Effects of steroids on germinal vesicle breakdown in vitro of intact follicles in the japanese whiting, Sillago japonica, a marine teleost

Camp. Biochem. Phy&l. Vol. %A, No. 2, pp. 257-261, 1990

03W9629190s3.00 + 0.00 0 I990 Pergamon Press plc

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EFFECTS OF STEROIDS ON GERMINAL VESICLE BREAKDOWN IN VlTl?O OF INTACT FOLLICLES IN THE JAPANESE WHITING, SILLAGO JA~O~rCA, A MARINE TELEOST MXHIYA M~T~UYAMA, YOSH~HIDEHANAKI and SHUHEIMATSUURA Department of Fisheries, Faculty of Agriculture, Kyushu University, Fukuoka 812, Japan (Received 23 November 1989) Abstract--l.

The effects of various steroids on germinal vesicle breakdown (GVBD) of follicle-enclosed oocytes of the Japanese whiting were examined by using an in vitro incubation technique. 2. Of the stimulatory steroids, 17a,20fi-dihydroxy-4-pregnen-3-one was the most effective, inducing 72% GVBD at concentrations as low as 0.1 ng/ml. The next most potent steroid was 20~-dihydroprogesterone~ inducing 33% GVBD at concentrations of 0.1 ngjml. 3. The specificity for steroid-stim~ated GVBD in Japanese whiting is consistent with several previous studies involving saimonids and various other teleosts, and suggests a possible physiolo~cal role for a 20~-dihydroprogestin in oocyte maturation of Japanese whiting.

INTRODUCTION

In teteosts, as in most other vertebrate groups, the process of oocyte final maturation is under the control of gonadotropin released by the pituitary gland, and is mediated via steroids secreted by the follicular cells of the oocytes (for review, see Goetz, 1983; Nagahama, 1987). Investigations have shown that oocytes of teleost can be induced to undergo oocyte final maturation in &ro in the presence of steroids or gonadotropins. However, much of this work has focused on salmonid species (Jalabert, 1976; Duffey and Goetz, 1980; Sower and Shreck, 1982; Nagahama et al., 1983; Yamauchi et al., 1983), and some freshwater species (Iwamatsu 1978, 1980; Goetz and Theofan, 1979; Nagahama et al., 1983; Pankhurst, 1985; Goetz and Cetta, 1985; Upadhyaya and Haider, 1986). In these species, 17~,20/?-dihydroxp4-pregnen-3-one (17r,20/?-P) is the most effective steroid inducer of oocyte maturation in uitro. This steroid has also been identified as the natural maturation-inducing hormone in the amago salmon ~~co~hy~chus rhodurus (Nagahama and Adachi, 1985). However in two freshwater teleosts, the Indian catfish ~e~~ro~~e~stes fos~ilis (Goswami and Sundararaji, 1974) and the zebrafish ~rachyda~io rerio (Van Ree et al., 1977), corticosteroids are the most effective steroids for inducing oocyte maturation. In contrast, comparativety little is known concerning steroid mediation of oocyte maturation in marine teleosts, including mummichog Funduius heteroclitus (Wallace and Selman, 1978; Greeley et al., I986), striped mullet Mugil cephalus (Wanshu and Thomas, 1987), red sea bream Pagrus major (Adachi et al., 1988), Atlantic croaker Micropogonius unduiutus __.” -.

.-_1_ Author to whom proofs should be sent: Micbiya ~atsuy~ma, Department of Fisheries, Faculty of Agriculture, Ky~shu University, Fukuoka 812, Japan.

(Trant and Thomas, 1988) and tobinumeri-dragonet Repornucenus beniteguri (Zhu et al., 1989). Although, in these fishes, 17cr,2Op-Pa,gain appears to be one of the most effective inducer of oocyte maturation in vitro, in Atlantic croaker, closely related C21 steroid 17c1,20/&21-trihydroxy-4-prenen-3-one (17a,20/?,21-P) has been proposed as the maturation-inducing steroid (MIS). The Japanese whiting, Sillago jffpon~cu, is a marine teleost which spawns every day between 19~2400 hr during the spawning season. from June to September (Kashiwagi et al., 1984). Recent studies have demonstrated that this fish has diurnal rhythm of oocyte development (Kobayashi et al., 1988) and the secretion of two major ovarian steroid hormones in teleosts, estradioi-178 and l?a,20/3-P (Matsuyama et cd., 1990). However, it is still unclear whether 17a,20fl-P is the natural MIS OF not in Japanese whiting. The present study used in vitro incubation techniques to determine the relative effectiveness of the eleven steroids in inducing oocyte maturation in follicle~nclosed oocytes of the Japanese whiting ip2vitro in an additional marine species considering germinal vesicle breakdown (GVBD) as the criterion of oocyte maturation, and to provide preliminary evidence concerning the relationship between the structure of steroids and their ability to initiate Japanese whiting oocyte maturation in vitro. MATERIALS

AND METHODS

Adult Japanese whiting were caught by angling from the coast near the Fisheries Research Laboratory of Kyushu University, Tsuyazaki, Fukuoka Prefecture, Japan, and transferred to the laboratory. Fish were kept in a round 1000 liter tank with running 26°C sea water and natural photoperiod, and fed twice a day with frozen mysid. In the presence of the males, females usually spawn in the early evening every day during the breeding season. Ovaries were sampled between OSO~-10~ hr when the largest oocytes

257

MICHIYAMATSUYAMA ef al

258

completed vitellogenesis (Matsuyama et al., 1990) and GVBD could be induced by steroids in only such oocytes (Kobayashi et al., 1988). The present study was conducted in August, 1989. Fish were killed by decapitation, their ovaries removed and placed in ice-cold Leibovitz’s L-15 culture medium (GIBCO), buffered with 0.02 M HEPES, pH adjusted to 7.5 with 1 N NaOH. Gentamycin sulfate (200 mg/l, Sigma) were added at the time of setting up the assay. Fully grown, postvitellogenic folliculated oocytes were isolated from ovarian tissue and 10 oocytes were placed in each well of plastic tissue culture plates (24 wells, Falcon) containing I ml medium in the continuous presence or absence of various concentrations of steroids. Steroids were dissolved in ethanoi. Ten microliters of hormone solution was added to the well to give the final concentration. Ten microliters of ethanol was added as a control for the steroid hormones. All steroids besides 17a,20&21-P (obtained from Stelaroid) were purchased from Sigma. Table 1shows the steroids used in this study, the shorthand notation by which they have been referred to in the text. The oocytes were incubated for 20 hr at 25°C in a temperature controlled incubator in an atmosphere of 100% air. All treatments were tested in triplicate. Following the incubation period, the medium was replaced with clearing solution of ethanol, formalin, and glacial acetic acid (6: 3: 1) (Goetz and Bergman, 1978), and the oocytes were inspected for GVBD under a dissecting microscope.

transparent and increase their size due to hydration. Subsequent to GVBD, ovulation is observed in all oocytes which had undergone GVBD. A detailed time course study on oocyte maturation in vim and in vitro will be reported in a separate paper. Table 2 shows the percentage GVBD induced by various steroids. Three 20fl-dihydroprogetins, 17cr,2Ofl-P, 20/?-P, and 17c(,2Ofi,21-P were able to induce GVBD in prematuration oocytes at concentrations as low as 0.1 ng/ml. Of these progestins, 17u,2Ofi-P was the most effective, inducing 72.2% GVBD at a concentration of 0.1 ng/ml. At this concentration, 208-P and 17u,20~,21-P induced 33.3 and 6.7% GVBD, respectively. Progesterone and DOC were relatively less stimulatory and were effective at a concentration of 1.0 ngjml. Of the stimulatory steroids tested, 17a-hydroxyprogesterone (17a-P) and pregnenolone were the least effective and induced GVBD at a relatively high concentration (10 ng/ml). In every incubation tested, cortisol, testosterone, 11-ketotestosterone and estradiol-17/J were totally ineffective in inducing GVBD. No GVBD occurred in the hormone-free medium. DISCUSSION

The results of the present study demonstrated that a number of different steroids at relatively high doses (10 ng/ml )--including pregnenolone, DOC and various progestins-can initiate maturation of Japanese whiting oocytes in vitro. Similar results have been reported in many teleost species examined to date. Such an ubiquitous response of oocytes to various steroids is probably due to the metabolism of many steroids to more active forms by some component of the follicular investments as suggested by Greeley et al. (1986). Although the results of the present study demonstrated that a number of different steroids can initiate maturation of Japanese whiting folliclesenclosed oocytes in vifro, there appears to be a common pattern in specificities of the steroids tested, i.e. 20~-dihydroprogestins appear to be far more effective at inducing maturation intact follicles than other steroids, especially at low and physiologically more relevant doses. 20/3-Hydroxyl progestins are apparently the most effective MIS in vitro in teleosts which have been studied: salmonids (see Canario and Scott, 1988), yellow perch (Goetz and Bergman, 1978; Goetz and Theofan, 1979), rock bass (Goetz and

RESULTS

The yolk of prematuration oocytes at the start of the incubation is translucent, and the germinal vesicles (GV) are all in a central position of the oocytes. The migration of the GV to the oocyte periphery becomes apparent 4-8 hr after exposure to steroids and the process of GVBD is completed by 8-12 hr. Oocytes undergoing GVBD are always Table 1. Systematic names and abbreviations of the steroids used in this study Steroid

Abbreviations

3B-Hydroxy-S-pregnen-20-one 4~Pregnen-3,2Odione 17~Hydroxy-4-pregnen-3,20-dione 208-Hydroxy-4-pregnen-3-one 17a.2O~-Dihydroxy-4-pregnen-3-ona 17~,20~,2l-Trihydroxy-4-pregnen-3-one 21-Hydroxy-4-pregnen-3,20-dione I lJLl7a.2l-Trihydroxy-4-pregnen-3.20-dione 178 -Hydroxy-4-androsten-3-one I7fl-Hydroxy-4-androsten-3,l I-dione 3.17B-Dihydroxy-1.3.5]10]-estratrien

Pre~eno~one Progesterone 171--P M/?-P 17a,ZOfl-P 17a,2O/J,2l-P DOC Cortisol Testosterone I I-Ketotestosterone Estradiol-17fl

Table 2. In citro effects of various steroids on the percentage of Silrogo joponica oocytes to complete GVBD Concentration Treatment Pregnenolone Progesterone l7or-P 200-P 17a,ZOp-P 17a,20~,21-P DOC Cortisol Testosterone I I-Ketotestosterone Estradiol-l7/3

fng/ml)

100

IO

I

66.7 + 19.3 88.9+ 11.1 83.3 + 16.7 93.3 f 6.7 100 91.7 of 8.3 91.7 k 8.3 0 0 0 0

51.1 k 24.8 38.9 + 20.0 80.6 * 10.0 91.7 + 8.3 100 88.9 + II.1 75.0 f 14.4 0 0 0 0

0 4.7 + 6.7 0 65.5 & 7.8 loo 44.4 * 15.5 16.7 ?r:8.3 0 0 0 0

0.1

0 0 0 33.3 * 33.3 72.2 + 14.7 6.7 k 6.7 0 0 0 0 0

Control All data represent the mean + SEM of three replicates from a single donor fish.

0.01

0

0 0 0 0 0 0 0 : 0 0 0

In vitro oocyte maturation in Japanese whiting Cetta, 1985) goldfish (Jalabert, 1976; Nagahama et al., 1983), medaka (Iwamatsu, 1980), Indian catfish (Mystus vittatus; Upadhyaya and Haider, 1986), mummichog (Greeley et al., 1986), striped mullet (Wanshu and Thomas, 1987), Atlantic croaker (Trant and Thomas, 1988), and tobinumeri-dragonet (Zhu et cd., 1989). In those teleosts, 17a,2Ofl-P is typically the more effective in the 20/J-hydroxyl progestins at initiating oocyte maturation in vitro except mummichog and Atlantic croaker-20B-P and 17a,20/$21-P are at least the equal of 17cr,208-P, respectively. Furthermore, 17a,20/?-P has been shown to be the natural maturation-inducing steroid in the amago salmon (Nagahama and Adachi, 1985). In addition to the importance of a 20B-hydroxyl group to the maturation-inducing ability of a steroid in Japanese whiting, additional steroid structurefunction relationships are suggested by the present study. For instance, the present results suggest that aromatization of the A ring abolishes the maturationinducing ability of a steroid (i.e. estradiol-178). Cl8 steroids, including estradiol-17/J, are generally totally ineffective in inducing oocyte maturation in most other teleosts examined to date (Goswami and Sundararaj, 1974; Goetz and Bergman, 1978; Young et cd., 1982; Goetz and Cetta, 1985; Upadhyaya and Haider, 1986; Greeley et al., 1986; Adachi et al., 1988; Zhu et al., 1989), with the apparent exception of the zebra fish (Van Ree et al., 1977) and the goldeye (Pankhurst, 1985). In several teleosts, Cl9 steroids, especially testosterone, have been shown to induce GVBD only at high concentrations (Goetz and Bergman, 1978; Young et al., 1982; Goetz and Cetta, 1985; Pankhurst, 1985; Greeley et al., 1986; Trant and Thomas, 1988; Adachi et al., 1988). In two catfish, H. fossilis (Goswami and Sundararaj, 1974) and M. &tutus (Upadhyaya and Haider, 1986), and in tobinumeri-dragonet (Zhu et al., 1989), testosterone is not effective in inducing oocyte maturation, which is in good agreement with the present study as well. The present results further suggest that a hydroxyl group at the 17-crposition does not appear necessary. Similar results are found in mummichog (Grceley et al., 1986) which has a steroid without this group (20/?-P) been shown to be as biologically active as 17a,20/3-P. 17a,20/?,21-P has recently been isolated and positively identified in maturing ovary of Atlantic croaker (Trant et al., 1986). In the 21-hydroxylated C21 steroids tested in the present study, cortisol was totally ineffective in inducing oocy te maturation of Japanese whiting, but DOC and 17a,20/?,21-P showed relatively high maturationinducing ability. The role of the 21-hydroxyl group in the lateral chain is unknown. However, it has been shown in rainbow trout (Canario and Scott, 1988) and .4tlantic croaker (Trant and Thomas, 1988), that 17a,20/?,21-P is as active as 17a,2OB-P in inducing in vitro oocyte maturation, suggesting that in these two species, the effect of the 21-hydroxyl group appears to be negligible. We therefore suggest that the present results reflect the relative importance of a 20p-hydroxyl group for the maturation-inducing activity. The concentration of steroid that induced GVBD in 50% of the Japanese whiting oocytes (median effective dose: MED,,) ranged from 0.01 to

259

0.1 ng/ml for 17a,20/?-P. Comparative measurements from salmoniform fish (Duffey and Goetz, 1980; Nagahama et al., 1983; Jalabert and Fostier, 1984; Canario and Scott, 1988), yellow perch (Goetz and Theofan, 1979), goldfish (Jalabert, 1976; Nagahama et al., 1983), rockbass (Goetz and Cetta, 1985) goldeye (Pankhurst, 1985), catfish (Upadhyaya and Haider, 1986), Atlantic croaker (Trant and Thomas, 1988), striped mullet (Wanshu and Thomas, 1987) and mummichog (Greeley et al., 1986) lie in the range 0.2 to 340 ng/ml. Comparison of the MEDSo for 17a,208-P-induced GVBD in fishes studied previously versus Japanese whiting oocytes suggest that Japanese whiting oocytes are more sensitive to 17a,208-P. In fact, the serum peak level of 17a,208-P in Japanese whiting is very low (0.1 ng/ml; Matsuyama et al., 1990) compared with the 30-400 ng/ml reported in salmonids (Young et al., 1983; Scott et al., 1983; Yamauchi et al., 1984; Dye et al., 1986; Fitzpatrick et al., 1986; Truscott et al., 1986) and in cyprinids (Shimizu et al., 1985; Kobayashi et al., 1987; LevaviZermonsky and Yaron, 1986). Thus, this difference between species may be due to a difference in oocyte sensitivity to follicular 17cr,2O#l-Pconcentration; it is likely that the Japanese whiting oocytes can undergo GVBD at lower levels of follicular 17a,208-P than those of other fishes which have been studied. The MED, value for 17a,2Ofi-P in the Japanese whiting oocytes is fairly consistent with the result from red sea bream, a daily spawning marine teleost like Japanese whiting (Adachi et al., 1988). This highest sensitivity to 17a,20B-P in fully-grown oocytes might be a characteristic of marine fish with daily spawning cycle. In most fish species ovulation does not follow in vitro steroid-induced final oocyte maturation, with the apparent exception of the yellow perch (Goetz and Theofan, 1979; Berndtson et al., 1989). As described above, Japanese whiting has a regular spawning cycle of 24 hr during the spawning season and oocytes of this species ovulate predictably in vitro following maturation-inducing steroids stimulation. These features make Japanese whiting an interesting model for the study of the endocrine control of ovulation as well as oocyte maturation, and this well incubation procedure of Japanese whiting oocytes will provide interesting informations on hormonal control of oocyte maturation and ovulation of teleost in future. In conclusion, the present study showed that 17a,20/?-P was the most effective maturationinducing hormone for culture of Japanese whiting oocyte in vitro. Our recent study on the diurnal rhythm of oocyte development and serum levels of 17a,2Op-P in Japanese whiting demonstrated that 17a,2OB-P showed a cyclical change in the day with a peak during maturation of the largest oocytes (Matsuyama et al., 1990), indicating that this steroid plays important roles in oocyte maturation of Japanese whiting. Considering these data taken together, the bioassay results in the present study suggest that 17a,20/?-P might be the natural MIS of Japanese whiting. However, since supportive physiological and biochemical data are lacking, further such studies are necessary to determine the true MIS of Japanese whiting.

MICHIYAMATSUYAMA et al.

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