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.     

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.     

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.     

Correlative histochemical studies on preneoplastic and neoplastic lesions in the kidney of rats treated with nitrosamines

Renal tubular lesions induced in male rats by two different carcinogens, N-nitrosomorpholine (NNM) and N-ethyl-N-hydroxyethylnitrosamine (EHEN), using a limited exposure "stop" protocol were investigated histochemically to demonstrate phenotypic cellular changes. The parameters measured included basophilia, glycogen content and the activity of the enzymes glucose-6-phosphatase (G6PASE), glycogen synthetase (SYN), glycogen phosphorylase (PHO), glucose-6-phosphate dehydrogenase (G6PDH), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), succinate dehydrogenase (SDH), alkaline phosphatase (ALP), acid phosphatase (ACP) and gamma-glutamyl transpeptidase (gamma-GT). The lesions observed were predominantly of either basophilic or oncocytic types. In each case, tubular lesions (altered tubules) appeared to give rise to epithelial tumors (epitheliomas) with the same cellular phenotype. Basophilic tubules and epitheliomas proved to be strongly positive for GAPDH and G6PDH while demonstrating a reduction or loss of G6PASE, ALP, ACP, gamma-GT, and SDH compared with controls and the surrounding proximal or distal tubules. In addition, large basophilic epitheliomas demonstrated an increase in both SYN and PHO activities. In contrast, most oncocytic tubules and oncocytomas characterized by abundant densely granular cytoplasm showed a reduction in the activity of G6PDH, but were intensely positive for SDH. However, a few oncocytic lesions demonstrated a decrease in both SDH and G6PDH activity. Rarely, decreased SDH and elevated G6PDH activities were observed in altered tubules resembling oncocytic tubules. It remains to be clarified whether these tubules represent a variation of the oncocytic lesions or, perhaps, another type of tubular lesion. The results indicate that basophilic and oncocytic epithelial tumors differ in their cytochemical pattern and histogenesis. In line with earlier suggestions, the basophilic tumors apparently originate from the proximal renal tubules, while the oncocytomas develop from the distal parts of the nephron. The basophilic tumors are characterized by an increased pentose phosphate pathway and glycolysis, with a corresponding reduction in mitochondrial respiration. However, the majority of the oncocytomas show an increased activity of the mitochondrial enzyme SDH, and a marked decrease in the activity of the key enzyme of the pentose phosphate pathway.     

Successful artificial insemination in the giant panda (Ailuropoda melanoleuca) at ueno zoo

Successful breeding of the giant panda (Ailuropoda melanoleuca) following artificial insemination was achieved at the Ueno Zoo in 2 consecutive years (1985 and 1986). The first cub, born in June 1985, unfortunately died 43 hours after birth from being crushed by the mother panda; the second cub, born in June 1986, has been growing in good health. Electroejaculation and artificial insemination procedures were performed after immobilization with diazepam (0.1 mg/kg) and ketamine HCL (4.0–5.0 mg/kg). Semen of the male panda was collected by electroejaculation using a rectal probe with a diameter of 2.0 cm and with eight rings as electrodes. Stimulation of the male was given with 3 V (30–40 mA) over a 5-sec period with 5-sec intervals. The female panda exhibited estrus between late February and early March in 1985 and also between mid-january and early February 1986. Increased excretion of urinary total estrogen showed coincidentally at maximum behavioral estrus, and a gradual rise of pregnanediol level was followed by artificial insemination. The gestational length for the first pregnancy was 110 days and that of the second 121 days.