Orthotopic models of pediatric brain tumors in zebrafish.

High-throughput screens (HTS) of compound toxicity against cancer cells can identify thousands of potential new drug-leads. But only limited numbers of these compounds can progress to expensive and labor-intensive efficacy studies in mice, creating a 'bottle neck' in the drug development pipeline. Approaches that triage drug-leads for further study are greatly needed. Here we provide an intermediary platform between HTS and mice by adapting mouse models of pediatric brain tumors to grow as orthotopic xenografts in the brains of zebrafish. Freshly isolated mouse ependymoma, glioma and choroid plexus carcinoma cells expressing red fluorescence protein were conditioned to grow at 34 °C. Conditioned tumor cells were then transplanted orthotopically into the brains of zebrafish acclimatized to ambient temperatures of 34 °C. Live in vivo fluorescence imaging identified robust, quantifiable and reproducible brain tumor growth as well as spinal metastasis in zebrafish. All tumor xenografts in zebrafish retained the histological characteristics of the corresponding parent mouse tumor and efficiently recruited fish endothelial cells to form a tumor vasculature. Finally, by treating zebrafish harboring ERBB2-driven gliomas with an appropriate cytotoxic chemotherapy (5-fluorouracil) or tyrosine kinase inhibitor (erlotinib), we show that these models can effectively assess drug efficacy. Our data demonstrate, for the first time, that mouse brain tumors can grow orthotopically in fish and serve as a platform to study drug efficacy. As large cohorts of brain tumor-bearing zebrafish can be generated rapidly and inexpensively, these models may serve as a powerful tool to triage drug-leads from HTS for formal efficacy testing in mice.

Isoforms of soluble alpha-tubulin in oocytes and brain of the frog (genus Rana): changes during oocyte maturation.

Rana oocytes have previously been shown to contain much more soluble tubulin than does the brain, suggesting different assembly and disassembly dynamics of frog oocyte tubulin compared to that in brain. By using centrifugation, SDS-PAGE, two-dimensional gel electrophoresis and Western blots, probed with anti-alpha-tubulin monoclonal antibodies, polymorphic alpha-tubulins (isoforms) were compared in brains and follicle-enclosed oocytes of northern (Rana pipiens) and southern (R. berlandieri) frogs. Oocyte tubulin in both species had isoforms with greater ranges of isoelectric point (pI) than those of brain tubulins; in particular, the oocyte tubulin pIs ranged further into the acidic region of the isoelectric-focusing gels than corresponding brain tubulin. This difference may, in part, be responsible for the previously reported assembly differences between oocyte tubulin (undetectable assembly) and brain tubulin (high assembly). Isoforms of alpha-tubulin with relatively acidic pI were more abundant in northern frog brain and oocyte soluble extracts than in analogous extracts from southern frogs. Furthermore, additional acidic alpha-tubulin isoforms were found in progesterone-treated oocytes (i.e., eggs), indicating increased heterogeneity of acidic alpha-tubulin isoforms during oocyte meiotic maturation. Among northern frog oocyte soluble components fractionated on Superose-6b columns, tubulin complexes with apparent molecular mass of about 1800 kDa were found to contain acidic alpha-tubulin isoforms while the putative oligomeric tubulins with an apparent molecular mass of about 250 kDa contained an additional relatively basic alpha-tubulin isoform. The acidic alpha-tubulin isoforms, therefore, are proposed to be associated with cold-adaptable cells of brain and oocytes, and may also be involved in stabilization of large soluble tubulin complexes in oocytes of the northern frog.

Soluble tubulin complexes in oocytes of the common leopard frog, Rana pipiens, contain gamma-tubulin.

Oocytes of the leopard frog, Rana pipiens, contain soluble tubulin which was previously shown to exist predominantly in megadalton (MDa) fractions and that fails to readily assemble in vitro. In order to further characterize these tubulin complexes, DEAE Sepharose chromatography, Sephacryl S-300 size exclusion columns and specific immunoprecipitation were used. The results revealed the presence of alpha-, beta-, and gamma-tubulin associated with several other proteins in the soluble fraction of Rana pipiens ovarian oocytes. These Rana oocyte tubulin complexes appear to be analogous to those recently reported in Xenopus ovulated eggs as gamma-tubulin ring complexes. This seems true since both size (estimates, i.e. approximately 2MDa) and protein components are similar. Furthermore, both alpha- and gamma-tubulin antibodies immunoprecipitated identical protein bands from Rana oocyte soluble fraction. These putative Rana gamma-tubulin ring proteins include 107, 97, 95, 90 and 75 kDa components which are similar in size to those found in Xenopus and other species. Rana appears to belong to a select group in which gamma-tubulin complexes contain significant alpha- and beta-tubulin (i.e., Xenopus and sheep), while other species such as Drosophila, Aspergillus, Saccharomyces, human cells and many other mammalian cells tested lack the other tubulin components. The heterogeneity in both size and protein components of Rana oocyte gamma-tubulin ring complexes may reflect different states of tubulin complex assembly. The lower vertebrate oocyte is hypothesized to act as a repository and prestaging point for the assembly of gamma-tubulin ring complexes which will become the maternal contribution to the centrosomes of the embryo. While the gamma-tubulin ring complexes of vertebrate eggs have been described previously, this is the first report biochemically characterizing soluble gamma-tubulin complexes in vertebrate ovarian oocytes.

Microinjection of anti-alpha-tubulin antibody (DM1A) inhibits progesterone-induced meiotic maturation and deranges the microtubule array in follicle-enclosed oocytes of the frog, Rana pipiens.

Microinjection of anti-alpha-tubulin (Dm1A) inhibited progesterone-induced meiotic maturation in large follicle-enclosed oocytes of the frog, Rana pipiens. DM1A (46 nl; 10 mg/ml) injection significantly increased the ED50 value for progesterone as determined by germinal vesicle dissolution (GVD) bioassay. By contrast, low doses of microinjected DM1A (46 nl; 2.5 mg/ml), anti-actin (clone KJ43A), anti-cytokeratin (C-11), anti-intermediate filament antibody (IFA), generic IgG (46 nl; 20 mg/ml) or sodium azide (46 nl; 1 mg/ml), an antibody preservative, were without inhibitory effect in this bioassay. Microinjected, affinity-purified DM1A (46 nl; 7.5 mg/ml) was also inhibitory, but preabsorption with pure tubulin prior to injection significantly reduced the inhibitory effect. DM1A injection had no effect on centrifugation-induced germinal vesicle migration (GVM). Previous work indicated that drugs (e.g. demecolcine and nocodazole), which destabilise microtubules, enhance both centrifugation-induced GVM and progesterone-induced GVD in Rana oocytes. Taking these results together, it is suggested that DM1A injection may have differential effects on microtubules in this cell. Thus, while the majority of microtubules were apparently depolymerised by DM1A (46 nl; 10 mg/ml) injection, a small subpopulation appeared to be stabilised as bundles. Confocal immunofluorescence microscopy of follicle-enclosed oocytes after DM1A injection revealed a major loss of microtubules throughout the cell; however, apparent sparse bundles of microtubules arranged in an approximately 600 microns shell were associated with the injectate region 24 h post-injection. By contrast, control follicle-enclosed oocytes topically labelled with DM1A post-fixation had extensive microtubule arrays similar to those previously reported in Xenopus oocytes. Intracellular recording after DM1A injection and progesterone treatment yielded an intermediate membrane potential (Vm = -31.8 mV) compared with control (immature) DM1A-injected cells (Vm = -44.7 mV) or potassium balanced salt solution (KBS)-injected cells matured with progesterone (Vm = -13.9 mV). These results suggest that DM1A injection does not completely inhibit electrophysiological changes initiated by progesterone. Working hypotheses are proposed that suggest a role for microtubules in the action of progesterone which normally lifts the prophase I block in the Rana follicle-enclosed oocyte.

The major soluble tubulins are found in mega dalton (MDa) fractions in fully-grown oocytes and eggs but not in brain of the frog, Rana pipiens.

During progesterone-induced oocyte maturation, the cell is converted from prophase I to metaphase II, which is known to involve microtubule changes. We hypothesize that progesterone affects the polymeric state of tubulins, isoform dynamics, as well as tubulin synthesis and degradation. To test the former, leopard frog (Rana pipiens) oocytes were treated with progesterone, homogenized and centrifuged at 16,000 x g. The resulting pellet and supernatant fractions were analyzed using immunoblots probed with anti-alpha-tubulin antibody DM1A and anti-beta-tubulin antibody DM1B. The results indicate that the majority of tubulins are in supernatant fractions (soluble cellular component) and further indicate changes in size of pelletable tubulins with time post-progesterone treatment. To characterize the supernatant tubulins, Superose-6b columns were used to fractionate 16,000 x g supernatant materials. The results indicate tubulins in fractions of up to 5 MDa (equivalent in size to c. 50 tubulin dimers or 5,000 kDa) predominate, while tubulin dimers and monomers of 110 kDa and 55 kDa, respectively, are relatively minor components. The tubulin in MDa fraction pools in the oocyte soluble component seem to be tissue specific, since the reverse patterns were found in either frog brain or rat (Rattus norvegicus) brain tissue under the same conditions. Microtubule poisons, taxol and nocodazole, were used to test the dynamics of tubulin in MDa fractions and the results were opposite to that expected, i.e., taxol, a microtubule stabilizer, decreased tubulin in large MDa fractions while nocodazole, a microtubule destabilizer, increased it. Preliminary data also indicated that progesterone treatment alters the tubulin size classes (size-shift) in comparison with that in immature oocytes. These results, in combination with other research reports, suggest that tubulin in MDa fractions may either be associated with other cellular components or act as intermediates in microtubule dynamics, perhaps via oligomerization, during oocyte maturation and early development.

Fibronectin peptide DRVPHSRNSIT and fibronectin receptor peptide DLYYLMDL arrest gastrulation of Rana pipiens.

Gastrulation is characterized by dramatic cell migration which is thought to require the interaction of cell adhesion molecules with extracellular molecules. We have tested two novel peptides, a fibronectin peptide and a fibronectin receptor peptide, for their effects on gastrulation of the leopard frog Rana pipiens. The fibronectin peptide DRVPHSRNSIT corresponds to residues 1373-1383 of the cell-binding domain of fibronectin; the receptor peptide DLYYLMDL corresponds to residues 124-131 of beta 1 subunit of a variety of integrins including alpha 5 beta 1. Either of these peptides significantly inhibited gastrulation after being microinjected into mid-blastulae. These results indicate that these sequences may correspond to the ligand/receptor interaction sites of fibronectin and its receptor(s).

Evidence of a myoepithelial cell in tick salivary glands.

Type III acini from feeding female Dermacentor variabilis varied in size during in vitro and in vivo fluid production. As the type III acinus enlarged, its lumen enlarged and the adlumenal cell became thinner. As the acinus contracted, its lumen became smaller while the adlumenal cell became wider. Actin was demonstrated in salivary glands using an immunoblot technique. Actin was localized in the adlumenal cells of type III acini with fluorescent microscopy using rhodamine-phalloidin and with electron microscopy using heavy meromyosin to decorate actin filaments. Pre-treatment of salivary glands with cytochalasin D abolished fluorescence in adlumenal cells subsequently treated with rhodamine-phalloidin. These results support the hypothesis that the adlumenal cell in type III acini functions as a myoepithelial cell.

Posttranslational modifications and assembly characteristics of goldfish tubulin.

Cell-free extracts from goldfish brain, ovarian follicles, testes and the cell line, ATCC CCL-71, were analyzed for posttranslationally modified tubulins. All samples, with the exception of that from brain where the reverse was true, contained more tyrosinated than detyrosinated alpha-tubulin. Additionally, extracts from brain and testes exhibited acetylated alpha-tubulin whereas this isoform was not visualized on blots of cell-free preparations from follicles and CCL-71 cells. Assembly of brain and ovary tubulin was induced with taxol. Brain tubulin, partially purified through three cycles of assembly/disassembly was associated with a variety of putative microtubule-associated proteins (MAPs), most of which had a high molecular mass. There were very few cold stable microtubules in brain preparations whereas, for ovary, purification of tubulin was hampered by significant losses of cold stable polymer. Comparison of brain and ovary showed there was no correlation between the extent of alpha-tubulin detyrosination or acetylation and cold stability of microtubules. Moreover, cycled tubulin from ovary contained acetylated tubulin even though this was not observed on blots of cell-free extracts from ovary or from follicles. Cultured goldfish cells contained extensive arrays of microtubules, many of which originated from discrete organizing centers. The results reveal the widespread distribution of posttranslationally modified tubulins in goldfish tissues, the differing assembly/disassembly characteristics of tubulin from brain versus ovary, and the presence of putative neural MAPs, mostly with a high molecular mass.

Taxol-induced assembly of brain and testis tubulins, and ovarian follicle tubulin dynamics in the frog (genus Rana), in vitro.

1. Tubulins from brain and testes of the frog (genus Rana) were purified by taxol and cyclic assembly-disassembly. 2. Attempts to purify ovarian tubulins by this same method were unsuccessful. 3. Oocyte fractions isolated in microtubule-stabilizing detergent buffer were analyzed by SDS-PAGE and Western blots were probed with monoclonal antibodies to tubulins. 4. In the fully-grown ovarian follicle (diameter approx. 1.7 mm), the majority of tubulin was present in the supernatant fraction. 5. Tubulin was routinely detected in the medium speed (16,000 g) prophase I oocyte pellet. 6. Nocodazole treatment of ovarian follicles in vitro reduced the pelletable tubulin detected, while taxol increased tubulin in the pellet and decreased the supernatant tubulin. 7. These results indicate that taxol is useful in the purification of brain and testes tubulin from Rana tissue; while oocyte tubulin appears to respond to taxol in vivo, another method will be required for in vitro assembly and purification of ovarian tubulin.

Effects of acrylamide on germinal vesicle migration and dissolution in oocytes of the frog, Rana pipiens.

We have demonstrated that when Rana oocytes are treated with 10 mM acrylamide, germinal vesicle migration (GVM) is promoted while meiosis reinitiation by progesterone is inhibited. A number of other specific alterations result from the acrylamide treatment: (i) A dense band of fibrillar material appears adjacent to the oolemma in acrylamide-treated oocytes. Furthermore, (ii) the fibrillar material reacts with an intermediate filament antibody using immunogold techniques applied to transmission electron microscopy. Moreover, (iii) acrylamide inhibits progestogen-induced annulate lamellae breakdown. In addition, (iv) the cortical mitochondria-rich layer appears to be thickened by acrylamide, which also (v) affects oocyte microvillar retraction and organization. Finally, (vi) electrophysiological measurement of membrane voltage indicates that acrylamide does not significantly affect cell viability during the incubation period used in this study. In summary, acrylamide exerts profound effects on the physiological event of GVM, and these are consistent with the hypothesis that changes in the cytoskeleton are a contributing factor in meiosis reinitiation.

Effect of microtubule reactive drugs on steroid- and centrifugation-induced germinal vesicle migration during goldfish oocyte meiosis.

During the process of progestogen-induced meiotic maturation in the goldfish oocyte, the oocyte nucleus (germinal vesicle, GV) migrates to the sperm entry site or micropyle at the animal pole. Following GV migration (GVM) to the micropyle, the nuclear membrane undergoes dissolution (GVD) and the cell enters metaphase I in preparation to generate the first polar body. Microtubule destabilizing drugs including colcemid, nocodazole and vinblastine were found to elicit GVM, mimicking the process which occurs just prior to the prophase I-metaphase I transition during steroid induced oocyte meiotic maturation. In addition, these drugs enhanced the induction of GVM by 17 alpha, 20 beta dihydroxy-4-pregnen-3-one, a potent, naturally occurring meiotogenic steroid in this species. By contrast, taxol, a microtubule stabilizing drug, was found to inhibit steroid induced GVM. A new assay for centrifugation induced GVM was applied to the goldfish oocyte in order to assess effects of steroids and drugs on GVM, without the complication of GVD or the restrictions imposed by the slow time course of naturally occurring GVM. The effective centrifugal force (ECF) required to elicit GVM in 50% of the oocytes (ECF50) decreased significantly after short incubations (1-5 hr) of oocytes with either 17 alpha,20 beta dihydroxy-4-pregnen-3-one or microtubule disrupting drugs (i.e., colcemid, nocodazole, or vinblastine). A working hypothesis, modeled after the effects of microtubule disrupting agents on intermediate filament arrays in somatic cells, is proposed in which a small number of microtubules or other polymeric tubulin units are responsible for maintaining a cytoskeletal array.(ABSTRACT TRUNCATED AT 250 WORDS)

Estradiol-17 beta Silastic implants suppress oocyte development in the brook trout, Salvelinus fontinalis.

A technique for long-term implantation (i.e., 6 months) of steroid-containing Silastic capsules in brook trout was developed along with a method for longitudinal study of ovarian dynamics in individual females. Estradiol-17 beta implantation, extending from spring during early oogenesis through November, suppressed overall ovarian development in a dose-dependent manner, as determined by follicle size analysis and gonosomatic index, and reduced the peak of plasma estradiol-17 beta normally observed prior to ovulation in the fall. Frozen sections of follicles revealed that estradiol implants retarded the progression of vitellogenesis from primary to secondary stages in the largest size class of follicles. In particular, sections of ovarian biopsies from animals with three estradiol implants had only small follicles in September which cytologically were no more advanced than those in May. By contrast, animals receiving steroid-free implants had significantly larger follicles in September which entered the secondary stage of vitellogenesis and became increasingly competent to respond to progestogen treatment in vitro through October and November. Image analysis of ovarian biopsies indicated that estradiol implants deranged the normal group-synchronous growth pattern of the brook trout ovary and tended to produce asynchronous growth, especially in animals with a single estradiol implant.

Germinal vesicle migration and dissolution in Rana pipiens oocytes: effect of steroids and microtubule poisons.

Germinal vesicle migration (GVM) as evidenced by the appearance of the germinal vesicle at the animal pole surface was induced by nocadazole and demecolcine (colcemid). Nocodazole significantly lowered the progesterone ED50 for germinal vesicle dissolution (GVD). Both demecolcine and nocodazole enhanced centrifugation-induced GVM (i.e., lowered ooplasmic viscoelasticity) after 6-h incubation, and both potentiated the effect of progesterone in this assay. Estradiol, by contrast, inhibited GVM induced by demecolcine in both follicle-enclosed and denuded oocytes. Estradiol was also found to inhibit the normal enhancement of centrifugation-induced GVM by demecolcine or progesterone. Taxol was found to have effects that were generally opposite to those of demecolcine and nocodazole. Taxol inhibited centrifugation-induced GVM either alone or in the presence of progesterone. In addition, taxol significantly increased the progesterone ED50 for GVD induction. Taken together the available data support the hypothesis that microtubules play a role in maintaining the internal position of the germinal vesicle in the prematuration oocyte and that changes occur in the oocyte cytoskeleton during maturation.

A study of goldfish oocyte meiosisin vitro: effects of 2,4-dinitrophenol and adenosine-5-triphosphate.

Germinal vesicle migration (GVM) and/or dissolution (GVD) were measured in goldfish oocytes, treated with 17α, 20ß dihydroxyprogesterone (DHP) and other compounds considered to effect the cytoskeleton and oxidative phosphorylation,in vitro. Administration of DHP reinitiated meiotic maturation, increasing GVM and GVD in goldfish oocytes. Addition of 2,4-dinitrophenol (DNP) to the incubation medium significantly inhibited DHP-induced GVM and GVD. The DNP effect was found to be partially reversible after 24 h and could be reversed fully after a further delay of approximately 24h. Treatment of goldfish oocytes with demecolcine (DE; a colchicine derivative also known as colcemid) induces GVM to the micropyle without effecting GVD; while Cytochalasin-B which inhibits microfilament polymerization impairs both GVM and GVD. Administration of DNP, significantly inhibited DE-induced GVM, suggesting that GVM as well as GVD are dependent upon the process of oxidative phosphorylation. Addition of adenosine-5' -triphosphate (ATP) at low concentrations (0.01-0.1 mM) did not effect DHP-induced or DNP-inhibited GVD in goldfish oocytes. The present results are consistent with the idea that migration of the oocyte nucleus during meiosis reinitiation has an energy requirement and involves participation by the cytoskeleton.

Effects of cytochalasin B on steroid-induced oocyte meiosis and centrifugally induced nuclear movement in the goldfish Carassius auratus.

In goldfish, 17 alpha, 20 beta-dihydroxyprogesterone (DHP) induced oocyte nucleus or germinal vesicle migration (GVM) and dissolution (GVD) in a dose-related fashion. Administration of cytochalasin B (CB) in the presence of DHP inhibited the steroid-induced GVM and GVD after 24 and 48 h of incubation. The presence of CB alone, at concentrations below 25 micrograms/mL, had no effect on GVM or GVD. Furthermore, CB, either alone or in combination with DHP, elicited significant increases in follicular diameter after 24 and 48 h of incubation. To test the effect of CB on ooplasmic viscoelasticity, fully grown follicles were centrifuged and the centrifugally induced germinal vesicle (GV) displacement was determined. Pretreatment (24 h) of follicles with high doses of CB (25 and 50 micrograms/mL) increased the movement of GV in a centripetal direction. However, at lower concentrations (0.005-5 micrograms/mL), CB treatment was without an effect on the centrifugally induced GV movement in the oocyte. The present study suggests involvement of microfilaments or other cytoskeletal components, sensitive to CB, in the mechanisms of GVM and GVD in goldfish oocytes. In addition, a simple technique has been described for testing ooplasmic viscoelasticity determined by movement of the GV under a centrifugal force.

Effect of insulin on meiosis reinitiation induced in vitro by three progestogens in oocytes of the goldfish (Carassius auratus).

Three progestogens were tested for their ability to elicit meiosis reinitiation as evidenced by germinal vesicle dissolution (GVD) in goldfish (Carassius auratus) follicle-enclosed oocytes in vitro. In two independent experiments 17 alpha-20 beta-dihydroxyprogesterone (DHP) was most active followed in turn by 17 alpha-hydroxyprogesterone (HP) and progesterone (P). Values of the effective dose for a 50% response for GVD induced by P and HP were significantly reduced by the addition of Na-insulin, however, insulin had no significant effect on DHP incubates. Meiotogenic activity was potentiated by Na-insulin in the following heirarchy: P greater than HP greater than DHP. These results indicate that insulin, which had little meiotogenic activity alone, was capable of differentially enhancing progestogen activity in this system. The mechanism by which Na-insulin augments progestogen GVD-inducing activity is unknown, but may include decrease of progestogen degradation, increase in progestogen biosynthesis, and direct insulin effects on the oocyte. The results suggest that insulin may play a physiological role in goldfish oocyte meiosis reinitiation by enhancing the activity of certain progestogens, which by themselves are not potent meiotogens.

Electrophysiology of in vitro insulin- and progesterone-induced reinitiation of oocyte meiosis in Rana pipiens.

Induction of meiosis in Rana pipiens oocytes in vitro was studied using intracellular electrophysiological recordings and a morphological count of nuclear dissolution. In type III (i.e., defolliculated theca-free, with follicle cells) and type IV (i.e., denuded lacking follicle cells) Rana oocytes (Schuetz and Lessman, '82), Na+-insulin evoked nuclear or germinal vesicle dissolution (GVD), apparent reinitiation of meiosis, and marked reductions in cellular membrane potential and membrane current. Electrophysiological indications of the reinitiation of oocyte meiosis were most strongly apparent in the marked reduction (ca.98%) of membrane current. The overall GVD activity of insulin was reduced but not totally absent in type IV oocytes compared to type III cells that received similar hormone treatment, confirming previously published findings that the follicle wall enhances insulin-induced GVD. Results confirm insulin GVD activity in this system and demonstrate that insulin-induced reinitiation of meiosis is associated with changes in membrane-associated parameters that are indistinguishable from those induced by progesterone. These results raise interesting questions concerning the cellular mechanism by which two chemically dissimilar hormones (i.e., steroid vs. protein) have similar or even identical effects on a cell such as the oocyte. The findings presented are consistent with the concept that more than one hormone may be involved in meiotic maturation of the oocyte.

Role of follicle wall in meiosis reinitiation induced by insulin in Rana pipiens oocytes.

The involvement of the ovarian follicle wall in insulin induction of Rana pipiens oocyte maturation in vitro was examined. Complete removal of the follicle wall significantly decreased, but did not obliterate, oocyte maturation (i.e., germinal vesicle breakdown, GVBD) induced by insulin. Dose-response studies of GVBD induction revealed that oocytes within intact follicles were at least 100 times more sensitive to insulin than denuded oocytes. Addition of cyanoketone, a steroid biosynthesis inhibitor, to intact follicles also suppressed insulin-induced GVBD. Inhibitory effects of either follicle wall removal or cyanoketone were not observed when denuded oocytes were treated with progesterone. Addition of either progesterone or pregnenolone to insulin-treated denuded oocytes augmented the oocyte GVBD response compared to either steroid alone and essentially replaced the effect of the follicle wall. In summary, steroidogenesis in the follicle wall appears to be a major factor contributing to the ability of insulin to induce GVBD. However, whether insulin stimulates follicle wall steroidogenesis or simply augments the biological activity of endogenous basal steroid levels is unresolved. The in vitro results show that oocyte maturation can be modulated by the combined actions of several hormones. Such steroid-insulin interactions may also be relevant to understanding the control of oocyte maturation in amphibians and other vertebrates, including mammals, under physiological conditions in vivo.