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The influence of colchicine on ovulation in vitro in the frog, Rana pipiens
GENERAL
AND
COMPARATIVE
The
2, 384-393 (1962)
ENDOCRINOLOGY
Influence
of Colchicine on Ovulation in the Frog, Rana pipiensl
in Vitro
PAUL A. WRIGHT Department
of Zoology,
University
of New
Received April
Hampshire,
Durham,
New
Hampshire
23, 1962
Colchicine (10.‘M) potentiates pituitary-induced ovulation in vitro by a facto) of about 3, a phenomenon noted previously by other workers. Colehiceine, lacking one of the colchicine methoxy radicals, had a much reduced potentiating activity on pituitary-induced ovulation. Colchicine augmented ovulation induced by progesterone by a factor of about 3, increased ovulatory activity of testosterone by a factor of only 1.25, and did not potentiate ovulation indured b.y cort.isone and hydrocortisone at all. Testosterone further augmented ovulation induced by mixtures of pituitar) extract and colchicine, but progesterone, cortisone, corticosterone, hydrocortisone, and pregnenolone did not. Colchicine reversed a part of the inhibition imposed by dicumarol on pituitary-induced ovulation, but was ineffective against inhibition by nicotinic acid analogs. A possible explanation for the action of colchicine is discussed. INTRODUCTION
Demonstration in 1943 by McPhail and Wilbur of a pronounced potentiating action of colchicine on pituitary-induced ovulation in vivo and in vitro in the frog, was an unexpected physiological activity of a compound famous at t,hat time only for arresting mitoses. McPhail and Wilbur left their results unexplained but went on to show (McPhail and Wilbur, 1944) that colchicine did not enhance activity of exogenously administered gonadotropin in rabbits or mice, or the action of testosterone on seminal vesicles and ventral prostate of castrated rats. On the other hand, colchicine had been found to bot,h mimic and potentiate androgenic activity in bitterlings (Havas, 1939) and to have a seemingly androgenic affect on chick embryos (Higbee, 1940). The possibility that colchicine might also have androgenic effects in frogs, coupled with recent successesin inducing ovulation with a variety ‘Aided Institutes
by a grant (A-2986) of Health.
from the National
of steroids, including androgens, in Rana pi$ens (Langan, 1941; Burgers and Li, 1960; Wright, 1961), prompted our reassessment of the role of colchicine and a colchicine analog on pituitary-induced ovulation in vitro, as well as an examination of colchicine’s effect on ovulation as induced by steroids and by combinations of pituitary factors and steroids. It also seemed of interest to investigate what effect the augmenting action of colchicine might have on the pronounced inhibit.ion of ovulation induced by cert,ain vitamin antimetabolites. MATERIALS
AND METHODS
Extra large jumbo Rana pipiens from Vermont and Wisconsin were used exclusively. Animals were shipped to the laboratory biweekly and stored in a little water in enamel pans under refrigeration. Concentrated pituitary extracts were made by grinding severai whole glands (to avoid individual variation in potency) in a small mortar and taking up in a small volume of dist,illed water. Exactly eqcivalent dosages were then supplied to the various ovulatory preparations on
any one day from this concentrated stock. Fol these experiments it was unnecessary to sensitize the animals by pituitary injection prior to removal of the ovarian tissue. Excised ovaries were placed in phosphate-bic;lrhonate-hufferrd Ringer’s solution and cut. into lohcs of approximately equal size. The ovulatory (in vitro) preparations consisted of: (1) a single ovarian lobe, containing 150-175 ova, immersed in 50 ml Ringer’s fluid in a standard covered Petri dish, (2) pituitary extract and/or steroid. and (3) any material (colchicine, antimetaholite, etc.) to he tested. The most effective strength of pituitary extract was the equivalent of 0.5 gland in January and 0.25 gland in March, and at least one preparation was included in each experiment (pituitary control) to determine the relative or “standard” rcsponsireness of ovarian tissue from day to day. I’nfortunately, sensitivity of any given ovarian tissue is not an entirely predictable entity and must be determined in this way for each animal used. Another preparation (Ringer control) for earh experimental run contained only ovarian tissue and Ringer’s fluid as a check against possible spontaneous ovulation without further added stimulus. These controls showed no activity in any of these tests, and ovulation observed in experimental preparations was therefore attributable to whatever factors were added. All tests, run at laboratory temperatures of 22”-24°C. extended over 22-24 hours, at the end of which time a count was made of the number of eggs ovulated. Since ovarian lobes contained approximately equal numbers of eggs (15&175), ovulatory counts in any one experiment indicate adequately the relative effectiveness of the various media to which the ovarian tissue was exposed. EXPERIMENTAL
AND
RESULTS
The potentiating effect of lo-” M colchitine on pituitary-induced ovulation in. vitro is plainly evident from the data in Table 1 and is comparable to that. reported for whole ovaries by McPhail and Wilbur. It may also be seen (Table 1) that colchiceine” is much less effective in potentiating ovulation, increasing the response to pituitary extract by a factor of only about 1.25. On two occasions (January 5 and *January 13) colchiceine did not potentiate pituitary-induced ovulation at all, but rather appeared to be inhibitory. It is significant ’ Colchiceine differs from colchicine replacement of an hydroxyl group for radical.
only by a methoxy
TABLE 1 EFFECT OF COLCHICI.UE .wt) COIL~HICEISE PITUITARY-INDXJCED OVGLATIO?; in Vitro
Pituitary
OS
alolle~
Pituitary + colrhicinen
Pituitary + colchice~ne~
l/5 l/5 r/7 l/7 l/13 l/l3 l/14 l/l4
0 120 3 17 102 24 29 55
98 144 120 15’2 163 154 110 139
85 20 60 22 43 56 80 74
Tot.als
350
Date
1080
445
a Pituitary concentration was the equivalent of 0.5 triturated gland in 50 ml Ringer’s fluid; colchitine and colchiceine concent,rat,ions were 10e5 M.
that ovarian lobes, whether stimulat.ed by pituitary extract alone or by combinations of pituitary factors and either colchicine or colchiceine, began extruding ova at TABLE 2 EFFECT OF COLCHICINE ON OVULATION in Vitro INDUCED BY MIXTURES OF PITUITARY EXTRACT AND STEROIDS Number Date
p;,;it;;y
of ova released in meoarations Pituitary + Pituitary + colchicine= steroida Progesterone
2/29 3/l 3/‘ 3/3 3/4 Totals
0 87 34 37 14 172
containinn Pituitary co!c.c.&;
i5
56 123 117 71 133
525
245
500
Testosterone
series
2126 3/22 3133 3/‘24 3/24
56 4 22 15 0
84 51 62 83 44
75 32 47 100 22
00 70 87 75 71
Totals
99
324
276
402
0 Pituitary 0.25 triturated concentration
+ +
series 55 65 54 61 10
-12 168 141 00
a
concentration was t.he equivalent of gland in 50 ml Ringer’s fluid, steroid was 0.05 mg, colchicine was 10” IIZ.
COLCHICIY-I?
EFFECT
ON
about the same time (bet,ween 8 and 10 hours after assembly). Hence, the increased response in preparations containing colchicine was due to more rapid release of ova after the process had started. A number of tests were made to ascertain a possible effect of colchicine on ovulation as induced by combinat.ions of pituitary factors and various nonestrogenic steroids, two series of which have been reported in Table 2. Several steroids (Wright, 1961) potentiate the ovulatory action of pituitary factors in vitro by multipliers of about 1.25 to 2.0, and it was therefore of particular interest to discover what effect the simultaneous inclusion of
EFFECT
Number
Date
1 mg
of ova released
OVULATION
in preparations
Steroid
0.01 mg
0.1 mg
12 4 19 0
in Vitro
containing
alone
+ colchirinea
1 mg
0.1 mg
0.01 mg
43 23 29 i
55 66 25 15
45 98 59 15
74 70 58 38
102
161
Progesterone 3/l 3/2 3/3 3/4
391
OVULATION
steroids would have on the augmenting effect of colchicine. Progesterone added to the mixture of pituitary factors and colchicine (Table 2) was without effect but testosterone further potentiated the action of colchicine. In a repetition of this experiment, corticosterone, hydrocortisone, cortisone, and pregnenolone, in the same dosages, were also without effect. All of these compounds bear a greater structural resemblance to progesterone than they do to testosterone. The effect of colchicine on ovulation induced by steroids is shown in Table 3. Colchicine potentiates progesterone-induced ovulation by nearly the same factor of 3
TABLE 3 ON STEROID-INDUCED
OF COLCHICINE
Steroid
FHOG
series
8 17 27 2 -
Totals
35
54
2/26 3/22 3/23 3/24 3/24
45 0 21 42 13
48 0 29 34 16
Testosterone
Totals
61 14 27 38 25
45 30 54 21 38
165
153
--
--
121
127
128
2126 3/4
18 19 53
Totals
90
39
3/3 3/6 3/7
36 15 4
18 20 44
Totals
55
3/2
acetate
12 9 18 --
190
series 74 2 14 38 0
-
Cortisone
2li
73 1 38 20 25 157
series
4 9
2 1 6
16
4 4 12
9
29
20
11 3 4
24 8 27
23 6 31
10 5 6
18
59
60
21
--
Hydrocortisone
series
--
a Colchicine
concentrat,ion
82 was 10-s M.
392
U-RIGHT
obtained using pituitary extracts, but augments the testosterone-induced process by a factor of only 1.25. No immediate explanation can be offered for the fact that colchicine did not potentiate ovulation induced by cortisone or hydrocortisone (Table 3). Inhibition of pituitary-induced ovulation by 10eFM dicumarol is clearly indicated in Table 4 as well as pa&al removal of this TABLE 4 EFFECT OF COLCHICINE ON IXHIBITION ~V~JL~TIOX BY CERTAIN VITAMIN ANTIMETABOLITES
OF
is no chemical reaction between colchicine and dicumarol. Presumably, then, colchitine has part,ially reversed an effect of dicumarol on ovarian tissue, or, possibly, on pituitary extract. Colchicine is ineffective in removing any of the inhibition imposed by analogs of nicotinic acid (pyridine-3-sulfonic acid and a-picolinic acid, Table 4). In this instance, nicotinic acid does reverse about. 307% of ovulatory inhibition imposed by its analogs (Wright et al., 1960). In our experience, steroids did not alter inhibition imposed by either dicumarol or analogs of nicotinir acid. DISCUSSION
Nrmher
Date
Pituitary alone-
of ova released Pituitary inhibitor’
+
Dicumxrol l/22 l/26 l/26 l/27 I/27 2/4
5 26 14 3 8 29
0 0 i 0 0 0
Totals
85
i
in preparations Pituitary colchicinen
+
containing Pituitary colcbicine inhibitora
series 70 94 67 12 68 83 $24
Pyridine+sulfonic
arid
33 8 20 0 59 0 120
series
l/15 l/l6 l/l’7
13 23 32
0 0 0 -
48 78 61
0 0 0
Totals
68
0
187
0
l/1.5 l/16 l/I7
I6 l!) 4!)
0 0 1
60 92
0 0 0
Totals
74
I
1!m
0
cu-picolink
+ +
acid
series -17
” Pituitary concentration was the equivalent, of 0.5 triturated gland in 50 ml Ringer’s fluid, colchicine was 10-E M, sntimetaholites were 5 X 1O-4 M.
inhibition by colchicine in some experiments. The mechahism for dicumarol inhibition is not known and has not been reported previously, but, in our experience, cannot be reversed by simultaneous inclusion of vitamin K in the ovulatory preparations in uitro. To our knowledge, there
Potentiation of induced ovulation by colchicine is dramatic beyond question, but the observation is tempered by the knowledge that the drug prevents cleavage of ova after fertilization (McPhail and Wilbur, 1943). Ova exposed to colchicine orient normally following fertilization but, being unable to complete the usual maturation divisions, arc inhibited from further development. Whether halting of cell division and potentiation of ovulation are in any way connected remains an open question, but the lack of this information should not be a deterrent to consideration of possible explanations for colchicine’s influence on ovulation. There arc several arguments in favor of considering colchicine in a physiological category with certain steroids. The work of Havas (1939) and Higbee (1940) has suggested an androgenic effect on hitterlings and embryonic chicks, respectively. The fact that. McPhail and Wilbur (1944) could not demonstrate similar activity in rabbits and mice may well signify an important difference in responsiveness of mammalian and lower vertebrate tissues. Chemically, colchicine is not steroid, but its ring structure bears some resemblance to the basic steroid configuration. It is we11 known, of course, that diethylstilbestrol and its relatives are not steroid, but are nonetheless pot.ent, estrogens. Colchicine is much more soluble than steroids in aqueous media, and could have different physiological potency on that account.
COLCHICINE
EFFECT
Colchicine’s activity is similar to, although more pronounced than, that of many steroids in potentiating pituitaryinduced ovulation, but it is dissimilar in that these same steroids induce ovulation independently of pituitary factors whereas colchicine cannot. Apparently the methoxy radicals of colchicine, or the particular pattern they assume, are important to the activity of the drug, for the loss of only one methoxy group, as in colchiceine, renders the compound far less effective in potentiating ovulation. It seems particularly significant that colchicine bolsters the ovulation-inducing activity of testosterone by a factor of only 1.25 (Table 3), but increases the effectiveness of progesterone by the same factor of approximately 3 obtained with pituitary ext,racts (Table 1). This observation may have some relationship to the fact that colchicine appears to have some androgenic potentiality. If so, it might well augment significantly the action of progesterone but not that of testosterone, another androgen. This explanation, on the other hand, would not justify the fact that colchicine seems to potentiate ovulation induced by mixtures of pituitary extract and testosterone but has no effect on ovulatory activity of pituitary extract-progesterone combination (Table 2). The influence of steroids on mitosis has not been entirely clarified, but it appears that some nonestrogenic steroids, in common with colchicine, may be mitosisinhibiting and others may be mitosisstimulating (Bass, 1959). To what extent steroids may play a normal role in frog ovulation is also uncertain (Wright, 1961; Wright and Flathers, 1961), but, there is convincing evidence that they could well participate. In consideration of data provided in this paper, it may not be an overextension to postulate a steroid in amphibian ovary or adrenal (perhaps one
ON
FROG
393
OVCLdTION
with methoxy groups) which has the marked ovulation-accelerating property of coIchicine but lacks its mitosis-arresting activity. ACKNOWLEDGMENTS Grateful appreciation is expressed to Miss Helen L. Greenwood and Miss Marilyn S. Sherman and t,o Mrs. Ann R. White for exceptional technical assistance, and to Dr. P. L. Perlman of Schering Corporation for many of the steroids used. REFERENCES A. D. (1959). Chemical influences on cell division and development. Ann. Rev. PhysioE.
BASS,
21,
49-68.
A. C. J., AND Lr, C. H. (1960). Amovulation in zlitro induced by mampituitary hormones and progesterone.
BURGERS,
phibian malian
Endocrinology
66, 255-259.
L. (1939). Influence of colchicine on the sexually induced colour change of Rhodeus amarus. Nature 143, 869-810. HIDBEE, E. (1940). Some results of colchicine injections. Science 92, 80. LANGAN, W. B. (1941). Ovulatory responses of Rana pipieiens to mammalian gonadotropic factors and sex hormones. Proc. Sot. Exptl. BioE. HAVAS,
Med.
47,
59-61.
M. K., AND WILBUR, K. M. (1943). The stimulating act’ion of colchicine on pituitary-induced ovulation of the frog. J. Phar-
MCPHAIL,
mncol.
Exptl.
Therap.
78,
304-313.
K., AND WILBUR, K. M. (1944). Absence of potentiation of gonadotropin and steroid function in mammals by colchicine.
MCPHAIL,
M.
Endocrinology WRIGHT,
35,
196-197.
P. A. (1961). Induction of ovulation in in Rana pipiens with steroids. Gen. Comp.
vitro Endocrinol. WRIQHT, P.
Facilitation
1, 20-23.
A., AND FLATHERS, of pituitary-induced
A. R. (1961). frog ovula-
in early fall. Proc. Sot. Med. 106, 346347. WRIGHT, P. A., FLATHERS, A. R., GREENWOOD. H. L., AND SHERMAN, M. S. (1960). Influence of tion
Exptl.
by
progesterone
Biol.
certain B-vitamin antimetabolites on ovulation in vitro in the frog, Rana pipiens. Proc. Sot. Exptl.
Biol.
Med.
105,
211-212.
AND
COMPARATIVE
The
2, 384-393 (1962)
ENDOCRINOLOGY
Influence
of Colchicine on Ovulation in the Frog, Rana pipiensl
in Vitro
PAUL A. WRIGHT Department
of Zoology,
University
of New
Received April
Hampshire,
Durham,
New
Hampshire
23, 1962
Colchicine (10.‘M) potentiates pituitary-induced ovulation in vitro by a facto) of about 3, a phenomenon noted previously by other workers. Colehiceine, lacking one of the colchicine methoxy radicals, had a much reduced potentiating activity on pituitary-induced ovulation. Colchicine augmented ovulation induced by progesterone by a factor of about 3, increased ovulatory activity of testosterone by a factor of only 1.25, and did not potentiate ovulation indured b.y cort.isone and hydrocortisone at all. Testosterone further augmented ovulation induced by mixtures of pituitar) extract and colchicine, but progesterone, cortisone, corticosterone, hydrocortisone, and pregnenolone did not. Colchicine reversed a part of the inhibition imposed by dicumarol on pituitary-induced ovulation, but was ineffective against inhibition by nicotinic acid analogs. A possible explanation for the action of colchicine is discussed. INTRODUCTION
Demonstration in 1943 by McPhail and Wilbur of a pronounced potentiating action of colchicine on pituitary-induced ovulation in vivo and in vitro in the frog, was an unexpected physiological activity of a compound famous at t,hat time only for arresting mitoses. McPhail and Wilbur left their results unexplained but went on to show (McPhail and Wilbur, 1944) that colchicine did not enhance activity of exogenously administered gonadotropin in rabbits or mice, or the action of testosterone on seminal vesicles and ventral prostate of castrated rats. On the other hand, colchicine had been found to bot,h mimic and potentiate androgenic activity in bitterlings (Havas, 1939) and to have a seemingly androgenic affect on chick embryos (Higbee, 1940). The possibility that colchicine might also have androgenic effects in frogs, coupled with recent successesin inducing ovulation with a variety ‘Aided Institutes
by a grant (A-2986) of Health.
from the National
of steroids, including androgens, in Rana pi$ens (Langan, 1941; Burgers and Li, 1960; Wright, 1961), prompted our reassessment of the role of colchicine and a colchicine analog on pituitary-induced ovulation in vitro, as well as an examination of colchicine’s effect on ovulation as induced by steroids and by combinations of pituitary factors and steroids. It also seemed of interest to investigate what effect the augmenting action of colchicine might have on the pronounced inhibit.ion of ovulation induced by cert,ain vitamin antimetabolites. MATERIALS
AND METHODS
Extra large jumbo Rana pipiens from Vermont and Wisconsin were used exclusively. Animals were shipped to the laboratory biweekly and stored in a little water in enamel pans under refrigeration. Concentrated pituitary extracts were made by grinding severai whole glands (to avoid individual variation in potency) in a small mortar and taking up in a small volume of dist,illed water. Exactly eqcivalent dosages were then supplied to the various ovulatory preparations on
any one day from this concentrated stock. Fol these experiments it was unnecessary to sensitize the animals by pituitary injection prior to removal of the ovarian tissue. Excised ovaries were placed in phosphate-bic;lrhonate-hufferrd Ringer’s solution and cut. into lohcs of approximately equal size. The ovulatory (in vitro) preparations consisted of: (1) a single ovarian lobe, containing 150-175 ova, immersed in 50 ml Ringer’s fluid in a standard covered Petri dish, (2) pituitary extract and/or steroid. and (3) any material (colchicine, antimetaholite, etc.) to he tested. The most effective strength of pituitary extract was the equivalent of 0.5 gland in January and 0.25 gland in March, and at least one preparation was included in each experiment (pituitary control) to determine the relative or “standard” rcsponsireness of ovarian tissue from day to day. I’nfortunately, sensitivity of any given ovarian tissue is not an entirely predictable entity and must be determined in this way for each animal used. Another preparation (Ringer control) for earh experimental run contained only ovarian tissue and Ringer’s fluid as a check against possible spontaneous ovulation without further added stimulus. These controls showed no activity in any of these tests, and ovulation observed in experimental preparations was therefore attributable to whatever factors were added. All tests, run at laboratory temperatures of 22”-24°C. extended over 22-24 hours, at the end of which time a count was made of the number of eggs ovulated. Since ovarian lobes contained approximately equal numbers of eggs (15&175), ovulatory counts in any one experiment indicate adequately the relative effectiveness of the various media to which the ovarian tissue was exposed. EXPERIMENTAL
AND
RESULTS
The potentiating effect of lo-” M colchitine on pituitary-induced ovulation in. vitro is plainly evident from the data in Table 1 and is comparable to that. reported for whole ovaries by McPhail and Wilbur. It may also be seen (Table 1) that colchiceine” is much less effective in potentiating ovulation, increasing the response to pituitary extract by a factor of only about 1.25. On two occasions (January 5 and *January 13) colchiceine did not potentiate pituitary-induced ovulation at all, but rather appeared to be inhibitory. It is significant ’ Colchiceine differs from colchicine replacement of an hydroxyl group for radical.
only by a methoxy
TABLE 1 EFFECT OF COLCHICI.UE .wt) COIL~HICEISE PITUITARY-INDXJCED OVGLATIO?; in Vitro
Pituitary
OS
alolle~
Pituitary + colrhicinen
Pituitary + colchice~ne~
l/5 l/5 r/7 l/7 l/13 l/l3 l/14 l/l4
0 120 3 17 102 24 29 55
98 144 120 15’2 163 154 110 139
85 20 60 22 43 56 80 74
Tot.als
350
Date
1080
445
a Pituitary concentration was the equivalent of 0.5 triturated gland in 50 ml Ringer’s fluid; colchitine and colchiceine concent,rat,ions were 10e5 M.
that ovarian lobes, whether stimulat.ed by pituitary extract alone or by combinations of pituitary factors and either colchicine or colchiceine, began extruding ova at TABLE 2 EFFECT OF COLCHICINE ON OVULATION in Vitro INDUCED BY MIXTURES OF PITUITARY EXTRACT AND STEROIDS Number Date
p;,;it;;y
of ova released in meoarations Pituitary + Pituitary + colchicine= steroida Progesterone
2/29 3/l 3/‘ 3/3 3/4 Totals
0 87 34 37 14 172
containinn Pituitary co!c.c.&;
i5
56 123 117 71 133
525
245
500
Testosterone
series
2126 3/22 3133 3/‘24 3/24
56 4 22 15 0
84 51 62 83 44
75 32 47 100 22
00 70 87 75 71
Totals
99
324
276
402
0 Pituitary 0.25 triturated concentration
+ +
series 55 65 54 61 10
-12 168 141 00
a
concentration was t.he equivalent of gland in 50 ml Ringer’s fluid, steroid was 0.05 mg, colchicine was 10” IIZ.
COLCHICIY-I?
EFFECT
ON
about the same time (bet,ween 8 and 10 hours after assembly). Hence, the increased response in preparations containing colchicine was due to more rapid release of ova after the process had started. A number of tests were made to ascertain a possible effect of colchicine on ovulation as induced by combinat.ions of pituitary factors and various nonestrogenic steroids, two series of which have been reported in Table 2. Several steroids (Wright, 1961) potentiate the ovulatory action of pituitary factors in vitro by multipliers of about 1.25 to 2.0, and it was therefore of particular interest to discover what effect the simultaneous inclusion of
EFFECT
Number
Date
1 mg
of ova released
OVULATION
in preparations
Steroid
0.01 mg
0.1 mg
12 4 19 0
in Vitro
containing
alone
+ colchirinea
1 mg
0.1 mg
0.01 mg
43 23 29 i
55 66 25 15
45 98 59 15
74 70 58 38
102
161
Progesterone 3/l 3/2 3/3 3/4
391
OVULATION
steroids would have on the augmenting effect of colchicine. Progesterone added to the mixture of pituitary factors and colchicine (Table 2) was without effect but testosterone further potentiated the action of colchicine. In a repetition of this experiment, corticosterone, hydrocortisone, cortisone, and pregnenolone, in the same dosages, were also without effect. All of these compounds bear a greater structural resemblance to progesterone than they do to testosterone. The effect of colchicine on ovulation induced by steroids is shown in Table 3. Colchicine potentiates progesterone-induced ovulation by nearly the same factor of 3
TABLE 3 ON STEROID-INDUCED
OF COLCHICINE
Steroid
FHOG
series
8 17 27 2 -
Totals
35
54
2/26 3/22 3/23 3/24 3/24
45 0 21 42 13
48 0 29 34 16
Testosterone
Totals
61 14 27 38 25
45 30 54 21 38
165
153
--
--
121
127
128
2126 3/4
18 19 53
Totals
90
39
3/3 3/6 3/7
36 15 4
18 20 44
Totals
55
3/2
acetate
12 9 18 --
190
series 74 2 14 38 0
-
Cortisone
2li
73 1 38 20 25 157
series
4 9
2 1 6
16
4 4 12
9
29
20
11 3 4
24 8 27
23 6 31
10 5 6
18
59
60
21
--
Hydrocortisone
series
--
a Colchicine
concentrat,ion
82 was 10-s M.
392
U-RIGHT
obtained using pituitary extracts, but augments the testosterone-induced process by a factor of only 1.25. No immediate explanation can be offered for the fact that colchicine did not potentiate ovulation induced by cortisone or hydrocortisone (Table 3). Inhibition of pituitary-induced ovulation by 10eFM dicumarol is clearly indicated in Table 4 as well as pa&al removal of this TABLE 4 EFFECT OF COLCHICINE ON IXHIBITION ~V~JL~TIOX BY CERTAIN VITAMIN ANTIMETABOLITES
OF
is no chemical reaction between colchicine and dicumarol. Presumably, then, colchitine has part,ially reversed an effect of dicumarol on ovarian tissue, or, possibly, on pituitary extract. Colchicine is ineffective in removing any of the inhibition imposed by analogs of nicotinic acid (pyridine-3-sulfonic acid and a-picolinic acid, Table 4). In this instance, nicotinic acid does reverse about. 307% of ovulatory inhibition imposed by its analogs (Wright et al., 1960). In our experience, steroids did not alter inhibition imposed by either dicumarol or analogs of nicotinir acid. DISCUSSION
Nrmher
Date
Pituitary alone-
of ova released Pituitary inhibitor’
+
Dicumxrol l/22 l/26 l/26 l/27 I/27 2/4
5 26 14 3 8 29
0 0 i 0 0 0
Totals
85
i
in preparations Pituitary colchicinen
+
containing Pituitary colcbicine inhibitora
series 70 94 67 12 68 83 $24
Pyridine+sulfonic
arid
33 8 20 0 59 0 120
series
l/15 l/l6 l/l’7
13 23 32
0 0 0 -
48 78 61
0 0 0
Totals
68
0
187
0
l/1.5 l/16 l/I7
I6 l!) 4!)
0 0 1
60 92
0 0 0
Totals
74
I
1!m
0
cu-picolink
+ +
acid
series -17
” Pituitary concentration was the equivalent, of 0.5 triturated gland in 50 ml Ringer’s fluid, colchicine was 10-E M, sntimetaholites were 5 X 1O-4 M.
inhibition by colchicine in some experiments. The mechahism for dicumarol inhibition is not known and has not been reported previously, but, in our experience, cannot be reversed by simultaneous inclusion of vitamin K in the ovulatory preparations in uitro. To our knowledge, there
Potentiation of induced ovulation by colchicine is dramatic beyond question, but the observation is tempered by the knowledge that the drug prevents cleavage of ova after fertilization (McPhail and Wilbur, 1943). Ova exposed to colchicine orient normally following fertilization but, being unable to complete the usual maturation divisions, arc inhibited from further development. Whether halting of cell division and potentiation of ovulation are in any way connected remains an open question, but the lack of this information should not be a deterrent to consideration of possible explanations for colchicine’s influence on ovulation. There arc several arguments in favor of considering colchicine in a physiological category with certain steroids. The work of Havas (1939) and Higbee (1940) has suggested an androgenic effect on hitterlings and embryonic chicks, respectively. The fact that. McPhail and Wilbur (1944) could not demonstrate similar activity in rabbits and mice may well signify an important difference in responsiveness of mammalian and lower vertebrate tissues. Chemically, colchicine is not steroid, but its ring structure bears some resemblance to the basic steroid configuration. It is we11 known, of course, that diethylstilbestrol and its relatives are not steroid, but are nonetheless pot.ent, estrogens. Colchicine is much more soluble than steroids in aqueous media, and could have different physiological potency on that account.
COLCHICINE
EFFECT
Colchicine’s activity is similar to, although more pronounced than, that of many steroids in potentiating pituitaryinduced ovulation, but it is dissimilar in that these same steroids induce ovulation independently of pituitary factors whereas colchicine cannot. Apparently the methoxy radicals of colchicine, or the particular pattern they assume, are important to the activity of the drug, for the loss of only one methoxy group, as in colchiceine, renders the compound far less effective in potentiating ovulation. It seems particularly significant that colchicine bolsters the ovulation-inducing activity of testosterone by a factor of only 1.25 (Table 3), but increases the effectiveness of progesterone by the same factor of approximately 3 obtained with pituitary ext,racts (Table 1). This observation may have some relationship to the fact that colchicine appears to have some androgenic potentiality. If so, it might well augment significantly the action of progesterone but not that of testosterone, another androgen. This explanation, on the other hand, would not justify the fact that colchicine seems to potentiate ovulation induced by mixtures of pituitary extract and testosterone but has no effect on ovulatory activity of pituitary extract-progesterone combination (Table 2). The influence of steroids on mitosis has not been entirely clarified, but it appears that some nonestrogenic steroids, in common with colchicine, may be mitosisinhibiting and others may be mitosisstimulating (Bass, 1959). To what extent steroids may play a normal role in frog ovulation is also uncertain (Wright, 1961; Wright and Flathers, 1961), but, there is convincing evidence that they could well participate. In consideration of data provided in this paper, it may not be an overextension to postulate a steroid in amphibian ovary or adrenal (perhaps one
ON
FROG
393
OVCLdTION
with methoxy groups) which has the marked ovulation-accelerating property of coIchicine but lacks its mitosis-arresting activity. ACKNOWLEDGMENTS Grateful appreciation is expressed to Miss Helen L. Greenwood and Miss Marilyn S. Sherman and t,o Mrs. Ann R. White for exceptional technical assistance, and to Dr. P. L. Perlman of Schering Corporation for many of the steroids used. REFERENCES A. D. (1959). Chemical influences on cell division and development. Ann. Rev. PhysioE.
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