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Blockade of progesterone-induced in vitro ovulation of Rana pipiens with chorionic gonadotropin
GEKERAL
AND
COMPARATIVE
Blockade
ENDOCRINOLOGY
(1961)
1, 492-493
of Progesterone-Induced Ram
pipiens
RICHARD Endocrinology Wyeth
with
in Vitro
Chorionic
A. EDGREN
CARTER
Section, Research and Development Luboratories, Philadelphia, Pennsylvania
Received
August
of
Gonadotropin
D. LINNEA
AND
Ovulation
DizGsions.
30, 1961
In vitro progesterone-induced ovulation of ovarian fragments of meadow frog* (Ram pipiens Schreber) is inhibited by chorionic gonadotropin, which is, itself, largely ineffective. In contrast, ovulation induced by frog pituitary ovulating hormones, which are effective by themselves, has been reported to be facilitated by progesterone. INTRODUCTION
Wright (1961) has recently shown that in vitro ovulation induced in fragments of ovaries of Rana pipiens Schreber with frog pituitary extract may be facilitated by simultaneous exposure of the ovary to progesterone. Individually, pituitary gonadotropin and progesterone are effective in stimulating ovulation. This observation raises the question of whether progesterone might also bc synergistic with inactive chorionic gonadotropin (CC,) in stimulating in vitro ovulation. The following experiments were designed to rxplorc this possibility. METHODS Erogs were pithed, opened immediately, thr ovaries removed and divided into fragments of approximately 100-200 eggs each. Each fragment, was placed in n 60 mm plastic culture dish containing 9 ml of frog Ringer’s solution. Materials to bc studied wprc added in solution 01 suspension in a small amount of Ringer’s and the I-olume of liquid brought to a consistent 10 ml. Readings were taken after 22-24 hr of exposure to the hormones at room temperature (22-25°C). The ovulated eggs were counted after gentle agitation, and those remaining in the ovarian fragment were teased apart and counted. Percentage of eggs ovulated is cmploycd for comparison. We shall not attempt a defense of the use of this statistic, other than to say that it would appear to give a clear picture of the effects of these
substances. Both the progesterone and the CG employed were obtained from Mann Research Laboratories. The experiments were conducted in April 1961 on frogs purchased from a dealer; they were reported to have been collected in Wisconsin. RESULTS
AND
DISCUSSION
Two experiments have been completed. In the first, 0.1 mg of progesterone was tested alone and in combination with a TABLE THE EFFECT OF A SERIES CHORI~NIC GONAIX~TROPIS PROGESTERONE-IKUUCEU
0F Ram
OVULATION
1 OF DOSES
(CG) in
OF ON
vdm
pipien.?
Treatment Progesterone (ws) 0 0. I 0. I 0.1 0. I 0. I 0. 1 0.1 0.1 0. 1 a The employed
cc; (11-J
Tot,:, I
0 0
0 92.;
0.1
18.8 Z-l.1 20.5
0 .x I .v 3 0 10.0 30 .o 100.0 :300.0
data are pooled from experiments the ovaries of two frogs.
35.0 16.5 1.6 0 .3 0 that
broad range of doses of CG (Table 1). The lower doses of CG had no obvious
OVULATIOS
effect upon progesterone-induced ovulation. The combination of progesterone 0.1 mg and CG 3.0 IU produced the greatest proportion of ovulation, but the great variability in this type of test hardly inspires confidence that this represents facilitation. TABLE THE
2
EFFECTS OF CHORIONIC GONAIWTROPIN OS PROGESTERONE-ISDWED in citro OVULATION OF Rana pipiensa I’rogestcrone
cc; (IL”) 0 0 OR 0.3 3.0 30.0
300.0
0 1.1 7 0 G.fi 4.7 0 .8 0
0.03
0.3
"4.3
38.2
“0.0
37.4
'5.8
27.0
22.9 9.2 0
30 .3 Il.!) 0.2
(CG)
lmg) 3.0 :C?.; 23.3 23.7 31.3 11 .5 1.4
30.0 5.8
11.1 14.3
17.0 11.1 0. 7
a Each figure represents percentage of eggs ovulated from four experiments emplo.ving one frog each. The ovaries from each frog were divided into 30 segments, so that each frog received the entire range of treatments. The percentage values are based upon egg masses that varied from 379 to 61.5 with :I mean of 475 eggs.
On the other hand, the higher dosesof CG clearly blocked ovulation; doses above 10 IU of CG produce a progressively decreasing percentage of ovulation.
193
BLOCK
In order to confirm this study, a single balanced experiment was designed and rcplicated four t.imes with ovarian fragments from four frogs. The effects of progesterone were studied at doses extending from 0.03 to 30 mg and CG at clots from 0.03 to 300 IL. TheAe m::terials were used alone and in all possible combinations (Table 21. The CG itself was largely ineffective in inducing ovulation, although lower doses of the hormone appeared to stimulate the release of 5-10s of the eggs. Progesterone, alone, was quite active, producing some 20-30% ovulation at dosesbetween 0.03 and 3.0 nqq. The 30 mg dose of progesterone was less effective. In combinations, CC appeared to be largely without effect at doses below 30 II!. Thirty IU of CG caused a marked depression in progesterone-induced ovulation, and the process was almost entirely blocked by the 300 IU dose. At the intermediate levels of CG there is an apparent increase in ovulation with 30.0 mg of progesterone. This increase is not statistically significant, and resulted from a series of ovarian fragments that were more responsive than one would have predicted from the other dat:t. REFERENCE WRIGHT, P. A. ,litro in Rnnn
(1961). pipiens
E~/rloc~inol.
1. 20-23.
Induction of ovulation iri with steroids. Gen. Clomp
AND
COMPARATIVE
Blockade
ENDOCRINOLOGY
(1961)
1, 492-493
of Progesterone-Induced Ram
pipiens
RICHARD Endocrinology Wyeth
with
in Vitro
Chorionic
A. EDGREN
CARTER
Section, Research and Development Luboratories, Philadelphia, Pennsylvania
Received
August
of
Gonadotropin
D. LINNEA
AND
Ovulation
DizGsions.
30, 1961
In vitro progesterone-induced ovulation of ovarian fragments of meadow frog* (Ram pipiens Schreber) is inhibited by chorionic gonadotropin, which is, itself, largely ineffective. In contrast, ovulation induced by frog pituitary ovulating hormones, which are effective by themselves, has been reported to be facilitated by progesterone. INTRODUCTION
Wright (1961) has recently shown that in vitro ovulation induced in fragments of ovaries of Rana pipiens Schreber with frog pituitary extract may be facilitated by simultaneous exposure of the ovary to progesterone. Individually, pituitary gonadotropin and progesterone are effective in stimulating ovulation. This observation raises the question of whether progesterone might also bc synergistic with inactive chorionic gonadotropin (CC,) in stimulating in vitro ovulation. The following experiments were designed to rxplorc this possibility. METHODS Erogs were pithed, opened immediately, thr ovaries removed and divided into fragments of approximately 100-200 eggs each. Each fragment, was placed in n 60 mm plastic culture dish containing 9 ml of frog Ringer’s solution. Materials to bc studied wprc added in solution 01 suspension in a small amount of Ringer’s and the I-olume of liquid brought to a consistent 10 ml. Readings were taken after 22-24 hr of exposure to the hormones at room temperature (22-25°C). The ovulated eggs were counted after gentle agitation, and those remaining in the ovarian fragment were teased apart and counted. Percentage of eggs ovulated is cmploycd for comparison. We shall not attempt a defense of the use of this statistic, other than to say that it would appear to give a clear picture of the effects of these
substances. Both the progesterone and the CG employed were obtained from Mann Research Laboratories. The experiments were conducted in April 1961 on frogs purchased from a dealer; they were reported to have been collected in Wisconsin. RESULTS
AND
DISCUSSION
Two experiments have been completed. In the first, 0.1 mg of progesterone was tested alone and in combination with a TABLE THE EFFECT OF A SERIES CHORI~NIC GONAIX~TROPIS PROGESTERONE-IKUUCEU
0F Ram
OVULATION
1 OF DOSES
(CG) in
OF ON
vdm
pipien.?
Treatment Progesterone (ws) 0 0. I 0. I 0.1 0. I 0. I 0. 1 0.1 0.1 0. 1 a The employed
cc; (11-J
Tot,:, I
0 0
0 92.;
0.1
18.8 Z-l.1 20.5
0 .x I .v 3 0 10.0 30 .o 100.0 :300.0
data are pooled from experiments the ovaries of two frogs.
35.0 16.5 1.6 0 .3 0 that
broad range of doses of CG (Table 1). The lower doses of CG had no obvious
OVULATIOS
effect upon progesterone-induced ovulation. The combination of progesterone 0.1 mg and CG 3.0 IU produced the greatest proportion of ovulation, but the great variability in this type of test hardly inspires confidence that this represents facilitation. TABLE THE
2
EFFECTS OF CHORIONIC GONAIWTROPIN OS PROGESTERONE-ISDWED in citro OVULATION OF Rana pipiensa I’rogestcrone
cc; (IL”) 0 0 OR 0.3 3.0 30.0
300.0
0 1.1 7 0 G.fi 4.7 0 .8 0
0.03
0.3
"4.3
38.2
“0.0
37.4
'5.8
27.0
22.9 9.2 0
30 .3 Il.!) 0.2
(CG)
lmg) 3.0 :C?.; 23.3 23.7 31.3 11 .5 1.4
30.0 5.8
11.1 14.3
17.0 11.1 0. 7
a Each figure represents percentage of eggs ovulated from four experiments emplo.ving one frog each. The ovaries from each frog were divided into 30 segments, so that each frog received the entire range of treatments. The percentage values are based upon egg masses that varied from 379 to 61.5 with :I mean of 475 eggs.
On the other hand, the higher dosesof CG clearly blocked ovulation; doses above 10 IU of CG produce a progressively decreasing percentage of ovulation.
193
BLOCK
In order to confirm this study, a single balanced experiment was designed and rcplicated four t.imes with ovarian fragments from four frogs. The effects of progesterone were studied at doses extending from 0.03 to 30 mg and CG at clots from 0.03 to 300 IL. TheAe m::terials were used alone and in all possible combinations (Table 21. The CG itself was largely ineffective in inducing ovulation, although lower doses of the hormone appeared to stimulate the release of 5-10s of the eggs. Progesterone, alone, was quite active, producing some 20-30% ovulation at dosesbetween 0.03 and 3.0 nqq. The 30 mg dose of progesterone was less effective. In combinations, CC appeared to be largely without effect at doses below 30 II!. Thirty IU of CG caused a marked depression in progesterone-induced ovulation, and the process was almost entirely blocked by the 300 IU dose. At the intermediate levels of CG there is an apparent increase in ovulation with 30.0 mg of progesterone. This increase is not statistically significant, and resulted from a series of ovarian fragments that were more responsive than one would have predicted from the other dat:t. REFERENCE WRIGHT, P. A. ,litro in Rnnn
(1961). pipiens
E~/rloc~inol.
1. 20-23.
Induction of ovulation iri with steroids. Gen. Clomp