Influence of Combined Microinjection of Gene Engineering Construct and Endonuclease on Preimplantation Development of Mouse Embryos in vitro

We studied the influence of combined microinjection of a gene engineering construct and site-specific endonuclease SalIin the pronucleus on preimplantation development of (CBA × C57BL)F₁ mouse embryos in vitro. The rate of survival of the embryos was estimated according to their capacity to develop until the blastocyst stage and hatch from zona pellucida. The results obtained suggest that the microinjection of exogenous DNA jointly with endonuclease SalI at concentrations from 0.1 to 0.01 U/l decreased reliably the rate of survival, as compared to the control (p < 0.05 and p < 0.01, respectively). However, a decrease of endonuclease SalI concentration in the injection mixture to 0.01 U/l enhanced the capacity of mouse embryos to develop until the blastocyst stage and hatch from zona pellucida, as compared to the embryos microinjected with exogenous DNA and endonuclease SalI at a higher concentration.     

Effects of cryopreservation of pronuclear-stage rabbit zygotes on the morphological survival, blastocyst formation, and full-term development after DNA microinjection

The objectives of this study were to examine the freezing sensitivity of pronuclear-stage rabbit zygotes and to produce transgenic rabbits using the cryopreserved zygotes. Zygotes were cryopreserved either by one of two vitrification protocols or by one of the two conventional freezing protocols. The morphological survival rates of zygotes subjected to two-step freezing in 1.5 M ethylene glycol and 0.1 M sucrose (74%) or to vitrification in 7.2 M ethylene glycol and 1.0 M sucrose (81%) were higher than those subjected to freezing in 1.5 M DMSO (46%) or to vitrification in a mixture of 2.0 M DMSO, 1.0 M acetamide, and 3.0 M propylene glycol (41%). But the in vitro development into blastocysts of zygotes cryopreserved by vitrification (17%) or to a lesser extent by freezing (52%) was impaired, when compared to that of fresh control zygotes (89%). Next, a fusion gene composed from bovine aS1-casein promoter and a human GH structural gene (2.8 kb) was microinjected into the pronucleus of rabbit zygotes frozen-thawed in ethylene glycol and sucrose. Then, the presence of exogenous DNA in the genome of newborn offspring was determined by PCR. The post-injection survival of frozen zygotes (97%) was the same as that of fresh control zygotes (96%). However, of 18 offspring derived from 414 frozen-thawed and DNA-injected zygotes, no transgenic rabbits were produced. Of 52 offspring derived from 403 DNA-injected fresh zygotes, 3 transgenic rabbits were found. Here we report the first rabbit offspring resulting from zygotes cryopreserved at the pronuclear-stage, although the cryopreservation procedure employed must be improved if zygotes are to be used for systematic production of transgenic rabbits.     

Egg-to-embryo transition is driven by differential responses to Ca(2+) oscillation number

Ca(2+) oscillations and signaling represent a basic mechanism for controlling many cellular events. Activation of mouse eggs entrains a temporal series of Ca(2+)-dependent events that include cortical granule exocytosis, cell cycle resumption with concomitant decreases in MPF and MAP kinase activities, and recruitment of maternal mRNAs. The outcome is a switch in cellular differentiation, i.e., the conversion of the egg into the zygote. By activating mouse eggs with experimentally controlled and precisely defined Ca(2+) transients, we demonstrate that each of these events is initiated by a different number of Ca(2+) transients, while their completion requires a greater number of Ca(2+) transients than for their initiation. This combination of differential responses to the number of Ca(2+) transients provides strong evidence that a single Ca(2+) transient-driven signaling system can initiate and drive a cell into a new developmental pathway, as well as can account for the temporal sequence of cellular changes associated with early development.     

SHORT COMMUNICATION: Double pronuclei injection of DNA into zygotes increases yields of transgenic mouse lines

Transgenic mice are increasingly used for gene function and regulation studies of mammalian genes. A major limitation is the necessity to produce a large number of founder animals to obtain one line with the desired expression pattern. We developed a method, the 'double pronuclei injection', that doubles the yield of transgenic mouse lines obtained from each injection session, thereby reducing the time, effort and costs of generating transgenic mice. Three transgenic vectors were microinjected into the male and female pronuclei of zygotes. Approximately half of the resulting born mice were transgenic. This represented a 60% increase in the yield of founders per injected zygote, and a 100% increase in the yield of transgenic mice per born animal, when compared to yields obtained using single pronucleus injection. This method should prove useful for generating large numbers of transgenic mice for gene regulation studies and for conditional gene ablation     

Nuclear cysteine-protease involved in male chromatin remodeling after fertilization is ubiquitously distributed during sea urchin development

Previously we have identified a cysteine-protease involved in male chromatin remodeling which segregates into the nuclei of the two blastomeres at the first cleavage division. Here we have investigated the fate of this protease during early embryogenesis by immunodetecting this protein with antibodies elicited against its N-terminal sequence. As shown in this report, the major 60 kDa active form of this protease was found to be present in the extracts of chromosomal proteins obtained from all developmental stages analyzed. In morula and gastrula the 70 kDa inactive precursor, which corresponds to the major form of the zymogen found in unfertilized eggs, was detected. In plutei larvas, the major 60 kDa form of this enzyme was found together with a higher molecular weight precursor (90 kDa) which is consistent with the less abundant zymogen primarily detected in unfertilized eggs. As reported here, either the active protease or its zymogens were visualized in most of the embryonic territories indicating that this enzyme lacks a specific pattern of spatial-temporal developmental segregation. Taken together our results indicate that this protease persists in the embryo and is ubiquitously distributed up to larval stages of development, either as an active enzyme and/or as an inactive precursor. These results suggest that this enzyme may display yet unknown functions during embryonic development that complement its role in male chromatin remodeling after fertilization.     

In vitro decondensation of the sperm chromatin in Holothuria tubulosa (sea cucumber) not affecting proteolysis of basic nuclear proteins

Sea urchin and sea star oocyte extracts contain proteolytic activities that are active against sperm basic nuclear proteins (SNBP). This SNBP degradation has been related to the decondensation of sperm chromatin as a possible model to male pronuclei formation. We have studied the presence of this proteolytic activity in Holothuria tubulosa (sea cucumber) and its possible relationship with sperm nuclei decondensation. The mature oocyte extracts from H. tubulosa contain a proteolytic activity to SNBP located in the macromolecular fraction of the egg‐jelly layer. SNBP degradation occurred both on sperm nuclei and on purified SNBP, histones being more easily degraded than protein Ø_o (sperm‐specific protein). SNBP degradation was found to be dependent on concentration, incubation time, presence of Ca²⁺, pH, and this activity could be a serine‐proteinase. Thermal denaturalization of the oocyte extracts (80°C, 10–15 min) inactivates its proteolytic activity on SNBP but does not affect sperm nuclei decondensation. These results would suggest that sperm nuclei decondensation occurs by a mechanism different from SNBP degradation. Thus, the sperm nuclei decondensation occurs by a thermostable factor(s) and the removal of linker SNBP (H1 and protein Ø_o) will be a first condition in the process of sperm chromatin remodeling.     

Centrosome attachment to the C. elegans male pronucleus is dependent on the surface area of the nuclear envelope

A close association must be maintained between the male pronucleus and the centrosomes during pronuclear migration. In C. elegans, simultaneous depletion of inner nuclear membrane LEM proteins EMR-1 and LEM-2, depletion of the nuclear lamina proteins LMN-1 or BAF-1, or the depletion of nuclear import components leads to embryonic lethality with small pronuclei. Here, a novel centrosome detachment phenotype in C. elegans zygotes is described. Zygotes with defects in the nuclear envelope had small pronuclei with a single centrosome detached from the male pronucleus. ZYG-12, SUN-1, and LIS-1, which function at the nuclear envelope with dynein to attach centrosomes, were observed at normal concentrations on the nuclear envelope of pronuclei with detached centrosomes. Analysis of time-lapse images showed that as mutant pronuclei grew in surface area, they captured detached centrosomes. Larger tetraploid or smaller histone::mCherry pronuclei suppressed or enhanced the centrosome detachment phenotype respectively. In embryos fertilized with anucleated sperm, only one centrosome was captured by small female pronuclei, suggesting the mechanism of capture is dependent on the surface area of the outer nuclear membrane available to interact with aster microtubules. We propose that the limiting factor for centrosome attachment to the surface of abnormally small pronuclei is dynein.