EFFECTS OF INDOMETHACIN AND PROSTAGLANDINS ON OVULATION OF GOLDFISH
N. E. STACEY and S. PANDEY Department of Zoology, University of British Columbia, Vancouver, B.C., Canada, and Department of Zoology, Patna University, Patna 5, India.
ABSTRACT A technique is described whereby elevated temperature and HCG injection yield a high percentage of ovulation in gravid goldfish. Indomethacin'(l0 ug/g; i.p. injection) completely inhibits ovulation if given within 6 hours following HCG (4 Ill/g); the unovulated oocytes develop rapidly into corpora atretica. PGE1, PGE2, and PGF,, (5 pg/g; i.p. injection) induce ovulation in fish treated with indomethacin and HCG; PGE2 was most effective when given 11 hours after HCG. The results suggest that the ovulatory action of prostaglandins following HCG stimulation is at the level of the ovary and that it is restricted to a period between 7 and 12 hours after the gonadotropin injection.
ACKNOWLEDGEMENTS: This work was supported by a University of British Columbia graduate fellowship to N.E.S., a Canadian International Development Agency Research Associateship to S.P., and an N.R.C. Grant-in-aid of Research to Dr. W.S. Hoar. We are grateful to Dr.J. Pike from the Upjohn Company for the generous gift of prostaglandins and to Dr. W.S. Hoar and Dr. N.R. Liley for reading the manuscript. Accepted April 1, 1975
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VOL. 9 NO. 4
597
PROSTAGLANDINS
INTRODUCTION A number of recent studies suggest that prostaglandins (PG's) play a role in the process of ovulation (see reviews 1, 2). Indomethacin (IM), an inhibitor of PG biosynthesis, blocks ovulation in both rats (3, 4) and rabbits (5), an effect which is reversed by treatment with PG's (6, 7). To date, published accounts of these phenomena have been restricted to studies of mamnals. In this paper, we present evidence that PG's are involved in the ovulation of goldfish; IM is shown to block the ovulatory response to HCG, and PG's are shown to overcome this blockade.
METHODS AND RESULTS Stocks of female comet goldfish were obtained from Hartz Mountain Pet Supply (Richmond, B.C., Canada) and kept in running dechlorinated tap water at 10-13°C on a 16 h photoperiod (8 a.m. to 12 p.m.). Frozen brine shrimp or trout pellets were provided ad Zibitwn at least once daily. Under these conditions, fish become gravid but do not ovulate. To induce ovulation, gravid females are warmed gradually to 200C on Day 1. Spontaneous ovulation usually occurs during the early morning of Day 3 (never on Day 2) and may be checked by gently pressing the abdomen to release a stream of eggs from the ovipore. If fish, warmed to 2OoC, are injected intraperitoneally (i.p.) with 4 IU/g HCG (dissolved in 0.6% NaCl) on the afternoon of Day 2, virtually 100% will ovulate on Day 3. HCG will also induce ovulation in fish kept at lo-13oC, but several days of treatment are usually required. The combined treatment with elevated temperature and HCG injection is a standard and reliable technique for the induction of ovulation. Experiment I In this experiment, intended to examine the effects of IM on ovulation, 20 gravid female goldfish were removed from the cold water stock tanks at 9 a.m. on Day 1, divided randomly into 2 groups (10 fish/ 15 gallon tank) and slowly warmed to 200C. At 10 a.m. on Day 2, each fish was anaesthetized in MS222 (tricaine methanesulphonate, Sandoz), weighed and injected i.p. with either (i) Group I, 10 pg/g IM-Sigma (5 pi/g) suspended in 0.6% NaCl (4 drops Tween 80/100 ml saline), or (ii) Group II, an equivalent volume of Tween 80 in saline. At 3 p.m. on Day 2 each fish was netted and injected i.p. with 4 IU/g HCG (Sigma). At 10 a.m. on Day 3, none of the IM treated fish had ovulated while 8 of 10 control fish ovulated normally (Table I). No adverse side effects were noted following IM treatment and the majority of the fish had the flaccid abdomen and large ovipore characteristic of ovulated goldfish. Two Group I fish were used for histology. On dissection, their ovaries contained large numbers of what appeared to be ovulated eggs, since they were clear, not opaque as in pre-ovulatory stages. Although the ova had not been released from the follicles, the ovaries had responded to the warming-HCG treatment.
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TABLE I EFFECT OF INDOMETHACIN (IM) ON OVULATIONOF HCG-TREATEDGOLDFISH
Group
","5(gE) - . .
Treatment on Day 2 10 a.m. 3 p.m.
No. fish ovulating on Day 3
24.0 +1.5 21.5 k1.1
saline
HCG (4 ID/g)
On each of Days 5 and 7, two Group I fish and one of the unovulated Group II fish received 4 IU/g HCG. In both cases, only the Group II female had ovulated the following day. On each of Days 9 and 11, two Group I females received 4 IU/g HCG; in both cases one of the two had ovulated the following day. These limited results suggest the injection of an IM suspension has a long-lasting inhibitory effect on HCG-induced ovulation (at least 6 days). Histological examination of the ovaries of all Group I females killed from Day 6 onward revealed large numbers of ~1and 6 stage corpora atretica (8). Even fish killed on Day 3 showed a thickening of the granulosa and zona radiata and rupture of the yolk vesicles. By Day 6, the granulosa had thickened greatly and many granulosa cells had entered the ooplasm to digest and absorb the yolk; degeneration of yolk vesicles and yolk granules was advanced. By Day 8 the atretic follicles were noticeably smaller and had begun to lose their spherical shape, trends which continued through Days 10-12. In contrast, ovaries of Group II females contained large numbers of stage I post-ovulatory corpora lutea (9), hollow structures formed of hypertrophied granulosa cells surrounding the atria1 cavity, formerly containing the oocyte. It would seem, therefore, that the presence of the clear oocytes which could not be stripped by gentle pressure on the abdomen (i.e., the intraovarian oocytes observed in dissection the morning of Day 3) indicated the comnencement of corpora atretica development (luteinization). Experiment II In the second experiment, designed to test the effects of PG's on ovulation of IM-treated fish, females were warmed and treated as in Experiment I with either IM and HCG (Groups I, II, III, and IV) or saline and HCG (Group V). In addition, they were netted at 7 p.m. on Day 2 and injected with either PGF2, (Group I, 8 fish), PGEl (Group II, 6 fish), PGE2 (Group III, 8 fish), or buffered saline (Group IV, 6 fish and Group V, 5 fish). All PG's were prepared imnediately prior to injection,
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PROSTAGLANDINS
TABLE II EFFECT OF INDOMETHACIN (IM) AND PROSTAGLANDINS ON OVULATION OF HCG-TREATED GOLDFISH
Group
Treatment on Day 2 10 a.m. 3 p.m. 7 p.m.
No. fish ovulating on Day 3
***
0
(i=8)
25.6 k2.4
IM*
HCG**
PGF2
$6)
28.8 k3.0
IM
HCG
PGE,
0
III (n=8)
23.1 51.9
IM
HCG
PGE,
0
$6)
20.7 kO.6
IM
HCG
saline
0
$=5)
27.6 +2.4
saline
HCG
saline
5
1 *
= 10 ug/g;
PGEl and PGE2 with buffered (tromethamine All PG's were
** = 4 IU/g;
***
= all PG's injected at 1 pg/g
by dissolving in 95% ethanol (1 mg/O.l ml) and diluting 9X saline (20 mg Na2C0s/100 ml 0.6% NaCl), and PGF2, salt) simply by dissolving in the buffered saline (1 mg/ml). injected i.p. at 1 ug/g (5 pi/g).
When checked at 10 a.m. on Day 3, none of the IM and HCG-treated fish receiving PG's or the 6 receiving IM, HCG, and saline had ovulated; all 5 control females receiving saline and HCG ovulated normally (Table II). Although other possibilities existed, we interpreted these results to mean that the PG injection was timed incorrectly in relation to the HCG injection and that possibly an insufficient PG dose was used. A pilot experiment was then run, duplicating the procedure of Experiment II, except that fish were injected with PGE2 (5 vg/g) at either 6 p.m. or 10 p.m. on Day 2, or 2 a.m. or 6 a.m. on Day 3. The 2 a.m. injection was most effective; 2 of 3 fish ovulated normally. None of 3 fish injected at 6 p.m. Day 2 ovulated and 1 of 3 fish injected at 10 p.m. Day 2 and 3 of 3 fish injected at 6 a.m. Day 3 produced very small numbers of eggs. Based on these results, the third experiment was carried out.
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Experiment III
In this experiment, fish were again warmed and injected .. with . _ IM and HCG (Groups I to V) or saline and HCG (Group VI) as described in Experiment I. In addition, PGE2 was injected at 10 p.m. on Day 2 (Group I), at 2 a.m. on Day 3 (Group II), and 6 a.m. on Day 3 (Group III). At 2 a.m. on Day 3, Group IV was injected with PGE1, Group V with PGFza, and Group VI with buffered saline. All PG's were prepared immediately before injection and given i.p. at 5 pg/g. Fish were checked for ovulation at 2 p.m. on Day 3. Ovulations were quantified volumetrically by squeezing the eggs into a plastic vial and drawing them into a plastic syringe. The ovaries of all unovulated fish were checked to ensure that they had responded to HCG; i.e., that they contained large numbers of clear oocytes. Four fish which had ovaries containing only opaque oocytes are not included in the results. Of the injection times tested, only PG administration at 2 a.m. induced ovulation of a normal volume of eggs (Table III). None of the 6 fish given PGE2 at 10 p.m. on Day 2 ovulated and the 3 responding fish of the 7 injected at 6 a.m. on Day 3 ovulated very few eggs (-c 0.1 ml). At the dosage employed, there appeared to be no difference between the ovulating potencies of the three PG's tested. Experiment IV In Experiment IV, by giving IM at various times after HCG treatment, we hoped to find a correspondence between the time at which IM would fail to block HCG-induced ovulation and the time at which PG's have been shown to restore HCG-induced ovulation following IM blockade. Thirty-eight gravid female goldfish were warmed on Day 1 and divided into 6 groups. At 3 p.m. on Day 2, each was netted, weighed, and injected i.p. with 4 Ill/g HCG. In addition, females in Group I were injected i.p. with 10 vg/g IM. At 9 p.m. and 12 p.m. Day 2, and 3 a.m. and 6 a.m. Day 3, females in Groups II, III, IV and V respectively were injected i.p. with 10 ug/g IM; females in Group VI received an equivalent volume of saline vehicle at 12 p.m. Day 2. At 1 p.m. on Day 3, all fish were checked for ovulation and the ovaries of all unovulated fish examined; 5 unovulated fish had not responded to HCG and are not included in the results. The results of this experiment (see Table IV) show that IM is completely effective in blocking ovulation when given up to 6 h after HCG treatment (9 p.m. Day 2). However, 9 h after HCG treatment, IM is only partially effective in blocking ovulation and by 3 a.m. Day 3 (12 h after HCG) it is without effect. All ovulated females in Group IV and Group V had ovulated some eggs when checked at the time of IM injection. It is not known whether ovulation had been completed at those times (3 a.m., 6 a.m. Day 3) as eggs were not removed for measuring until 1 p.m. Day 3.
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%
t
I
HCG HCG
IM
saline
25.2 k3.5
27.6 +l.O +++
HCG
IM
24.6 k1.0
++ = 4 IU/g;
HCG
IM
26.6 k2.6
HCG++ HCG
IMt
saline
PGF,,
PGE,
PGE, PGE,
= all PG's injected at 5 ug/g
PGE2+++
Treatment on Day 2 Day 3 10 a.m. 2 a.m. 3 p.m. 10 p.m. 6 a.m.
IM
25.4 k1.9
23.2 k1.2
wt (9) + S.E.
= 10 pg/g;
(x=6,
III (n=7)
(:=6)
Group
5
6
5
3
7
0
No. fish ovulating on Day 3
EFFECT OF PROSTAGLANDINS ON OVULATION OF GOLDFISH TREATED WITH INDOMETHACIN AND HCG
TABLE III
***
***
****
3.4/2.2/2.0/1.2/l.:
*** 3.2/3.4/0.8/
3.8/0.6/
2.8/0.7/0.2/
Volume of ovulated eggs (ml) * = < 0.1 ml
(:!6)
III (n=6)
$6)
Group
Day 2 9 p.m. p.m.
HCG
HCG
HCG
HCG
HCG+IM
3 p.m.
saline
IM
12
Treatment on 3
IM
a.m.
Day 6 3
IM
a.m.
4
5
5
0
0
No. fish ovulating on Day 3
INHIBITION OF OVULATIONBY INDOMETHACIN INJECTEDAT VARIOUS TIMES AFTER HCG
TABLE IV
0.7/0.5/0.5/0.4
4.1/2.8/2.2/0.5/0.5
4.3/4.3/2.4/2.1/1.0
2.3/0.9/*
Volume of ovulated eggs (ml) * = co.1 ml
I
1
2
E
j!
%
PROSTAGLANDINS
DISCUSSION The results of the experiments described in this paper constitute _. the first published account of possible prostaglandin involvement in the ovulatory process of submammalian vertebrates. We have demonstrated that indomethacin, an inhibitor of prostaglandin synthesis, blocks ovulation in intact, gravid goldfish treated with HCG. Indomethacin is totally effective if given 5 h before, coincident with, or 6 h after HCG; indomethacin blocks only some of the fish when given 9 h after HCG and is without effect on ovulation when given 12 h after HCG. Furthermore, we have shown that exogenous prostaglandins overcome indomethacin blockade and that, at least in the case of PGE2, the time of the prostaglandin injection is important. We found no obvious differences in the ovulatory potencies of PGE1, PGEz, and PGFncr. We feel that the above reported effects of indomethacin and prostaglandins on ovulation in the goldfish suggest an ovulatory role for prostaglandins at the level of the ovary. This is not to suggest that prostaglandins play no role in gonadotropin secretion; in fact, the design of our experiments (in which prostaglandins are given only to indomethacin-treated fish, and ovulations are the only data) precludes the possibility of obtaining positive evidence for such a function. While indomethacin may have interfered with the spontaneous release of gonadotropin, the histological demonstration of follicular luteinization indicates that the ovaries had responded to the HCG treatment and that the failure to ovulate was in fact due to an inhibition of follicu.lar rupture. From the results of mammalian studies, two hypotheses (not necessarily mutually exclusive) have been advanced to explain the role(s) of prostaglandins in ovulation. One contends that prostaglandins act indirectly to induce ovulation by stimulating release of an ovulatory surge of gonadotropin (3, 10). The second hypothesis suggests prostaglandins induce ovulation through some direct action on the ovary (1, 2, 6, 7, 11). Though the precise mechanism remains to be elucidated, the ovulatory role of prostaglandins at the ovarian level seems to be confined to the process of follicular rupture. Thus, indomethacin was shown to inhibit ovulation in response to LH or HCG in the rabbit, and although the ova were retained within the follicles, histologically normal luteinization occurred and both the acute steroidogenic response and that of the accompanying pseudopregnancy were unaffected (12). The retention of the ova may result from some alteration in preovulatory follicular swelling, an inhibition of ovarian smooth muscle contraction, or some as yet unknown effect (13, 14). Diaz-Infante et aZ. (7) have shown recently that indomethacin inhibits, while PGF2, reinitiates, ovarian contractility in the rabbit. In mammals, it appears that ovulatory levels of gonadotropin (endogenous or injected) initiate follicular maturation leading to elevated follicular prostaglandin levels prior to the time of ovulation. In the estrous rabbit, such a situation has been well documented (15). From 5 h to 9 h after injection of HCG, levels of PGF and PGE in Graafian follicles were elevated from 10 times to 60 times and from 3 times to
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PROSTAGLANDINS
15 times respectively over the levels prior to and one hour after HCG injection (15); indomethacin (20 pg/g) given 30 minutes prior to HCG completely abolished this effect (14). The failure of injected prostaglandins to restore ovulation completely in all indomethacin treated goldfish at the most effective injection time (2 a.m. on Day 3) may have been due to insufficient dosage; although the dosage of prostaglandin was quite large, the amount reaching the follicle may have been low. We feel the problem is more likely one of inappropriately-timed injections. The total ineffectiveness of prostaglandins administered at 7 p.m. in Experiment II (4 h after HCG) and at 10 p.m. in Experiment III (7 h after HCG) suggest that maturational steps preceding follicular rupture had not been completed. Indomethacin injected at this time (9 p.m., 6 h after HCG; Experiment IV) is still completely effective in blocking ovulation. However, as indomethacin given at 12 p.m. (9 h after HCG) is only partially effective in blocking ovulation, it would seem that the eggs of some fish have matured to a preovulatory stage in which endogenous prostaglandins are effective in inducin follicular rupture. This process is complete by 3 a.m. (12 h after HCGB , as indomethacin given at this time has no effect on ovulation; in fact, all fish given indomethacin at 3 a.m. had already ovulated at least a few eggs. As only 3 h separate the time at which indomethacin first failed to inhibit ovulation (12 p.m., 9 h after HCG , the most effective time for prostaglandin-induced ovulation (2 a.m. 1 , and the first time at which ovulated eggs could be removed (3 a.m., 12 h after HCG), it is proposed that one role of prostaglandin in goldfish ovulation is at the level of the ovary and is restricted to the final stages of oocyte maturation. As well, the marginal effects of the 6 a.m. prostaglandin injection in Experiment III suggest that HCG-stimulated follicles which have been blocked with indomethacin will respond to exogenous prostaglandins within a fairly restricted period, and that by 15 h after HCG, follicular sensitivity to prostaglandins has decreased considerably.
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REFERENCES 1.
Labhsetwar, A.P. Prostaglandins and the Reproductive Cycle, Fed. Proc. 33:61, 1974.
2.
Pharriss, B.B. and J.E. Shaw. Rev. Physiol. 36:391, 1974.
3.
Orczyk, G.P. and H.R. Behrman. Ovulation Blockade by.Aspirin or Indomethacin - in oivo Evidence for a Role of Prostaglandin in Gonadotropin Secretion, Prostaglandins 1:3, 1972.
4.
Armstrong, D.T. and D.L. Grinwich. Blockade of Spontaneous and LHinduced Ovulation in Rats by Indomethacin, an Inhibitor of Prostaglandin Biosynthesis. Prostaglandins 1:21, 1972.
5.
Grinwich, D.L., T. Kennedy and D.T. Armstrong. Dissociation of Ovulatory and Steroidogenic Actions of Luteinizing Hormone in Rabbits with Indomethacin, an Inhibitor of Prostaglandin Biosynthesis, Prostaglandins 1:89, 1972.
6.
Tsafriri, A., H.R. Lindner, U. Zor and A. Lamprecht. Physiological Role of Prostaglandins in the Induction of Ovulation, Prostaglandins 2:1, 1972.
7.
Diaz-Infante, A. Jr., K.H. Wright and E.E. Wallach. Effects of Indomethacin and Prostaglandin Fpcron Ovulation and Ovarian Contractility in the Rabbit, Prostaglandins 5:567, 1974.
8.
Bretschneider, L.H. and J.J. de Wit. Sexual Endocrinology of Nonmammalian Vertebrates, Elsevier, Amsterdam, 1947.
9.
Khoo, K.H. Steroidogenesis and the Role of Steroids in the Endocrine Control of Oogenesis and Vitellogenesis in the Goldfish, Carassius auratus, Ph.D. Thesis, University of British Columbia, 1974.
10.
Harms, P.G., S.R. Ojeda and S.M. McCann. Prostaglandin-induced Release of Pituitary Gonadotropins: Central Nervous System and Pituitary Sites of Action, Endocrinology 94:1459, 1974.
11.
Sato, T., K. Taya, T. Jyujo and M. Igarishi. Ovulation Block by Indomethacin, an Inhibitor of Prostaglandin Synthesis: A Study of its Site of Action in Rats, J. Reprod. Fert. 39:33, 1974.
12.
Armstrong, D.T., Y.S. Moon and D.L. Grinwich. Possible Role of Prostaglandins in Ovulation, Advances in the Biosciences 9:709, 1973.
13.
Armstrong, D.T., D.L. Grinwich, Y.S. Moon and J. Zamecnik. Inhibition of Ovulation in Rabbits by Intrafollicular Injection of Indomethacin and Prostaglandin F Antiserum, Life Sci. 14:129, 1974.
606
Prostaglandins in Reproduction, Ann.
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14.
Yang, N.S.T., J.M. Marsh and W.J. LeMaire. Prostaglandin Changes Induced by Ovulatory Stimuli in Rabbit Graafian Follicles. The Effect of Indomethacin, Prostaglandins 4:395, 1973.
15.
LeMaire, W.J., N.S.T. Yang, H.R. Behrman and J.M. Marsh. Preovulatory Changes in the Concentration of Prostaglandins in Rabbit Graafian Follicles, Prostaglandins 3:367, 1973.
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N. E. STACEY and S. PANDEY Department of Zoology, University of British Columbia, Vancouver, B.C., Canada, and Department of Zoology, Patna University, Patna 5, India.
ABSTRACT A technique is described whereby elevated temperature and HCG injection yield a high percentage of ovulation in gravid goldfish. Indomethacin'(l0 ug/g; i.p. injection) completely inhibits ovulation if given within 6 hours following HCG (4 Ill/g); the unovulated oocytes develop rapidly into corpora atretica. PGE1, PGE2, and PGF,, (5 pg/g; i.p. injection) induce ovulation in fish treated with indomethacin and HCG; PGE2 was most effective when given 11 hours after HCG. The results suggest that the ovulatory action of prostaglandins following HCG stimulation is at the level of the ovary and that it is restricted to a period between 7 and 12 hours after the gonadotropin injection.
ACKNOWLEDGEMENTS: This work was supported by a University of British Columbia graduate fellowship to N.E.S., a Canadian International Development Agency Research Associateship to S.P., and an N.R.C. Grant-in-aid of Research to Dr. W.S. Hoar. We are grateful to Dr.J. Pike from the Upjohn Company for the generous gift of prostaglandins and to Dr. W.S. Hoar and Dr. N.R. Liley for reading the manuscript. Accepted April 1, 1975
PROSTAGLANDINS APRIL 1975
VOL. 9 NO. 4
597
PROSTAGLANDINS
INTRODUCTION A number of recent studies suggest that prostaglandins (PG's) play a role in the process of ovulation (see reviews 1, 2). Indomethacin (IM), an inhibitor of PG biosynthesis, blocks ovulation in both rats (3, 4) and rabbits (5), an effect which is reversed by treatment with PG's (6, 7). To date, published accounts of these phenomena have been restricted to studies of mamnals. In this paper, we present evidence that PG's are involved in the ovulation of goldfish; IM is shown to block the ovulatory response to HCG, and PG's are shown to overcome this blockade.
METHODS AND RESULTS Stocks of female comet goldfish were obtained from Hartz Mountain Pet Supply (Richmond, B.C., Canada) and kept in running dechlorinated tap water at 10-13°C on a 16 h photoperiod (8 a.m. to 12 p.m.). Frozen brine shrimp or trout pellets were provided ad Zibitwn at least once daily. Under these conditions, fish become gravid but do not ovulate. To induce ovulation, gravid females are warmed gradually to 200C on Day 1. Spontaneous ovulation usually occurs during the early morning of Day 3 (never on Day 2) and may be checked by gently pressing the abdomen to release a stream of eggs from the ovipore. If fish, warmed to 2OoC, are injected intraperitoneally (i.p.) with 4 IU/g HCG (dissolved in 0.6% NaCl) on the afternoon of Day 2, virtually 100% will ovulate on Day 3. HCG will also induce ovulation in fish kept at lo-13oC, but several days of treatment are usually required. The combined treatment with elevated temperature and HCG injection is a standard and reliable technique for the induction of ovulation. Experiment I In this experiment, intended to examine the effects of IM on ovulation, 20 gravid female goldfish were removed from the cold water stock tanks at 9 a.m. on Day 1, divided randomly into 2 groups (10 fish/ 15 gallon tank) and slowly warmed to 200C. At 10 a.m. on Day 2, each fish was anaesthetized in MS222 (tricaine methanesulphonate, Sandoz), weighed and injected i.p. with either (i) Group I, 10 pg/g IM-Sigma (5 pi/g) suspended in 0.6% NaCl (4 drops Tween 80/100 ml saline), or (ii) Group II, an equivalent volume of Tween 80 in saline. At 3 p.m. on Day 2 each fish was netted and injected i.p. with 4 IU/g HCG (Sigma). At 10 a.m. on Day 3, none of the IM treated fish had ovulated while 8 of 10 control fish ovulated normally (Table I). No adverse side effects were noted following IM treatment and the majority of the fish had the flaccid abdomen and large ovipore characteristic of ovulated goldfish. Two Group I fish were used for histology. On dissection, their ovaries contained large numbers of what appeared to be ovulated eggs, since they were clear, not opaque as in pre-ovulatory stages. Although the ova had not been released from the follicles, the ovaries had responded to the warming-HCG treatment.
598
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TABLE I EFFECT OF INDOMETHACIN (IM) ON OVULATIONOF HCG-TREATEDGOLDFISH
Group
","5(gE) - . .
Treatment on Day 2 10 a.m. 3 p.m.
No. fish ovulating on Day 3
24.0 +1.5 21.5 k1.1
saline
HCG (4 ID/g)
On each of Days 5 and 7, two Group I fish and one of the unovulated Group II fish received 4 IU/g HCG. In both cases, only the Group II female had ovulated the following day. On each of Days 9 and 11, two Group I females received 4 IU/g HCG; in both cases one of the two had ovulated the following day. These limited results suggest the injection of an IM suspension has a long-lasting inhibitory effect on HCG-induced ovulation (at least 6 days). Histological examination of the ovaries of all Group I females killed from Day 6 onward revealed large numbers of ~1and 6 stage corpora atretica (8). Even fish killed on Day 3 showed a thickening of the granulosa and zona radiata and rupture of the yolk vesicles. By Day 6, the granulosa had thickened greatly and many granulosa cells had entered the ooplasm to digest and absorb the yolk; degeneration of yolk vesicles and yolk granules was advanced. By Day 8 the atretic follicles were noticeably smaller and had begun to lose their spherical shape, trends which continued through Days 10-12. In contrast, ovaries of Group II females contained large numbers of stage I post-ovulatory corpora lutea (9), hollow structures formed of hypertrophied granulosa cells surrounding the atria1 cavity, formerly containing the oocyte. It would seem, therefore, that the presence of the clear oocytes which could not be stripped by gentle pressure on the abdomen (i.e., the intraovarian oocytes observed in dissection the morning of Day 3) indicated the comnencement of corpora atretica development (luteinization). Experiment II In the second experiment, designed to test the effects of PG's on ovulation of IM-treated fish, females were warmed and treated as in Experiment I with either IM and HCG (Groups I, II, III, and IV) or saline and HCG (Group V). In addition, they were netted at 7 p.m. on Day 2 and injected with either PGF2, (Group I, 8 fish), PGEl (Group II, 6 fish), PGE2 (Group III, 8 fish), or buffered saline (Group IV, 6 fish and Group V, 5 fish). All PG's were prepared imnediately prior to injection,
APRIL
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VOL. 9 NO. 4
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PROSTAGLANDINS
TABLE II EFFECT OF INDOMETHACIN (IM) AND PROSTAGLANDINS ON OVULATION OF HCG-TREATED GOLDFISH
Group
Treatment on Day 2 10 a.m. 3 p.m. 7 p.m.
No. fish ovulating on Day 3
***
0
(i=8)
25.6 k2.4
IM*
HCG**
PGF2
$6)
28.8 k3.0
IM
HCG
PGE,
0
III (n=8)
23.1 51.9
IM
HCG
PGE,
0
$6)
20.7 kO.6
IM
HCG
saline
0
$=5)
27.6 +2.4
saline
HCG
saline
5
1 *
= 10 ug/g;
PGEl and PGE2 with buffered (tromethamine All PG's were
** = 4 IU/g;
***
= all PG's injected at 1 pg/g
by dissolving in 95% ethanol (1 mg/O.l ml) and diluting 9X saline (20 mg Na2C0s/100 ml 0.6% NaCl), and PGF2, salt) simply by dissolving in the buffered saline (1 mg/ml). injected i.p. at 1 ug/g (5 pi/g).
When checked at 10 a.m. on Day 3, none of the IM and HCG-treated fish receiving PG's or the 6 receiving IM, HCG, and saline had ovulated; all 5 control females receiving saline and HCG ovulated normally (Table II). Although other possibilities existed, we interpreted these results to mean that the PG injection was timed incorrectly in relation to the HCG injection and that possibly an insufficient PG dose was used. A pilot experiment was then run, duplicating the procedure of Experiment II, except that fish were injected with PGE2 (5 vg/g) at either 6 p.m. or 10 p.m. on Day 2, or 2 a.m. or 6 a.m. on Day 3. The 2 a.m. injection was most effective; 2 of 3 fish ovulated normally. None of 3 fish injected at 6 p.m. Day 2 ovulated and 1 of 3 fish injected at 10 p.m. Day 2 and 3 of 3 fish injected at 6 a.m. Day 3 produced very small numbers of eggs. Based on these results, the third experiment was carried out.
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Experiment III
In this experiment, fish were again warmed and injected .. with . _ IM and HCG (Groups I to V) or saline and HCG (Group VI) as described in Experiment I. In addition, PGE2 was injected at 10 p.m. on Day 2 (Group I), at 2 a.m. on Day 3 (Group II), and 6 a.m. on Day 3 (Group III). At 2 a.m. on Day 3, Group IV was injected with PGE1, Group V with PGFza, and Group VI with buffered saline. All PG's were prepared immediately before injection and given i.p. at 5 pg/g. Fish were checked for ovulation at 2 p.m. on Day 3. Ovulations were quantified volumetrically by squeezing the eggs into a plastic vial and drawing them into a plastic syringe. The ovaries of all unovulated fish were checked to ensure that they had responded to HCG; i.e., that they contained large numbers of clear oocytes. Four fish which had ovaries containing only opaque oocytes are not included in the results. Of the injection times tested, only PG administration at 2 a.m. induced ovulation of a normal volume of eggs (Table III). None of the 6 fish given PGE2 at 10 p.m. on Day 2 ovulated and the 3 responding fish of the 7 injected at 6 a.m. on Day 3 ovulated very few eggs (-c 0.1 ml). At the dosage employed, there appeared to be no difference between the ovulating potencies of the three PG's tested. Experiment IV In Experiment IV, by giving IM at various times after HCG treatment, we hoped to find a correspondence between the time at which IM would fail to block HCG-induced ovulation and the time at which PG's have been shown to restore HCG-induced ovulation following IM blockade. Thirty-eight gravid female goldfish were warmed on Day 1 and divided into 6 groups. At 3 p.m. on Day 2, each was netted, weighed, and injected i.p. with 4 Ill/g HCG. In addition, females in Group I were injected i.p. with 10 vg/g IM. At 9 p.m. and 12 p.m. Day 2, and 3 a.m. and 6 a.m. Day 3, females in Groups II, III, IV and V respectively were injected i.p. with 10 ug/g IM; females in Group VI received an equivalent volume of saline vehicle at 12 p.m. Day 2. At 1 p.m. on Day 3, all fish were checked for ovulation and the ovaries of all unovulated fish examined; 5 unovulated fish had not responded to HCG and are not included in the results. The results of this experiment (see Table IV) show that IM is completely effective in blocking ovulation when given up to 6 h after HCG treatment (9 p.m. Day 2). However, 9 h after HCG treatment, IM is only partially effective in blocking ovulation and by 3 a.m. Day 3 (12 h after HCG) it is without effect. All ovulated females in Group IV and Group V had ovulated some eggs when checked at the time of IM injection. It is not known whether ovulation had been completed at those times (3 a.m., 6 a.m. Day 3) as eggs were not removed for measuring until 1 p.m. Day 3.
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%
t
I
HCG HCG
IM
saline
25.2 k3.5
27.6 +l.O +++
HCG
IM
24.6 k1.0
++ = 4 IU/g;
HCG
IM
26.6 k2.6
HCG++ HCG
IMt
saline
PGF,,
PGE,
PGE, PGE,
= all PG's injected at 5 ug/g
PGE2+++
Treatment on Day 2 Day 3 10 a.m. 2 a.m. 3 p.m. 10 p.m. 6 a.m.
IM
25.4 k1.9
23.2 k1.2
wt (9) + S.E.
= 10 pg/g;
(x=6,
III (n=7)
(:=6)
Group
5
6
5
3
7
0
No. fish ovulating on Day 3
EFFECT OF PROSTAGLANDINS ON OVULATION OF GOLDFISH TREATED WITH INDOMETHACIN AND HCG
TABLE III
***
***
****
3.4/2.2/2.0/1.2/l.:
*** 3.2/3.4/0.8/
3.8/0.6/
2.8/0.7/0.2/
Volume of ovulated eggs (ml) * = < 0.1 ml
(:!6)
III (n=6)
$6)
Group
Day 2 9 p.m. p.m.
HCG
HCG
HCG
HCG
HCG+IM
3 p.m.
saline
IM
12
Treatment on 3
IM
a.m.
Day 6 3
IM
a.m.
4
5
5
0
0
No. fish ovulating on Day 3
INHIBITION OF OVULATIONBY INDOMETHACIN INJECTEDAT VARIOUS TIMES AFTER HCG
TABLE IV
0.7/0.5/0.5/0.4
4.1/2.8/2.2/0.5/0.5
4.3/4.3/2.4/2.1/1.0
2.3/0.9/*
Volume of ovulated eggs (ml) * = co.1 ml
I
1
2
E
j!
%
PROSTAGLANDINS
DISCUSSION The results of the experiments described in this paper constitute _. the first published account of possible prostaglandin involvement in the ovulatory process of submammalian vertebrates. We have demonstrated that indomethacin, an inhibitor of prostaglandin synthesis, blocks ovulation in intact, gravid goldfish treated with HCG. Indomethacin is totally effective if given 5 h before, coincident with, or 6 h after HCG; indomethacin blocks only some of the fish when given 9 h after HCG and is without effect on ovulation when given 12 h after HCG. Furthermore, we have shown that exogenous prostaglandins overcome indomethacin blockade and that, at least in the case of PGE2, the time of the prostaglandin injection is important. We found no obvious differences in the ovulatory potencies of PGE1, PGEz, and PGFncr. We feel that the above reported effects of indomethacin and prostaglandins on ovulation in the goldfish suggest an ovulatory role for prostaglandins at the level of the ovary. This is not to suggest that prostaglandins play no role in gonadotropin secretion; in fact, the design of our experiments (in which prostaglandins are given only to indomethacin-treated fish, and ovulations are the only data) precludes the possibility of obtaining positive evidence for such a function. While indomethacin may have interfered with the spontaneous release of gonadotropin, the histological demonstration of follicular luteinization indicates that the ovaries had responded to the HCG treatment and that the failure to ovulate was in fact due to an inhibition of follicu.lar rupture. From the results of mammalian studies, two hypotheses (not necessarily mutually exclusive) have been advanced to explain the role(s) of prostaglandins in ovulation. One contends that prostaglandins act indirectly to induce ovulation by stimulating release of an ovulatory surge of gonadotropin (3, 10). The second hypothesis suggests prostaglandins induce ovulation through some direct action on the ovary (1, 2, 6, 7, 11). Though the precise mechanism remains to be elucidated, the ovulatory role of prostaglandins at the ovarian level seems to be confined to the process of follicular rupture. Thus, indomethacin was shown to inhibit ovulation in response to LH or HCG in the rabbit, and although the ova were retained within the follicles, histologically normal luteinization occurred and both the acute steroidogenic response and that of the accompanying pseudopregnancy were unaffected (12). The retention of the ova may result from some alteration in preovulatory follicular swelling, an inhibition of ovarian smooth muscle contraction, or some as yet unknown effect (13, 14). Diaz-Infante et aZ. (7) have shown recently that indomethacin inhibits, while PGF2, reinitiates, ovarian contractility in the rabbit. In mammals, it appears that ovulatory levels of gonadotropin (endogenous or injected) initiate follicular maturation leading to elevated follicular prostaglandin levels prior to the time of ovulation. In the estrous rabbit, such a situation has been well documented (15). From 5 h to 9 h after injection of HCG, levels of PGF and PGE in Graafian follicles were elevated from 10 times to 60 times and from 3 times to
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15 times respectively over the levels prior to and one hour after HCG injection (15); indomethacin (20 pg/g) given 30 minutes prior to HCG completely abolished this effect (14). The failure of injected prostaglandins to restore ovulation completely in all indomethacin treated goldfish at the most effective injection time (2 a.m. on Day 3) may have been due to insufficient dosage; although the dosage of prostaglandin was quite large, the amount reaching the follicle may have been low. We feel the problem is more likely one of inappropriately-timed injections. The total ineffectiveness of prostaglandins administered at 7 p.m. in Experiment II (4 h after HCG) and at 10 p.m. in Experiment III (7 h after HCG) suggest that maturational steps preceding follicular rupture had not been completed. Indomethacin injected at this time (9 p.m., 6 h after HCG; Experiment IV) is still completely effective in blocking ovulation. However, as indomethacin given at 12 p.m. (9 h after HCG) is only partially effective in blocking ovulation, it would seem that the eggs of some fish have matured to a preovulatory stage in which endogenous prostaglandins are effective in inducin follicular rupture. This process is complete by 3 a.m. (12 h after HCGB , as indomethacin given at this time has no effect on ovulation; in fact, all fish given indomethacin at 3 a.m. had already ovulated at least a few eggs. As only 3 h separate the time at which indomethacin first failed to inhibit ovulation (12 p.m., 9 h after HCG , the most effective time for prostaglandin-induced ovulation (2 a.m. 1 , and the first time at which ovulated eggs could be removed (3 a.m., 12 h after HCG), it is proposed that one role of prostaglandin in goldfish ovulation is at the level of the ovary and is restricted to the final stages of oocyte maturation. As well, the marginal effects of the 6 a.m. prostaglandin injection in Experiment III suggest that HCG-stimulated follicles which have been blocked with indomethacin will respond to exogenous prostaglandins within a fairly restricted period, and that by 15 h after HCG, follicular sensitivity to prostaglandins has decreased considerably.
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REFERENCES 1.
Labhsetwar, A.P. Prostaglandins and the Reproductive Cycle, Fed. Proc. 33:61, 1974.
2.
Pharriss, B.B. and J.E. Shaw. Rev. Physiol. 36:391, 1974.
3.
Orczyk, G.P. and H.R. Behrman. Ovulation Blockade by.Aspirin or Indomethacin - in oivo Evidence for a Role of Prostaglandin in Gonadotropin Secretion, Prostaglandins 1:3, 1972.
4.
Armstrong, D.T. and D.L. Grinwich. Blockade of Spontaneous and LHinduced Ovulation in Rats by Indomethacin, an Inhibitor of Prostaglandin Biosynthesis. Prostaglandins 1:21, 1972.
5.
Grinwich, D.L., T. Kennedy and D.T. Armstrong. Dissociation of Ovulatory and Steroidogenic Actions of Luteinizing Hormone in Rabbits with Indomethacin, an Inhibitor of Prostaglandin Biosynthesis, Prostaglandins 1:89, 1972.
6.
Tsafriri, A., H.R. Lindner, U. Zor and A. Lamprecht. Physiological Role of Prostaglandins in the Induction of Ovulation, Prostaglandins 2:1, 1972.
7.
Diaz-Infante, A. Jr., K.H. Wright and E.E. Wallach. Effects of Indomethacin and Prostaglandin Fpcron Ovulation and Ovarian Contractility in the Rabbit, Prostaglandins 5:567, 1974.
8.
Bretschneider, L.H. and J.J. de Wit. Sexual Endocrinology of Nonmammalian Vertebrates, Elsevier, Amsterdam, 1947.
9.
Khoo, K.H. Steroidogenesis and the Role of Steroids in the Endocrine Control of Oogenesis and Vitellogenesis in the Goldfish, Carassius auratus, Ph.D. Thesis, University of British Columbia, 1974.
10.
Harms, P.G., S.R. Ojeda and S.M. McCann. Prostaglandin-induced Release of Pituitary Gonadotropins: Central Nervous System and Pituitary Sites of Action, Endocrinology 94:1459, 1974.
11.
Sato, T., K. Taya, T. Jyujo and M. Igarishi. Ovulation Block by Indomethacin, an Inhibitor of Prostaglandin Synthesis: A Study of its Site of Action in Rats, J. Reprod. Fert. 39:33, 1974.
12.
Armstrong, D.T., Y.S. Moon and D.L. Grinwich. Possible Role of Prostaglandins in Ovulation, Advances in the Biosciences 9:709, 1973.
13.
Armstrong, D.T., D.L. Grinwich, Y.S. Moon and J. Zamecnik. Inhibition of Ovulation in Rabbits by Intrafollicular Injection of Indomethacin and Prostaglandin F Antiserum, Life Sci. 14:129, 1974.
606
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14.
Yang, N.S.T., J.M. Marsh and W.J. LeMaire. Prostaglandin Changes Induced by Ovulatory Stimuli in Rabbit Graafian Follicles. The Effect of Indomethacin, Prostaglandins 4:395, 1973.
15.
LeMaire, W.J., N.S.T. Yang, H.R. Behrman and J.M. Marsh. Preovulatory Changes in the Concentration of Prostaglandins in Rabbit Graafian Follicles, Prostaglandins 3:367, 1973.
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