Active caspase-3 and ultrastructural evidence of apoptosis in spontaneous and induced cell death in bovine in vitro produced pre-implantation embryos.

In this study we investigated chronological onset and involvement of active caspase-3, apoptotic nuclear morphology, and TUNEL-labeling, as well as ultrastructural evidence of apoptosis, in both spontaneous and induced cell death during pre-implantation development of bovine in vitro produced embryos. Pre-implantation embryos (2-cell to Day 8 blastocysts) were cultured with either no supplementation (untreated) or with 10 microM staurosporine for 24 hr (treated). Embryos were subjected to immunohistochemical staining of active caspase-3, TUNEL-reaction for detection of DNA degradation and DAPI staining for detection of apoptotic nuclear morphology, and subjected to fluorescence microscopy. Additionally, treated and untreated blastocysts were fixed and processed for ultrastructural identification of apoptosis. Untreated embryos revealed no apoptotic features at 2- and 4-cell stages. However, active caspase-3 and apoptotic nuclear morphology were observed in an untreated 8-cell stage, and TUNEL-labeling was observed from the 16-cell stage. Blastomeres concurrently displaying all apoptotic features were present in a few embryos at 16-cell and morula stages and in all blastocysts. All three features were observed from the 8-cell stage in treated embryos, and blastomeres with apoptotic features appeared more numerous in treated than in untreated embryos. Ultrastructural evidence of apoptosis occurred with a comparable distribution pattern as apoptotic features detected by fluorescence microscopy in both treated and untreated blastocysts. Activation of caspase-3 is likely involved in both spontaneous and induced apoptosis in bovine pre-implantation embryos, and immunohistochemical staining of active caspase-3 may be used in combination with other markers to identify apoptosis in pre-implantation embryos.

Attempts towards derivation and establishment of bovine embryonic stem cell-like cultures.

Current knowledge on the biology of mammalian embryonic stem cells (ESC) is stunningly sparse in light of their potential value in studies of development, functional genomics, generation of transgenic animals and human medicine. Despite many efforts to derive ESC from other mammalian species, ESC that retain their capacity for germ line transmission have only been verified in the mouse. However, the criterion of germ line transmission may not need to be fulfilled for exploitation of other abilities of these cells. Promising results with human ESC-like cells and adult stem cells have nourished great expectations for their potential use in regenerative medicine. However, such an application is far from reality and substantial research is required to elucidate aspects of the basic biology of pluripotent cells, as well as safety issues associated with the use of such cells in therapy. In this context, methods for the derivation, propagation and differentiation of ESC-like cultures from domestic animals would be highly desirable as biologically relevant models. Here, we review previously published efforts to establish bovine ESC-like cells and describe a procedure used in attempts to derive similar cells from bovine Day 12 embryos.

Ultrastructure and cell death of in vivo derived and vitrified porcine blastocysts.

Morphological and molecular signs of injury and cell death and subsequent regeneration following vitrification of porcine blastocysts were evaluated by light (LM) and transmission electron microscopy (TEM) as well as TUNEL/propidium iodide (PI) nuclear staining followed by confocal microscopy (CSM). In vivo derived blastocysts were assigned to one of the following four groups: Controls-(1) fixed immediately after collection (C0h) and (2) after 24 hr culture in vitro (C24h) and vitrified embryos-(3) fixed immediately after vitrification and warming (V0h), and (4) after 24 hr of culture upon warming after vitrification (V24h). Observation by LM and TEM showed that the V0h embryos displayed collapse of the blastocoele cavity (BC) and cell swelling, a general distension or shrinkage of mitochondria and massive increase in the amount of vesicles, vacuoles, and secondary lysosomes (SLs). Approximately 2/3 of the V24h embryos had recovered, whereas the remaining 1/3 were degenerated. Recovered embryos displayed almost normal blastocyst morphology, except for a widening of the perivitelline space, accumulation of debris and partial distension of mitochondria, whereas degenerated embryos were disintegrated into a poorly defined mass of cells and debris including cells with abundant degeneration of mitochondria and other organelles. Both recovered and degenerated embryos displayed a persistent abundance of presence of small membrane bounded vesicles, vacuoles, and SLs. Evaluation of TUNEL/PI stained embryos showed only occasional appearance of TUNEL positive nuclei with typical apoptotic morphology in controls (C0h 0.67%, C24h 1.22%) and in the V0h embryos (0.93%). The percentage of apoptotic nuclei in embryos at V24h was significantly higher than in all other groups (2.64%). Vitrified embryos showed significantly increased appearance of DNA fragmented nuclei without typical morphological features of apoptosis (V0h 1.43%, V24h 4.30%) compared with controls (C0h 0.26%, C24h 0.45%). The observed morphological changes and increased DNA fragmentation observed immediately after vitrification and warming probably reflects a direct damaging effect of vitrification. During 24 hr of culture a portion of the embryos was able to regenerate and along with the regenerative process, apoptosis--a possible pathway for elimination of damaged cells--became evident.

Consequences of in vivo development and subsequent culture on apoptosis, cell number, and blastocyst formation in bovine embryos.

Bovine embryos produced in vitro differ considerably in quality from embryos developed in vivo. The in vitro production system profoundly affects the competence to form blastocysts, the number of cells of the total embryo and of the inner cell mass (ICM), and the incidence of apoptosis. To our knowledge, the effects of different postfertilization regimens before and after completion of the fourth embryonic cell cycle on these aspects have not yet been investigated. In the present study, we assessed the blastulation rate by stereomicroscopy and the cell number of the total embryo, of the ICM, and of the cells with apoptotic changes by confocal laser-scanning microscopy after staining with propidium iodide and TUNEL. Two groups of embryos were developed in heifers, after superovulation, until 45 or 100 h postovulation (po) and, after collection on slaughter, were further cultured in vitro until Day 7 po. A third and fourth group comprised embryos that were produced entirely in vitro or in vivo. The results indicate that passage in vivo of the fourth cell cycle does not prevent acceleration of the formation of the blastocoele in vitro but may be the critical factor contributing to a higher cell number in the total blastocyst and its ICM. The lower quality of in vitro-produced embryos can be attributed to the ICM having less viable cells because of a lower number of cells and a higher incidence of apoptosis that appears to be determined before completion of the fourth cell cycle.

Chronology of apoptosis in bovine embryos produced in vivo and in vitro.

The postimplantation developmental potential of embryos can be affected by various forms of cell death, such as apoptosis, at preimplantation stages. However, correct assessment of apoptosis is needed for adequate inference of the developmental significance of this process. This study is the first to investigate the independent chronological occurrence of apoptotic changes in nuclear morphology and DNA degradation (detected by the TUNEL reaction) and incidences of nuclei displaying these features at various preimplantation stages of bovine embryos produced both in vivo and in vitro. Different elements of apoptosis were observed at various developmental stages and appeared to be differentially affected by in vitro production. Nuclear condensation was observed from the 6-cell stage in vitro and the 8-cell stage in vivo, whereas the TUNEL reaction was first observed at the 6-cell stage in vitro and the 21-cell stage in vivo. Morphological signs of other forms of cell death were also observed in normally developing embryos produced both in vivo and in vitro. The onset of apoptosis seems to be developmentally regulated in a stage-specific manner, but discrete features of the apoptotic process may be differentially regulated and independently modulated by the mode of embryo production. Significant differences in indices of various apoptotic features were not evident between in vivo- and in vitro-produced embryos at the morula stage, but such differences could be observed at the blastocyst stage, where in vitro production was associated with a higher degree of apoptosis in the inner cell mass.

Efficacy of porcine gonadotropins for repeated stimulation of ovarian activity for oocyte retrieval and in vitro embryo production and cryopreservation in Siberian tigers (Panthera tigris altaica).

A comparison of the amino acid sequences demonstrated that Siberian tiger gonadotropins are more homologous with those of porcine than any other commercially available preparation. The present study measured the efficacy of repeated ovarian stimulation with purified porcine gonadotropins on the follicular, hormonal, and immunogenic responses in Siberian tigers as well as on the ability of oocytes retrieved by laparoscopic follicular aspiration to fertilize and cleave in vitro. Controlled rate and vitrification cryopreservation methods were also compared for their ability to support ongoing cleavage following thawing of presumptive 2- to 4-cell tiger embryos generated in vitro. Vitrification supported continued embryonic cleavage in vitro while controlled rate freezing did not. Stereological microscopy indicated an excellent ovarian response with the recovery of quality cumulus-oocyte complexes that apparently fertilized and cleaved in vitro. However, ultrastructural and physiological examination revealed abnormal and unnatural responses such as the failure of some cumulus-oocyte complexes to reach maturity and progestagen levels to approach normalcy. At the same time, analyses of blood for antibodies failed to detect an immune reaction to these foreign gonadotropins in an assay that tested positive for the chorionic gonadotropin-stimulated domestic cat. Together, these observations suggest that porcine gonadotropins may be effective for the ovarian stimulation of tigers but that some modifications to administration protocols are needed to produce a more natural response.