Induction of maturation in follicle-enclosed oocytes: the response to gonadotropins at different stages of follicular development

Antral follicles, isolated from either nontreated or pregnant mare's serum gonadotropin (PMSG)-primed 27-day-old rats, were incubated in the absence or the presence of either luteinizing hormone (LH), follicle-stimulating hormone (FSH), or forskolin. The effect of these agents on oocyte maturation and cyclic adenosine 3',5'-monophosphate (cAMP) accumulation was studied and compared. Both gonadotropins, LH and FSH, as well as forskolin, effectively induced maturation of oocytes enclosed by large antral follicles isolated from PMSG-primed rats. On the other hand, we found that maturation of oocytes enclosed by small antral follicles, isolated from nonprimed and PMSG-primed rats, could be induced by either FSH or forskolin but not by LH. cAMP determinations revealed that, in spite of the inability of LH to induce oocyte maturation, elevated concentrations of the nucleotide were detectable in small antral follicles exposed to this gonadotropin. Since granulosa cells isolated from the large but not the small antral follicles were stimulated by LH to generate cAMP, the elevation of cAMP concentrations in the small antral follicle apparently represented the response of the theca cells to this gonadotropin. Since it is the ability of the granulosa cells to interact with the hormone that determines whether or not oocyte maturation will occur, we suggest that the granulosa, but not the theca cells, mediate LH action to induce oocyte maturation.     

Evidence for an ascorbate shuttle for the transfer of reducing equivalents across chromaffin granule membranes

Adrenal chromaffin granules must shuttle reducing equivalents from the cytosol inward to reduce ascorbic acid oxidized during norepinephrine biosynthesis by intragranular dopamine-beta-hydroxylase. A transmembrane electron shuttle between the external (cytosolic) and intragranular ascorbate pools was demonstrated in vitro in intact bovine chromaffin granules undergoing tyramine- or dopamine-stimulated dopamine-beta-hydroxylase turnover. Incubation of intact chromaffin granules with tyramine results in a time-dependent decrease in reduced intragranular ascorbate and production of octopamine. The rate of ascorbate oxidation is a function of the extragranular concentrations of tyramine over the range 50 microM to 2 mM and is 95% inhibited by addition of the dopamine-beta-hydroxylase inhibitor disulfiram. The stoichiometry of octopamine synthesized/ascorbate oxidized closely approximates unity. The presence of extragranular dopamine also induces oxidation of intragranular ascorbate which is inhibited by blocking dopamine transport with reserpine. On the other hand, incubation with octopamine, which is also transported by the granules, causes no net decrease in reduced intragranular ascorbate. The presence of 400 microM extragranular ascorbate abolishes the observed tyramine-induced intragranular ascorbate oxidation. The addition of ascorbate extragranularly 30 min after addition of tyramine reverses the oxidation of intragranular ascorbate. The measurement of [14C]ascorbate distribution ratios in granule pellets and supernatants indicates that there is no transmembrane transport of ascorbate. Extravesicular NADH had no significant effect on matrix ascorbate levels during beta-hydroxylation. These data provide new in vitro evidence that chromaffin granules shuttle reducing equivalents inwardly from an extra- to an intravesicular ascorbate pool and that cytosolic ascorbate is the source of the intragranular reducing equivalents required during norepinephrine biosynthesis.     

DETERMINATION OF GUSTATORY SUCROSE THRESHOLD IN WATER BY USE OF A LINEAR SUCROSE GRADIENT

A novel experimental method was developed which allows the determination of the threshold concentration of sucrose by use of a linear sucrose gradient in water. With this method a continuous tasting of the test-liquid is possible. A panel of 15 persons experienced in taste-testing was used. Three gradients of different steepness were applied: 0 to 1.5% (w/w) sucrose in 2 min (I), 3 min (II) and 4 min (III). The results of the new method were compared with those of the standard method (DIN). With gradients I and II we found values which were significantly higher than those of the standard method (I: 0.49% (w/w); II: 0.46% (w/w); DIN: 0.31% (w/w)), whereas with gradient III the same threshold value was found as with the DIN-Method (III: 0.32% (w/w)).     

A cell culture assay for follicle-stimulating hormone

Cultured rat ovarian granulosa cells respond to follicle-stimulating hormone (FSH) by synthesizing and secreting plasminogen activator. The specificity of the response for FSH prompted us to explore the use of this system as an in vitro bioassay for FSH. The release of FSH by pituitary cell cultures has been examined by this method, as have been preparations of FSH of known biological activity. The results indicate that the granulosa cell system allows accurate, rapid, and convenient determinations of FSH activity. Furthermore, the method obviates metabolic clearance problems associated with whole animal assays and it is extremely sensitive: as little as 10(-15) mol (approximately 100 micronIU) of FSH can be detected.     

Nucleotide sequence of the Pseudomonas fluorescens signal peptidase II gene (lsp) and flanking genes.

The lsp gene encoding prolipoprotein signal peptidase (signal peptidase II) is organized into an operon consisting of ileS and three open reading frames, designated genes x, orf149, and orf316 in both Escherichia coli and Enterobacter aerogenes. A plasmid, pBROC128, containing a 5.8-kb fragment of Pseudomonas fluorescens DNA was found to confer pseudomonic acid resistance on E. coli host cells and to contain the structural gene of ileS from P. fluorescens. In addition, E. coli strains carrying pBROC128 exhibited increased globomycin resistance. This indicated that the P. fluorescens lsp gene was present on the plasmid. The nucleotide sequences of the P. fluorescens lsp gene and of its flanking regions were determined. Comparison of the nucleotide sequences of the lsp genes in E. coli and P. fluorescens revealed two highly conserved domains in this enzyme. Furthermore, the five genes which constitute an operon in E. coli and Enterobacter aerogenes were found in P. fluorescens in the same order as in the first two species.     

Effect of neurotensin on pancreatic function in man

The effect of neurotensin on submaximally-stimulated hepatobiliary and pancreatic secretion was studied in 6 healthy subjects. An intravenous infusion of neurotensin 1.4 ± 0.3 pmol/kg/min, designed to reproduce plasma neurotensin immunoreactivity levels within the physiological range, produced a significant increase in pancreatic bicarbonate output. Plasma concentrations of pancreatic polypeptide rose by 83 ± 16 pmol/l and were associated with a small reduction in trypsin, but no significant change in bilirubin outputs.     

Mitochondrial regulation in sea urchins. I. Mitochondrial ultrastructure transformations and changes in the ADP:ATP ratio at fertilization.

Mitochondrial ultrastructural transformations have been examined in intact eggs and embryos from three sea urchins, Arbacia punctulata, Strongylocentrotus purpuratus, and Lytechinus pictus. Following fertilization, naturally occurring ultrastructural transformations (designated as condensed to orthodox) were observed to occur in mitochondria of all three families. The ratios of the soluble ADP and ATP pools were examined in eggs of S. purpuratus before and after fertilization in order to test whether the ultrastructural transformations reflect a decrease in relative size of the ADP pool following fertilization. Our data indicate that there is a decrease in the ADP:ATP ratio at fertilization. These findings and their implications are discussed with respect to presently accepted theories on mitochondrial regulation.     

Delimitation of the maritime boundary in the gulf of Maine area

The compulsory dispute settlement regime included in the 1982 Law of the Sea Convention is recognized as one of the most comprehensive in a modern international convention. Yet, in the recent application of this regime, the question has arisen as to whether the procedural prerequisites associated with the LOS Convention's compulsory dispute settlement mechanism are so arduous as to avoid binding and compulsory jurisdiction in most instances. This article addresses that question by examining, in particular, the reasoning of the Southern Bluefin Tuna arbitration tribunal, which found Article 281 of Section 1 of the LOS Convention to bar jurisdiction to the compulsory dispute settlement mechanism prescribed by the Convention, and offers suggestions as to how states might distinguish or overcome the barriers imposed by the Southern Bluefin Tuna tribunal in future cases.