Seymour H. Pomerantz

The Role of Nonsteroidal Regulators in Control of Oocyte and Follicular Maturation

Publisher Summary This chapter discusses the role of nonsteroidal regulators in control of oocyte and follicular maturation. The ovarian follicle is bathed in a fluid rich in steroid hormones as well as nonsteroidal regulators which interact to serve to control its maturation, responsiveness to gonadotropins, as well as to lead to control of the maturation of its oocyte. The orderly maturation of an ovarian follicle and its oocyte is controlled by pituitary LH and FSH in concert with local intrafollicular regulators. The principal nonsteroidal follicular regulators are an oocyte maturation inhibitor, a luteinization inhibitor, a luteinization stimulator, FSH receptor binding inhibitor, and inhibin-F. OMI is a polypeptide

Tyrosinase activity and abundance in Cloudman melanoma cells

Rabbit anti-tyrosinase antibodies were used to study the abundance, processing, and degradation of tyrosinase in murine (Cloudman) melanoma cells. The polyclonal antibodies precipitated low-molecular-weight (68,000 and 70,000) and high-molecular-weight (78,000 and 80,000) tyrosinases that had a precursor-product relationship. Cells with high basal tyrosinase activity had high levels of newly synthesized tyrosinase. Cells with low tyrosinase activity synthesized less tyrosinase and degraded the enzyme at a faster rate than cells with high tyrosinase activity. Melanotropin (melanocyte stimulating hormone), dibutyryl cyclic adenosine monophosphate, and isobutylmethylxanthine caused an increase in the abundance of newly synthesized tyrosinase that was directly proportional to the increase in enzyme activity. This enzyme was not a phosphoprotein. Other changes in the culture conditions that increased the level of tyrosinase activity increased the abundance of newly synthesized enzyme. It is thus concluded that the level of tyrosinase activity in Cloudman melanoma cells is a direct reflection of the abundance of enzyme protein.

L-Tyrosine-3, 5-3H Assay for Tyrosinase Development in Skin of Newborn Hamsters

Tyrosinase in hamster skin, measured by the production of 3HOH from L-tyrosine-3,5-3H, increases about sevenfold in specific activity from the 14-day fetus to day 6 of life. Albino hamsters exhibit no activity, but enzyme is present in the ventral (white) areas of the Syrian hamster. 3,4-Dihydroxy-L-phenylalanine is required as a cosubstrate.

Purification and properties of tyrosinases from Vibrio tyrosinaticus

Two tyrosinase isoenzymes (EC1. 10.3.1) have been highly purified from Vibrio tyrosinaticus . This represents the first characterization of tyrosinases from eubacteria. The enzymes have molecular weights of 41,000 and 38,500, closer to the tyrosinase of Neurospora crassa (33,000) than to the mammalian enzymes from mouse and hamster melanoma (55,000 and 70,000). The bacterial tyrosinases are also different from the mammalian enzymes in that the former possess basic isoelectric points. The Vibrio tyrosinases do not cross-react with antiserum against a hamster melanoma tyrosinase. The K m values for l -tyrosine are 3.1 m m for both isoenzymes while the K m value for l -DOPA is 36 m m for one and 67 m m for the other. The values for tyrosine are tenfold, and those for dopa about 100-fold, larger than the corresponding K m values for the tumor enzymes. Apoenzyme was prepared by treatment with diethyldithiocarbamate, and activity was partially or wholly restored by addition of Cu 2+ , Mn 3+ , Cd 2+ , or Fe 2+ . The one isoenzyme examined has some activity on d -tyrosine and very slight activity against d, l - m -tyrosine. No activity was detected with catechol or l -phenylalanine.

Intrafollicular control of oocyte maturation in the PIG

SummaryThe mammalian oocyte becomes arrested at the diplotene stage of the first meiotic division during prenatal or early postnatal life. It remains arrested in meiosis until shortly before ovulation when the surge of gonadotropin induces resumption and completion of meiosis to the metaphase II stage. When oocytes are harvested from medium-sized or large follicles of pig and other species and cultured, they resume meiosis spontaneously indicating that the follicles exert an inhibitory influence on meiosis. To analyze the control of meiosis by follicular components, culture of isolated pig oocytes in the presence of follicular cells or follicular fluid (FF1) has been used as a model in this laboratory. An oocyte maturation inhibitor (OMI) has been isolated and partially purified by ultrafiltration and gel chromatography of FF1 and shown to be a polypetide with a molecular weight in the order of 2000 daltons. Physiological characterization has shown that the effect of OMI in vitro is reversible and that it can be overcome by luteinizing hormone (LH). The action of OMI requires the presence of cumulus cells surrounding the oocyte since it was found that denuded oocytes, stripped of cumulus cells, do not respond to OMI. Furthermore, when cumulus-enclosed oocytes were cultured, OMI inhibited the differentiation of the cumulus cells in terms of morphology and progesterone secretion in a dose-related manner. The inhibition of cumulus differentiation by OMI was reversible and could be overcome by LH. The results indicate that the effect of partially purified OMI upon meiosis may be mediated by the cumulus cells.

Serum concentrations of β-melanocyte-stimulating hormone in human pregnancy

Abstract β-Melanocyte-stimulating hormone (β-MSH) has been measured in pregnant women with the use of a sensitive tube radioimmunoassay technique which does not require prior extraction. This peptide rises progressivley throughout pregnancy with its highest concentration at term. Measurable quantities exceeding maternal levels were observed in both cord blood and amniotic fluid and elevated levels of β-MSH were found in lactating women. The role and possible chorionic origin of β-MSH remain to be determined, as well as the possible clinical use.

Monophenolase activity of avocado polyphenol oxidase

Abstract Polyphenol oxidase of avocado mesocarp catalyses (a) the orthohydroxylation of monophenols like l -tyrosine, d -tyrosine, tyramine and p -cresol, and (b) the oxidation of the corresponding o -dihydroxyphenols to quinones. The rate of step b is much greater than that of step a. The hydroxylation of monophenols occurs after a lag period. DOPA or ascorbate effectively eliminate the lag but not dl -6-methyltetrahydropteridine or tetrahydrofolic acid. At 1.66 × 10 −4 M, α,α-dipyridyl has no effect, while diethyldithiocarbamate at this concentration inhibits the hydroxylation reaction by 90%. The tyrosinase activity of avocado polyphenol oxidase is inactivated in the course of the reaction; this inactivation occurs faster and is more pronounced in the presence of exogenously added DOPA. This inactivation is partially prevented by a large excess of ascorbate. The K m values indicate that tyramine, dopamine, p -cresol and 4-methyl catechol are better substrates for avocado polyphenol oxidase than tyrosine or DOPA.

Action of Porcine Follicular Fluid Oocyte Maturation Inhibitor in Vitro: Possible Role of the Cumulus Cells

To study the mode of action of porcine follicular fluid oocyte maturation inhibitor isolated cumulus-enclosed or mechanically denuded pig oocytes were used. Two types of culture media were employed, a complex containing 15% pig serum (TC199A) and a defined, minimal medium (BMOC). The maturation of cumulus-enclosed oocytes was comparable in the two types of culture media, but only in the complex medium did the cumulus cells remain functional in terms of morphology and progesterone secretion. The low molecular weight portion of follicular fluid partially inhibited oocyte meiosis and cumulus progesterone secretion in 199A. No inhibition of oocyte maturation was seen when follicular fluid was added to BMOC. Since denuded oocytes did not respond to follicular fluid in either culture medium, it is suggested that the cumulus cells may mediate the action of the follicular fluid oocyte maturation inhibitor.

A sensitive new assay for the oxidation of 3,4-dihydroxy L-phenylalanine by tyrosinase.

Abstract A sensitive new assay for the oxidation of 3,4-dihydroxy- l -phenylalanine (dopa) employs [2,5,6-3H]dopa and involves the measurement of 3H+ released from the 6-position when the dihydroindole ring is formed. At its lower limit of sensitivity the assay will measure the oxidation of about 0.8 nmol min−1 of dopa and also permits the measurement of this activity in homogenates and turbid solutions. Use of the radiometric assay proves that the slow step in the overall conversion of dopa to dopachrome is the oxidation of 2,3-dihydro-5,6-dihydroxyindole-2-carboxylate to dopachrome.

Actions of hormones and other factors upon oocyte maturation.

Oocyte maturation is controlled by a combination of hormonal and local follicular factors. Osmolarity, pH, and perhaps Ca2+ concentration of the surrounding medium are also important. Follicular fluid contains a low molecular weight OMI which acts to keep the oocyte from maturing. Luteinizing hormone added to cultured cumulus enclosed porcine oocytes can reverse the inhibitory action of OMI. The level of OMI in the follicular fluid appears to decrease as the follicle matures. Addition of FSH and prolactin to cultured granulosa cells stimulates OMI secretin whereas addition of testosterone or dihydrotesterone brings about a decrease in OMI secretion. Elevated LH in vivo may bring about oocyte maturation before ovulation by (a) an antagonist action on OMI; (b) stimulating the synthesis of testosterone by theca cells and thus inhibiting the synthesis of OMI by granulosa cells; and (c) action on the granulosa cells to promote luteinization which may also cause a decrease in OMI synthesis. The hastened oocyte maturation associated with follicular atresia could be due to a decline in OMI due to granulosa cell death and/or elevated follicular androgens.