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In the teleost Fundulus heteroclitus, serotonin (5-HT) reversibly inhibits oocyte maturation induced in vitro by the maturation-inducing steroid (MIS) 17,20beta-dihydroxy-4-pregnen-3-one (17,20betaP). The 5-HT inhibition of 17,20betaP-induced meiotic maturation was examined in ovarian follicles at different developmental stages or isolated at different times during the follicular cycle. Steroid treatment of late vitellogenic and early maturing follicles (1.2- to 1.7-mm diameter) promoted oocyte maturation in a size-dependent manner, and this maturation was inhibited by 5-HT in follicles of < 1.6- to 1.7-mm diameter. Thus, the 5-HT inhibition progressively decreased as follicles developed the ability to mature in the absence of 17,20betaP. The effectiveness of 5-HT to increase follicular cAMP remained similar within the same developmental stages, indicating that the reduction of 5-HT inhibitory action was not related to the competence of 5-HT to activate inhibitory signals in the oocyte. During the follicular cycle, fully grown follicles (1.3- to 1.4-mm diameter) showed a decreased maturational competence in response to gonadotropin or MIS stimulation after the follicular recruitment into maturation and spawning occurred, which coincided with an increase of the effectiveness of 5-HT at inhibiting 17,20betaP-induced maturation. In further experiments, preincubation of follicles with hCG was found to reduce 5-HT inhibitory action, but when follicles were incubated with either hCG in the presence of a steroidogenesis inhibitor or estradiol-17beta (E2), the 5-HT inhibition was unaffected. These findings suggest that 5-HT inhibition of the MIS-induced meiotic maturation is not under direct gonadotropin or E2 regulation but that it might be regulated in vivo by changes in the competence of the oocytes to undergo oocyte maturation after MIS stimulation.

In vitro steroidogenesis of ovarian follicles incubated with radioactive precursors or a Fundulus heteroclitus pituitary extract (FPE) was investigated. Steroids were extracted from both the medium and follicular tissue and fractionated by liquid or thin-layer chromatography. A similar pattern of steroid metabolites was obtained with either [14C]pregnenolone or [14C]progesterone as exogenous precursor. Several metabolites comigrated with known reference steroids and thus were tentatively identified. Some have been previously reported to induce germinal vesicle breakdown (GVBD) in this and other species, particularly 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (DHP). [3H]DHP added to intact follicles or denuded oocytes was also extensively metabolized. All the DHP metabolites produced by the intact follicle were tested for biological activity. Three of the metabolites were almost as effective inducers of GVBD as DHP itself, and two were tentatively identified as 5 alpha-pregnan-3 alpha,17 alpha,20 beta-triol and 5 alpha-pregnan-3 beta,17 alpha,20 beta-triol. However, DHP was the most potent and the quickest inducer of GVBD, indicating that its maturational action is not due to metabolic conversion to a more active form. In addition, we found two very active fractions after HPLC analysis of steroid extracts from FPE-stimulated follicles: one that corresponded to and was further identified (mass spectroscopy) as DHP and a second tentatively identified as the DHP metabolite 5 alpha-pregnan-3 beta,17 alpha,20 beta-triol. This study provides strong evidence that DHP plays the major role as a maturation inducing-steroid in F. heteroclitus, even though DHP is not the only active steroid produced by maturing follicles.

Oocytes of marine and estuarine teleosts often undergo pronounced volume increases during the maturation phase of development that precedes ovulation and fertilization. To examine the physiological correlates of these volume increases, prematuration follicles of the saltmarsh teleost, Fundulus heteroclitus, were cultured in vitro with a maturation-inducing steroid (17 alpha-hydroxy-20 beta-dihydroprogesterone). Mean follicle volume rose significantly (75%) during a 40-h incubation period. Similar to the situation previously found in vivo, uptake of water by the maturing follicle was responsible for this volume increase in vitro, with the water content increasing from 62% to 78% of the total follicle mass. The follicle contents of two probable osmotic effectors--Na+ and K(+)--also rose, the increase in K+ being twice that of Na+. The influx of K+ even exceeded water uptake, resulting in a net increase in the concentration of this cation. It thus appears that the influx of these cations, in particular K+, is a major cause of the uptake of osmotically obligated water and subsequent volume increase experienced by maturing F. heteroclitus follicles. In a search for operant mechanisms, it was found that follicle hydration, but not maturation, was strictly dependent on external K+ in a concentration-dependent manner. The mechanism by which K+ accumulates in the follicle was insensitive to ouabain, so that a typical Na+, K(+)-ATPase mechanism does not appear to be involved. The ability of external K+ to promote follicle hydration was gradually lost during the maturation process as the oocyte dissociated from the surrounding granulosa cells in preparation for ovulation.(ABSTRACT TRUNCATED AT 250 WORDS)

A procedure was developed for studying vitellogenin (VTG) incorporation by vitellogenic oocytes of Fundulus heteroclitus in vitro. Since homologous VTG can be obtained from this animal only with great difficulty, the use of [32P]VTG from Xenopus laevis was explored as an alternative. Vitellogenic as well as maturational-stage oocytes were found to sequester X. laevis [32P]VTG from the medium, and incorporation was found to be linear with time for at least up to 12 hr. Once incorporated into the oocyte, [32P]VTG did not appear to undergo turnover. The effect of different [32P]VTG concentrations on incorporation indicated that the uptake mechanism was saturable. Unlabeled F. heteroclitus VTG and X. laevis VTG were also found to compete effectively with X. laevis [32P]VTG, whereas bovine serum albumin did not. These results represent the first documentation of a successful culture system for receptor-mediated VTG incorporation by teleost oocytes.

The protein content of various size follicles was measured in Fundulus heteroclitus and indicated four phases of increase relative to follicle volume: Phases I (previtellogenic; estimated to be less than 0.01 mg/mm3), II (vitellogenic; 0.20 mg/mm3), III (early maturation; 0.03 mg/mm3), and IV (late maturation; 0 mg/mm3). A pronounced and rapid size increase occurs during maturation due to hydration, but protein uptake, which was also documented cytologically, contributes to about 16% of the volume increment during early maturation. Protein incorporation appears to stop abruptly at the time of germinal vesicle breakdown, most likely reflecting an altered physiological state of the oocyte. SDS-polacrylamide gel electrophoretic patterns of various size follicles indicated that five major protein bands (molecular weights = 122, 103, 45, 26, and 20 k) accumulate during vitellogenesis and presumably are proteolytically derived from a 200-kDa vitellogenin precursor. During maturation, the 122- and 45-kDa proteins disappear and several new, lower molecular weight bands appear. Proteolysis of specific yolk proteins may thus help generate part of the osmotic pressure gradient required for water uptake during oocyte maturation.

The effects of several steroids on the maturation of follicle-enclosed oocytes of the mummichog Fundulus heteroclitus in vitro were examined. At a relatively high concentration (1.0 microgram/ml), a number of different steroids, including pregnenolone, 17 alpha-hydroxypregnenolone, corticosterone, cortisol, 11-deoxycorticosterone, 11-deoxycortisol, androstenedione, testosterone, progesterone, 17 alpha-hydroxyprogesterone, 20 beta-dihydroprogesterone, and 17 alpha-hydroxy-20 beta-dihydroprogesterone, were able to induce germinal vesicle breakdown (GVBD) in prematuration oocytes. Cholesterol, 17 beta-estradiol, 11-ketotestosterone, and 11 beta-hydroxytestosterone were totally ineffective. In general, 11-oxysteroids tended to be less effective than their 11-deoxysteroid counterparts. Two 20 beta-dihydroprogestins--17 alpha-hydroxy-20 beta-dihydroprogesterone and 20 beta-dihydroprogesterone--were the most potent maturation-inducing steroids, initiating 50% GVBD at 1 ng/ml in follicles obtained from ovaries containing mature or maturing follicles in vivo, or at 2.5-4.0 ng/ml in follicles from ovaries lacking mature or maturing oocytes in vivo. These results are consistent with several previous studies involving salmonids and various other teleosts, and suggest a possible physiological role for a 20 beta-dihydroprogestin in the resumption of meiotic maturation in F. heteroclitus.

Isolated ovarian follicles from several species were cultured to develop an in vitro bioassay system for Fundulus heteroclitus gonadotropin. An extract of F. heteroclitus pituitaries, when tested in heterologous systems using follicles from Rana pipiens. Xenopus laevis, and Carassius auratus, was ineffective in provoking either germinal vesicle breakdown or steroid production. In a homologous system using F. heteroclitus follicles, F. heteroclitus pituitary extract was capable of inducing both germinal vesicle breakdown and steroid production in a dose-dependent fashion. Testosterone, estradiol-17 beta, and 17 alpha-hydroxy,20 beta-dihydroprogesterone were detected in both the culture media and the follicle extracts after F. heteroclitus pituitary extract stimulation. The steroidogenic responses resulting from the pituitary extract stimulation were dependent on the size and stage of follicular development. Only large vitellogenic follicles (1.2-1.4 mm diameter) were able to produce 17 alpha-hydroxy,20 beta-dihydroprogesterone and testosterone. Small vitellogenic follicles (less than 1.2 mm) were unresponsive to stimulation by F. heteroclitus pituitary extracts as scored by either germinal vesicle breakdown or production of 17 alpha-hydroxy, 20 beta-dihydroprogesterone and testosterone. However, estradiol-17 beta production was detected in follicles of a much wider size range: Follicles as small as 0.8 mm diameter were responsive to F. heteroclitus pituitary extract stimulation and produced a large quantity of estradiol-17 beta. There was a marked seasonal sensitivity of F. heteroclitus follicles to pituitary extract stimulation in vitro. Follicles obtained from fish outside of the breeding season (January) were less responsive to stimulation by pituitary extract or steroid. The same preparation of pituitary extract was capable of provoking germinal vesicle breakdown in follicles obtained in May. Pituitary extracts prepared during October through January were also less potent than those prepared during the breeding season (February through September). We conclude that F. heteroclitus gonadotropin(s) shows a noticeable species specificity and that F. heteroclitus follicles exhibit both a season- and a size-dependent responsiveness to gonadotropin(s). Hence, with a judicious use of the appropriate types of F. heteroclitus ovarian follicles, we have been able to demonstrate that in vitro oocyte maturation and steroid production are sensitive, homologous bioassays for F. heteroclitus gonadotropin(s).