acid (tAMCHA) on PdBU-induced ovulatory efficiency. Ovaries were perfused with 200 nM PdBU in the presence and absence of tranexamic acid. Ovulatory efficiency was determined as described in Materials and Methods. Each value is expressed as a mean. *, P < 0.05 compared to PdBU alone. Discussion In the present study a rabbit ovarian in vitro perfusion model was used to investigate the role of the calcium phospholipid-dependent PKC pathway in the process of follicular rupture. A potent tumor-promoting phorbol ester, phorbol 12,13-dibutyrate, activates PKC by substituting for DG in the presence of calcium and phospholipid (25). PdBU induced ovulation in the extracorporeal perfused rabbit ovary in the absence of hCG. However, ovulatory efficiency was less then that usually achieved with hCG. Simultaneously, 4cY-phorbol12,13-didecanoate, an inactive phorbol ester (25), failed to induce follicular rupture even at high doses. Phorbol ester-induced ovulatory efficiency was dose related, with the peak value occurring at 200 nM PdBU. The PKC system has been implicated in the regulation of blood pressure, possibly mediated via angiotensin-II (26). Since, mean ovarian arterial pressure did not differ in phorbol ester and control perfusion
ROLE
OF PKC
experiments, it is unlikely that phorbol ester induces ovulation in this system by elevating ovarian arterial blood pressure. Arachidonic acid metabolites, in particular PGF2,, and prostacyclin, are considered possible intermediaries between gonadotropin secretion and ovulation in the rabbit (27, 28). In the present experiments, dose-dependent increases in PGEZ, and 6-keto-PGFr, perfusate concentrations PGL were observed after PdBU exposure. PdBU at a dose of 600 nM produced a continued elevation of PG levels, but did not alter ovulatory efficiency compared to 200 nM PdBU. These results suggest that phorbol ester-induced ovulation may not occur as a direct result of increased PG production, although it is possible that only threshold levels of PGs are required. The precise mode of PG involvement in follicular rupture continues to be investigated. Phorbol esters have been associated with increased PG synthetase activity and increased arachidonic acid accumulation (11, 29). Although PGs may be involved in the release or activation of collagenolytic enzymes within the follicle, blockade of PG synthesis does not inhibit tPA secretion (30,31). Nonetheless, indomethacin (a PG synthetase inhibitor) prevents ovarian collagen breakdown and follicular rupture in PMSG-primed rats, and PGF*, can induce ovulation in the rabbit in the absence of gonadotropins (32, 33). The exact relationship among ovulation, tPA, and PGs remains to be clearly established. The data from these experiments suggest that although PGs may be intermediaries in the process of ovulation, they may not be absolutely essential for PKC-induced follicle rupture. Perfusate progesterone levels were unchanged after the administration of 200 nM PdBU compared to levels in control ovaries. Since the highest ovulatory efficiency was achieved with this dose of phorbol ester, these experiments suggest that under physiological conditions, CAMP may serve as the major second messenger for steroidogenesis, rather than PKC. Although it has been reported that phorbol esters can regulate granulosa cell progesterone production, the magnitude of progesterone production was much less in response to phorbol esters than in response to LH (11). In this study perfusate levels of progesterone were significantly elevated after treatment with 600 nM PdBU compared with 200 nM PdBU and 0 nM PdBU (control). It has been shown that GnRH may mediate steroidogenesis through the PKC pathway (12). The increase in progesterone observed in this experiment may correspond to the physiological role of GnRH in ovarian granulosa cell steroidogenesis. The PKC inhibitors staurosporine and calphostin-C were used in the perfusion model to study the role of the PKC signal pathway in gonadotropin-induced ovulation. Staurosporine, a potent inhibitor of the catalytic domain of the PKC molecule, exhibits a lo-25% cross-reactivity with the CAMP pathway (34). Calphostin-C, a more specific PKC inhibitor, blocks the DG-binding region of PKC, but does not affect PKA at the concentrations used in this experiment (34). In these experiments, both inhibitors blocked hCG-induced ovulation in a dose-related manner. Larger doses of calphostin-C were required to achieve equivalent levels of inhibition as with staurosporine, a finding that is generally consistent
IN
OVULATION
Endo. 1992 Voll31 -No 4
with the relative potency of the compounds in vitro (34). Our findings are similar to those obtained by others using staurosporine in the rat model (19). Our experiments support the concept that PKC may be involved in gonadotropin-induced follicular rupture in the rabbit. Although LH has been shown to increase the synthesis and metabolism of phosphoinositides in rat granulosa cells from mature follicles (3), this observation is controversial, as others have shown that LH does not increase inositol phospholipid metabolism in rat granulosa cells (12). Recently, Guderman et al. (15) have provided evidence for dual coupling of the murine LH receptor to both adenylate cyclase and phosphoinositide breakdown and calcium mobilization. Since the EDs0 for PKC stimulation was much higher than that required for activation of the CAMP pathway, LH may use two signal transduction pathways to control separate cellular functions (15). Little information is available on the effect of LH on inositol phospholipid metabolism in the rabbit. Other purported intermediaries of ovulation, including bradykinin and angiotensin-II, have been shown to activate the PKC pathway in several cell populations (26, 35, 36). Consequently, PKCmediated follicular rupture may occur directly via hCG activation or result from PKC’s role as a second messenger for other intermediaries whose release may be triggered by gonadotropins. Perfusate PGF2,, levels were unaffected by either calphostin-C or staurosporine after gonadotropin stimulation. This finding provides further evidence that PGs may not be absolutely essential for PKC-mediated ovulation. Instead, PGs, such as PGF2,,, may function as amplifiers of the gonadotropin message. Progesterone levels were not reduced after hCG-treated ovaries were exposed to staurosporine and calphostin-C. This finding is consistent with the accepted role for CAMP in LH-induced steroidogenesis. The CAMP pathway has also been shown to be an important signal transduction system in determining oocyte maturation in the rabbit (6). Since the PKC inhibitors used in these experiments had no effect on hCG-induced oocyte maturation, these experiments suggest that the PKC pathway is not involved in gonadotropin-mediated oocyte maturation. Phorbol esters have been shown to stimulate the secretion of tPA release as well as decrease the secretion of plasminogen activator inhibitor from human umbilical vein endothelial cells, an effect potentiated by CAMP (16-18). Phorbol ester, therefore, may induce follicular rupture via initiation of the proteolytic enzyme cascade. We have previously reported that tranexamic acid inhibits PGFz,-induced ovulation in a dose-related manner in the in vitro perfused rabbit ovary (33). Tranexamic acid inhibits the conversion of plasminogen to plasmin by plasminogen activator (33). In the present experiments tranexamic acid inhibited phorbol ester-induced ovulation, implying that the PKC pathway has a direct effect on activation of the proteolytic enzyme cascade. In conclusion, these experiments demonstrate that the calcium phospholipid-dependent PKC pathway has a major role in gonadotropin-induced follicular rupture in the rabbit. Inhibition of PdBU-induced ovulation by tranexamic acid
ROLE OF PKC IN OVULATION indicates that PKC-mediated follicular rupture from activation of plasminogen activator.
may result
Acknowledgments The authors wish to thank Ono Pharmaceutical Co. (Osaka, Japan) for the generous gift of the PGs. We thank Dr. Susan J. Atlas for her help in statistical analysis. We also would like to thank Mr. Ramesh Ghodgaonkar and Ms. Brenda Schryer for PG assays. We appreciate the technical assistance of Ms. Bev Smith, Ms. Kirsten Grell, and Mr. Robert Wesley, and we thank Ms. Fran Karas for help in the preparation of the manuscript.
References EE, Atlas SJ, Dharmarajan AM, Oski JA, Santulli R 1989 1. Wallach The periovulatory interval: physiological and endocrinologic implications. In: Yoshinaga K, Mori T (eds) Development of Preimplantation Embryos and Their Environment. Alan R Liss, New York, pp 87-100 Y, Dharmarajan AM, Kaufman G, Kokia E, Adashi EY, 2. Takehara The effect of interleukin-10 on ovulation, oocyte maturation, and fertilization in the in vitro perfused rabbit ovary. 39th Annual Meeting of the Society for Gynecologic Investigation, San Antonio TX, 1992 (Abstract 242) 3. Davis JS, Weakland LL, West LA, Farese RV 1986 Luteinizing hormone stimulates the formation of inositol trisphosphate and cyclic AMP in rat granulosa cells. Biochem J 238:597-604 4. Adashi EY, Resnick CE, Jastorff B 1990 Blockade of granulosa cell differentiation by an antagonistic analog of adenosine 3’,5’-cyclic monophosphate (CAMP): central but non-exclusive intermediary role of CAMP in follicle-stimulating hormone action. Mol Cell Endocrinol 72:1-11 5. LeMaire WJ, Marsh JM 1975 Interrelationships between prostaglandins, cyclic AMP and steroids in ovulation. J Reprod Fertil [Suppl] 22:53-74 6. Hosoi Y, Yoshimura Y, Atlas SJ, Adachi T, Wallach EE 1989 Effects of dibutyryl cyclic AMP on oocyte maturation and ovulation in the perfused rabbit ovary. J Reprod Fertil 85:405-411 S, Beers WH 1976 Studies on the role of plasminogen 7. Strickland activator in ovulation. J Biol Chem 251:5694-5702 PV, Hedin L, Janson PO 1986 The role of cyclic adenosine 8. Holmes 3’,5’-monophosphate in the ovulatory process of the in vitro perfused rabbit ovary. Endocrinology 118:2195-2202 9. BrHnnstrom M, Koos RD, LeMaire WJ, Janson PO 1987 Cyclic adenosine 3’,5’-monophosphate-induced ovulation in the perfused rat ovary and its mediation by prostaglandins. Biol Reprod 37:10471053 10. Yoshimura Y, Hosoi Y, Atlas SJ, Ghodgaonkar R, Dubin NH, Wallach EE, The effect of CAMP on hCG-induced ovulation, oocyte maturation, and PG production. 33rd Annual Meeting of the Society for Gynecologic Investigation, Toronto, Ontario, Canada, 1986 (Abstract 183) ester regulation of rat granulosa 11. Kawai Y, Clark MR 1985 Phorbol ceil prostaglandin and progesterone accumulation. Endocrinology 116:2320-2326 in 12. Leung PCK, Wang J 1989 The role of inositol lipid metabolism the ovary. Biol Reprod 40:703-708 LL, Farese RV, West LA 1987 Luteinizing 13. Davis JS, Weakland hormone increases inositol trisphosphate and cytosolic free CA’+ in isolated bovine luteal cells. J Biol Chem 262:8515-8521 14. Davis JS, Weakland LL, Weiland DA, Farese RV, West LA 1987 Prostaglandin FZo stimulates phosphatidylinositol 4,5-bisphosphate hydrolysis and mobilizes intracellular Ca’+ in bovine luteal cells. Proc Nat1 Acad Sci USA 84:3728-3732 15 Guderman T, Birnbaumer M, Birnbaumer L 1992 Evidence for dual coupling of the murine luteinizing hormone receptor to aden-
16.
17.
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22.
23. 24.
25.
26. 27.
28
29
30
31. 32.
33.
34.
35.
36.
1809
ylyl cyclase and phosphoinositide breakdown and Ca*’ mobilization. J Biol Chem 267:4479-4488 Levin EG, Marotti KR, Sante11 L 1989 Protein kinase C and the stimulation of tissue plasminogen activator release from human endothelial cells. I Biol Chem 264:16030-16036 Sante11 L, Levin-EG 1988 Cyclic AMP potentiates phorbol ester stimulation of tissue plasminogen activator release and inhibits secretion of plasminogen activator inhibitor-l from human endothelial cells. J Biol Chem 263:16802-16808 Grulich-Henn J, Muller-Berghaus G 1990 Regulation of endothelial tissue plasminogen activator and plasminogen activator inhibitor type 1 synthesis by diacylglycerol, phorbol ester, and thrombin. Blut 61:38-44 Guerre Jr EF, Clark MR, Muse KN, Curry Jr TE 1991 lntrabursal administration of protein kinase or proteinase inhibitors: effects on ovulation in the rat. Fertil Steril 56:126-133 Lambertsen Jr CJ, Greenbaum DF, Wright KH, Wallach EE 1976 In vitro studies of ovulation in the perfused rabbit ovary. Fertil Steril 27:178-187 Kobayashi Y, Wright KH, Santulli R, Wallach EE 1981 Ovulation and ovum maturation in the rabbit ovary perfused in vitro. Biol Reprod 24:483-490 Dharmarajan AM, Yoshimura Y, Sueoka K, Atlas SJ, Dubin NH, Ewing LL, Zirkin BR, Wallach EE 1988 Progesterone secretion by corpora lutea of the isolated perfused rabbit ovary during pseudopregnancy. Biol Reprod 38:1137-1143 AhrCn K, Janson PO, Selstam G 1971 Perfusion of ovaries in vitro and in vi& Acta Endocrinol (Copenh) 158:285-305 Dubin NH, Ghodeaonkar RB, King TM 1979 Role of urosta!zlandin production.in spogtaneous anh oxytocin-induced uteine coctractile activity in in vitro pregnant rat uteri. Endocrinology 105:47-51 Kikkawa U, Takai Y, Tanaka Y, Miyake R, Nishizuka Y 1983 Protein kinase C as a possible receptor protein of tumor-promoting phorbol esters. J Biol Chem 258:11442-11445 Scholz H, Kurtz A 1990 Role of protein kinase C in renal vasoconstriction caused by angiotensin 11: Am J Physiol 259:C421-C426 Kitai H. Kobavashi Y. Santulli R, Wright KH, Wallach EE 1985 The reldtionshfp between prostaglandingand histamine in the ovulatory process as determined with the in vitro perfused rabbit. Fertil Steril43:646-651 Yoshimura Y, Dharmarajan AM, Gips S, Adachi T, Hosoi Y, Atlas SJ, Wallach EE 1988 Effects of prostacyclin on ovulation and microvasculature of the in vitro perfused rabbit ovary. Am J Obstet Gynecol 159:977-982 Rodway ME, Leung PCK 1990 lnositol lipid metabolism and calcium signaling in rat ovarian cells. In: Gibori G (ed) Signalling and Gene Expression in the Ovary. Springer-Verlag, New York, pp 2538 Espey L, Shimada H, Okamura H, Mori T 1985 Effect of various agents on ovarian plasminogen activator activity during ovulation in pregnant mare’s serum gonadotropin-primed immature rats. Biol Reprod 32:1087-1094 Reich R, Miskin R, Tsafriri A 1985 Follicular plasminogen activator: involvement in ovulation. Endocrinology 116:516-521 Reich R, Tsafriri A 1984 Mechanisms involved in follicular rupture in the rat. In: McKerns KW, Naor NZ (eds) Hormonal Control of the Hypothalamo-Pituitary-Gonadal Axis. Plenum Press, New York, pp 337-353 Miyazaki T, Dharmarajan AM, Atlas SJ, Katz E, Wallach EE 1991 Do-prostaglandins lead’ to ovulation in the rabbit by stimulating uroteolvtic enzvme activitv? Fertil Steril 55:1183-l 188 ?amaoi
The Role of Protein Kinase-C in Gonadotropin-Induced Ovulation in the in Vitro Perfused Rabbit Ovary* G. KAUFMAN,
A. M. DHARMARAJAN,
Department of Gynecology and Obstetrics, Baltimore, Maryland 21205
Y. TAKEHARA, Johns Hopkins
C. S. CROPP,
University
AND
E. E. WALLACH
School of Medicine,
ABSTRACT Tumor-promoting phorbol esters are believed to affect ovarian granulosa cell progesterone and prostaglandin (PG) production and possibly ovulation by activating protein kinase-C (PKC). The effects of phorbol esters and PKC inhibitors on ovulation, progesterone, and PG production were examined in an in vitro perfused rabbit ovary. The effect of tranexamic acid, an inhibitor of the conversion of plasminogen activator to plasmin, on phorbol ester-induced ovulation was also examined. Phorbol l&13-dibutyrate (PdBU), a PKC stimulator, induced ovulation in a dose-related manner in the absence of gonadotropins (56%, 200 nM PdBU; O%, 0 nM PdBU; P< 0.05). Perfusate progesterone levels were increased only after 600 nM PdBU treatment, and perfusate PGF2,., PGE,, and 6-keto-PGF],, were increased in a dose-dependent fashion (P < 0.05). Staurosporine, a potent inhibitor of the catalytic domain of PKC, and calphostin-C, a specific inhibitor of the diacyl-
glycerol-binding region, inhibited hCG-induced ovulation in a doserelated manner. Gonadotropin-induced ovulation decreased from 73% without staurosporine to 19% with 1.0 pM staurosporine (P < 0.01). Calphostin-C reduced ovulatory efficiency from 60% to 24% (P < 0.01). However, neither inhibitor decreased progesterone or PGF,, production by ovaries exposed to hCG. hCG-induced oocyte maturation was also unaffected by exposure to either staurosporine or calphostin-C. Tranexamic acid reduced phorbol ester-induced ovulatory efficiency from 67% to 37% (P < 0.05). These findings demonstrate that the calciumdependent PKC pathway is instrumental in gonadotropin-mediated follicular rupture in the rabbit. Although PGs may play an important role in ovulation, they do not appear to be directly responsible for PKC-mediated follicular rupture. (Endocrinology 131: 1804-1809, 1992)
E
Recently, the calcium phospholipid-dependent PKC pathway has been implicated as a second major signal transduction mechanism for hormone-mediated ovarian function (11, 12). The PKC pathway requires the hydrolysis of phosphatidylinositol 4,5-bisphosphate into diacylglycerol (DG) and inositol triphosphate by phospholipase-C. DG activates PKC, which, in turn, phosphorylates specific intracellular proteins. GnRH, LH, and prostaglandin FZn(PGF*,), substancesthat have been implicated in ovulation in the in vitro perfused rabbit ovary, have been shown to increasephosphoinositide metabolism in rat granulosa cells and bovine and rat luteal cells (3, 12-14). Recent evidence also indicates that the murine LH receptor may be capable of activating both PKC and adenylate cyclase, although with different EDsovalues (15). GnRH, PGF2,,,and possibly gonadotropins also mediate steroid hormone production through the PKC pathway (12). A potent phorbol ester, 12-0-tetradecanoyl-phorbol 13-acetate, which activates PKC, can regulate rat granulosa cell progesterone and PG production (11). Phorbol estersare also able to stimulate tissue plasminogen activator (tPA) mRNA production and decreasethe secretion of plasminogen activator inhibitor-l from human endothelial cells, an effect that is potentiated by CAMP (16-18). Plasminogen activator is thought to activate the proteolytic enzyme cascade,eventually resulting in the production of collagenaseand follicular rupture (1). Recently, Guerre, Jr., et al. (19) observed that staurosporine, a PKC inhibitor, inhibits PMSG-induced ovulation in the rat (19). Taken together, these findings implicate a possible role for phospholipid-PKC signal transduction in mediating gonadotropin-induced follicular rupture. The objectives of the study were to investigate: 1) the
of the extracorporeal perfused rabbit ovary to hCG consistently results in ovulation (1). Many substancesappear to be involved locally in the ovulatory process, including proteolytic enzymes (i.e. plasmin and collagenase), arachidonic acid metabolites, histamine, bradykinin, angiotensin-II, oxygen free radicals, and cytokines (1, 2). However, the intracellular signal transduction pathways by which the LH surge or gonadotropin stimulus results in follicular rupture
XPOSURE
remain
unclear.
It is well established that gonadotropins (FSH, LH, and hCG) induce steroidogenesisand cell differentiation through the CAMP-adenylate cyclase pathway (3-5). Oocyte maturation
as well
as gonadotropin-induced
follicular
rupture
may
also be dependent on CAMP (6-9). In contrast, we have observed that (Bu)~cAMP fails to induce ovulation in the absence of gonadotropins (6), and CAMP inhibits hCG-induced ovulation in the in vitro perfused rabbit ovary (10). Although most studies have focused on the CAMP pathway, little attention has been given to protein kinase-C (PKC) signal transduction. Received May 7, 1992. Address requests for reprints to: E. E. Wallach, M.D., Department of Gynecology and Obstetrics, Johns Hopkins Medical Institutions, 600 North Wolfe Street, Houck 264A, Baltimore, Maryland 21205. Address all correspondence to: A. M. Dharmarajan, Ph.D., Department of Gynecology and Obstetrics, Johns Hopkins Medical Institutions, 600 North Wolfe Street, Park 82-202, Baltimore, Maryland 21205. *This work was supported by NIH Grant HD-19430 (to E.E.W.), Population Center Grant HD-06268 (to A.M.D.), the SmithKline Beecham Fellowship (to Y.T.), and the Rockefeller Foundation (to A.M.D.). Presented in part at the 36th Annual Meeting of Society for Gynecologic Investigation, San Diego, CA, March 15-18, 1989.
1804
ROLE
OF PKC
effect of phorbol 12,13-dibutyrate (PdBU), a stimulator of PKC, on ovulation and progesterone and PG production in the absence of gonadotropins; 2) the effect of staurosporine, a potent inhibitor of PKC, on gonadotropin-induced ovulation and progesterone and PG production; 3) the effect of calphostin-C, a specific PKC inhibitor, on gonadotropininduced ovulation and progesterone and PG production; and 4) the effect of tranexamic acid [trans-4-(aminomethyl)cyclohexanecarboxylic acid], an inhibitor of the conversion of plasminogen to plasmin, on PdBU-induced ovulation. To this end, an in vitro perfused rabbit ovary model was used to investigate the effects of these agents on ovarian function in a carefully controlled and monitored environment.
Materials
and Methods
Animals New Zealand White mature female rabbits, weighing 3.0-4.5 kg, were used. All rabbits were caged individually for at least 3 weeks before use. Animals were given water and an unrestricted diet of rabbit chow. Rabbits were anesthetized with iv sodium pentobarbital (32 mg/kg), treated with heparin sulfate (120 U/kg) for anticoagulation, and then subjected to laparotomy. Ovaries were excluded from the study if they were immature or if two or more of the follicles were hemorrhagic at the time of laparotomy. Experimental ovaries were excluded if the paired contralateral control unstimulated ovary ovulated, possibly due to an endogenous LH surge before ovariectomy. All studies were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals.
In vitro perfusion The cannulation procedure and perfusion apparatus have been described in detail previously (20-22). Each ovarian artery was isolated and cannulated in situ after ligation of major anastomotic connections. The ovary with its cannulated vascular pedicle and supportive connective tissue was removed. The number of mature follicles (>1.5 mm in diameter) observed on the surface of each ovary was recorded, and the ovary was immediately placed in the perfusion chamber. The perfusion system consists of a chamber containing the ovary, an oxygenator, a reservoir, and a pulsatile roller pump that maintains perfusate flow at 1.5 ml/min, the approximate blood flow to the rabbit ovary (23). The oxygenator was gassed with 95% 0,.5% CO,. Ovaries were perfused for 10.5 h at 37 C in 150 ml medium 199 (Gibco, Grand Island, NY) supplemented with heparin sulfate (200 U/liter), insulin (20 U/liter), streptomycin sulfate (5b mg/liter), and penicillin G (75 mg/liter). Perfusate samules were collected from the arterial cannula 0, 0.5, 1, 2, 4, 6, 8, and 10 h after hCG or PdBU administration and replaced with an equal volume of fresh medium. Samples were stored at -20 C for later determination of I’G and progesterone concentrations. The ovary was monitored for fresh ovulation points every half-hour. A follicle was considered to be ruptured when the cumulus containing an ovum was observed protruding from the ovarian surface. Ovulatory efficiency was calculated using the following formula: [number of ovulated ova/number of mature follicles] X 100 (20, 21).
Experimental
IN OVULATION
1805
ovaries were treated with 6 PM 4cu-phorbol 12,13-didecanoate, and observations were compared to those of control ovaries. Samples were collected for determination of progesterone and PG concentrations, and ovulatory efficiency was calculated as described previously. In selected experiments mean arterial pressure was determined by a manometer attached to the arterial cannula in both control and treated ovaries. Effects of staurosporine and calphostin-C on hCG-induced ovulation, progesterone and PG production, and oocyte maturation. These experiments were designed to investigate the effect of PKC inhibition on hCGinduced ovulation and progesterone and PGs production. Thirty-six rabbits were used for this experiment. Staurosporine (Sigma; 0.5 mg) and calphostin-C (Kamiya Biomedical, Thousand Oaks, CA; 1.0 mg) were dissolved in 1.0 ml medium 199 or dimethylsulfoxide, respectively. Inhibitor or vehicle alone was dissolved in 150 ml perfusion medium to concentrations of 0.1, 0.5, and 1.0 PM staurosporine or 0.2, 1.0, and 2.0 PM calphostin-C before the onset of the experiment. One hundred international units of hCG were administered via the arterial cannula at 0 h, and samples were collected for determination of progesterone and PG concentrations. Ovulatory efficiency was calculated, and ovulated ova surrounded by their cumulus mass were carefully recovered from the perfusion chamber with a glass pipette. Cumulus cells were removed with gentle agitation after exposure to hyaluronidase (0.34 U/ml; Sigma Chemical Co.). The denuded oocytes were assessed for the presence or absence of germinal vesical breakdown. At the conclusion of the experiment, unruptured oocyte-cumulus complexes were obtained by rupturing the remaining mature intact follicles with a 24-gauge needle. These ova were also assessed cytologically for germinal vesical breakdown. Effect of tranexamic acid on PdBU-induced ovulation. This experiment was designed to investigate the effect of inhibition of the early steps in proteolytic enzyme cascade on phorbol ester-induced ovulation. Six rabbits were used for this experiment. Tranexamic acid (Sigma) was added to the perfusate at a concentration of 10 mM. The contralateral ovary was perfused without tranexamic acid. Thirty minutes after the onset of the perfusion, PdBU (0.2 PM) was added to the perfusate. Ovulatory efficiency was calculated as described above.
Progesterone
and PG RIAs
Progesterone concentrations in perfusate samples were measured using a solid phase kit (Diagnostic Products Corp., Los Angeles, CA). Progesterone antibody is bound covalently to the inner surface of polypropylene assay tubes. All samples and standards were assayed in duplicate. The intra- and interassay variations were 7.5% and 6.6%, respectively. Perfusate concentrations of I’GF>,,, PGE,, and 6-ketoPGFi,,, a metabolite of prostacyclin, were measured by methods previously described (24). PGF2,. and PGEz antibodies each cross-react less than 2% with the other I’G classes tested. The 6-keto-PGF,,, antibody cross-reacts less than 0.1% with the primary I’Gs. For each prostanoid measured, the intraassay coefficient of variation was less than 8%, and samples from each experiment were assayed simultaneously to avoid error due to interassay variation.
Statistical
analysis
Data for PG and progesterone levels were evaluated by analysis of variance, with P < 0.05 considered significant. Comparison of ovulatory efficiencies was performed using x2 analysis, and P < 0.05 was considered significant. Dose-response studies were designed using a balanced block method; the order of the ovaries for each experiment was determined by a random number table.
design
Effect of phorbol ester on ovulation and progesterone and PG production. This experiment was designed to investigate the effect of PKC stimulation on ovulation and progesterone and PG production in the absence of hCG. Twenty-one rabb& were used for this experiment, One millieram of PdBU (Siema Chemical Co., St. Louis, MO) was dissolved in 1 o. v ml 100% ethyl alcohol. Ovaries were placed in the perfusion chamber at -0.5 h, and PdBU (to a final concentration of 0.02, 0.1, 0.2, or 0.6 1~) or vehicle alone was administered via arterial cannula at 0 h. Four
Results Effect of PdBU on ovulation the absence of hCG
and progesterone
and PG levels in
PdBU induced ovulation in the in vitro perfused rabbit ovary in a dose-related manner in the absence of gonadotro-
ROLE
1806
OF PKC
pins (Fig. 1). Ovulatory efficiency varied from 0% for control and 20 nM PdBU-treated ovaries to 56% for 200 nM PdBUtreated ovaries. 4Lu-Phorbol 12,13-didecanoate, an inactive phorbol ester, did not stimulate ovulation in the absenceof hCG even at dosesas high as 6 PM (Fig. 1). Perfusate PGF2,, levels significantly increased from 143.3 f 26.2 pg/ml for 0 nM PdBU-treated ovaries (control) to 893.8 f 175.9 pg/ml after 200 nM PdBU treatment (P < 0.05; Fig. 2). PGE2and 6keto-PGF,,, also increased significantly after 200 nM PdBU administration compared to levels in control ovaries (P < 0.05; Fig. 2). PG concentrations were significantly elevated after 600 nM PdBU administration (P < 0.05; Fig. 2). Perfusate levels of progesterone were unchanged after treatment with 200 nM PdBU compared to control values (Fig. 3). However, perfusate progesterone levels increasedsignificantly after 600
IN OVULATION
Endo. 1992 Vol 131. No 4
nM PdBU treatment (8.2 f 1.4 rig/ml) compared to the control level (3.0 + 0.4 rig/ml; P < 0.05; Fig. 3). Mean arterial pressure was unaffected after exposure of the ovaries to 200 nM PdBU (n = 4; 63 mm mercury) compared with 0 nM PdBU (control; n = 4; 60 mm mercury). Effect of staurosporine on hCG-induced and PG levels, and oocyte maturation
ovulation,
progesterone
hCG-induced ovulation was inhibited by staurosporine compared to that in hCG-treated controls (P < 0.01; see Table 1). Ovulatory efficiency was reduced from 73% for ovaries perfused with hCG alone to 48% for hCG plus 0.5 PM staurosporine-treated ovaries and 19% for hCG plus 1.0 PM staurosporine-treated ovaries. Staurosporine did not affect perfusate progesterone levels (which varied from 13.4 + 3.2 to 26.2 f 13.4 rig/ml) and did not inhibit perfusate PG levels in response to hCG (data not shown). Staurosporine had no effect on hCG-induced oocyte maturation (seeTable 3). Effect of calphostin-C on hCG-induced and PG levels, and oocyte maturation
0
20
100
PdBU
200
600
(nM)
‘6 phorbol 12,13did.canoat,
FIG. 1. Ovulatory efficiency (percent) of PdBU- and 4ol-phorbol12,13didecanoate-treated ovaries compared to control ovaries perfused with medium containing vehicle. Ovaries were treated with the indicated concentrations of phorbol ester, and ovulatory efficiency was calculated as described in Materials and Methods. Each value is expressed as a mean. The values represent 4-12 ovaries/group. *, P < 0.01 us. 0 nM PdBU (control). P < 0.001 for all values taken together.
"
ovulation,
progesterone
hCG-induced ovulation was also inhibited in a dosedependent manner by calphostin-C compared to that in hCG-treated controls (P < 0.05; Table 2). Ovulatory efficiency was reduced from 60% for ovaries perfused with hCG alone to 31% for hCG plus 1.0 PM-calphostin-C-treated ovaries and 24% for hCG plus 2.0 PM calphostin-C-treated ovaries. Calphostin-C did not affect perfusate progesterone levels (which varied from 5.9 + 1.0 to 3.0 + 1.6 rig/ml) and did not inhibit hCG-stimulated PG production (data not shown), nor did calphostin-C influence hCG-induced oocyte maturation (Table 3).
10000-
*
E 2 UJ 5
FIG. 2. Effects of the indicated PdBU concentrations on perfusate PGF,,,, PGE,, and 6-keto-PGF1, levels. Control perfusions (0 nM PdBU) contained vehicle. Measurements of PGs were performed as described in Materials and Methods. The values are expressed as the mean + SE and represent six to nine ovaries per group. *, P < 0.05 VS. 0 nM PdBU (control).
6000
-
n q
ii J .-c
OnM PdBU 200 nM PdBU 600 nM PdBU
6000
-
4000
-
z 2 z 5; 2 n
PGF
2a
PGE
6-keto-PGF 2
la
ROLE
OF PKC
IN OVULATION TABLE 3. Percent germinal vesicle breakdown (GVBD) of ovulated and follicular oocytes perfused with hCG alone or with hCG plus staurosporine or calphostin-C Staurosporine
Ovulated oocytes GVBD (%) Follicular oocytes GVBD (%)
0.1
0.5
1.0
100
100
96
100
100
100
87
100
Calphostin-C 0
Ovulated oocytes GVBD (W) Follicular oocytes GVBD (%) PdBU
(nM)
FIG. 3. Effects of the indicated PdBU concentrations on perfusate progesterone levels. Progesterone values were determined as described in Materials and Methods. The values are expressed as the mean f SE and represent six to nine ovaries per group. *, P < 0.05 us. control.
TABLE in uitro
1. Effect of staurosporine on hCG-induced perfused rabbit ovary
0
10 10 45 33 73
2. Effect of calphostin-C perfused rabbit ovary
0.1
0.5
1.0
8 8
1
7
7
35 19 54
44 21 48
4 31 6 19”
on hCG-induced Calphostin-C
Perfused ovaries (no.) Ovaries ovulated (no.) Mature follicles (no.) Ovulations (no.) Ovulatory efficiency (%)
ovulation in the (PM)
0
0.2
1.0
2.0
11 11
6 6 28 16 57
6 6 51 16 31”
6 5 42 10 24”
57 34 60
Ovaries were perfused with calphostin-C (0.2, 1.0, and 2.0 pM) or vehicle (0 pM) and stimulated to ovulate by hCG (100 IU) 30 min after the onset of perfusion, as described in Materials and Methods. P < 0.001 for all values taken together. ” P < 0.05 compared to control. Effect of tranexamic
acid on PdBU-induced
(MM) 1.0
2.0
90
79
93
70
82
90
95
81
Ovaries were perfused with either staurosporine or calphostin-C, at the indicated concentrations, and hCG (100 IU). Controls (0 PM) received vehicle alone. Ovulated and follicular oocytes were retrieved and denuded, as described in Materials and Methods. The presence or absence of GVBD was assessed.
(PM)
Ovaries were perfused with staurosporine (0, 0.1, 0.5, and 1.0 PM) and stimulated to ovulate by hCG (100 IU) 30 min after the onset of perfusion, as described in Materials and Methods. P < 0.001 for all values taken together. ’ P < 0.01 compared to control. TABLE in vitro
0.2
ovulation in the
%XJrOSpOrine
Perfused ovaries (no.) Ovaries ovulated (no.) Mature follicles (no.) Ovulations (no.) Ovulatory efficiency (%)
(PM)
0
ovulation
Tranexamic acid significantly inhibited phorbol ester-induced ovulation. Ovulatory efficiency was reduced from 67% to 37% (P < 0.05) after treatment of PdBU-exposed ovaries with 10 mM tranexamic acid (see Fig. 4).
PdBU
PdBU
+ 10 mM
tAMCHA
FIG. 4. Effect of 10 mM tranexamic acid (tAMCHA) on PdBU-induced ovulatory efficiency. Ovaries were perfused with 200 nM PdBU in the presence and absence of tranexamic acid. Ovulatory efficiency was determined as described in Materials and Methods. Each value is expressed as a mean. *, P < 0.05 compared to PdBU alone. Discussion In the present study a rabbit ovarian in vitro perfusion model was used to investigate the role of the calcium phospholipid-dependent PKC pathway in the process of follicular rupture. A potent tumor-promoting phorbol ester, phorbol 12,13-dibutyrate, activates PKC by substituting for DG in the presence of calcium and phospholipid (25). PdBU induced ovulation in the extracorporeal perfused rabbit ovary in the absence of hCG. However, ovulatory efficiency was less then that usually achieved with hCG. Simultaneously, 4cY-phorbol12,13-didecanoate, an inactive phorbol ester (25), failed to induce follicular rupture even at high doses. Phorbol ester-induced ovulatory efficiency was dose related, with the peak value occurring at 200 nM PdBU. The PKC system has been implicated in the regulation of blood pressure, possibly mediated via angiotensin-II (26). Since, mean ovarian arterial pressure did not differ in phorbol ester and control perfusion
ROLE
OF PKC
experiments, it is unlikely that phorbol ester induces ovulation in this system by elevating ovarian arterial blood pressure. Arachidonic acid metabolites, in particular PGF2,, and prostacyclin, are considered possible intermediaries between gonadotropin secretion and ovulation in the rabbit (27, 28). In the present experiments, dose-dependent increases in PGEZ, and 6-keto-PGFr, perfusate concentrations PGL were observed after PdBU exposure. PdBU at a dose of 600 nM produced a continued elevation of PG levels, but did not alter ovulatory efficiency compared to 200 nM PdBU. These results suggest that phorbol ester-induced ovulation may not occur as a direct result of increased PG production, although it is possible that only threshold levels of PGs are required. The precise mode of PG involvement in follicular rupture continues to be investigated. Phorbol esters have been associated with increased PG synthetase activity and increased arachidonic acid accumulation (11, 29). Although PGs may be involved in the release or activation of collagenolytic enzymes within the follicle, blockade of PG synthesis does not inhibit tPA secretion (30,31). Nonetheless, indomethacin (a PG synthetase inhibitor) prevents ovarian collagen breakdown and follicular rupture in PMSG-primed rats, and PGF*, can induce ovulation in the rabbit in the absence of gonadotropins (32, 33). The exact relationship among ovulation, tPA, and PGs remains to be clearly established. The data from these experiments suggest that although PGs may be intermediaries in the process of ovulation, they may not be absolutely essential for PKC-induced follicle rupture. Perfusate progesterone levels were unchanged after the administration of 200 nM PdBU compared to levels in control ovaries. Since the highest ovulatory efficiency was achieved with this dose of phorbol ester, these experiments suggest that under physiological conditions, CAMP may serve as the major second messenger for steroidogenesis, rather than PKC. Although it has been reported that phorbol esters can regulate granulosa cell progesterone production, the magnitude of progesterone production was much less in response to phorbol esters than in response to LH (11). In this study perfusate levels of progesterone were significantly elevated after treatment with 600 nM PdBU compared with 200 nM PdBU and 0 nM PdBU (control). It has been shown that GnRH may mediate steroidogenesis through the PKC pathway (12). The increase in progesterone observed in this experiment may correspond to the physiological role of GnRH in ovarian granulosa cell steroidogenesis. The PKC inhibitors staurosporine and calphostin-C were used in the perfusion model to study the role of the PKC signal pathway in gonadotropin-induced ovulation. Staurosporine, a potent inhibitor of the catalytic domain of the PKC molecule, exhibits a lo-25% cross-reactivity with the CAMP pathway (34). Calphostin-C, a more specific PKC inhibitor, blocks the DG-binding region of PKC, but does not affect PKA at the concentrations used in this experiment (34). In these experiments, both inhibitors blocked hCG-induced ovulation in a dose-related manner. Larger doses of calphostin-C were required to achieve equivalent levels of inhibition as with staurosporine, a finding that is generally consistent
IN
OVULATION
Endo. 1992 Voll31 -No 4
with the relative potency of the compounds in vitro (34). Our findings are similar to those obtained by others using staurosporine in the rat model (19). Our experiments support the concept that PKC may be involved in gonadotropin-induced follicular rupture in the rabbit. Although LH has been shown to increase the synthesis and metabolism of phosphoinositides in rat granulosa cells from mature follicles (3), this observation is controversial, as others have shown that LH does not increase inositol phospholipid metabolism in rat granulosa cells (12). Recently, Guderman et al. (15) have provided evidence for dual coupling of the murine LH receptor to both adenylate cyclase and phosphoinositide breakdown and calcium mobilization. Since the EDs0 for PKC stimulation was much higher than that required for activation of the CAMP pathway, LH may use two signal transduction pathways to control separate cellular functions (15). Little information is available on the effect of LH on inositol phospholipid metabolism in the rabbit. Other purported intermediaries of ovulation, including bradykinin and angiotensin-II, have been shown to activate the PKC pathway in several cell populations (26, 35, 36). Consequently, PKCmediated follicular rupture may occur directly via hCG activation or result from PKC’s role as a second messenger for other intermediaries whose release may be triggered by gonadotropins. Perfusate PGF2,, levels were unaffected by either calphostin-C or staurosporine after gonadotropin stimulation. This finding provides further evidence that PGs may not be absolutely essential for PKC-mediated ovulation. Instead, PGs, such as PGF2,,, may function as amplifiers of the gonadotropin message. Progesterone levels were not reduced after hCG-treated ovaries were exposed to staurosporine and calphostin-C. This finding is consistent with the accepted role for CAMP in LH-induced steroidogenesis. The CAMP pathway has also been shown to be an important signal transduction system in determining oocyte maturation in the rabbit (6). Since the PKC inhibitors used in these experiments had no effect on hCG-induced oocyte maturation, these experiments suggest that the PKC pathway is not involved in gonadotropin-mediated oocyte maturation. Phorbol esters have been shown to stimulate the secretion of tPA release as well as decrease the secretion of plasminogen activator inhibitor from human umbilical vein endothelial cells, an effect potentiated by CAMP (16-18). Phorbol ester, therefore, may induce follicular rupture via initiation of the proteolytic enzyme cascade. We have previously reported that tranexamic acid inhibits PGFz,-induced ovulation in a dose-related manner in the in vitro perfused rabbit ovary (33). Tranexamic acid inhibits the conversion of plasminogen to plasmin by plasminogen activator (33). In the present experiments tranexamic acid inhibited phorbol ester-induced ovulation, implying that the PKC pathway has a direct effect on activation of the proteolytic enzyme cascade. In conclusion, these experiments demonstrate that the calcium phospholipid-dependent PKC pathway has a major role in gonadotropin-induced follicular rupture in the rabbit. Inhibition of PdBU-induced ovulation by tranexamic acid
ROLE OF PKC IN OVULATION indicates that PKC-mediated follicular rupture from activation of plasminogen activator.
may result
Acknowledgments The authors wish to thank Ono Pharmaceutical Co. (Osaka, Japan) for the generous gift of the PGs. We thank Dr. Susan J. Atlas for her help in statistical analysis. We also would like to thank Mr. Ramesh Ghodgaonkar and Ms. Brenda Schryer for PG assays. We appreciate the technical assistance of Ms. Bev Smith, Ms. Kirsten Grell, and Mr. Robert Wesley, and we thank Ms. Fran Karas for help in the preparation of the manuscript.
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