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Prostaglandin F2a, an ovulatory intermediate in the in vitro perfused rabbit ovary model
PROSTAGLANDINS
PROSTAGLANDIN
F2a, AN OVULATBRY INTERMEDIATE
IN THE IN VITRO PERFUSED RABBIT OVARY MODEL by Sheldon Schlaff, Yoshimune Kobayashi,2 Karen H. Wright Rosemary Santulli and Edward E. Wallach Breskman Endocrine Laboratory Department of Obstetrics and Gynecology Pennsylvania Hospital and University of Pennsylvania School of Medicine Philadelphia, Pennsylvania
ABSTRACT PGF2, has been proposed as a mediator of mammalian ovulation. To elucidate further the role of PGF2, in the process of ovulation, PGF and PGF2a metabolite were measured by radioimmunoassay in the perfusate of an -in vitro perfused rabbit ovary preparation. Perfusion medium samples were collected over a 10 to 12 hour period from ovaries perfused with tissue culture Ml99 (total volume 150 ml, sample volume 3 ml) to which varying amounts of hCG had been added. [The with PGFlar antisera a 40% cross reaction ',:z total PGF was measured with this antisera.] metabolite showed a linear Both PGF and PGF2a increase with time and numbers of ovulations. PGF media accumulation was 575 pg/ovary/ovulation/hr and PGF2a metabolite accumulation was 367 Medium prostaglandin content pg/ovary/ ovulation/hr. could be correlated with numbers of ovulations, ovulatory efficiency (number of ovulations/ total follicles) These data best fit a model but not total follicles. producing units of independent ovulatory PGF2a without recruitment or 'interaction between them. We infer tha PGF and PGF2, metabolites in this system index of the ovulation can be used as a direct process.
JULY 1983VOL.26NO.l
111
PROSTAGLANDINS
INTRODUCTION Elucidation of the biochemical events leading to ovulation at the level of the ovary presents a major Prostaglandins have been implibiological challenge. cated in this process acting locally at the ovarian level. Support for this concept includes the observation that indomethacin blocks ovulation in the intact rabbit(l), rat(2) and monkey(3) and in the in vitro PGF2, can perfused rabbit ovary preparation.(4) overcome this blockade of ovulation in the in vitro perfused rabbit ovary(4) in the- intact and also monkey(3). PGE2 in this in vitro model does not overcome indomethacin blocked-ovuion in contrast to Furthermore, intrafollicular the (5). PGF2a intact rabbit injection of PGF antiserum in the completely inhibits subsequent ovulation -in vivo(6). Total follicular content of PGE and PGF have been measured in the rabbit and rat(7),(8), but circulating levels of PGF and its stable metabolite (13, 14dihydro, IS-keto PGF2,) during the ongoing ovulatory order to have not been reported. In process substantiate that a compound serves as an agent in any particular process requires measurement of that compound and correlation with concurrent physiological events. prostaglandin measurement of ovarian Quantitative as an ovulatory production may serve to identify PGF intermediate. These data could then be used to evaluate conditions and experimental the effect of various metabolite PGF or PGF perturbants on chemical production. The in vitro perfused rabbit ovary model is nations since the ideally suited for- such determiinfluences and from systemic ovary is isolated fluid for of perfusion collection sequential performed under measurement can be prostaglandin carefully controlled conditions without disturbing the This experiment was designed to ovulatory process. determine if Prostaglandin F and F2a metabolite are produced by the ovulating ovary and to determine if a correlation exists between PG production and ovulation. MATERIALS AND METHODS The in vitro perfused rabbit ovary system has been describedpreviously(9)and utilizes excised ovaries from A laparotomy was 3-4.5 kg New Zealand white rabbits. The performed under sodium pentobarbital anesthesia. ovarian arteries were located and cannulated -in situ.
112
JULY 1983VOL. 26 NO.1
PROSTAGLANDINS
The ovary was initially flushed with cold Medium 199 supplemented with insulin, heparin, streptomycin, and penicillin without added protein carrier, and the ovary excised and placed in the perfusion system. This consisted of a silastic tubing oxygenator, a Buchler roller pump and a perfusion chamber. The total volume of perfusate in the system was 150 ml. A flow of 1.5 ml/minute was maintained. Samples of perfusion fluid (3 were removed prior to the beginning of the ml) perfusion, at the onset of perfusion and at least 2 hour intervals throughout the perfusion for prostaglandin determinations. Throughout ovarian perfusion, observations were made continually for occurrence of ovulation. Several experimental doses of HCG were used, 15 to 50 IU/ml, to obtain a range of ovulations in order to correlate the number of ovulations with prostaglandin medium content. Without added HCG no ovulations occurred in control experiments. The 50 IU/150 ml dose represented an approximate plateau level for numbers of ovulations. Eight of the ten HCG stimulated ovaries ovulated (range O-7 ovulations). The HCG stimulated ovaries that did not ovulate received, in one case 15 IU, the lowest dose of HCG in this experimental series and in the other indomethacin (0.5 ugm/ml) plus HCG (5OIU). PGF and dihydroketo PGF2a were both measured in a Antisera for PGFz~ and its double antibody system. from metabolite were obtained Clinical Assays, Total PGF was measured Cambridge, Massachusetts. because of a 40% cross-reaction of anti-PGF2a with 0.1% PGE or PGF2a metabolite PGF2,. Less than antisera. Antiserum to with PGF cross reacted was more PGF dihydroketo specific than PGF2a antiserum and demonstrated less than 0.1% cross-reaction this with PGF2a, or PGE2. Because of PGFla, specifity, chromatography was not performed on medium samples. Using the method of Jaffe and Behrman(lO), one ml of Medium 199 was extracted with 5 ml petroleum ether and the organic phase was poured off after sample freezing by dry ice immersion. To the thawed aqueous phase was added 3 ml of a solvent mixture containing ethyl acetate, isopropanol and 0.1 M HCl, 3:3:1 by This was vortexed for 60 seconds followed by volume. addition of 2 ml of ethyl acetate and 3 ml of water. phase and the organic centrifuged The tubes were transferred to a glass tube and compressed air dried at Sample room temperature over a 6-12 hour period. recovery was assessed by prior addition of tritiated (obtained from New England Nuclear, Boston, PGF2a
JULY 1983VOL.26NO.l
113
PROSTAGLANDINS MA., or Clinical Assays, Cambridge, MA.). Extraction efficiency was reproducible and ranged from 65-75% for both PGF2, and dihydroketo PGF2,. Samples were reconstituted from dryness with 1 ml of 0.05 M Tris, pH 7.4, with 1 mg/ml pigskin gelatin and 0.01% sodium azide. Aliquots were incubated with tritiated prostaglandin and antisera at 4°C. overnight followed by addition of 90 ug of rabbit gamma globulin and 100-150 ul of undiluted goat antirabbit gamma globulin antisera (obtained from Pocono Rabbit Farms, Canadensis, PA). Raw data from a standard curve was corrected for extraction efficiency and perfusion volume change. Assay sensitivity for both assays was approximately 20 pg/ml as judged by 95% confidence limits of buffer sample controls without added prostaglandin. PGF and dihydroketo PGF2, were quantitatively recovered from Medium 199 when added to test tubes at a level of 200 pg/ml with parallel dose response curves for native and extracted material. carried out to test the Control experiments were recovery with the perfusion system using PGF2, (200 pg/ml) and HCG 50 IU/150 ml added to the perfusion bath This resulted in quantitative without any ovaries. recovery of (102-110%) corrected for when PGF2a extraction efficiency even over a ten hour period. Again, parallel dose response cures were obtained with native PGF, and extracted material. Without added HCG, two ovaries from two different animals did not ovulate nor show any significant increase in PGF content from the baseline which were initially at 5.8 and 37 It should be stressed that no protein carrier pg/ml. this particular for was necessary in system prostaglandin recovery but in other experiments variable loss of steroids was observed during perfusion unless carrier was added. RESULTS In each of the experiments, concentration of both PGF and dihydroketo PGF2, rose without evidence of any lag phase (Figure 1). This data could be best fit by a linear increase of prostaglandin content with time. were 0.78-0.99 in 8 (Correlation coefficients A lag phase experiments, p
JULY 1983VOL.26 NO.1
PROSTAGLANDINS
0
12
4
6
8
IO 0
IOO-
I- *WU
E EE >' I_g a
z
y= 3.7x+58.1
0
r = 0.81
p( 0.025
40-
20(0) (0) I 0
I
12
(2) I
(4) 0
(5) I
(6) I
4
6
8
IO
TIME HOURS AFTER HCG ADDED TO PERFUSATE
>
Figure 1 PGF and dihydroketo PGF2, as a function of time in a representative experiment after HCG was added to the perfusate. Prostaglandin production is linear without evidence of a lag phase. The numbers in parenthesis are the numbers of ovulations noted at the appropriate time points. JULY
1983 VOL.26NO.
I
115
PROSTAGLANDINS Dihydroketo and PGF production can PGF2a clearly be correlated with one another at each time point (Figure 2). The rate of increase of PGF2a metabolite is constant and 72% of the rate of increase of PGF ( b dihydroketo PGF2,/ PGF = slope = 0.72). to other The other 28% of PGF may be converted prostaglandin products or alternatively a rate limiting or equilibrium step of conversion of PGF to dihydroketo PGF may exist such that in the system, 72% was the In any case, the major maximum conversion possible. portion of PGF2a correlated very well metabolite The major PGF measured with PGF levels in the medium. presumed to be PGF2a since in physiological was from PGF2a. systems dihydroketo PGFz~ -is derived
=
loo-
E \ 5
80-
20 PGF
40
CONCENTRATION
60
80
100
(pg/ml)
metabolite Relationship of Figure 2 PGF2a PGF and dihydroketo productiof;- to PGF production. In PGF2, were compared at identical time points. this experiment at least 72% of the PGF2, metabolite accumulating with gonadotrophin stimulation was derived from PGF based on the slope (0.72).
116
JULY
1983 VOL. 26 NO. 1
PROSTAGLANDINS
0
I
2
TOTAL
3
4
5
6
7
OVULATIONS
PGF and PGF2, metabolite production as a The slope of rise total ovulation points. of each prostaglandin plotted vs. time derived from all the experiments is graphed vs. total ovulations in each experiment. There is a high correlation between PGF and ovulations and accumulation metabolite PGF2a achieved. JULY
1983 VOL. 26 NO. 1
117
PROSTAGLANDINS The media accumulation rates of PGF (8 ovaries) and dihydroketo PGF2, (6 ovaries) were correlated with ovulation (Figure 3). Media accumulation was 575 pg/hr/ovulation of PGF and 367 pg/hr/ovulation of The ratio of dihydroketo PGF/PGF PGF2a metabolite. accumulation was 0.63 based on the ratio of the slopes as shown in Fig. 3 underlining the significant coversion of PGF to its metabolite. These accumulation rates may be closely related to ovarian production rates, since during arterial perfusion, ovarian production would be expected to be cleared from the ovary. It is theoretically ovarian possible, however, that an compartment could exist that has a slow rate of equilibrium with the extracellular fluid. Medium PGF content did not show a significant increase over 10 hours perfusion when Indomethacin was used with 50 IU hCG (0.9 + 2.5 pg/ml/hr, r=0.54 & 0.63, 2 experiments, 2 rabbits; 2 ovaries). In contrast to with Indomethacin, the absence of ovulations six ovulations occurred in in each of the two control ovaries (2 experiments, 2 rabbits, 2 ovaries) with hCG alone with a significant increase in PGF accumulation (10.0 f 4.2 pg/ml/hr p
JULY
1983 VOL. 26 NO. 1
PROSTAGLANDINS
producing PGF2,. No data could be derived from these experiments to indicate that the increase in associated with each ovulation results in PGF2a further increase in ovulations. In such a recruitment process a nonlinear or exponential rate of increase in ovulations would be expected as PGF increased. The involvement of prostaglandins in the ovulatory process has been demonstrated in several species. PGF production is increased in follicles of HCG treated rabbits (7) as ovulation is approached and in human ovarian tissue exposed to gonadotropins (12). Increased contractility of ovarian smooth musculature has been associated with PGF2a both in vivro and in vitro in the rabbit (13). In addit?bnnF2. can stimulate ovulation in the in vitro perfused rabbit ovary in the absence of gonadotropins (13). protaglandin could be While basal production derived from non-follicular sources such as the vascular pedicle or fat, the significant increase of accumulation with ovulation was probably derived from the follicle. We infer this from: the increase in prostaglandin accumulation with increasing ovulation, the absence of significant prostaglandin accumulation without ovulation in ovaries without hCG or treated with indomethacin, and the absence of hCG receptors in vascular pedicle or fat. No ovulations occured without hCG stimulation. The effect of changing biochemical conditions with pharmacologic agents on ovulations and its effect on perfusion medium prostaglandins may serve to establish a quantitative marker. This may help elucidate some of the intermediate biochemical steps in the ovulatory process.
JULY 1983VOL.26NO.l
119
PROSTAGLANDINS References 1)
Diaz-Infante, A. Jr., K.H. Wright, and E.E. Wallach. Effects of Indomethacin and PGF2, on Ovulation and Ovarian Contractility in the Rabbit. Prostaglandins 5:567, 1974.
2)
Tsafriri, A., H.R. Lindner, U. Zor, and S. A. Lamprecht. Physiological Role of Prostaglandins on the Induction of Ovulation. Prostaglandins 2:1, 1972.
3)
Wallach, E.E., R. Bronson, Y. Hamada, K.H. Wright, and V.C. Stevens. Effectiveness of PGF2, in Restoration of hMG-hCG Induced Ovulation in Indomethacin Treated Rhesus Monkeys. Prostaglandins 1975. -10:129,
4)
Hamada, Y., K.H. Wright, and E.E. Wallach. Reversal of Indomethacin Blocked Ovulation PGF2,. Fertil. Steril. -30:702,1978.
5)
Hamada, Y., R.A. Bronson, K.H. Wright, and E.E. Wallach. Ovulation in the Perfused Rabbit Ovary: The Influence of Prostaglandins and Prostaglandin Inhibitors. Biol . Rprod. <-17:58, 1977.
6)
Armstrong, D.T., D.L. Grinwich, Y.S. Moon, and J. Inhibition of Ovulation in Rabbits by Zamecnick. Intrafollicular Injection of Indomethacin and PGF Antiserum. Life Sci. -14:129, 1974.
7)
N.S.J., J.M. Marsh, and W.J. LeMaire. Yang, Prostaglandin Changes Induced by Ovulatory Stimuli The Effect of Indoin Rabbit Graafian Follicles: methacin. Prostaglandins 4:395, 1973.
8)
LeMaire, W.J., Post Ovulatory Prostaqlandins Prostaglandins
9)
Lambersteen, C.J., D.F. Greenabum, K.H. Wright, and In vitro studies of ovulation in the E.E. Wallach. Fertil. Steril. 27: 178, perfused rabbit ovary. 1976.
In Vitro by -
Pre and R. Linder, and J.M. Marsh. Changes in the Concentration of in Rat Graafian Follicles. 9:221, 1975
18)
Prostaglandin E, A, F Jaffe, B.M. and H.R. Behrman. Academic in "Methods of Hormone Radioimmunoassy." Press, New York, 1974, p. 9.
11)
Needleman, P., S.D. Bronsom, A. Wychie, and M. Cardiac and renal PG12. J. Clin. Spivakoff. Invest. -61:839, 1978.
120
JULY 1983 VOL. 26 NO. 1
PROSTAGLANDINS 12)
Plunkett, E.R., Y.S.' Moon, J. Zamecnik, and D.T. Preliminary Evidence of a Role for Armstrong. Prostaglandin F in Human Follicular Function. Am. J. Obstet. Gynecol. 123:391, 1975.
13)
Virutamasen, P., K.H. Wright, and E.E. Wallach. on Ovarian Effects of Prostaglandins E and F Contractility in the Rabbit. Obstet. Gynecol. 132:728, 1978.
Editor: Harold R. Behrman Received: 7-27-81 Revised: 3-l-83 Accepted : 4-25-83
1.
Supported by NIH Grant HD-05948 and the Breskman Foundation.
2.
Population
JULY
Council
1983 VOL. 26 NO. 1
Fellow
in Reproductive
Biology.
121
PROSTAGLANDIN
F2a, AN OVULATBRY INTERMEDIATE
IN THE IN VITRO PERFUSED RABBIT OVARY MODEL by Sheldon Schlaff, Yoshimune Kobayashi,2 Karen H. Wright Rosemary Santulli and Edward E. Wallach Breskman Endocrine Laboratory Department of Obstetrics and Gynecology Pennsylvania Hospital and University of Pennsylvania School of Medicine Philadelphia, Pennsylvania
ABSTRACT PGF2, has been proposed as a mediator of mammalian ovulation. To elucidate further the role of PGF2, in the process of ovulation, PGF and PGF2a metabolite were measured by radioimmunoassay in the perfusate of an -in vitro perfused rabbit ovary preparation. Perfusion medium samples were collected over a 10 to 12 hour period from ovaries perfused with tissue culture Ml99 (total volume 150 ml, sample volume 3 ml) to which varying amounts of hCG had been added. [The with PGFlar antisera a 40% cross reaction ',:z total PGF was measured with this antisera.] metabolite showed a linear Both PGF and PGF2a increase with time and numbers of ovulations. PGF media accumulation was 575 pg/ovary/ovulation/hr and PGF2a metabolite accumulation was 367 Medium prostaglandin content pg/ovary/ ovulation/hr. could be correlated with numbers of ovulations, ovulatory efficiency (number of ovulations/ total follicles) These data best fit a model but not total follicles. producing units of independent ovulatory PGF2a without recruitment or 'interaction between them. We infer tha PGF and PGF2, metabolites in this system index of the ovulation can be used as a direct process.
JULY 1983VOL.26NO.l
111
PROSTAGLANDINS
INTRODUCTION Elucidation of the biochemical events leading to ovulation at the level of the ovary presents a major Prostaglandins have been implibiological challenge. cated in this process acting locally at the ovarian level. Support for this concept includes the observation that indomethacin blocks ovulation in the intact rabbit(l), rat(2) and monkey(3) and in the in vitro PGF2, can perfused rabbit ovary preparation.(4) overcome this blockade of ovulation in the in vitro perfused rabbit ovary(4) in the- intact and also monkey(3). PGE2 in this in vitro model does not overcome indomethacin blocked-ovuion in contrast to Furthermore, intrafollicular the (5). PGF2a intact rabbit injection of PGF antiserum in the completely inhibits subsequent ovulation -in vivo(6). Total follicular content of PGE and PGF have been measured in the rabbit and rat(7),(8), but circulating levels of PGF and its stable metabolite (13, 14dihydro, IS-keto PGF2,) during the ongoing ovulatory order to have not been reported. In process substantiate that a compound serves as an agent in any particular process requires measurement of that compound and correlation with concurrent physiological events. prostaglandin measurement of ovarian Quantitative as an ovulatory production may serve to identify PGF intermediate. These data could then be used to evaluate conditions and experimental the effect of various metabolite PGF or PGF perturbants on chemical production. The in vitro perfused rabbit ovary model is nations since the ideally suited for- such determiinfluences and from systemic ovary is isolated fluid for of perfusion collection sequential performed under measurement can be prostaglandin carefully controlled conditions without disturbing the This experiment was designed to ovulatory process. determine if Prostaglandin F and F2a metabolite are produced by the ovulating ovary and to determine if a correlation exists between PG production and ovulation. MATERIALS AND METHODS The in vitro perfused rabbit ovary system has been describedpreviously(9)and utilizes excised ovaries from A laparotomy was 3-4.5 kg New Zealand white rabbits. The performed under sodium pentobarbital anesthesia. ovarian arteries were located and cannulated -in situ.
112
JULY 1983VOL. 26 NO.1
PROSTAGLANDINS
The ovary was initially flushed with cold Medium 199 supplemented with insulin, heparin, streptomycin, and penicillin without added protein carrier, and the ovary excised and placed in the perfusion system. This consisted of a silastic tubing oxygenator, a Buchler roller pump and a perfusion chamber. The total volume of perfusate in the system was 150 ml. A flow of 1.5 ml/minute was maintained. Samples of perfusion fluid (3 were removed prior to the beginning of the ml) perfusion, at the onset of perfusion and at least 2 hour intervals throughout the perfusion for prostaglandin determinations. Throughout ovarian perfusion, observations were made continually for occurrence of ovulation. Several experimental doses of HCG were used, 15 to 50 IU/ml, to obtain a range of ovulations in order to correlate the number of ovulations with prostaglandin medium content. Without added HCG no ovulations occurred in control experiments. The 50 IU/150 ml dose represented an approximate plateau level for numbers of ovulations. Eight of the ten HCG stimulated ovaries ovulated (range O-7 ovulations). The HCG stimulated ovaries that did not ovulate received, in one case 15 IU, the lowest dose of HCG in this experimental series and in the other indomethacin (0.5 ugm/ml) plus HCG (5OIU). PGF and dihydroketo PGF2a were both measured in a Antisera for PGFz~ and its double antibody system. from metabolite were obtained Clinical Assays, Total PGF was measured Cambridge, Massachusetts. because of a 40% cross-reaction of anti-PGF2a with 0.1% PGE or PGF2a metabolite PGF2,. Less than antisera. Antiserum to with PGF cross reacted was more PGF dihydroketo specific than PGF2a antiserum and demonstrated less than 0.1% cross-reaction this with PGF2a, or PGE2. Because of PGFla, specifity, chromatography was not performed on medium samples. Using the method of Jaffe and Behrman(lO), one ml of Medium 199 was extracted with 5 ml petroleum ether and the organic phase was poured off after sample freezing by dry ice immersion. To the thawed aqueous phase was added 3 ml of a solvent mixture containing ethyl acetate, isopropanol and 0.1 M HCl, 3:3:1 by This was vortexed for 60 seconds followed by volume. addition of 2 ml of ethyl acetate and 3 ml of water. phase and the organic centrifuged The tubes were transferred to a glass tube and compressed air dried at Sample room temperature over a 6-12 hour period. recovery was assessed by prior addition of tritiated (obtained from New England Nuclear, Boston, PGF2a
JULY 1983VOL.26NO.l
113
PROSTAGLANDINS MA., or Clinical Assays, Cambridge, MA.). Extraction efficiency was reproducible and ranged from 65-75% for both PGF2, and dihydroketo PGF2,. Samples were reconstituted from dryness with 1 ml of 0.05 M Tris, pH 7.4, with 1 mg/ml pigskin gelatin and 0.01% sodium azide. Aliquots were incubated with tritiated prostaglandin and antisera at 4°C. overnight followed by addition of 90 ug of rabbit gamma globulin and 100-150 ul of undiluted goat antirabbit gamma globulin antisera (obtained from Pocono Rabbit Farms, Canadensis, PA). Raw data from a standard curve was corrected for extraction efficiency and perfusion volume change. Assay sensitivity for both assays was approximately 20 pg/ml as judged by 95% confidence limits of buffer sample controls without added prostaglandin. PGF and dihydroketo PGF2, were quantitatively recovered from Medium 199 when added to test tubes at a level of 200 pg/ml with parallel dose response curves for native and extracted material. carried out to test the Control experiments were recovery with the perfusion system using PGF2, (200 pg/ml) and HCG 50 IU/150 ml added to the perfusion bath This resulted in quantitative without any ovaries. recovery of (102-110%) corrected for when PGF2a extraction efficiency even over a ten hour period. Again, parallel dose response cures were obtained with native PGF, and extracted material. Without added HCG, two ovaries from two different animals did not ovulate nor show any significant increase in PGF content from the baseline which were initially at 5.8 and 37 It should be stressed that no protein carrier pg/ml. this particular for was necessary in system prostaglandin recovery but in other experiments variable loss of steroids was observed during perfusion unless carrier was added. RESULTS In each of the experiments, concentration of both PGF and dihydroketo PGF2, rose without evidence of any lag phase (Figure 1). This data could be best fit by a linear increase of prostaglandin content with time. were 0.78-0.99 in 8 (Correlation coefficients A lag phase experiments, p
JULY 1983VOL.26 NO.1
PROSTAGLANDINS
0
12
4
6
8
IO 0
IOO-
I- *WU
E EE >' I_g a
z
y= 3.7x+58.1
0
r = 0.81
p( 0.025
40-
20(0) (0) I 0
I
12
(2) I
(4) 0
(5) I
(6) I
4
6
8
IO
TIME HOURS AFTER HCG ADDED TO PERFUSATE
>
Figure 1 PGF and dihydroketo PGF2, as a function of time in a representative experiment after HCG was added to the perfusate. Prostaglandin production is linear without evidence of a lag phase. The numbers in parenthesis are the numbers of ovulations noted at the appropriate time points. JULY
1983 VOL.26NO.
I
115
PROSTAGLANDINS Dihydroketo and PGF production can PGF2a clearly be correlated with one another at each time point (Figure 2). The rate of increase of PGF2a metabolite is constant and 72% of the rate of increase of PGF ( b dihydroketo PGF2,/ PGF = slope = 0.72). to other The other 28% of PGF may be converted prostaglandin products or alternatively a rate limiting or equilibrium step of conversion of PGF to dihydroketo PGF may exist such that in the system, 72% was the In any case, the major maximum conversion possible. portion of PGF2a correlated very well metabolite The major PGF measured with PGF levels in the medium. presumed to be PGF2a since in physiological was from PGF2a. systems dihydroketo PGFz~ -is derived
=
loo-
E \ 5
80-
20 PGF
40
CONCENTRATION
60
80
100
(pg/ml)
metabolite Relationship of Figure 2 PGF2a PGF and dihydroketo productiof;- to PGF production. In PGF2, were compared at identical time points. this experiment at least 72% of the PGF2, metabolite accumulating with gonadotrophin stimulation was derived from PGF based on the slope (0.72).
116
JULY
1983 VOL. 26 NO. 1
PROSTAGLANDINS
0
I
2
TOTAL
3
4
5
6
7
OVULATIONS
PGF and PGF2, metabolite production as a The slope of rise total ovulation points. of each prostaglandin plotted vs. time derived from all the experiments is graphed vs. total ovulations in each experiment. There is a high correlation between PGF and ovulations and accumulation metabolite PGF2a achieved. JULY
1983 VOL. 26 NO. 1
117
PROSTAGLANDINS The media accumulation rates of PGF (8 ovaries) and dihydroketo PGF2, (6 ovaries) were correlated with ovulation (Figure 3). Media accumulation was 575 pg/hr/ovulation of PGF and 367 pg/hr/ovulation of The ratio of dihydroketo PGF/PGF PGF2a metabolite. accumulation was 0.63 based on the ratio of the slopes as shown in Fig. 3 underlining the significant coversion of PGF to its metabolite. These accumulation rates may be closely related to ovarian production rates, since during arterial perfusion, ovarian production would be expected to be cleared from the ovary. It is theoretically ovarian possible, however, that an compartment could exist that has a slow rate of equilibrium with the extracellular fluid. Medium PGF content did not show a significant increase over 10 hours perfusion when Indomethacin was used with 50 IU hCG (0.9 + 2.5 pg/ml/hr, r=0.54 & 0.63, 2 experiments, 2 rabbits; 2 ovaries). In contrast to with Indomethacin, the absence of ovulations six ovulations occurred in in each of the two control ovaries (2 experiments, 2 rabbits, 2 ovaries) with hCG alone with a significant increase in PGF accumulation (10.0 f 4.2 pg/ml/hr p
JULY
1983 VOL. 26 NO. 1
PROSTAGLANDINS
producing PGF2,. No data could be derived from these experiments to indicate that the increase in associated with each ovulation results in PGF2a further increase in ovulations. In such a recruitment process a nonlinear or exponential rate of increase in ovulations would be expected as PGF increased. The involvement of prostaglandins in the ovulatory process has been demonstrated in several species. PGF production is increased in follicles of HCG treated rabbits (7) as ovulation is approached and in human ovarian tissue exposed to gonadotropins (12). Increased contractility of ovarian smooth musculature has been associated with PGF2a both in vivro and in vitro in the rabbit (13). In addit?bnnF2. can stimulate ovulation in the in vitro perfused rabbit ovary in the absence of gonadotropins (13). protaglandin could be While basal production derived from non-follicular sources such as the vascular pedicle or fat, the significant increase of accumulation with ovulation was probably derived from the follicle. We infer this from: the increase in prostaglandin accumulation with increasing ovulation, the absence of significant prostaglandin accumulation without ovulation in ovaries without hCG or treated with indomethacin, and the absence of hCG receptors in vascular pedicle or fat. No ovulations occured without hCG stimulation. The effect of changing biochemical conditions with pharmacologic agents on ovulations and its effect on perfusion medium prostaglandins may serve to establish a quantitative marker. This may help elucidate some of the intermediate biochemical steps in the ovulatory process.
JULY 1983VOL.26NO.l
119
PROSTAGLANDINS References 1)
Diaz-Infante, A. Jr., K.H. Wright, and E.E. Wallach. Effects of Indomethacin and PGF2, on Ovulation and Ovarian Contractility in the Rabbit. Prostaglandins 5:567, 1974.
2)
Tsafriri, A., H.R. Lindner, U. Zor, and S. A. Lamprecht. Physiological Role of Prostaglandins on the Induction of Ovulation. Prostaglandins 2:1, 1972.
3)
Wallach, E.E., R. Bronson, Y. Hamada, K.H. Wright, and V.C. Stevens. Effectiveness of PGF2, in Restoration of hMG-hCG Induced Ovulation in Indomethacin Treated Rhesus Monkeys. Prostaglandins 1975. -10:129,
4)
Hamada, Y., K.H. Wright, and E.E. Wallach. Reversal of Indomethacin Blocked Ovulation PGF2,. Fertil. Steril. -30:702,1978.
5)
Hamada, Y., R.A. Bronson, K.H. Wright, and E.E. Wallach. Ovulation in the Perfused Rabbit Ovary: The Influence of Prostaglandins and Prostaglandin Inhibitors. Biol . Rprod. <-17:58, 1977.
6)
Armstrong, D.T., D.L. Grinwich, Y.S. Moon, and J. Inhibition of Ovulation in Rabbits by Zamecnick. Intrafollicular Injection of Indomethacin and PGF Antiserum. Life Sci. -14:129, 1974.
7)
N.S.J., J.M. Marsh, and W.J. LeMaire. Yang, Prostaglandin Changes Induced by Ovulatory Stimuli The Effect of Indoin Rabbit Graafian Follicles: methacin. Prostaglandins 4:395, 1973.
8)
LeMaire, W.J., Post Ovulatory Prostaqlandins Prostaglandins
9)
Lambersteen, C.J., D.F. Greenabum, K.H. Wright, and In vitro studies of ovulation in the E.E. Wallach. Fertil. Steril. 27: 178, perfused rabbit ovary. 1976.
In Vitro by -
Pre and R. Linder, and J.M. Marsh. Changes in the Concentration of in Rat Graafian Follicles. 9:221, 1975
18)
Prostaglandin E, A, F Jaffe, B.M. and H.R. Behrman. Academic in "Methods of Hormone Radioimmunoassy." Press, New York, 1974, p. 9.
11)
Needleman, P., S.D. Bronsom, A. Wychie, and M. Cardiac and renal PG12. J. Clin. Spivakoff. Invest. -61:839, 1978.
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PROSTAGLANDINS 12)
Plunkett, E.R., Y.S.' Moon, J. Zamecnik, and D.T. Preliminary Evidence of a Role for Armstrong. Prostaglandin F in Human Follicular Function. Am. J. Obstet. Gynecol. 123:391, 1975.
13)
Virutamasen, P., K.H. Wright, and E.E. Wallach. on Ovarian Effects of Prostaglandins E and F Contractility in the Rabbit. Obstet. Gynecol. 132:728, 1978.
Editor: Harold R. Behrman Received: 7-27-81 Revised: 3-l-83 Accepted : 4-25-83
1.
Supported by NIH Grant HD-05948 and the Breskman Foundation.
2.
Population
JULY
Council
1983 VOL. 26 NO. 1
Fellow
in Reproductive
Biology.
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