Molecular events involved in the promotion and regulation of capacitation Dr Rachel Gibbons Clinical Embryologist King’s College Hospital.

Molecular events involved in the promotion and regulation of capacitation

Dr Rachel Gibbons

 Clinical Embryologist

King’s College Hospital

Points to be addressed

• What is sperm capacitation and why is it important?

• What mechanisms are involved in promoting and/or regulating capacitation and the acrosome reaction?

• Might capacitation defects be linked with reduced fertility?

• Can basic research results be applied to clinical practice to enhance fertility in vitro?

• Might environmental compounds affect fertility in vivo?

What is capacitation?

• Capacitation involves a physiological ‘switching on’ of sperm so that they can fertilize an egg. Capacitation takes time.

• The sperm must undergo many changes during capacitation..

• When released from the male reproductive tract, mammalian sperm will not be capable of fertilising an oocyte immediately.

• They must first undergo a process called capacitation.

Changes that occur during capacitation . .

• The loss of decapacitation factors

• Stimulation of G protein-coupled receptor pathways that lead to the production of cAMP which eventually leads to an increase in protein tyrosine phosphorylation

• Changes in intracellular ion concentrations e.g. a steady rise in intracellular Ca 2+

• Changes in membrane structure and fluidity

• These are referred to as ‘decapacitation factors’ (DF). During capacitation, DF molecules are lost and this allows the sperm to ‘switch on’.

• If DFs are added to capacitated suspensions, the sperm will be ‘switched off’ and revert to the uncapacitated state, hence the term decapacitation.

• Therefore, capacitation is reversible.

• When released from the male, mammalian sperm will have a number of different molecules present on their plasma

membrane that keep sperm uncapacitated.

Decapacitation Factors

Sperm have a long journey to reach the oocyte, so it is useful that they are not immediately able to fertilize; they can ‘switch on’ as they are moving towards the site of fertilization.

What is required for successful fertilization?

• Once capacitated, sperm can undergo the acrosome reaction if they contact an unfertilized egg.

• Sperm that undergo the acrosome reaction spontaneously, will not be able to attach to the egg and so will be non-

fertilizing.

• Ideally, capacitation should be regulated so that sperm capacitate but are prevented from undergoing spontaneous acrosome loss. That way, fertilizing potential will be

retained until an egg is in sight!

• Special molecules on the sperm plasma membrane bind to complementary proteins on the egg’s zona pellucida.

Special molecules for docking with the zona pellucida

The Acrosome Reaction

In normal fertilization, (1) the capacitated sperm binds to the zona pellucida, (2) undergoes the acrosome reaction, (3) penetrates the zona and then (4) fuses with the oocyte.

Investigating capacitation

How can one analyze treated sperm suspensions for effects on capacitation?

spontaneous acrosome reaction

induction of acrosome reaction

chlortetracycline (CTC) patterns *

in vitro fertilization *

biochemical parameters (e.g., cAMP production,

protein tyrosine phosphorylation) *

CTC fluorescence patterns

Pattern Functional state Acrosome

F uniform fluorescence

uncapacitated present

B fluorescence-free band in posterior head

capacitated(potentially fertile)

present

AR dim/absent fluorescence

capacitated absent

CTC fluorescence patterns in mouse sperm

F B

AR

CTC fluorescence patterns in human sperm

F B

AR

Mouse sperm decapacitation factor (DF)

• Mouse sperm have a DF with fucose residues that enable DF to bind to a receptor (DF-R) with fucose binding sites. A GPI anchor is involved in attachment of DF-R to the membrane.

• DF binding to its receptor results in activation of a calcium ATPase, resulting in maintenance of low intracellular calcium .

• During capacitation, DF is lost; this allows intracellular calcium to rise. Centrifugation removes the DF from

mouse sperm, resulting in accelerated capacitation.

ATPADP+PiCa2+

Ca2+

GPIanchor

Ca2+

ATPaseCa2+

Ca2+

DF

R

Plasma Membrane

Cytosol

R

Initial hypothesis for mechanism of action of DF

Fucose residues

Adding exogenous fucose to uncapacitated mouse sperm suspensions during the final 10 min of a 25 min preincubation period accelerates capacitation as evidenced by a significantly higher fertilization rate.

Treatment Fertilized oocytes/ Polyspermic total oocytes, oocytes

__________________________________________________________________

Control 95/189, 50.3% 2/95, 2.1%

5 mM fucose 255/343, 74.3%** 41/255, 16.1%__________________________________________________________________

**P<0.025 compared with control suspensions; oocytes fixed 75 min after sperm addition and evaluated for the presence of one or more decondensing sperm heads (n=4).

Enhanced fertilizing ability in the presence of exogenous fucose which can displace DF molecules

Recent experimental results

• The mouse DF-R has been purified, sequenced and found to have high homology with phosphatidylethanolamine-binding protein 1 (PEBP 1).

• An anti-peptide antiserum to PEBP 1 has been used for Western blotting and immunocytochemistry of both mouse and human sperm.

PEBP 1 localization - mouse

Uncapacitated

DF-depleted(spun at 30 min)

Capacitated(inc ~2 h)

Untreated DF-treated PI-PLC treated

• Intensity of PEBP 1/DF-R staining is capacitation state-dependent & reversible (note that adding DF to DF-depleted or capacitated cells changes the staining pattern to that of uncapacitated cells).

• PI-PLC treatment to remove GPI-anchored proteins essentially abolished staining.

• Mouse sperm DF can bind to capacitated human sperm and decapacitate them.

• Addition of exogenous fucose to human sperm suspensions accelerates capacitation.

Human sperm have a similar DF/DF-R system

PEBP 1 localization – human

Uncapacitated

Capacitated

Untreated PI-PLC treated

• PEBP 1/DF-R staining distribution is similar to that of mouse sperm but capacitation state-dependent changes are less obvious.

• PI-PLC treatment to remove GPI-anchored proteins again abolished staining.

Hypothesis for DFDF-R mechanism of action

DF-R/PEBP 1 plays its fundamental role in capacitation by causing alterations in the sperm plasma membrane in both head and flagellum. This has functional consequences for

many membrane-associated proteins, not just Ca2+-ATPase.

Possible links with infertility

• If DF molecules are not lost, sperm would never switch on.

• Sperm might have no or little DF, leading toprecocious capacitation and early acrosome loss.

• Such defects would not be associated with reduced sperm numbers, so could account for ‘unexplained’ infertility. On the next slide, all samples had reasonable sperm concentrations and

motility, but many had atypical proportions of capacitated and/or acrosome-reacted cells.

Mechanisms that promote & regulate capacitation

• When released into an appropriate environment, sperm usually begin to capacitate and then continue until some undergo spontaneous acrosome reactions; we have called these sperm ‘over-capacitated’.

• Biologically, this is undesirable since these sperm will be non-fertilizing.

Regulation of capacitation is needed to reduce spontaneous acrosome loss so that capacitated sperm maintain fertilizing potential.

Interesting small molecules

• FPP (fertilization promoting peptide) *• Adenosine *• Calcitonin *

All act as ‘first messengers’ to regulate capacitation, first stimulating cAMP and capacitation, then inhibiting cAMP and the spontaneous acrosome reaction. This prevents ‘over-capacitation’. (* found in seminal plasma)

• Adrenaline/noradrenaline

G protein coupled receptor pathway

• All of these molecules are first messengers, which means they act on the outside of the cell.

•This therefore implies the need for there to be an internal signal transduction pathway in order for them to have an effect

•This pathway is a GPCR pathway..

G protein coupled receptor pathway

First messenger

First messenger

StimulatoryG protein

InhibitoryG protein

Membrane adenylyl cyclase

cAMP

Protein kinase A

Phosphotyrosine kinases

Protein tyrosine phosphorylation

Capacitation

Components of signal transduction pathways able to regulate cAMP are present in the same regions

• Receptors for FPP, adenosine, adrenaline and calcitonin are found in both the acrosomal cap region and the flagellum.

• Some stimulatory and inhibitory G proteins and mACs, involved in relevant signalling pathways, are also found in these same places.

Receptors on sperm

(all GPCRs that regulate cAMP production)

• Adenosine: both stimulatory & inhibitory receptors are present and function in a capacitation state-

dependent manner

• Adrenergic: both stimulatory & inhibitory receptors are present and function in a capacitation state-dependent manner

• Calcitonin: only one population of receptors but first stimulate and then inhibit cAMP

Gs

Golf

Gi2

Mouse Human

Immunolocalization of G subunits

mAC isoform distribution in mouse sperm

mAC2

mAC3 *

mAC4

mAC8 *

Stimulatory A2A receptors in: (a) uncapacitated, (b) capacitated, (c) capacitated cells + DF – show localization seen in uncapacitated cells (a)

Inhibitory A1 receptors in: (d) uncapacitated and (e) capacitated cells

Localization of adenosine receptors: note that staining pattern is capacitation state-dependent

Relevance to successful fertilization?

Since these first messengers present in seminal plasma and body fluids can regulate mAC/cAMP pathways in vitro, they might also regulate sperm function in vivo. This would maximize the number of potentially fertilizing sperm in the vicinity of unfertilized oocytes.

Sperm used for ART are removed from seminal

plasma and so are no longer in contact with these peptides.

Possible links with infertility

• Defective receptors might allow sperm to acrosome-react precociously and so become non-

fertilizing.

• A lack of ‘first messengers’ might allow sperm to acrosome-react precociously.

• Treatment: These molecules could be added to sperm suspensions used for IVF and/or IUI to accelerate capacitation and ensure early penetration.

The presence/absence of DF bound to DF-R alters the activity of several G protein-coupled receptors

(GPCR), not just Ca2+-ATPase

• Adenosine receptors

• Calcitonin receptors

• Adrenergic receptors

Could exogenous compounds affect sperm function?

• Estrogenic xenobiotics, eg, genistein (in soybeans)

• Compounds structurally related to amphetamine

• Genistein at low nM concentrations in vitro accelerates capacitation and then stimulates the acrosome reaction in both mouse and human sperm. Human sperm appear to be even more responsive than mouse sperm.

• These low concentrations are well within the range found in human females 3 h after drinking 25 g of soy milk.

• Similar responses in vivo could result in sperm losing fertilizing ability before contacting an egg: already acrosome-reacted sperm cannot bind to the zona.

Mechanism of action

• These xenobiotics appear to stimulate capacitation and acrosome loss by causing unregulated cAMP production.

• These weakly estrogenic xenobiotics appear to act at the

sperm cell surface rather than via classical estrogen receptors.

• These effects observed in vitro, especially the rapid occurrence of spontaneous acrosome reactions, might have negative

implications for fertility in vivo.

• 2receptors that inhibit mAC/cAMP

Sperm also have two populations of adrenergic receptors:

• 1, 2 and 3 receptors that stimulate mAC/cAMP

Conclusions

• These effects observed in vitro, especially inhibition of the spontaneous acrosome reaction, might have positive

implications for fertility in vivo, especially in females who use drugs with structural similarities to amphetamine.

• Many individuals use amphetamine-related compounds for medical and/or social reasons: (1) khat leaves for recreational purposes, (2) amphetamine-related drugs for social (e.g., Ecstasy) or addictive (e.g., Dexedrine, Benzedrine) purposes & (3) amphetamine-related drugs for medical conditions (e.g., salbutamol for asthma).

• These could bind to adrenergic receptors on sperm and have a positive effect on fertility in vivo, even though this may not have been the intended result

• Signal transduction mechanisms that are able to regulate capacitation may provide clues for ways to enhance fertility in vitro.

Main conclusion – Sperm are very important!!!

• Defects in capacitation may account for fertility problems in some couples.

• Environmental compounds might affect fertility in vivo either in a positive or a negative way.