Induction of DNA replication in the germinal vesicle of the growing mouse oocyte.

Growing mouse oocytes are physiologically arrested in the G2 phase of prophase of the first meiotic division. Growing oocytes were isolated from ovaries of 9- to 12-day-old mice and fused with parthenogenetic one-cell eggs or two-cell embryos derived from fertilized eggs. Resulting hybrids were injected with Dig-11-dUTP and examined for DNA replication using immunofluorescence. Parthenogenetic one-cell eggs fused at telophase II, G1, and middle-to-late S phase, and also S-phase two-cell blastomeres, were able to trigger DNA synthesis in oocyte germinal vesicle (GV) in the majority of hybrids cultured to the end of the first cell cycle. Activation of replication in the GV occurred within 2-3 h after fusion of growing oocytes with S-phase eggs. We show indirectly that the reactivation of replication in GVs was not dependent on the breakdown of the GV envelope. Although GVs had the ability to renew DNA replication after fusion, the G2 blastomere nuclei were incapable of reinitiating DNA replication under the influence of S-phase one-cell eggs. We hypothesize that the nuclei of growing oocytes arrested in meiotic prophase are in a physiological state that is equivalent to replication-competent G1, and not G2, nuclei.

Delayed sperm incorporation into parthenogenetic mouse eggs: sperm nucleus transformation and development of resulting embryos.

In this study we examined the effect of experimentally induced asynchrony between male and female pronuclei on male pronucleus formation and developmental potential of the resulting mouse embryos. We demonstrate that when the interval between oocyte activation and sperm incorporation is up to 1.5-2 hr, the spermatozoa transform into normal pronuclei. These male pronuclei can replicate their chromosomes during the first embryonic cell cycle and are transcriptionally competent. During the first cleavage these "delayed" male pronuclei condense into discrete mitotic chromosomes and when resulting embryos are transplanted into oviducts of pregnant females at least some of them can develop to term. In contrast, when sperm nuclei are introduced into parthenogenetic eggs 3 hr or more after activation, their transformation into pronuclei is significantly impaired, and they neither replicate nor transcribe. During the first mitosis they form a group of condensed chromatin, which is displaced into one of the resulting blastomeres leading to formation of haploid/diploid mosaic embryos. These mosaic embryos have poor developmental potential: only a few can reach blastocyst stage in vitro and no full-term development of such embryos was observed after transfer into pregnant females. We conclude that the cytoplasmic factors that make possible the transformation of a sperm nucleus into a functional male pronucleus exhaust within 1.5-2 hr after fertilization and that the male genome which had skipped the first cell cycle cannot become a functional partner in the embryonic genome.

Nuclear envelope removal/maintenance determines the structural and functional remodelling of embryonic red blood cell nuclei in activated mouse oocytes.

Nuclei of embryonic red blood cells (e-RBC) from 12-day mouse fetuses are arrested in G0 phase of the cell cycle and have low transcriptional activity. These nuclei were transferred with help of polyethylene glycol (PEG)-mediated fusion to parthenogenetically activated mouse oocytes and heterokaryons were analysed for nuclear structure and transcriptional activity. If fusion proceeded 25-45 min after oocyte activation, e-RBC nuclei were induced to nuclear envelope breakdown and partial chromatin condensation, followed by formation of nuclei structurally identical with pronuclei. These 'pronuclei', similar to egg (female) pronuclei, remained transcriptionally silent over several hours of in vitro culture. If fusion was performed 1 h or later (up to 7 h) after activation, the nuclear envelope of e-RBC nuclei remained intact and nuclear remodelling was less spectacular (slight chromatin decondensation, formation of nucleolus precursor bodies). These nuclei, however, reinforced polymerase-II-dependent transcription within a few hours of in vitro culture. Our present experiments, together with our previous work, demonstrate that nuclear envelope breakdown/maintenance are critical events for nuclear remodelling in activated mouse oocytes and that somatic dormant nuclei can be stimulated to renew transcription at a time when the female pronucleus remains transcriptionally silent.

Transcription and DNA replication of sperm nuclei introduced into blastomeres of 2-cell mouse embryos.

The aim of this study was to investigate the behaviour of sperm nuclei in the cytoplasm of the 2-cell mouse embryo. To this end, we produced hybrids between anucleate fertilised oocyte fragments and blastomeres of the 2-cell embryos. When sperm nuclei at the stage of decondensation or recondensation were introduced into blastomeres the development of male pronuclei was usually retarded and they never reached the size of the blastomere nuclei. These abortive male pronuclei were unable to initiate transcription but they were capable of synthesising DNA. The majority of sperm nuclei introduced into blastomeres as early male pronuclei developed normally and reached the size of the blastomere nuclei. They synthesised DNA simultaneously with blastomere nuclei and were transcriptionally active. In addition they participated in the cleavage division of hybrid cells. This shows that the very early male pronucleus when transmitted from the oocyte cytoplasm to the blastomere cytoplasm can respond positively to the new cytoplasmic factors, i.e. it undertakes both DNA replication and transcription according to the time schedule characteristic of the second cell cycle.

Fusion with activated mouse oocytes modulates the transcriptional activity of introduced somatic cell nuclei.

We have analyzed the transcriptional activity of somatic cell nuclei fused with artificially activated mouse oocytes. Two types of somatic cells have been used: transcriptionally silent thymocytes, obtained from the thymus of newborn mice, and transcriptionally active murine erythroleukemia cells (MEL) from in vitro culture. Cells were fused with activated oocytes, either less than 1 h, or 3 h post-ethanol-treatment. When the fusion occurred early after activation (1 h or less), the transferred somatic cell nuclei reacted by nuclear envelope breakdown (NEBD), which exposed their chromatin to the cytoplasmic environment of the oocytes, and the reconstituted nuclei underwent significant ultrastructural remodeling. No transcriptional activity was ever detected in these reconstituted nuclei during the subsequent 3-4 h of culture of the resulting hybrid cells. In the case of MEL, this means that transcriptional activity ceased as soon as they entered the cytoplasm. In contrast, somatic nuclei which entered the cytoplasm of activated oocytes 3 h postactivation did not undergo NEBD and their remodeling was less pronounced. In contrast to the first group, these nuclei were transcriptionally active during the following 3-4 h of culture. In both cases, the female pronucleus remained transcriptionally silent.

Development-dependent localization of nuclear antigens in growing mouse oocytes.

We have analyzed the distribution of nuclear and nucleolar proteins during the period of oocyte's growth. Oocytes were isolated mechanically or enzymatically from ovaries of juvenile mice of various ages (from 1 to 28 days after birth). Small nuclear ribonucleoproteins (snRNPs), the splicing factor SC-35, and a protein linked to cell proliferation (p-120) were detected by indirect immunofluorescence. snRNP distribution is consistent with the prophase state of oocyte's nuclei, while SC-35 (and p-120) exhibit a "speckled" distribution throughout the entire period of growth. The number of speckles (or foci) appears maximal around 10 days after birth, i.e., in the period of maximal transcriptional activity, and is sensitive to alpha-amanitin treatment. On the other hand, the immunofluorescent distribution of of nucleolin and p-103 (a nucleolar marker of the granular component) is compared to the ultrastructural distribution of the granular component analyzed by electron microscopy on oocytes of the same age.

Sperm entry into fertilised mouse eggs.

We have investigated the oolemma block to polyspermy in the mouse. Zona-free and zona-intact eggs were fertilised and subsequently re-inseminated (the latter following zona pellucida removal). The 'perivitelline' block to polyspermy in zona-intact eggs renders motile sperm in the perivitelline space unable to bind to the oolemma. This is not connected with irreversible changes in the egg plasma membrane, because freshly added sperm can still fuse with such eggs freed from the zona. Fertilised eggs eventually lose the ability to fuse with sperm within 1 h, while still being able to bind many sperm.

Relationship between sperm nucleus remodelling and cell cycle progression of fragments of mouse parthenogenotes.

Nucleate and anucleate fragments of parthenogenetically activated mouse oocytes, as well as cybrids obtained by fusion of anucleate fragments (cytoplasts) of maturing and activated matured oocytes were fertilized at different time after activation. Remodelling of the sperm nucleus was studied by electron microscopy at 1.5 and 3 h after fertilization and, in addition, at 14 h in cybrids. Results show that 1) the nuclear envelope of the sperm nucleus can break down when the insemination takes place after the end of M-phase, but the capacity of the parthenote cytoplasm to remodel the sperm nucleus is restricted in time. 2) Male chromatin can decondense within the old, unbroken nuclear envelope, but in such cases formation of a male pronucleus, one of the two nuclei of zygote possessing inactive nucleoli, is never observed.

[The comparative characteristics of the peroxidase conjugates used in Soviet immunoenzyme test systems for detecting HIV antibodies]. / Sravnitel'naia kharakteristika peroksidaznykh kon''iugatov, primeneniaemykh v otechestvennykh immunofermentnykh test-sistemakh dlia vyiavleniia antitel k VICh.

Five lots of peroxidase conjugates were studied in enzyme immunoassay systems "Recombinant-HIV" and "Peptoscreen-2" for detection of antibody to HIV. The conjugates differed from each other by the source of generation and methods of preparation. The conjugates were studied in biochemical tests and ELISA. When panels of sera from HIV-infected children and adults were employed, the advantages of using the anti-immunoglobulin conjugate over protein A-peroxidase conjugate were demonstrated, as the former increased the sensitivity of the test systems to HIV antibody detection.

[Combined experimental use of vaccine against acute human encephalomyelitis and immunomodulators]. / Kombinirovannoe primenenie v éksperimente vaktsiny protiv ostrogo éntsefalomielita cheloveka s immunomoduliatorami.

Combined use of vaccine and immunomodulators such as ridostin, inosiplex and polyribonate against acute encephalomyelitis of humans (AEMHs) was studied. It was shown that low immunogenic doses of the vaccine did not provide a protective action against the virus of AEMHs while after administration of the vaccine in combination with the immunomodulators there was protection in all the groups of the animals exposed to the low immunogenic doses of the vaccine during the first immunization. It was noted in regard to all the combinations of the immunomodulators and vaccine used in the low immunogenic doses that the level of the increase in the titer of the virus-specific antibodies, the proliferative activity to the specific antigen and mitogens and of interferon induction depended on the immunomodulator type. At the same time, it was found that the marked production of interferon within the first 24 hours observed after the use of the combination of inosiplex, ridostin and the vaccine resulted in increased activity of natural killer cells and lower proliferative activity of cells and production of virus-specific antibodies. This was indicative of the necessity of choosing the immunomodulators, their doses and time of the administration in relation to the immunization.

Anucleate fragments of parthenogenetic eggs and of maturing oocytes contain complementary factors required for development of a male pronucleus.

Eight-h-old anucleate fragments of artificially activated mouse oocytes produced within 30 min after activation were fused with anucleate fragments of ovarian oocytes bisected at the GV stage and matured in vitro for 4-5 h. The resulting cybrids were inseminated soon after fusion and fixed 3-4 h or 14 h later. After 3-4 h of culture most of the sperm nuclei underwent decondensation. Early male pronuclei were observed sporadically. After overnight culture small or fully formed male pronuclei were present in monospermic and dispermic cybrids. The abortive nuclei, designated as stage IIa (earlier described by Borsuk and Tarkowski, 1989) were also observed and their occurrence was strongly correlated with the rate of polyspermy. In unfused (control) anucleate fragments of maturing and activated oocytes inseminated at the same time as the cybrids, male pronuclei were never observed. These results show that the cytoplasms of activated eggs and of maturing oocytes contain complementary factors required for the transformation of the sperm nucleus into the male pronucleus. The former has lost the capability to break down the sperm nucleus envelope but contains factors required for pronuclear growth. The latter is able only to initiate the process of transformation, i.e., to break down the nuclear envelope and decondense the denuded sperm chromatin. However, the activity of the factor responsible for nuclear envelope breakdown is limited because in cybrids penetrated by more than two spermatozoa, transformation of sperm nuclei was usually abnormal.

[The evaluation of a commercial EIA test system for determining the specific activity of antiherpetic preparations]. / Otsenka kommercheskoi test-sistemy IFA dlia opredeleniia spetsificheskoi aktivnosti protivogerpeticheskikh preparatov.

The data on the use of a commercial EIA test system for detection of antibodies in control of preparations against herpes simplex and cytomegaloviruses are presented. The enzyme immunoassay test system for antibody determinations to herpes simplex virus produced by the Odessa bacterial preparations enterprise was shown to be suitable for determination of the specific potency (antigenicity) of herpes simplex vaccine. The advantages of this method over the currently used neutralization test were established. Titration of commercial immunoglobulins detects lots with high litres of antibody to herpes simplex virus. For the same purpose, lots of commercial immunoglobulins were tested for antibodies to cytomegalovirus using a West Germany test-system (Behring). It is concluded that enzyme immunoassay test systems for antibody determinations may be used for screening of lots of immunoglobulins of special effects (against herpes simplex and cytomegalovirus infections) both at the stage of serum and final preparation screening.

[Immunogenicity and stability of a Soviet inactivated cultured vaccine against Japanese encephalitis]. / Immunogennost' i stabil'nost' otechestvennoi kul'tural'noi inaktivirovannoi vaktsiny protiv iaponskogo éntsefalita.

Six immunologically active vaccine batches inducing a specific antibody to Japanese encephalitis (JE) virus were obtained in serial manufacture of the preparation. In HI tests, the minimal antibody titre was 1:80, the maximal 1:320, neutralization index 1g was 3.7 to 5.2. The data on the stability of the antigenic potency of the vaccines in relation to the duration of storage at 4 degrees-6 degrees C are presented (the follow-up period 3 years). A certain relationship was found between the antigenic potency of the preparation and the titre of the initial infectious tissue culture virus. Also, a definite correlation was found between the initial immunogenic potency of the vaccines and their stability in storage. After 3 years of storage, three vaccine lots remained antigenically active, namely those which after manufacture had induced antihemagglutinins in titres 1:160 to 1:320. The antigenic activity of 6 vaccine batches prepared from the production strain Peking-1 (Nakayama serotype) was studied against the predominant strain of Jagar-10 serotype. All the freshly prepared vaccine batches were found to induce production of antihemagglutinins to both serotypes of JE virus, whereas virus-neutralizing antibodies were found only to the test strain Nakajama-NIH homologous to the vaccine Peking-1 strain. After 1 year of storage, four vaccine batches lost their capacity to induce production of antihemagglutinins to Jagar-01 strain, two batches induced antibody in low titres. This fact should be considered in evaluation of postvaccination immunity status in humans.

Transformation of sperm nuclei into male pronuclei in nucleate and anucleate fragments of parthenogenetic mouse eggs.

Our objective was to examine the ability of nucleate and anucleate fragments of artificially activated mouse eggs to transform sperm nucleus into male pronucleus. To this end, zona-free oocytes in metaphase II were activated by ethanol and bisected into halves (one with the spindle, the other anucleate) either within 10 to 20 min (series A) or 3 or 5 hr later (series B). In series A, the fragments were inseminated 3.5, and 8 h after activation, and in series B, 3 and 5 h after activation. Both nucleate and anucleate fragments lose the capability of transforming sperm nucleus into fully formed pronucleus sometime between 3 and 5 h after activation. In 8 h old parthenogenetic fragments, the majority of sperm nuclei remain unchanged or begin decondensation but never reach the stage of an early pronucleus. In over 1/3 of anucleate fragments of this age group, sperm nuclei develop defectively; chromatin decondenses inside the persisting nuclear envelope. In other experimental groups, the incidence of these abnormal sperm nuclei varies between 0 and 10%. In general, the anucleate fragments retain the capability to transform sperm nuclei (fully or partially) longer than their nuclear counterparts. This difference may be accounted for by a different level of substances required for pronuclear growth (extrachromosomal constituents of the germinal vesicle and nuclear lamins): high and constant in the cytoplasm of anucleate egg halves and low and progressively decreasing in the nucleate halves because of their putative uptake by the female pronucleus. However, the cytoplasmic factors responsible for the initial stages of transformation (nuclear envelope breakdown, chromatin decondensation) become eventually inactivated both in the presence and in the absence of a female pronucleus.

Behavior of sperm nuclei in intact and bisected metaphase II mouse oocytes fertilized in the presence of colcemid.

Our objective was to examine the developmental fate of sperm nuclei in oocytes fertilized under conditions of meiotic arrest. Therefore zona-free metaphase II oocytes and oocyte fragments (nucleate and anucleate) were fertilized in the presence of colcemid. In anucleate oocyte fragments, normal male pronuclei develop. In contrast, in intact oocytes and nucleate fragments sperm nuclei after initial decondensation undergo secondary condensation. This state is maintained as long as the oocytes are treated with colcemid. When the drug is removed 3 h after insemination, the meiotic spindle(s) is reconstructed, the second polar body(ies) is extruded, and a female pronucleus (or micronuclei) forms. At the same time the sperm nucleus decondenses again and transforms into a male pronucleus. In addition oocytes fertilized in the presence of colcemid could not be refertilized. These observations suggest that oocytes and oocyte fragments fertilized in the presence of colcemid undergo activation despite the failure of pronucleus formation. The inhibitory effect of colcemid on the formation of pronuclei is expressed only in the presence of oocyte chromosomes. We suggest that colcemid stabilizes factors responsible for chromosome condensation that are associated with oocyte chromosomes but not factors (whether the same or different) present in the cytoplasm.

[Use of inbred mice for assessing the neurovirulence of strains of the tick-borne encephalitis virus complex and the immunogenicity of a concentrated tick-borne encephalitis vaccine]. / Ispol'zovanie inbrednykh myshei dlia otsenki neirovirulentnosti shtammov virusov kompleksa kleshchevogo éntsefalita i immunogennosti kontsentrirovannoi vaktsiny kleshchevogo éntsefalita.

The sensitivity of different lines of inbred mice to strains of the tick-borne encephalitis complex viruses differing in their biological properties and the degree of attenuation was compared. The mice differing in their genetic characteristics and susceptibility to extraneurally inoculated virus were shown to have different immunological responsiveness to inoculation of an inactivated vaccine antigen.

Preimplantation development of gynogenetic diploid mouse embryos.

Diploid gynogenetic mouse embryos were produced in a three-step procedure: fertilization in vitro, suppression of the 2nd polar body formation by Cytochalasin B, and microsurgical removal of the male pronucleus. The operated eggs were transplanted to the oviduct of recipient females for 72 or 96 h. The overall recovery rate was 73%, but compacted morulae and blastocysts constituted only 28.6% of transplanted eggs. After 72 h blastocysts were rare (3.5%) but 24 h later their incidence increased to 21.2%. In eggs homozygous for T6 chromosome it was possible to prove karyologically that the male pronucleus was effectively removed and that the diploid genome was of purely maternal origin.

[Standardization of preparations intended for the serodiagnosis of arboviruses]. / Standartizatsiia preparatov, prednaznachenykh dlia serodiagnostiki arbovirusov.

As the result of our research work, 3 reference preparations have been first obtained and studied in accordance with all requirements of biological standardization. These preparations are the national standard of yellow fever antiserum and immune ascitic fluids (IAF) used as reference reagents: IAF to tick-borne encephalitis virus and IAF to Japanese encephalitis virus. The new preparations are stable, possess sufficient specific activity and can be used as standard preparations for the identification of the above-mentioned viruses.