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The effect of temperature on the clearance of intraperitoneally-injected gonadotropin in the goldfish Carassius auratus
Aquaculture, 19 (1980) 275-285 0 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
275
THE EFFECT OF TEMPERATURE ON THE CLEARANCE OF INTRAPERITONEALLY-INJECTED GONADOTROPIN IN THE GOLDFISH CARASSIUS AURATUS ALAN FRANK COOK and R.E. PETER Zoology Department,
University of Alberta, Edmonton,
Alta. T6G 2E9 (Canada)
(Accepted 9 September 1979)
ABSTRACT Cook, A.F. and Peter, R.E., 1980. The effect of temperature on the clearance of intraperitoneallp-injected gonadotropin in the goldfish, Carassius auratus. Aquacultore, 19: 275-285. The serum half-disappearance time (ts), metabolic clearance rate and volume of distribution of intraperitoneally administered carp gonadotropin (cGtH) and endogenous GtH levels were determined in sexually mature male and female goldfish, Carassius auratus maintained at 12 f 1°C or 20 2 1°C. The results indicated that the rate of serum uptake of the injected cGtH from the peritoneal cavity was greater at 20°C than at 12°C in sexually mature male goldfish. Although increased endogenous serum GtH levels and decreased values of serum t,,,, were associated with the elevated temperature, there was no difference in any of the parameters between sexually mature male and female goldfish acclimated to 12°C.
INTRODUCTION
The intraperitoneal (ip) administration of fish pituitary hormones (hypophysation) is used extensively to artifically spawn a number of commercially important teleost species (Pickford and Atz, 1957; Kuo and Nash, 1975; Chaudhuri, 1976). Although a number of studies have concerned the interrelationships between environmental factors and gonadotropin (GtH)-induced spawning (Hora, 1945; Bhowmick et al., 1977; Berniarz and Epler, 1977; Stacey et al., 1979), there are few describing the influence of temperature on the blood GtH levels following an ip injection, Crim and Evans (1976) used radioimmunoassay (RIA) to measure plasma GtH levels following a single ip injection of partially purified GtH in immature rainbow trout, Salmo gairdneri, maintained at three different temperatures. Their results indicated that the rate of clearance from the plasma decreased with decreasing ambient temperature and that detectable blood GtH levels could be found for up to 1 week following the ip injection of a large dose of GtH. The plasma GtH levels following the ip administration of carp pituitary extract (Jalabert et al., 1977) and highly purified salmon GtH
276
for the common carp, Cyprinus respectively. However these studies were not designed to study the effects of temperature on the plasma clearance of GtH. In the present study we report on the serum half-disappearance time (tlh), volume of distribution (Vi), and serum metabolic clearance rate (MCR), and GtH levels in sexually mature male goldfish, Carussius aura tus, maintained at 20 + 1°C and sexually mature male and female goldfish maintained at 12 + l”C, following a single ip injection of highly purified carp GtH (cGtH). (Jalabert
et al., 1978) have been determined
carpio, and coho salmon,
MATERIALS
Oncorhynchus
kisutch,
AND METHODS
Goldfish, Curussius uuratus, of the common or comet varieties were purchased from Grassyfork Fisheries Ltd., Martinsville, Indiana. All fish were maintained for a minimum of 28 days in 1500-l flow-through aquaria under a simulated natural photoperiod (Edmonton, Alberta, Canada) prior to experimental treatment. During this period the water temperature was maintained at 12 f 1°C. At random times during the daily photophase, the fish were fed a commercial trout chow (Ewos) ad libitum. In spite of the theoretical advantages of excluding the pituitary gland as a source of endogenous GtH production for assessing the disappearance characteristics of the exogenous cGtH by the single injection technique, this approach was rejected in view of pronounced hemodynamic (A.F. Cook, personal observation, 1978) and metabolic (Walker and Johansen, 1977) changes associated with goldfish hypophysectomy. A possible source of error with intact fish is the nonspecific component of the ip injection of cGtH; to obviate this possibility the effect of the injection per se was determined in the first experiment by including a number of control groups. After the initial acclimation period, sexually mature male fish were randomly divided into three groups, and reacclimated in 75-1 plastic aquaria maintained at 12 f 1°C under a light dark cycle consisting of 12 h light alternating with 12 h dark (lights on at 08.00 h). The fish were fed Tetramin at 09.00 h daily. Prior to injection or blood sampling the fish were anaesthetized by immersion in 0.05% tricaine methanesulfonate (Sigma, St. Louis, MO), until all visible movements ceased. After 10 days, all fish in group I were bled (pre-experiment sample) from the caudal vasculature between 14.00 and 15.00 h; the remaining fish were undisturbed at this time. The methods of anaesthesia, blood sampling and preparation of serum have been described previously Crim et al., 1976; Cook, 1979). After 7 days recovery, group I fish were given ip injections of 0.025 ,ug cGtH (highly purified glycoprotein cGtH, gift of B. Breton) per g body weight (BWt) (5 pg cGtH per ml teleost saline) with a 250 ~1 Hamilton syringe fitted with a disposable 27 guage X l/z” needle. This dose of cGtH is sufficient to cause a large increase in serum GtH levels (see below) and induce ovarian development (R.E. Peter, unpublished results, 1979), but is not large enough to induce ovulation (A.F. Cook and
277
R.E. Peter, unpublished results, 1979). Blood samples were taken at either 1, 3, 6,12,18, 24, 72 or 168 h post-injection. All samples were taken at the same time in the daily photophase as the presample (14.00-15.00 h), thus each fish in this group served as its own control. Vehicle-injected group II fish received an equivalent volume of teleost saline and were sampled identical to group I. The uninjected control group (group III) was sampled on the same schedule as groups I and II. Blood samples were assayed for GtH by RIA as described previously (Hontela and Peter, 1978,1979). No fish was sampled more than twice after injection or within a 48 h period. Following the last blood sample the fish were killed and the gonosomatic index (GSI) for each fish was calculated as follows: GSI = (gonadal weight/total BWt) X 100. Since there were no significant differences in the GtH values between the various control groups in this experiment (Fig.l), the values obtained from the self-control group (pre-experiment sample) were used as a measure of endogenous serum GtH levels in subsequent experiments. Two additional experiments were done, one using male fish at 20 + 1°C and the other using female fish at 12 f l”C, with the same protocol as described above, except that groups II and III were not included. To dissociate the disappearance pattern of the exogenous hormone from that of the total (endogenous and exogenous) GtH, the individual GtH values obtained from the pre-experimental sample were subtracted from the values obtained after the ip injection of cGtH. The values of exogenous serum GtH were then expressed as a percent of the injected dose per ml serum after standardization to a common BWt of 25 g. The disappearance of exogenous cGtH after the ip injection, expressed as a percent of the administered dose per ml serum, exhibited a linear disappearance pattern on a semi-logarithmic plot (Fig. 4). The disappearance profile of exogenous cGtH in the serum was expressed by the equation: x (t) = A ema* where the concentration of exogenous hormone at any time after the ip injection, x (t), is a function of the ordinate intercept A and the slope (Y. The values of these parameters and their estimates of error were determined using regression analysis of the log-transformed data as described by Normand and Fortier (1970). The serum &., MCR and Vi of exogenous cGtH were calculated as 0.693/a, a/A and l’/A (Tait and Burstein, 1964; Shipley and Clark, 1972). Statistical differences between groups were determined by analysis of variance and the Duncan multiple range test, or the Student’s t-test, where applicable (Steel and Torrie, 1960). RESULTS
The results of the experiment using sexually mature male goldfish (GSI = 3.41 f 0.09%) maintained at 12 * 1°C are shown in F&l. The serum GtH
278
50
c
I
-.----
s
-“\ . I i \ !
0
a
0.025 0
_.__-t_..-
uninjected
___*__-
self
tnlected cant
body
welghf
controls rols
Control
48
24
cGtH per 9
p.
vehicle-
72
y
168
HOURS
Fig.1. Serum gonadotropin levels in sexually mature male goldfish maintained at 12 + 1°C The ‘self-control’ group (open stars) was sampled 7 days prior to intraperitonal injection of 0.025 rcg highly purified carp gonadotropin (cGtH) per g body weight (solid squares) at the same time of day as the post-injection sampIe. The vehicle-injected control group (open squares) received 5 ~1 of teleost saline per g body weight. Values are mean t 1 SEM (N = 8 to 14 per sample).
levels did not differ significantly either within each of the control groups or between the control groups at any sample time (mean value 3.14 + 0.06 ng/ ml). Fish receiving the cGtH had significantly elevated (P < 0.01) serum GtH levels compared to the control values at the first sample after injection (1 h), and the maximum level (44.67 +- 3.72 ng/ml) was obtained 3 h postinjection. Serum GtH remained significantly greater than the control values until the 48 h post-injection sample; there were no significant differences in the serum GtH levels between the fish receiving cGtH and the self-control values for the remainder of the sampling period (Fig. 1). There were no differences in GSI values between groups (pooled GSI = 3.41 I 0.09%). Fig.2 illustrates the serum GtH levels in sexually mature male goldfish (GSI = 2.63 f 0.23%) maintained at 20 + 1°C. No significant differences in serum GtH levels were detected between sample times for the self-control values (mean value 11.63 + 1.02 ng/ml). Following injection of GtH, the maximum GtH level (67.91 f 3.12 ng/ml) was obtained at 1 h post-injection and declined until at 24 h post-injection there was no longer a significant difference from the presample values. The serum GtH profile for sexually mature female goldfish (GSI = 6.91 + 0.80%) maintained at 12 f 1°C (Fig. 3) is similar to that of mature male goldfish maintained at the same temperature (Fig. 1). The maximum serum
279
8
0
48
24
72
HOURS
Fig.2. Serum gonadotropin levels in sexually mature male goldfish maintained at 20 f. 1°C. The ‘self-control’ group (solid stars) was sampled 7 days prior to a single intraperitoneal injection of 0.025 pg cGtH per g body weight (solid squares). Values are mean * 1 SEM (N = 8).
i \
I
eil__!
i’4l/ ,--
_!-_.-.._._-.._i_.
-..------
,-----._--__+
I
A
I
0
8
24
48
72
”
s-
16%
HOURS
Fig.3. Serum gonadotropin levels in sexually mature female goldfish maintained at 12 + 1°C. Symbols are the same as described for Fig. 2. Values are mean f 1 SEM (N = 8).
280
GtH value (48.42 + 2.07 ng/ml) was not obtained until 3 h after the ip administration of cGtH. The GtH values of the cGtH-injected group remained significantly elevated over the self-control values until 48 h post-injection. As in the previous two experiments there were no significant differences in the self-control values throughout the sampling period (mean value 5.59 + 0.38 ng/ml). The disappearance of exogenous cGtH, expressed as a percent of the administered dose per ml serum, after a single ip injection in sexually mature male goldfish maintained at 20 + l”C, was characterized by a straight line on a semi-logarithmic plot (Fig. 4). The serum disappearance profiles of ip-administered cGtH, determined in sexually mature male and female gold-
0
4
6
12
16
20
24
HOURS
Fig.4. A semi-logarithmic plot of the metabolic clearance profile of immunoreactive GtH determined in sexually mature male goldfish maintained at 20 + 1°C. Each value is the mean (vertical bars are fr 1 SEM) of 8 to 14 samples obtained by subtracting the initial pre-injection control GtH value from the post-injection sample for each fish, and expressed as a percent of the injected dose after standardization to a common body weight of 25 g.
1
2.63 f 0.23c 3.41 f 0.09 6.91 i 0.30
Male Male Female
20 12 12
Temperature (“C)
11.63 * 1.02e 4.09 + 0.73 5.11 * 0.61
Serumb GtH (ng/mI)
I
,..
-
--
^
--
*a
I.
a 0.025 rg cGtH per g body weight. b Mean value determined from all the control serum GtH values. c AI1 data are mean f 1 SEM.
GSI (%)
Sex
9.83 f 1.12c 14.53 f 1.15 12.50 r 0.71 _
Distribution volume (ml)
6.03 ? 2.1gc 10.25 + 0.91 9.43 f 1.39
Serum half-disappearance time (h)
1.13 f O.lSC 0.98 * 0.11 0.92 +_0.16
Serum metabolic clearance rate (ml *h-l -25 g-l)
Comparison of presample GtH levels, and distribution volume, serum metabolic clearance rate and half-disappearance time calculated from measurements of serum GtH levels (Figs. 1, 2 and 3) following a single intraperitoneal injection* of glycoprotein cGtH, in the goldfish, Carassius auratus.
TABLE I
282
fish maintained at 12 + 1°C also exhibited well-defined single-order kinetics (graphs not shown). Table I summarizes the results obtained by analysis of the serum disappearance profiles of exogenous GtH, calculated from the data of Figs 1, 2 and 3. The vi of males maintained at 12 + 1°C was significantly greater (P < 0.01) than at 20 rt l”C, although the calculated values of Vi did not differ significantly of 12 f 1°C acclimated fish. The tlh was significantly less (P < 0.01) in male goldfish maintained at 20 + 1°C than at 12 f l”C, although the serum MCR did not differ significantly between either temperature or sex in these three experiments. There was no difference between the tl/, of male and female fish at 12 f l”C, the average value being 9.84 + 0.41 h (mean + 1 SEM). DISCUSSION
The values of MCR, ts and Vi reported in the present study are based on the serum disappearance profiles of ip-administered cGtH. However, these values are probably not representative of the dynamics of endogenous GtH in goldfish. Although the dosage used is small relative to that often used in hypophysation (Chaudhuri, 1976), the single ip injection caused serum cGtH levels to increase to a peak of 6-10 times the endogenous GtH levels and may therefore alter endogenous GtH clearance and distribution parameters. Also, the uptake of the injected cGtH from the peritoneal cavity to the circulatory system will alter values of tG and Vi (see below). In support of this, A.F. Cook and R.E. Peter (unpublished results, 1979) found that the values for tlh and Vi, determined by use of intraarterially-injected radioiodinated tracer cGtH in 25 g BWt female goldfish, varied between 3 and 13 min, and 0.4 and 1.4 ml, respectively. In addition, since sampling did not begin until 1 h post-injection in the present study, it is likely that the extrapolation of the exogenous serum GtH disappearance profile to the time injection (see Fig. 4) greatly overestimates the true Vi. The dynamics of the changing GtH levels indicate an effect of temperature on the MCR and Vi of ip-injected cGtH in goldfish. The tl/, for cGtH is 1.7 times greater in sexually mature male goldfish maintained at 20 f 1°C than at 12 ?r 1°C. The finding that the ip-injected cGtH MCR [defined as the volume of serum that is completely and irreversibly cleared of hormone per unit time (Tait and Burstein, 1964)] was not significantly greater at 20 + 1°C compared to 12 + l”C, is probably related to the significant decrease in Vi found in the 20 + 1°C acclimated fish. Since the maximum serum GtH levels following ip injection of cGtH occurred 2 h later and was smaller in magnitude in the 12 + 1°C fish compared to the 20 + 1°C fish, it is evident that the rate of uptake of the injected cGtH from the peritoneal cavity is slower at 12°C and contributes to the greater value of Vi in these fish. There were no significant differences between sexually mature male and female goldfish acclimated to 12 f 1°C in the serum MCR, ts and Vi of ip-injected cGtH.
283
Although a number of studies have utilized the ip site for the injection of GtH in eleosts (Nayyar et al., 1976; Upadhyay et al., 1978; de Montalembert et al., 1978; Gordon and Zohar, 1978; Jalabert et al., 1978; R.E. Peter, unpublished results, 1979; Stacey et al., 1979), a lack of information concerning post-injection circulating GtH levels has limited their interpretation. We have recently described the relationship between temperature and the latent time to ovulation after an ip injection of human chorionic gonadotropin (HCG) in goldfish (Stacey et al., 1979). The present study suggests that the increase in latency to ovulation at lower temperatures may, in part, represent delayed uptake of HCG from the peritoneal cavity to the circulatory system. Also, it has been shown that ip injections of a similar dose of cGtH at one time in the daily photoperiod can be more effective than at other times in causing ovarian development in goldfish (R.E. Peter, unpublished results, 1979). On the basis of the present results, it is clear that in such experiments the gonads of the fish are being exposed to markedly changing hormone levels during the post-injected period, and that temperature has a major role in determining the changes that occur. The present results confirm that warmer temperatures are associated with increased blood levels of endogenous GtH in goldfish (Gillet et al., 1977, 1978; Hontela and Peter, 1978), similar to other teleost species (Reviews: Billard et al., 1978; Peter and Crim, 1979). Our findings confirm and extend those of Crim and Evans (1976) (see Introduction) by showing that warmer temperatures are associated with a decrease in the blood half-disappearance time of exogenous GtH. The present study also suggests an increased rate of uptake of GtH from the injection site to the circulatory system at 20 f 1°C compared to 12 f 1°C. It is probable that the poor success in the hypophysation of a number of carp species (see Chaudhuri, 1976) may in part, be attributed to a failure to appreciate both the rapid uptake of hormone from the peritoneal cavity into the circulatory system and the rapid clearance of hormone from the blood after ip administration into fish at warm temperatures. ACKNOWLEDGEMENTS
This research was funded by a NSERC 1967 Science Scholarship A.F.C. and NSERC Grant A6371’to R.E.P.
to
REFERENCES Berniarz, K. and Epler, P., 1977. Investigations on inducing sexual maturity in the male eel Anguih anguilla L. J. Fish Biol., 10: 553-559. Bhomwick, R.M., Kowtal, G.V., Jana, R.K. and Gupta, S.D., 1977. Experiments on second spawning of major Indian carps in the same season by hypophysation. Aquaculture, 12: 149-155.
284 Billard, R., Breton, B., Fostler, A., Jalabert, B. and Weil, C., 1978. Endocrine control of the teleost reproductive cycle and its relation to external factors: salmonid and cyprinid models. In: P.J. Gaillard and H.H. Boer (Editors), Comparative Endocrinology. Elsevier/North-Holland Biomedical Press, Amsterdam, pp. 37-48. Chaudhuri, H., 1976. Use of hormones in induced spawning of carps. J. Fish. Res. Board Can., 33: 940-947. Cook, A.F., 1979. Some aspects of the dynamics of the glycoprotein gonadotropin in goldfish, Carassius auratus. M.Sc. thesis. University of Alberta, Edmonton, Alta, 152 pp. Crim, L.W. and Evans, D.M., 1976. Gonadotropic hormone treatment of rainbow trout (Safmo gairdneri): plasma hormone profile following a single injection. J. Fish. Res. Board Can., 33: 2841-2844. Crim, L.W., Peter, R.E. and Billard, R., 1976. Stimulation of gonadotropin secretion by intraventricular injection of hypothalamic extracts in the goldfish, Carassius auratus. Gen. Comp. Endocrinol., 30: 77-82. De Montalembert, G., Jalabert, B. and Bry, C., 1978. Precocious induction of maturation and ovulation in northern pike (Esox lucius). Ann. Biol. Anim. Biochim. Biophys., 18: 969-976. Gillet, C., Billard, R. and Breton, B., 1977. Effets de la temperature sur la taux de gonadotropine plasmatique et le spermatogeneses de Poisson rouge Carassius auratus. Can.J. Zool., 55: 242-245 (English abstract). Gillet, C., Breton, B. and Billard, R., 1978. Seasonal effects of exposure to temperature and photoperiod regimes on gonad growth and plasma gonadotropin in goldfish (Carassius auratus). Ann. Biol. Anim. Biochim. Biophys., 18: 1045-1050. Gordon, H. and Zohar, Y., 1978. Induced spawning of Sparus aurata (L.) by means of hormonal treatments. Ann. Biol. Anim. Biochim. Biophys., 18: 985-990. Hontela, A. and Peter, R.E., 1978. Daily cycles in serum gonadotropin levels in the goldfish: effects of photoperiod, temperature and sexual condition. Can. J. Zool., 56: 2430-2442. Hontela, A. and Peter, R.E., 1979. Effects of pineaiectomy, blinding and sexual condition on daily variations in serum gonadotropin levels in the goldfish. Gen. Comp. Endocrinol., in press. Hora, A.L., 1945. Analysis of factors influencing the spawning of major carps. Proc. Indian Natl. Sci. Acad. Part B, 11: 303-312. Jalabert, B., Breton, B., Brzuska, E., Fostier, A. and Wienianwski, J., 1977. A new tool for induced spawning: the use of 17a-hydroxy-20p-dihydroprogesterone to spawn carp at low temperature. Aquaculture, 10: 353-364. Jalabert, B., Goetz, F.W., Breton, B., Fostier, A. and Donaldson, E.M., 1978. Precocious induction of oocyte maturation and ovulation in coho salmon, Oncorkynchus kisutch. J. Fish. Res. Board Can., 35: 1423-1429. Kuo, C.-M. and Nash, C.E., 1975. Recent progress on the control of ovarian development and induced spawning of the grey mullet (Mugil cephalus L.). Aquaculture, 5: 19-29. Nayyar, S.K.. Kesharanath, P. and Sundararaj, B.I., 1976. Maintenance of spermatogenesis and seminal vesicles in the hypophysectomized catfish, Heteropneustes fossilis (Bloch): effects of ovine and salmon gonadotropin and testoterone. Can. J. Zool., 54: 285-292. Normand, M. and Fortier, C., 1970. Numerical versus analytical integration of hormonal disappearance data. Can. J. Physiol. Pharmacol., 48: 274-281. Peter, R.E. and Crim, L.W., 1979. Reproductive endocrinology of fishes: gonadal cycles and gonadotropin in teleosts. Annu. Rev. Physiol., 41: 323-335. Pickford, G.E. and Atz, J.W., 1957. The Physiology of the Pituitary Gland of Fishes. Zool. Sot. New York, New York, N.Y., 613 pp.
285 Shipley, R.A. and Clark, R.E., 1972. Tracer Methods for in vivo Kinetics: Theory and Applications. Academic Press, New York, NY. and London, pp. 1-44. Stacey, N.E., Cook, A.F. and Peter, R.E., 1979. Spontaneous and gonadotropin-induced ovulation in the goldfish, Carassius auratus L. : effects of external factors. J. Fish. Biol., 15: 349-361. Steel, R.G.D. and Torrie, J.H., 1960. Principles and Procedures of Statistics. McGraw-Hill, New York, N.Y., 412 pp. Tait, J.F. and Burstein, S., 1964. In vivo studies of steroid dynamics in man. In: G. Pincus, D.V. Thimann and E.B. A&wood (Editors), The Hormones. Vol. 5. Academic Press, New York, N.Y., pp. 441-557. Upadhyay, S.N., Breton, B. and Billard, R., 1978. Ultrastructural studies on experimentally induced vitellogenesis in juvenile rainbow trout (S&no gairdneri R.). Ann. Biol. Anim. Biochim. Biophys., 18: 1019-1026. Walker, R.M. and Johansen, P.H., 1977. The effects of hypophysectomy on liver glycogenolytic enzymes and starvation response in goldfish (Carassius auratus L.). Can. J. Zool. 55: 1297-1303.
275
THE EFFECT OF TEMPERATURE ON THE CLEARANCE OF INTRAPERITONEALLY-INJECTED GONADOTROPIN IN THE GOLDFISH CARASSIUS AURATUS ALAN FRANK COOK and R.E. PETER Zoology Department,
University of Alberta, Edmonton,
Alta. T6G 2E9 (Canada)
(Accepted 9 September 1979)
ABSTRACT Cook, A.F. and Peter, R.E., 1980. The effect of temperature on the clearance of intraperitoneallp-injected gonadotropin in the goldfish, Carassius auratus. Aquacultore, 19: 275-285. The serum half-disappearance time (ts), metabolic clearance rate and volume of distribution of intraperitoneally administered carp gonadotropin (cGtH) and endogenous GtH levels were determined in sexually mature male and female goldfish, Carassius auratus maintained at 12 f 1°C or 20 2 1°C. The results indicated that the rate of serum uptake of the injected cGtH from the peritoneal cavity was greater at 20°C than at 12°C in sexually mature male goldfish. Although increased endogenous serum GtH levels and decreased values of serum t,,,, were associated with the elevated temperature, there was no difference in any of the parameters between sexually mature male and female goldfish acclimated to 12°C.
INTRODUCTION
The intraperitoneal (ip) administration of fish pituitary hormones (hypophysation) is used extensively to artifically spawn a number of commercially important teleost species (Pickford and Atz, 1957; Kuo and Nash, 1975; Chaudhuri, 1976). Although a number of studies have concerned the interrelationships between environmental factors and gonadotropin (GtH)-induced spawning (Hora, 1945; Bhowmick et al., 1977; Berniarz and Epler, 1977; Stacey et al., 1979), there are few describing the influence of temperature on the blood GtH levels following an ip injection, Crim and Evans (1976) used radioimmunoassay (RIA) to measure plasma GtH levels following a single ip injection of partially purified GtH in immature rainbow trout, Salmo gairdneri, maintained at three different temperatures. Their results indicated that the rate of clearance from the plasma decreased with decreasing ambient temperature and that detectable blood GtH levels could be found for up to 1 week following the ip injection of a large dose of GtH. The plasma GtH levels following the ip administration of carp pituitary extract (Jalabert et al., 1977) and highly purified salmon GtH
276
for the common carp, Cyprinus respectively. However these studies were not designed to study the effects of temperature on the plasma clearance of GtH. In the present study we report on the serum half-disappearance time (tlh), volume of distribution (Vi), and serum metabolic clearance rate (MCR), and GtH levels in sexually mature male goldfish, Carussius aura tus, maintained at 20 + 1°C and sexually mature male and female goldfish maintained at 12 + l”C, following a single ip injection of highly purified carp GtH (cGtH). (Jalabert
et al., 1978) have been determined
carpio, and coho salmon,
MATERIALS
Oncorhynchus
kisutch,
AND METHODS
Goldfish, Curussius uuratus, of the common or comet varieties were purchased from Grassyfork Fisheries Ltd., Martinsville, Indiana. All fish were maintained for a minimum of 28 days in 1500-l flow-through aquaria under a simulated natural photoperiod (Edmonton, Alberta, Canada) prior to experimental treatment. During this period the water temperature was maintained at 12 f 1°C. At random times during the daily photophase, the fish were fed a commercial trout chow (Ewos) ad libitum. In spite of the theoretical advantages of excluding the pituitary gland as a source of endogenous GtH production for assessing the disappearance characteristics of the exogenous cGtH by the single injection technique, this approach was rejected in view of pronounced hemodynamic (A.F. Cook, personal observation, 1978) and metabolic (Walker and Johansen, 1977) changes associated with goldfish hypophysectomy. A possible source of error with intact fish is the nonspecific component of the ip injection of cGtH; to obviate this possibility the effect of the injection per se was determined in the first experiment by including a number of control groups. After the initial acclimation period, sexually mature male fish were randomly divided into three groups, and reacclimated in 75-1 plastic aquaria maintained at 12 f 1°C under a light dark cycle consisting of 12 h light alternating with 12 h dark (lights on at 08.00 h). The fish were fed Tetramin at 09.00 h daily. Prior to injection or blood sampling the fish were anaesthetized by immersion in 0.05% tricaine methanesulfonate (Sigma, St. Louis, MO), until all visible movements ceased. After 10 days, all fish in group I were bled (pre-experiment sample) from the caudal vasculature between 14.00 and 15.00 h; the remaining fish were undisturbed at this time. The methods of anaesthesia, blood sampling and preparation of serum have been described previously Crim et al., 1976; Cook, 1979). After 7 days recovery, group I fish were given ip injections of 0.025 ,ug cGtH (highly purified glycoprotein cGtH, gift of B. Breton) per g body weight (BWt) (5 pg cGtH per ml teleost saline) with a 250 ~1 Hamilton syringe fitted with a disposable 27 guage X l/z” needle. This dose of cGtH is sufficient to cause a large increase in serum GtH levels (see below) and induce ovarian development (R.E. Peter, unpublished results, 1979), but is not large enough to induce ovulation (A.F. Cook and
277
R.E. Peter, unpublished results, 1979). Blood samples were taken at either 1, 3, 6,12,18, 24, 72 or 168 h post-injection. All samples were taken at the same time in the daily photophase as the presample (14.00-15.00 h), thus each fish in this group served as its own control. Vehicle-injected group II fish received an equivalent volume of teleost saline and were sampled identical to group I. The uninjected control group (group III) was sampled on the same schedule as groups I and II. Blood samples were assayed for GtH by RIA as described previously (Hontela and Peter, 1978,1979). No fish was sampled more than twice after injection or within a 48 h period. Following the last blood sample the fish were killed and the gonosomatic index (GSI) for each fish was calculated as follows: GSI = (gonadal weight/total BWt) X 100. Since there were no significant differences in the GtH values between the various control groups in this experiment (Fig.l), the values obtained from the self-control group (pre-experiment sample) were used as a measure of endogenous serum GtH levels in subsequent experiments. Two additional experiments were done, one using male fish at 20 + 1°C and the other using female fish at 12 f l”C, with the same protocol as described above, except that groups II and III were not included. To dissociate the disappearance pattern of the exogenous hormone from that of the total (endogenous and exogenous) GtH, the individual GtH values obtained from the pre-experimental sample were subtracted from the values obtained after the ip injection of cGtH. The values of exogenous serum GtH were then expressed as a percent of the injected dose per ml serum after standardization to a common BWt of 25 g. The disappearance of exogenous cGtH after the ip injection, expressed as a percent of the administered dose per ml serum, exhibited a linear disappearance pattern on a semi-logarithmic plot (Fig. 4). The disappearance profile of exogenous cGtH in the serum was expressed by the equation: x (t) = A ema* where the concentration of exogenous hormone at any time after the ip injection, x (t), is a function of the ordinate intercept A and the slope (Y. The values of these parameters and their estimates of error were determined using regression analysis of the log-transformed data as described by Normand and Fortier (1970). The serum &., MCR and Vi of exogenous cGtH were calculated as 0.693/a, a/A and l’/A (Tait and Burstein, 1964; Shipley and Clark, 1972). Statistical differences between groups were determined by analysis of variance and the Duncan multiple range test, or the Student’s t-test, where applicable (Steel and Torrie, 1960). RESULTS
The results of the experiment using sexually mature male goldfish (GSI = 3.41 f 0.09%) maintained at 12 * 1°C are shown in F&l. The serum GtH
278
50
c
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a
0.025 0
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uninjected
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self
tnlected cant
body
welghf
controls rols
Control
48
24
cGtH per 9
p.
vehicle-
72
y
168
HOURS
Fig.1. Serum gonadotropin levels in sexually mature male goldfish maintained at 12 + 1°C The ‘self-control’ group (open stars) was sampled 7 days prior to intraperitonal injection of 0.025 rcg highly purified carp gonadotropin (cGtH) per g body weight (solid squares) at the same time of day as the post-injection sampIe. The vehicle-injected control group (open squares) received 5 ~1 of teleost saline per g body weight. Values are mean t 1 SEM (N = 8 to 14 per sample).
levels did not differ significantly either within each of the control groups or between the control groups at any sample time (mean value 3.14 + 0.06 ng/ ml). Fish receiving the cGtH had significantly elevated (P < 0.01) serum GtH levels compared to the control values at the first sample after injection (1 h), and the maximum level (44.67 +- 3.72 ng/ml) was obtained 3 h postinjection. Serum GtH remained significantly greater than the control values until the 48 h post-injection sample; there were no significant differences in the serum GtH levels between the fish receiving cGtH and the self-control values for the remainder of the sampling period (Fig. 1). There were no differences in GSI values between groups (pooled GSI = 3.41 I 0.09%). Fig.2 illustrates the serum GtH levels in sexually mature male goldfish (GSI = 2.63 f 0.23%) maintained at 20 + 1°C. No significant differences in serum GtH levels were detected between sample times for the self-control values (mean value 11.63 + 1.02 ng/ml). Following injection of GtH, the maximum GtH level (67.91 f 3.12 ng/ml) was obtained at 1 h post-injection and declined until at 24 h post-injection there was no longer a significant difference from the presample values. The serum GtH profile for sexually mature female goldfish (GSI = 6.91 + 0.80%) maintained at 12 f 1°C (Fig. 3) is similar to that of mature male goldfish maintained at the same temperature (Fig. 1). The maximum serum
279
8
0
48
24
72
HOURS
Fig.2. Serum gonadotropin levels in sexually mature male goldfish maintained at 20 f. 1°C. The ‘self-control’ group (solid stars) was sampled 7 days prior to a single intraperitoneal injection of 0.025 pg cGtH per g body weight (solid squares). Values are mean * 1 SEM (N = 8).
i \
I
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i’4l/ ,--
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-..------
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I
A
I
0
8
24
48
72
”
s-
16%
HOURS
Fig.3. Serum gonadotropin levels in sexually mature female goldfish maintained at 12 + 1°C. Symbols are the same as described for Fig. 2. Values are mean f 1 SEM (N = 8).
280
GtH value (48.42 + 2.07 ng/ml) was not obtained until 3 h after the ip administration of cGtH. The GtH values of the cGtH-injected group remained significantly elevated over the self-control values until 48 h post-injection. As in the previous two experiments there were no significant differences in the self-control values throughout the sampling period (mean value 5.59 + 0.38 ng/ml). The disappearance of exogenous cGtH, expressed as a percent of the administered dose per ml serum, after a single ip injection in sexually mature male goldfish maintained at 20 + l”C, was characterized by a straight line on a semi-logarithmic plot (Fig. 4). The serum disappearance profiles of ip-administered cGtH, determined in sexually mature male and female gold-
0
4
6
12
16
20
24
HOURS
Fig.4. A semi-logarithmic plot of the metabolic clearance profile of immunoreactive GtH determined in sexually mature male goldfish maintained at 20 + 1°C. Each value is the mean (vertical bars are fr 1 SEM) of 8 to 14 samples obtained by subtracting the initial pre-injection control GtH value from the post-injection sample for each fish, and expressed as a percent of the injected dose after standardization to a common body weight of 25 g.
1
2.63 f 0.23c 3.41 f 0.09 6.91 i 0.30
Male Male Female
20 12 12
Temperature (“C)
11.63 * 1.02e 4.09 + 0.73 5.11 * 0.61
Serumb GtH (ng/mI)
I
,..
-
--
^
--
*a
I.
a 0.025 rg cGtH per g body weight. b Mean value determined from all the control serum GtH values. c AI1 data are mean f 1 SEM.
GSI (%)
Sex
9.83 f 1.12c 14.53 f 1.15 12.50 r 0.71 _
Distribution volume (ml)
6.03 ? 2.1gc 10.25 + 0.91 9.43 f 1.39
Serum half-disappearance time (h)
1.13 f O.lSC 0.98 * 0.11 0.92 +_0.16
Serum metabolic clearance rate (ml *h-l -25 g-l)
Comparison of presample GtH levels, and distribution volume, serum metabolic clearance rate and half-disappearance time calculated from measurements of serum GtH levels (Figs. 1, 2 and 3) following a single intraperitoneal injection* of glycoprotein cGtH, in the goldfish, Carassius auratus.
TABLE I
282
fish maintained at 12 + 1°C also exhibited well-defined single-order kinetics (graphs not shown). Table I summarizes the results obtained by analysis of the serum disappearance profiles of exogenous GtH, calculated from the data of Figs 1, 2 and 3. The vi of males maintained at 12 + 1°C was significantly greater (P < 0.01) than at 20 rt l”C, although the calculated values of Vi did not differ significantly of 12 f 1°C acclimated fish. The tlh was significantly less (P < 0.01) in male goldfish maintained at 20 + 1°C than at 12 f l”C, although the serum MCR did not differ significantly between either temperature or sex in these three experiments. There was no difference between the tl/, of male and female fish at 12 f l”C, the average value being 9.84 + 0.41 h (mean + 1 SEM). DISCUSSION
The values of MCR, ts and Vi reported in the present study are based on the serum disappearance profiles of ip-administered cGtH. However, these values are probably not representative of the dynamics of endogenous GtH in goldfish. Although the dosage used is small relative to that often used in hypophysation (Chaudhuri, 1976), the single ip injection caused serum cGtH levels to increase to a peak of 6-10 times the endogenous GtH levels and may therefore alter endogenous GtH clearance and distribution parameters. Also, the uptake of the injected cGtH from the peritoneal cavity to the circulatory system will alter values of tG and Vi (see below). In support of this, A.F. Cook and R.E. Peter (unpublished results, 1979) found that the values for tlh and Vi, determined by use of intraarterially-injected radioiodinated tracer cGtH in 25 g BWt female goldfish, varied between 3 and 13 min, and 0.4 and 1.4 ml, respectively. In addition, since sampling did not begin until 1 h post-injection in the present study, it is likely that the extrapolation of the exogenous serum GtH disappearance profile to the time injection (see Fig. 4) greatly overestimates the true Vi. The dynamics of the changing GtH levels indicate an effect of temperature on the MCR and Vi of ip-injected cGtH in goldfish. The tl/, for cGtH is 1.7 times greater in sexually mature male goldfish maintained at 20 f 1°C than at 12 ?r 1°C. The finding that the ip-injected cGtH MCR [defined as the volume of serum that is completely and irreversibly cleared of hormone per unit time (Tait and Burstein, 1964)] was not significantly greater at 20 + 1°C compared to 12 + l”C, is probably related to the significant decrease in Vi found in the 20 + 1°C acclimated fish. Since the maximum serum GtH levels following ip injection of cGtH occurred 2 h later and was smaller in magnitude in the 12 + 1°C fish compared to the 20 + 1°C fish, it is evident that the rate of uptake of the injected cGtH from the peritoneal cavity is slower at 12°C and contributes to the greater value of Vi in these fish. There were no significant differences between sexually mature male and female goldfish acclimated to 12 f 1°C in the serum MCR, ts and Vi of ip-injected cGtH.
283
Although a number of studies have utilized the ip site for the injection of GtH in eleosts (Nayyar et al., 1976; Upadhyay et al., 1978; de Montalembert et al., 1978; Gordon and Zohar, 1978; Jalabert et al., 1978; R.E. Peter, unpublished results, 1979; Stacey et al., 1979), a lack of information concerning post-injection circulating GtH levels has limited their interpretation. We have recently described the relationship between temperature and the latent time to ovulation after an ip injection of human chorionic gonadotropin (HCG) in goldfish (Stacey et al., 1979). The present study suggests that the increase in latency to ovulation at lower temperatures may, in part, represent delayed uptake of HCG from the peritoneal cavity to the circulatory system. Also, it has been shown that ip injections of a similar dose of cGtH at one time in the daily photoperiod can be more effective than at other times in causing ovarian development in goldfish (R.E. Peter, unpublished results, 1979). On the basis of the present results, it is clear that in such experiments the gonads of the fish are being exposed to markedly changing hormone levels during the post-injected period, and that temperature has a major role in determining the changes that occur. The present results confirm that warmer temperatures are associated with increased blood levels of endogenous GtH in goldfish (Gillet et al., 1977, 1978; Hontela and Peter, 1978), similar to other teleost species (Reviews: Billard et al., 1978; Peter and Crim, 1979). Our findings confirm and extend those of Crim and Evans (1976) (see Introduction) by showing that warmer temperatures are associated with a decrease in the blood half-disappearance time of exogenous GtH. The present study also suggests an increased rate of uptake of GtH from the injection site to the circulatory system at 20 f 1°C compared to 12 f 1°C. It is probable that the poor success in the hypophysation of a number of carp species (see Chaudhuri, 1976) may in part, be attributed to a failure to appreciate both the rapid uptake of hormone from the peritoneal cavity into the circulatory system and the rapid clearance of hormone from the blood after ip administration into fish at warm temperatures. ACKNOWLEDGEMENTS
This research was funded by a NSERC 1967 Science Scholarship A.F.C. and NSERC Grant A6371’to R.E.P.
to
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285 Shipley, R.A. and Clark, R.E., 1972. Tracer Methods for in vivo Kinetics: Theory and Applications. Academic Press, New York, NY. and London, pp. 1-44. Stacey, N.E., Cook, A.F. and Peter, R.E., 1979. Spontaneous and gonadotropin-induced ovulation in the goldfish, Carassius auratus L. : effects of external factors. J. Fish. Biol., 15: 349-361. Steel, R.G.D. and Torrie, J.H., 1960. Principles and Procedures of Statistics. McGraw-Hill, New York, N.Y., 412 pp. Tait, J.F. and Burstein, S., 1964. In vivo studies of steroid dynamics in man. In: G. Pincus, D.V. Thimann and E.B. A&wood (Editors), The Hormones. Vol. 5. Academic Press, New York, N.Y., pp. 441-557. Upadhyay, S.N., Breton, B. and Billard, R., 1978. Ultrastructural studies on experimentally induced vitellogenesis in juvenile rainbow trout (S&no gairdneri R.). Ann. Biol. Anim. Biochim. Biophys., 18: 1019-1026. Walker, R.M. and Johansen, P.H., 1977. The effects of hypophysectomy on liver glycogenolytic enzymes and starvation response in goldfish (Carassius auratus L.). Can. J. Zool. 55: 1297-1303.