Apoptosis: Role and significance in seminiferous epitheliumDr.M.M.MisroProfessorDepartment of Reproductive Biomedicine1

APOPTOSIS: Can the cell be Programmed to suicide ?

INTRODUCTIONCell 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 developmentThe 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 ApoptosisImportant in normal physiology / developmentDevelopment: Immune systems maturation, Morphogenesis, Neural developmentAdult: Immune privilege, DNA Damage and wound repair.

Excess apoptosisNeurodegenerative diseases

Deficient apoptosisCancerAutoimmunity

What makes a cell decide to commit suicide?Withdrawal of positive (Growth) signalsgrowth 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 research1800sNumerous observation of cell death1908Mechnikov wins Nobel prize (phagocytosis) 1930-40Studies of metamorphosis1948-49Cell death in chick limb & exploration of NGF1955Beginning of studies of lysomes1965Necrosis & PCD described1971Term apoptosis coined1977Cell death genes in C. elegans1980-82DNA ladder observed & ced-3 identified1990Apoptosis 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. NecrosisCellular, chromatin condensation formation of apoptotic bodiesMembranes remain intactInitiated by a signal transduction processActive process, requires ATP, macromolecule synthesisCell is phagocytosed, no tissue reaction Ladder-like DNA fragmentation-200bpIn vivo, individual cells appear involved

No chromatin condensation or formation of apoptotic bodiesCell lyses, eliciting an inflammatory reaction Initiated by direct cell damage, mostly physicalPassive process, No macromolecule synthesis DNA fragmentation is random, or smeared-not ladder like-200 bpIn vivo, whole areas of the tissue are affectedNecrosisApoptosis

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

ANormal cell BApoptosis: Apoptotic bodies

Contrast ofApoptosis and necrosisApoptosisNecrosisDeath by apoptosis is a neat, orderly process

Biochemical characteristics of apoptosis :2.0kbp1.00.50.2180~200bp DNA ladderApoptosis induced by Cyto C

Lane 10 h 21 h 32 h 43 h 54 h 6Control 7Marker

Active site: CysteineCleavage site: Asparatic acid Cysteine Asparatic acid specific proteaseAps-Xxx Apoptosis is carried out by a proteolytic system caspase (1) Why called caspase?

Apoptosis can be divided into two phasesActivation 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 ProcaspasesProcaspaseNH2-terminal prodomain: Highly variableLarge 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. 3 groups of caspase:1apoptotic initiators: caspase-2, caspase-8, caspase-9 and Caspase-10 2apoptotic executioners: caspase-3, caspase-6caspase-7 and 14 (morphology change) 3inflammatory mediators: caspase-1, and caspase-11

Procaspases are activated by binding to adaptor proteinsThe caspase cascade involved in apoptosisA. Procaspase activation by proteolytic cleavage.B. Caspase cascade

Molecular Regulation of ApoptosisCaspasesInactivateActivate2) Caspases are synthesized as inactive zymogens

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 (Caspase-activated 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

MAJOR PLAYERS IN APOPTOSISCaspasesDeath signals e.g. TNF & TNFR p53 BAX Bcl-2 family

Summaryan 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.

TESTISMALE REPRODUCTIVE SYSTEM EPIDIDYMISTUNICA ALBUGINEAMediastinum containing RETE TESTISLOBULES

TESTISMALE REPRODUCTIVE SYSTEM SEMINIFEROUS TUBULESSEMINIFEROUS EPITHELIUM- complex stratified epithelium containing two basic cell populations:(1) SPERMATOGENIC CELLS(2) SERTOLI CELLSstem cells which regularly replicate and differentiate into mature sperm as they migrate toward the lumennonreplicating physical support cellsINTERSTITIAL CONNECTIVE TISSUE(1) LEYDIG CELLSproduce 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 (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).

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

b

a

d

c

e

f

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).

aecfdbncFig.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).

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. Chaki &Misro, Apoptosis, 2005

Chart1

20.22

50.253

25815

3523.425

403050

10080.3580

Pyknotic

Apoptotic

Dead

Percentage of cells

Chart2

20.22985.75.7000000

50.2539766330000

25815853.93.92.52.54.64.62.92.9

3523.425754.54.53.33.33.33.344

403050503.73.7662.72.73.53.5

10080.358020776.76.7552.52.5

Pyknotic

Apoptotic

Dead

Live

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

Sheet1

PyknoticApoptoticDeadLive

Control20.2298

1d Cr50.25397

3d Cr2581585

5d Cr3523.42575

7d Cr40305050

15d Cr10080.358020

Sheet2

Sheet3

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bFigure 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).

Chart1

45702002.91130

446619056.815

40601007.9722

4034353.2106.7

371715535

Preleptotin/Leptotin spermatocyte

Pachytene Spermatocyte

Round Spermatid

Cells/tubular cross section

Sheet1

Preleptotin/Leptotin spermatocytePachytene SpermatocyteRound Spermatid

Control4570200

5d4466190

10d4060100

15d403435

30d371715

Sheet2

Sheet3

Summary of the findingsLarge 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 ApoptosisOxidant StimuliNon-Oxidant StimuliIs it possible to Inhibit this induction of Apoptosis?H2O2HOCl, etcCytotoxic drugsPersistent stimulation with hormone (hCG)Withdrawal of growth factorsTNF-alphaOxidative stress

Apoptosis of germ cells in vitro through oxidant stimuliThe oxidant selected for the purpose is micro-molar concentrations of :H2O2 *

Cell viability of isolated testicular germ cells with or without H2O2 for 1 hour in the medium. More than 75% of the H2O2 exposed cells were found viable by trypan blue exclusion. C-Control. *p

Evaluation of oxidative stress in rat testicular germ cells post-treated with H2O2. 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 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

Assessment of total antioxidant capacity (TAC) in testicular germ cells with or without H2O2 (10 M). C-Control. *p

In situ end labelling (ISEL) of testicular germ cells post-treated with H2O2. 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 H2O2 exposed testicular germ cells. C-Control. *p

Rise (3-30 folds) in caspase-3 activity (a) and PARP cleavage in testicular germ cell following H2O2 treatment (b). C-Control. *P

Expression levels of Bcl-2 family members. Increase in pro-apoptotic proteins (Bax, Bid, Bak, Bad) and decrease in anti-apoptotic 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. *

Pathways of signal transduction in H2O2 induced testicular germ cell apoptosis. Western blots showing expressions of (intrinsic) caspase-9, cytochrome C (a) and (extrinsic) caspase-8, FasL, Fas (c) are supported by RT-PCR analysis of caspase-9 (b), caspase-8, FasL, Fas (d). C-Control.

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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.

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Summary of the findingsH2O2 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 stimulatedCaspase 3 activity was estimated 30 fold higher.

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Maheshwari & Misro, FEBS J 2009

Conclusion Continuous production of sperm cells through division is the hallmark of the male gonadIn 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

*testis-06

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