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8/3/2019 Sperm Ion Channels
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Molecular approaches for
understanding the
ion channels of sperm
Manjit PanigrahiPhD Scholar
Roll No. 1094
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Introduction
Sperm physiology, maturation and fertilization
Basic types of ion channels
Diversity of ion channels in sperm
Functional roles of ion channels in sperm
Molecular approach
conclusion
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Introduction
Sperm physiology, maturation and fertilization
Basic types of ion channels
Diversity of ion channels in sperm
Functional roles of ion channels in sperm
Molecular approach
conclusion
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³I feel like we have discovered theentrance into an ancient pyramid that
no one has ever been inside of before´
David Clapham
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Fertilization is a matter of life or death.
In animals of sexual reproduction ±
competent male and female gametes determine thegeneration of a new individual.
Ion channels are key elements in the dialogue betweensperm, its environment, and the egg.
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Understanding how sperm ion channels participate infertilization requires combining
Planar bilayer techniques
Measurement of intracellular Ca
2+
and intracellular pHusing fluorescent probes
Patch-clamp recordings
Molecular cloning and heterologous expression
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Introduction
Sperm physiology, maturation and fertilization
Basic types of ion channels
Diversity of ion channels in sperm
Functional roles of ion channels in sperm
Molecular approach
conclusion
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Spermatogenesis
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Introduction
Sperm physiology, maturation and fertilization
Basic types of ion channels
Diversity of ion channels in sperm
Functional roles of ion channels in sperm
Molecular approach
conclusion
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Membrane transport
Our area of
interest
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Voltage gating- sensetransmembrane potential(Cav, Kv, CatSper etc.)
Ligand gating - inresponse to specific ligands(ACh, 5-HT, GABA etc.)
Mechanical gating (CLCC1)
Channels open in response to a varietyof different stimuli
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Introduction
Sperm physiology, maturation and fertilization
Types of ion channels
Diversity of ion channels in sperm
Functional roles of ion channels in sperm
Molecular approach
conclusion
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1. CatSper 3,4
2. Cav
3. TRPC
4. Kir
5. Kv
6. IP3R
7. nAChR
1. TRPC
2. IP3R
3. Kir
1. CatSper 1,2
2. Cav
3. TRPC
4. Kv
5. IP3R
1. CatSper 2
2. Cav
3. TRPC
4. Kv
5. Kir
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Voltage gated Calcium channels (CaV )
N, P / Q, L and R type (HVA) - mouse sperm( Wennemuth et al . 2000)
T type (LVA) - Mouse spermatogenic cells
(Hagiwara and kawa, 1984)
CNG family - first sperm ion channel cloned andlocalized on flagella of sperm
( Weyand et al . 1994)
TRP channels ± flagella of sperm (Castellano et al . 2003)
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Catsper family
Catsper 1 & 2 localized to the flagella
( Ren et al . 2001, Quill et al 2001)
Catsper 3 & 4 to the head of sperm(Lobly et al . 2003, Jin et al . 2005)
K+ channels ± sperm flagella (Yeung and Cooper 2001)
nAChR cation channel (ligand gated )- Acrosome
region, midpiece of tail (Bray et al . 2002)
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Introduction
Sperm physiology, maturation and fertilization
Basic types of ion channels
Diversity of ion channels in sperm
Functional roles of ion channels in sperm
Molecular approach
conclusion
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During the journey of spermatozoa from epididymis tofemale reproductive tract - dramatic changes in theenvironmental osmolality
It develops efficient mechanism to efflux certain
osmolytes (e.g. K+ ions)
Capacitation is the changes that lead to hyperactivatedmotility of the spermatozoon ( e.g. CaV, Catsper etc.)
The AR allows sperm to penetrate the zona pellucidalayer and release of lytic enzymes for the fusion of eggand sperm head (e.g. CaV, TRPC etc.)
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(Navarro et al . 2008)
Mechanism of hyperactivated motility
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Mechanism of acrosome reaction
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Ion channels implicated in sperm function
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Strategies tostudy
ion channels
Patch-clamprecordings
Fluorescentindicators Molecular
approach
Planarbilayer
techniques
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Introduction
Sperm physiology, maturation and fertilization
Basic types of ion channels
Diversity of ion channels in sperm
Functional roles of ion channels in sperm
Molecular approach
conclusion
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Immunocytochemistry
RT-PCR
Northern blotting, Western blotting
Knocking out of gene with homologous recombination
Cloning, characterisation and heterologous expression
Expression profiling by realtime PCR
Insilico analysis
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Voltage-gated calcium (CaV) channels
7 different genes coding HVA CaVchannel 1 subunits
3 genes encoding LVA channels
1 subunits exhibits repeats
comprising 4domains Each domain contains 6 TM
helices ( S1- S6) surrounding acentral pore
(Caterall 2000, Caterall et al . 2003)
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Transient receptor potential-C (TRPC) channel
7 closely related genes
encoding TRPC channels inmammals (TRPC1-7)
Non-selectively permeableto cations- selectivity of calcium over sodium
Consists of 6 TM spanningsegment
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Cyclic nucleotide - gated (CNG) channel
6 different human genescoding for CNG channels
CNG channel genes fall intotwo different gene subfamilies(CNGA and CNGB)
Allow the passage of divalentcations, in particular Ca2+
Consists of 6 TM spanningsegment (S1-S6)
(Felix et al . 2005)
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CatSper channel
5 different genes encodingCatSper 1,2,3 ,4 &
Expressed in the plasmamembrane of principal piece of sperm tail
CatSper protein is a single 6 TMspanning repeat ± closest to Cav
channels
S4 segment acts as a voltagesensor
CatSper1¶s amino terminous -abundance of histidine residues
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While searching for Ca2+ channels ± A fragment of ESTcDNA was confirmed to be CatSper 1
(Ren et al ., 2001)
The search for any accesory protein by insilicoidentification ± Catsper 3, 4
Real-time PCR demonstrated ±
CatSper 1, 3 & 4 transcripts late stage spermatogenesis
CatSper 2 in early stage spermatogenesis
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Mice carrying null mutations for
CaV1.3 (Platzer et al., 2000) CaV2.2 (Ino et al ., 2001)
CaV2.3 (Saegusa et al ., 2000) CaV3.1 (Kim et al ., 2001) CaV3.2 (Chen et al ., 2003)
TRPC2 (Stowers et al ., 2002)
CNGA3 (Biel et al ., 1999)
The CaV1.2 null mutant is embryonic lethal(Seisenberger et al ., 2000)
CaV2.1 null pups die ~3-4 weeks after birth
(Jun et al ., 1999)
have normalfertility
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Gene deletion by homologous recombination of
S1 of CatSper1 (Ren et al ., 2001)
S1-S3 of CatSper2 (Quill et al ., 2003)
Pore region of CatSper 3
and CatSper 4 (Jin
et al ., 2007)
Sperm morphology, weight, life span, litter size, grossbehaviour, or mating behaviour was unaffected inCatsper1-4 null mice
Thus CatSper channels - only known ion channels thatspecifically affect mammalian male fertility
completemaleinfertility
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Introduction
Sperm physiology, maturation and fertilization
Basic types of ion channels
Diversity of ion channels in sperm
Functional roles of ion channels in sperm
Molecular approach
conclusion
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An ideal ion channel-active blocker could be taken either bymen or women to prevent fertilization as a contraceptive
(Zhang et al . 2005)
The CatSper gene can be a potential target for male infertiltyscreening
Molecular research is now focussing on identification andcharacterization of genes & proteins of ion channels
In future, navigation throughout sperm databases, proteomicsand other molecular tools may provide us with a bettermechanistic understanding of male gamete function
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