Apoptosis
Jalal Ghasemzadeh
Andrology lab
Yazd Reproductive Sciences Institute
 In humans, the rate of cell growth and cell death is
balanced to maintain the weight of the body.
Life cannot exist without cellular death
Cell death can occur via several processes (about 11 type):
1. Apoptosis
2. Necrosis
3. Autophagy
4. Entosis
5. Oncosis
6. Pyroptosis
.
.
.
11.
 Apoptosis = “normal” or “programmed” cell death
 Necrosis = “accidental” or “ordinary” cell death
 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.
 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).
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 ”.
2. Normal event in development of the nervous system
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
Apoptosis is a beneficial and important phenomenon:
 In animals
Embryonic Mouse Paw
Embryonic Chicken Foot
Tail absorption of the tadpole
 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).
 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
 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
 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
 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.
Apoptotic cells
-Eat me signals
 Takes about 30 - 60 min.
 In the tissue only about 5% cells is affected by the PCD.
 Physiological changes and phagocytosis is very fast.
APOPTOSIS
Extrinsic pathway (death receptor- mediated events)
Intrinsic pathway (mitochondria- mediated events)
 Death Receptors
 Death Ligands
 Adaptor Proteins
 Caspaseses
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
 To date, ten major caspases have been identified and broadly
categorized into:
 Signaling or Initiator caspases
 Effector or Executioner caspases
 Inflammatory caspases
 The other caspases that have been identified include:
Caspases
 Central role in cascade of apoptotic events is played by
caspase 3 (CPP32)
 “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.
 The best characterized receptors & ligands corresponding
death include:
Receptors
FasR (CD95/APO1)
DR3
DR4 (TRAIL-R1)
DR5 (TRAIL-R2)
TNFR1
TNFR2
Ligands
FasL
Apo3L
Apo2L
Apo2L
TNF-α
TNF-ß
and
 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
 Bcl-2 family proteins
 Cytochrom c
 Adaptor proteins
 Caspaseses
 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.
 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.
 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.
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.
DISC
(3,7)
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.
Abortive apoptosis
• Sakkas et al (1999) have proposed that presence of Faspositive 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.
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.
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
 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
• 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 Faspositive 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
 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.
Assay for apoptosis
 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
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
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.
TUNEL positive cell
TUNEL negative cell
TUNEL positive cell
TUNEL positive cell
TUNEL negative cell
TUNEL positive cell
Relationship between tunel & caspases
Membrane alterations
 During the process of apoptosis, one of the earliest events is
Externalization of Phosphatidyl serine (PS) from the inner
to the outer plasma membrane of apoptotic cells.
 These cells can be demonstrated by bound with FITClabeled Annexin V and detected with fluorescent
microscopy.
 Positive cell in the Annexin V assay beginning of the
apoptotic process
Membrane alterations
 The membranes of necrotic cells are labeled with annexin V.
Since loss of membrane integrity is a pathognomonic
feature of necrotic cell death, necrotic cells will stain with
specific membrane-impermeant nucleic acid dyes such as
propidium iodide and trypan blue.
 The membrane integrity of apoptotic cells can be
demonstrated by the exclusion of these dyes.
Fluorescein 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
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.
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
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
(Hyperplasia)
(Tissue atrophy)
‫با تشکر‬
Thanks for your attention