From oocyte maturation to the in vitro cell cycle: the history of discoveries of Maturation-Promoting Factor (MPF) and Cytostatic Factor (CSF)

This article briefly reviews the classical cell cycle studies using oocytes and zygotes of mainly amphibians in the past century. The discussions are focused on the investigations into the cytoplasmic factors that regulate meiosis during oocyte maturation and the initiation of mitosis during fertilisation, which were carried out in the author's lab between 1967 and 1987. This chronicle traces the development of the problems and the direction in which their solutions were attempted in the course of these investigations. The author tries to answer the following questions: why he decided to study oocyte maturation, how he discovered progesterone as a maturation-inducing hormone, how he discovered and characterised the cytoplasmic regulators of the cell cycle, Maturation-Promoting Factor (MPF) and Cyto-Static Factor (CSF), and how he invented the method of observing cell cycle processes in a cytoplasmic extract in vitro.     

New PCR-RFLP analysis for the mouse Tnfsf6gld gene caused by a point mutation in the Tnfsf6 [tumor necrosis factor (ligand) superfamily, member 6] locus.

A new polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis was developed for genetic typing of the mouse Tnfsf6gld mutation. An artificial restriction site was introduced to the mouse Tnfsf6gld mutation by PCR amplification using a modified primer. The three genotypes of the Tnfsf6 locus (Tnfsf6gld/Tnfsf6gld, Tnfsf6gld/+, and +Tnfsf6-gld/+Tnfsf6-gld) could be distinguished clearly and easily. This PCR-RFLP analysis was found to be useful for the identification of the mouse Tnfsf6gld mutation.     

S-100 Protein in Clonal Astroglioma Cells Is Released by Adrenocorticotropic Hormone and Corticotropin-Like Intermediate-Lobe Peptide

S-100 protein in clonal GA-1 and C6 rat glioma cell lines was released in serum-free medium supplemented with adrenocorticotropic hormone (ACTH). The induction of S-100 protein release by ACTH was dose-dependent, showing a half-maximal release at about 5 microM, and the S-100 protein concentration in the medium increased sharply within 3 min, but slightly during further incubation. The S-100 protein release was apparently accompanied by a decrease in the membrane-bound form of S-100 protein in the cell. The S-100 protein release was induced not by the ACTH1-24 fragment, which exhibits the known effects of ACTH, but by the ACTH18-39 fragment, which is designated as corticotropin-like intermediate-lobe peptide (CLIP). These results indicate that the C-terminal half of ACTH is responsible for the S-100 protein release. The enhancement of S-100 protein release by ACTH was also observed in normal rat glioblasts. The release induced by ACTH was apparently specific to S-100 protein, because little release of the cytoplasmic enzymes, creatine kinase, and enolase was observed under the same conditions. High concentrations (5 mM) of dibutyryl cyclic AMP or dibutyryl cyclic GMP were also found to induce S-100 protein release; however, catecholamines (epinephrine, norepinephrine, isoproterenol, and dopamine), acetylcholine, and glutamic acid did not enhance the release.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stabilization and enhancement of primary cytostatic factor (CSF) by ATP and NaF in amphibian egg cytosols

Amphibian zygotes microinjected with the cytoplasm or cytosol of unactivated eggs are arrested at metaphase of mitosis. The activity responsible for this effect has been designated primary "cytostatic factor (CSF)." Primary CSF disappears from the cytoplasm after egg activation, as well as from cytosols after addition of Ca2+. In the present study, using fresh cytosols of Rana pipiens eggs, a unit of CSF activity was defined as the dose required to arrest 50% of the recipients, and the specific activity of a cytosol was expressed in units per microgram protein. Specific activities of cytosols prepared with the one-step centrifugation method employed in the present study were double the activities in cytosols obtained by the previously described two-step procedure. During storage at 2 degrees C, CSF specific activity in cytosols fell rapidly within hours of extraction and disappeared completely within 2 days. However, if NaF and ATP were added to fresh cytosols, specific activities increased within hours and remained high for at least several days. Addition of gamma-S-ATP also significantly increased the longevity of the activity during storage at 2 degrees C. Further, it was found that primary CSF activity could be recovered by ATP additions to cytosols in which residual activity was still present, but no activity was recovered by ATP addition if cytosols had completely lost activity. When Ca2+ was added to cytosols to which NaF and ATP had been added, CSF was inactivated more slowly than in control cytosols without NaF and ATP additions. Therefore, it appears that maintenance of primary CSF activity in vitro requires protein phosphorylation and that protein dephosphorylation is involved with its inactivation. Also, we compared the sensitivities to primary CSF of Xenopus laevis and R. pipiens two-cell embryos. In order to arrest 50% of recipients, the concentration of primary CSF in Xenopus blastomeres was three times higher than in Rana blastomeres.     

Chromosome condensation activity in the cytoplasm of anucleate and nucleate fragments of mouse oocytes

The activity of maturation promoting factor (MPF) which causes chromosome condensation and subsequent oocyte maturation was investigated in mouse oocytes using polyethylene-glycol-mediated cell fusion technique. Fully grown oocytes were bisected at germinal vesicle (GV) stage or shortly after germinal vesicle breakdown (GVBD) into anucleate and nucleate fragments. After 2-3 or 15-17 hr of culture these fragments were fused with interphase blastomeres from two-cell embryos. It was found that almost all the anucleate oocyte fragments cultured for a short term (2-3 hr), regardless of whether they were produced at GV stage or after GVBD, induced premature chromosome condensation in the blastomere nuclei, whereas only about 20% of those cultured for a long term (15-17 hr) could do so. On the other hand, the nucleate fragments always retain the cytoplasmic activity to induce chromosome condensation. Thus we suggested that the MPF initially could appear in mouse oocytes independently of the GV, that the mixing of GV material with the oocyte cytoplasm following GVBD had no effect on the activity of MPF in anucleate fragments, and that oocyte chromosomes or some components associated with them could play a significant role in maintaining the MPF activity.     

The localisation of an Mr 74,000 major chromatin antigen on native salivary chromosomes of Drosophila melanogaster

An antigen making a major contribution to the immune response to Drosophila melanogaster chromatin resides primarily on a nonhistone charge-class family of proteins of Mr 74,000. Immunofluorescence detects this antigen at interbands, puffs and diffuse bands of D. melanogaster salivary chromosomes isolated without exposure to acid fixatives, and on nucleoplasmic ribonucleoprotein droplets. In the electron microscope, gold labelling reveals the binding of monoclonal antibodies specific for the antigen at chromosomal loci generally bearing putative ribonucleoprotein (RNP) particles. However, the locus 3C 11–12 is remarkable in that it bears putative RNP particles but is virtually unlabelled, suggesting protein specificity at different active loci.     

Transformation of sperm nuclei to metaphase chromosomes in the cytoplasm of maturing oocytes of the mouse

Zona-free oocytes of the mouse were inseminated at prometaphase I or metaphase I of meiotic maturation in vitro, and the behavior of the sperm nuclei within the oocyte cytoplasm was examined. If the oocytes were penetrated by up to three sperm, maturation continued during subsequent incubation and became arrested at metaphase II. Meanwhile, each sperm nucleus underwent the following changes. First, the chromatin became slightly dispersed. By 6 h after insemination, this dispersed chromatin had become coalesced into a small mass, from which short chromosomal arms later became projected. Between 12 and 18 h after insemination, each mass of chromatin became resolved into 20 discrete metaphase chromosomes. In contrast, if oocytes were penetrated by four to six sperm, oocyte meiosis was arrested at metaphase I, and each sperm nucleus was transformed into a small mass of chromatin rather than into metaphase chromosomes. If oocytes were penetrated by more than six sperm, the maternal chromosomes became either decondensed or pycnotic, and the sperm nuclei were transformed into larger masses of chromatin. As control experiments, immature and fully mature metaphase II oocytes were inseminated. In the immature oocytes, which were kept immature by exposure to dibutyryl cyclic AMP, no morphological changes in the sperm nucleus were observed. On the other hand, in the fully mature oocytes, which were activated by sperm penetration, the sperm nucleus was transformed into the male pronucleus. Therefore, the cytoplasm of the maturing oocyte develops an activity that can transform the highly condensed chromatin of the sperm into metaphase chromosomes. However, the capacity of an oocyte is limited, such that it can transform a maximum of three sperm nuclei into metaphase chromosomes. Furthermore, the presence of more than six sperm causes a loss of the ability of the oocyte to maintain the maternal chromosomes in a metaphase state.     

Non-cytotoxic activity of pyran copolymer-induced macrophages associated with potentiation of tumour vaccine in recipient mice

Summary Mice inoculated with both L1210 murine tumour vaccine and pyran copolymer were more resistant to L1210 than those inoculated with either of these agents alone. Rabbit anti-mouse thymocyte globulin and silica reduced the augmented resistance of these mice, suggesting the involvement of activated anti-tumour T cells and macrophages in the augmented resistance. We studied the activation of these two cells separately and examined the possible contribution of pyran copolymer-induced peritoneal cells to the augmented resistance to an inoculation of live tumour. Pyran copolymer-induced peritoneal cells endowed the tumour vaccine-primed mice, but not unprimed mice, with resistance to implanted L1210 and, among those peritoneal cell populations, macrophages but not T cells were responsible for this effect since the activity was associated with a cell population which was (1) adherent to nylon wool columns, (2) sensitive to silica and (3) insensitive to anti-Thy 1.2 antibody plus complement. The pyran copolymer-induced peritoneal cells had very little antiproliferative activity when tested against L1210 in vitro and mice inoculated with these peritoneal cells did not survive a challenge of live L1210 cells much longer (<1 day) than L1210 inoculated control mice. Furthermore, the survival of L1210 vaccine-primed mice inoculated with one-tenth the amount of live L1210 (10²) was still much shorter than that of mice primed with L1210 vaccine plus pyran copolymer and challenged with ten times as many (10³) live L1210 cells. Therefore, direct tumouricidal activity was probably not a major factor in the in vivo immunological augmenting activity of the pyran copolymer-induced macrophages.