The Effects of Exogenous Gonadotropins on Ovarian Adenylate Cyclase Activity CHRISTINE L. NUGENT, A. LOPATA, AND M. K. GOULD* Department of Obstetrics and Gynecology, Queen Victoria Memorial Hospital, Monash University, Melbourne, Victoria, Australia; and ^Department of Biochemistry, Monash University, Clayton, Victoria, Australia ABSTRACT. Adenylate cyclase was assayed in pellets prepared by centrifuging for 10 min at 600 x g homogenates of ovaries of immature rats treated with PMS and hCG. Activity was detected in the absence of any stimulatory agents and increased markedly in the presence of fluoride. Dose-dependent activity occurred in vitro in response to LH ranging from 0.2 to 10 /u.g/ml of incubate but not at higher concentrations. Marked changes in adenylate cyclase activities were observed with preparations from ovaries excised at various times after gonadotropin treatment. These changes, measured as the response to both fluoride and LH were observed to occur in four main stages. An initial
G
ONADOTROPINS are known to control various metabolic processes in the ovary including ovarian differentiation, growth and steroid hormone secretion. One of the early steps in gonadotropin action appears to be stimulation of the adenylate cyclase-cyclic AMP system (1). This system has been shown to be present in ovarian tissues of several species (2-4) including rat luteal ovary preparations (5). Although the sensitivity of the adenylate cyclase in these luteal ovaries was not tested, both LH and FSH stimulated adenylate cyclase activity in ovarian homogenates from immature rats (6). Evidence has been presented for the involvement of cyclic AMP in the control of ovarian steroidogenesis in several species. Slices of bovine corpus luteum and interstitial cells of rabbit ovary elaborate progestational steroids when incubated in the presence of cyclic AMP or LH (7,8). Time sequence studies have shown that LH produced cyclic AMP accumulation prior to any increased synthesis of progestins (2). In addition both the cyclic nucleotide and its dibutyryl derivative (N6-O2'-dibutyryl cyclic Received March 6, 1975.
decrease occurred V2 to 1 day after administration of hCG. Activity then increased and remained significantly elevated from day 3 to 7. A second but more dramatic rise was observed on day 8 and this enhanced level of activity remained elevated until day 13. A decreased level of activity occurred on day 15. Unstimulated activity remained low for the 16 day period studied although significant rises were observed on day 3 and days 8 to 15 after the administration of hCG. We have suggested that these changes in the adenylate cyclase activity modulate the level of cyclic AMP in the luteal cells and thereby induce changes in the activity of enzymes involved in progestin biosynthesis. (Endocrinology 97: 581, 1975)
3',5'-AMP) have been found to stimulate progestin synthesis and produce morphological luteinization in tissue cultures of granulosa cells harvested from rhesus monkey and porcine ovaries (9). These effects were similar to those obtained with the addition of LH and FSH to the culture medium. Evidence presented recently indicates that the process of luteinization might also be held in check by cyclic AMP (10). Injection of cyclic AMP or the dibutyryl derivative, directly into Graafian follicles of estrous rabbits inhibited luteinization in response to LH whereas injection of phosphodiesterase induced luteinization in a number of follicles. The mechanisms by which cyclic AMP may mediate LH action is not clear although evidence of a cyclic AMPdependent protein kinase in the bovine ovary (11) indicates that these mechanisms involve, in part or wholly, cyclic AMPstimulated phosphorylation of cellular proteins. The object of the present study was to determine the time sequence of the response of the adenylate cyclase system in the ovaries of prepubertal rats following injections of PMS and hCG. Using an in 581
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vitro system the response of the enzyme was studied with respect to the basal, or unstimulated activity, and both LH and fluoride stimulated activity.
Endo • 1975 Vol 97 • No 3
weighed in tared vials, the tissues were then dried for 3 days at 104 C, cooled in a desiccator and re-weighed. Preparation of the adenylate cyclase fraction
Materials and Methods Materials Pregnant mare's serum (PMS) was obtained from Schering Pty. Ltd., human chorionic gonadotropin (hCG) was from Organon Laboratories, and bovine LH (NIH-LH B7) was supplied by the National Institutes of Health. a-32Plabelled ATP was purchased from the Radiochemical Centre, Amersham. Unlabelled ATP, cyclic AMP, creatine phosphate (CP), creatine kinase (CK), glucose-6-phosphodehydrogenase, hexokinase and NADP were obtained from the Boehringer-Mannheim Corporation. Bovine serum albumin, fraction V, was from the Commonwealth Serum Laboratories. "Trizma" base and "Trizma" HC1 were purchased from the Sigma Chemical Company, and the aluminum oxide, activity 1, neutral, for column chromatography, was from Merck. The chromatography columns were similar to those described by White and Zenser (12) and were plugged with glass wool. Chemicals used in the assays of adenylate cyclase, ATP and CP, were of "Analar" grade. Solvents and fluors for the scintillation fluid were obtained from the Packard Instrument Co., Inc. Pretreatment of rats Superovulation was induced in 23- to 28-dayold Wistar strain rats by sc injection of 75 IU of PMS and 37.5 IU of hCG 64 h later. These animals were then kept under a constant lighting schedule of 12 h light and 12 h darkness. The rats were killed by cervical dislocation one day prior to, and 0, lA, 1, 3, 5, 7, 8, 9, 13 and 15 days after the injection of hCG. Vaginal smears were taken daily from the first day of vaginal opening to determine the duration of pseudopregnancy. Ovarian and uterine weights Both wet and dry weights of the ovaries and uterine horns were determined for representative groups of rats for each experimental day. Immediately after the animals were killed their ovaries and uterine horns were excised and
Groups of 3 to 6 rats were killed between 10 and 11 AM on the morning of each experimental day. The ovaries were excised free of surrounding fat and were pooled and weighed. They were then homogenized in 4.5 vol of ice-cold 0.25M sucrose in a Kontes-Dual homogenizer using 3 strokes of the teflon pestle at 800 rpm. The homogenate was washed into a weighed centrifuge tube with ice-cold 0.25M sucrose and diluted to contain 1 part of ovarian tissue in 10 parts of sucrose. It was then centrifuged at 600 x g for 10 min and the pellet was resuspended in icecold, 0.25M sucrose to give a concentration of 20 mg of fresh ovarian tissue/200 /x\. This preparation was prepared fresh each day and was used within 2 h from the time the rats were killed. All assays were performed in quadruplicate. In order to compare adenylate cyclase activity on different days in terms of a standard weight of ovarian membrane fraction the dry weights of the 200 /A aliquots were determined in quadruplicate on each experimental day. It was therefore possible to calculate adenylate cyclase activity in equivalent amounts of ovarian tissue and to express the results as nmol of cyclic AMP produced per gram of wet (or dry) ovary per minute. Assay of adenylate cyclase The incubation medium for the assay of adenylate cyclase consisted of: 50 HIM Tris HC1 (Tris (hydroxymethyl) aminomethane HCl) buffer at pH 7.6, 5 HIM MgCl2, 1 mM cyclic AMP, 0.1% BSA, 1.8 mM a-(32P)-ATP (2-8 cpm/pmol) and either 10 mM NaF or 10 /xg LH. An ATP regenerating system consisting of 10 mM CP and 2-4 U CK was also added. The final volume of the incubation mixture containing 200 (A of the 600 x g pellet preparation was 0.6 ml. The 600 x g pellet preparation was added to the incubation medium to start the reaction and the mixtures were incubated at 30 C for periods of up to 25 min. Fifty microliters of a solution containing 0.2 mg cyclic AMP in 50 mM Tris HCl buffer at pH 7.6 was then added to the incubation tubes. The reactions were stopped
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OVARIAN ADENYLATE CYCLASE ACTIVITY by immersing the tubes in boiling water for 3 min. The tubes were then centrifuged to remove denatured protein and the supernatant was assayed for cyclic AMP according to the method described by White and Zenser (12). This involved running the supernatant completely on to columns of aluminum oxide followed by 3.5 ml of 50 HIM Tris HC1 buffer at pH 7.6 and collecting a single 3.0 ml eluate fraction. Duplicates of 1.0 ml of this fraction were mixed with 10 ml of Bray's scintillation medium (13) and counted for 32P in a Packard Tri-Carb liquid scintillation spectrometer. Reaction blanks were obtained from incubations to which aliquots of boiled enzyme had been added. It was found that the activity of these blanks was up to 5% of the stimulated and 30% of the lowest basal adenylate cyclase values. The ability of the alumina columns to separate cyclic AMP was tested repeatedly. The recovery of [32P]cyclic AMP applied to the columns was 86 to 90%. When 32P-labelled ATP was applied to the columns a maximum of only 0.005% could be detected in the eluate fraction corresponding to cyclic AMP. When 5'-AMP was applied to the columns less than 0.1% could be eluted. Inorganic 32P is strongly bound by the alumina (12). The purity of the 32P-labelled cyclic AMP isolated by passing the incubation mixtures from the LH, NaF or control experiments, through columns of aluminum oxide was established by column chromatography on Dowex 1-C1 ion exchange resin, crystallization to constant specific activity and paper chromatography. Degradation of cyclic 3',5'-AMP The degradation of radioactive cyclic AMP during incubation of adenylate cyclase preparations from ovaries of gonadotropin treated rats was studied. Tritiated cyclic AMP (19 x 103 cpm) was incubated in the adenylate cyclase assay medium in the presence of unlabelled ATP and either 0.25 mM or 1.0 niM cyclic AMP or 10 mM theophylline. Inclusion of 1 mM cyclic AMP in the adenylate cyclase incubation medium prevented the breakdown of tritiated cyclic AMP for at least 18 min. However, 10 mM theophylline failed to completely inhibit the breakdown of tritiated cyclic AMP and was only as effective as the addition of 0.25 mM cyclic AMP. The ability of 1 mM cyclic AMP to protect radioactive cyclic AMP from hydrolysis during
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incubations of adenylate cyclase preparations was tested in the presence of 10 mM NaF or 10 fj.g LH. It was shown that the level of tritiated cyclic AMP remained constant for at least 18 min of incubation in the presence of these agents. Maintenance of ATP as substrate for the adenylate cyclase reaction The concentrations of ATP and CP were estimated in incubations of the adenylate cyclase assay mixture using the method of Lamprecht and Trautschold (14,15). The concentration of ATP in the medium used for assaying adenylate cyclase was determined throughout a 25 min incubation period in both the presence and absence of an ATP-regenerating system. The regenerating system consisted of 10 mM CP and 1-2 U of CK. It was shown that in the presence of the regenerating system there was a gradual but significant decrease in the concentration of ATP whereas in the absence of the regenerating system the concentration of ATP fell rapidly and could not be detected after 25 min of incubation. In an attempt to determine the conditions for a maximum rate of regeneration of ATP during adenylate cyclase assays a study was made of the ability of various levels of the regenerating system to maintain ATP. ATP and CP levels were estimated in incubations of the adenylate cyclase mixture containing one of the following combinations of the regenerating system: system A, 10 mM CP, 1-2 U CK; system B, 10 mM CP 2-4 U CK; system C, 20 mM CP, 2-4 U CK. Regenerating system B (2-4 U CK, 10 mM CP) was used for the assay of adenylate cyclase in the present studies since at this level the regenerating system efficiently maintained ATP and its concentration in the assay medium was found to be 1.25 mM after 20 min of incubation. Moreover under these conditions CP was not limiting as substrate for the regenerating enzyme.
Results Effects of PMS and hCG on the ovaries of immature rats Changes in ovarian and uterine weights brought about by treating immature rats with 75 IU PMS and 37.5 IU of hCG are shown in Fig. 1. Maximum ovarian wet and dry weights were found on day 3 after the hCG injection. The ovarian dry weight then
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recorded to determine whether the immature ovaries secrete estrogen in response to the gonadotropin treatment. As may be seen the uterine horns reached maximum wet and dry weights on day 5 after the hCG injection. The wet weight decreased markedly by day 7 and then more gradually up to day 15. There was a similar but less striking change in the dry weight. Changes in adenylate cyclase activity
5 7 9 Day after hCG injection
11
13
15
FIG. 1. Changes in wet and dry weights of ovaries and uterine horns of immature rats injected with PMS and hCG. Ovarian wet (o o) and dry (• •) weights; uterine wet (a D) and dry (• •) weights.
remained elevated with no further significant change up to day 15. On the other hand the wet weight decreased from day 3 to 7 and then its level appeared to plateau up to day 15. Uterine wet and dry weights were
Changes in the response of the adenylate cyclase enzyme in ovarian tissue from gonadotropin treated immature rats are shown in Fig. 2. Unstimulated, or basal, activity remained low (0.92 to 3.53 nmol/g ovary/min) throughout the 16-day period studied. Significantly increased activities were found on day 3 (P < 0.001) and on days 8 to 15 (0.01 > P > 0.001). LH-stimulated activity was significantly greater (P < 0.001) than basal activity on day 0 of treatment and on days 3 to 15. Fluoride-stimulated activity was significantly greater than both basal and LH-stimulated activities at all times.
so
i
40
FIG. 2. Basal and stimulated adenylate cyclase activity measured in homogenates of ovaries that were removed at various times after the treatment of immature rats with PMS and hCG. The 600 x g fractions of these homogenates, representing 20 mg of fresh ovarian tissues, were incubated for 12 min under basal conditions (D), in the presence of 10 /xg LH (0), or in the presence of 10 mM NaF (•). Each assay was performed in quadruplicate and the results are shown as the mean ± 1 SD.
ra 20
•2\ -2
-1
0
2
3
4
5
6
7
13
14
Day after hCC injection
PMS
hCG
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OVARIAN ADENYLATE CYCLASE ACTIVITY
The response of the adenylate cyclase to LH was characterized by an initial rise from 1.29 nmol/g ovary/min XV2 days after PMS to 2.40 nmol/g ovary/min 2V2 days after PMS. It should be noted that a significant decrease in this response occurred half a day after the injection of hCG which was followed by a significant rise above all previous values to 6.61 nmol/g ovary/min on day 3. This significantly elevated activity persisted until day 7. A second and dramatic increase occurred by day 8 and a maximum activity of 16.38 nmol/g ovary/min was found on day 9. This enhanced activity was present up to day 13 but fell significantly on day 15 to 4.43 nmol/g ovary/min. As may be seen from Fig. 2 changes in fluoride-stimulated activity were similar but more striking than the LH-stimulated activity. Two and a half days after PMS the fluoride-stimulated activity was 8.90 nmol/g ovary/min. A significant decrease in the response of the adenylate cyclase was apparent after the injection of hCG. The first significant increase occurred 1 day after administration of hCG. It then rose to 20.21 nmol/g ovary/min by day 3 and remained elevated until day 7. At this stage as in the case of LH, there was a dramatic increase in the fluoride-stimulated activity. This greatly enhanced activity, of 42.30 to 48.72 nmol/g ovary/min, was found to be present up to day 13 but decreased thereafter to a level of 23.36 nmol/g ovary/min which was not significantly different from that measured on day 7.
585
3-0
i 20
K)
0-1
10
10 LH(pg/ml)
100
1000
FlG. 3. Dose-response curve for LH-stimulated adenylate cyclase in luteal ovary preparations from PMShCG treated immature rats. The adenylate cyclase activity of the 600 x g pellet was determined in the presence of various concentrations of luteinizing hormone. Each point is the mean ± 1 SD of triplicate samples.
of activity was reached was 10 /Ag/ml and the activity did not alter for doses of LH up to 500 /x,g/ml. At these maximal doses the activity was 2.71 nmol/g ovary/min. Half maximal stimulation was obtained with 1.5 (xg LH/ml incubation mixture. Time-course studies
Ovarian homogenates prepared from the gonadotropin-treated immature rats 7 days after the injection of hCG were incubated in the presence of 10 /xg LH or 10 mM fluoride or without these agents. The rate Effects of LH on adenylate cyclase activity of production of cyclic AMP was measured in vitro after 6, 12 and 18 min of incubation. As may A 600 x g pellet preparation of ovaries be seen in Fig. 4 the basal activity as well taken from gonadotropin treated rats was as the LH and fluoride stimulated activities incubated under the conditions described in were linear over this period, the rates being Materials and Methods but in the presence 1.61, 5.40 and 18.10 nmol/g ovary/min, of various concentrations of LH. Figure 3 respectively. shows the sigmoidal dose response curve Discussion obtained when the concentration of LH in the medium varied from 0.2 to 500 //,g/ml. The method for measuring adenylate cyThe concentration at which a constant level clase employed in the present study was
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NUGENT, LOPATA AND GOULD
FIG. 4. Adenylate cyclase activity as a function of time in preparations of ovaries removed from immature rats 7 days after the injection of hCG. Fractions of the 600 x g pellet representing 20 nig of fresh tissue were incubated under basal conditions (O O reg coeff 0.032, corr coeff 0.884) or in the presence of 10 mM NaF ( D — • — D , reg coeff 0.362, corr coeff 0.945) or 10 Mg LH (• •, reg coeff 0.109, corr coeff 0.873).
initially described by White and Zenser (12) for the assay of the enzyme in plasma membrane fractions isolated from rat kidney. The experiments described in this paper have validated this method for measuring adenylate cyclase activity in ovarian homogenates. Several workers have used an ATPregenerating system in the assay of adenylate cyclase but did not establish that the system actually maintained ATP levels during the time intervals of incubation (5,16). It was reported that the 600 x g pellet of the rat luteal- ovary contained a complex of highly active "ATPase" enzymes consisting of adenosine triphosphatase, adenosine diphosphatase and nucleotidase (5). These enzymes rapidly degrade ATP added as substrate for the adenylate cyclase reaction. Our data show that although "ATPase" enzymes are active in rat luteal ovary preparations, ATP may be conserved as substrate for the adenylate cyclase enzyme by the inclusion of QK and GP as an ATP-regenerating system. This regenerating system functions efficiently to.maintain adequate concentrations of ATP for at least 20 min. of incubation in the presence of an ovarian preparation equivalent to 20 mg of fresh tissue. Under these conditions basal adenylate cyclase activity could be measured in ovaries of immature rats treated with either, PMS or PMS and hCG. Moreover, the same conditions have been em-
Enilo • 1975 Vol 97 • No .1
ployed to demonstrate the sensitivity of ovarian adenylate cyclase to different dosages of LH in the incubation medium and to study the response of the enzyme to fluoride and LH in vitro. The response of the adenylate cyclase system in the ovaries of immature rats treated with gonadotropins can be described in four main phases. 1. The ovulatory phase (day 0 to day V2 after hCG) when there is an apparent fall in the adenylate cyclase activity measured as the fluoride and LH stimulated activities. 2. The phase of corpus luteum formation and growth (day 1 to day 7 after hCG) during which both the fluoride and LH stimulated activities increase gradually to reach a maximum level 3 days after the injection of hCG. This level of activity is sustained for a further 4 days. A consistent increase in the basal, or unstimulated, adenylate cyclase activity also occurs during this period although the increase has not been found to be statistically significant. 3. The phase of enhanced ovarian adenylate cyclase activity, which lasts from day 8 to 13 after the injection of hCG. This period is characterized by a dramatic increase in both the fluoride and LH stimulated activities and corresponds to the first significant increase in the basal adenylate cyclase activity. 4. The phase of declining ovarian adenylate cyclase activity (from day 13 after hCG) during which the response of the enzyme measured as the basal and stimulated activities dropped to the levels found in phase 2. A measured rise or fall in the stimulated adenylate cyclase activity could be due to a change in the content of enzyme in the ovary, to a change in the sensitivity of the enzyme, or to changes in both these factors. Thus the initial fall in the adenylate cyclase activity observed in phase 1 may be related to ovulation which involves loss of oocytes, follicular cells and fluid from the ovary. Moreover, a fall in the sensitivity of the adenylate cyclase measured as a decrease in the ratio of stimulated to basal enzyme activity appears to have occurred
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OVARIAN ADENYLATE CYCLASE ACTIVITY during this period. Following the injection of PMS and hCG into immature rats an increase in the ovarian adenylate cyclase content and sensitivity contribute to the rise in the enzyme activity observed during phases 2 and 3. Increased sensitivity of the adenylate cyclase, measured as the in vitro response of the enzyme to LH and fluoride appears to predominate during days 3 to 7 after hCG injection (phase 2). The significant increase in basal activity (e.g., from a mean value of 1.65 nmol cAMP/g ovary/min during days 3 to 7) up to a mean value of 3.22 nmol cAMP/g ovary/min (P < 0.001) during days 8 to 13 after hCG injection, was accompanied by a fall in the sensitivity of the enzyme. This decrease in the adenylate cyclase sensitivity was judged by a fall in the fluoride:basal activity (e.g., from 23:1 on day 7 to a ratio of 14:1 on day 9) and a similar fall in the LH:basal ratio (e.g., from 8:1 on day 7 to a ratio of 5:1 on day 9). These results suggest that synthesis of the enzyme may be the main factor contributing to the enhanced activity observed during days 8 to 13 after hCG injection. In ovarian tissues from various species (3,4,9,17) LH appears to activate the adenylate cyclase system as an early step in the stimulation of steroid biosynthesis. The cyclic AMP generated is considered to be the intermediate which activates the enzymes of the biosynthetic pathway (1). Luteinizing hormone has also been shown to stimulate progestin synthesis in the luteal ovary of the gonadotropin primed rat both in vivo and in vitro (18). In view of these findings and the present data indicating a significant increase of adenylate cyclase
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activity in the ovaries of immature rats treated with gonadotropins, particularly between days 3 to 7 and 8 to 13 after the treatment, it is suggested that the changes in adenylate cyclase activity modulate the level of cyclic AMP in luteal cells and are associated with changes in steroidogenesis. References I! .Niswender, G. D., K. M. J. Menon, and R. B. Jaffe, Fertil Steril 23: 432, 1972. 2. Marsh, J. M., R. W. Butcher, K. Savard, and E. W. Sutherland,; Biol Chem 241: 5436, 1966. 3. Kuehl, F. A., Jr., J. L. Humes, J. Tarnoff, V. J. Cirillo, and E. J. Ham, Science 169: 883, 1970. 4. Dorrington, J. H., and B. Bagget, Endocrinology 83: 989, 1969. 5. Stansfield, D. A., and D. J. Franks, Biochim Biophys Ada 242: 606, 1971. 6. Fontaine, Y. A., E. Fonatine-Bertrand, N. DelerueLebelle, and C. Salmon,/ Physiol (Paris) 63: 49A, 1971. 7. Marsh, J. M., and K. Savard, Steroids 8: 133, 1966. 8. Dorrington, J. H., and R. Kilpatrick, Biochem J 104: 725, 1967. 9. Channing, C. P., Recent Progr Horrn Res 26: 589, 1970. 10. Lemaire, W. J., T. Mills, Y. Ito, and J. M. Marsh, Biol Reprod 6: 109, 1972. 11. Kuo, J. F., and P. Greengard, Proc Natl Acad Sci USA 64: 1349, 1971. 12. White, A. A., and T. V. Zenser, Anal Biochem 41: 372, 1971. 13. Bray, G. A., Anal Biochem 1: 279, 1960. 14. Lamprecht, W., and I. Trautschold, In Bergmeyer, H. U. (ed.), Methods of Enzymatic Analysis, Academic Press, New York, 1965, p. 543. 15. , and P. Stein, ibid., p. 610. 16. Streeto, J. M., and W. J. Reddy, Anal Biochem 21: 416, 1971. 17. Marsh, J. M.J Biol Chem 245: 1596, 1970. 18. Armstrong, D. T., J. O. O'Brien, and R. O. Greep, Endocrinology 75: 488, 1964.
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G
ONADOTROPINS are known to control various metabolic processes in the ovary including ovarian differentiation, growth and steroid hormone secretion. One of the early steps in gonadotropin action appears to be stimulation of the adenylate cyclase-cyclic AMP system (1). This system has been shown to be present in ovarian tissues of several species (2-4) including rat luteal ovary preparations (5). Although the sensitivity of the adenylate cyclase in these luteal ovaries was not tested, both LH and FSH stimulated adenylate cyclase activity in ovarian homogenates from immature rats (6). Evidence has been presented for the involvement of cyclic AMP in the control of ovarian steroidogenesis in several species. Slices of bovine corpus luteum and interstitial cells of rabbit ovary elaborate progestational steroids when incubated in the presence of cyclic AMP or LH (7,8). Time sequence studies have shown that LH produced cyclic AMP accumulation prior to any increased synthesis of progestins (2). In addition both the cyclic nucleotide and its dibutyryl derivative (N6-O2'-dibutyryl cyclic Received March 6, 1975.
decrease occurred V2 to 1 day after administration of hCG. Activity then increased and remained significantly elevated from day 3 to 7. A second but more dramatic rise was observed on day 8 and this enhanced level of activity remained elevated until day 13. A decreased level of activity occurred on day 15. Unstimulated activity remained low for the 16 day period studied although significant rises were observed on day 3 and days 8 to 15 after the administration of hCG. We have suggested that these changes in the adenylate cyclase activity modulate the level of cyclic AMP in the luteal cells and thereby induce changes in the activity of enzymes involved in progestin biosynthesis. (Endocrinology 97: 581, 1975)
3',5'-AMP) have been found to stimulate progestin synthesis and produce morphological luteinization in tissue cultures of granulosa cells harvested from rhesus monkey and porcine ovaries (9). These effects were similar to those obtained with the addition of LH and FSH to the culture medium. Evidence presented recently indicates that the process of luteinization might also be held in check by cyclic AMP (10). Injection of cyclic AMP or the dibutyryl derivative, directly into Graafian follicles of estrous rabbits inhibited luteinization in response to LH whereas injection of phosphodiesterase induced luteinization in a number of follicles. The mechanisms by which cyclic AMP may mediate LH action is not clear although evidence of a cyclic AMPdependent protein kinase in the bovine ovary (11) indicates that these mechanisms involve, in part or wholly, cyclic AMPstimulated phosphorylation of cellular proteins. The object of the present study was to determine the time sequence of the response of the adenylate cyclase system in the ovaries of prepubertal rats following injections of PMS and hCG. Using an in 581
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5S2
vitro system the response of the enzyme was studied with respect to the basal, or unstimulated activity, and both LH and fluoride stimulated activity.
Endo • 1975 Vol 97 • No 3
weighed in tared vials, the tissues were then dried for 3 days at 104 C, cooled in a desiccator and re-weighed. Preparation of the adenylate cyclase fraction
Materials and Methods Materials Pregnant mare's serum (PMS) was obtained from Schering Pty. Ltd., human chorionic gonadotropin (hCG) was from Organon Laboratories, and bovine LH (NIH-LH B7) was supplied by the National Institutes of Health. a-32Plabelled ATP was purchased from the Radiochemical Centre, Amersham. Unlabelled ATP, cyclic AMP, creatine phosphate (CP), creatine kinase (CK), glucose-6-phosphodehydrogenase, hexokinase and NADP were obtained from the Boehringer-Mannheim Corporation. Bovine serum albumin, fraction V, was from the Commonwealth Serum Laboratories. "Trizma" base and "Trizma" HC1 were purchased from the Sigma Chemical Company, and the aluminum oxide, activity 1, neutral, for column chromatography, was from Merck. The chromatography columns were similar to those described by White and Zenser (12) and were plugged with glass wool. Chemicals used in the assays of adenylate cyclase, ATP and CP, were of "Analar" grade. Solvents and fluors for the scintillation fluid were obtained from the Packard Instrument Co., Inc. Pretreatment of rats Superovulation was induced in 23- to 28-dayold Wistar strain rats by sc injection of 75 IU of PMS and 37.5 IU of hCG 64 h later. These animals were then kept under a constant lighting schedule of 12 h light and 12 h darkness. The rats were killed by cervical dislocation one day prior to, and 0, lA, 1, 3, 5, 7, 8, 9, 13 and 15 days after the injection of hCG. Vaginal smears were taken daily from the first day of vaginal opening to determine the duration of pseudopregnancy. Ovarian and uterine weights Both wet and dry weights of the ovaries and uterine horns were determined for representative groups of rats for each experimental day. Immediately after the animals were killed their ovaries and uterine horns were excised and
Groups of 3 to 6 rats were killed between 10 and 11 AM on the morning of each experimental day. The ovaries were excised free of surrounding fat and were pooled and weighed. They were then homogenized in 4.5 vol of ice-cold 0.25M sucrose in a Kontes-Dual homogenizer using 3 strokes of the teflon pestle at 800 rpm. The homogenate was washed into a weighed centrifuge tube with ice-cold 0.25M sucrose and diluted to contain 1 part of ovarian tissue in 10 parts of sucrose. It was then centrifuged at 600 x g for 10 min and the pellet was resuspended in icecold, 0.25M sucrose to give a concentration of 20 mg of fresh ovarian tissue/200 /x\. This preparation was prepared fresh each day and was used within 2 h from the time the rats were killed. All assays were performed in quadruplicate. In order to compare adenylate cyclase activity on different days in terms of a standard weight of ovarian membrane fraction the dry weights of the 200 /A aliquots were determined in quadruplicate on each experimental day. It was therefore possible to calculate adenylate cyclase activity in equivalent amounts of ovarian tissue and to express the results as nmol of cyclic AMP produced per gram of wet (or dry) ovary per minute. Assay of adenylate cyclase The incubation medium for the assay of adenylate cyclase consisted of: 50 HIM Tris HC1 (Tris (hydroxymethyl) aminomethane HCl) buffer at pH 7.6, 5 HIM MgCl2, 1 mM cyclic AMP, 0.1% BSA, 1.8 mM a-(32P)-ATP (2-8 cpm/pmol) and either 10 mM NaF or 10 /xg LH. An ATP regenerating system consisting of 10 mM CP and 2-4 U CK was also added. The final volume of the incubation mixture containing 200 (A of the 600 x g pellet preparation was 0.6 ml. The 600 x g pellet preparation was added to the incubation medium to start the reaction and the mixtures were incubated at 30 C for periods of up to 25 min. Fifty microliters of a solution containing 0.2 mg cyclic AMP in 50 mM Tris HCl buffer at pH 7.6 was then added to the incubation tubes. The reactions were stopped
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OVARIAN ADENYLATE CYCLASE ACTIVITY by immersing the tubes in boiling water for 3 min. The tubes were then centrifuged to remove denatured protein and the supernatant was assayed for cyclic AMP according to the method described by White and Zenser (12). This involved running the supernatant completely on to columns of aluminum oxide followed by 3.5 ml of 50 HIM Tris HC1 buffer at pH 7.6 and collecting a single 3.0 ml eluate fraction. Duplicates of 1.0 ml of this fraction were mixed with 10 ml of Bray's scintillation medium (13) and counted for 32P in a Packard Tri-Carb liquid scintillation spectrometer. Reaction blanks were obtained from incubations to which aliquots of boiled enzyme had been added. It was found that the activity of these blanks was up to 5% of the stimulated and 30% of the lowest basal adenylate cyclase values. The ability of the alumina columns to separate cyclic AMP was tested repeatedly. The recovery of [32P]cyclic AMP applied to the columns was 86 to 90%. When 32P-labelled ATP was applied to the columns a maximum of only 0.005% could be detected in the eluate fraction corresponding to cyclic AMP. When 5'-AMP was applied to the columns less than 0.1% could be eluted. Inorganic 32P is strongly bound by the alumina (12). The purity of the 32P-labelled cyclic AMP isolated by passing the incubation mixtures from the LH, NaF or control experiments, through columns of aluminum oxide was established by column chromatography on Dowex 1-C1 ion exchange resin, crystallization to constant specific activity and paper chromatography. Degradation of cyclic 3',5'-AMP The degradation of radioactive cyclic AMP during incubation of adenylate cyclase preparations from ovaries of gonadotropin treated rats was studied. Tritiated cyclic AMP (19 x 103 cpm) was incubated in the adenylate cyclase assay medium in the presence of unlabelled ATP and either 0.25 mM or 1.0 niM cyclic AMP or 10 mM theophylline. Inclusion of 1 mM cyclic AMP in the adenylate cyclase incubation medium prevented the breakdown of tritiated cyclic AMP for at least 18 min. However, 10 mM theophylline failed to completely inhibit the breakdown of tritiated cyclic AMP and was only as effective as the addition of 0.25 mM cyclic AMP. The ability of 1 mM cyclic AMP to protect radioactive cyclic AMP from hydrolysis during
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incubations of adenylate cyclase preparations was tested in the presence of 10 mM NaF or 10 fj.g LH. It was shown that the level of tritiated cyclic AMP remained constant for at least 18 min of incubation in the presence of these agents. Maintenance of ATP as substrate for the adenylate cyclase reaction The concentrations of ATP and CP were estimated in incubations of the adenylate cyclase assay mixture using the method of Lamprecht and Trautschold (14,15). The concentration of ATP in the medium used for assaying adenylate cyclase was determined throughout a 25 min incubation period in both the presence and absence of an ATP-regenerating system. The regenerating system consisted of 10 mM CP and 1-2 U of CK. It was shown that in the presence of the regenerating system there was a gradual but significant decrease in the concentration of ATP whereas in the absence of the regenerating system the concentration of ATP fell rapidly and could not be detected after 25 min of incubation. In an attempt to determine the conditions for a maximum rate of regeneration of ATP during adenylate cyclase assays a study was made of the ability of various levels of the regenerating system to maintain ATP. ATP and CP levels were estimated in incubations of the adenylate cyclase mixture containing one of the following combinations of the regenerating system: system A, 10 mM CP, 1-2 U CK; system B, 10 mM CP 2-4 U CK; system C, 20 mM CP, 2-4 U CK. Regenerating system B (2-4 U CK, 10 mM CP) was used for the assay of adenylate cyclase in the present studies since at this level the regenerating system efficiently maintained ATP and its concentration in the assay medium was found to be 1.25 mM after 20 min of incubation. Moreover under these conditions CP was not limiting as substrate for the regenerating enzyme.
Results Effects of PMS and hCG on the ovaries of immature rats Changes in ovarian and uterine weights brought about by treating immature rats with 75 IU PMS and 37.5 IU of hCG are shown in Fig. 1. Maximum ovarian wet and dry weights were found on day 3 after the hCG injection. The ovarian dry weight then
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recorded to determine whether the immature ovaries secrete estrogen in response to the gonadotropin treatment. As may be seen the uterine horns reached maximum wet and dry weights on day 5 after the hCG injection. The wet weight decreased markedly by day 7 and then more gradually up to day 15. There was a similar but less striking change in the dry weight. Changes in adenylate cyclase activity
5 7 9 Day after hCG injection
11
13
15
FIG. 1. Changes in wet and dry weights of ovaries and uterine horns of immature rats injected with PMS and hCG. Ovarian wet (o o) and dry (• •) weights; uterine wet (a D) and dry (• •) weights.
remained elevated with no further significant change up to day 15. On the other hand the wet weight decreased from day 3 to 7 and then its level appeared to plateau up to day 15. Uterine wet and dry weights were
Changes in the response of the adenylate cyclase enzyme in ovarian tissue from gonadotropin treated immature rats are shown in Fig. 2. Unstimulated, or basal, activity remained low (0.92 to 3.53 nmol/g ovary/min) throughout the 16-day period studied. Significantly increased activities were found on day 3 (P < 0.001) and on days 8 to 15 (0.01 > P > 0.001). LH-stimulated activity was significantly greater (P < 0.001) than basal activity on day 0 of treatment and on days 3 to 15. Fluoride-stimulated activity was significantly greater than both basal and LH-stimulated activities at all times.
so
i
40
FIG. 2. Basal and stimulated adenylate cyclase activity measured in homogenates of ovaries that were removed at various times after the treatment of immature rats with PMS and hCG. The 600 x g fractions of these homogenates, representing 20 mg of fresh ovarian tissues, were incubated for 12 min under basal conditions (D), in the presence of 10 /xg LH (0), or in the presence of 10 mM NaF (•). Each assay was performed in quadruplicate and the results are shown as the mean ± 1 SD.
ra 20
•2\ -2
-1
0
2
3
4
5
6
7
13
14
Day after hCC injection
PMS
hCG
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OVARIAN ADENYLATE CYCLASE ACTIVITY
The response of the adenylate cyclase to LH was characterized by an initial rise from 1.29 nmol/g ovary/min XV2 days after PMS to 2.40 nmol/g ovary/min 2V2 days after PMS. It should be noted that a significant decrease in this response occurred half a day after the injection of hCG which was followed by a significant rise above all previous values to 6.61 nmol/g ovary/min on day 3. This significantly elevated activity persisted until day 7. A second and dramatic increase occurred by day 8 and a maximum activity of 16.38 nmol/g ovary/min was found on day 9. This enhanced activity was present up to day 13 but fell significantly on day 15 to 4.43 nmol/g ovary/min. As may be seen from Fig. 2 changes in fluoride-stimulated activity were similar but more striking than the LH-stimulated activity. Two and a half days after PMS the fluoride-stimulated activity was 8.90 nmol/g ovary/min. A significant decrease in the response of the adenylate cyclase was apparent after the injection of hCG. The first significant increase occurred 1 day after administration of hCG. It then rose to 20.21 nmol/g ovary/min by day 3 and remained elevated until day 7. At this stage as in the case of LH, there was a dramatic increase in the fluoride-stimulated activity. This greatly enhanced activity, of 42.30 to 48.72 nmol/g ovary/min, was found to be present up to day 13 but decreased thereafter to a level of 23.36 nmol/g ovary/min which was not significantly different from that measured on day 7.
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3-0
i 20
K)
0-1
10
10 LH(pg/ml)
100
1000
FlG. 3. Dose-response curve for LH-stimulated adenylate cyclase in luteal ovary preparations from PMShCG treated immature rats. The adenylate cyclase activity of the 600 x g pellet was determined in the presence of various concentrations of luteinizing hormone. Each point is the mean ± 1 SD of triplicate samples.
of activity was reached was 10 /Ag/ml and the activity did not alter for doses of LH up to 500 /x,g/ml. At these maximal doses the activity was 2.71 nmol/g ovary/min. Half maximal stimulation was obtained with 1.5 (xg LH/ml incubation mixture. Time-course studies
Ovarian homogenates prepared from the gonadotropin-treated immature rats 7 days after the injection of hCG were incubated in the presence of 10 /xg LH or 10 mM fluoride or without these agents. The rate Effects of LH on adenylate cyclase activity of production of cyclic AMP was measured in vitro after 6, 12 and 18 min of incubation. As may A 600 x g pellet preparation of ovaries be seen in Fig. 4 the basal activity as well taken from gonadotropin treated rats was as the LH and fluoride stimulated activities incubated under the conditions described in were linear over this period, the rates being Materials and Methods but in the presence 1.61, 5.40 and 18.10 nmol/g ovary/min, of various concentrations of LH. Figure 3 respectively. shows the sigmoidal dose response curve Discussion obtained when the concentration of LH in the medium varied from 0.2 to 500 //,g/ml. The method for measuring adenylate cyThe concentration at which a constant level clase employed in the present study was
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NUGENT, LOPATA AND GOULD
FIG. 4. Adenylate cyclase activity as a function of time in preparations of ovaries removed from immature rats 7 days after the injection of hCG. Fractions of the 600 x g pellet representing 20 nig of fresh tissue were incubated under basal conditions (O O reg coeff 0.032, corr coeff 0.884) or in the presence of 10 mM NaF ( D — • — D , reg coeff 0.362, corr coeff 0.945) or 10 Mg LH (• •, reg coeff 0.109, corr coeff 0.873).
initially described by White and Zenser (12) for the assay of the enzyme in plasma membrane fractions isolated from rat kidney. The experiments described in this paper have validated this method for measuring adenylate cyclase activity in ovarian homogenates. Several workers have used an ATPregenerating system in the assay of adenylate cyclase but did not establish that the system actually maintained ATP levels during the time intervals of incubation (5,16). It was reported that the 600 x g pellet of the rat luteal- ovary contained a complex of highly active "ATPase" enzymes consisting of adenosine triphosphatase, adenosine diphosphatase and nucleotidase (5). These enzymes rapidly degrade ATP added as substrate for the adenylate cyclase reaction. Our data show that although "ATPase" enzymes are active in rat luteal ovary preparations, ATP may be conserved as substrate for the adenylate cyclase enzyme by the inclusion of QK and GP as an ATP-regenerating system. This regenerating system functions efficiently to.maintain adequate concentrations of ATP for at least 20 min. of incubation in the presence of an ovarian preparation equivalent to 20 mg of fresh tissue. Under these conditions basal adenylate cyclase activity could be measured in ovaries of immature rats treated with either, PMS or PMS and hCG. Moreover, the same conditions have been em-
Enilo • 1975 Vol 97 • No .1
ployed to demonstrate the sensitivity of ovarian adenylate cyclase to different dosages of LH in the incubation medium and to study the response of the enzyme to fluoride and LH in vitro. The response of the adenylate cyclase system in the ovaries of immature rats treated with gonadotropins can be described in four main phases. 1. The ovulatory phase (day 0 to day V2 after hCG) when there is an apparent fall in the adenylate cyclase activity measured as the fluoride and LH stimulated activities. 2. The phase of corpus luteum formation and growth (day 1 to day 7 after hCG) during which both the fluoride and LH stimulated activities increase gradually to reach a maximum level 3 days after the injection of hCG. This level of activity is sustained for a further 4 days. A consistent increase in the basal, or unstimulated, adenylate cyclase activity also occurs during this period although the increase has not been found to be statistically significant. 3. The phase of enhanced ovarian adenylate cyclase activity, which lasts from day 8 to 13 after the injection of hCG. This period is characterized by a dramatic increase in both the fluoride and LH stimulated activities and corresponds to the first significant increase in the basal adenylate cyclase activity. 4. The phase of declining ovarian adenylate cyclase activity (from day 13 after hCG) during which the response of the enzyme measured as the basal and stimulated activities dropped to the levels found in phase 2. A measured rise or fall in the stimulated adenylate cyclase activity could be due to a change in the content of enzyme in the ovary, to a change in the sensitivity of the enzyme, or to changes in both these factors. Thus the initial fall in the adenylate cyclase activity observed in phase 1 may be related to ovulation which involves loss of oocytes, follicular cells and fluid from the ovary. Moreover, a fall in the sensitivity of the adenylate cyclase measured as a decrease in the ratio of stimulated to basal enzyme activity appears to have occurred
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OVARIAN ADENYLATE CYCLASE ACTIVITY during this period. Following the injection of PMS and hCG into immature rats an increase in the ovarian adenylate cyclase content and sensitivity contribute to the rise in the enzyme activity observed during phases 2 and 3. Increased sensitivity of the adenylate cyclase, measured as the in vitro response of the enzyme to LH and fluoride appears to predominate during days 3 to 7 after hCG injection (phase 2). The significant increase in basal activity (e.g., from a mean value of 1.65 nmol cAMP/g ovary/min during days 3 to 7) up to a mean value of 3.22 nmol cAMP/g ovary/min (P < 0.001) during days 8 to 13 after hCG injection, was accompanied by a fall in the sensitivity of the enzyme. This decrease in the adenylate cyclase sensitivity was judged by a fall in the fluoride:basal activity (e.g., from 23:1 on day 7 to a ratio of 14:1 on day 9) and a similar fall in the LH:basal ratio (e.g., from 8:1 on day 7 to a ratio of 5:1 on day 9). These results suggest that synthesis of the enzyme may be the main factor contributing to the enhanced activity observed during days 8 to 13 after hCG injection. In ovarian tissues from various species (3,4,9,17) LH appears to activate the adenylate cyclase system as an early step in the stimulation of steroid biosynthesis. The cyclic AMP generated is considered to be the intermediate which activates the enzymes of the biosynthetic pathway (1). Luteinizing hormone has also been shown to stimulate progestin synthesis in the luteal ovary of the gonadotropin primed rat both in vivo and in vitro (18). In view of these findings and the present data indicating a significant increase of adenylate cyclase
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activity in the ovaries of immature rats treated with gonadotropins, particularly between days 3 to 7 and 8 to 13 after the treatment, it is suggested that the changes in adenylate cyclase activity modulate the level of cyclic AMP in luteal cells and are associated with changes in steroidogenesis. References I! .Niswender, G. D., K. M. J. Menon, and R. B. Jaffe, Fertil Steril 23: 432, 1972. 2. Marsh, J. M., R. W. Butcher, K. Savard, and E. W. Sutherland,; Biol Chem 241: 5436, 1966. 3. Kuehl, F. A., Jr., J. L. Humes, J. Tarnoff, V. J. Cirillo, and E. J. Ham, Science 169: 883, 1970. 4. Dorrington, J. H., and B. Bagget, Endocrinology 83: 989, 1969. 5. Stansfield, D. A., and D. J. Franks, Biochim Biophys Ada 242: 606, 1971. 6. Fontaine, Y. A., E. Fonatine-Bertrand, N. DelerueLebelle, and C. Salmon,/ Physiol (Paris) 63: 49A, 1971. 7. Marsh, J. M., and K. Savard, Steroids 8: 133, 1966. 8. Dorrington, J. H., and R. Kilpatrick, Biochem J 104: 725, 1967. 9. Channing, C. P., Recent Progr Horrn Res 26: 589, 1970. 10. Lemaire, W. J., T. Mills, Y. Ito, and J. M. Marsh, Biol Reprod 6: 109, 1972. 11. Kuo, J. F., and P. Greengard, Proc Natl Acad Sci USA 64: 1349, 1971. 12. White, A. A., and T. V. Zenser, Anal Biochem 41: 372, 1971. 13. Bray, G. A., Anal Biochem 1: 279, 1960. 14. Lamprecht, W., and I. Trautschold, In Bergmeyer, H. U. (ed.), Methods of Enzymatic Analysis, Academic Press, New York, 1965, p. 543. 15. , and P. Stein, ibid., p. 610. 16. Streeto, J. M., and W. J. Reddy, Anal Biochem 21: 416, 1971. 17. Marsh, J. M.J Biol Chem 245: 1596, 1970. 18. Armstrong, D. T., J. O. O'Brien, and R. O. Greep, Endocrinology 75: 488, 1964.
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