Sperm Capacitation2

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What is capacitation?

• It is a process that allows the acrosome reaction to occur, So the sperm can penetrate the zona pellucida of the oocyte.

• The physiological changes that confer on the sperm the ability to fertilize are called Capacitation.

Introduction

• After leaving the testis, mammalian spermatozoa are morphologically differentiated but have acquired neither progressive motility nor the ability to fertilize a metaphase II arrested egg.

• During epididymal transit, sperm acquire the ability to move progressively; but they are still fertilization incompetent.

Int.Cont.• Fertilization capacity is gained after

residence in the female tract for a finite time.

• Capacitation is also correlated with changes in sperm motility patterns, designated as sperm hyperactivation.

• Capacitation causes head changes (acrosome reaction) and tail changes (motility changes).

Capacitation consists of at least two components:

1- An initial membrane alteration, that allows spermatozoa to undergo the second phase, which is the fusion of plasma membrane with outer acrosomal membrane.

-The first phase is referred to as capacitation.

- The second phase is referred to as acrosome reaction(AR).

Original Models of CapacitationEpididymal Capacitated

FemaleTract+

Ejaculated

SeminalPlasma+

Decapacitation Factors

Sperm tail changes after capacitation

What cause sperm capacitation?

• In vivo the oviduct and uterus fluids capacitate the spermatozoa in different animals.

• In vitro, both caudal or ejaculated sperm capacitate when incubate under a variety of conditions in defined madia.

In Vitro Sperm capacitationThe sperm cell is programmed to undergo

capacitation when it is incubated in the appropriate medium.

1-Role of media constituents. - Serum albumin.

- Calcium.

- Bicarbonate.

2- Effectors and intracellular messengers. - Cyclic AMP metabolism.

- Protein tyrosine phosphrylation.

In Vitro capacitation Cont.

- pH.

- Membrane potential.

- Free radicals.

- Heparin.

- Glucose.

Ejaculated spermatozoa

• Contain decapacitated agent.

Initial Events in Capacitation

• Decapacitation factor removal– Time dependent– Induced by binding of a capacitating agent

• Glycosaminoglycans - heparin or heparan sulfate– Components of extracellular matrix of uterine and

oviduct cells

– Induced by cholesterol removal• Acceptor is albumin

– Sequence not clear

Ejaculated SpermD F

Plasma Membrane

Cytoplasm

Nucleus

Acrosome

D F

Cytoplasm

Nucleus

Acrosome

GAG

Cholesterol

Chol-AcceptorD FD FD F

D F

Heparin

Time?

Albumin

Role of media constituents

-Serum albumin ( usually bovine serum albumin) BSA.

. BAS during in vitro capacitation remove cholestrol from sperm plasma membrane.

BSA A1 BSA-chol.Sperm plasma membrane

Cholestrol efflux

Role of media constituents

-Calcium:increase in intracellular Ca during capacitation.

Capacitate sperm with heparin requires extracellular calcium.

When medium Ca decline the time required for capacitation increases.

Sperm plasma membrane

Cholesterol effluxBSA BSA-chol. Ca

Ca

A2

A1

Ejaculated SpermD F

Plasma Membrane

Cytoplasm

Nucleus

Acrosome

D F

Cytoplasm

Nucleus

Acrosome

GAG

Cholesterol

Chol-AcceptorD FD FD F

D F

Heparin

Time?

Albumin

ATP ADP(+(

Decapacitation Factors

( Seminal Plasma) DF

Ca2+

•Ejaculated Sperm•>> Active Ca2+ ATPase

•Removal DF>> Decreased Ca2+-ATPase

Low Ca

(-)

Heparin and Cholesterol Removal

Ca2+ Ca2+

Na+

Ca2+

H+

Increasing Ca

Role of media constituents

-Bicarbonate:Transmembrane movement of HCO3 responsible for increase in intracellular pH that is observed during capacitation.

SPM Cholesterol efflux

BSA BSA-chol.A1 Ca

Ca

A2+

HCO3

HCO3

A3 AC

+ +

Intracellular second messengers

• Intracellular messenger mediating capacitation will be considered from two respective.

- The regulatory system that appear to be common among different species.

- The regulatory process that may be unique to one or more species.

Intracellular second messenger cont.

-Cyclic AMP metabolism:The protein kinase A (PK-A) activity increases during sperm capacitation as a result of elevation intracellular cAMP.

- The mode of regulation of cAMP metabolism during sperm capacitation may be integrated with changes in Ca and HCO3 movement.

S.P.M. Cholesterol efflux

BSAA1 BSA-chol.

+

HCO3

HCO3

A3

Ca

A2

Ca

+ +

AC

cAMPPDE

+

PK-A

5`AMP

Role of cAMP in sperm capacitation

B1

- Protein tyrosine phosphorylation

• Protein tyrosine phosphorylation mediates a variety of cellular function .

• The increase in protein tyrosine phosphorylation is dependent on the presence of BSA, Ca and NaHCO3 in the medium.

• The role of protein tyrosine phosphorylation in sperm capacitation is regulated through a PK-A.

Cholesterol efflux

BSAA1 BSA-chol.

+

HCO3

HCO3

A3

Ca

A2

Ca

+ +

AC

cAMPPDE

+

PK-A

5`AMP

S.P.M.

PTK

+

+

-Ptyr-Ptase

-

Protein tyrosine phosphrylation

B1

B2

Intracellular pH

• The pH increase during sperm capacitation.

• Increase pH during bovine sperm capacitation by heparin.

Cholesterol efflux

BSAA1 BSA-chol.

+

HCO3

HCO3

A3

Ca

A2

Ca

+ +

AC

cAMPPDE

+

PK-A

PTK

+

+

-Ptyr-Ptase

-

Protein tyrosine phosphrylation

B1

B2

5`AMP

S.P.M.

RecheparinCa

cAMP

pHB3

- Membrane potential

• Capacitation is accompanied by the hyperpolarization of the sperm plasma membrane.

• Membrane hyperpolarization is due to an enhanced k permeability.

Cholesterol efflux

BSAA1 BSA-chol.

+

HCO3

HCO3

A3

Ca

A2

Ca

+ +

AC

cAMPPDE

+

PK-A

PTK

+

+

-

-

B1

B2

RecCa

cAMP

pHB3 5`AMP

Ptyr-Ptase

Protein tyrosine phosphrylation

S.P.M.

heparin

KK

hyperpolarization

+B4

Capacitation

-Free radicals

• The free radicals has a role in sperm lipid peroxidation and sperm viability.

• Superoxide anion cause capacitation and hyperactivationof the spermatozoa.

• Reactive oxygen regulate protein tyrosine phosphorelation of several protein.

Cholesterol efflux

BSAA1 BSA-chol.

+

HCO3

HCO3

A3

Ca

A2

Ca

+ +

AC

cAMPPDE

+

PK-A

PTK

+

+

-

-

B1

B2

RecCa

cAMP

pHB3

heparin

KK

hyperpolarization

+B4

Capacitation

5`AMP

Protein tyrosine phosphrylation

Ptyr-Ptase

S.P.M.

O2

NOB5a

B5b

-Heparin

• Bovine in vitro sperm capacitation can be accomplished in media containing heparin.

• The active capacitating agent in the oviduct fluid is though to be a heparin-like glycosaminoglycan.

• The glycosaminoglycan may promote capacitation by binding to and removing seminal plasma protein.

• Heparin increases cAMP synthesis,elevate pH and regulate the capacitation-associated changes.

Ejaculated SpermD F

Plasma Membrane

Cytoplasm

Nucleus

AcrosomeAcrosome

D F

Cytoplasm

Nucleus

AcrosomeAcrosome

GAG

Cholesterol

Chol-AcceptorD FD FD F

D F

Heparin

Time?

Albumin

Cholesterol efflux

BSAA1 BSA-chol.

+

HCO3

HCO3

A3

Ca

A2

Ca

+ +

AC

cAMPPDE

+

PK-A

PTK

+

+

-

-

B1

B2

RecCa

cAMP

pHB3

heparin

KK

hyperpolarization

+B4

Capacitation

5`AMP

Protein tyrosine phosphrylation

Ptyr-Ptase

O2

NOB5a

B5b

S.P.M.

+

B6

-Glucose

• Glucose has inhibitory or stimulatory actions on capacitation is controversial and is species dependent.

• In bovine glucose inhibits heparin-induced capacitation in vitro.

• Capacitation medium for mouse sperm, which contains glucose has no inhibatory effects .

Cholesterol efflux

BSAA1 BSA-chol.

+

HCO3

HCO3

A3

Ca

A2

Ca

+ +

AC

cAMPPDE

+

PK-A

PTK

+

+

-

-

B1

B2

RecCa

cAMP

pHB3

heparin

KK

hyperpolarization

+B4

Capacitation

5`AMP

Protein tyrosine phosphrylation

Ptyr-Ptase

O2

NOB5a

B5b

+

B6

S.P.M.

glucose Gluc.

pyruvate

2ATP 2NADH 2H

-

B7

The Acrosome ReactionPlasma Membrane

Acrosome

Fused PlasmaMembrane and

Outer AcrosomalMembrane

Nucleus

AcrosinHyaluronidase