Sperm Ion Channels

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Molecular approaches for

understanding the

ion channels of sperm

Manjit PanigrahiPhD Scholar

Roll No. 1094



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



Introduction





Molecular approach

conclusion



³I feel like we have discovered theentrance into an ancient pyramid that

no one has ever been inside of before´

David Clapham



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.



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



Introduction





Molecular approach

conclusion



Spermatogenesis



Introduction





Molecular approach

conclusion



Membrane transport

Our area of

interest



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



Introduction


Types of ion channels



Molecular approach

conclusion



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



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)



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)



Introduction





Molecular approach

conclusion



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.)



(Navarro et al . 2008)

Mechanism of hyperactivated motility



Mechanism of acrosome reaction



Ion channels implicated in sperm function



Strategies tostudy

ion channels

Patch-clamprecordings

Fluorescentindicators Molecular

approach

Planarbilayer

techniques



Introduction





Molecular approach

conclusion



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



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)



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



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)



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



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



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



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



Introduction





Molecular approach

conclusion



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

Sperm Ion Channels

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