Molecular approaches for understanding the ion channels of sperm

Manjit Panigrahi PhD 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 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

I feel like we have discovered the entrance 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 the generation of a new individual.  Ion channels are key elements in the dialogue between sperm, its environment, and the egg.

Understanding how sperm ion channels participate in fertilization requires combining  Planar bilayer techniques  Measurement of intracellular Ca2+ and intracellular pH using fluorescent probes  Patch-clamp recordings  Molecular cloning and heterologous expression

 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

Spermatogenesis

 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

Membrane transport

Our area of interest

Channels open in response to a variety of different stimuli

Voltage gating- sense transmembrane potential (Cav, Kv, CatSper etc.)

Ligand gating - in response to specific ligands (ACh, 5-HT, GABA etc.) Mechanical gating (CLCC1)

 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

1. 2. 3. 4. 5. 6. 1. 2. 3. 4. 5. CatSper 1,2 Cav TRPC 7.

CatSper 3,4 Cav TRPC Kir Kv IP3R nAChR 1. 2. 1. CatSper 2 Cav TRPC Kv Kir 3. TRPC IP3R Kir

Kv 2. IP3R 3. 4. 5.

   

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 and localized 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 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

During the journey of spermatozoa from epididymis to female reproductive tract - dramatic changes in the environmental osmolality It develops efficient mechanism osmolytes (e.g. K+ ions) to efflux certain

Capacitation is the changes that lead to hyperactivated motility of the spermatozoon ( e.g. CaV, Catsper etc.) The AR allows sperm to penetrate the zona pellucida layer and release of lytic enzymes for the fusion of egg and sperm head (e.g. CaV, TRPC etc.)

Mechanism of hyperactivated motility

(Navarro et al. 2008)

Mechanism of acrosome reaction

Ion channels implicated in sperm function

Planar bilayer techniques

Patch-clamp recordings

Strategies to study ion channels Fluorescent indicators

Molecular approach

 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

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 CaV channel 1 subunits  3 genes encoding LVA channels  subunits exhibits repeats comprising 4domains
1

 Each domain contains 6 TM helices ( S1- S6) surrounding a central pore (Caterall 2000, Caterall et al. 2003)

Transient receptor potential-C (TRPC) channel

 7 closely related genes encoding TRPC channels in mammals (TRPC1-7)  Non-selectively permeable to cations- selectivity of calcium over sodium  Consists of 6 TM spanning segment

Cyclic nucleotide - gated (CNG) channel

 6 different human genes coding for CNG channels  CNG channel genes fall into two different gene subfamilies (CNGA and CNGB)  Allow the passage of divalent cations, in particular Ca2+  Consists of 6 TM spanning segment (S1-S6) (Felix et al. 2005)

CatSper channel

 5 different genes encoding CatSper 1,2,3 ,4 &  Expressed in the plasma membrane of principal piece of sperm tail  CatSper protein is a single 6 TM spanning repeat closest to Cav channels  S4 segment acts as a voltage sensor  CatSper1s amino terminous abundance of histidine residues

While searching for Ca2+ channels A fragment of EST cDNA was confirmed to be CatSper 1 (Ren et al., 2001) The search for any accesory identification Catsper 3, 4 Real-time PCR demonstrated  CatSper 1, 3 & 4 transcripts late stage spermatogenesis  CatSper 2 in early stage spermatogenesis protein by insilico

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)

have normal fertility

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)

 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)

complete male infertility

 Sperm morphology, weight, life span, litter size, gross behaviour, or mating behaviour was unaffected in Catsper1-4 null mice  Thus CatSper channels - only known ion channels that specifically affect mammalian male fertility

 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

An ideal ion channel-active blocker could be taken either by men or women to prevent fertilization as a contraceptive
(Zhang et al. 2005)

The CatSper gene can be a potential target for male infertilty screening Molecular research is now focussing on identification and characterization of genes & proteins of ion channels In future, navigation throughout sperm databases, proteomics and other molecular tools may provide us with a better mechanistic understanding of male gamete function