Daniel Szöllösi

De novo formation of centrioles in parthenogenetically activated, diploidized rabbit embryos

Summary— In rabbit oocytes activated parthenogenetically by repetitive electric pulses, centrioles develop de novo in blastocysts. Centrioles were not observed in earlier stages of development, not until the blastocoele is formed. Up to the morula stage (between 8–32 cells), a filamentous, electron‐dense material develops and aggregates with a small vesicle fraction within the well developed Golgi apparatus. A spherical to ovoid electron dense mass forms, which is comparable to the deuterosome or to the blepharoplast. The quantity of the electron dense material enlarges and it seems to give rise to the centriole “generating complex”. Centrioles arise in all three differentiated cell types of the blastocysts, the mural and polar trophoblasts and the embryonal cell mass at the same time. Some of the forming centrioles in parthenotes have a co‐linear arrangement, as in control blastocysts. It is not yet known whether the co‐linearly arranged centrioles represent a maturation phase, prior to the formation of the usual diplosome, with centrioles oriented perpendicularly to each other. Nor is it known whether the forming centrioles are functioning as the polar organizer of the mitotic spindle or if they can perform any other centriolar function.

Chromatin behaviour under influence of puromycin and 6-DMAP at different stages of mouse oocyte maturation.

Preovulatory mouse oocytes were cultured in vitro up to each subsequent stages of maturation: germinal vesicle (GV), germinal vesicle breakdown (GVBD), groups of not yet individualized bivalents, circular bivalents, late prometaphase I, metaphase I, anaphase I and telophase I. The stages were identified in living oocytes by fluorescence microscopy using Hoechst 33342 as a specific vital dye. Oocytes from each stage of development developed in vitro and ovulated metaphase II oocytes were subsequently cultured in the presence of puromycin or 6-dimethylaminopurine (6-DMAP), an inhibitor of protein phosphorylation. The effects on chromatin of these drugs were studied during and at the end of culture by fluorescence and electron microscopy. We found that puromycin and 6-DMAP stop meiosis when applied at all stages of oocyte maturation, except for metaphase II. Oocytes at this stage are activated by puromycin. Reaction of the oocytes to the two drugs is different at GV and at metaphase II. All of the other stages react to the drugs by chromatin compaction, which can be followed by chromatin decondensation to form a nucleus. Our results suggest that late prophase chromatin condensation, bivalent individualization and retention of their individuality, as well as individualization of monovalents from telophase and retention of their individuality at metaphase II, are dependent on protein phosphorylation. The events occurring between metaphase I and telophase I are independent of protein synthesis and phosphorylation. The events occurring between metaphase II and formation of the nucleus are independent of protein synthesis.

Sperm penetration into immature mouse oocytes and nuclear changes during maturation: an EM study.

The ultrastructure of oocyte and sperm nuclei was studied in mouse ovarian oocytes inseminated in vitro and cultured for 1 1/2 and 3 h in a medium containing dbcAMP or lacking the maturation inhibitor. In oocytes blocked at the germinal vesicle (GV) stage, certains maturation‐linked changes were noted. Sperm apposition and sperm‐oocyte fusion were similar to that during fertilization of ovulated oocytes. The sperm nucleus and its nuclear envelope remained intact after penetrating into the ovarian oocyte. One and a half h after removal of the drug (time 0 of maturation) the germinal vesicle (GV) and sperm nucleus remained intact. In oocytes maturing for 3 h, the nuclear envelopes of the GV and sperm nucleus had fragmented. The NE of the oocyte formed quadruple membranes while the NE of the sperm remained as flat vesicles. Oocyte chromatin condensed to form chromosomes, whereas at the same time the sperm chromatin was in the process of decondensation and was surrouded by fragments of the sperm NE. The sperm chromatin, composed of DNA complexed with protamines, consisted of thin fibrils; the individual fibrils measured 3.8 nm in diameter. Near the penetrated spermatozoa only occasional Mts were detected which were not related to the proximal centriole which was recognizable in the neck‐piece of the flagellum. Thus in mouse oocytes the introduced sperm centriole is not capable of behaving as a centrosome and organizing microtubules in the form of an aster.

Activation of paternally derived regulatory mechanism in early mouse embryo

A stage-specific occurrence of retrovirus-like elements (intracisternal particles) is regularly observed within the endoplasmic reticulum of early mouse embryos. A quantitative electron microscopic study was carried out on hybrid embryos issued from the matings between high- and low-producer mouse strains of intracisternal particles. The low-producer character behaved as dominant in all these crosses, and this enabled us to demonstrate the activation of paternally introduced regulatory mechanism in two-cell embryos and morulae. There was no sign of maternal effect in the results of reciprocal crosses.

Cytoplasmic Changes in the Mammalian Oocytes During the Preovulatory Period

The terms meiotic maturation, oocyte maturation, and meiotic resumption usually refer to the reinitiation of nuclear events in oocytes originally arrested at the diplotene stage during fetal life (the dictyate stage). This process leads to the completion of the first meiotic division, i.e., the expulsion of the first polar body. Meiotic processes are arrested anew in the oocytes at metaphase II in mammals and probably in most vertebrates. This is the condition in which penetration of the fertilizing spermatozoon takes place and when the oocyte attains its full developmental capacity (Mangia and Canipari 1977; Baker 1979).

Virus-like particles and related expressions in mammalian oocytes and preimplantation stage embryos

The presence of virus-like particles in the oocytes and preimplantation stage embryos of several species has perplexed mammalian embryologists since their occurrence was first detected by electron microscopy (1, 2). Interest in this phenomenon was renewed in the early 1970s when three laboratories independently reported the presence of particles resembling RNA tumor viruses in the early cleavage stage embryos of several strains of mice (3, 4, 5). These studies showed that virus-like particle expression was stage-specific, was not the result of exogenous infection and, in spite of relatively large numbers of particles, did not interfere with normal development. Collectively, the observations led to the conclusion that virus-like particle expression was a scheduled event in the normal developmental program of the embryo.

Centrosomes with striated rootlets in rabbit zygotes

An electron microscopic study of the rabbit zygote has shown the presence of numerous paracrystalline structures (PSs) around the pronuclei. The majority of these structures are situated in the narrow space between pronuclei. The PSs during interphase are associated with small dense knobs, and filamentous material; some of them, namely those situated in the internuclear space, are also associated with striated rootlets. The PS and its appendages form a complex which nucleates microtubules during interphase and phase M. The structure of these complexes changes with the cell cycle. Striated rootlets disappear at G2/M. Dense knobs and filamentous material separate from the PS, become loose and associate with numerous microtubules at the poles of the first mitotic spindle. PSs and their associated structures are considered to be a newly discovered morphological form of the centrosome.

The Use of Isopropyl-N-phenylcarbamate as a Potential Contraceptive

The most successful contraceptive practices, exclusive of the physical methods, affect the complex hypothalamic—hypophyseal—gonadal feedback systems. The intervention could theoretically occur at any point of this feedback loop. Steroid supplementation reduces the level of gonadotrophin-secretory activity and via it gonadal steroid production. The inconveniences of this method to the user and the user’s undesirable physiological responses to this method have become known during recent decades of contraceptive practice (for a review, see Regulation de la Fecondite,Vol. 83, INSERM, Paris, 1979). Steroid-based contraceptives certainly influence gamete production and, as well, the hormone-secretory activity of the gonads themselves and various protein hormones of the central nervous system.