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Effects of third ventricle injection of prostaglandins on gonadotropin secretion in goldfish, Carassius auratus
GENERAL
AND
COMPARATIVE
Effects
ENDOCRINOLOGY
30, 451-456 (1976)
of Third Ventricle Injection of Prostaglandins Gonadotropin Secretion in Goldfish, Carassius auratd
on
R. E. PETER AND R. BILLARD~ Department
of
Zoology. University of Alberta, Edmonton, Alberta T6G 2E9, Canada Accepted July 26, 1976
The effects of injection of prostaglandin (PG) El, PGEz, and PGF,, into the third ventricle on serum gonadotropin (GTH) concentrations in the goldfish were tested. Blood samples were taken at 30 min postinjection for radioimmunoassay of serum GTH. PG dosages of 0.5 and 1.0 pg were ineffective. However, PGE, and PGF, at the 2.0~pg dosage significantly suppressed serum GTH. PGE, at a 2.0~pg dosage had no effect. There were no effects on serum GTH when 2.0 pg of PGE,, PGE,, or PGF,, were injected intraperitoneally. The results indicate that PGE, and PGF,, suppress gonadotropin secretion by some action, presumably on the hypothalamus. However, action of PGE, and PGF,, by diffusion from the site of iniection to some other brain site or the pituitary cannot be eliminated in the present study.-
A number of studies have shown that some prostaglandins (PG) of the E series can influence gonadotropin secretion, particularly luteinizing hormone (LH), in the laboratory rat by an effect directly on the hypothalamus (Harms et al., 1973, 1974; Labhsetwar and Zolovick, 1973; Spies and Norman, 1973). Of the various PG’s tested, the hypothalamic site of action seems to be most responsive to PGE, (Harms et al., 1973, 1974; Ojeda et al., 1974). Studies by Chobsienget al. (1975), Eskayet al. (1975), and Ojeda et al. (1975) indicate that PGE, acts to stimulate LH-releasing hormone (LH-RH) secretion. However, a direct pituitary site of action in stimulation of gonadotropin secretion by PG has also been shown both in vitro (Sato et al., 1975; Makino, 1973; Ratner et al., 1974) and in viva (Sato et al., 1975; Harms et al., 1974). However, on the basis of required dosages, the pitui-
tary site of action is less responsive than the hypothalamic site. In teleosts there is no evidence available indicating an influence of PG on gonadotropin (GTH) secretion. However, a gonadotropin releasing factor has been shown to be present in hypothalamic extracts of carp (Breton et al., 1971, 1972b, 1975; Breton and Weil, 1973), goldfish (Crim et al., 1976), and rainbow trout (Breton et al., 1972b). In addition, synthetic LH-RH stimulates GTH secretion in carp (Breton and Weil, 1973), brown trout (Crim and Cluett, 1974), and goldfish (Crim et al., 1976). In the present work the effects on GTH secretion of PGE1, PGE2, and PGFzu injected into the third ventricle of goldfish were tested. Sexually mature female goldfish were used in the experiments to test whether the expected stimulation of GTH secretion would induce ovulation.
’ Supported by National Research Council of Canada Grant A6371 to R.E.P. ’ Permanent address: Laboratoire de Physiologie des Poissons, Institut National de la Recherche Agronomique, 78350 Jouy-en-Josas, France.
Goldfish, common or comet variety, used in the study were purchased in April 1975 from Grassyfork Fisheries, Co., Inc., Martinsville, Ind. Prior to use in
451 Copyright All rights
@ 1976 by Academic Press. Inc. of reproduction in any form reserved.
MATERIALS
AND METHODS
452
PETER
AND
experiments the fish were held for about 120 days on a simulated natural photoperiod at 10-14” in 1500-liter flow-through aquaria. Just before the experiments commenced the photoperiod was about 16 hr light-8 hr dark. For the experiments the fish were transferred to 96-liter flow-through aquaria regulated to 12-13” and held at a 16-hr light-8-hr dark photoperiod. The fish were fed Purina trout chow (Ralston Purina Co., St. Louis, MO.) ad /i&rum throughout the preexperimental and the experimental periods.
BILLARD
nula, was then injected into the cannula. Injections were done with a 5- or lo-~1 Hamilton syringe fitted with a short piece of PE 20 tubing on the needle. Following the injection, the cannula was recapped and the fish was returned to the aquarium. At 30 min following the injection a blood sample of about 0.2 to 0.3 @I was taken from the caudal vasculature. The fish were anesthetized prior to the blood sampling. The injections and sampling were done between 5 and 6.5 hr after the lights came on in each case. The blood samples were stored over chipped ice for about 2 hr Experiment 1 prior to centrifugation at 2500 rpm for 30 min. Serum was pipetted into micro test tubes and quick-frozen A total of 37 females, sexually mature, and weighover dry ice. The serum samples were stored at -20” ing 62.52 g (SD = 8.68) were selected for the experiment. Each fish was identified by colored threads at- until analyzed. The PG injections done on Day 3 (run 1) after cantached to the fms and a numbered tag attached to the left operculum (size 1 Monel tag, National Band and nulation were 0.5 pg of either PGE,, PGE,, or PGF,, in 1 ~1 of PS. The injections on Day 5 (run 2) and Day Tag Co., Newport, KY.). A cannula was implanted 7 (run 3) were, respectively, 1 and 2 pg of PGE,, into the third ventricle of each fish by the following procedure: The fish were prepared for stereotaxic im- PGE,, and PGF*, in 1 ~1 of PS. PGE, and PGE, soplantation following the technique of Peter (1970) as lutions were made fresh on the day of use. PGF,, modified by Peter and Gill (1975), except that the solutions were in some cases stored overnight at 4” parietal bone flap was completely removed. Before before use. The PGF,, used was in the form of the mounting the fish in the sterotaxic apparatus, one or tromethamine salt. For each run the fish in each group were randomly selected from among all those sometimes two optical screws, 3 mm in length, were cannulated. anchored in bone on each side lateral to the region At 9 days postcannulation the fish were again anesfrom which the parietal bone flap had been removed. thetized, and a small volume of methylene blue dye The cannula (described below) was then implanted into the third ventricle, dorsal to the anterior part of was injected through the cannula. The fish were then the nucleus lateral tuberis pars posterioris using the autopsied and the accuracy of placement of the cantechnique of Peter (1970) as modified by Peter and nula checked. Fish in which the cannula tip had been Gill (1975) (coordinates used + 0.7, M, D 3.0). The placed out through the ventral side of the brain (N = 4) were rejected from the experiment. The ovary was space in the cranium above the brain was filled with dissected out and weighed, and its condition was freshly made freshwater fish physiological solution noted. Fish that ovulated during the experiment (N = (PS). Following this, a scale was taken from posterior to the opercular region, and a groove cut in it. The 2) or were postovulatory (N = 1) or immature (N = 1) scale was then fitted around the cannula so as to were rejected from the experiment. The two fish that cover the hole in the cranium. Dental cement (Jet ovulated during the experiment were in either the PS or PGF,, groups during the three runs of the experiacrylic, type 2, class 1, Lang Dental Mfg. Co., Inc., ment. One fish, found to have a kidney infection at Chicago, Ill.) was then spread around the cannula, autopsy was also rejected from the experiment. The completely over the hole in the cranium and around gonosomatic index of the 28 fish remaining in the exthe screw heads. Once the cement dried, the cannula was firmly anchored in position, and the fish was re- periment was 15.60 (SD = 3.37). moved from the stereotaxic apparatus. A cap made of Cnnnrrln preparation. The cannula consisted basia piece of PE 10 tubing (Intramedic polyethylene tub- cally of an 11-13-mm length of stainless-steel 30ing, Clay Adams, Inc., Parsippany, N. J.) sealed at gauge tubing, with a dead space of about 0.2 ~1. As one end was placed over the exposed tip of the can- an anchoring device a piece of stainless steel 22-gauge nula and the fish was returned to the aquarium. hypodermic tubing about 3-4 mm in length was fitted At 3, 5, and 7 days after implantation of the can- over the 3(rgauge tubing, and glued to about the midnula the fish were each caught by hand and placed in dle of it with cu-cyanoacrylate (Krazy Glue, Krazy anesthetic solution (tricaine methanesulfonate, 1: 1000 Glue, Inc., Chicago, Ill.). Stainless-steel wire (0.2dilution). After nearly or just ceasing opercular mm diameter) was then wrapped around the piece of movements in the anesthetic solution, each fish was 22-gauge tubing from one end to the other so that it removed to lie on a dampened towel, and the cap on just overlapped onto the 30-gauge tubing. The ends of the cannula was removed. A total of 1.2 ~1 of PS, or the wire were twisted together, cut to about 3-mm the combination of 1 ~1 of PG solution, followed by length, and left projecting perpendicular to the lon0.2 ~1 of PS to flush the PG solution from the can- gitudinal axis of the tubing.
PROSTAGLANDINS
Experiment
AND
GONADOTROPIN
IN
GOLDFISH
453
2
A total of 28 sexually mature female goldfish weighing 53.12 g (SD = 9.67) were selected for the experiment. The fish were acclimated to the same conditions as in Experiment 1. The fish were divided into four groups. Each group was identified by a fin clip. The fish in each group were anesthetized together, and injected intraperitoneally with either 1 ~1 PS, or 1 ,ul PS containing either 2 ,ug PGE,, PGEZ, or PGF,,. As in Experiment 1, blood was sampled from the caudai vasculature at 30-min postinjection. The blood samples were treated as in Experiment 1.
Gonadotropin Radioimmunoassay The samples from Experiments 1 and 2 were assayed together in duplicate according to the technique of Crim et al. (1976). Briefly, the radioimmunoassay procedure involves the competition between the goldfish GTH antigen, and 1311-1abeled carp GTH for binding sites on rabbit antibody to carp GTH. A standard curve was developed by competition of the labeled hormone with purified carp GTH. Precipitation of the antibody-bound carp or goldfish GTH was by a second antibody system of normal rabbit serum and goat anti-rabbit y-globulin. Parallelism between dilutions of goldfish serum and goldfish crude pituitary extract and carp GTH standards indicates suitability of the assay. An assay system similar to this has been developed previously and applied to goldfish by Breton et al. (1972a). The Student’s t test was used to test significance of the results.
RESULTS
Figure 1 presents the results from Experiment 1. There were no significant differences in serum GTH concentrations in run 1 or in run 2 in which the effects of third ventricle injection of 0.5 or 1.0 pg, respectively, of PGEl, PGEz, and PGF,, were tested. In run 3 in which 2.0-pg doses of PG were tested, there was a significant decrease (P < 0.025) in serum GTH compared to the PS-treated group following PGE, or PGF,, injection. The 2.0~pg dose of PGEl had no significant effect on serum GTH. Although not indicated in Fig. 1, the serum GTH concentration of the PS group was significantly higher in run 3 compared to run 1 (P c 0.05). The serum GTH for the PS group in run 2 was intermediate between the values found in runs 1 and 3.
FIG. 1. The effects of third ventricle injection of PGE,, PGE,, and PGF,, on serum gonadotropin concentrations of female goldfish at 30 min postinjection. The dosages used in runs 1, 2, and 3 were, respectively, 0.5, 1.0, and 2.0 fig. The values are means 2 SE. **, Significant at P < 0.025.
Experiment 2. Figure 2 summarizes the results of Experiment 2. There were no significant effects due to the intraperitoneal injection of 2.Opg of PGE1, PGEz, or PGF*, on serum GTH concentrations. However, again PGE, tended to cause an increase in serum GTH concentration. DISCUSSION
The results indicate that there is an effect by PGE2 or PGF,, injected into the third ventricle of goldfish to suppress serum GTH concentrations. The results cannot be attributed to some nonspecific effect, such
454
PETER AND BILLARD
PS
2.opJ =I
2.99
2QfiQ
PGE2
PW2
*
FIG. 2. The effects of intraperitoneal injection of 2.0 pg of PGE,, PGE,, and PGF,, on serum gonadotropin concentrations of female goldfish at 30 min postinjection. The values are means -C SE.
as a mechanical or an osmotic disturbance at the injection site, since the same dosage of PGE, (2.0 ,ug) injected into the third ventricle had no effects on serum GTH. Also, injections of 0.5 or 1.0 pg of PGEl, PGEz, or PGF,, were without significant effect. Furthermore, this effect cannot be attributed to a systemic action of PGE, and PGFza , since serum GTH concentrations were not significantly changed by intraperitoneal injection of a 2.0~pg dosage. Thus, the observed changes in serum GTH concentrations must have been the result of a change in the secretion rate of GTH by the pituitary gland. The above results are in contrast to findings in which plasma LH levels are increased by injection of PGE, into the third ventricle of the female rat (Harms et al., 1973, 1974; Ojeda et al., 1974) or the lateral cerebral ventricle of the male rat (Eskay et al., 1975). However, the nonsignificant increases in serum GTH concentration in the goldfish treated intraventricularly with low doses (0.5 and 1.0 pg) or intraperitoneally with a high dose (2.0 pg) of PGE, may be suggestive of a stimulatory effect. Since these experiments were done on sexually mature females, perhaps a clear stimulatory effect may occur with fish at a different stage of sexual maturity or under different temperature conditions. Higher temperatures could not be used in the present experiments since the fish would ovulate under such conditions. In further contrast
with the present results, PGE, injected into the third ventricle has also been found to increase plasma LH concentrations in the female rat (Spies and Norman, 1973; Ojeda et al., 1974) and the male rat (Ojeda et al., 1974), whereas no effects of PGE, were noted in the goldfish. Also, high doses of PGF,, administered systemically are effective in increasing plasma LH in female rats (Batta et al., 1974; Taya et al., 1975). In the present study, PGFza was found to suppress GTH secretion. These contrasting results suggest that some major differences probably exist between the mechanisms controlling GTH secretion in teleosts and mammals. Since only one postinjection sampling time (30 min) was used in the present study, the time course of the suppression of serum GTH concentration remains to be investigated. In the rat the plasma LH concentration is elevated at 15, 30, and 60 min following PGEz injection into the third ventricle (Harms et al., 1973, 1974; Ojeda et ul., 1974). In the carp, plasma GTH is elevated from about 2 min for up to 25 min foilowing a single intravenous injection of hypothalamic extract or synthetic LH-RH (Breton and Weil, 1973). In the goldfish, serum GTH concentration is increased at 20 min following injection of hypothalamic extract or synthetic LH-RH into the third ventricle of fish held at 12-14” (Crim et al., 1976). Thus, the 30-min sampling time used in the present study should be well within the range of the response time for a shortterm increase or decrease in GTH secretion, considering that the fish were held at 12-13” during the experiments. The significant increase in serum GTH of the PS-injected group for run 3 compared to run 1 in Experiment 1 cannot be fully explained. As the serum GTH value for the PS group in run 2 was intermediate between the values from runs 1 and 3, and, since each group in each run of Experiment 1 was randomly selected from all the cannulated fish, the increase in serum GTH was gradual for all the fish during the course of
PROSTAGLANDINS
AND
GONADOTROPIN
the experiment. An explanation for the gradual increase is that it may reflect the cumulative effects of the stress of repeated handling and blood sampling of the fish. However, an effect Of StreSS Oil gonadotropin secretion in fish has not previously been reported. Even though this gradual increase occurred it is not likely that it influenced the responsiveness of the fish to PG, since PGF,, tended to suppress gonadotropin levels at the lowest dosage (see Fig. 1) and in a number of the animals (N = 5) in the intermediate dose group. The group average for the intermediate dose of PGF,, (1 .O ;ELg) was similar to the PS-injected group because three animals, one in particular, With relatively higher GTH values offset the animals with the lower values. The mechanism for sunnression of GTH secretion in goldfish by intraventricularly injected PGE, and PGF,, is unknown. Since there is a releasing factor for GTH in the teleost hypothalamus (Breton et al., 1971, 1972b, 1975; Breton and Weil, 1973; Crim et al., 1975; for review of earlier work see Peter, 1973) the action of PGE, and PGF,, may be on the secretion of the releasing factor, similar to the situation in the rat, although in the rat the effect is stimulatory (see introductory section). The inhibition of releasing factor secretion, or some other action by PGE, and PGF,, to suppress GTH secretion in the goldfish, may be a component of a similar mechanism, postulated by Dodd (1975), for the inhibition of GTH secretion and the prevention of premature ovulation in the female dogfish, until the appropriate conditions are available for egg laying. On the other hand, although the site of action of PGE, and PGFzo, was assumed to be the hypothalamus, diffusion to some brain site outside the hypothalamus, or to the pituitary, cannot be eliminated in the present study. ACKNOWLEDGMENTS We wish to thank Dr. E. Burzawa-Gerard for the gift of purified carp gonadotropin and Dr. L. Crim for the antibody to the carp gonadotropin used in the
IN
455
GOLDFISH
radioimmunoassay procedure. The prostaglandins used in the study were a gift from Dr. J. E. Pike, Upjohn Company. We wish to thank Miss Janet Bystrom for technical assistance. The visit by Dr. R. Billard to the University of Alberta was made possible
by an exchangefellowship between the National Research Council of Canada and Centre National de la Recherche Scientifique of France.
REFERENCES Batta, S. K., Zanisi, M., and Martini, L. (1974). Prostaglandins and gonadotropin secretion. Neuroendocrinology
14, 224-232.
Breton, B., Billard, R., Jalabert, B., and Kann, G. (1972a). Dosage radioimmunologique des gonadotropines plasmatiques chez Carassius auratus, au tours du nycthembre et pendant l’ovulation. Gen.
Comp.
Endocrinol.
18, 463-468.
Breton, B., Jalabert, B., Billard, R., and Weil, c. (1971). Stimulation in vitro de la liberation d’hormone gonadotrope hypophysaire par un facteur hypothalamique chez la carpe Cyprinus carpio L. C.R. Acad. Sci. Ser. D 273, 2591-2594. Breton, B., Jalabert, B., and Weil, C. (1975). Caracterisation partielle d’un facteur hypothalamique de liberation des hormones gonadotropes chez la carpe (Cyprinus carpio L.) etude in vitro. Gen. Comp. Endocrinol. 25, 405-415. Breton, B., and Weil, C. (1973). Effets du LWFSHRH synthetique et d’extraits hypothalamiques de carpe sur la secretion d’hormone gonadotrope in vivo chez la carpe (Cyprinus carpio L.). C.R. Acad.
Sci.
Ser. D 277, 2061-2064.
Breton, B., Weil, C., Jalabert, B., and Billard, R. (1972b). Activite reciproque des facteurs hypothalamiques de belier (Oris aries) et de poissons teleosteens sur la secretion in vitro des hormones c-HG et LH respectivement par les hypophyses de carpe et de belier. C.R. Acad. Sci. Ser. D 274, 2530-2533. Chobsieng, P., Naor, Z., Kock, Y., Zor, U., and Linder, H. R. (1975). Stimulatory effect of prostaglandin E, on LH release in the rat: Evidence for hypothalamic site of action. Neuroendocrinology
17, 12-17.
Crim, L. W., and Cluett, D. M. (1974). Elevation of plasma gonadotropin concentration in response to mammalian gonadotropin releasing hormone (GRH) treatment of the male brown trout as determined by radioimmunoassay. Endocr. Res. Commun. 1, 101-110. Crim, L. W., Peter, R. E., and Billard, R. (1976). Stimulation of gonadotropin secretion by intraventricular injection of hypothalamic extracts in the goldfish, Carassius aurarus. Gen. Comp. Endocrinol. (in press). Dodd, J. M. (1975). The hormones of sex and repro-
456
PETER
AND
duction and their effects in fish and lower chordates: Twenty years on. Amer. 2001. 15, Suppl. 1, 137-171. Eskay, R. L., Warberg, J., Mical. R. S., and Porter, J. C. (1975). Prostaglandin E,-induced release of LHRH into hypophysial portal blood. Endocrinology 97, 816-824. Harms, P. G., Ojeda, S. R., and McCann, S. M. involvement in (1973). Prostaglandin hypothalamic control of gonadotropin and prolactin release. Science 181, 760-761. Harms, P. G., Ojeda, S. R., and McCann, S. M. (1974). Prostaglandin-induced release of pituitary gonadotropins: Central nervous system and pituitary sites of action. Endocrinology 94, 14591464. Labhsetwar, A. P., and Zolovick, A. (1973). Hypothalamic interaction between prostaglandins and catecholamines in promoting gonadotropin secretion for ovulation. Nature New Eiol. 246, 55-56. Makino, T. (1973). Study of the intracellular mechanism of LH release in the anterior pituitary. Amer.
J. Obsret.
Gynecol.
115, 606-614.
Ojeda, S. R., Harms, P. G., and McCann, S. M. (1974). Effect of third ventricular injections of prostaglandins (PG’s) on gonadotropin release in conscious free moving male rats. Prostaglundins 8, 545-552. Ojeda, S. R., Wheaton, J. E., and McCann, S. M.
BILLARD
(1975). Prostaglandin E,-induced release of luteinizing hormone-releasing factor (LRF). Neuroendocrinology
17, 283-287.
Peter, R. E. (1970). Hypothalamic control of thyroid gland activity and gonadal activity in the goldfish, Carassius
auratus.
Gen.
Comp.
Endocrinol.
334-356. Peter, R. E. (1973). Neuroendocrinology Amer.
Zool.
14,
of teleosts.
13, 743-755.
Peter, R. E., and Gill, V. E. (1975). A stereotaxic atlas and technique for forebrain nuclei of the goldfish, Carassius
auratus.
J. Comp.
Neural.
159,69-102.
Ratner, A., Wilson, M. C., Srivastava, L., and Peake, G. T. (1974). Stimulatory effects of prostaglandin E, on rat anterior pituitary cyclic AMP and luteinizing hormone release. Prostaglandins 5, 165-171. Sato, T., Hirono, M.. Jyiyo, T., Iesaka, T., Taya, K., and Igarashi, M. (1975). Direct action of prostaglandins on the rat pituitary. Endocrinology 96, 45-49. Spies, H. G., and Norman, R. L. (1973). Luteinizing hormone release and ovulation induced by the intraventricular infusion of prostaglandin E, into pentobarbital blocked rats. Prostaghndins 4, 131-141. Taya, K., Sato, T., and Igarashi, M. (1975). Effect of prostaglandin F,, upon ovulation and LH, FSH and prolactin secretion in chlorpromazine blocked rats. Endocrinol. Japan. 22, 131-136.
AND
COMPARATIVE
Effects
ENDOCRINOLOGY
30, 451-456 (1976)
of Third Ventricle Injection of Prostaglandins Gonadotropin Secretion in Goldfish, Carassius auratd
on
R. E. PETER AND R. BILLARD~ Department
of
Zoology. University of Alberta, Edmonton, Alberta T6G 2E9, Canada Accepted July 26, 1976
The effects of injection of prostaglandin (PG) El, PGEz, and PGF,, into the third ventricle on serum gonadotropin (GTH) concentrations in the goldfish were tested. Blood samples were taken at 30 min postinjection for radioimmunoassay of serum GTH. PG dosages of 0.5 and 1.0 pg were ineffective. However, PGE, and PGF, at the 2.0~pg dosage significantly suppressed serum GTH. PGE, at a 2.0~pg dosage had no effect. There were no effects on serum GTH when 2.0 pg of PGE,, PGE,, or PGF,, were injected intraperitoneally. The results indicate that PGE, and PGF,, suppress gonadotropin secretion by some action, presumably on the hypothalamus. However, action of PGE, and PGF,, by diffusion from the site of iniection to some other brain site or the pituitary cannot be eliminated in the present study.-
A number of studies have shown that some prostaglandins (PG) of the E series can influence gonadotropin secretion, particularly luteinizing hormone (LH), in the laboratory rat by an effect directly on the hypothalamus (Harms et al., 1973, 1974; Labhsetwar and Zolovick, 1973; Spies and Norman, 1973). Of the various PG’s tested, the hypothalamic site of action seems to be most responsive to PGE, (Harms et al., 1973, 1974; Ojeda et al., 1974). Studies by Chobsienget al. (1975), Eskayet al. (1975), and Ojeda et al. (1975) indicate that PGE, acts to stimulate LH-releasing hormone (LH-RH) secretion. However, a direct pituitary site of action in stimulation of gonadotropin secretion by PG has also been shown both in vitro (Sato et al., 1975; Makino, 1973; Ratner et al., 1974) and in viva (Sato et al., 1975; Harms et al., 1974). However, on the basis of required dosages, the pitui-
tary site of action is less responsive than the hypothalamic site. In teleosts there is no evidence available indicating an influence of PG on gonadotropin (GTH) secretion. However, a gonadotropin releasing factor has been shown to be present in hypothalamic extracts of carp (Breton et al., 1971, 1972b, 1975; Breton and Weil, 1973), goldfish (Crim et al., 1976), and rainbow trout (Breton et al., 1972b). In addition, synthetic LH-RH stimulates GTH secretion in carp (Breton and Weil, 1973), brown trout (Crim and Cluett, 1974), and goldfish (Crim et al., 1976). In the present work the effects on GTH secretion of PGE1, PGE2, and PGFzu injected into the third ventricle of goldfish were tested. Sexually mature female goldfish were used in the experiments to test whether the expected stimulation of GTH secretion would induce ovulation.
’ Supported by National Research Council of Canada Grant A6371 to R.E.P. ’ Permanent address: Laboratoire de Physiologie des Poissons, Institut National de la Recherche Agronomique, 78350 Jouy-en-Josas, France.
Goldfish, common or comet variety, used in the study were purchased in April 1975 from Grassyfork Fisheries, Co., Inc., Martinsville, Ind. Prior to use in
451 Copyright All rights
@ 1976 by Academic Press. Inc. of reproduction in any form reserved.
MATERIALS
AND METHODS
452
PETER
AND
experiments the fish were held for about 120 days on a simulated natural photoperiod at 10-14” in 1500-liter flow-through aquaria. Just before the experiments commenced the photoperiod was about 16 hr light-8 hr dark. For the experiments the fish were transferred to 96-liter flow-through aquaria regulated to 12-13” and held at a 16-hr light-8-hr dark photoperiod. The fish were fed Purina trout chow (Ralston Purina Co., St. Louis, MO.) ad /i&rum throughout the preexperimental and the experimental periods.
BILLARD
nula, was then injected into the cannula. Injections were done with a 5- or lo-~1 Hamilton syringe fitted with a short piece of PE 20 tubing on the needle. Following the injection, the cannula was recapped and the fish was returned to the aquarium. At 30 min following the injection a blood sample of about 0.2 to 0.3 @I was taken from the caudal vasculature. The fish were anesthetized prior to the blood sampling. The injections and sampling were done between 5 and 6.5 hr after the lights came on in each case. The blood samples were stored over chipped ice for about 2 hr Experiment 1 prior to centrifugation at 2500 rpm for 30 min. Serum was pipetted into micro test tubes and quick-frozen A total of 37 females, sexually mature, and weighover dry ice. The serum samples were stored at -20” ing 62.52 g (SD = 8.68) were selected for the experiment. Each fish was identified by colored threads at- until analyzed. The PG injections done on Day 3 (run 1) after cantached to the fms and a numbered tag attached to the left operculum (size 1 Monel tag, National Band and nulation were 0.5 pg of either PGE,, PGE,, or PGF,, in 1 ~1 of PS. The injections on Day 5 (run 2) and Day Tag Co., Newport, KY.). A cannula was implanted 7 (run 3) were, respectively, 1 and 2 pg of PGE,, into the third ventricle of each fish by the following procedure: The fish were prepared for stereotaxic im- PGE,, and PGF*, in 1 ~1 of PS. PGE, and PGE, soplantation following the technique of Peter (1970) as lutions were made fresh on the day of use. PGF,, modified by Peter and Gill (1975), except that the solutions were in some cases stored overnight at 4” parietal bone flap was completely removed. Before before use. The PGF,, used was in the form of the mounting the fish in the sterotaxic apparatus, one or tromethamine salt. For each run the fish in each group were randomly selected from among all those sometimes two optical screws, 3 mm in length, were cannulated. anchored in bone on each side lateral to the region At 9 days postcannulation the fish were again anesfrom which the parietal bone flap had been removed. thetized, and a small volume of methylene blue dye The cannula (described below) was then implanted into the third ventricle, dorsal to the anterior part of was injected through the cannula. The fish were then the nucleus lateral tuberis pars posterioris using the autopsied and the accuracy of placement of the cantechnique of Peter (1970) as modified by Peter and nula checked. Fish in which the cannula tip had been Gill (1975) (coordinates used + 0.7, M, D 3.0). The placed out through the ventral side of the brain (N = 4) were rejected from the experiment. The ovary was space in the cranium above the brain was filled with dissected out and weighed, and its condition was freshly made freshwater fish physiological solution noted. Fish that ovulated during the experiment (N = (PS). Following this, a scale was taken from posterior to the opercular region, and a groove cut in it. The 2) or were postovulatory (N = 1) or immature (N = 1) scale was then fitted around the cannula so as to were rejected from the experiment. The two fish that cover the hole in the cranium. Dental cement (Jet ovulated during the experiment were in either the PS or PGF,, groups during the three runs of the experiacrylic, type 2, class 1, Lang Dental Mfg. Co., Inc., ment. One fish, found to have a kidney infection at Chicago, Ill.) was then spread around the cannula, autopsy was also rejected from the experiment. The completely over the hole in the cranium and around gonosomatic index of the 28 fish remaining in the exthe screw heads. Once the cement dried, the cannula was firmly anchored in position, and the fish was re- periment was 15.60 (SD = 3.37). moved from the stereotaxic apparatus. A cap made of Cnnnrrln preparation. The cannula consisted basia piece of PE 10 tubing (Intramedic polyethylene tub- cally of an 11-13-mm length of stainless-steel 30ing, Clay Adams, Inc., Parsippany, N. J.) sealed at gauge tubing, with a dead space of about 0.2 ~1. As one end was placed over the exposed tip of the can- an anchoring device a piece of stainless steel 22-gauge nula and the fish was returned to the aquarium. hypodermic tubing about 3-4 mm in length was fitted At 3, 5, and 7 days after implantation of the can- over the 3(rgauge tubing, and glued to about the midnula the fish were each caught by hand and placed in dle of it with cu-cyanoacrylate (Krazy Glue, Krazy anesthetic solution (tricaine methanesulfonate, 1: 1000 Glue, Inc., Chicago, Ill.). Stainless-steel wire (0.2dilution). After nearly or just ceasing opercular mm diameter) was then wrapped around the piece of movements in the anesthetic solution, each fish was 22-gauge tubing from one end to the other so that it removed to lie on a dampened towel, and the cap on just overlapped onto the 30-gauge tubing. The ends of the cannula was removed. A total of 1.2 ~1 of PS, or the wire were twisted together, cut to about 3-mm the combination of 1 ~1 of PG solution, followed by length, and left projecting perpendicular to the lon0.2 ~1 of PS to flush the PG solution from the can- gitudinal axis of the tubing.
PROSTAGLANDINS
Experiment
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GONADOTROPIN
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GOLDFISH
453
2
A total of 28 sexually mature female goldfish weighing 53.12 g (SD = 9.67) were selected for the experiment. The fish were acclimated to the same conditions as in Experiment 1. The fish were divided into four groups. Each group was identified by a fin clip. The fish in each group were anesthetized together, and injected intraperitoneally with either 1 ~1 PS, or 1 ,ul PS containing either 2 ,ug PGE,, PGEZ, or PGF,,. As in Experiment 1, blood was sampled from the caudai vasculature at 30-min postinjection. The blood samples were treated as in Experiment 1.
Gonadotropin Radioimmunoassay The samples from Experiments 1 and 2 were assayed together in duplicate according to the technique of Crim et al. (1976). Briefly, the radioimmunoassay procedure involves the competition between the goldfish GTH antigen, and 1311-1abeled carp GTH for binding sites on rabbit antibody to carp GTH. A standard curve was developed by competition of the labeled hormone with purified carp GTH. Precipitation of the antibody-bound carp or goldfish GTH was by a second antibody system of normal rabbit serum and goat anti-rabbit y-globulin. Parallelism between dilutions of goldfish serum and goldfish crude pituitary extract and carp GTH standards indicates suitability of the assay. An assay system similar to this has been developed previously and applied to goldfish by Breton et al. (1972a). The Student’s t test was used to test significance of the results.
RESULTS
Figure 1 presents the results from Experiment 1. There were no significant differences in serum GTH concentrations in run 1 or in run 2 in which the effects of third ventricle injection of 0.5 or 1.0 pg, respectively, of PGEl, PGEz, and PGF,, were tested. In run 3 in which 2.0-pg doses of PG were tested, there was a significant decrease (P < 0.025) in serum GTH compared to the PS-treated group following PGE, or PGF,, injection. The 2.0~pg dose of PGEl had no significant effect on serum GTH. Although not indicated in Fig. 1, the serum GTH concentration of the PS group was significantly higher in run 3 compared to run 1 (P c 0.05). The serum GTH for the PS group in run 2 was intermediate between the values found in runs 1 and 3.
FIG. 1. The effects of third ventricle injection of PGE,, PGE,, and PGF,, on serum gonadotropin concentrations of female goldfish at 30 min postinjection. The dosages used in runs 1, 2, and 3 were, respectively, 0.5, 1.0, and 2.0 fig. The values are means 2 SE. **, Significant at P < 0.025.
Experiment 2. Figure 2 summarizes the results of Experiment 2. There were no significant effects due to the intraperitoneal injection of 2.Opg of PGE1, PGEz, or PGF*, on serum GTH concentrations. However, again PGE, tended to cause an increase in serum GTH concentration. DISCUSSION
The results indicate that there is an effect by PGE2 or PGF,, injected into the third ventricle of goldfish to suppress serum GTH concentrations. The results cannot be attributed to some nonspecific effect, such
454
PETER AND BILLARD
PS
2.opJ =I
2.99
2QfiQ
PGE2
PW2
*
FIG. 2. The effects of intraperitoneal injection of 2.0 pg of PGE,, PGE,, and PGF,, on serum gonadotropin concentrations of female goldfish at 30 min postinjection. The values are means -C SE.
as a mechanical or an osmotic disturbance at the injection site, since the same dosage of PGE, (2.0 ,ug) injected into the third ventricle had no effects on serum GTH. Also, injections of 0.5 or 1.0 pg of PGEl, PGEz, or PGF,, were without significant effect. Furthermore, this effect cannot be attributed to a systemic action of PGE, and PGFza , since serum GTH concentrations were not significantly changed by intraperitoneal injection of a 2.0~pg dosage. Thus, the observed changes in serum GTH concentrations must have been the result of a change in the secretion rate of GTH by the pituitary gland. The above results are in contrast to findings in which plasma LH levels are increased by injection of PGE, into the third ventricle of the female rat (Harms et al., 1973, 1974; Ojeda et al., 1974) or the lateral cerebral ventricle of the male rat (Eskay et al., 1975). However, the nonsignificant increases in serum GTH concentration in the goldfish treated intraventricularly with low doses (0.5 and 1.0 pg) or intraperitoneally with a high dose (2.0 pg) of PGE, may be suggestive of a stimulatory effect. Since these experiments were done on sexually mature females, perhaps a clear stimulatory effect may occur with fish at a different stage of sexual maturity or under different temperature conditions. Higher temperatures could not be used in the present experiments since the fish would ovulate under such conditions. In further contrast
with the present results, PGE, injected into the third ventricle has also been found to increase plasma LH concentrations in the female rat (Spies and Norman, 1973; Ojeda et al., 1974) and the male rat (Ojeda et al., 1974), whereas no effects of PGE, were noted in the goldfish. Also, high doses of PGF,, administered systemically are effective in increasing plasma LH in female rats (Batta et al., 1974; Taya et al., 1975). In the present study, PGFza was found to suppress GTH secretion. These contrasting results suggest that some major differences probably exist between the mechanisms controlling GTH secretion in teleosts and mammals. Since only one postinjection sampling time (30 min) was used in the present study, the time course of the suppression of serum GTH concentration remains to be investigated. In the rat the plasma LH concentration is elevated at 15, 30, and 60 min following PGEz injection into the third ventricle (Harms et al., 1973, 1974; Ojeda et ul., 1974). In the carp, plasma GTH is elevated from about 2 min for up to 25 min foilowing a single intravenous injection of hypothalamic extract or synthetic LH-RH (Breton and Weil, 1973). In the goldfish, serum GTH concentration is increased at 20 min following injection of hypothalamic extract or synthetic LH-RH into the third ventricle of fish held at 12-14” (Crim et al., 1976). Thus, the 30-min sampling time used in the present study should be well within the range of the response time for a shortterm increase or decrease in GTH secretion, considering that the fish were held at 12-13” during the experiments. The significant increase in serum GTH of the PS-injected group for run 3 compared to run 1 in Experiment 1 cannot be fully explained. As the serum GTH value for the PS group in run 2 was intermediate between the values from runs 1 and 3, and, since each group in each run of Experiment 1 was randomly selected from all the cannulated fish, the increase in serum GTH was gradual for all the fish during the course of
PROSTAGLANDINS
AND
GONADOTROPIN
the experiment. An explanation for the gradual increase is that it may reflect the cumulative effects of the stress of repeated handling and blood sampling of the fish. However, an effect Of StreSS Oil gonadotropin secretion in fish has not previously been reported. Even though this gradual increase occurred it is not likely that it influenced the responsiveness of the fish to PG, since PGF,, tended to suppress gonadotropin levels at the lowest dosage (see Fig. 1) and in a number of the animals (N = 5) in the intermediate dose group. The group average for the intermediate dose of PGF,, (1 .O ;ELg) was similar to the PS-injected group because three animals, one in particular, With relatively higher GTH values offset the animals with the lower values. The mechanism for sunnression of GTH secretion in goldfish by intraventricularly injected PGE, and PGF,, is unknown. Since there is a releasing factor for GTH in the teleost hypothalamus (Breton et al., 1971, 1972b, 1975; Breton and Weil, 1973; Crim et al., 1975; for review of earlier work see Peter, 1973) the action of PGE, and PGF,, may be on the secretion of the releasing factor, similar to the situation in the rat, although in the rat the effect is stimulatory (see introductory section). The inhibition of releasing factor secretion, or some other action by PGE, and PGF,, to suppress GTH secretion in the goldfish, may be a component of a similar mechanism, postulated by Dodd (1975), for the inhibition of GTH secretion and the prevention of premature ovulation in the female dogfish, until the appropriate conditions are available for egg laying. On the other hand, although the site of action of PGE, and PGFzo, was assumed to be the hypothalamus, diffusion to some brain site outside the hypothalamus, or to the pituitary, cannot be eliminated in the present study. ACKNOWLEDGMENTS We wish to thank Dr. E. Burzawa-Gerard for the gift of purified carp gonadotropin and Dr. L. Crim for the antibody to the carp gonadotropin used in the
IN
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radioimmunoassay procedure. The prostaglandins used in the study were a gift from Dr. J. E. Pike, Upjohn Company. We wish to thank Miss Janet Bystrom for technical assistance. The visit by Dr. R. Billard to the University of Alberta was made possible
by an exchangefellowship between the National Research Council of Canada and Centre National de la Recherche Scientifique of France.
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14, 224-232.
Breton, B., Billard, R., Jalabert, B., and Kann, G. (1972a). Dosage radioimmunologique des gonadotropines plasmatiques chez Carassius auratus, au tours du nycthembre et pendant l’ovulation. Gen.
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Breton, B., Jalabert, B., Billard, R., and Weil, c. (1971). Stimulation in vitro de la liberation d’hormone gonadotrope hypophysaire par un facteur hypothalamique chez la carpe Cyprinus carpio L. C.R. Acad. Sci. Ser. D 273, 2591-2594. Breton, B., Jalabert, B., and Weil, C. (1975). Caracterisation partielle d’un facteur hypothalamique de liberation des hormones gonadotropes chez la carpe (Cyprinus carpio L.) etude in vitro. Gen. Comp. Endocrinol. 25, 405-415. Breton, B., and Weil, C. (1973). Effets du LWFSHRH synthetique et d’extraits hypothalamiques de carpe sur la secretion d’hormone gonadotrope in vivo chez la carpe (Cyprinus carpio L.). C.R. Acad.
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Breton, B., Weil, C., Jalabert, B., and Billard, R. (1972b). Activite reciproque des facteurs hypothalamiques de belier (Oris aries) et de poissons teleosteens sur la secretion in vitro des hormones c-HG et LH respectivement par les hypophyses de carpe et de belier. C.R. Acad. Sci. Ser. D 274, 2530-2533. Chobsieng, P., Naor, Z., Kock, Y., Zor, U., and Linder, H. R. (1975). Stimulatory effect of prostaglandin E, on LH release in the rat: Evidence for hypothalamic site of action. Neuroendocrinology
17, 12-17.
Crim, L. W., and Cluett, D. M. (1974). Elevation of plasma gonadotropin concentration in response to mammalian gonadotropin releasing hormone (GRH) treatment of the male brown trout as determined by radioimmunoassay. Endocr. Res. Commun. 1, 101-110. Crim, L. W., Peter, R. E., and Billard, R. (1976). Stimulation of gonadotropin secretion by intraventricular injection of hypothalamic extracts in the goldfish, Carassius aurarus. Gen. Comp. Endocrinol. (in press). Dodd, J. M. (1975). The hormones of sex and repro-
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duction and their effects in fish and lower chordates: Twenty years on. Amer. 2001. 15, Suppl. 1, 137-171. Eskay, R. L., Warberg, J., Mical. R. S., and Porter, J. C. (1975). Prostaglandin E,-induced release of LHRH into hypophysial portal blood. Endocrinology 97, 816-824. Harms, P. G., Ojeda, S. R., and McCann, S. M. involvement in (1973). Prostaglandin hypothalamic control of gonadotropin and prolactin release. Science 181, 760-761. Harms, P. G., Ojeda, S. R., and McCann, S. M. (1974). Prostaglandin-induced release of pituitary gonadotropins: Central nervous system and pituitary sites of action. Endocrinology 94, 14591464. Labhsetwar, A. P., and Zolovick, A. (1973). Hypothalamic interaction between prostaglandins and catecholamines in promoting gonadotropin secretion for ovulation. Nature New Eiol. 246, 55-56. Makino, T. (1973). Study of the intracellular mechanism of LH release in the anterior pituitary. Amer.
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Ojeda, S. R., Harms, P. G., and McCann, S. M. (1974). Effect of third ventricular injections of prostaglandins (PG’s) on gonadotropin release in conscious free moving male rats. Prostaglundins 8, 545-552. Ojeda, S. R., Wheaton, J. E., and McCann, S. M.
BILLARD
(1975). Prostaglandin E,-induced release of luteinizing hormone-releasing factor (LRF). Neuroendocrinology
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Peter, R. E. (1970). Hypothalamic control of thyroid gland activity and gonadal activity in the goldfish, Carassius
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Peter, R. E., and Gill, V. E. (1975). A stereotaxic atlas and technique for forebrain nuclei of the goldfish, Carassius
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Ratner, A., Wilson, M. C., Srivastava, L., and Peake, G. T. (1974). Stimulatory effects of prostaglandin E, on rat anterior pituitary cyclic AMP and luteinizing hormone release. Prostaglandins 5, 165-171. Sato, T., Hirono, M.. Jyiyo, T., Iesaka, T., Taya, K., and Igarashi, M. (1975). Direct action of prostaglandins on the rat pituitary. Endocrinology 96, 45-49. Spies, H. G., and Norman, R. L. (1973). Luteinizing hormone release and ovulation induced by the intraventricular infusion of prostaglandin E, into pentobarbital blocked rats. Prostaghndins 4, 131-141. Taya, K., Sato, T., and Igarashi, M. (1975). Effect of prostaglandin F,, upon ovulation and LH, FSH and prolactin secretion in chlorpromazine blocked rats. Endocrinol. Japan. 22, 131-136.