Nuclear transfer of freeze-dried somatic cells into enucleated sheep oocytes.

Lyophilization has been used since long time to preserve yeast and bacteria strains. Subsequently, a great deal of efforts has been dedicated to the preservation in a dry state of red blood cells and platelets. However, despite more than 30 years passed by, no significant progress has been achieved. Recently, it has been reported that freeze-dried mice spermatozoa were able to generate normal offspring following injection into the mature mice oocytes. In this work, we prompted to apply the lyophilization protocol developed for mice spermatozoa to sheep somatic cells (lymphocytes and granulosa cells). More than 350 enucleated sheep oocytes were injected with granulosa cells, and freeze dried using the protocol developed for mice sperm cells. Transplanted nuclei organized large pronuclei with fragmented DNA, but none of them entered the first mitosis. In the second part of the experiments, trehalose and EGTA were found to reduce significantly the extent of nuclear damage (65% and 55% intact nuclei in lymphocyte and granulosa cells, respectively) following freeze drying. Granulosa cells lyophilized with EGTA/trehalose and stored at room temperature for 3 years were used for nuclear transfer, and the injected oocytes were cultured in vitro for 7 days. Approximately 16% of the oocyte injected with freeze-dried cells developed into blastocysts. To conclude, we demonstrated for the first time that nucleated cells maintain genomic integrity after prolonged storage in a dry state, and we were able to achieve early embryonic development following injection of these cells into enucleated sheep oocytes.

Genetic rescue of an endangered mammal by cross-species nuclear transfer using post-mortem somatic cells.

Since the advent of procedures for cloning animals, conservation biologists have proposed using this technology to preserve endangered mammals. Here we report the successful cloning of a wild endangered animal, Ovis orientalis musimon, using oocytes collected from a closely related, domesticated species, Ovis aries. We injected enucleated sheep oocytes with granulosa cells collected from two female mouflons found dead in the pasture. Blastocyst-stage cloned embryos transferred into sheep foster mothers established two pregnancies, one of which produced an apparently normal mouflon. Our findings support the use of cloning for the expansion of critically endangered populations.

Mammalian oocyte therapies.

In assisted human reproduction, the cytoplasm of oocytes recovered from follicles is often abnormal. Its lower quality, especially in older patients, may be responsible for certain chromosomal abnormalities or developmental arrest. Thus, the deficiency of some vital molecules, which are necessary for oocyte maturation, can be the cause of infertility in women. Moreover, mutated mitochondrial DNA (mtDNA) that is located in the oocyte cytoplasm might be transmitted to offspring. With the advance of new micromanipulation techniques like the oocyte nucleus replacement or cytoplasmic transfer, some of these abnormalities could be theoretically eliminated. In this review, we briefly discuss some of these approaches and their potential use in assisted human reproduction.

An attempt to reduce polyspermic penetration in lamb oocytes.

The incidence of polyspermy in lamb oocytes matured and fertilized in vitro is very high and this results in a reduced developmental potential of embryos arising from them. We have attempted to produce oocytes more resistant to this fertilization anomaly. The oocytes from prepubertal lambs 7-12 weeks old were matured in a medium supplemented with various blood sera and oviductal fluid and fertilized in vitro. Significantly higher monospermic penetration was found in a medium supplemented with BSA--3 mg/ml (63.9%) and OF--20% concentration (55.8%). Lower monospermy was recorded in the presence of 10% LS (44.6%) or 10% SS (40.8%), and particularly in a medium with 10% FCS (26.9%). In contrast, high monospermy (78.7%) was observed in oocytes from adult donors matured and fertilized in an identical system. In another set of experiments we estimated whether polyspermy can be reduced by improvement of the cytoplasmic maturation of prepubertal oocytes using a two-step maturation protocol. After artificial arrest of the maturation for 24 h with a specific cdk inhibitor--BL-I, 50 miocroM--more than 80% oocytes from prepubertal and adult donors did not resume meiosis. When incubated thereafter in a drug-free medium for another 24 h, the oocytes of both categories progressed to MII in the rate comparable with control (80% to 90% MII). However, after fertilization no significant differences in the level of monospermic penetration was recorded between the arrested group (59.8%) and control (58.8%), both matured in the presence BSA, and 46.6% and 52.3% after treatment with OF. Also, no significant difference was observed between the arrested and control oocytes from adult donors (72.6% and 84.8%, respectively). These results suggest that high polyspermy in prepubertal oocytes is caused by developmental imperfection and can't be fully eliminated either by modifying the composition of culture media or by prolongation of the culture interval.

Effect of protein supplement source on porcine oocyte maturation and subsequent embryonic development after parthenogenetic activation.

The aim of this study was to compare the effect of purified GPBoS and commonly used FCS on porcine oocyte maturation and subsequent embryonic development after their parthenogenetic activation. COCs were obtained from dissected follicles and cultured for 18, 24, 30, 36, 42 and 48 h in M-199 medium either with GPBoS or FCS. After 24 h with GPBoS, 91% of oocytes reached MI stage while in the medium supplemented with FCS, only 29% of oocytes reached the same stage (P < 0.05). The majority of oocytes from the FCS group (61%) reached MI stage approximately 6 h later. In the time periods between 36 to 48 h both groups of oocytes reached the same stage of maturation. After 48 h of culture the oocytes with extruded polar bodies were activated by a single electric pulse and then cultured with 4 mM 6-DMAP. Activated oocytes were cultured in PZM-3 medium supplemented with 3 mg/ml of BSA. After 7 days, the development and the quality of embryos were evaluated. The results showed that the maturation of oocytes in the presence of GPBoS significantly increased their subsequent developmental ability when compared with FCS supplementation (27% vs. 19% of blastocysts, P < 0.05). However, differential staining revealed that once blastocysts were formed in either group, they had the same total cell number (40 vs. 41) and also the ICM/total cell ratio (0.27 vs. 0.29).

Kinetics of MPF and histone H1 kinase activity differ during the G2- to M-phase transition in mouse oocytes.

Maturation promoting factor (MPF) is universally recognized as the biological entity responsible for driving the cell cycle from G2- to M-phase. Histone H1 kinase activity is widely accepted as a biochemical indicator of p34cdc2 protein kinase complex activity and therefore MPF activity. In this paper we present results which indicate that during the G2- to M-phase transition in mouse oocytes the dynamic of p34cdc2 related histone H1 kinase activity differs markedly from the biological activity of MPF as measured by classical cell fusion procedures. MPF is activated just before germinal vesicle breakdown (GVBD) whereas histone H1 kinase is activated 5-7 h later coincident with the formation of the definitive first metaphase plate. The biological activity of MPF is merely reduced to about 50% of control levels by a short period of protein synthesis inhibition (1-2 h) and completely suppressed after a prolonged period of inhibition (4-5 h). By contrast, inhibition of protein synthesis in mouse oocytes results in a rapid and complete suppression of histone H1 kinase activity. Therefore, biological MPF and histone H1 kinase activity should not be used in an interchangeable manner during the G2- to M-phase transition in mouse oocytes.

Early changes in embryonic nuclei fused to chemically enucleated mouse oocytes.

Mouse oocytes were chemically enucleated by subjecting them to etoposide and cycloheximide treatment during the first meiotic division (Fulka, Jr. and Moor, Mol. Reprod. Dev. 34:427-430, 1993) and thereafter electrofused to karyoplasts prepared from: (i) two-cell stage embryos at the G2-phase; (ii) four-cell stage blastomeres (S- or G2-phase); or (iii) embryonic stem (ES) cells. In the first series of experiments we used fusion conditions which do not induce egg activation to define the series of nuclear changes that are initiated immediately following fusion. Although fusion is evident within 5-10 min of induction, nuclei remain visible for up to 20 min prior to chromatin condensation and the formation of metaphase plates (60-90 min post fusion). After activation, the anaphase-telophase transition is completed within 1-2 h, followed thereafter by cleavage of 75% of reconstituted eggs into two equal nucleated blastomeres, irrespective of the origin of the nuclei used for fusion. We conclude from the first study that a protocol involving fusion without activation, followed 90 min later by activation, is likely to be optimal for nuclear transplantation using MII-phase cytoplasts. In the second series of experiments the above optimized protocol was used to study the effects of different cell cycle combinations on chromosome organization in eggs reconstituted by nuclear transplantation. Both G1- and S-phase karyoplasts fused to MII-phase cytoplasts exhibited spindle abnormalities in all eggs studied. Characteristic abnormalities in these cell cycle combinations included chromatin fragmentation and joining or aggregations of chromatin.(ABSTRACT TRUNCATED AT 250 WORDS)

A reversible block at the G1/S border during cell cycle progression of mouse embryos.

Late 2-cell stage mouse embryos were cultured in M-199 plus 100 micrograms/ml Na pyruvate 25 micrograms/ml gentamycin and 0.3% BSA with or without mimosine (200 microM, 150 microM, 100 microM and 50 microM) for a short (4-5 h) or long (18-20 h) culture period; after drug removal subsequent embryo development was evaluated. Late 2-cell stage mouse embryos treated with mimosine were blocked at the 4-cell stage. Autoradiographic studies show that mimosine inhibits cell cycle progression in mouse embryos at the G1/S boundary. The onset of DNA replication occurs within 15 min of releasing the embryos from mimosine block. Embryos pretreated with mimosine at 200 microM and 150 microM for 4-5 h progress after 3-4 days in culture to hatched blastocyst (71% and 79%, respectively) compared with control (90%). However a longer pretreatment (18-20 h) with mimosine at 200 microM was significantly detrimental to the subsequent developmental progression to hatched blastocyst (2% vs 81%, p < or = 0.05); the proportion of degenerated embryos was significantly increased with mimosine at 200 microM and 150 microM compared with control (57% and 28% vs 4%, p < or = 0.05) after 3-4 days in culture. Preliminary studies with mimosine treatment at 100 microM and 50 microM for 18-20 h show that 70% and 37% of the embryos were blocked at 4-cell stage, respectively. These results indicate that mimosine inhibits cell cycle progress in mouse embryos at the G1/S border and thus induces a reversible arrest in a dose- and time-dependent manner.

Nucleus replacement in Mammalian oocytes.

Our contribution discusses the potential use of cell therapies (nucleus replacement) in mammalian oocytes. It is assumed that these approaches may be used, for example, for the elimination of mutated maternally transmitted mitochondrial DNA (mtDNA) as well as for the reconstruction of normal oocytes from oocytes that are developmentally compromised. Moreover, it is speculated that the replacement of germinal vesicles by somatic cells may result in cells of the haploid genome: the production of germ cells from somatic cells. The preliminary results obtained in our laboratories are discussed in this article.

Accumulation of the proteolytic marker peptide ubiquitin in the trophoblast of mammalian blastocysts.

Ubiquitination is a universal protein degradation pathway in which the molecules of 8.5-kDa proteolytic peptide ubiquitin are covalently attached to the epsilon-amino group of the substrate's lysine residues. Little is known about the importance of this highly conserved mechanism for protein recycling in mammalian gametogenesis and fertilization. The data obtained by the students and faculty of the international training course Window to the Zygote 2000 demonstrate the accumulation of ubiquitin-cross-reactive structures in the trophoblast, but not in the inner cell mass of the expanding bovine and mouse blastocysts. This observation suggests that a major burst of ubiquitin-dependent proteolysis occurs in the trophoblast of mammalian peri-implantation embryos. This event may be important for the success of blastocyst hatching, differentiation of embryonic stem cells into soma and germ line, and/or implantation in both naturally conceived and reconstructed mammalian embryos.