Comparison of gene expression during preimplantation development between diploid and haploid mouse embryos

Reliable estimation and improvement of the developmental potential of in vitro production (IVP) embryos requires functional criteria of embryo quality. Antiapoptotic and mitogenic effects of insulin-like growth factor I (IGF-I), applied during bovine IVP, were studied. Day 6.5 blastocysts were fixed and processed for TUNEL to detect apoptotic cells, for immunocytochemical detection of proliferating cell nuclear antigen (PCNA), and for propidium iodide (PI) staining to detect all nuclei. Laser scanning confocal microscopy was used to determine apoptotic (TUNEL/PI) and proliferative (PCNA/PI) indices. Addition of IGF-I to the culture but not to the maturation medium increased the morula/blastocyst yield (P = 0.03), but the cleavage rate was not affected. During culture, IGF-I significantly lowered the apoptotic index by decreasing the number of apoptotic cells per embryo and elevated the total cell number of the blastocysts. The frequency of blastocysts with apoptotic cells was not affected. IGF-I increased the proportion of blastocysts with apoptotic cells in the inner cell mass area only by reducing apoptosis in the trophectoderm area. The PCNA index was not affected by IGF-I. A positive correlation observed between apoptotic and PCNA-positive cells was significant in groups stimulated with IGF-I during in vitro culture. Of TUNEL-positive cells, 30%-40% per embryo were also positive for PCNA. This colocalization may indirectly suggest an activation of DNA repair process in TUNEL-positive cells in response to DNA fragmentation. IGF-I reduces apoptosis in bovine IVP embryos. The requirement of IGF-I is more critical during embryo culture than during oocyte maturation. Our data suggest that an assay for TUNEL in conjunction with cell proliferation analysis can provide useful information about the quality of IVP embryos.     

Phage display and structural studies reveal plasticity in substrate specificity of caspase‐3a from zebrafish

The regulation of caspase‐3 enzyme activity is a vital process in cell fate decisions leading to cell differentiation and tissue development or to apoptosis. The zebrafish, Danio rerio, has become an increasingly popular animal model to study several human diseases because of their transparent embryos, short reproductive cycles, and ease of drug administration. While apoptosis is an evolutionarily conserved process in metazoans, little is known about caspases from zebrafish, particularly regarding substrate specificity and allosteric regulation compared to the human caspases. We cloned zebrafish caspase‐3a (casp3a) and examined substrate specificity of the recombinant protein, Casp3a, compared to human caspase‐3 (CASP3) by utilizing M13 bacteriophage substrate libraries that incorporated either random amino acids at P5‐P1′ or aspartate fixed at P1. The results show a preference for the tetrapeptide sequence DNLD for both enzymes, but the P4 position of zebrafish Casp3a also accommodates valine equally well. We determined the structure of zebrafish Casp3a to 2.28Å resolution by X‐ray crystallography, and when combined with molecular dynamics simulations, the results suggest that a limited number of amino acid substitutions near the active site result in plasticity of the S4 sub‐site by increasing flexibility of one active site loop and by affecting hydrogen‐bonding with substrate. The data show that zebrafish Casp3a exhibits a broader substrate portfolio, suggesting overlap with the functions of caspase‐6 in zebrafish development.     

Apoptosis in zebrafish development

Apoptosis (programmed cell death) is important in normal biological processes and in pathogenesis in vertebrates. This review focuses on some of the prominent features of apoptosis during fish development. Caspases and other apoptosis-regulating genes have been cloned from zebrafish (Danio rerio) and other fish species. Elucidation of in vivo functions of apoptosis is focused on development, morphogenesis and sex differentiation. In an attempt to elucidate cause and effect relationships between caspase and development, transgenic zebrafish overexpressing procaspase-3 were generated. Stress-induced apoptosis in zebrafish embryos can be monitored by whole mount TUNEL staining and caspase assay. Thus, zebrafish is a useful experimental model animal for investigation of apoptosis in vivo.     

The perplexed and confused mutations affect distinct stages during the transition from proliferating to post-mitotic cells within the zebrafish retina

To identify and study genes essential for vertebrate retinal development, we are screening zebrafish embryos for mutations that disrupt retinal histogenesis. Key steps in retinogenesis include withdrawal from mitosis by multipotent neuroepithelial cells, specification to particular cell types, migration to the appropriate laminar positions, and molecular and morphological differentiation. In this study, we have identified two recessive mutations that affect the transition of proliferating neuroepithelial cells to postmitotic retinal cells. Both the perplexed and confused mutant phenotypes were initially detectable when the first retinal neuroepithelial cells began to leave the cell cycle. At this time, each mutant retina showed increased cell death and a lack of morphological differentiation. Cell death was found to be apoptotic in both perplexed and confused retinas based on TUNEL analysis and activation of caspase-3. TUNEL-phosphoRb-BrdU colocalization studies indicated that the perplexed mutation caused death in cells transitioning from a proliferative to postmitotic state. For the confused mutation, TUNEL-phosphoRb-BrdU analysis revealed that only a subset of postmitotic cells were induced to activate apoptosis. Mosaic analysis demonstrated that within the retina the perplexed mutation functions noncell-autonomously. Furthermore, whole lens or eye cup transplantations indicated that the retinal defect was intrinsic to the retina. Mosaic analysis with confused embryos showed this mutation acts cell-autonomously. From these studies, we conclude that the perplexed and confused genes are essential at distinct stages during the transition from proliferating to postmitotic cells within the zebrafish retina.     

Apoptosis and in vitro development of preimplantation porcine embryos derived in vitro or by nuclear transfer

Apoptosis occurs during preimplantation development in both in vivo- and in vitro-produced embryos, and it may contribute to embryonic loss. The present study investigated the development of porcine nuclear transfer (NT) embryos reconstructed by using fetal fibroblasts as compared to embryos produced by in vitro fertilization (IVF). The onset and the frequency of apoptosis in NT and IVF embryos were examined via morphological and nuclear changes and TUNEL assay. The NT blastocysts had a similar number of nuclei as compared to IVF blastocysts and appeared to be morphologically similar. Relative to IVF embryos, the NT embryos had a lower cleavage rate (42.7% vs. 71.0%) and a lower developmental rate (11.1% vs. 28.6%) to the blastocyst stage. The earliest positive TUNEL signals were detected in the NT embryos on Day 5 of culture. The percentage of cells undergoing apoptosis in the NT embryos was higher than that of the IVF embryos and increased with time in vitro. Some of the abnormal morphological changes observed during early development related to apoptosis. Cytoplasmic fragmentation, developmental arrest, and nuclear condensation were typical characteristics of embryos undergoing apoptosis. Some mechanisms of the apoptotic pathway were triggered by changes in the NT embryos. The developmental rates of NT embryos might be improved by identifying specific apoptotic pathways and then intervening in these pathways to improve development.     

Quinazolinone exposure induces apoptosis and changes expressions of Fas/FasL and C-Flip in embryonic mice testicles

Quinazolinones represent a class of sedative and anticancer drugs. Quinazolinones-based compounds have ability to suppress prostate tumor growth via apoptosis. Apoptosis is very common in embryos and adults of normal and injured mammalian testes. Effects a new derivative of quinazolinone (4(3H) quinazolinone-2-ethyl-2-phenyl ethyl (QEPE)), on the testis of Balb/C mice embryos were investigated. QEPE was able to reduce number of germ cells and diameter of seminiferous tubules. TUNEL assay analysis indicated that reduction correlated with an increase in the number of apoptotic cell. Furthermore, electron microscope observations confirmed typical apoptotic morphologies characterized by chromatin fragmentation. Finally, RT-PCR analysis showed QEPE increases the levels of Fas/Fasl and decreases C-Flip mRNAs in the testis of exposed embryos.     

Staining apoptosis in paraffin sections. Advantages and limits

OBJECTIVE: To describe the advantages and limits of apoptosis detection on paraffin sections by TdT-mediated dUTP nick end labeling (TUNEL). STUDY DESIGN: Two hundred sixty-five paraffin-embedded samples from malignant and benign human tissue were analyzed by TUNEL. Also, biparametric analysis of apoptosis and proliferation index (MIB-1), apoptosis, cytokeratin or leukocyte common antigen was performed. RESULTS: Our preliminary conclusions are as follows. The limits are that this labelling method might detect cells that have not shown DNA fragmentation specific for apoptosis only. The technique is extremely sensitive to the degree of proteolytic digestion. TUNEL identifies nuclei in areas of necrosis. Indeed, the staining of necrotic areas of tissue with the in situ labelling method should not cause confusion since simple morphologic examination of tissues will suffice to identify areas of necrotic cells. The advantages are that TUNEL is a method of simplifying the identification of apoptotic nuclei in routinely processed tissue sections, maintaining topography. It allows retrospective studies and biparametric analysis of cell death and proliferation on the same sample. Furthermore, with biparametric stain, it could better identify the origin (epithelial, mesenchymal, and so on) of apoptotic cells. CONCLUSION: TUNEL is a good method of detecting apoptotic nuclei in fixed, embedded tissue sections, but, because of the limits of the method, the results should be interpreted in conjunction with apoptosis assessment by routine light microscopy.     

Early detection of staurosporine-induced apoptosis by comet and annexin V assays

Comet, TUNEL, and annexin V assays were used to identify DNA fragmentation and plasma membrane alterations occurring during staurosporine-induced apoptosis in Chinese hamster ovary cells. TUNEL assay detected apoptotic cells after 6 h treatment. The occurrence of annexin V immunofluorescence staining after 1 h treatment confirms that exposure of phosphatidylserine (PS) residues is an early biochemical feature of apoptosis. According to intensity, three annexin staining patterns were distinguished, related to different steps in the apoptotic process. The detection of highly damaged cells by the comet assay after 3 h treatment occurred earlier than the detection of DNA modifications by the TUNEL assay, but later than the exposure of PS residues. However, late apoptotic cells, otherwise characterized by plasma membrane disruption and high annexin V staining, were not detected by the comet assay. In this case, comet assay modified by omitting electrophoresis (halo assay) was more sensitive for an accurate quantification of the apoptotic fraction. Accepted: 2 June 1999     

Analysis of DNA fragmentation of in vitro cultured bovine blastocysts using TUNEL

Programmed cell death (apoptosis) characteristically affects the single cells of blastocysts whereas necrosis affects cluster of cells in both the inner cell mass (ICM) and the trophectoderm (TE). This study uses the trophectodermrminal deoxynucleotidyl transferase (TdT) mediated dUTP nick-end labeling (TUNEL) assay as a way of evaluating the proportion of apoptotic cells and, thus, bovine blastocyst quality during in vitro culture at Days 6,7, and 8. Furthermore, parthenogenetic blastocysts were compared to in vitro fertilized blastocysts at Day 7. Confocal microscopy was used to generate three-dimensional reconstructions of the blastocysts. Apoptosis was observed in both early (Day 6) and late (Day 8) developing blastocysts. The dead cell index (DCI, total number of apoptotic nuclei/total number of nuclei) tend to increase as the in vitro culture time increases, and apoptosis is proportionately higher in the ICM than in the TE. The ratio of ICM to TE cells remains relatively constant even as the blastocysts cell number increases (Day 6 = 11.9 +/- 2.2, Day 7 = 11.2 +/- 0.5, Day 8 = 11.7 +/- 0.4). The overall cell number is significantly reduced in parthenogenetic blastocysts compared to Day 7 in vitro produced blastocysts (P = 0.037). The parthenogenetic blastocysts also show an increase of apoptosis over Day 7 controls. The decrease in cell number in the parthenogenetic blastocysts may be due to the increase of apoptotic nuclei observed. Based on these results we found the TUNEL assay to be a useful method for evaluating in vitro culture conditions of pre-implantation bovine embryos.     

Heat shock-induced apoptosis in preimplantation bovine embryos is a developmentally regulated phenomenon

Apoptosis is a form of cell death that can function to eliminate cells damaged by environmental stress. One stress that can compromise embryonic development is elevated temperature (i.e., heat shock). For the current studies, we hypothesized that heat shock induces apoptosis in bovine embryos in a developmentally regulated manner. Studies were performed to 1) determine whether heat shock can induce apoptosis in preimplantation embryos, 2) test whether heat-induced apoptosis is developmentally regulated, 3) evaluate whether heat shock-induced changes in caspase activity parallel patterns of apoptosis, and 4) ascertain whether exposure to a mild heat shock can protect embryos from heat-induced apoptosis. As determined by TUNEL reaction, exposure of bovine embryos > or =16 cells on Day 5 after insemination to 41 or 42 degrees C for 9 h increased the percentage of cells undergoing apoptosis. In addition, there was a duration-dependent increase in the proportion of blastomeres that were apoptotic when embryos were exposed to temperatures of 40 or 41 degrees C, which are more characteristic of temperatures experienced by heat-stressed cows. Heat shock also increased caspase activity in Day 5 embryos. However, heat shock did not induce apoptosis in 2- or 4-cell embryos, nor did it increase caspase activity in 2-cell embryos. The apoptotic response of 8- to 16-cell-stage bovine embryos to heat shock depended upon the day after insemination that heat shock occurred. When 8- to 16-cell embryos were collected on Day 3 after insemination, heat shock of 41 degrees C for 9 h did not induce apoptosis. In contrast, when 8- to 16-cell embryos were collected on Day 4 after insemination and exposed to heat shock, there was an increase in the percentage of cells undergoing apoptosis. Exposure of 8- to 16-cell embryos at Day 4 to a mild heat shock of 40 degrees C for 80 min blocked the apoptotic response to a subsequent, more-severe heat shock of 41 degrees C for 9 h. In conclusion, apoptosis is a developmentally acquired phenomenon that occurs in embryos exposed to elevated temperature, and it can be prevented by induced thermotolerance.     

Detection of apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling and acridine orange in Drosophila embryos and adult male gonads

In Drosophila, vast numbers of cells undergo apoptosis during normal development. In addition, excessive apoptosis can be induced in response to a variety of stress or injury paradigms, including DNA damage, oxidative stress, nutrient deprivation, unfolded proteins and mechanical tissue damage. Two of the most commonly used methods to label apoptotic cells in Drosophila are terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) for fixed tissues and acridine orange (AO) staining for live embryos or tissues. Here, we describe protocols for labeling apoptotic cells in Drosophila embryos and adult male gonads. Slightly modified protocols can also be applied for other Drosophila tissues. The AO protocol is quick, simple and allows real-time imaging of doomed cells in live tissues. However, it is difficult to combine with conventional counterstains or Ab labeling. On the other hand, this functionality is readily afforded by the TUNEL protocol, which permits the detection of apoptotic cells in fixed tissues. These staining procedures can be completed in 1-2 d.     

Critical evaluation of techniques to detect and measure cell death--study in a model of UV radiation of the leukaemic cell line HL60.

The reliability of eight distinct methods (Giemsa staining, trypan blue exclusion, acridine orange/ethidium bromide (AO/EB) double staining for fluorescence microscopy and flow cytometry, propidium iodide (PI) staining, annexin V assay, TUNEL assay and DNA ladder) for detection and quantification of cell death (apoptosis and necrosis) was evaluated and compared. Each of these methods detects different morphological or biochemical features of these two processes. The comparative analysis of the 8 techniques revealed that AO/EB (read in fluorescence microscopy) provides a reliable method to measure cells in different compartments (or pathways) of cell death though it is very time consuming. PI staining and TUNEL assay were also sensitive in detecting very early signs of apoptosis, but do not allow precise quantification of apoptotic cells. These three methods were concordant in relation to induction of apoptosis and necrosis in HL60 cells with the various UV irradiation time periods tested. Both AO/EB (read by flow cytometry) and annexin V-FITC/PI failed to detect the same number of early apoptotic cells as the other three methods. Trypan blue is valueless for this purpose. Giemsa and DNA ladder might be useful as confirmatory tests in some situations.     

斑马鱼胚胎发育中细胞凋亡的研究进展

细胞凋亡是细胞在基因调控下发生的主动消亡过程,在脊椎动物胚胎发育过程中非常重要。斑马鱼作为一种十分理想的发育分子生物学研究模型,在有关细胞凋亡在诸如形态发生、性别分化等方面功能之活体在位研究中日益受到重视。目前,斑马鱼胚胎发育中主要凋亡通路研究已进行了不少工作,特别是caspase及其它凋亡调控基因在斑马鱼中已被成功克隆,通过转基因斑马鱼胚胎中胁迫诱导细胞凋亡并研究其信号通路以及斑马鱼胚胎形态发生的异常改变,为阐明这些凋亡调控基因与发育之间的关系提供了一个强有力的手段。     

The zebrafish/tumor xenograft angiogenesis assay

Here we describe a method to study tumor angiogenesis in zebrafish (Danio rerio) based on the injection of proangiogenic mammalian tumor cells into the perivitelline space of zebrafish embryos at 48 h post-fertilization. Within 24-48 h, proangiogenic tumor grafts induce a neovascular response originating from the developing subintestinal vessels. This can be observed at macroscopic and microscopic levels after whole-mount alkaline phosphatase staining of wild-type zebrafish embryos, or by fluorescence microscopy in transgenic VEGFR2:G-RCFP embryos in which endothelial cells express the green fluorescent protein under the control of the VEGFR2/KDR promoter. Angiogenesis inhibitors added to the injected cell suspension or to the fish water prevent tumor-induced neovascularization. The assay is rapid and inexpensive, representing a novel tool for investigating tumor angiogenesis and for antiangiogenic drug discovery. Also, gene inactivation by antisense morpholino oligonucleotides injection in zebrafish embryos may allow the identification of genes involved in tumor angiogenesis.     

Late-gestation ventricular myocardial reduction in fetuses of hyperglycemic CD1 mice is associated with increased apoptosis

BACKGROUND : Previous work in our laboratory showed reduced myocardium and dilated ventricular chambers in gestation day (GD) 17 hearts that were collected from hyperglycemic CD1 mouse dams. Pre-breeding maternal immune stimulation, using Freund's complete adjuvant (FCA), diminished the severity of these fetal heart lesions. The following experiments were performed to detect possible changes in fetal heart apoptotic cell death, under hyperglycemic conditions and with or without maternal immune stimulation. METHODS : Female CD1 mice were injected with 200 mg/kg of streptozocin (STZ) to induce insulin-dependent diabetes mellitus. Half of these mice received prior FCA injection. Fetal hearts were collected on GD 17 and myocardial apoptotic cells were quantified using flow cytometry. A panel of apoptosis regulatory genes (Bcl2, p53, Casp3, Casp9, PkCe) was then examined in the fetal myocardium using RT-PCR. RESULTS : Early apoptotic cells and late apoptotic/necrotic cells were significantly increased in fetal hearts from STZ or STZ+FCA dams. Pre-treatment with FCA reduced late apoptotic/necrotic cells to control level, suggesting some cell death protection was rendered by FCA. Paradoxically in the face of such increased cell death, the expression of pro-apoptotic genes Casp3 and Casp9 was decreased by diabetes, while the anti-apoptotic gene Bcl2 was increased. CONCLUSIONS : Maternal hyperglycemia causes dys-regulated apoptosis of fetal myocardial cells. Such effect may be prevented by maternal immune stimulation. Birth Defects Res (Part B) 86:409–415, 2009. © 2009 Wiley-Liss, Inc.     

Cloning of a novel human caspase-9 splice variant containing only the CARD domain

Caspase-9 plays a key role in the intrinsic apoptotic pathway and currently two splice variants (caspase-9-alpha and -beta) have been identified. The present study cloned and characterized a novel caspase-9 splice variant, hereby designated Casp9-gamma. Casp9-gamma is generated from an additional alternative 3' splice site in the fourth exon of caspase-9, resulting in a 58-nucleotide fragment insertion compared with the full-length caspase-9-alpha. The fragment introduces an in-frame stop codon, and the resulting open reading frame (ORF) is preterminated. The Casp9-gamma comprises the deduced 154 amino acid residues containing only the caspase recruitment domain (CARD) and does not contain the large and small subunits. The Casp9-gamma does not promote apoptosis when overexpressed in mammalian cells. Moreover, it inhibits the cleavage of procaspase-3 mediated by proapoptotic member Bax or apoptosis inductor staurosporine. Therefore, Casp9-gamma may function as an endogenous apoptotic inhibitor by interfering with the CARD-CARD interaction between Apaf-1 (apoptotic protease activating factor-1) and procaspase-9. In addition, Casp9-gamma does not enhance NF-kappaB activation in transfected 293T cells, conflicting with previous evidence that the isolated CARD of caspase-9 activates NF-kappaB in ND7 cells. This suggests that the procaspase-9-mediated NF-kappaB activation in response to cellular stresses is cell type-specific through an unidentified mechanism.     

Apoptotic bone cells may be engulfed by osteoclasts during alveolar bone resorption in young rats

The alveolar bone is a suitable in vivo physiological model for the study of apoptosis and interactions of bone cells because it undergoes continuous, rapid and intense resorption/remodelling, during a long period of time, to accommodate the growing tooth germs. The intensity of alveolar bone resorption greatly enhances the chances of observing images of the extremely rapid events of apoptosis of bone cells and also of images of interactions between osteoclasts and osteocytes/osteoblasts/bone lining cells. To find such images, we have therefore examined the alveolar bone of young rats using light microscopy, the TUNEL method for apoptosis, and electron microscopy. Fragments of alveolar bone from young rats were fixed in Bouin and formaldehyde for morphology and for the TUNEL method. Glutaraldehyde-formaldehyde fixed specimens were processed for transmission electron microscopy. Results showed TUNEL positive round/ovoid structures on the bone surface and inside osteocytic lacunae. These structures--also stained by hematoxylin--were therefore interpreted, respectively, as osteoblasts/lining cells and osteocytes undergoing apoptosis. Osteoclasts also exhibited TUNEL positive apoptotic bodies inside large vacuoles; the nuclei of osteoclasts, however, were always TUNEL negative. Ultrathin sections revealed typical apoptotic images--round/ ovoid bodies with dense crescent-like chromatin--on the bone surface, corresponding therefore to apoptotic osteoblasts/lining cells. Osteocytes also showed images compatible with apoptosis. Large osteoclast vacuoles often contained fragmented cellular material. Our results provide further support for the idea that osteoclasts internalize dying bone cells; we were however, unable to find images of osteoclasts in apoptosis.     

Evaluation of viability and apoptosis in horse embryos stored under different conditions at 5 degrees C

The aim of this study was to evaluate the viability (percentage of dead cells) and the incidence of DNA fragmentation of horse embryos after storage in three different media at 5 degrees C for 6 and 24 h. Forty embryos were stored in Emcare Holding Solution for 6 and 24 h, in Hams'F10 or Vigro Holding Plus for 24 h at 5 degrees C (n = 9-10 per group) and 10 embryos were evaluated immediately after collection. First, embryos were stained, immediately after collection or following storage, to detect dead cells (DAPI) and, subsequently, DAPI-stained embryos were fixed and stained to detect DNA fragmentation (TUNEL). Finally, all the fixed embryos were re-stained with DAPI to determine the total number of cells. The percentage of cells stained with both TUNEL and DAPI or TUNEL-only or DAPI-only were determined. The percent of dead cells (DAPI-labelled) per embryo increased with duration of storage, but no differences were detected between the storage media. The percentage of early apoptotic cells (TUNEL+/DAPI-) in fresh and stored embryo for 6 h or 24 h did not differ significantly (P > 0.05). There was a significant correlation between the percentage of cells labelled by TUNEL and DAPI (R = 0.87) (P < 0.001). These results suggest that cooled storage increases cell death but this does not appear to occur by induction of apoptosis and that DAPI staining proves to be a quick and reliable method for assessing embryo viability.     

Dose- and time-dependent effects of TNFalpha and actinomycin D on cell death incidence and embryo growth in mouse blastocysts

This study was undertaken to obtain information about characteristics of different types of induced apoptosis in preimplantation embryos. Freshly isolated mouse blastocysts were cultured in vitro with the addition of two apoptotic inductors--TNFalpha and actinomycin D--at various doses and times. The average number of nuclei and the percentage of dead cells were evaluated in treated embryos. Classification of dead cells was based on morphological assessment of their nuclei evaluated by fluorescence microscopy, the detection of specific DNA degradation (TUNEL assay), the detection of active caspase-3 and cell viability assessed by propidium iodide staining. The addition of both apoptotic inductors into culture media significantly increased cell death incidence in blastocysts. Their effects were dose and time dependent. Lower concentrations of inductors increased cell death incidence, usually without affecting embryo growth after 24 h culture. Higher concentrations of inductors caused wider cell damage and also retarded embryo development. In all experiments, the negative effect of actinomycin D on blastomere survival and blastocyst growth was greater than the effect of TNFalpha. Furthermore, the addition of actinomycin D into culture media increased cell death incidence even after 6 h culture. Differences resulted probably from diverse specificity of apoptotic inductors. The majority of dead cells in treated blastocysts were of apoptotic origin. Morphological and biochemical features of apoptotic cell death induced by both TNFalpha and actinomycin D were similar and had homologous profile. In blastomeres, similarly to somatic cells, the biochemical pathways of induced apoptosis included activation of caspase-3 and internucleosomal DNA fragmentation.     

Cell-specific localisation of apoptosis in the bovine ovary at different stages of the oestrous cycle

Apoptosis was localized in all ovarian cell types of 23 cows in various stages of the oestrous cycle, using the detection of active caspase-3, in situ end labelling (TUNEL) and DNA fluorescent staining (DAPI). Very few apoptotic cells were found in primordial, primary, secondary and vital tertiary follicles. In contrast, apoptosis in atretic tertiary follicles was much more frequent, and high apoptotic scores were recorded when using the TUNEL technique and lower scores with the caspase-3 assay. Cystic atretic follicles showed in general a higher apoptotic score than obliterative atretic follicles, with intermediate to high scores in granulosa cells and lower scores in theca cells. In corpora lutea, large and small lutein cells had intermediate to high scores using the caspase-3 assay, and intermediate to low scores using the TUNEL assay. Irrespective of the detection method, the scores were higher in lutein cells than in the capsular stroma cells. In all ovarian structures examined, variations in apoptotic scores were seen in the different cycle stages, suggesting a cycle-dependent influence on apoptosis, although correlations with plasma progesterone concentrations were low.