Apoptosis: Role and

significance in seminiferous
epithelium

Dr.M.M.Misro
Professor
Department of Reproductive
Biomedicine
1

APOPTOSIS:
Can the cell be
Programmed to
?suicide

INTRODUCTION
Cell death by injury
-Mechanical damage
-Exposure to toxic chemicals

Cell death by suicide

-Internal signals
-External signals

?Why
should a cell commit suicide
Apoptosis is needed for proper development
The resorption of the tadpole tail
The formation of the fingers and toes of the fetus
The sloughing off of the inner lining of the uterus
The formation of the proper connections between neurons in the brain
Apoptosis is needed to destroy cells
Cells infected with viruses
Cells of the immune system
Cells with DNA damage
Cancer cells

Importance of Apoptosis
Important in normal physiology / development
Development: Immune systems maturation,
Morphogenesis, Neural development
Adult: Immune privilege, DNA Damage and wound
repair.

Excess apoptosis
Neurodegenerative diseases

Deficient apoptosis
Cancer
Autoimmunity

?What makes a cell decide to commit suicide

Withdrawal of positive (Growth) signals
growth factors for neurons
Interleukin-2 (IL-2)

Receipt of negative (Death) signals

increased levels of oxidants within the cell
damage to DNA by oxidants
death activators :

Tumor necrosis factor alpha (TNF-)

Lymphotoxin (TNF-)
Fas ligand (FasL)

History of cell death / apoptosis research

1800s

Numerous observation of cell death

1908

Mechnikov wins Nobel prize (phagocytosis)

1930-40

Studies of metamorphosis

1948-49

Cell death in chick limb & exploration of NGF

1955

Beginning of studies of lysomes

1965

Necrosis & PCD described

1971

Term apoptosis coined

1977

Cell death genes in C. elegans

1980-82

DNA ladder observed & ced-3 identified

1990

Apoptosis genes identified, including bcl-2,

fas/apo1 & p53, ced-3 sequenced
(Richerd et.al., 2001)

PRIZED Horvitz, and Sulston share Physiology or Medicine Nobel

2002
for their discoveries concerning genetic regulation
of organ development and programmed cell death

Apoptosis vs. Necrosis

Necrosis
No chromatin
condensation or formation
of apoptotic bodies
Cell lyses, eliciting an
inflammatory reaction
Initiated by direct cell
damage, mostly physical
Passive process, No
macromolecule synthesis
DNA fragmentation is
random, or smeared-not
ladder like-200 bp
In vivo, whole areas of the
tissue are affected

Apoptosis
Cellular, chromatin
condensation formation of
apoptotic bodies
Membranes remain intact
Initiated by a signal
transduction process
Active process, requires ATP,
macromolecule synthesis
Cell is phagocytosed, no tissue
reaction
Ladder-like DNA
fragmentation-200bp
In vivo, individual cells appear
involved

Morphological and biochemical

characteristics of apoptosis
: Morphology changes

Early : Chromosome condensation, cell body shrink

Later : Blebbing and Nucleus and cytoplasm fragment
Apoptotic bodies
At last: Phagocytosed

A Normal cell

B Apoptosis: Apoptotic bodies

Contrast ofApoptosis and

necrosis

Apoptosis

Necrosis

Death by apoptosis is a neat, orderly

process

Biochemical characteristics of
200bp DNA ladder~180

: apoptosis

Apoptosis induced
by Cyto C

2.0kbp
1.0
0.5
0.2

Lane 1 0 h
h 2 1
h 3 2
h 4 3
h 5 4
Control 6
Marker 7

Apoptosis is carried out by

a proteolytic system
caspase
?Why called caspase( 1)

Active site: Cysteine

Cleavage site:
Asparatic acid

Cysteine
Asparatic acid
specific protease
Aps-Xxx

Apoptosis can be divided into two

phases
Activation phase
The cell responds to death signals that
.commit it to undergoing self-destruction
Execution phase
.The death sentence is carried out
Apoptosis cells are recognized by
phagocytes because they carry exposed
.markers, called eat me signals
The best studied eat me signal is the
presence of phosphatidylserine molecules in
.the outer leaflet of PM of apoptotic cells

?How to activate caspases

All caspases expressed as proenzymes
Procaspases
NH2-terminal prodomain: Highly variable

Procaspase

Large subunit (20kD)

Small subunit
(10kD)
?How are procaspases activated to initiate the caspase cascade
The activation is triggered by adaptor proteins that bring
.multiple copies of specific procaspases
:groups of caspase 3
apoptotic initiators: caspase-2, caspase-8, caspase-9 and
Caspase-10
apoptotic executioners: caspase-3, caspase-6 caspase-7 2
and
14 (morphology change)
inflammatory mediators: caspase-1,

and caspase-11 3

Procaspases are activated by binding to

adaptor proteins
The caspase cascade
involved in apoptosis
A. Procaspase activation by
. proteolytic cleavage
B. Caspase cascade

Molecular Regulation of Apoptosis

Caspases

Caspases are cysteine proteases that cleave over( 1

100 substrates to induce cell death
Nuclear lamins
Inactivate
DNA repair enzymes
cytoskeleton
other caspases (Caspase Cascade)
Caspase-activated DNAase (CAD)

Activate

Caspases are synthesized as inactive zymogens( 2

The target proteins of caspase are the

:following
More than a dozen protein kinase, including
FAK, PKC, and Raf1. FAK disrupt cell
.adhesion for the apoptotic cell
Lamins. Cleavage of lamins leads to the
disassembly of the nuclear lamina and
.shrinkage of the nucleus
Proteins required for cell structure. Such as
actin, and gelsilin. Cleavage and inactivation of
.these proteins lead to changes in cell shape
Induce cell display signals, marked it for
.phagocytosis

The inhibitor of CAD (Caspaseactivated Dnase, an endonuclease).

Cleavage of CAD inhibitor lead to
activation of CAD, once activated, CAD
translocates from the cytosol to the
.nucleus severing DNA into fragments
Enzymes involved in DNA repair.
Which are inactivated by caspase cleavage.
DNA repair is a homeostatic activity that is
.inappropriate in an apoptotic cell

Apoptosis: Pathways
Extrinsic Pathway
Death
Ligands

Death
Receptors

Intrinsic Pathway
DNA
damage
& p53

Mitochondria/
Cytochrome C

Initiator
Caspase 8
Effector
Caspase 3
Initiator
Caspase 9

PCD

MAJOR PLAYERS IN
APOPTOSIS

Caspases
Death signals e.g. TNF & TNFR
p53
BAX
Bcl-2 family

DNA damage

p53

p21

Cell Cycle
Arrest

DNA
Repair

Nuclear
Mitochondrial
BAX (proapoptotic)

Cyt C
Apaf-1

bcl- 2
Anti-apoptotic

DNA nucleosomal
200 bp fragments

Initiator
Caspase-8E / 9I

Apsartate- AA
proteolysis

Effector
Caspase- 3

CAD
(DNA-ase)

Summary
an important process of cell death
can be initiated extrinsically through death ligands
(e.g. TRAIL, FasL) activating initiator caspase 8 through
induced proximity.
can be initiated intrinsically through DNA damage (via
cytochrome c) activating initiator caspase 9 through
oligomerization.
Initiator caspases 8 and 9 cleave and activate
effector caspase 3, which leads to cell death.

MALE REPRODUCTIVE SYSTEM

TUNICA ALBUGINEA
TESTIS

EPIDIDYMIS

con Medias
tain
t
ing inum
RE
TES TE
TIS

LOBULES

MALE REPRODUCTIVE SYSTEM

TESTIS
SEMINIFEROUS TUBULES
SEMINIFEROUS EPITHELIUM
complex stratified epitheliumcontaining two basic cell
:populations
SPERMATOGENIC CELLS) 1(
stem cells which regularly replicate
and differentiate into mature sperm
as they migrate toward the lumen
SERTOLI CELLS) 2(
nonreplicating physical support cells
INTERSTITIAL CONNECTIVE
TISSUE
LEYDIG CELLS) 1(
produce and release testosterone

TESTICULAR GERM CELLS

.Testis is an immunoprotected site
.Well suited for antigen development on germ cells
Germ cells get support and nourishment from Sertoli cells which
.has the capacity to maintain only an adequate number of such cells
Upon initiation of spermatogenesis at puberty, early germ cells
apoptotic wave occurs, to remove abnormal germ cells and to
maintain a proper ratio between maturing germ cells and Sertoli
.cells (Koji 2001)
Up to 75% of the spermatogenic cells are eliminated during the
.maturation (Bartke, 1995; Billig et. al. 1995)

Initiation of Germ Cell Apoptosis (GCA)

Spontaneous germ cell death, a frequent event occurring during normal
.spermatogenesis (Blanco-Rodriguez, 1996)
:Germ cells are also very sensitive to a wide spectrum of apoptotic stimuli
Including deficiency of survival signals caused by either hormone
deprivation/alteration or by impairment of their signaling pathways
(Sinha-Hikim, 1995; Zirkin, 1999; Lee et al, 1997)
Exposure to environmental stresses such as ionizing radiations .2
(Embree-Ku et al, 2002 & Richburg et al, 2000), mild hypo- and
hyperthermia or experimental cryptorchidism (Chaki & Misro, 2003;
.Ogi et al & Yin et al, 1998), oxidative stress (Maneesh et al, 2005)

.1

Apoptotic cells are recognized by Sertoli cells through the binding of their
membrane receptor, class B scavenger receptor type I, to phosphatidylserine
which appears on the surface of apoptotic germ cells and they are then rapidly
.eliminated by phagocytosis

The significance of germ cell apoptosis

According to some only spermatogonia exceeding the supportive
.capacity of Sertoli cells are eliminated to prevent overcrowding
Others suggest that spermatogonia elimination may represent an early
selection of abnormal cells before the onset of meiosis (Rodriguez et.
.al., 1997)
As a safeguard to genetic integrity of the male gamete and the
synchronization between the spermatogonial and the spermatocyte
.cycles
In specific conditions like ischemia or cryptorchidism, the rate of
.germ cell apoptosis increases significantly (Misro et al, 2003)

Molecular Mechanism of Germ Cell Apoptosis

It has been shown that the intracellular balance of BclxL and
Bax proteins is crucial for early apoptotic wave in seminiferous
.epithelium (Rodriguez, 1997)
Fas/FasL system has been shown to be the major inducer of
GCA under pathological conditions (viz., cryptorchid-, heated-,
drug treated, irradiated- or hormone-deprived testes) (Lee et. al.,
.1999), through caspase 8 activity
p53 protein is another widely described regulator of both cell
.proliferation and apoptosis
p53 plays a role in mediating both spontaneous and injury-
.induced Germ cell apoptosis

p53 involves in the regulation of apoptosis in mitotically active

spermatogonia as well as in meiotic or postmeiotic germ cells
.(Richburg et. al., 2000; Beumer et al., 1998)
Caspases activity is also up-regulated during GCA, caspase 2
activity contributes to the initial wave of germ cell apoptosis
.during the first round of spermatogenesis (Zheng et al, 2006)
AP-1 transcription factors (c-jun, c-fos, Jun D) involved in the
Sertoli cell-mediated control of germ cell apoptosis (Suomalainen
.et al, 2004)
JNK participates in the regulation of mouse spermatogenesis
(Phelan et al., 1999), whereas ERK1/2 is induced in sperm
.maturation (Lu et al., 1999) and capacitation (Luconi et al., 1998)

JNK induced apoptosis in response to stress or survival factor

deprivation in part by inducing FasL expression (Maclaren et al.,
.2000)
Testicular IR injury stimulates IL-1 expression, leading
activation of the JNK pathway and ultimately E-selectin
expression and neutrophil recruitment to the testis (Jeffery et al.,
.2003)
ROS produced by the recruited neutrophils perturb Bcl-2
family members in the germ cells and thus initiate apoptosis via
.the mitochondrial pathway
However, the fine molecular events controlling testicular germ
.cell apoptosis are still being investigated

Stress during experimental

cryptorchidism

Fig.1 Histological
sections of rat testes
stained with
hematoxylin and
eosin. Representative
section from control
testes shows normal
spermatogenesis (a).
No significant
alterations in
spermatogenesis by
1d (b), large number
of pyknotic cells along
with multinucleated
giant cells by 3d (c)
and by 5d (d),
disruption of
spermatogenic
epithelium,
prevalence of giant
cells and pyknotic
germ cells continued
by 7d (e) and
vacuolated
seminiferous
epithelium with a
single layer of germ
cells close to the
basement membrane
by 15d (f) of
cryptorchidism (n=6
testes against each
.panel, x400)

nc
Fig.3 In situ end labeling
(ISEL) of DNA in testicular
sections. A representative
section from the negative
control
(nc)
and
non
cryptorchid control (a) testes
displaying
hardly
any
staining in the germ cells,
positive staining (arrow) of
the nuclei in the germ cells
is seen following 1d, 3d and
5d of cryptorchidism (b, c,
d), Few giant cells stain
positive for apoptosis by 7d
of cryptorchidism (e) and
almost all cells are showing
positive staining by 15d of
cryptorchidism
(f).
(n=6
testes against each panel,
.x400)

Pyknotic vs Apoptotic cells in tubule

sections/ live vs dead cells isolated in vitro
)% (

Chaki &Misro, Apoptosis, 2005

120

Pyknotic
Apoptotic
Dead
Live

100
80
60
40
20
0

Control

1d Cr

3d Cr

5d Cr

7d Cr

15d Cr

Fig.4 Percentage of cells pyknotic versus apoptotic in testicular

sections and live versus dead among those isolated in vitro. While
live cells dwindled in number a significant rise in the number of
. apoptotic cells is seen by 15 days of cryptorchidism

d
250
Cells/tubular cross section

Figure 3
Histological
sections of rat testes
stained with hematoxylin
and eosin. Representative
section from the control
testes
shows
normal
spermatogenesis
(a).
Pyknotic nuclei in germ
cells
close
to
the
basement membrane can
be resolved by 5d (b),
increased vacuolation of
the
seminiferous
epithelium
in
some
tubules with the rise
number
of
pyknotic
(arrowhead) cells by 10d
(c), decrease in tubular
diameter and number of
germ cells present with in
the seminiferous tubule
by 15d (d) and further
depletion in germ cell
number and increase in
pyknotic cells by 30d (e)
of estradiol treatment.
Status on prevalence of
germ
cells
during
different
periods
of
estradiol treatment (f).
(n=6 testes against each
.panel, x400)

Preleptotin/Leptotin
sperm atocyte
Pachytene
Sperm atocyte
Round Spermatid

200
150
100
50

Control

5d

10d

15d

30d

Summary of the findings

Large scale removal of germ cells

.through apoptosis
Removal was facilitated through
.formation of giant cells
Various apoptotic effectors are
.activated
.Rise in oxidative stress
Decrease in intra-testicular
.testosterone
Apoptosis 2005, 10:395-405

Induction of Apoptosis

Oxidant
Stimuli
HOCl,
etc
Cytotoxic drugs
H 2O 2

Non-Oxidant
Stimuli
Persistent stimulation with
hormone (hCG)
Withdrawal of growth
factors
TNF-alpha

Oxidative stress

Is it possible to Inhibit this

induction of Apoptosis

41

Apoptosis of germ cells in vitro

through oxidant stimuli
:The oxidant selected for the purpose is micro-molar concentrations of

H2O2

42

Cell viability of isolated testicular germ cells with or without H2O2

for 1 hour in the medium. More than 75% of the H 2O2 exposed
cells were found viable by trypan blue exclusion. C-Control.
*p<0.01
43

Evaluation of oxidative stress in rat testicular germ cells post-treated with H 2O2. A dose dependent increase in lipid peroxidation as
measured by TBARS formation (a), transient rise followed by a steep decline in glutathione-s-transferase activity at the highest
44
concentration of H2O2 (b), significant decline in superoxide dismutase (SOD) activity coinciding with transcript levels representing Mn
SOD but not of Cu/Zn SOD (c) and catalase activity along with its transcripts (d). C-Control. *p<0.01, **p<0.00

Assessment of total antioxidant capacity (TAC) in

testicular germ cells with or without H2O2 (10 M). CControl. *p<0.05

45

c
C

10

H2O2 (M)

1000
bp

500
bp

In situ end labelling (ISEL) of testicular germ cells post-treated with H 2O2.
Marked rise in percentage of ISEL positive cells (a) was observed in the
treated (right panel) compared to untreated cells (left panel) (b) x 400.
Ladder assay showing DNA fragmentation in H 2O2 exposed testicular46 germ

a
b
H2O2 (M)
116 kDa
89 kDa

10

PARP

Rise (3-30 folds) in caspase-3 activity (a) and PARP cleavage in

testicular germ cell following H2O2 treatment (b). C-Control.
47
*P<0.01

a
H2O2 (M)

H2O2 (M)

10

10

23 kDa

Bax

482 bp

Bax

27 kDa

Bcl-2

293 bp

Bcl-2

539 bp

-Actin

22 kDa

Bid

30 kDa

Bak

25 kDa

Bad

42 kDa

-Actin

Expression levels of Bcl-2 family members. Increase in proapoptotic proteins (Bax, Bid, Bak, Bad) and decrease in antiapoptotic protein Bcl-2 at the highest (10 M H2O2)
concentration (a) was observed. Expression of Bax and Bcl-2
as detected by RT-PCR (b). C-Control.
48

a
H2O2 (M)

b
C

10

46 kDa

H2O2 (M)
Caspase-9

aCaspase-9 539 bp

15 kDa

Cyt. C

42 kDa

-Actin

10

H2O2 (M)
Caspase-8

40 kDa

FasL

48 kDa

Fas

42 kDa

10
Caspase-9
-Actin

c
55 kDa

132 bp

17 kDa

H2O2 (M)

-Actin

10

123 bp

Caspase 8

238 bp

FasL

351 bp

Fas

539 bp

-Actin

Pathways of signal transduction in H2O2 induced testicular germ cell

apoptosis. Western blots showing expressions of (intrinsic) caspase-9,
49
cytochrome C (a) and (extrinsic) caspase-8, FasL, Fas (c) are supported by

a
H2O2 (M)

H2O2 (M) C

10

10

53 kDa

p53

54 kDa
46 kDa

JNK

42 kDa

-Actin

54 kDa
46 kDa

pJNK

b
H2O2 (M)
397 bp
539 bp

38 kDa

pP38

65 kDa

NF-B

10
p53

-Actin 42 kDa

Actin

Other pathways of signal transduction in H2O2 induced

testicular germ cell apoptosis. Dose dependent increase in
expression of p53 protein (a) coinciding with marked rise in
transcript levels in all treated groups (b) and change in
expression levels of proteins (JNK, p-JNK, p-p38, NF-B) was
observed (c). C-Control.
50

Summary of the findings

H2O2 at concentrations 1-10 M was found to induce
. apoptosis in testicular germ cells
Induction of apoptosis was associated with a significant
increase in lipid peroxidation and a concomitant
decrease in SOD & catalase activity
Markers for signalling pathways like, Extrinsic (Fas, FasL
& caspase 8) and Intrinsic (Bid, Bak, Bad, Bax &
.caspase 9) are found up-regulated
C-jun N-terminal kinase and P38 phophorylated forms are
also stimulated
.Caspase 3 activity was estimated 30 fold higher
51

Maheshwari & Misro, FEBS J 2009

Conclusion

Continuous production of sperm cells through division is the

hallmark of the male gonad
In the process, cells which are not fit need to be removed from
the system without affecting the process itself. Apoptosis, is
thus becomes one of the effective ways of removing cells from
. the seminferous epithelium
Induction of apoptosis in the seminiferous epithelium can be
achieved by different means, but the question yet to be
answered is whether or not we shall be able to program a
particular cell type to undergo programmed cell death. This
would also facilitate in the development of a molecular method
of regulation of male fertility and population control