Charles A. Lessman

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.

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.

Estradiol-17β 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-17β 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-17β 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 groupsynchronous growth pattern of the brook trout ovary and tended to produce asynchronous growth, especially in animals with a single estradiol implant.

Detection of juvenile hormone I, II and III in adult monarch butterflies (Danaus plexippus)

Abstract Hemolymph of Danaus plexippus (monarch butterfly) was analyzed for juvenile hormone titer by gas liquid chromatography-mass spectrometry (GC-MS) in the selected ion monitoring mode. Laboratory reared, reproductively active, adult males and females contained JH I, II and III. JH II predominated with titers ranging from 2.3 to 11 ng/ml hemolymph. Titers of JH I and JH III were approx. 1 order of magnitude lower than those of JH II. JH I, II and III acids were also detected, but at levels generally lower than the corresponding JHs. JH titers in field collected reproductively inactive, gregarious adult monarchs, were 1–2 orders of magnitude lower than those of laboratory reared reproductively active monarchs. JH II was again the predominant JH in these animals.

Juvenile hormone metabolism, binding and esterase activities in the haemolymph of the adult monarch butterfly (Danaus p. plexippus L.: Lepidoptera)

The interaction of [3H]juvenile hormone I (JHI) with haemolymph of the adult monarch butterfly, Danaus p. plexippus L. was investigated. Whole haemolymph bound [3H]JHI preferentially in vitro compared to bovine serum albumin or buffer alone using a charcoal binding assay. [3H]JHI was converted to polar metabolites by adult haemolymph as assessed by an octane: methanol-H2O partition assay and thin layer chromatography. Phenyl methyl sulphonyl fluoride (PMSF), a potent esterase inhibitor, was effective in decreasing the conversion of [3H]JHI to polar metabolites, thus suggesting the presence of JH esterase (JHE) activity. Post eclosion JHE activity assessed in vitro increased in females, peaking at day 6 post eclosion; while in males there was an initial decrease followed by a small peak at day 6. The fluctuations observed were not attributable to haemolymph volume changes. [3H]JHI metabolism assessed in vivo at 40, 140, 240 and 1380 min post injection, tested at four different stages or periods of the life cycle, indicated that the greatest metabolism of [3H]JHI occurred at day 3 post eclosion in both sexes followed respectively by January males and females, June-July males and females, September females and finally September male wild caught adults. JH binding assays carried out on haemolymph fractions separated by Sephadex G-100 column chromatography indicated a peak of JH binding activity corresponding to 29,500 ± 6000 mol. wt (x ± SEM, n = 6). Bioassay indicated that the bulk of endogenous JH was associated with this peak of JH binding activity. JHE activity assessed in these same fractions indicated two major peaks one > 100,000 mol. wt and the other at 79,000 ± 5000 mol. wt (x ± SEM, n = 6). The fractions of the smaller mol. wt peaks were found to inactivate both [3H]JHI and endogenous JH biological activity when assessed using the Galleria wax test.

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. The net effect of progestogen or microtubule disrupting drugs would be to collapse or reorganize the array to allow GVM.

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, 20β 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.

Interaction of juvenile hormone III with cricket (Gryllus domesticus) ovary in vitro as assessed by high performance liquid chromatography

Abstract 1. 1. A study of the interaction of 3H-juvenile hormone III (JH III) with ovarian tissue of the cricket, Gryllus domesticus, was carried out in vitro. 2. 2. High performance liquid chromatography (HPLC) was used to follow the radiolabel in both media and tissue extracts. 3. 3. At 0.5, 2, 4, 8 and 12 hr incubation, tissue extracts contained low amounts (i.e., 10% or less of the total) of the radiolabel while the media extracts contained decreasing amounts of intact JH III and increasing amounts of radiolabeled metabolites. 4. 4. The conversion of JH III to polar metabolites was time- and tissue-dependent. 5. 5. The major metabolite did not co-migrate with JH acid, JH diol or JH acid-diol; but may represent a conjugated form of the JH acid-diol. 6. 6. No significant conversion occurred in the presence of large (ovulated) terminal oocytes which lacked follicle wall components.