Resumption of oocyte meiosis in mammals: on models, meiosis activating sterols, steroids and EGF-like factors.

De novo synthesis of meiosis activating sterols (MAS) was stimulated by LH- and AY-9944 in rat cultured follicles and cumulus oocyte complexes (COCs), but could not be measured in denuded oocytes. Thus, MAS synthesized by the somatic compartment of the follicle could serve as a signal for the resumption of meiosis. Nevertheless, the delay in germinal vesicle breakdown (GVB) after MAS or AY-9944 stimulation as compared with gonadotropins, obtained by several groups, remains the strongest evidence against the suggested role of MAS as an essential mediator of LH in meiosis resumption. Recently several studies using mammalian COCs in culture have implied that steroids, like in fish and amphibians, serve as signals in mediating the LH/hCG stimulation of meiosis. However, in these studies there was no clear distinction between the requirement for steroids for the acquisition of meiotic competence, oocyte and follicle wellbeing or as a signal for meiotic resumption. Further, some of the authors overlooked earlier studies showing that blocking ovarian or follicular steroidogenesis does not affect GVB, the first step of meiosis resumption. Finally, in vivo and in vitro studies in the rat confirm and extend recent studies showing that locally produced and released EGF-like factors, such as epiregulin, seem to mediate at least part of the LH/hCG actions on oocyte maturation and release of ova at ovulation.

Tyrosinases of murine melanocytes with mutations at the albino locus.

Tyrosinase (monophenol monooxygenase; monophenol, L-dopa:oxygen oxidoreductase, EC 1.14.18.1) is a key enzyme in the synthesis of melanin. Reduced levels of tyrosinase play an important role in albinism. The data described here show differences in the expression and characteristics of tyrosinase in cutaneous murine melanocytes grown in culture from normal wild-type strains (C/C); from three albino locus mutants: himalayan (ch/ch), chinchilla (cch/cch), and albino (c/c); and from the double-mutant heterozygous pink-eyed chinchilla (cchp/cp). Our results suggest that the diminished pigmentation in all mutants is due to abnormal posttranslational modification of the enzyme: the levels of mRNA for tyrosinase in wild-type, himalayan, and pink-eyed chinchilla melanocytes are similar; the himalayan mutation confers a deficiency in N-linked glycosylation, which results in an extremely unstable enzyme that is also temperature sensitive; the chinchilla and albino mutations confer susceptibility to proteolytic cleavage; the pink-eye dilution confers a reduction in the levels of immunoprecipitable tyrosinase, and what little enzyme there is fails to be translocated from the trans-Golgi network to melanosomes. The kinetics of activation and inhibition of the enzyme by the cofactor dopa are unique for the mutants tested and differ from those of tyrosinase from wild-type melanocytes. The findings support the conclusion that the albino locus in mice encodes the structural gene of tyrosinase.

Isolation and sequence of a cDNA clone for human tyrosinase that maps at the mouse c-albino locus.

Screening of a lambda gt11 human melanocyte cDNA library with antibodies against hamster tyrosinase (monophenol, L-dopa:oxygen oxidoreductase, EC 1.14.18.1) resulted in the isolation of 16 clones. The cDNA inserts from 13 of the 16 clones cross-hybridized with each other, indicating that they were from related mRNA species. One of the cDNA clones, Pmel34, detected one mRNA species with an approximate length of 2.4 kilobases that was expressed preferentially in normal and malignant melanocytes but not in other cell types. The amino acid sequence deduced from the nucleotide sequence showed that the putative human tyrosinase is composed of 548 amino acids with a molecular weight of 62,610. The deduced protein contains glycosylation sites and histidine-rich sites that could be used for copper binding. Southern blot analysis of DNA derived from newborn mice carrying lethal albino deletion mutations revealed that Pmel34 maps near or at the c-albino locus, the position of the structural gene for tyrosinase.

Inhibition of progesterone-mediated maturation of oocytes of Xenopus laevis by oocyte maturation inhibitor from pig follicular fluid: development of a routine assay for the inhibitor with Xenopus oocytes.

We describe an assay for oocyte maturation inhibitor (OMI) using the progesterone-mediated maturation of Xenopus oocytes. The test fraction used was a partially purified fraction from pig follicular fluid, which gave consistent inhibition of maturation in the pig oocyte assay. In the toad assay, oocytes (30-50) from each toad were pretested to determine whether satisfactory maturation was achieved because widespread animal variation was observed. Toads whose oocytes showed greater than 60% maturation in the pretest could be used directly. Toads whose oocytes showed less than 60% maturation were injected with pregnant mares serum gonadotropin (PMSG) in order to increase progesterone-mediated maturation. The dose and time after injection of PMSG before harvesting oocytes, dose and duration of progesterone exposure, and order of exposure of oocytes to OMI and progesterone were important variables. Opposing effects of OMI and progesterone were seen in oocytes from toads receiving 60 IU PMSG. In the routine assay we use animals whose oocytes show greater than 60% maturation in the pretest or animals treated with 12 IU of PMSG 4 to 7 days before use. Oocytes are exposed to the OMI fraction for 1 hr, progesterone is added, and incubations continued until controls reach maximum maturation (5 to 8 hr). The inhibition of toad oocyte maturation by OMI is reversible. The toad and mammalian oocyte assays were compared using more highly purified fractions of OMI and gave identical results.

Oocyte maturation inhibitor.

The preovulatory surge of gonadotropins induces within the mature Graafian follicle a series of changes culminating in the release of a fertilizable ovum. These include resumption of the meiotic division, a process held in abeyance from a short time after birth, and the progression of the oocyte from the dictyate stage to the metaphase of the second meiotic division. Here the role of a follicular factor, oocyte maturation inhibitor (OMI), in preventing resumption of meiosis by ova of antral follicles prior to the surge of gonadotropins has been reviewed. The suggested involvement of OMI in regulation of meiosis is based on the following observations: (1) fully grown mammalian oocytes explanted from their follicles undergo meiotic maturation spontaneously, whereas follicle-enclosed ova remain immature until stimulated; (2) co-culture of oocytes isolated from their follicles with follicular granulosa cells, granulosa cell extract and follicular fluid inhibits the spontaneous maturation; (3) the inhibition of oocyte maturation by OMI is reversible and in several of the models employed can be removed by the addition of the physiological trigger of meiosis, luteinizing hormone (LH). The current state of OMI characterization and purification has been described and the involvement of additional factors, such as cyclic AMP, in the regulation of meiosis discussed.

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.

Regulation of the development of meiotic competence and of the resumption of oocyte maturation in the rat.

The first meiotic maturation division of mammalian oocytes is initiated in the embryo or during the early postnatal period. However, when the germ cells reach the diplotene stage the meiotic process is arrested. Meiosis is normally kept in abeyance up to a short period prior to ovulation, when the process is resumed in preovulatory follicles. Resumption of meiosis is studied in mammalian oocytes mainly in two dissimilar in vitro models, isolated oocytes maturing spontaneously in culture and hormone-induced maturation of follicle-enclosed oocytes. A third approach, namely, co-culture of oocytes with follicular constituents was adopted in order to test the role of follicular components in the control of meiosis. Such studies demonstrated an inhibitory action of granulosa cells, granulosa-cell conditioned medium and of follicular fluid (FF1) upon the spontaneous maturation of co-cultured oocytes. By contrast, theca tissue was without effect on meiosis. Addition of luteinizing hormone (LH) to co-cultures of rat granulosa cells and rat oocytes induced resumption of meiosis, as it does in vivo or in vitro in follicle-enclosed oocytes. It is therefore suggested that within antral follicles meiosis is held in abeyance by a granulosa cell product, the inhibitor of oocyte maturation (OMI). Further studies led to the conclusion that OMI is not species specific, that its production by granulosa cells is enhanced by follicle stimulating hormone (FSH) and that its concentration in FF1 is dependent upon the development of the follicle and not the stage of the oestrous cycle. OMI appears to be a peptide of less than 2000 Da. Its action on the oocyte appears to be mediated, at least partially, by cumulus cells and is potentiated by cyclic AMP. Since OMI activity has been demonstrated only in antral follicles, we examined the development of the ability of rat oocytes to undergo spontaneous maturation during their growth phase in preantral follicles. We have found that the ability of rat oocytes to resume maturation ('meiotic competence') is acquired between days 20-26 post partum. By the use of hypophysectomy on day 15 of life and by treatment with hormones and inhibitors we demonstrated that the acquisition of meiotic competence is dependent upon FSH stimulation and that it is mediated, at least partially, by ovarian oestrogen production. The findings that oocytes from preantral follicles are meiotically incompetent suggests that the physiological role of follicular OMI is limited only to antral follicles i.e. when the oocytes acquire meiotic competence.(ABSTRACT TRUNCATED AT 400 WORDS)

Regulation of tyrosinase in human melanocytes grown in culture.

Tyrosinase, the enzyme that controls the synthesis of melanin, is a unique product of melanocytes. Normal and malignant human melanocytes grown in culture were used to study the factors that regulate the expression of tyrosinase. Immunoprecipitation experiments showed that newly synthesized tyrosinase appeared as a protein with an apparent molecular weight of 70,000 that was processed to a protein with an apparent molecular weight of 80,000. Neither tunicamycin nor 2-deoxy-D-glucose inhibited this conversion, suggesting that O-glycosylation is the major biochemical event in the posttranslational modification of tyrosinase. Agents that stimulated the proliferation of normal melanocytes also stimulated tyrosinase activity. Melanocytes with low levels of tyrosinase activity synthesized less tyrosinase, processed the enzyme more slowly, and degraded it more rapidly than melanocytes with high levels of tyrosinase activity. We conclude that tyrosinase activity in cultures of human melanocytes derived from different donors is determined predominantly by its abundance.

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.

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.

Tyrosine aminotransferase in AKR/J albino and C57BL/6J black mouse skin.

L-Tyrosine aminotransferase is present in a high speed supernatant fraction of skin homogenate of AKR/J albino and C57BL/6J black mice. The conversion of tyrosine to p-hydroxyphenylpyruvate was shown to be catalyzed by an aminotransferase by the following observations: the reaction was partially dependent on the presence of low concentrations of alpha-ketoglutarate; catalase was ineffective in increasing the yield of p-hydroxyphenylpyruvate; there was potent inhibition by typical inhibitors of pyridoxal phosphate enzymes and of rat liver tyrosine aminotransferase; there was no inhibition by inhibitors of L-amino acid oxidase; and there was no oxidation of L-leucine, the best substrate for rat kidney L-amino acid oxidase. The aminotransferase was stimulated by mercaptoethanol and was inhibited by high concentrations of alpha-ketoglutarate. The apparent Km for tyrosine was 5 X 10(-3) M and the molecular weight, determined by sucrose density gradient centrifugation, was 150-200,000. Dopa was also transaminated by the crude enzyme. No tyrosine aminotransferase could be detected in extracts of hamster melanoma.