Fluctuations in gonadotropin and ovarian steroids during the annual cycle and spawning of the common carp

Fish Physiology and Biochemistry vol. 2 nos. 1-4 pp 75-86 (1986) Kugler Publications, Amsterdam/Berkeley Fluctuations in gonadotropin and ovarian steroids during the annual cycle and spawning of the common carp Zvi Yaron and Berta Levavi-Zermonsky Department of Zoology, The George S. Wise Faculty of Life Sciences, TeI-A viv University, Tel A viv 69978, Israel Keywords: Carp, ovary, vitellogenesis, spawning-induction, gonadotropin, 17a, 20/3-dihydroxy-4-pregnen-3-one, estradiol, testosterone, annual cycle, germinal vesicle Abstract The main objective of the paper is to describe the annual changes in hormones associated with reproduction in the female carp under the conditions prevailing in the Israeli fish culture. Fish were sampled monthly throughout 1984; gonadosomatic index (GSI) was calculated and the diameter of ovarian follicles was measured. Gonadotropin (GTH) content in the pituitary and the circulating GTH, estradiol, testosterone and 17a, 20~3-dihydroxy-4-pregnen-3-one (17, 20-P) were determined by specific radioimmunoassays. GTH, estradiol and testosterone showed a bimodal annual pattern. The late summer peak was associated with initial vitellogenesis while the peak in spring occurred just before spawning, which took place in April-May. A resting phase in ovarian activity was noted in June and July. The levels of 17, 20-P were very low compared with those occurring during spawning induction. The paper summarizes a previous study by our laboratory on the changes in circulating hormones, as related to oocyte stages, in female carp induced to spawn by a GTH-calibrated pituitary extract. This study associates the short but prominent peak in 17, 20-P with the presence of follicles with maturing oocytes in the ovary. A correlation was found between the percentage of oocytes with eccentric germinal vesicle initially present in ovarian biopsies of females carp and their spawning success after hypophysation. The paper describes simple means to ensure successful induction of spawning in carp by utilizing a calibrated pituitary extract and prior selection of females that would respond to the induction treatment. Introduction Annual fluctuations in gonadotropin (GTH) content of the pituitary and plasma in relation to temperature and gonadosomatic index (GSI) have been described in the European and Chinese races of the common carp (Billard et al. 1978; Zhao et al. 1984). In the European race estradiol levels were also measured. There is a marked difference between these carp populations in the timing of the annual peaks of the measured parameters. In Europe the peak of pituitary GTH occurs during spawning in June-July, whereas in China it occurs in February-March, just prior to spawning. The difference in the timing may be attributed to the difference in the climatic conditions. However, various races of the common carp may also show genetic differences in their reproductive parameters such as the GSI, age of maturity (Hulata et al. 1975), or estradiol level in the circulation (Weft et al. 1980). The 'Dor 70' carp and the progeny of its cross with other European races may reach maturity in Israel within a year (Hulata et al. 1975), and thus differ considerably from carp populations in Europe.

76 The present report describes the fluctuations in the hormonal profile as related to ovarian growth in the common carp (Dor 70 x Yugoslavian races) under the conditions prevailing in the Israeli fish culture. The report also summarizes our previous study on the fluctuations in GTH and certain steroids during spawning induction and the related morphological changes in ovarian follicles. In addition, we report here on the application of the ovarian biopsy technique for determining the suitability of female carp to spawning induction. Materials and methods Water temperature Temperature records were kindly furnished by Mr. A. Halevy, Fish and Aquaculture Research Station, Dor, about 16 km north ofgan Shmuel. These were daily maxima and minima of the temperature measured during 1984 in midwater of an earthen pond about 1 m deep. The weekly minima and maxima were averaged to produce a weekly mean temperature. Fish and tissues sampling About 20 fish were obtained at monthly intervals (generally in the fourth week of the month) during 1984 from the commercial crop of Kibbutz Gan Shmuel, 51 km north of Tel Aviv. Two samples were obtained in April (15th and 29th). The fish were offspring of a cross between female 'Dor 70' race and males of a Yugoslavian race (Wohlfarth et al. 1980); this is the most common combination of carp cultivated in Israel. The fish were transported alive to the laboratory; their body and gonadal weights were recorded. Blood samples (3-6 ml) were taken from the caudal vessels and the serum was separated and stored at -20 C until assayed. The pituitary was removed and stored in absolute ethanol at -20 C. An ovarian sample was fixed in ethanol: formaldehyde: acetic acid (6:3:1) for measuring oocyte diameter. Repeated examinations of the follicles before and after 1, 2, 800, or 1000 h in the fixative indicated that their diameter did not change after fixation. Gonadotropin determination Carp gonadotropin (cgth) in pituitary glands and serum samples was determined by a double-antibody radioimmunoassay (RIA); the details of the assay were as published elsewhere (Levavi- Zermonsky and Yaron, 1986) except that the standard cgth was obtained from Dr. B. Breton, Rennes. The anti cgth was a gift from Dr. V. Lichtenberg, Hamburg. The sensitivity of the standard curve was 78 pg/tube. Determination of steroids Estradiol was determined by RIA as detailed in Yaron et al. (1977). The sensitivity of the standard curve was 4.1 pg/tube and that of the assay was 48.3 pg/ml for serum samples of 0.8 ml. The progestogen, 17 u, 20 /%dihydroxy-4-pregnen-3-one (17, 20-P) was measured by RIA using an antibody kindly given by Dr. A.P. Scott (Lowestoft, U.K.). The sensitivity of the standard curve was 8.5 pg/tube and that of the assay was 89.5 pg/ml for serum samples of 0.8 ml. The details of these assays and their validation for carp serum are as in Levavi- Zermonsky and Yaron (1986). Circulating testosterone was determined by RIA using monoclonal antibodies for testosterone (Batch F/2) obtained from Dr. N. Moav (Ness Ziona, Israel). The antibody had less than 0.1 7o cross-reaction with either androsterone, dehydroepiandrosterone, 11 /3- hydroxytestosterone, 11-keto-testosterone, progesterone, 17, 20-P or estradiol. The sensitivity of the standard curve was 9 pg/tube and that of the assay was 100 pg/ml for serum samples of 0.8 ml. Suitability o f female carp for spawning induction Twenty-five females, kept separately from males in a plastic-covered earthen pond since the previous breeding season, were transferred on April 19, 1983

77 to the hatchery and housed in 450 L polypropylene tanks supplied with running well water (22 C) and adequate aeration. Two females, differing in size or other external features, were housed in each tank in order to enable individual identification. At noon of the following day an ovarian sample was taken from each female by biopsy, and the oocytes stages were determined after clearing the tissue sample (details in Levavi-Zermonsky and Yaron, 1986). The females were injected intramuscularly with a priming dose of a calibrated carp pituitary extract (Dag Shan Ltd., Beit Shah, Israel; see Yaron et al. 1984) containing immunoreactive GTH (IR-cGTH; 0.1 mg/kg bw). Eleven hours later the main dose of the extract, containing IR-cGTH (0.5 mg/kg), was injected. The following morning (April 21) each female was lifted and stripping was attempted. A similar experiment was repeated on March 1-2, 1984. Ovarian events and hormonal profile during spawning induction Ten females (4.5 _+ 0.42 kg bw), kept as above, were selected for spawning induction by prior examination of their ovarian stage, and were transferred into the hatchery. On June 9, 1984 they were injected with a priming dose of the calibrated extract containing IR-cGTH (0.07 mg/kg) and l lh later with the main dose (0.35 mg/kg). Blood was sampled and the ovary was biopsied at intervals, starting 3h before priming and ending with stripping. The ovarian tissue or ovulated eggs were cleared and the oocyte stages were recorded. IRcGTH, estradiol and 17, 20-P were measured in the serum samples. This experiment is part of a wider study reported elsewhere (Levavi-Zermonsky and Yaron, 1986). All non-percent data are expressed as group mean + SEM. Results Annual cycle High water temperature of 28-30 C was maintain- ed in the fishponds along the coastal plain in Israel until mid September 1984 and only then decreased gradually. Lowest temperature of 13-14 C occurred in January-February and December (Fig. 1A). Two generations of follicles could be easily recognized under the dissecting microscope in total preparations of ovarian samples: 'small' follicles not exceeding 0.15 mm, which occurred throughout the year, and vitellogenic follicles which populated the ovary only from August until May (Fig. 1B). Initial ovarian growth occurred in August. Microscopic examination of ovarian sections from the July sample showed peripheral vacuoles in only a few oocytes which conform to stage 2 of Gupta (1975). Increase in number of these early vitellogenic oocytes occurred in August, but only in September was there evidence of yolk granules deposited among the vacuoles (stage 3 of Gupta 1975). Vitellogenic follicles continued their growth until February when they reached their maximal diameter of 1.10 + 0.01 mm (vitellogenic phase). The follicles retained their maximal diameter until spawning in April-May (postvitellogenic phase). The GSI increased gradually from August until February - March and started to decrease, due to spawning, from April to May (Fig. 1C). The values of oocyte diameter and GSI presented for May 1984 were taken from 15 females; five before spawning, with GSI of 9.35 + 2.2, and 10 females which had already spawned with GSI of 0.87 + 0.07 exhibiting only 'small' follicles (Fig. 1B, C). During the selection of the fish for the study an effort was made to collect fish of around 1 kg body weight. This was not always possible; fish considerably smaller were included in the samples of January, end of April and May (Fig. 1D). The significant difference in body weight between fish in the September to December samples and those of the January sample indicates that the latter belong to a younger population which had been spawned the previous year (Spring 1983). The summer and autumn samples consisted of fish older than one year (i.e. had been spawned in Spring 1982). The circulating estradiol levels were low in August but showed a transient peak in September (Fig. 2A). The levels in January were low or undetectable. A gradual increase in estradiol levels

78 A TEMP o C I I I,,, I I I i I I I I I FOLLICLE mm 10-0.6 0.2 10- C 17 19 20 2O GSI 6-2- I I I I I - ~ l I I D p- BODY WEIGHT 1.0 kg 0,5 n N n l:i n NII J F M A M J J A S 0 N D Fig. t. Annual fluctuations in fishpond temperature along the coastal plain of Israel during 1984 and parameters associated with reproduction in female carp (Dot 70 x Yugoslavian races). Fish were obtained monthly from the commercial crop of Kibbutz Gan Shmuel; two samples were oaken in April (15th, 29th) A. Weekly means of temperature recorded throughout 1984 in an earthen pond at the Fish and Aquaculture Research Station Dor, about 16 km north of Gan Shmuel. B. Diameter of ovarian follicles; at least 40 follicles w~re examined in each female, number of fish as in Fig. IC. Open bars: vitellogenic oocytes; hatched bars denotes the presence of non-vitellogenic 'small' oocytes throughout the year. Among the fish sampled in May some were postovulatory with 'small' follicles only and others had not ovulated. (dashed-line histogram). C. Gonadosomatic index (GSI). D. Mean body weight of fish in each sample, number of fish as in Fig. IC. started thereafter and a comparatively high level was maintained well after spawning. At the end of 1984, when females were about 18 months old the gradual increase in estradiol apparently commenced in November (Fig. 2A). The fluctuations in testosterone levels were in a pattern similar to that of estradiol; the autumn and spring peaks occurred in the same months (September and March). However, in January and the end of April the levels in most fish were below the limit of detection. The levels of this steroid increased between April and June (Fig. 2B). The levels of 17, 20-P were very low in the monthly samples all year round (Fig. 2C). A prominent increase in circulating IR-cGTH occurred from July to August. Unfortunately, IRcGTH in September and November could be measured only in single specimens, hence, no reliable data can be presented for these months. A low level was maintained from January through mid April

79 E 2 ng/.h 0.7 0.4 A 21 6 19 13 13 A / 19 0.1 11 1 11 17 I I T ng/ml 0.5 0.3 B 7 /4% 6 16 / 13 m 17,20-P ng/ml 0.1 0"9 f 0.3 I I I I I I I I I I I I 13 C 18 ~5 ~...,,~,J,o ~. i"rl - "=' % 14 ~ 13 21 18 I I I 1 I I t I I 1 I 1, 14 D 7 IR-cGTH ng/ml I 1.4- Z 12 16 8- "~7"~" '~" -.,~ 10,. f'"c-----c--,r,,, 13 18 5 /,0/ 0 I 1 I 1 1_ IR-cGTH mg./pit m 0.8-0.2- i i4." # e * m ee I I l J F M 16 12 T t8,,...... * 11 11.J~/o, 10 # " '.~... ~..."I... ~..,o.." " * ~ I I I I I I I I I A M J J A S O N D Fig. 2. Annual fluctuations in certain hormones associated with reproduction in female carp. A. Circulating estradiol-17/3 (E2); IB. Circulating testosterone (T); C. Circulating 17 ez, 20/3-dihydroxy-4-pregnen-3-one (17, 20-P); D. Immunoreactive carp gonadotropin (IRcGTH) in serum.e. Content of IR-cGTH in aqueous extracts of pituitary glands.

80 240 PRIMING 1 INDUCTION 195 "~ 150 n C "r i u 105 60 /! I / / lo ~! I E2/~ /,.x /-/ 10 7 4 rrl I,o..I 15 ],~Q0 m O9,._.,,... 12 18 23 01 03 07 hr Fig. 3. Fluctuations in IR-cGTH, E2 and 17, 20-P in the circulation of female carp induced to spawn by a calibrated pituitary extract. Priming dose of the extract, containing 0.07 mg IR-cGTH/kg body weight was administered at noon (left arrow) and the main induction dose containing 0.35 mg 1R-cGTH/kg body weight was given 1 lh later (right arrow). All females ovulated and were stripped the next morning. (Data from Levavi-Zermonsky and Yaron, 1986). and a significant increase occurred thereafter (Fig. 2D). The pituitary content of IR-cGTH was lowest in September and increased in October. In fish collected at the beginning of the year an increase occurred in February and peaked in March and early April. The pituitary IR-cGTH dropped dramatically after spawning. Eight spent females collected in May had 0.210 _+ 0.031 mg 1R-cGTH per gland while 3 females which had not spawned showed a much higher content (0.647 + 0.135 rag/gland; p < 0.001). Suitability o f female carp for spawning induction Table 1 indicates that females initially having more than 65 70 oocytes at stage II (eccentric germinal vesicles) spawned successfully after hypophysation in both experiments. After incubation according to Rothbard (1981), more than 90% of the eggs hatched normally. Females initially having less than 26 70 of their oocytes at stage II failed to respond to the treatment and did not ovulate. Hormonal profile and ovarian events during spawning induction Circulating level of IR-cGTH increased from 2.32 + 0.41 to 81.03 _+ 5.01 ng/ml (n = 10) following the administration of the priming dose, and remained at that level until the administration of the main dose (Fig. 3). A higher concentration (231 +

81 % 10 3 5 3 5( nvln i h lira t'vv i re'l-ha iv....! II III IV V I II III IVY I I! III IV v 09 18 23 01 03 hr Fig. 4. Percentage of oocyte maturational stages in ovarian biopsies taken at intervals from carp before and during induction of spawning. Fish and treatment as in Fig. 3. Each female was biopsied on alternate points. Figures above the histograms denote number of fish biopsied at each point. Maturational stages were defined as: Stage 1 - central germinal vesicle (GV); Stage II - eccentric GV; Stage 111 - peripheral GV; Stage IV - GV breakdown; Stage V - ovulated egg. (Data from Levavi-Zermonsky and Yaron, 1986). 22.93 ng/ml) was reached within 2 h after the injection of the main dose and remained at a similar level for 6h. Estradiol concentration increased from 0.62 + 0.13 ng/ml before priming to 8.68 _ t.01 ng/ml at 2300h. However, the level decreased temporarily to 3.64 _+ 0.98 ng/ml following the injection of the main dose. The level of 17, 20-P did not change at all "after the priming injection and started to increase only upon the administration of the main dose of the calibrated pituitary extract. It reached a very high peak (111.02 _+ 14.59 ng/ml) at 0300h, after which it returned to the baseline (Fig. 3). Initially, the ten females chosen for the experiment contained oocytes at stages I and II (16% and 84%, respectively; Fig. 4). Three females examined 6h after priming contained 21% oocytes with peripheral GV (stage III). Just prior to the administration of the main dose at 2300h, the 5 females examined contained no oocytes at stage I; 64% of the oocytes were at stage III while 21% had already undergone GV breakdown (GVBD). Ovulated eggs appeared 2h after injection of the main dose and all females had ovulated before 0300h (Fig. 4). All females were successfully stripped between 0700h and 0800h and released a large number of eggs. After fertilization, rinsing and swelling according to Rothbard (1981), the average volume of spawn per female was 2,000 300 ml which roughly corresponds to 1.5 million eggs. About 90o/o of the zygotes hatched successfully. Discussion Annual cycle The fish utilized for the study of the annual cycle were taken from the commercial crop of a fish farm. The procedure of harvesting the fish involves a severe stress of netting, crowding and 'pumping' the animals out of the sump into the transporting tank. Moreover, blood was taken from the fish after further transportation into the laboratory in Tel Aviv (51 km away). It is possible that the levels of the hormones determined in these samples do not reflect the natural situation of the fish at the time of collection. Indeed, the highest level of estradiol measured in the annual cycle of the local carp (0.64 ng/ml) is considerably lower than the peak reported in carp reared in an aquarium in France (about 15 ng/ml; Billard et al 1978). However, the fish population used in the present study had enjoyed, until their harvest, a seminatural environment in which they would naturally spawn (Fig. 1B). In addition, the levels of estradiol, testosterone and 17, 20-P measured by Kime and Dolben (1985) in carp prior to spawning induction are in agreement with those reported in the present study. Moreover, fish of the broodstock transferred carefully into the hatchary and kept under optimal conditions for several days before spawning induction, showed estradiol levels not much different from those recorded before spawning in the March sample (Figs. 2A, 3). This comparison may indicate that although some endocrine parameters in the carp, such as cortisol level, may change drastically due to handling stress (Ilan and Yaron 1976) other para-

82 meters, such as GTH or estradiol, may not change appreciably during the first few hours of stress. Diurnal fluctuations in the levels of GTH and ovarian steroids have been reported in the trout (Zohar et al. 1982). The baseline and the pattern of the fluctuations are characteristic to each reproductive phase. Inevitably, the monthly sampling practiced in the present study on the annual cycle of the carp can only reflect the baseline level in each month. Bearing these limitations in mind it is possible to outline an annual cycle of the parameters associated with reproduction of the carp in the Israeli fish culture. Two peaks in IR-cGTH, testosterone and estradiol could be discerned during the annual cycle. The late summer peaks (September) of testosterone and estradiol coincided with the initial appearance of yolk granules in the growing oocytes. This is in agreement with the current view that estrogens, and to a lesser extent also testosterone, induce the hepatic synthesis of vitellogenin in fish (reviewed by Ng and Idler 1983, Fostier et al. 1983). The increase in circulating IR-cGTH preceded the abovementioned steroids peaks by one month. One may expect such a relationship between increased GTH levels and the GTH-dependent steroid synthesis. It should be noted, however, that the stimulatory effect of GTH on ovarian steroidogenesis is manifested in a few hours rather than several weeks when examined either in vivo (Weil et al. 1980; Fig. 3) or in vitro (Zohar et al. 1982; Bogomolnaya and Yaron 1984). Another increase in estradiol began in November (in older fish) or in January-February (in younger fish). In March, both estradiol and testosterone reached peak levels together with a high content of pituitary IR-cGTH which was retained in preovulatory females in Early April. At that time the oocytes had already completed the vitellogenic process and had reached their final diameter. It is possible that the peak in pituitary GTH in spring is associated with the induction of final maturation and ovulation (Figs. 1, 2). It should be emphasized that hypophysation of carp with postvitellogenic oocytes is followed by an increase in estradiol level (Fostier et al. 1979; Weil et al. 1980; Manning and Kime 1984; Kime and Dolben 1985; Fig. 3). It would appear that, in this respect, carp differ from salmonids in which estradiol secretion, in response to GTH, is reduced considerably after the completion of vitellogenesis (Scott and Sumpter 1983; Young et al. 1983a; Kagawa et al. 1983a). The situation in the carp is rather similar to that found in Catostomus commersoni where estradiol level does not decrease prior to ovulation (Scott et al. 1984). The levels of 17, 20-P (Fig. 2C) were extremely low throughout the year compared with the dramatic surge observed during final maturation induced by hypophysation (Fig. 3). Since the surge is extremely short and totally disappears after a few hours, it is unlikely that it will be reflected in the monthly samples. A single peak in both circulating GTH and estradiol was reported in adult carp females in France. The GTH peak occurred in July, at the normal spawning time, although no ovulation was observed in these females. This was followed by an increase in estradiol which lasted until November and probably reflected the beginning of a new reproductive cycle; vitellogenesis in these fish was complete by October (Billard et at. 1978). In addition, carp females studied in Poland showed high circulating GTH during and beyond the spawning season. These high levels were correlated with the vitellogenic phase which was complete by December (Bieniarz et al. 1978). Both studies indicate a short vitellogenic phase which commences immediately after spawning in June-July and is complete by October to December. In the carp population in Israel, the spawning in ponds is two months earlier than in Europe (April-May), but the vitellogenic phase starts in August, like their European counterparts. However the vitellogenic phase in the Israeli carp commences only after a resting period of two months, June and July. Hence, in Israel, the vitellogenic phase is prolonged. The older fish examined in December had not yet reached maximal follicular diameter and did not differ from those of the younger fish examined in January. Maximal oocyte diameter was attained only in February, at least two months later than in Europe. Hence, the postvitellogenic phase in Israel is apparently shorter than in Europe. Carp studied

83 in China show a single peak of circulating and pituitary GTH which corresponds to the decrease in GSI during spawning (Zhao et al 1984). In our study, the pattern of GTH content in the pituitary did not coincide with that found in the serum (Fig. 2D, E). The moderate but significant increase in circulating GTH which occurred in April coincided with a drop in the pituitary content of the hormone. The further increase between July and August occurred when pituitary content of this hormone was minimal. However, the single determinations of IR-cGTH in September and November do not allow an adequate estimation as to the extent of this peak. The IR-cGTH content in the carp pituitary during 1984 showed a bimodal annual pattern; moderate levels were maintained in October- December and corresponded to the increase in GSI; and a peak occurred in March-April, just prior to spawning. A similar bimodal pattern but with somewh~tt different timing was observed also during 1982 in the carp population of the same origin (Yaron et al 1984). Studies on the effect of LH-RH on GTH secretion from carp pituitary have shown seasonal variation in the response, or 'sensitivity' to the hypothalamic peptide; the response to LH-RH being stronger during the breeding season in summer (Weil et al. 1975). In a further study (Weil 1981) the 'global secretion' of GTH, in response to LH-RH treatment, showed two annual peaks, one in March and the other in September. If, indeed, the pituitary content of GTH is reflected by the 'global secretion', then the bimodal pattern in the pituitary GTH content of carp in France does not differ fundamentally from that of their counterparts in Israel. Suitability o f female carp for spawning induction Carp ovaries in Israel contain fully-grown oocytes (a diameter of about 1 mm) as from February on (Fig. IB). However, the success of spawning induction does not depend only on follicular diameter but also on the position of the germinal vesicle within the oocyte. Females responded to hypophysation only if initially more than 65 7o of their oocytes had already started GV migration (Table 1). In vitro experiments have shown that GVBD is attained faster, and in a larger proportion, after stimulation with Table 1. Suitability of female carp for spawning induction 21-22.3.1983 (total) 25 n (Fish) 7o oocytes at stage Successful stripping 10 18.5 81.5 Unsuccessful stripping 15 90.0 10.0 1-2.3.1984 (total) 20 Successful stripping 15 34.4 65.6 Unsuccessful stripping 5 74.0 26.0 In the two experiments ovarian biopsies consisting of > 40 follicles were taken from female carp. Oocytes were cleared in ethanol: formaldehyde: acetic acid (6:3:1) and their maturational stages were determined under a dissecting microscope. The fish were administered a priming dose of calibrated carp pituitary extract containing 0.1 mg/kg IR-cGTH, and I lh later the main inducion dose containing 0.5 mg/kg IR-cGTH. Stripping was attempted 7-10h after the second injection. No response, partial or abnormal emmission of eggs were considered as 'unsuccessful stripping'. pituitary extract if the percentage of oocytes with migratory or peripheral GV is, initially, higher (Epler 1981). The procedure of oocyte examination in ovarian biopsies of female carp destined for spawning is already applied in several hatcheries in Israel as a routine. By employing this method only suitable females are selected for spawning induction, while others are returned to the pond until their oocytes show further progress, namely, initial migration of GV. Ovarian events and hormonal profile during spawning induction The results presented here indicate that injection of a priming dose to female carp resulted in an increase in circulating IR-cGTH and a further migrat!on of the GV until a peripheral position is attained (Figs. 3, 4). Migration of GV from a central to an eccentric position had been found previously in carp injected with 1 mg/kg of uncalibrated carp pituitary homogenate (Bieniarz et al. 1979). In the I I1

84 present study estradiol level increased considerably after priming, but no change occurred in the level of 17,20-P, which remained as low as measured during the annual cycle. The injection of the main dose resulted in a further increase in circulating IRcGTH, far beyond the level measured in the annual cycle. The level attained after the second injection (231 + 23 ng/ml) was rather similar to that determined in carp during actual spawning under natural conditions in China (256 ng/ml; Zhao et al. 1984). As a result of the second injection of the pituitary extract, further development occurred in the oocytes, namely GVBD and ovulation. Females in the same experiment injected with the priming dose only did not show this development (Levavi- Zermonsky and Yaron 1986). These morphological changes in the oocytes were associated also with profound alterations in the steroid profile. The level of 17,20-P increased dramatically while the level of estradiol temporarily decreased (Fig. 3). These results differ principally from those obtained in carp by Kime and Dolben (1985) experimenting in a similar protocol. In their two experiments only one fish showed any increase in the level of 17,20-P following hypophysation. However, all fish exhibited an increase in 17 c~hydroxyprogesterone (17-P; 2-13 ng/ml). Based on these data, it was concluded that 17-P may be the maturational steroid in carp, taking into account, however, that 'small but physiologically active amounts' of 17,20-P may be locally produced in the ovary. The present results show that in all females tested a very prominent peak of 17,20-P occurred 3h prior to ovulation. The magnitude of this peak (111.02 _+ 14.59 ng/ml) is about 20 times higher than that recorded for 17-P by Kime and Dolben (1985). Since only partial ovulation was recorded in their experiments, it seems unlikely that the steroid profile outlined by these authors fully represents the normal sequence of steroidal changes in spawning carp. The temporary decrease in estradiol level, which occurs concomitantly with the increase in 17,20-P, indicates a possible shift in the steroidogenic pattern, basically similar to that observed in salmonids and the goldfish (Scott et al. 1982, 1983; Zohar et al. 1982; Young et al. 1983b). This shift should in- volve a reduction in 17,20 desmolase and the induction of 20/3-reductase, with or without reduction in aromatase activity. If such a shift does actually occur in the carp, it should be reflected by increased production of 17,20-P and also its immediate precursor, 17-P, as reported in the trout (Scott and Baynes 1982), in the goldfish (Kagawa et al. 1983b; Peter et al. 1984) and in the carp (Kime and Dolben 1985). In contrast to the sequence of steroidal succession in the trout, estradiol level in the carp is only temporarily reduced and elevated levels occur during ovulation and spawning, and probably remain high for weeks thereafter. This is reflected in the annual cycle, where the levels of these steroids decreased only in July, 8-10 weeks after spawning. The physiological significance of the high postspawning estradiol levels is not clear. It is possibly associated with oogonial proliferation which follows immediately after the spawning season. Acknowledgements Part of this research was supported by grants (AQ-18 and AQ 4 III) from the National Council of Research and Development, Israel and the GKSS, Geesthacht-Tesperhude, F.R.G. We wish to thank Dr. A.P. Scott (Lowestoft) and Dr. V. Lichtenberg (Hamburg) for the kind gift of antibodies, and Dr. B. Breton (Rennes) for the standard cgth. We thank Kibbutz Gan Shmuel for the generous supply of carp used in this research. Special thanks are due to the team of the Fish Breeding Center, Gan Shmuel, for their enthusiastic support. We also thank Mrs. R. Suzeen and Mrs. S. Rahav for the graphic work, and finally Ms. Alissa Bogomolnaya and Mrs. Hassia Kedem for their continuous assistance throughout the project. References cited Abraham, G.E. 1974. Radioimmunoassay of steroids in biological materials. Acta Endocrinol. 75: (Suppl. 183), 23-28. Bieniarz, K., Epler, P., Breton, B. and Thuy, L.N. 1978. The annual reproduction cycle in adult carp in Poland: ovarian state and serum gonadotropin level. Ann. Biol. anita. Bioch. Biophys. 18: 917-921.

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