Comparison of the level(s) of DNA damage using Comet assay in bovine oocytes subjected to selected vitrification methods.

It was suggested that the cryodamage to oocytes' DNA has been responsible for the compromised developmental competence of cryopreserved oocytes. Vitrification of bovine oocytes affected not only cellular components, but also nuclear material. A significant rate of DNA fragmentation was found in bovine frozen or vitrified oocytes analysed by Comet assay regardless of cryopreservation method. Our method of vitrification using droplet system after gentle pre-equilibration treatment is one of the most effective cryopreservation methods employed for bovine oocytes so far, making it possible to develop 30% blastocyst stage embryos. In this study, the extent of DNA damage in bovine oocytes vitrified using three vitrification methods (droplet system, Open Pulled Straw and traditional vitrification in 0.25 ml insemination straws) was compared using Comet assay. Vitrification in droplet system and Open Pull Straws vitrification did not result in detectable cryoinjuries of DNA of bovine oocytes. On the contrary, DNA fragmentation was found in four of 26 oocytes vitrified in 0.25 ml straws (15.4%, p

Developmental potential of selectively enucleated immature mouse oocytes upon nuclear transfer.

Selective enucleation (SE) was applied to germinal vesicle (GV) oocytes by removing the chromatin attached to nuclear envelope, and leaving the liquid contents of GV in the cytoplast. However, after reconstruction with 1/8 blastomeres or fetal fibroblasts (FFs) neither the maturation efficiency nor the frequency of normal (asymmetric) division was improved as compared with completely enucleated (CE) oocytes. Chromosomal aberrations introduced with somatic nuclei were not rescued in SE oocytes either. On the other hand, timing of maturation division in SE GV oocytes, but not in CE GV oocytes, reconstructed with GV-karyoplasts was like in the control. After maturation and fertilization in vitro, SE oocytes reconstructed with 1/8 blastomeres developed nucleolated donor pronuclei, contrary to CE oocytes. The latter could be rescued with nucleoli-containing nucleus, but not anucleolate nucleus, from a 1/2 blastomere. SE oocytes reconstructed with FFs contained nucleolated pronuclei upon activation, unlike CE GV oocytes. These experiments show that the ooplast nucleolar material and/or embryonic nucleolus are indispensable for pronuclei formation. SE oocytes reconstructed with 1/8 blastomeres or FFs failed to cleave after activation or in vitro fertilization. Control GV oocytes enucleolated before fertilization seized cleavage at the 6-cell stage, as oppose to intact GV oocytes, which in 50.9% yielded morulae/blastocysts. These results suggest that ooplast nucleolar material is essential for the cleavage divisions. Activation of cumulus-enclosed SE GV oocytes matured in hormone-supplemented medium and fused to 1/2 blastomere-karyoplasts, yielded morulae, and blastocysts in 45.5% and 23.4% of reconstructed oocytes, respectively.

The possible role of cell cycle stage in mammalian cloning.

Progress in mammalian cloning started from cloning embryos (of mice, rats, rabbits, sheep, goats, pigs, cattle and rhesus monkeys) and culminated in obtaining clones of sheep, cattle, pigs and mice from adult somatic cells. Knowing the relationship between the cell cycles of the recipient and the donor of cell nucleus in embryonic cloning by nuclear transfer one can adjust the phases of the cell cycle properly. Metaphase II recipients accept G1 (in most species) or G2 donors (in the mouse). Interphase recipients can harbour nuclei in all stages of cell cycle. Relatively little is known about somatic cloning. Two attitudes are applied: either the donor is in the G0 phase or the recipient is in a prolonged MII phase.

Development of bovine embryos on Vero/BRL cell monolayers (mixed co-culture).

The objective of this study was to test a new co-culture system of bovine embryos, which we call "mixed co-culture." This system consists of culturing embryos on cell monolayers composed of both Vero and BRL cells (Vero/BRL). Cumulus-oocyte complexes from ovaries of slaughtered cows were matured and fertilized in vitro. The presumptive zygotes were cultured with Vero/BRL (Group 1), BRL (Group 2) or Vero (Group 3) cell monolayers, in 40 microL drops of Menezo B2 medium supplemented with 10% FBS and antibiotics. The development of the presumptive zygotes was compared on Day 2 [48 h post insemination (pi)] and Day 7 (168 h pi). On Day 2, there was no difference between the groups. On Day 7, the highest percentage of compacted morulae/blastocysts was observed in mixed co-culture of Vero/BRL cells: 40% versus 36% on BRL versus 27% on Vero cell monolayers. The differences were statistically significant (P < or = 0.05). Among compacted morulae/blastocysts, blastocysts prevailed in mixed co-culture: 67% on Vero/BRL as compared with 55% on BRL and 27% on Vero cell monolayers. The differences were highly statistically significant (P < or = 0.01). The results suggest that Vero/BRL cells improve the development of bovine embryos.

In vitro and in vivo development of bovine embryos from zygotes and 2-cell embryos microinjected with exogenous DNA.

The objectives of these experiments were: 1) to determine an effective culture method for production of transferable bovine embryos following exogenous DNA microinjection; 2) to determine the effect of these methods on the ability of the injected zygotes and 2-cell embryos to develop in vivo; and, 3) to compare development of embryos microinjected as zygotes or 2-cell embryos. DNA fragments encoding bovine growth hormone (bGH), bGH-10Delta6, and a bGH antagonist, bGH-M8 (5) were used. A total of 639 zygotes and 153 2-cell embryos were injected. Zygotes and 2-cell embryos microinjected with bGH-M8 were incubated for 6 days in oviducts of intermediate recipients (rabbits or sheep) or co-cultured in vitro with bovine oviduct epithelial cells. Zygotes and 2-cell embryos microinjected with bGH-10Delta6 were co-cultured in vitro only. The most effective method for the production of transferable bovine embryos following exogenous DNA microinjection was via in vitro co-culturing with bovine epithelial cells. For example, 32.3% of the bGH-M8 and 33.5% of the bGH-10Delta6 microinjected zygotes reached the morula/blastocyst stage while 48.4% and 63.0% of the 2-cell embryos injected with bGH-M8 and bGH-10Delta6, respectively, developed to the morula/blastocyst stage. The percentage of blastocysts obtained for control, non-injected zygotes and 2-cell embryos was 34.5% and 69.6%, respectively. The developmental rate to the morula/blastocyst stage was approximately 20% greater for embryos obtained from microinjected 2-cell embryos relative to microinjected zygotes. However, there was no significant difference in pregnancy rates following transfer of these blastocysts to cow uteri.

Grand-paternal age and the development of autism-like symptoms in mice progeny.

Advanced paternal age (APA) contributes to the risk of autism spectrum disorders (ASDs) in children. In this study, we used a mouse model to investigate the effects of APA on behavioral features related to autistic syndromes (that is, social deficits, communication impairments and stereotypic/repetitive behaviors). We also examined whether such effects are transmitted across generations. To do this, males aged 15 months (APA) and 4 months (control) were bred with 4-month-old females, and the resulting offspring (F1) and their progeny (F2; conceived by 4-month-old parents) were tested for the presence and severity of ASD-like behaviors. Our results indicate that APA resulted in offspring that displayed distinctive symptoms of ASD. We found that both F1 conceived from old fathers and F2 derived from old grandfathers displayed increased ultrasound vocalization (USV) activity, decreased sociability, increased grooming activity and increased anxiety-like responses. Moreover, such abnormalities were partially transmitted to the second generation of mice, having APA grandfathers. In conclusion, our study suggests that the risk of ASD could develop over generations, consistent with heritable mutations and/or epigenetic alterations associated with APA.

Interspecies somatic cell nuclear transfer: a salvage tool seeking first aid.

Much emphasis is currently given to the use of Interspecific Somatic Cell Nuclear Transfer (ISCNT) as a potential salvage tool for endangered animals. In this short review we present a survey on all data published so far on ISCNT, including abstract communication in international meetings. From the analysis of these data it appears that the results obtained are very preliminary and often confusing on the real stage of the embryonic development obtained. Moreover, the acronym ISCNT is improperly used because in many reports the nuclei and oocyte donor are not within the same species, but belong to different order and sometimes taxa, therefore, we classified all the ISCNT reports by allocating cell and oocyte donors to their respective order/species/class. The efficiency of cloning is low in all species owing to incomplete nuclear reprogramming of differentiated cells under the current procedures. ISCNT, however, poses additional hurdles which are rarely addressed in previously published work, and on which we focus in this review: mt/genomic DNA compatibility; embryonic genome activation of the donor nucleus by the recipient oocyte; availability of suitable foster mothers for ISCNT embryos. All these issues are discussed here, and possible solutions for the successful application of somatic cell nuclear transfer to endangered animals are also put forth.

Preimplantation development of microsurgically obtained haploid and homozygous diploid mouse embryos and effects of pretreatment with Cytochalasin B on enucleated eggs.

Haploid embryos were obtained by microsurgical removal of one pronucleus, followed by doubling of the haploid chromosome set with Cytochalasin B (CB), either at the first or second mitosis. This procedure provides a source of fully homozygous diploid embryos which were grown in vitro or in vivo. The effect of CB treatment before and during operation on the course of enucleation and further development of embryos was studied. Out of 81 eggs made diploid at 2-cell stage and transplanted into the oviducts of immature or pseudopregnant recipients 27 morulae and blastocysts were recovered, but not a single case of implantation occurred by the eighth or ninth day of development. After 72-80 h of in vitro culture, most of the homozygous embryos were morulae but after an additional 24 h the majority of them transformed into blastocysts. The rate of development of homozygotes was markedly better than that of haploids, which progressed beyond morula stage. The immediate survival rate of operated eggs was dependent on whether or not the eggs were pre-incubated and the enucleation was performed in the presence of CB. In the former case the immediate survival rate was nearly twice as high as in the absence of CB, but more of the treated eggs underwent fragmentation and early developmental arrest.

Haploid mouse embryos obtained by microsurgical removal of one pronucleus.

A pronucleus can be microsurgically removed from the fertilized mouse egg. Out of 145 haploid eggs obtained by this method and transplanted into the oviduct of pseudopregnant recipients, 36 multicellular embryos were recovered on the 4th or 5th day. On the 4th day all embryos were morulae composed of 8-50 cells, with the majority containing 8-16 cells. After an additional 24 h in vivo or in vitro the cell number increased considerably, sometimes up to as many as 80. Out of 36 multicellular embryos only one developed into a blastocyst while the others remained at the morula stage. Karyological investigations confirmed that the embryos were haploid and revealed that all were gynogenetic. Possible reasons for the absence of the androgenones and for the scarcity of blastocysts are discussed.

Preimplantation development of rabbit embryos after transfer of embryonic nuclei into different cytoplasmic environment.

The development of nuclear-transfer oocytes and zygotes was tested in the rabbit. Metaphase II oocytes and zygotes in the early pronuclear stage were treated with a cytoskeletal inhibitor (cytochalasin D), enucleated, and subsequently fused either with single blastomeres from eight- and 16-cell stages (oocytes and zygotes) or with pronuclei-containing karyoplasts (zygotes only). Also, nonenucleated zygotes were fused with 1/8 blastomeres. Fusion was performed by means of an electric field. Development of reconstituted embryos was monitored mainly in vitro, but a certain number of embryos developed from oocytes and zygotes receiving nuclei from eight-cell stages were also transferred into pseudopregnant does. Development of nuclear-transfer oocytes was distinctly better than that of nuclear-transfer zygotes, since 16.9% and 9.5% oocytes vs. 8.1% and 3.7% zygotes carrying eight- and 16-cell nuclei, respectively, developed to the blastocyst stage. Two advanced but already dead fetuses were found after transfer of 27 four-cell embryos obtained after fusion of oocytes with 1/8 blastomeres. No implantations were observed after transfer of 25 four-cell embryos developed from enucleated zygotes receiving eight-cell nuclei. These findings indicate that, in the rabbit, some nuclei from 16-cell embryos are still capable of promoting at least preimplantation development. Comparison between the developmental abilities of oocyte- and zygote-derived nuclear-transfer embryos also suggests that the cytoplasmic environment of recipient cell is more crucial for the development of reconstituted embryos than the stage of introduced nuclei (at least up to the 16-cell stage). The majority of pronuclear exchange embryos (69.9%) and 40% of nonenucleated zygotes receiving eight-cell nuclei were able to develop to the blastocyst stage. This latter observation indicates, similarly as with mouse, a supporting role of residual pronuclei for participation of an eight-cell nucleus in the development of reconstituted zygotes.

Effects of electric field on fusion rate and survival of 2-cell rabbit embryos.

Electric-field-induced blastomere fusion was studied in 2-cell rabbit embryos. Field strengths (1 to 3kV cm-1) and durations (35 to 1000 microseconds) were chosen so as to provide the right balance between fusion rate, viability and developmental capacity of embryonic cells. Maximum plasma membrane tolerance of 2-cell rabbit embryos was observed at about 3kV cm-1 for 1000 microseconds. All surviving 'fused' embryos were able to develop in vitro and most of them formed expanded blastocysts. Observation of 'fused' embryos immediately after fusion and during the whole cell cycle showed that 27.7% of the two diploid nuclei remained separated in the hybrid cell. More than one metaphase plate was formed at the onset of mitosis causing direct cleavage into three or four 'cells'. In the remaining embryos the two diploid nuclei seemed to form a common metaphase plate and cleaved into two equal blastomeres. After transfer to recipient does, 54.4% of these tetraploid embryos developed beyond implantation. Between day 11 and 20, ten live and morphologically fully normal embryos were recovered. Nine embryos were uniformly tetraploid and one recovered on day 18 was a diploid/tetraploid mosaic. The remaining implantation sites contained either abnormal, very retarded embryos or indefinable embryo remnants. After transfer of 'nonfused' embryos treated with 3kV cm-1, 49% gave birth to normal live young. These results suggest that the electric field can be applied successfully in a relatively wide strength and duration range without causing any visible teratogenic effect on treated embryos. Thus, tetraploid embryos can develop normally at least until two-thirds of pregnancy, but the question whether they are able to survive till term remains open.

[Somatic cloning in mammals]. / Klonowanie somatyczne ssaków.

Somatic cloning in mammals became possible due to refinement of the nuclear transfer technique consisting of using nuclear donor cells in GO or activating reconstituted oocytes few hours after nuclear transfer (post-activation). Sheep, goats, pigs, cattle and mice have been cloned this way; the latter two species - of both sexes. Cloning of transgenic mammals producing human therapeutic proteins in milk (or urine) can find application in pharmacology and medicine. Somatic cloning of a goat producing human antithrombine III in its milk has already been achieved.

Behaviour of thymocyte nuclei in non-activated and activated mouse oocytes.

Cells originating from the thymus of newborn mice were fused with mouse oocytes using polyethylene glycol. The behaviour of thymocyte nuclei was studied in non-activated metaphase II oocytes, and in oocytes activated in vitro with ethanol. In non-activated oocytes all thymocyte nuclei undergo premature chromosome condensation with individualization of chromosomes; the chromosomes form separate groups in the cytoplasm, or are assembled around the metaphase II spindle, or located on the extra-spindle. In activated oocytes thymocyte nuclei start to develop along a pronucleus-like pathway (decondensation, visualization of nucleoli, swelling) and increase up to 200 times in volume during 24 h culture in vitro, eventually reaching the size of a fully grown pronucleus. Activation/fusion timing seems to be critical for the full remodelling of thymocyte nuclei. Nuclei introduced before (10-30 min) or shortly after (up to 60 min) activation often grow larger than the female pronucleus. Those introduced into oocytes long before activation (greater than 30 min) undergo premature condensation with subsequent reformation of nuclei that are sometimes deficient (as indicated by the presence of micronuclei), or of hybrid character. Nuclei introduced late after activation (greater than 60 min) are mostly doomed to retarded development. The implications of the present observations for nuclear transfer experiments in mammals are discussed.

Nuclear transfer from teratocarcinoma cells into mouse oocytes and eggs.

A spontaneous ovarian teratocarcinoma was isolated from a LT/Sv mouse female and converted into an ascites tumor from which embryonal carcinoma (EC) cells were dissociated. Non-enucleated and enucleated, activated oocytes were fused with EC cells and either cultured in vitro or transferred into ligated oviducts of Swiss/A females. The nucleocytoplasmic hybrids cultured in vitro up to 22 h were examined cytologically at various time intervals. EC nuclei showed morphological remodelling in the foreign cytoplasm. EC chromosomes and female pronuclear chromosomes together formed a common metaphase. The nucleocytoplasmic hybrids developed in vivo were analyzed cytologically between the first and third day after oviduct transfer. The majority of embryos developed abnormally and, in a few instances, they had passed several cleavage divisions and reached, at best, a developmental stage resembling a premature morula. Fertilized, enucleated eggs were fused with EC cells or microinjected with EC nuclei. The resulting nucleocytoplasmic hybrids were either cultured in vitro or in vivo up to the fourth day. Enzyme tests were carried out on the nuclear transplant embryos, using electrophoretic variants of glucose phosphate isomerase (GPI) in order to distinguish between EC nuclei (GPI-A) and recipient eggs (GPI-B). The EC-specific GPI could be detected in about one third of the embryos analyzed and, in several instances, also together with the egg-specific GPI. Most of them were arrested during early cleavage divisions. Some embryos cleaved abnormally or mimicked normal embryogenesis. In a few instances, development resulted in embryos that resembled late preimplantation embryos.

Effects of preactivation of ooplasts or synchronization of blastomere nuclei in G1 on preimplantation development of rabbit serial nuclear transfer embryos.

Blastomeres from eight-cell-stage rabbit embryos have been fused with enucleated metaphase II oocytes (ooplasts) or with ooplasts that were preactivated before fusion. Preactivation of ooplasts before nuclear transfer (NT) raises the rate of preimplantation development from 15% to 56%, which remains elevated in the next series of NT (48.6% and 47.2% in the second and third rounds, respectively). Transfer of eight-cell embryos from the third round to the recipient resulted in the birth of normal young. Synchronization of blastomere nuclei in the G1 phase with nocodazole before fusion results in 42% morula/blastocyst formation. However, in the second generation of NT embryos, the yield drops to as low as 17%, indicating deleterious effects of the second nocodazole treatment on blastomeres. The calculated number of clones per one round of cloning was 4.5, 3.9, and 3.8 in subsequent series; the highest number of morulae and blastocysts that developed from individual donor embryos after three rounds were 26 and 27, respectively.

Development of isolated sheep inner cell masses/embryonic discs in vitro.

Ovine inner cell masses (ICMs)/embryonic discs cultured in vitro, in conditions copying those in which mouse embryonic stem cells (ESCs) arise from mouse blastocysts, give rise to ectodermal colonies. Day 10-11 ICMs/epiblasts produce ectodermal colonies sufficiently often (55-60%) for it to be considered worthwhile trying to generate presumed ESCs from them. Younger ICMs can only be taken into account if culture conditions can be improved so that ICM/ectodermal cells are more numerous. Older embryonic discs (12-13 day) are inconvenient because of the problem of endoderm overgrowing ectoderm. Secondary cultures of ectodermal colonies form epithelial or mesenchymal cells, which can be passaged at least seven times (50 days).

Case manager follow-up to failed appointments and subsequent service utilization.

Case manager responses to failed appointments were monitored for 83 seriously mentally ill persons in a rural community mental health center. Case manager actions taken were grouped into four categories of follow-up from most intensive to least intensive: home visit, phone call, letter, and no follow-up. On the whole, case managers most frequently did not follow-up missed appointments (56.7%), followed up by letters (21.3%), and telephone calls (18.7%), and home visits (3.3%). Analyses revealed that home visits were most intensive and all clients who were visited following failed appointments did not fail the subsequent appointment. Clients who received telephone calls or letters were about equally likely to fail the subsequent appointment, but were much more likely to attend the subsequent appointment than were clients who received no follow-up to the failed appointment. Interestingly, clients who failed appointments and received no follow-up were much more likely to need emergency services rather than a regular appointment as their next contact with the clinic.