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Oocyte maturation in the mummichog (Fundulus heteroclitus): Effects of steroids on germinal vesicle breakdown of intact follicles in vitro
GENERAL
AND COMPARATIVE
ENDOCRINOLOGY
62, 281-289 (1986)
Oocyte Maturation in the Mummichog (fundulus heteroclitus): Effects of Steroids on Germinal Vesicle Breakdown of Intact Follicles in Vitro MARK
S. GREELEY,
JR., DAN R. CALDER, MALCOLM AND ROBIN A. WALLACE
H. TAYLOR,*
HANS
HoIs,?
Whitney Laboratory, University of Florida, St. Augustine, Florida 32086; *School of Life and Health Sciences, University of Delaware, Newark, Delaware 19716; and +State University of Utrecht, 8 Padualuan, 3508 TB Utrecht, The Netherlands Accepted December 15, 1985 The effects of several steroids on the maturation of follicle-enclosed oocytes of the mummichog Fundalas heteroclitus in vitro were examined. At a relatively high concentration (1 .O pgiml), a number of different steroids, including pregnenolone, 17a-hydroxypregnenolone, corticosterone, cortisol, 1I-deoxycorticosterone. 1I-deoxycortisol, androstenedione. testosterone, progesterone, 17o-hydroxyprogesterone, 20@-dihydroprogesterone, and 17ahydroxy-20P-dihydroprogesterone, were able to induce germinal vesicle breakdown (GVBD) in prematuration oocytes. Cholesterol, 17P-estradiol, 11-ketotestosterone, and 1lp-hydroxytestosterone were totally ineffective. In general, 1I-oxysteroids tended to be less effective than their 11-deoxysteroid counterparts. Two 20@-dihydroprogestins-17o(hydroxy-20lSdihydroprogesterone and 20P-dihydroprogesterone-were the most potent maturation-inducing steroids, initiating 50% GVBD at 1 &ml in follicles obtained from ovaries containing mature or maturing follicles in viva, or at 2.5-4.0 rig/ml in follicles from ovaries lacking mature or maturing oocytes in laivo. These results are consistent with sevi era1 previous studies involving salmonids and various other teleosts, and suggest a possible physiological role for a 20lSdihydroprogestin in the resumption of meiotic maturation in F. heteroclitus. 0 1986 Academic Press. Inc.
The oocytes of teleost fish are arrested in meiotic prophase I during most of their development and growth, subsequently resuming meiotic maturation at the end of this period in response to an appropriate hormonal effector. Numerous steroids have been reported to be capable of stimulating teleost oocytes to resume meiotic maturation in vitro (for review, see Goetz, 1983). However, much of this work has focused on only one group of teleosts of particular economic significance-the salmonids. In many salmonids, including rainbow trout, Salrno gairdneri (Jalabert, 1976; Nagahama et al., 1983), brook trout, Sulvelinus fontinalis (Duffey and Goetz, 1980), coho salmon, Oncorhynchus kisutch (Sower and Shreck, 1982), amago salmon, Oncorhynchus rhodurus, and the ayu, Pleco-
glossus altivelis (Nagahama et al. 1983), one steroid, 17a-hydroxy-20@dihydroprogesterone (17a-OH-20@diPROG), appears to be the most effective at inducing maturation in vitro. This steroid has also been identified in the blood and plasma of several salmonids (Scott et nl., 1982; IIirose et al., 1983; Kagawa et al., 1983), is produced by salmonid follicles irz vitro (Fostier et al. ~ 1981; Suzuki et al., 1981; Nagahama and Kagawa, 1982; Young et al., 19831, and, just recently, has been identified as ‘the natural maturation-inducing steroid in the amago salmon (Nagahama and Adachi, 1985). In cantrast, comparatively little is kriown concerning steroid mediation of oocyte maturation in nonsalmonid teleosts, for to date only a few of the innumerable nonsal281 0016-6480186 $1.50 Copyright D 1986 by Academic PKSS, Inc. All rights of reproduction in any form reserved
282
GREELEY
monid teleost species have been examined in this respect. In some, including medaka, Oryzias latipes (Iwamatsu, 1978, 1980), yellow perch, Perca flavescens (Goetz and Theofan, 1979), goldfish, Carassius auratus (Nagahama et al.,, 1983), goldeye, Hiodon alosoides (Pankhurst , 1985)) and rock bass, Ambloplites rupestris (Goetz and Cetta, 1985), 17a-OH-20@diPROG again appears to be one of the most effective initiators of oocyte maturation in vitro. However, in other nonsalmonid teleosts, notably the Indian catfish, Heteropneustes fossilis (Goswami and Sundararaj, 1974) and the zebrafish, Brachydanio rerio (van Ree et al., 1977), corticosteroids have been reported to be more effective than progestins. We herein examine the relative ability of several physiological steroids, including various progestins and corticosteroids, to induce maturation in vitro of intact follicles from the cyprinodontid teleost Fundulus heteroclitus. In an earlier investigation, progesterone and 1 l-deoxycorticosterone were shown to be equally effective initiators of follicle maturation in vitro (Wallace and Selman, 1978), but comparable data concerning the effectiveness of 17aOH-20P-diPROG are lacking. Thus the purposes of the present study are several: (1) to determine which of these steroids would be most appropriate for future studies involving maturation of F. heteroclitus follicles in vitro, (2) to provide preliminary evidence concerning the relationship between the structure of steroids and their ability to initiate F. heteroclitus follicle maturation in vitro, and (3) to compare the relative effectiveness of these steroids, especially the various progestins and corticosteroids, in initiating follicle maturation in vitro in an additional nonsalmonid species. Evidence is presented that, in F. heteroclitus as in salmonids and a few other teleost species, two 20/3-dihydroprogestins, 17or-OH-20@diPROG and 20P-dihydroprogesterone (20@diPROG), are the most ef-
ET AL.
fective physiological steroids for initiating follicle maturation in vitro. MATERIALS
AND METHODS
Fish and biochemicals. Fundulus were collected during their breeding season-February through September at this location (M. S. Greeley, H. Hols, and R. A. Wallace, manuscript in preparation)-from salt marshes in the vicinity of the Whitney Laboratory near St. Augustine in northeastern Florida, and subsequently held in an outdoor concrete holding tank. To encourage and support vitellogenesis, fish were fed a liberal diet including mullet roe. Biochemicals, including steroids, were obtained from Sigma. Follicles. Fish were killed by decapitation and their ovaries removed. Ovaries were gently pulled apart with watchmakers’ forceps while immersed in FO solution (Wallace and Selman, 1978), and prematuration follicles (see description below) were isolated from connective tissue and other follicles. Culture techniques were essentially those of Wallace and Selman (1978). differing primarily in that a commercially available culture medium (L-15 with glutamine, diluted to 75% with distilled water, and containing 50 mgiml gentamycin) was substituted for a previously employed proprietary medium, FO (FO was still used for maintaining the ovaries during dissection). Although FO is adequate for most culture purposes, 75% L-15 proved superior in the present instance by extending the size range of usable follicles, as compared with FO (Fig. 1). For the purposes of this study, prematuration follicles were considered to be those vitellogenic follicles, 1.2-1.4 mm in diameter, that demonstrated no obvious visual indications of yolk clarification, which indicates the onset of maturation in this species (see Fig. 2). Such follicles underwent 100% germinal vesicle breakdown (GVBD) in response to steroid in a preliminary experiment with 75% L-15 culture media, but did not mature in vitro without the addition of exogenous steroid (Fig. 1). Maturing follicles are defined as those which still contain a germinal vesicle (GV), but are distinctly clearing and thus considered to be in the early stages of maturation. In contrast to prematuration follicles, follicles designated as maturing on the basis of observed yolk clearing underwent maturation in vitro with or without the addition of exogenous steroid to the incubation medium. Mature follicles are herein defined rather broadly, so as to include both preovulatory follicles lacking a germinal vesicle and ovulated eggs. Initial screening of steroids. Prematuration follicles from 6 to 10 ovaries, containing neither mature follicles nor maturing follicles in viva, were pooled for each set of steroid trials. From each pool, individual aliquots of IO-15 follicles were withdrawn and placed in 10x35mm plastic petri dishes containing 3 ml of
Fundulus
OOCYTE
IOO-
ao-
60 9
1
1 0 8
I
I
:
40-
i
i
/
/
:’
283
cyclic ovarian environment of a follicle and the concentrations of 17~0H-ZIP-diPROG and 20p-diPROG required to induce maturation, additional p001s of prematuration follicles were constructed, this time from ovaries with or without maturing follicles in !&o. Aliquots of follicles from these pools were then tested against various concentrations of each of the above steroids.
RESULTS
I
/’
/’
in Vitro
I i
: t
20-
MATURATION
Maturation
in Vitro
In F. heteroclitus, the yolk of prematuration follicles is somewhat translucent, I 0.8 0.9 I .o /_I 1.2 I .3 I .4 I .5 rather than totally opaque as in many other teleosts, and the nucleus or GV-which Follicle diameter (mm) migrates from the interior of the follicle to, FIG. 1. Comparison of FO and 75% L-15 media for F. hetevoclitrrs foIIicle culture in vitro. Follicles from the periphery prior to the resumption of 3.8 mm in diameter (early vitellogenesis) through 1.4 maturation -is highly visible. In consemm in diameter (fully grown, prematuration follicles) quence, important changes within the foland 1.5 mm in diameter (early maturation, yolk especlearing but GV still present) from a single ovary were licle during the course of maturation, cially GVBD, are readily observed without incubated with or without 17a-hydroxy-20p-dihydroprogesterone (0.1 &ml) in either FO or 75% L-15. the necessity of chemical clearing or disEarly-vitellogenic follicles (0.8-0.9 mm in diameter) section of the follicle. could not be induced to undergo GVBD under any The first visual indications of the onset conditions. Some mid-vitellogenic follicles (1 .O- 1.1 of in vitro follicle maturation in F. here+ mm in diameter) did undergo GVBD in response to the clitus include an accelerated clarification of steroid, but only in 75% L-15. Late vitellogenic follicles (1.2- 1.4 mm in diameter) could be induced by the yolk, and an increase in the,size of the the steroid to undergo GVBD in either FO or 75% follicle due to hydration (Wallace and L-15, although more consistently in the latter medium. Selman, 1978, 1983, both events ‘occurring Both in FO and 75% L-15, only those folIicles 1.5 mm 16 hr following exposure to in diameter and clearing in viva underwent GVBD in approximately a stimulatory steroid (Fig. 2). Between 2 r~irru with ar without (controls) the addition of steroid to the culture medium. and 24 hr the nucleus begins to brea down, a process normally completed by 28 medium. Steroids of appropriate concentration (either hr. Subsequent to GVBD, the follicle un1.0, 0.1, or 0.01 &ml) were added in 10 ~1 ethanol; dergoes additional size increases due to ‘hycontrols contained medium and ethanol alone. Unless dration, and oil droplets continue to cootherwise stated in the text, incubations were conalesce. Other than increasing atresia, howducted for 40 hr at 21°C in a temperature-controlled ever, few further changes occur in the incubator. At the end of this period, follicles were scored for the occurrence of GVBD by utilizing a dis- follicle after 40 hr-the length ‘of the secting scope and transmitted’ light to facilitate present incubations. scoring. In nature, certain typical’ post&VI3 Each steroid was tested in three to five separate events within the F. heteroditus ,folticle, trials, each trial as described above. The resultssuch as ad’djtional oil droplet Coalescence being similar in separate trials-were then pooled for final presentation. Atretic follicles were disregarded and hydration, occasionally fail to occur as when analyzing results. the oocyte ovulates from surrounding ‘folDose-response CUYV~S.In order to (1) obtain prelicular investments1 immediately f$o&ng cise dose-response curves for the purpose of comGVBD (M. S. Greeley, $3. Hols, D. 13. paring the effectiveness of 17~~qH-20P-diPROG and Calder, and R. A. Wallace, manuscript in 20+3-diPROG at inducing maturation in F. heteroclitus follicles, and (2) examine the relationship between the preparation). We therefore considekd the 0
.-.'--.
----
:-
.-...-.
- . ..-.-.
i
284
GREELEY Preovulotory follicle
1.7-
z G z s a Y ,” 2
Gv;;./i\. 1.6Clearing begun
.’ /
1.5Steroid
v
, .4 -,JT.P:_ ----a--t 0
I
-.--.--.I 8
I
I 16
I
I 24
Time
_ .---I
I 32
c.o:::: I
I 40
I
I 48
ihr)
FIG. 2. Time course of F. hete~~2itus follicle size changes during maturation in vitro initiated by 17~ hydroxy-20P-dihydroprogesterone (0.1 pgiml). Follicle diameters represent the mean for 30 follicles obtamed from a pool derived from five fish which contained neither mature nor maturing follicles in viva. Solid line represents follicles incubated with steroid; dashed line represents control follicles incubated in the absence of exogenous steroid.
occurrence of GVBD to be, in this species, the most consistent and reliable indicator of follicle maturation in viva or in vitro. Ovulation rarely follows maturation of the F. heteroclitus follicle in vitro. Occasionally, however, oocytes induced to resume maturation in vitro begin to ovulate from their follicular investments subsequent to GVBD, but fail to complete ovulation successfully. These oocytes eventually die. In scoring results, such oocytes were counted among those having undergone GVBD, rather than being deleted from consideration as are other atretic follicles, since they had completed GVBD prior to the failed ovulation. Initial
Screening
of Steroids
The F. heteroclitus ovary is very cyclic, oocytes maturing at regular intervalswith lunar or semilunar periodicitythroughout a lengthy breeding season in nature (Taylor and DiMichelle, 1980; M. S. Greeley, H. Hols, and R. A. Wallace, manuscript in preparation) and, to some extent, under laboratory conditions (Taylor, 1984). It was suspected when beginning these studies that the responsive-
ET AL.
ness of F. heteroclitus prematuration oocytes to steroid stimulation of meiotic maturation might also cycle in a similar fashion. Therefore, in an attempt to standardize results, all initial experiments were performed utilizing only prematuration follicles from ovaries in the same stage-in particular, those ovaries which did not already contain any mature or maturing follicles in vivo. Two progestins, 20P-diPROG and 17c~OH-20@diPROG, were the most potent maturation-inducing steroids tested during initial steroid screening experiments, being capable of initiating GVBD at concentrations as low as 0.01 pg/ml (Table 1). Although not nearly as effective, many other steroids were also able to initiate maturation in F. heteroclitus follicles, at least at the relatively high concentration of 1.0 bg/ml (Table 1). However, some of these, including androstenedione and the 1 l-oxygenated corticosteroids (cortisol and corticosterone), were ineffective at concentrations lower than 1.0 pg/ml. Others, such as ll-ketotestosterone and 1 lp-hydroxytestosterone, were either only partially effective at 1.0 pg/ml, or, in the case of cholesterol, 17l%estradiol, and estrone, totally ineffective (Table 1). Follicle
Sensitivity
The relative effectiveness of both 17~3OH-20@diPROG and 20@diPROG in stimulating GVBD in prematuration follicles was further examined in follicles from ovaries in two very different reproductive states: (1) ovaries containing mature or maturing follicles in vivo (approximately equivalent to ovaries obtained in nature from fish during their spring-tide spawning peaks) and (2) ovaries containing neither mature nor maturing follicles in vivo (approximately equivalent to ovaries from wild fish between spring-tide spawning peaks). Prematuration follicles from the former ovaries were found to be much more sensi-
Fundulus
EFFECTS
OF SEVERAL
OOCYTE
STFXOIDS
ON
in Vitro
MATURATION
TABLE 1 GVBD OFF. heteroclitus
1.0 kg/ml Steroid Cholesterol 17P-Estradiol Estrone Androstenedione Testosterone 1I-Ketotestosterone 1l&OH-Testosterone Pregnenolone 17a-OH-Pregnenolone Cortisol Corticosterone 1 I-Deoxycortisol 1 I-Deoxycorticosterone Progesterone 17cr-OH-Progesterone 20@-Dihydroprogesterone 17cL-OH-20P-Dihydroprogesterone
285
FOLLICLES
in
Vitro
0.1 kg/ml
0.01 &g/ml
M
%GVBD
n
%GVBD
n
%GVB5
2s 29 27 30 2s 29 25 25 24 26 44 26 28 28 51 51 69
0 0 0 73 84 21 4 100 92 88 93 70 a9 93 94 82 92
33 29 33 27 32 27 32 46 50 30 42 57 59 71
-
46 45 34 34 45 56 42 45 45 38 60
0 0 9 0 0 0 0 2 2 61 44
tive to steroid stimulation of maturation in vitro than were follicles from the latter ovaries (Fig. 3). Although 100% of the prematuration follicles from the former ovaries underwent maturation in vitro with either 17a-OH-20l3-diPROG or 20P-diPROG at doses as low as 0.005 pgiml (5 ngiml, Fig. 3), some follicles from the latter ovaries failed to respond in vitro even at relatively high steroid concentrations (0.1-0.01 kg/ml, Table 1 and Fig. 3). In fact, while 2.5-4.0 rig/ml of these steroids was required to cause GVBD in 50% of the prematuration follicles obtained from ovaries containing neither mature nor maturing follicles in vivo, only 1 rig/ml was required to attain comparable results with prematuration follicles obtained from ovaries containing mature or maturing follicles in vivo. Relative Effectiveness of the 20fHlihydroprogestins Both 17cx-OH-20@-diPROG and 2OP-diPROG ,were equally effective at inducing maturation in F. heteroclitus follicles, although 20pldiPROG tended to be slightly
0 62 0 0 75 63 3 13 70 70 64 91 97 81
more effective than 17at-OH-20@-diPROG with the least responsive follicles (Table 1 and Fig. 3). The time courses of follicle
FIG. 3. Comparative dose-response curves for 20P-dihydroprogesterone and 17cu-hydroxy-20&d&ydroprogesterone initiation of F. heteroclirus follicle maturation in vitro. Each point represents a percentage GVBD, after 40-hr incubations in 75% L-15, of 32 I 1 follicles tested in three separate trials. Two pools of prematuration follicles (1.2- 1.4 mm in diameter) for each trial were derived from 6 to 10 ovaries apiece: one pool of follicles from ovaries akeady containing mature or maturing follicles in I&O, and one pool of follicles from ovaries containing neither mature nor maturing follicles ifi viva.
286
GREELEY
maturation in V&O, stimulated by either 17ol-OH-20P-diPROG or 20@-diPROG, were also essentially identical (Fig. 4). DISCUSSION
The results of the present study demonstrate that a number of different steroids at relatively high doses (1.0 kg/ml)-ineluding testosterone and various progestins and corticosteroidszcan initiate maturation of F. heteroclitus follicles in vitro. Such a ubiquitous respon-se of a follicle to various steroids at high doses is not unusual among the teleosts examined to date (Goetz, 1983), and is probably due, at least in part, to the metabolism of many steroids to more active forms by some component of the follicular investments. However, in F. heteroclitus as well as many other teleosts, there is one particular class of steroids-the 20@dihydroprogestinsthat appears to be far more effective at inducing maturation in intact follicles than other steroids, especially at lower and physiologically more relevant doses.
100
1
80 i
-_ ,_ -__ 0
8
I6
24 Time
32
46
48
(hr)
FIG. 4. Time course of GVSiI in F. heteroclitus follicles in vitro initiated by either 20@dihydroprogesterone or 17oi-hydroxy-aOp-dihydroprogesterone (0.1 pgiml). Each treatment group was composed of 35 prematuration follicles (1.2-1.4 mti in diameter tirematur&ion size) taken from a single pool derived from three ovaries which contained neither mature nor ma: turing follicles in vivo.
E;T AL.
20&Dihydroprogestins are apparently the most effective maturation-inducing steroids in vitro in several teleosts, including rainbow trout, S. gairdtieri (Jalabert, 1976; Nagahama et al., 1983), yellow perch, P. jluvescens (Goetz and Theofan, 1979), brook trout, S. fontinalis (Duffey and Goetz, 1980), coho salmon, 0. kisutch (Sower and Shreck, 1982), amago salmon, 0. rhodurus, ayu, P. altivelis, goldfish, C. auratus (Nagahama et al., 1983), rock bass, A. rupestris (Goetz and Cetta, 1985), medaka, 0. latipes (Iwamatsu, 1978, 1980), and now the mummichog, F. heteroclitus. In those teleosts in which both 17a-OH20P-diPROG and 20@-diPROG have been examined in sufficient detail-the present results being a notable exception17crOH-2O@diPRdG is typically the more effective of the two at initiating follicle maturation in vitro. Furthermore, 17a-OH-20l3diPROG has recently been shown to be the natural maturation-inducing steroid in the amago salmon (Nagahama and Adachi, 1985). Thus, this particular 20@dihydroprogestin is at this time the best candidate for a universal maturation-inducing steroid in the teleosts-if such exists-despite the occasional apparent exception such as the Indian catfish (Goswami and Sundararaj, 1974). As indicated above, the present results are somewhat unusual in that 20l3-diPROG is at least the equal of 17a-OH-20/3-diPROG at initiating follicle maturation in vitro in F. heteroclitus. In fact, the similarities of the respective time courses and dose-response curves argue that the modes of action of both steroids are identical. However, the physiological significance of this observation is not immediately apparent. Both steroids could be converted to another as yet unidentified active steroid in the follicle cells prior to oocyte stimulation, or both could be structurally mimicking the action of the other or another steroid in directly facilitating oocyte maturation. We therefore suggest the
Fmdulus
OOCYTE
MATURATION
present results reflect the relative importance of a 2OP-hydroxyl group for the maturation-inducing activity of a steroid, and defer for the present the question of whether either 17cx-OH-2OP-diPROG or 2Op-diPROG is actually a natural maturation-inducing steroid in this fish. In addition to the apparent importance of a 20@-hydroxl group to the maturation-inducing ability of a steroid in F. heteroc&us, additional, but admittedly preliminary, steroid structure-function relationships are suggested by the present results. For instance, a hydroxyl group at the 17~~ position does not appear necessary. Similar results are found in amphibians, where a 17ol-hydroxyl group is even reported actually to inhibit the maturation-inducing activity of a steroid (Morrill and Bloch, 1977). In F. heteroclitus, a hydroxyl or ketone at the 11 position appears to inhibit steroid activity (i.e., 11-oxygenated testosterone and corticosteroid derivatives); such also seems to be the case in several other teleosts (see Goetz, 1983). The present results further suggest that aromatization of the A ring abolishes the maturation-inducing ability of a steroid (i.e., the estrogens); similar results have been reported for most other teleosts examined to date, with the apparent exception of the zebrafish, B. rerio (van Ree et al., 1977), in which es&one, but not 17p-estradiol or estrio& stimulates maturation. We caution that these steroid structure-function relationships are necessarily tenuous. The F. heteroclitus follicle has proven notoriously resistant to defolliculation; thus we are currently unable to distinguish direct from indirect steroid effects. The effective concentrations of progesterone, DOG, and cortisol in the preliminary screening experiment of the present study were approximately 10 times, higher than reported in a previous study with F. hetevloclitus (Wallace and Selman, 1978). The follicles employed in the previous study were obtained’from ovaries that con-
in Vitro
287
tained follicles in all stages of development, including maturation; thus, those follicles may have been primed or sensitized in vivs by endogenous gonadotropins or steroids I In contrast, follicles employed in the screening portion of the present study were selected from .FunduZus ovaries lacking mature or maturing oocytes in viva, and thus were apparently relatively insensitive to stimulation, as evidenced by (1) controls never undergoing GVBD, and (2) at least some oocytes in nearly all incubations failing to undergo GVBD even with the addition of steroids to the culture medium. Additional trials of 17a-OH-20@diPROG and 20P-diPRQG at various concentrations were therefore conducted with pools of follicles derived from ovaries either with or without actively maturing follicles in v&o. The purposes of this set of experiments were (1) to examine further the potential effects of the prematuration follicle’s environment on the sensitivity of the follicle to steroid stimulation of maturation, and (2) to determine more accurately the comparative effectiveness of 17m-OH-20@-diPROG and 2Op-diPROG. The present results show the F. heterocfitus prematuration follicle to be relatively sensitive to steroid stimulation when the ovary from which it was obtained contained other follicles undergoing maturation in viva, and relatively insensitive when the ovary did not contain any maturing follicles in Co. This suggests that the follicle is in some manner “primed” in the former ovaries, possibly by endogenous gonadotropin or steroids. To date, the potential for differential responsibeness of’ apparently similar follicles to steroid s~irn~la~~on at different stages of an ovarian cycle has been poorly addressed in teleosts. This is unfortunate, as the reasons for such variation in follicle sensitivity, if understood, could colntribute great,ly to an understanding of the physiological mechanisms underlying the hormonal coNntrol of both follicle maturation and reproductCve cycles
288
GREELEY
in general, and thus deserve greater attention than heretofore accorded. In conclusion, we report that 20P-diPROG and 17a-OH-20P-diPROG are potent maturation-inducing steroids for culture of Fundulus oocytes in vitro. These resuits suggest that a steroid with a similar structure might be the natural maturationinducing steroid of F. heteroclitus, possibly even 17a-OH-20P-diPROG itself since (1) this steroid has already been identified as the maturation-inducing steroid in one teleast, the amago salmon (Nagahama and Adachi, 1985), and (2) this steroid has recentlv been identified bv radioimmunoassay in media in which F. hetevoclitus follicles were stimulated to mature by Fundulus pituitary extracts (Lin et al. 1985). ACKNOWLEDGMENTS This work was supported 8202512toR.A.W.
by NSF Grant PCM
REFERENCES Duffey, R. J., and Goetz, E W. (1980). The in vitro effects of 17a-hydroxy-20P-dihydroprogesterone on germinal vesicle breakdown in brook trout (Salvelinus fontinalis) oocytes. Gen. Camp. Endocrinol. 41, 563-565. Fostier, A., Jalabert, B., Campbell, C., Terqui, M., and Breton, B. (1981). CinCtique de lib&ration in vitro de 17a-hydroxy-20P-dihydroprogesterone par des follicles de Truite arc-en-ciel, Salmo gairdnerii. C.R. Acad. Sci. Ser. 3 292, 777-780. Goetz, E W. (1983). Hormonal control of oocyte final maturaiion and ovulation in fishes. In “Fish Physiology” (W. S. Hoar, D. J. Randall? and E. M. Donaldson, eds.), Vol. 9, pp. 117-170. Academic Press, New York. Goetz, F. W., and Cetta, F. (1985). Zn vitro steroid stimulation of final maturation in oocytes of rock bass (Ambloplites rupestris). Reprod. Nutr. Develop. 25, 33-38. Goetz, E W., and Theofan, G. (1979). In vitro stimulation of germinal vesicle breakdown and ovulation of yellow perch (Perca flavescens) oocytes. Effects of 17a-hydroxy-20p-dihydroprogesterone and prostaglandins. Gen. Cornp. Endocrinol. 37, 273-285.
Goswami, S. V., and Sundararaj, B. I. (1974). Zn vitro maturation and ovulation of oocytes of the catfish, Heteropneustes fossilis (Bloch): Effects of mammalian hypophyseal hormones, catfish pitui-
ET AL. tary homogenate, steroid precursors and metabolites and gonadal and adrenocortical steroids. J. Exp. 2001. 178, 467-478. Hirose, K., Nagahama, Y., Adachi, S., and Wakabayashi, K. (1983). Changes in serum concentrations of gonadotropin, 17&,20@dihydroxy-4-pregnene-3-one during synthetic LH-RH-induced final oocyte maturation and ovulation in the Ayu Plecoglossus altivelis. Bull. Japan. Sot. Sci. Fisheries 49, 1165- 1169. 1wamatsu, T. (1978). Studies on oocyte maturation of the Medaka, Oryzias latipes. V. On the structure of steroids that induce maturation in vitro. J. Exp. Zool.
204, 401-408.
Iwamatsu, T. (1980). Studies on oocyte maturation of the Medaka, Oryzias latipes. VIII. Role of follicular constituents in gonadotropin- and steroidinduced maturation of oocytes in vitro. J. Exp. Zool.211,231-239.
Jalabert, B. (1976). In vitro oocyte maturation and ovulation in rainbow trout (Salmo gairdneri), northern pike (.&ox lucius), and goldfish (Carassius auuatus). J. Fish. Res. Board Canad. 33, 974-988. Kagawa, H.,
Young, G., and Nagahama, Y. (1983). Changes in plasma hormone levels during gonadal maturation in female goldfish Cavassius auratus. Bull. Japan. Sot. Sci. Fish. 49, 1783-1787. Lin, Y.-W. i., LaMarca, M. J., and Wallace, R. A. (1985). Bioassay for Fund&s gonadotropins: In vitro oocyte maturation and steroid production by isolated ovarian follicles. Amer. Zoo/. 25, 133A. Morrill, G. A., and Bloch, E. (1977). Structure-function relationships of various steroids relative to induction of nuclear breakdown and ovulation in isolated amphibian oocytes. 1. Steroid Biochem. 8, 133-139. Nagahama, Y., and Adachi, S, (1985). Identification of maturation-inducing steroid in a teleost, the amago salmon (Oncorhynchus rhodurus). Dev. Biol. 109, 428-435. Nagahama, Y., Hirose, K., Young, G., Adachi, S., Suzuki, K., and Tamaoki, B.-l. (1983). Relative in vitro effectiveness of 17a,20p-dihydroxy-4pregnen-3-one and other pregnene derivatives on germinal vesicle breakdown in oocytes of ayu (Plecoglossus altivelis), amago salmon (Oncorhynchus rhodurus), rainbow trout (Salmo gairdneri), and goldfish (Carassius auratus). Gen. Camp. Endocrinol. 51, 15-23. Nagahama, Y., and Kagawa, H. (1982). In vitro steroid production in the postovulatory fdllicles of the amago salmon, Oncorhynchus rhodurus. in response to salmon gonadotropin. J. Exp. Zool. 219, 105-109. Pankhurst, N. W. (1985). Final maturation and ovulation of oocytes of the goldeye, Hiodon alosoides
Fundulus OOCYTE
MATURATION
(Rafinesque), in vitro. Canad. J. Zool. 63, 1003-1009. Scott, A. P., Sheldrick, E. L., and Flint, A. P. F. (1982). Measurement of 17a,20B-dihydroxy-4pregnen-3-one in plasma of trout (Salvo gairdneri Richardson): Seasonal changes and response to salmon pituitary extract. Gen. Comp. Endocrinol. 46, 444-451. Sower, S. A., and Shreck, C. B. (1982). In vitro induction of final maturation of oocytes from coho salmon. Trans. Amer. Fish. Sot. 111, 399-403. Suzuki, K.. Tamaoki. B.-I., and Nagahama, Y. (1981). In vitro synthesis of an inducer for germinal vesicle breakdown of fish oocytes, 17a,20B-dihydroxy-4-pregnen-3-one, by ovarian tissue preparation of amago salmon (Oncorhynchus rhodurus). Gen. Cornp. Endocrinol. 45, 533-53.5. Taylor, M. H. (1984). Lunar synchronization of fish reproduction. Trans Amer. Fish Sot. 113, 484-493. Taylor, M. II., and DiMichele, L. (1980). Ovarian
in Vitro
289
changes during the lunar spawning cycle of Funda/us heteroclitus. Copeia, pp. 118-125. van Ree, G. E., Lok, D., Bosman, G. J. C. G. M. (1977). In vitro induction of nuclear breakdown in oocytes of the zebrafish, Brachydanio rerio (Ham. Buch.). Effects of the composition of the medium and of protein and steroid hormones. Proc. K. Ned. Akad. Wet. Ser. C 80, 353-313. Wallace, R. A., and Selman, K. (1978). Oogenesis in Fundulus heteroclitus. I. Preliminary observations on oocyte maturation in viva and in vitro. Dev. Biol. 62, 354-369. Wallace, R. A., and Selman, K. (1985). Major protein changes during vitellogenesis and maturation of Fundulus oocytes. Dev. Biol, 110, 492-498. Young, G., Ueda, H., and Nagahama. Y. (1983). Estradiol-17B and 17a,20j3-Dihydroxy-4-pregnen-3one production by isolated ovarian follicles of amago salmon (Oncovhync~zus rhodurus) in response to mammalian pituitary and placental hormones and salmon gonadotropin. Gen. Colzp. Endocrinol. 52, 329-335.
AND COMPARATIVE
ENDOCRINOLOGY
62, 281-289 (1986)
Oocyte Maturation in the Mummichog (fundulus heteroclitus): Effects of Steroids on Germinal Vesicle Breakdown of Intact Follicles in Vitro MARK
S. GREELEY,
JR., DAN R. CALDER, MALCOLM AND ROBIN A. WALLACE
H. TAYLOR,*
HANS
HoIs,?
Whitney Laboratory, University of Florida, St. Augustine, Florida 32086; *School of Life and Health Sciences, University of Delaware, Newark, Delaware 19716; and +State University of Utrecht, 8 Padualuan, 3508 TB Utrecht, The Netherlands Accepted December 15, 1985 The effects of several steroids on the maturation of follicle-enclosed oocytes of the mummichog Fundalas heteroclitus in vitro were examined. At a relatively high concentration (1 .O pgiml), a number of different steroids, including pregnenolone, 17a-hydroxypregnenolone, corticosterone, cortisol, 1I-deoxycorticosterone. 1I-deoxycortisol, androstenedione. testosterone, progesterone, 17o-hydroxyprogesterone, 20@-dihydroprogesterone, and 17ahydroxy-20P-dihydroprogesterone, were able to induce germinal vesicle breakdown (GVBD) in prematuration oocytes. Cholesterol, 17P-estradiol, 11-ketotestosterone, and 1lp-hydroxytestosterone were totally ineffective. In general, 1I-oxysteroids tended to be less effective than their 11-deoxysteroid counterparts. Two 20@-dihydroprogestins-17o(hydroxy-20lSdihydroprogesterone and 20P-dihydroprogesterone-were the most potent maturation-inducing steroids, initiating 50% GVBD at 1 &ml in follicles obtained from ovaries containing mature or maturing follicles in viva, or at 2.5-4.0 rig/ml in follicles from ovaries lacking mature or maturing oocytes in laivo. These results are consistent with sevi era1 previous studies involving salmonids and various other teleosts, and suggest a possible physiological role for a 20lSdihydroprogestin in the resumption of meiotic maturation in F. heteroclitus. 0 1986 Academic Press. Inc.
The oocytes of teleost fish are arrested in meiotic prophase I during most of their development and growth, subsequently resuming meiotic maturation at the end of this period in response to an appropriate hormonal effector. Numerous steroids have been reported to be capable of stimulating teleost oocytes to resume meiotic maturation in vitro (for review, see Goetz, 1983). However, much of this work has focused on only one group of teleosts of particular economic significance-the salmonids. In many salmonids, including rainbow trout, Salrno gairdneri (Jalabert, 1976; Nagahama et al., 1983), brook trout, Sulvelinus fontinalis (Duffey and Goetz, 1980), coho salmon, Oncorhynchus kisutch (Sower and Shreck, 1982), amago salmon, Oncorhynchus rhodurus, and the ayu, Pleco-
glossus altivelis (Nagahama et al. 1983), one steroid, 17a-hydroxy-20@dihydroprogesterone (17a-OH-20@diPROG), appears to be the most effective at inducing maturation in vitro. This steroid has also been identified in the blood and plasma of several salmonids (Scott et nl., 1982; IIirose et al., 1983; Kagawa et al., 1983), is produced by salmonid follicles irz vitro (Fostier et al. ~ 1981; Suzuki et al., 1981; Nagahama and Kagawa, 1982; Young et al., 19831, and, just recently, has been identified as ‘the natural maturation-inducing steroid in the amago salmon (Nagahama and Adachi, 1985). In cantrast, comparatively little is kriown concerning steroid mediation of oocyte maturation in nonsalmonid teleosts, for to date only a few of the innumerable nonsal281 0016-6480186 $1.50 Copyright D 1986 by Academic PKSS, Inc. All rights of reproduction in any form reserved
282
GREELEY
monid teleost species have been examined in this respect. In some, including medaka, Oryzias latipes (Iwamatsu, 1978, 1980), yellow perch, Perca flavescens (Goetz and Theofan, 1979), goldfish, Carassius auratus (Nagahama et al.,, 1983), goldeye, Hiodon alosoides (Pankhurst , 1985)) and rock bass, Ambloplites rupestris (Goetz and Cetta, 1985), 17a-OH-20@diPROG again appears to be one of the most effective initiators of oocyte maturation in vitro. However, in other nonsalmonid teleosts, notably the Indian catfish, Heteropneustes fossilis (Goswami and Sundararaj, 1974) and the zebrafish, Brachydanio rerio (van Ree et al., 1977), corticosteroids have been reported to be more effective than progestins. We herein examine the relative ability of several physiological steroids, including various progestins and corticosteroids, to induce maturation in vitro of intact follicles from the cyprinodontid teleost Fundulus heteroclitus. In an earlier investigation, progesterone and 1 l-deoxycorticosterone were shown to be equally effective initiators of follicle maturation in vitro (Wallace and Selman, 1978), but comparable data concerning the effectiveness of 17aOH-20P-diPROG are lacking. Thus the purposes of the present study are several: (1) to determine which of these steroids would be most appropriate for future studies involving maturation of F. heteroclitus follicles in vitro, (2) to provide preliminary evidence concerning the relationship between the structure of steroids and their ability to initiate F. heteroclitus follicle maturation in vitro, and (3) to compare the relative effectiveness of these steroids, especially the various progestins and corticosteroids, in initiating follicle maturation in vitro in an additional nonsalmonid species. Evidence is presented that, in F. heteroclitus as in salmonids and a few other teleost species, two 20/3-dihydroprogestins, 17or-OH-20@diPROG and 20P-dihydroprogesterone (20@diPROG), are the most ef-
ET AL.
fective physiological steroids for initiating follicle maturation in vitro. MATERIALS
AND METHODS
Fish and biochemicals. Fundulus were collected during their breeding season-February through September at this location (M. S. Greeley, H. Hols, and R. A. Wallace, manuscript in preparation)-from salt marshes in the vicinity of the Whitney Laboratory near St. Augustine in northeastern Florida, and subsequently held in an outdoor concrete holding tank. To encourage and support vitellogenesis, fish were fed a liberal diet including mullet roe. Biochemicals, including steroids, were obtained from Sigma. Follicles. Fish were killed by decapitation and their ovaries removed. Ovaries were gently pulled apart with watchmakers’ forceps while immersed in FO solution (Wallace and Selman, 1978), and prematuration follicles (see description below) were isolated from connective tissue and other follicles. Culture techniques were essentially those of Wallace and Selman (1978). differing primarily in that a commercially available culture medium (L-15 with glutamine, diluted to 75% with distilled water, and containing 50 mgiml gentamycin) was substituted for a previously employed proprietary medium, FO (FO was still used for maintaining the ovaries during dissection). Although FO is adequate for most culture purposes, 75% L-15 proved superior in the present instance by extending the size range of usable follicles, as compared with FO (Fig. 1). For the purposes of this study, prematuration follicles were considered to be those vitellogenic follicles, 1.2-1.4 mm in diameter, that demonstrated no obvious visual indications of yolk clarification, which indicates the onset of maturation in this species (see Fig. 2). Such follicles underwent 100% germinal vesicle breakdown (GVBD) in response to steroid in a preliminary experiment with 75% L-15 culture media, but did not mature in vitro without the addition of exogenous steroid (Fig. 1). Maturing follicles are defined as those which still contain a germinal vesicle (GV), but are distinctly clearing and thus considered to be in the early stages of maturation. In contrast to prematuration follicles, follicles designated as maturing on the basis of observed yolk clearing underwent maturation in vitro with or without the addition of exogenous steroid to the incubation medium. Mature follicles are herein defined rather broadly, so as to include both preovulatory follicles lacking a germinal vesicle and ovulated eggs. Initial screening of steroids. Prematuration follicles from 6 to 10 ovaries, containing neither mature follicles nor maturing follicles in viva, were pooled for each set of steroid trials. From each pool, individual aliquots of IO-15 follicles were withdrawn and placed in 10x35mm plastic petri dishes containing 3 ml of
Fundulus
OOCYTE
IOO-
ao-
60 9
1
1 0 8
I
I
:
40-
i
i
/
/
:’
283
cyclic ovarian environment of a follicle and the concentrations of 17~0H-ZIP-diPROG and 20p-diPROG required to induce maturation, additional p001s of prematuration follicles were constructed, this time from ovaries with or without maturing follicles in !&o. Aliquots of follicles from these pools were then tested against various concentrations of each of the above steroids.
RESULTS
I
/’
/’
in Vitro
I i
: t
20-
MATURATION
Maturation
in Vitro
In F. heteroclitus, the yolk of prematuration follicles is somewhat translucent, I 0.8 0.9 I .o /_I 1.2 I .3 I .4 I .5 rather than totally opaque as in many other teleosts, and the nucleus or GV-which Follicle diameter (mm) migrates from the interior of the follicle to, FIG. 1. Comparison of FO and 75% L-15 media for F. hetevoclitrrs foIIicle culture in vitro. Follicles from the periphery prior to the resumption of 3.8 mm in diameter (early vitellogenesis) through 1.4 maturation -is highly visible. In consemm in diameter (fully grown, prematuration follicles) quence, important changes within the foland 1.5 mm in diameter (early maturation, yolk especlearing but GV still present) from a single ovary were licle during the course of maturation, cially GVBD, are readily observed without incubated with or without 17a-hydroxy-20p-dihydroprogesterone (0.1 &ml) in either FO or 75% L-15. the necessity of chemical clearing or disEarly-vitellogenic follicles (0.8-0.9 mm in diameter) section of the follicle. could not be induced to undergo GVBD under any The first visual indications of the onset conditions. Some mid-vitellogenic follicles (1 .O- 1.1 of in vitro follicle maturation in F. here+ mm in diameter) did undergo GVBD in response to the clitus include an accelerated clarification of steroid, but only in 75% L-15. Late vitellogenic follicles (1.2- 1.4 mm in diameter) could be induced by the yolk, and an increase in the,size of the the steroid to undergo GVBD in either FO or 75% follicle due to hydration (Wallace and L-15, although more consistently in the latter medium. Selman, 1978, 1983, both events ‘occurring Both in FO and 75% L-15, only those folIicles 1.5 mm 16 hr following exposure to in diameter and clearing in viva underwent GVBD in approximately a stimulatory steroid (Fig. 2). Between 2 r~irru with ar without (controls) the addition of steroid to the culture medium. and 24 hr the nucleus begins to brea down, a process normally completed by 28 medium. Steroids of appropriate concentration (either hr. Subsequent to GVBD, the follicle un1.0, 0.1, or 0.01 &ml) were added in 10 ~1 ethanol; dergoes additional size increases due to ‘hycontrols contained medium and ethanol alone. Unless dration, and oil droplets continue to cootherwise stated in the text, incubations were conalesce. Other than increasing atresia, howducted for 40 hr at 21°C in a temperature-controlled ever, few further changes occur in the incubator. At the end of this period, follicles were scored for the occurrence of GVBD by utilizing a dis- follicle after 40 hr-the length ‘of the secting scope and transmitted’ light to facilitate present incubations. scoring. In nature, certain typical’ post&VI3 Each steroid was tested in three to five separate events within the F. heteroditus ,folticle, trials, each trial as described above. The resultssuch as ad’djtional oil droplet Coalescence being similar in separate trials-were then pooled for final presentation. Atretic follicles were disregarded and hydration, occasionally fail to occur as when analyzing results. the oocyte ovulates from surrounding ‘folDose-response CUYV~S.In order to (1) obtain prelicular investments1 immediately f$o&ng cise dose-response curves for the purpose of comGVBD (M. S. Greeley, $3. Hols, D. 13. paring the effectiveness of 17~~qH-20P-diPROG and Calder, and R. A. Wallace, manuscript in 20+3-diPROG at inducing maturation in F. heteroclitus follicles, and (2) examine the relationship between the preparation). We therefore considekd the 0
.-.'--.
----
:-
.-...-.
- . ..-.-.
i
284
GREELEY Preovulotory follicle
1.7-
z G z s a Y ,” 2
Gv;;./i\. 1.6Clearing begun
.’ /
1.5Steroid
v
, .4 -,JT.P:_ ----a--t 0
I
-.--.--.I 8
I
I 16
I
I 24
Time
_ .---I
I 32
c.o:::: I
I 40
I
I 48
ihr)
FIG. 2. Time course of F. hete~~2itus follicle size changes during maturation in vitro initiated by 17~ hydroxy-20P-dihydroprogesterone (0.1 pgiml). Follicle diameters represent the mean for 30 follicles obtamed from a pool derived from five fish which contained neither mature nor maturing follicles in viva. Solid line represents follicles incubated with steroid; dashed line represents control follicles incubated in the absence of exogenous steroid.
occurrence of GVBD to be, in this species, the most consistent and reliable indicator of follicle maturation in viva or in vitro. Ovulation rarely follows maturation of the F. heteroclitus follicle in vitro. Occasionally, however, oocytes induced to resume maturation in vitro begin to ovulate from their follicular investments subsequent to GVBD, but fail to complete ovulation successfully. These oocytes eventually die. In scoring results, such oocytes were counted among those having undergone GVBD, rather than being deleted from consideration as are other atretic follicles, since they had completed GVBD prior to the failed ovulation. Initial
Screening
of Steroids
The F. heteroclitus ovary is very cyclic, oocytes maturing at regular intervalswith lunar or semilunar periodicitythroughout a lengthy breeding season in nature (Taylor and DiMichelle, 1980; M. S. Greeley, H. Hols, and R. A. Wallace, manuscript in preparation) and, to some extent, under laboratory conditions (Taylor, 1984). It was suspected when beginning these studies that the responsive-
ET AL.
ness of F. heteroclitus prematuration oocytes to steroid stimulation of meiotic maturation might also cycle in a similar fashion. Therefore, in an attempt to standardize results, all initial experiments were performed utilizing only prematuration follicles from ovaries in the same stage-in particular, those ovaries which did not already contain any mature or maturing follicles in vivo. Two progestins, 20P-diPROG and 17c~OH-20@diPROG, were the most potent maturation-inducing steroids tested during initial steroid screening experiments, being capable of initiating GVBD at concentrations as low as 0.01 pg/ml (Table 1). Although not nearly as effective, many other steroids were also able to initiate maturation in F. heteroclitus follicles, at least at the relatively high concentration of 1.0 bg/ml (Table 1). However, some of these, including androstenedione and the 1 l-oxygenated corticosteroids (cortisol and corticosterone), were ineffective at concentrations lower than 1.0 pg/ml. Others, such as ll-ketotestosterone and 1 lp-hydroxytestosterone, were either only partially effective at 1.0 pg/ml, or, in the case of cholesterol, 17l%estradiol, and estrone, totally ineffective (Table 1). Follicle
Sensitivity
The relative effectiveness of both 17~3OH-20@diPROG and 20@diPROG in stimulating GVBD in prematuration follicles was further examined in follicles from ovaries in two very different reproductive states: (1) ovaries containing mature or maturing follicles in vivo (approximately equivalent to ovaries obtained in nature from fish during their spring-tide spawning peaks) and (2) ovaries containing neither mature nor maturing follicles in vivo (approximately equivalent to ovaries from wild fish between spring-tide spawning peaks). Prematuration follicles from the former ovaries were found to be much more sensi-
Fundulus
EFFECTS
OF SEVERAL
OOCYTE
STFXOIDS
ON
in Vitro
MATURATION
TABLE 1 GVBD OFF. heteroclitus
1.0 kg/ml Steroid Cholesterol 17P-Estradiol Estrone Androstenedione Testosterone 1I-Ketotestosterone 1l&OH-Testosterone Pregnenolone 17a-OH-Pregnenolone Cortisol Corticosterone 1 I-Deoxycortisol 1 I-Deoxycorticosterone Progesterone 17cr-OH-Progesterone 20@-Dihydroprogesterone 17cL-OH-20P-Dihydroprogesterone
285
FOLLICLES
in
Vitro
0.1 kg/ml
0.01 &g/ml
M
%GVBD
n
%GVBD
n
%GVB5
2s 29 27 30 2s 29 25 25 24 26 44 26 28 28 51 51 69
0 0 0 73 84 21 4 100 92 88 93 70 a9 93 94 82 92
33 29 33 27 32 27 32 46 50 30 42 57 59 71
-
46 45 34 34 45 56 42 45 45 38 60
0 0 9 0 0 0 0 2 2 61 44
tive to steroid stimulation of maturation in vitro than were follicles from the latter ovaries (Fig. 3). Although 100% of the prematuration follicles from the former ovaries underwent maturation in vitro with either 17a-OH-20l3-diPROG or 20P-diPROG at doses as low as 0.005 pgiml (5 ngiml, Fig. 3), some follicles from the latter ovaries failed to respond in vitro even at relatively high steroid concentrations (0.1-0.01 kg/ml, Table 1 and Fig. 3). In fact, while 2.5-4.0 rig/ml of these steroids was required to cause GVBD in 50% of the prematuration follicles obtained from ovaries containing neither mature nor maturing follicles in vivo, only 1 rig/ml was required to attain comparable results with prematuration follicles obtained from ovaries containing mature or maturing follicles in vivo. Relative Effectiveness of the 20fHlihydroprogestins Both 17cx-OH-20@-diPROG and 2OP-diPROG ,were equally effective at inducing maturation in F. heteroclitus follicles, although 20pldiPROG tended to be slightly
0 62 0 0 75 63 3 13 70 70 64 91 97 81
more effective than 17at-OH-20@-diPROG with the least responsive follicles (Table 1 and Fig. 3). The time courses of follicle
FIG. 3. Comparative dose-response curves for 20P-dihydroprogesterone and 17cu-hydroxy-20&d&ydroprogesterone initiation of F. heteroclirus follicle maturation in vitro. Each point represents a percentage GVBD, after 40-hr incubations in 75% L-15, of 32 I 1 follicles tested in three separate trials. Two pools of prematuration follicles (1.2- 1.4 mm in diameter) for each trial were derived from 6 to 10 ovaries apiece: one pool of follicles from ovaries akeady containing mature or maturing follicles in I&O, and one pool of follicles from ovaries containing neither mature nor maturing follicles ifi viva.
286
GREELEY
maturation in V&O, stimulated by either 17ol-OH-20P-diPROG or 20@-diPROG, were also essentially identical (Fig. 4). DISCUSSION
The results of the present study demonstrate that a number of different steroids at relatively high doses (1.0 kg/ml)-ineluding testosterone and various progestins and corticosteroidszcan initiate maturation of F. heteroclitus follicles in vitro. Such a ubiquitous respon-se of a follicle to various steroids at high doses is not unusual among the teleosts examined to date (Goetz, 1983), and is probably due, at least in part, to the metabolism of many steroids to more active forms by some component of the follicular investments. However, in F. heteroclitus as well as many other teleosts, there is one particular class of steroids-the 20@dihydroprogestinsthat appears to be far more effective at inducing maturation in intact follicles than other steroids, especially at lower and physiologically more relevant doses.
100
1
80 i
-_ ,_ -__ 0
8
I6
24 Time
32
46
48
(hr)
FIG. 4. Time course of GVSiI in F. heteroclitus follicles in vitro initiated by either 20@dihydroprogesterone or 17oi-hydroxy-aOp-dihydroprogesterone (0.1 pgiml). Each treatment group was composed of 35 prematuration follicles (1.2-1.4 mti in diameter tirematur&ion size) taken from a single pool derived from three ovaries which contained neither mature nor ma: turing follicles in vivo.
E;T AL.
20&Dihydroprogestins are apparently the most effective maturation-inducing steroids in vitro in several teleosts, including rainbow trout, S. gairdtieri (Jalabert, 1976; Nagahama et al., 1983), yellow perch, P. jluvescens (Goetz and Theofan, 1979), brook trout, S. fontinalis (Duffey and Goetz, 1980), coho salmon, 0. kisutch (Sower and Shreck, 1982), amago salmon, 0. rhodurus, ayu, P. altivelis, goldfish, C. auratus (Nagahama et al., 1983), rock bass, A. rupestris (Goetz and Cetta, 1985), medaka, 0. latipes (Iwamatsu, 1978, 1980), and now the mummichog, F. heteroclitus. In those teleosts in which both 17a-OH20P-diPROG and 20@-diPROG have been examined in sufficient detail-the present results being a notable exception17crOH-2O@diPRdG is typically the more effective of the two at initiating follicle maturation in vitro. Furthermore, 17a-OH-20l3diPROG has recently been shown to be the natural maturation-inducing steroid in the amago salmon (Nagahama and Adachi, 1985). Thus, this particular 20@dihydroprogestin is at this time the best candidate for a universal maturation-inducing steroid in the teleosts-if such exists-despite the occasional apparent exception such as the Indian catfish (Goswami and Sundararaj, 1974). As indicated above, the present results are somewhat unusual in that 20l3-diPROG is at least the equal of 17a-OH-20/3-diPROG at initiating follicle maturation in vitro in F. heteroclitus. In fact, the similarities of the respective time courses and dose-response curves argue that the modes of action of both steroids are identical. However, the physiological significance of this observation is not immediately apparent. Both steroids could be converted to another as yet unidentified active steroid in the follicle cells prior to oocyte stimulation, or both could be structurally mimicking the action of the other or another steroid in directly facilitating oocyte maturation. We therefore suggest the
Fmdulus
OOCYTE
MATURATION
present results reflect the relative importance of a 2OP-hydroxyl group for the maturation-inducing activity of a steroid, and defer for the present the question of whether either 17cx-OH-2OP-diPROG or 2Op-diPROG is actually a natural maturation-inducing steroid in this fish. In addition to the apparent importance of a 20@-hydroxl group to the maturation-inducing ability of a steroid in F. heteroc&us, additional, but admittedly preliminary, steroid structure-function relationships are suggested by the present results. For instance, a hydroxyl group at the 17~~ position does not appear necessary. Similar results are found in amphibians, where a 17ol-hydroxyl group is even reported actually to inhibit the maturation-inducing activity of a steroid (Morrill and Bloch, 1977). In F. heteroclitus, a hydroxyl or ketone at the 11 position appears to inhibit steroid activity (i.e., 11-oxygenated testosterone and corticosteroid derivatives); such also seems to be the case in several other teleosts (see Goetz, 1983). The present results further suggest that aromatization of the A ring abolishes the maturation-inducing ability of a steroid (i.e., the estrogens); similar results have been reported for most other teleosts examined to date, with the apparent exception of the zebrafish, B. rerio (van Ree et al., 1977), in which es&one, but not 17p-estradiol or estrio& stimulates maturation. We caution that these steroid structure-function relationships are necessarily tenuous. The F. heteroclitus follicle has proven notoriously resistant to defolliculation; thus we are currently unable to distinguish direct from indirect steroid effects. The effective concentrations of progesterone, DOG, and cortisol in the preliminary screening experiment of the present study were approximately 10 times, higher than reported in a previous study with F. hetevloclitus (Wallace and Selman, 1978). The follicles employed in the previous study were obtained’from ovaries that con-
in Vitro
287
tained follicles in all stages of development, including maturation; thus, those follicles may have been primed or sensitized in vivs by endogenous gonadotropins or steroids I In contrast, follicles employed in the screening portion of the present study were selected from .FunduZus ovaries lacking mature or maturing oocytes in viva, and thus were apparently relatively insensitive to stimulation, as evidenced by (1) controls never undergoing GVBD, and (2) at least some oocytes in nearly all incubations failing to undergo GVBD even with the addition of steroids to the culture medium. Additional trials of 17a-OH-20@diPROG and 20P-diPRQG at various concentrations were therefore conducted with pools of follicles derived from ovaries either with or without actively maturing follicles in v&o. The purposes of this set of experiments were (1) to examine further the potential effects of the prematuration follicle’s environment on the sensitivity of the follicle to steroid stimulation of maturation, and (2) to determine more accurately the comparative effectiveness of 17m-OH-20@-diPROG and 2Op-diPROG. The present results show the F. heterocfitus prematuration follicle to be relatively sensitive to steroid stimulation when the ovary from which it was obtained contained other follicles undergoing maturation in viva, and relatively insensitive when the ovary did not contain any maturing follicles in Co. This suggests that the follicle is in some manner “primed” in the former ovaries, possibly by endogenous gonadotropin or steroids. To date, the potential for differential responsibeness of’ apparently similar follicles to steroid s~irn~la~~on at different stages of an ovarian cycle has been poorly addressed in teleosts. This is unfortunate, as the reasons for such variation in follicle sensitivity, if understood, could colntribute great,ly to an understanding of the physiological mechanisms underlying the hormonal coNntrol of both follicle maturation and reproductCve cycles
288
GREELEY
in general, and thus deserve greater attention than heretofore accorded. In conclusion, we report that 20P-diPROG and 17a-OH-20P-diPROG are potent maturation-inducing steroids for culture of Fundulus oocytes in vitro. These resuits suggest that a steroid with a similar structure might be the natural maturationinducing steroid of F. heteroclitus, possibly even 17a-OH-20P-diPROG itself since (1) this steroid has already been identified as the maturation-inducing steroid in one teleast, the amago salmon (Nagahama and Adachi, 1985), and (2) this steroid has recentlv been identified bv radioimmunoassay in media in which F. hetevoclitus follicles were stimulated to mature by Fundulus pituitary extracts (Lin et al. 1985). ACKNOWLEDGMENTS This work was supported 8202512toR.A.W.
by NSF Grant PCM
REFERENCES Duffey, R. J., and Goetz, E W. (1980). The in vitro effects of 17a-hydroxy-20P-dihydroprogesterone on germinal vesicle breakdown in brook trout (Salvelinus fontinalis) oocytes. Gen. Camp. Endocrinol. 41, 563-565. Fostier, A., Jalabert, B., Campbell, C., Terqui, M., and Breton, B. (1981). CinCtique de lib&ration in vitro de 17a-hydroxy-20P-dihydroprogesterone par des follicles de Truite arc-en-ciel, Salmo gairdnerii. C.R. Acad. Sci. Ser. 3 292, 777-780. Goetz, E W. (1983). Hormonal control of oocyte final maturaiion and ovulation in fishes. In “Fish Physiology” (W. S. Hoar, D. J. Randall? and E. M. Donaldson, eds.), Vol. 9, pp. 117-170. Academic Press, New York. Goetz, F. W., and Cetta, F. (1985). Zn vitro steroid stimulation of final maturation in oocytes of rock bass (Ambloplites rupestris). Reprod. Nutr. Develop. 25, 33-38. Goetz, E W., and Theofan, G. (1979). In vitro stimulation of germinal vesicle breakdown and ovulation of yellow perch (Perca flavescens) oocytes. Effects of 17a-hydroxy-20p-dihydroprogesterone and prostaglandins. Gen. Cornp. Endocrinol. 37, 273-285.
Goswami, S. V., and Sundararaj, B. I. (1974). Zn vitro maturation and ovulation of oocytes of the catfish, Heteropneustes fossilis (Bloch): Effects of mammalian hypophyseal hormones, catfish pitui-
ET AL. tary homogenate, steroid precursors and metabolites and gonadal and adrenocortical steroids. J. Exp. 2001. 178, 467-478. Hirose, K., Nagahama, Y., Adachi, S., and Wakabayashi, K. (1983). Changes in serum concentrations of gonadotropin, 17&,20@dihydroxy-4-pregnene-3-one during synthetic LH-RH-induced final oocyte maturation and ovulation in the Ayu Plecoglossus altivelis. Bull. Japan. Sot. Sci. Fisheries 49, 1165- 1169. 1wamatsu, T. (1978). Studies on oocyte maturation of the Medaka, Oryzias latipes. V. On the structure of steroids that induce maturation in vitro. J. Exp. Zool.
204, 401-408.
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Jalabert, B. (1976). In vitro oocyte maturation and ovulation in rainbow trout (Salmo gairdneri), northern pike (.&ox lucius), and goldfish (Carassius auuatus). J. Fish. Res. Board Canad. 33, 974-988. Kagawa, H.,
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