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Apoptosis Jalal Ghasemzadeh Andrology lab Yazd Reproductive Sciences Institute.

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Presentation on theme: "Apoptosis Jalal Ghasemzadeh Andrology lab Yazd Reproductive Sciences Institute."— Presentation transcript:

1 Apoptosis Jalal Ghasemzadeh Andrology lab Yazd Reproductive Sciences Institute

2 بسم الله الرحمن الرحیم

3 In humans, the rate of cell growth and cell death is balanced to maintain the weight of the body.

4 Life cannot exist without cellular death

5 Cell death can occur via several processes ( about 11 type ) : 1. Apoptosis 2. Necrosis 3. Autophagy 4. Entosis 5. Oncosis 6. Pyroptosis. 11.

6 Apoptosis = “normal” or “programmed” cell death Necrosis = “accidental” or “ordinary” cell death

7  The word ‘‘apoptosis’’ comes from the ancient Greek, meaning the: ‘‘falling of petals from a flower’’ or ‘‘of leaves from a tree in autumn’’  The term apoptosis ( a-po-toe-sis ) was first used in a now-classic paper by Kerr et al 1972 to describe a morphologically distinct form of cell death.

8 Apoptosis or programmed cell death ( PCD ) is a mode of cell death that occurs under normal physiological conditions and the cell is an active participant in its own demise ( “cellular suicide” ). It is important for the development of multicellular organism ( embryonic development ) and homeostasis of their tissues ( adult ).

9 Apoptosis is a beneficial and important phenomenon:  In embryo 1. During embryonic development, help to digit formation. Lack of apoptosis in humans can lead to webbed fingers called “ syndactyly ”.

10 2. Normal event in development of the nervous system

11 Apoptosis is a beneficial and important phenomenon:  In adult Normal cell turn over Tissue homeostasis Induction and maintenance of immune tolerance Development of the nervous system Endocrine-dependent tissue atrophy Elimination of activated, damaged and abnormal cells

12 Apoptosis is a beneficial and important phenomenon:  In animals Embryonic Chicken Foot Embryonic Mouse Paw Tail absorption of the tadpole

13 Apoptosis is the physiological cell death which unwanted or useless cells are eliminated during development and other normal biological processes. Necrosis is the pathological cell death which occurs when cells are exposed to a serious physical or chemical insult (hypoxia, hyperthermia, ischemia).

14 There are many observable morphological and biochemical differences between necrosis and apoptosis:  Morphological features Necrotic cells Apoptotic cells  Volume enlargement  Volume reduction  Swelling of cytoplasm  Shrinking of cytoplasm & mitochondria  No loss of membrane integrity  Loss of membrane integrity  No vesicle formation  Formation of apoptotic bodies  Condensation of chromatin & DNA fragmentation

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16  Biochemical features Necrotic cells  Loss of regulation of ion homeostasis  No energy requirement ( passive process, also occurs at 4 °C ) Apoptotic cells  Tightly regulated process  Energy ( ATP ) - dependent ( active process, doesn’t occur at 4 °C )  Release of various factors into cytosol by mitochondria  Activation of caspase cascade

17  Physiological significance Necrosis  Affects groups of contiguous cells  Evoked by non-physiological disturbances (lytic viruses, hypoxia)  Phagocytosis by macrophages  Significant inflammatory response Apoptosis  Affects single cells or small clusters of cells  Induced by physiological stimuli(lack of growth factors, DNA damage)  Rapidly phagocytized by adjacent epithelial cells or macrophages  No inflammatory response

18  There is essentially no inflammatory reaction associated with the process of apoptosis nor with the removal of apoptotic cells because:  Apoptotic cells do not release their cellular constituents into the surrounding interstitial tissue.  They are quickly phagocytized by surrounding cells thus likely preventing secondary necrosis.  The engulfing cells do not produce anti-inflammatory cytokines.

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20 Apoptotic cells -Eat me signals

21  Takes about 30 - 60 min.  In the tissue only about 5% cells is affected by the PCD.  Physiological changes and phagocytosis is very fast.

22 APOPTOSIS

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25 Caspases = Cysteinyl aspartate specific proteases A family of intracellular cysteine proteases that play a pivotal role in the initiation and execution of apoptosis. At least 14 different members of caspases in mammalian cells have been identified All are synthesized as inactive proenzymes ( zymogen ) with 32-56 kDa

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28 “ Death receptors” that are members of the tumor necrosis factor (TNF) receptor superfamily. Death receptors have a cytoplasmic domain of about 80 amino acids called the “death domain”. This death domain plays a critical role in transmitting the death signal from the cell surface to the intracellular signaling pathways.

29  The best characterized receptors & ligands corresponding death include : FasR (CD95/APO1) FasL DR3 Apo3L DR4 (TRAIL-R1) Apo2L DR5 (TRAIL-R2) Apo2L TNFR1 TNF-α TNFR2 TNF-ß Ligands Receptors

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31  Binding of trimeric FasL to Fas  Trimerization and clustering of Fas  Recruitment of Fas-associated death domain (FADD) to Fas  Recruitment of caspase-8 to FADD  Formation of Death-Inducing Signaling Complex (DISC )  Activation of caspase-8 (autoactivation)  Activation of effector caspases  Apoptosis

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33 The stimuli that initiate the intrinsic pathway produce intracellular signals such as radiation (DNA damage), absence of certain growth factors, hormones and cytokines. All of these stimuli cause changes in the mitochondrial outer membrane permeabilization (MOMP) Release of pro-apoptotic proteins such as cytochrome c, Smac/DIABLO, AIF, endonuclease G and CAD from the inter-membrane space into the cytosol. Cytochrome c binds and activates Apaf-1 as well as procaspase-9, forming an “apoptosome”. Caspase-9 activation, subsequent caspase-3 activation and cell death.

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35 The control & regulation of apoptotic mitochondrial events occurs through members of the Bcl-2 family of proteins  Anti-apoptotic proteins include Bcl-2, Bcl-x, Bcl-XL, Bcl-w  Pro-apoptotic proteins include Bax, Bak, Bid, Bad, Bim, Bik  The main mechanism of action of the Bcl-2 family of proteins is the regulation of cytochrome c release from the mitochondria via alteration of mitochondrial membrane permeability.

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37  Apoptosis plays two primary roles in spermatogenesis: It is essential in limiting the population of germ cells to a number that can be supported by Sertoli cells. Selective depletion of abnormal germ cells, i.e. cells with abnormal morphology, altered biochemical function or DNA damage.

38 Fas System in sperm apoptosis The main factor postulated to be implicated in sperm apoptosis is the cell surface protein, Fas (CD95 or APO1) Fas is a type I membrane protein that belongs to the tumor necrosis factor receptor family. Binding of Fas ligand (FasL) or agonistic anti-Fas antibody to Fas, kills cells by apoptosis.

39 DISC (3,7)

40 Abortive apoptosis It has been shown that any defects in the remodeling of cytoplasm during spermatogenesis and presence of cytoplasmic retentions with increased expression of apoptotic markers like Fas, caspase1, P53 and P21 may lead “abortive apoptosis”. A phenomenon in which defective sperm cells escape PCD and are present in the ejaculate.

41 Abortive apoptosis Sakkas et al (1999) have proposed that presence of Fas- positive sperm in ejaculate, is an indicative of sperm cells which are labeled for apoptosis. they escape apoptosis because: 1) There are too many abnormal spermatozoa for the available Fas-L (Fas ligand produced by sertoli cell) 2) May signaling through Fas system is not functional.

42 Abortive apoptosis According to this model, these spermatozoa that escape the process, may indicate the presence of combination of abnormalities including histone-protamine substitution, DNA remodeling and also cytoplasmic and membrane anomalies. Abortive apoptosis can explain why a high percentage of spermatozoa with DNA damage and abnormal forms spermatozoa which show markers of apoptosis have been seen in patients with abnormal semen parameters.

43 Abortive apoptosis Regarding to this evidence, there is not complete clearance of abnormal spermatozoa through apoptosis. Abortive apoptosis in sperm associated with: - abnormal morphologies - aberrant biochemical functions - nuclear DNA damage

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45  The markers of apoptosis expressed by a varying proportions of ejaculated sperm and are including: 1.Externalization of Phosphatidyl serine (PS) to the sperm outer membrane leaflet 2.Activated caspases 3.Loss of the integrity of the mitochondrial membrane potential (MMP) 4.DNA fragmentation

46 Production of ejaculated spermatozoa that possess apoptotic markers (such as Fas positivity and DNA damage) indicate that in some men with abnormal sperm parameters, an ‘abortive apoptosis’ has taken place. In men with normal sperm parameters, the percentage of Fas- positive spermatozoa is low. Samples with low sperm concentration are more likely to have a high proportion of Fas-positive spermatozoa. The apoptotic mechanism of spermatozoa is already triggered before ejaculation. After ejaculation, spermatozoa do not become apoptotic spontaneously

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48 Male infertility appears to be positively correlated with increased levels of sperm with apoptotic markers. In the natural selection process, spermatozoa with damaged DNA would have a low chance of achieving conception with an oocyte. All data indicate that increased levels of sperm apoptosis during in vitro fertilization (IVF) correlate to impaired embryo morphology at early cleavage stages, failure to progress to the blastocyst stage in culture and decreased pregnancy rates.

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50 Assay for apoptosis

51  Apoptosis assays, based on methodology, can be classified into six major groups: 1. Cytomorphological alterations 2. DNA fragmentation 3. Detection of caspases, cleaved substrates, regulators and inhibitors 4. Membrane alterations 5. Detection of apoptosis in whole mounts 6. Mitochondrial assays

52 Cytomorphological alterations The evaluation of hematoxylin and eosin-stained tissue sections with light microscopy does allow the visualization of apoptotic cells. This method detects the later events of apoptosis. TEM is considered the gold standard to confirm apoptosis: (1) electron-dense nucleus (2) nuclear fragmentation (3) intact cell membrane (4) disorganized cytoplasmic organelles (5) large clear Vacuoles (6) blebs at the cell surface

53 DNA fragmentation DNA fragmentation occurs in the later phase of apoptosis. TUNEL (Terminal dUTP Nick End-Labeling) assay quantifies the incorporation of deoxyuridine triphosphate (dUTP) at single and double stranded DNA breaks in a reaction catalyzed by the template-independent enzyme, terminal deoxynucleotidyl transferase (TdT). Incorporated dUTP is labeled such that breaks can be quantified either by flowcytometry, fluorescent microscopy, or light microscopy.

54 TUNEL positive cell TUNEL negative cell

55 TUNEL positive cell

56 TUNEL negative cell

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58 Relationship between tunel & caspases

59 Membrane alterations

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61 F luorescein isothyocyanate (FITC)- conjugated Annexin V + PI The vital dye propidium iodide (PI) should be used in combination with annexin V. By using an additional labeling with the vital dye propidium iodide (PI), it is possible to distinguish viable, apoptotic and necrotic sperm populations at the same time

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63 Detection of Apoptosis in Whole Mounts Apoptosis can also be visualized in whole mounts of embryos or tissues using dyes such as acridine orange (AO), Nile blue sulfate (NBS), and neutral red (NR). Since these dyes are acidophilic, they are concentrated in areas of high lysosomal and phagocytotic activity.

64 Mitochondrial Assays Mitochondrial assays and cytochrome c release allow the detection of changes in the early phase of the intrinsic pathway. The mitochondrial outer membrane (MOM) collapses during apoptosis, allowing detection with afluorescent cationic dye. Cytochrome c release from the mitochondria can also be assayed using fluorescence and electron microscopy in living or fixed cells. Apoptotic or anti-apoptotic regulator proteins such as Bax, Bid, and Bcl-2 can also be detected using fluorescence and confocal microscopy

65 Aberrant cell death can lead to many human diseases:  Decreased apoptosis Cancer, Autoimmune disorders  Excessive apoptosis Neurodegenerative and immunodeficiency (AIDS) disorders, Ischemia NOTE: Properties of carcinogenic agents (chemical agents as well as radiations) are the growth-inhibition power and the ability to induce cell death. These properties are widely used in anticancer chemo- and radiotherapies

66 (Tissue atrophy) (Hyperplasia)

67 با تشکر Thanks for your attention


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