Identification of potassium-dependent and -independent components of the apoptotic machinery in mouse ovarian germ cells and granulosa cells

Recent studies with thymocytes have suggested a critical role for intracellular potassium in the regulation of apoptosis. In this study, we examined the pathways of K(+) regulation during ovarian cell death. In initial studies, fluorographic analysis demonstrated a significant loss of K(+) during apoptosis stimulated by doxorubicin in oocytes and trophic hormone deprivation in granulosa cells. In oocytes, suppression of potassium efflux by potassium-enriched medium prevented condensation, budding, and fragmentation, although it did not block DNA degradation, suggesting the existence of potassium-independent nucleases in oocytes. Culture of granulosa cells in potassium-enriched medium inhibited internucleosomal DNA cleavage, although high-molecular weight DNA cleavage was apparent, suggesting that the nuclease or nucleases responsible for generating 50-kilobase (kb) fragments in these cells is potassium independent. To address this directly, isolated granulosa cell nuclei were stimulated to autodigest their DNA, and internucleosomal, but not large-fragment, cleavage was completely blocked by 150 mM potassium. We next examined whether the proapoptotic caspases are targets for potassium regulation. In cell-free assays, processing of pro-interleukin-1beta and proteolysis of cellular actin by recombinant caspase-1 and caspase-3, respectively, were suppressed by the presence of 150 mM potassium. Other monovalent ions (NaCl, LiCl) exerted a similar effect in these cell-free assays. Thus, in oocytes and granulosa cells, potassium efflux appears to occur early in the cell death program and may regulate a number of apoptotic events including caspase activity and internucleosomal DNA cleavage. However, there also exist novel potassium-independent pathways in both ovarian germ cells and somatic cells that signal certain apoptotic events, such as large-fragment DNA cleavage.     

Epidermal growth factor and basic fibroblast growth factor suppress the spontaneous onset of apoptosis in cultured rat ovarian granulosa cells and follicles by a tyrosine kinase-dependent mechanism.

Recent biochemical studies have suggested that apoptotic cell death is the molecular mechanism underlying the degeneration of ovarian follicles during atresia. Using a sensitive autoradiographic method for the detection of DNA fragmentation, we studied apoptosis in ovarian granulosa cells or intact follicles placed in serum-free culture as model systems to elucidate the hormonal regulation of atresia. Immature rats (25 days old) were primed for 2 days with 10 IU equine CG to induce a homogeneous population of mature preovulatory follicles. Granulosa cells isolated from these follicles contained predominantly intact high mol wt DNA. However, a time-dependent, spontaneous onset of internucleosomal DNA fragmentation characteristic of apoptotic cell death occurred in granulosa cells during culture. Treatment of granulosa cells with epidermal growth factor (EGF), transforming growth factor-alpha (TGF alpha), or basic fibroblast growth factor (bFGF) inhibited the spontaneous onset of apoptotic DNA cleavage found during culture by 40-60%. In contrast, insulin-like growth factor I, insulin, TGF beta and tumor necrosis factor-alpha were ineffective. Likewise, activation of the protein kinase A or C pathways with forskolin or phorbol 12-myristate 13-acetate, respectively, did not prevent the onset of DNA fragmentation, although inclusion of a tyrosine kinase inhibitor (genistein) completely blocked the ability of EGF, TGF alpha, and bFGF to suppress apoptosis in granulosa cells. Similar to cultured granulosa cells, a spontaneous onset of apoptosis was also observed to occur in isolated preovulatory follicles during culture. Furthermore, treatment of follicles with EGF or bFGF inhibited the spontaneous initiation of apoptosis, and the suppressive effects of these growth factors were also attenuated by co-treatment with genistein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Multiparametric study of atresia in ewe antral follicles: histology, flow cytometry, internucleosomal DNA fragmentation, and lysosomal enzyme activities in granulosa cells and follicular fluid

The differential quantitative participation of apoptosis and necrosis in ewe antral follicles of two different sizes, separated in four stages of atresia using macroscopic, histologic, and esteroid quantification methods was assessed. Annexin V binding and propidium iodide (PI) uptake was used to detect healthy live cells (Annexin V negative/PI negative), early apoptotic cells (Annexin V+/PI-), and necrotic or late apoptotic cells (PI+). Additionally we used internucleosomal DNA fragmentation as a quantitative estimate of apoptosis. Presence and distribution of lysosomal enzymes in follicular fluid and granulosa cells was used as a measure of necrotic cell death. DNA flow cytometry and gel electrophoresis were positively correlated with the progression of atresia, small atretic follicles tend to have higher percentages of internucleosomal cleaved DNA than follicles >6 mm. Annexin/PI binding also indicates that apoptosis and necrosis increase with atresia progression, generally apoptosis outweighs necrosis in small follicles. Acid phosphatase and glucosaminidase in follicular fluid of 3-6 mm follicles showed no significant modifications between healthy and initially atretic follicles, and only a small, but significant increase in activity in advancedly atretic follicles. On the contrary, lysosomal enzyme activity in follicles >6 mm showed positive correlation between atresia stages and the activities of acid phosphatase and glucosaminidase in follicular fluid. A similar size-differential behavior was found in free or membrane-bound lysosomal enzyme activity of granulosa cells. Necrosis, but principally apoptosis, were present during all stages of follicular maturation indicating that growth and maturation of ovarian follicles involves a continuous renewal of granulosa cells, regulated by apoptosis. Mechanisms regulating this equilibrium may participate in the final destiny, whether ovulation or atresia of ovarian follicles.     

Apoptosis in human monocytic THP.1 cells involves several distinct targets of N-tosyl-L-phenylalanyl chloromethyl ketone (TPCK)

N-Tosyl-L-phenylalanyl chloromethyl ketone (TPCK), a chymotrypsin-like serine protease inhibitor, affected apoptosis in human monocytic THP.1 cells differently dependent on both the concentration used and the apoptotic stimulus. TPCK (50 - 75 microM) induced both biochemical and ultrastructural changes characteristic of apoptosis, including proteolysis of poly (ADP-ribose) polymerase (PARP) and lamins together with formation of large kilobase pair fragments of DNA, particularly of 30 - 50 and 200 - 300 kilobase pairs in length but without internucleosomal cleavage of DNA. The induction of apoptosis by TPCK also involved the processing of CPP32 and Mch 3 to their catalytically active subunits. Benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethyl ketone (Z-VAD.FMK), an ICE-like protease inhibitor, completely prevented all the biochemical and morphological changes induced by TPCK demonstrating the involvement of ICE-like proteases in the execution phase of apoptosis. Lower concentrations of TPCK (5 - 20 microM) prevented internucleosomal cleavage of DNA induced by other apoptotic stimuli. TPCK (10 microM) inhibited cell death induced by etoposide but potentiated that induced by cycloheximide demonstrating that it differentially affected apoptosis in THP.1 cells dependent on the stimulus used. These results are consistent with at least three distinct TPCK targets, one being important for cell survival, the second in facilitating internucleosomal cleavage of DNA and the third in the modulation of apoptosis induced by different apoptotic stimuli.     

Morphological and biochemical identification of apoptosis in small, medium, and large bovine follicles and the effects of follicle-stimulating hormone and insulin-like growth factor-I on spontaneous apoptosis in cultured bovine granulosa cells

The first objective of this study was to determine whether the death of bovine granulosa cells (GC) isolated from small ( 8 mm) follicles during follicular atresia occurs by apoptosis. The second objective was to establish an in vitro model system to elucidate the developmental (GC from follicles of different sizes) and hormonal (FSH and insulin-like growth factor-I [IGF-I]) regulation of bovine GC apoptosis during follicular atresia. Bovine ovaries were obtained from a nearby slaughterhouse. Follicles were classified by morphometric criteria as healthy or atretic. Apoptosis in GC from follicles of different sizes was analyzed by both morphological and biochemical methods. Bovine GC were cultured for 48 h at a density of 5 x 10(6) cells/ml in serum-free media at 39 degrees C to determine the effects of FSH and IGF-I on apoptosis. The results showed that apoptosis occurred in GC from all sizes of follicles. Apoptosis in GC was also detected in some healthy follicles. Degenerate GC displayed the morphological characteristics of apoptosis, including nuclei with marginated chromatin, a single condensed nucleus, multiple nuclear fragments, and/or membrane-bound structures containing variable amounts of chromatin and/or cytoplasm (apoptotic bodies). All GC classified as apoptotic on the basis of their morphology contained fragmented DNA measured by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) technique. Cells that had undergone apoptosis were observed mainly in GC and in scattered theca cells. Throughout the GC layer, apoptotic cell death was more prevalent among antral GC than among mural GC. Interestingly, morphological results showed that no apoptosis occurred in cumulus cells. A time-dependent, spontaneous onset of apoptosis occurred in GC from small, medium, and large follicles during in vitro serum-free culture. The rate of DNA fragmentation in the culture of GC from small follicles was higher than that from medium and large follicles. FSH attenuated apoptotic cell death in GC from medium follicles more effectively than in those from small follicles. IGF-I also suppressed apoptosis in cultured GC from small follicles. In conclusion, this study showed that 1) GC death during bovine follicular development and atresia occurs by apoptosis; 2) apoptosis occurs in GC and theca cells; however, apoptosis does not occur in cumulus cells even in atretic antral follicles; 3) GC from all small, medium, and large follicles undergo spontaneous onset of apoptosis when cultured under serum-free conditions; and 4) FSH and IGF-I can attenuate apoptosis in cultured bovine GC.     

Arachidonic acid promotes glutamate-induced cell death associated with necrosis by 12- lipoxygenase activation in glioma cells

Glutamate induced glutathione (GSH) depletion in C6 rat glioma cells, which resulted in cell death. This cell death seemed to be apoptosis through accumulation of reactive oxygen species (ROS) or hydroperoxides representing cytochrome c release from mitochondria and internucleosomal DNA fragmentation. A significant increase of 12-lipoxygenase enzyme activity was observed in the presence of arachidonic acid (AA) under GSH depletion induced by glutamate. AA promoted the glutamate-induced cell death, which reduced caspase-3 activity and diminished internucleosomal DNA fragmentation. Furthermore, AA reduced intracellular NAD, ATP and membrane potentials, which indicated dysfunction of the mitochondrial membrane. Protease inhibitors such as N-alpha-tosyl-L-phenylalanine chloromethyl ketone (TPCK) and 3, 4-dichloroisocumarin (DCI) but no Ac-DEVD, a caspase inhibitor, suppressed the glutamate-induced cell death. AA reduced the inhibitory effect of TPCK and DCI on the glutamate-induced cell death. These results suggest that AA promotes cell death by inducing necrosis from caspase-3-independent apoptosis. This might occur through lipid peroxidation initiated by ROS or lipid hydroperoxides generated during GSH depletion in C6 cells.     

Progesterone receptor-mediated inhibition of apoptosis in granulosa cells isolated from rats treated with human chorionic gonadotropin

Almost all ovarian follicles undergo atresia during follicular development. However, the number of corpora lutea roughly equals the number of preovulatory follicles in the ovary. Because apoptosis is the cellular mechanism behind follicle and luteal cell demise, this suggests a change in apoptosis susceptibility during the periovulatory period. Sex steroids are important regulators of follicular cell survival and apoptosis. The aim of the present work was to study the role of progesterone receptor-mediated effects in the regulation of granulosa cell apoptosis. The levels of internucleosomal DNA fragmentation were evaluated in rat granulosa cells before and after induction of the nuclear progesterone receptor, using hCG treatment to eCG-primed rats to mimic the naturally occurring LH surge. Granulosa cells isolated from hCG-treated rats showed a several-fold increase in the expression of progesterone receptor mRNA and a 47% decrease (P < 0.01) in DNA fragmentation after 24 h incubation in serum-free medium compared to granulosa cells isolated from rats treated with eCG only. The effect of hCG treatment in vivo was dose-dependently reversed in vitro by addition of antiprogestins (Org 31710 or RU 486) to the culture medium, demonstrated by increased DNA fragmentation as well as increased caspase-3 activity. Addition of antiprogestins to granulosa cells isolated from immature or eCG-treated rats did not result in increased DNA fragmentation. The results suggest that progesterone receptor-mediated effects are involved in regulating the susceptibility to apoptosis in LH receptor-stimulated preovulatory rat granulosa cells.     

Identification and characterization of glucocorticoid-regulated nuclease(s) in lymphoid cells undergoing apoptosis

Apoptosis is a physiological process by which selected cells are deleted from a population in response to specific regulatory signals. A hallmark of apoptosis is the internucleosomal degradation of DNA prior to cell death. We are studying glucocorticoid-induced lymphocytolysis as a model system for apoptosis within the immune system. In rat thymocytes, the internucleosomal DNA cleavage which occurs following glucocorticoid treatment is both time- and dose-dependent, and is blocked by the glucocorticoid antagonist RU 486, indicating that this effect is mediated by the glucocorticoid receptor. Similar experiments using glucocorticoid-responsive (wt) and glucocorticoid-resistant (nt⁻) S49.1 lymphoma cell lines confirm that internucleosomal DNA degradation and cell death are glucocorticoid receptor-mediated events and thus reflect the direct effects of glucocorticoids on lymphocytes. In an effort to identify the nuclease(s) responsible for the DNA degradation, we have developed two assays to detect nucleases whose activity is altered by glucocorticoid treatment. The first assay involves electrophoresing extracts of nuclear protein from control and glucocorticoid-treated lymphoid cells into SDS-polyacrylamide gels containing [³²P]DNA within the gel matrix. This assay is used to estimate the molecular mass of the nuclease, based on the observed in situ nuclease activity. The second assay uses HeLa nuclei as a substrate to detect internucleosomal cleavage activity present in nuclear extracts of control and glucocorticoid-treated lymphoid cells. Using these assays we have identified a novel Ca²⁺, Mg²⁺-dependent nuclease with an apparent molecular weight of 18 kDa in both S49 wt cells and rat thymocytes treated with glucocorticoids. Furthermore, nuclear extracts of glucocorticoid-treated, but not control, rat thymocytes and S49 wt cells were capable of cleaving HeLa chromatin at internucleosomal sites. In an effort to determine the identity of the nuclease capable of internucleosomal cleavage of DNA, nuclear extracts from dex-treated rat thymocytes were fractionated by gel filtration chromatography under non-denaturing conditions, and the fractions were analyzed using the [³²P]DNA SDS-PAGE and HeLa nuclei assays. When analyzed under native conditions, the 18 kDa nuclease described previously appears to exist as a 25 kDa protein which may be part of a high molecular weight complex. Interestingly, only the 25 kDa form of the protein was associated with internucleosomal DNA cleavage activity where as the high molecular weight form of the enzyme was devoid of this activity.     

Changes in expression of anti-apoptotic protein, cFLIP, in granulosa cells during follicular atresia in porcine ovaries

Follicular selection is performed in mammalian ovaries, as most follicles undergo atresia during follicular development and growth. Follicular regression is indicated to begin with granulosa cell apoptosis. To reveal the molecular mechanisms of the selection, we examined the changes in the levels of cellular-Flice like inhibitory protein (cFLIP) expression in porcine granulosa cells. cFLIP is the homologue of intracellular apoptosis inducer (procaspase-8/Flice), and has two alternative splicing isoforms: cFLIP short form (cFLIP(S)) and long form (cFLIP(L)). By competing with caspase-8, cFLIP inhibits apoptosis initiated by death receptors. The changes in the levels of cFLIP(S) and cFLIP(L) mRNA and protein expression in granulosa cells were determined by RT-PCR and Western blotting, respectively. cFLIP(L) mRNA and protein were highly expressed in granulosa cells of healthy follicles and decreased during atresia. cFLIP(S) mRNA levels in granulosa cells were low and showed no change among the stages of follicular development, and its protein level was extremely low. We examined the changes in the localization of cFLIP mRNAs in pig ovaries by in situ hybridization and found that cFLIP(L) is abundant in granulosa cells of healthy follicles in comparison with those of atretic follicles. Immunohistochemical analyses demonstrated that the cFLIP protein is highly expressed in the granulosa cell of healthy follicles but weakly expressed in that of atretic follicles. We presumed that cFLIP, especially cFLIP(L), plays an anti-apoptotic role in the granulosa cells of healthy follicles of pig ovaries, and that cFLIP could be a major survival factor that determines whether growth or atresia occurs in porcine follicles.     

Pro- and Anti-Apoptotic Effects of an Inhibitor of Chymotrypsin-Like Serine Proteases

The irreversible inhibitor of chymotrypsin-like serine proteases, N-tosyl –L-phenylalanine chloromethylketone (TPCK), was shown to prevent internucleosomal DNA cleavage caused by inducers of apoptosis. The pro-apoptotic properties of TPCK have been studied less thoroughly. The aim of the present study was to investigate the pro- and anti-apoptotic activities of TPCK on HL-60 cells and compare them with the actions of the mitochondrial electron transport inhibitor antimycin A (AMA). The results showed that TPCK alone caused activation of cell cycle checkpoints, mitochondrial cytochrome c release, caspase-3 activation, and chromatin condensation. Caspase-8 was not required for cytochrome c release but was crucial to caspase-3 activation. TPCK synergistically enhanced AMA-induced cytochrome c release and caspase-3 activation while completely blocking AMA-induced internucleosomal DNA fragmentation for at least 8 hours. Rather than blocking AMA-induced DNA fragmentation, the general serine protease inhibitor 4-(2-aminoethyl)-benzenesulphonyl fluoride (AEBSF) actually enhanced it. The pro-apoptotic effect of TPCK may be due to activation of cell cycle checkpoints via inhibition of the proteasome. The apoptotic pathways activated by TPCK and AMA probably converge at the level of the mitochondria. The mode by which TPCK prevents internucleosomal DNA fragmentation is probably not through serine protease inhibition.     

Coordinate involvement of cysteine protease and nuclease in the executive phase of plant apoptosis

We have developed an oat cell-free apoptosis system to investigate the execution mechanisms of plant apoptosis. Cell extracts derived from oat tissues undergoing toxin (victorin)-induced apoptosis caused nuclear collapse and internucleosomal DNA fragmentation in isolated nuclei. Pharmacological studies revealed that cysteine protease, which is E-64-sensitive but insensitive to caspase-specific inhibitors, is a crucial component in the morphological change of isolated nuclei, and that nuclease and the cysteine protease act cooperatively to induce the apoptotic DNA laddering. Interestingly, this finding is contrasted with those in well-studied animal cell-free systems in which an apoptotic endonuclease is solely responsible for the DNA fragmentation.     

Action of apoptotic endonuclease DNase gamma on naked DNA and chromatin substrates

The internucleosomal cleavage of genomic DNA is a biochemical hallmark of apoptosis. DNase gamma, a Mg2+/Ca2+-dependent endonuclease, has been suggested to be one of the apoptotic endonucleases, but its biochemical characteristic has not been fully elucidated. Here, using recombinant DNase gamma, we showed that DNase gamma is a Mg2+/Ca2+-dependent single-stranded DNA nickase and has a high activity at low ionic strength. Under higher ionic strength, such as physiological buffer conditions, the endonuclease activity of DNase gamma is restricted, but its activity is enhanced in the presence of linker histone H1, which explains DNA cleavage at linker regions of apoptotic nuclei.     

Activation of internucleosomal DNA cleavage in human CEM lymphocytes by glucocorticoid and novobiocin. Evidence for a non-Ca2(+)-requiring mechanism(s)

Internucleosomal DNA cleavage is the key molecular event of the cytolytic phase of glucocorticoid-induced lymphocytolysis. We find that novobiocin, the topoisomerase II inhibitor, is a potent inducer of in vivo internucleosomal DNA cleavage in human CEM lymphocytes. This in vivo effect is very rapid, time- and dose-dependent, requires cellular integrity, and does not require de novo protein synthesis. Recently our data (Alnemri, E. S., and Litwack, G. (1989) J. Biol. Chem. 264, 4104-4111) suggested that activation of DNA cleavage in CEM-C7 lymphocytes by glucocorticoids is independent of calcium uptake. Similarly, the novobiocin effect is also independent of calcium uptake and does not occur in isolated CEM nuclei or in CEM cells treated previously with the divalent cation ionophore A23187. Internucleosomal DNA cleavage induced by novobiocin or glucocorticoid generates blunt-ended double-stranded DNA fragments possessing 3'-hydroxyls and 5'-phosphates. As demonstrated by gel retardation analysis and DNase I footprinting, novobiocin causes the disruption and unfolding of an in vitro reconstituted mononucleosome so that it becomes more susceptible to DNase I cleavage. Our data suggest that 1) novobiocin rapid activation of internucleosomal DNA cleavage and chromatin changes in CEM lymphocytes are molecular features of apoptosis or programmed cell death. 2) CEM lymphocytes apparently do not express a Ca2(+)-dependent endonuclease. 3) The mechanism(s) of glucocorticoid or novobiocin-induced DNA cleavage in CEM lymphocytes involves activation of a constitutive non Ca2(+)-dependent endonuclease. We propose that the majority of nuclear chromatin is maintained in a highly compact and charge-neutralized state and that disruption of this highly ordered structure, directly by novobiocin or indirectly by glucocorticoid, may lead to the exposure and unmasking of internucleosomal linker DNA regions which are substrates for a constitutive non-Ca2(+)-dependent endonuclease.     

Age-associated up-regulation of EGR1 promotes granulosa cell apoptosis during follicle atresia in mice through the NF-κB pathway

Follicular atresia is the main process responsible for the loss of follicles and oocytes from the ovary, and it is the root cause of ovarian aging. Apoptosis of granulosa cells (GCs) is the cellular mechanism responsible for follicular atresia in mammals. Recent advances have highlighted fundamental roles for EGR1 in age-related diseases via the induction of apoptosis. In the present study, we found that the expression of EGR1 was significantly increased in aged mouse ovaries compared with young ovaries. Immunohistochemical analysis revealed strongly positive EGR1 staining in atretic follicles, especially in apoptotic granulosa cells. We further showed that EGR1 up-regulation in mouse primary granulosa cells inhibited cell proliferation and promoted apoptosis. In addition, the promotion of apoptosis in GCs by EGR1 increases over time and with reactive oxygen species (ROS) stimulation. Our mechanistic study suggested that EGR1 regulates GC apoptosis in a mitochondria-dependent manner and that this mainly occurs through the NF-κB signaling pathway. In conclusion, our results suggested that age-related up-regulation of EGR1 promotes GC apoptosis in follicle atresia during ovarian aging.     

Determination of onset of apoptosis in granulosa cells of the preovulatory follicles in the bonnet monkey (Macaca radiata): correlation with mitogen-activated protein kinase activities

During reproductive life, only a selected few ovarian follicles mature and ovulate, while the vast majority of follicles undergo a degenerative process called atresia. Recent studies have indicated that follicular atresia is mediated through apoptosis of follicular granulosa cells. The objectives of the present study were to determine the time of onset of apoptosis in granulosa cells of preovulatory follicles and to evaluate the consequences of gonadotropin withdrawal on mitogen-activated protein (MAP) kinase activities. Bonnet monkeys (Macaca radiata) undergoing controlled ovarian stimulation cycles were utilized for stimulation of multiple follicles, and granulosa cells were retrieved from preovulatory follicles at 24, 48, 72, and 96 h after stopping gonadotropin treatment. Serum and follicular fluid estradiol concentrations were highest at 24 h but declined precipitously (P < 0.05) to reach the lowest concentrations at 96 h; however, progesterone concentrations during this period did not increase, indicating the absence of luteinization. Quantitative analysis of genomic DNA by 3'-end labeling revealed the presence of low-molecular-weight fragments from 48 h onward, but by agarose gel electrophoresis, DNA laddering could be visualized only after 72 h. Messenger RNA expression for Bax, caspase-2, and caspase-3 increased with the onset of apoptosis. Immunoblot analysis of MAP kinases in lysates of granulosa cells (48-72 h) indicated increased (P < 0.05) levels of phosphorylated extracellular response kinase-1 and -2, Jun N-terminal kinase (JNK)-1 and -2, and p38. However, in vitro kinase assay data indicated that only phospho-JNK and -p38 activities were higher at 72 h compared to 24 h. These results demonstrate that granulosa cells of preovulatory follicles undergo apoptosis and that increased activities of phospho-JNK and -p38 are correlated with apoptosis in the primate.     

Apoptosis in granulose cells induced by intrafollicular peptide

Ovarian follicular fluid peptide (OFFP) purified from sheep ovaries has been earlier shown to induce degeneration of ovarian follicles in mice. In the present study, whether the effect of OFFP on granulosa cells was similar to apoptosis was studied using three parameters. Immature mice injected with pregnant mare serum gonadotropin on day 0 were administered with 10 or 20 μg of OFFP on day 1 and autopsied on day 2. The granulosa cells were collected from the ovarian follicles. The presence of apoptotic bodies were observed by staining the cells with acridine orange. DNA profiles of DAPI-stained cells analysed by flow cytometry also revealed apoptotic response to OFFP. Furthermore, agarose gel electrophoresis of low molecular weight DNA fraction extracted from the cells of OFFP-treated animals confirmed ladder formation and induction of apoptosis and not necrosis in granulosa cells. In conclusion, all the three parameters indicated apoptotic changes in granulosa cells of ovarian follicles in .mice treated with OFFP. The effect of OFFP seems to be exerted directly on the granulosa cells showing its autocrine role in the process of follicular atresia. This is discussed in the light of other intra/extra ovarian factors.     

Cellular Events in Fas/APO-1-Mediated Apoptosis in JURKAT T Lymphocytes

In the present study we investigated the Fas-mediated cellular events using the human leukemic T cell line, JURKAT. Ligation of the Fas receptor with a monoclonal antibody (IgM) resulted in the rapid (within 3 h) induction of apoptosis and was characterized by a sequence of distinct morphological and biochemical events. Thus, plasma membrane blebbing, condensation of the chromatin, and formation of high-molecular-weight (HMW) DNA fragments were the earliest events observed (by 45 min). They were followed by cleavage of DNA into oligonucleosomal-length fragments (laddering pattern) and the formation of apoptotic bodies, and finally, rounding of the apoptotic cells and complete cleavage of DNA into oligonucleosomal-length fragments occurred. The mitochondria remained structurally intact up to the stage of oligonucleosomal-length DNA cleavage, and the ability of the cells to exclude trypan blue was not compromised throughout the time course of the experiments. In contrast to many other model systems, apoptosis in JURKAT cells after anti-Fas treatment did not require the presence of extracellular Ca²⁺ or Mg²⁺ and was only partially inhibited by Zn²⁺. In addition, Fas-mediated apoptosis was unaffected by the presence of free radical scavengers or inhibitors of protein phosphatases, protein kinases, and nitric oxide synthesis. However, the serine protease inhibitors, N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) and 3,4-dichloroisocoumarin (DCI) prevented anti-Fas-induced apoptosis in JURKAT cells. Low concentrations of these inhibitors blocked oligonucleosomal-length, but not HMW, DNA fragmentation. The latter required a higher concentration of TPCK or DCI to block. In addition, low concentrations of DCI also prevented Fas-mediated plasma membrane blebbing. In summary, our results suggest that proteolysis plays a central role in Fas-mediated apoptosis and that distinct proteolytic enzymes are involved in HMW DNA fragmentation, and oligonucleosomal-length DNA fragmentation, as well as in plasma membrane blebbing.     

Characterization of apoptosis-like endonuclease activity in avian thymocytes.

In the current study the internucleosomal DNA cleavage activity associated with apoptosis was investigated in avian thymocytes. Thymocyte nuclear proteins from glucocorticoid-treated chickens were incubated with chicken red blood cell (cRBC) nuclei, and DNA degradation was analyzed by agarose gel electrophoresis and fluorescence-activated flow cytometry. The thymocyte nuclear extract contained an endonuclease activity that degraded cRBC chromatin at internucleosomal sites as detected by agarose gel electrophoresis. Flow cytometry analysis of cRBC nuclei that were treated with thymocyte nuclear proteins demonstrated a loss of cellular DNA as a function of the amount of added nuclease activity. Furthermore, it was demonstrated that the thymocyte nuclear extract contained a nuclease activity that was capable of degrading radiolabelled naked 32P-DNA into acid soluble DNA fragments. All three assay methods demonstrate that the thymocyte nuclease activity can be inhibited by EDTA, zinc ions and the nuclease inhibitor aurintricarboxylic acid. Based on the analysis of cofactor requirement of this nuclease activity and its susceptibility to inhibitors, the endonuclease activity present in avian apoptotic thymocytes appears to be identical to the mammalian counterpart.