Lecture V, Part I: Gamete Lecture V, Part I: Gamete Transport & Acrosomal Transport & Acrosomal

ExocytosisExocytosis

(Fertilization-Part 1)(Fertilization-Part 1)

Dugal Stermer: Of Birds and Bees, A Sexual Study. Collins Publishers, San Francisco, 1995.

Of Bees, Birds and…Copulating Buicks

Bringing Gametes Together:Bringing Gametes Together:• Sperm transport through bovine uterus & oviduct takes 4-16 hours

• Defective/dead spermatozoa pass through oviduct in ~15 minutes (Saacke RG, 2004).

Hurdles:

Spermatozoon:

1. Cervix/uterus2. Uterotubal junction3. Isthmus/Sperm reservoir

Oocyte:

1. Follicle wall2. Infundibulum3. Isthmus

1. Vagina, Cervix & Uterus1. Vagina, Cervix & UterusSemen deposition:Semen deposition: Vaginal (cow, sheep, primates)Vaginal (cow, sheep, primates)Cervical (mouse)Cervical (mouse)Intrauterine (sow, mare)Intrauterine (sow, mare)

Cervix: Cervix: Spermatozoa migrate along the walls & folds, Spermatozoa migrate along the walls & folds, not through the lumennot through the lumen

Challenges:Challenges:Acidic Ph in vagina (high lactate secretion)Acidic Ph in vagina (high lactate secretion)Retrograde flow of semenRetrograde flow of semenLong uterine body (4 cm) and Long uterine body (4 cm) and horns (20-40 cm; bovine).horns (20-40 cm; bovine).Leukocytic infiltration of uterus Leukocytic infiltration of uterus (sperm phagocytosis)(sperm phagocytosis)

Solutions:Solutions: Cervical mucus serves as a vehicle for sperm and a filter for seminal Cervical mucus serves as a vehicle for sperm and a filter for seminal plasma plasma Uterine muscle contractions increase in the late follicular phaseUterine muscle contractions increase in the late follicular phaseTiming of immune response to sperm is delayed (leukocyte infiltration Timing of immune response to sperm is delayed (leukocyte infiltration occurs only after coitus)occurs only after coitus)

Seminal PlasmaSeminal Plasma

Produced by male sex accessory glandsProduced by male sex accessory glandsAlkaline Ph for neutralization of vaginal Alkaline Ph for neutralization of vaginal environmentenvironmentFormation of vaginal /copulatory plug by Formation of vaginal /copulatory plug by semenogelin & protease-nexin 1 (PN1-KO semenogelin & protease-nexin 1 (PN1-KO male mice have watery plug, are subfertile)male mice have watery plug, are subfertile)Protease inhibitors, prostaglandins, immuno-Protease inhibitors, prostaglandins, immuno-suppressors are present in suppressors are present in seminal plasmaseminal plasmaProtective coating of SP-proteinsProtective coating of SP-proteinsInduction of CD removal (boar)Induction of CD removal (boar)Sp proteins necessary for sperm binding to Sp proteins necessary for sperm binding to oviductal sperm reservoir (e.g. BSP1A)oviductal sperm reservoir (e.g. BSP1A)

Acquisition of MotilityAcquisition of MotilityMotility is suppressed in the epididymis (low pH, low lactate, low Motility is suppressed in the epididymis (low pH, low lactate, low ATP)ATP)

Products of male accessory glands may induce motilityProducts of male accessory glands may induce motility

Upon sperm deposition, testis specific, soluble adenylate cyclase Upon sperm deposition, testis specific, soluble adenylate cyclase induces cAMP elevation induces cAMP elevation (Ca2+ Mn2+ is cofactor, not Mg2+; Phosphodiesterase [PDE] is an antagonist)(Ca2+ Mn2+ is cofactor, not Mg2+; Phosphodiesterase [PDE] is an antagonist)

Unidentified regulatory element responds by activating protein Unidentified regulatory element responds by activating protein kinase A (PKA).kinase A (PKA).

PKA is sequestered by A-kinase anchoring proteins (AKAPs) in PKA is sequestered by A-kinase anchoring proteins (AKAPs) in acrosome (AKAP110) and fibrous sheath (AKAP 84).acrosome (AKAP110) and fibrous sheath (AKAP 84).

Possible PK-A target: Possible PK-A target: c-rosc-ros tyrosine kinase: acquired in the initial tyrosine kinase: acquired in the initial segment of epididymis; KO results in immotile spermatozoa.segment of epididymis; KO results in immotile spermatozoa.

2: Uterotubal Junction2: Uterotubal Junction

Challenge:Challenge: folds in mucosa folds in mucosa (dead ends), viscous fluid, (dead ends), viscous fluid, removal of seminal plasma, removal of seminal plasma, removal of defective/slow removal of defective/slow spermatozoa.spermatozoa.

Solution: waves of Solution: waves of contractions.contractions.

3. Oviductal Sperm Reservoir3. Oviductal Sperm ReservoirBinding of spermatozoa to oviductal epithelium in utero-tubal Binding of spermatozoa to oviductal epithelium in utero-tubal junction or isthmusjunction or isthmus

Described first by Yanagimachi and Chang (1963)Described first by Yanagimachi and Chang (1963)

Present in cows, pigs, hamsters, sheep, hares, mares (human???)Present in cows, pigs, hamsters, sheep, hares, mares (human???)

Forms when spermatozoa bind to fucosylated ligands resembling Lewis trisaccharide on the surface of oviductal epithelium.

Proposed Functions:Proposed Functions:

Maintenance of spermatozoa between the onset of oestrus and Maintenance of spermatozoa between the onset of oestrus and ovulationovulation

Synchronization of Sperm & Egg Transport In OviductSynchronization of Sperm & Egg Transport In Oviduct

Prevention of polyspermy Prevention of polyspermy

Capacitation and hyperactivationCapacitation and hyperactivation

Sperm Reservoir: Why Timing Sperm Reservoir: Why Timing MattersMatters

Time difference between Time difference between mating and ovulationmating and ovulation

In cattle AI, earlier In cattle AI, earlier insemination reduces insemination reduces fertilization rates, but fertilization rates, but increases embryo qualityincreases embryo quality

Oviductal Sperm Reservoir: Cont’dOviductal Sperm Reservoir: Cont’d

Binding of spermatozoa:Binding of spermatozoa:

Carbohydrate recognition (lectin-like molecules on the Carbohydrate recognition (lectin-like molecules on the sperm head. Oviductal mucosa protects spermatozoa sperm head. Oviductal mucosa protects spermatozoa against aging and damage.against aging and damage.

Release:Release:

A change it the sperm surface, rather than a change in A change it the sperm surface, rather than a change in oviductal epitheliumoviductal epithelium

Only hyperactivated spermatozoa can detachOnly hyperactivated spermatozoa can detach

Seminal Plasma Glycoproteins That Seminal Plasma Glycoproteins That Could Mediate Could Mediate

Sperm-Oviduct/Reservoir InteractionsSperm-Oviduct/Reservoir Interactions

BSP=BSP=BBovine ovine SSeminal eminal PPlasma proteins produced by seminal lasma proteins produced by seminal vesicles: vesicles:

PDC-109 (BSP-A1/A2)PDC-109 (BSP-A1/A2)BSP-A3BSP-A3BSP-30kDaBSP-30kDa

Present in ejaculated, but not in epididymal spermatozoaPresent in ejaculated, but not in epididymal spermatozoa

Research by Suarez SS, et al.,Research by Suarez SS, et al.,

PDC109/BSP-A1/A2PDC109/BSP-A1/A2

Binds to sperm acrosomal plasma Binds to sperm acrosomal plasma membrane upon ejaculation (purified BSP membrane upon ejaculation (purified BSP binds to epididymal sperm acrosomes in binds to epididymal sperm acrosomes in vitro)vitro)

Stabilizes sperm plasmalemma, perhaps Stabilizes sperm plasmalemma, perhaps prevents premature capacitationprevents premature capacitation

Facilitates sperm binding to oviductal Facilitates sperm binding to oviductal epithelium epithelium in vivoin vivo (purified BSP enables (purified BSP enables epididymal sperm binding to oviductal epididymal sperm binding to oviductal epithelial explants in vitro).epithelial explants in vitro).

Partial loss of BSP during capacitation Partial loss of BSP during capacitation coincides with sperm release from sperm coincides with sperm release from sperm reservoirreservoir

Gwathmey et al., 2003, Gwathmey et al., 2003, Biol. Reprod.Biol. Reprod. 69:809-815 69:809-815

Spermadhesin AQN1Spermadhesin AQN1

Acrosomal Acrosomal surface protein surface protein originating from originating from seminal plasmaseminal plasma

Lost during Lost during sperm sperm detachment from detachment from sperm reservoirsperm reservoir

Sperm Challenges In OviductSperm Challenges In Oviduct

1. Capacitation

2. Hyperactivation

3. Chemotaxis/recognition

4. Cumulus penetration

5. Sperm-Zona Binding

6. Acrosome Reaction

7. Egg penetration (pre-fertilization)

8. Fertilization

Capacitation & HyperactivationCapacitation & Hyperactivation

Oviductal epithelium does not seem to release Oviductal epithelium does not seem to release sperm by reducing binding sites for sperm on the sperm by reducing binding sites for sperm on the epithelial cells; instead, changes in sperm cause epithelial cells; instead, changes in sperm cause the release.the release.

In this sense, epididymal sperm maturation can In this sense, epididymal sperm maturation can be defined as the acquisition of competence to be defined as the acquisition of competence to udenrgo C&Hudenrgo C&H

Capacitation*Capacitation*Set of changes in the sperm plasma Set of changes in the sperm plasma membrane that enables a cell to membrane that enables a cell to acquire fertilizing potential/undergo acquire fertilizing potential/undergo acrosome reactionacrosome reaction

Probably triggered by oviductal Probably triggered by oviductal secretion near the time of ovulationsecretion near the time of ovulation

Requires removal of seminal fluid Requires removal of seminal fluid (has decapacitating activity).(has decapacitating activity).

Asynchronous and Continuous: Asynchronous and Continuous: Only a percentage of spermatozoa Only a percentage of spermatozoa are capacitated and those are are capacitated and those are continuously replaced. continuously replaced.

Capacitated state lasts 50-240 Capacitated state lasts 50-240 minutes. Post-capacitated cells die minutes. Post-capacitated cells die unless they undergo acrosome unless they undergo acrosome reaction.reaction.

*Chang, 1951, Austin 1951, observed that rat spermatozoa cannot penetrate *Chang, 1951, Austin 1951, observed that rat spermatozoa cannot penetrate the egg immediately after coitus and need ~2 h in the female tract to acquire the egg immediately after coitus and need ~2 h in the female tract to acquire such an ability.such an ability.

Capacitation Capacitation II.II.

Initiated in cervix by seminal plasma Initiated in cervix by seminal plasma removal, completed in isthmus during removal, completed in isthmus during detachment from sperm reservoir by detachment from sperm reservoir by female-derived factors.female-derived factors.

Female factors: promote tyrosine Female factors: promote tyrosine phopshorylation, are NOT species specificphopshorylation, are NOT species specific

Sperm Changes:Sperm Changes:

Cholesterol efflux (albumin/BSA serves as Cholesterol efflux (albumin/BSA serves as sterol acceptor sterol acceptor in vitroin vitro) increases ) increases intracellular pH, tyrosine phosphorylation, intracellular pH, tyrosine phosphorylation, plasma sperm membrane fluidity, plasma sperm membrane fluidity, destabilization and fusibility (externalization destabilization and fusibility (externalization of PM receptors).of PM receptors).

Acrosome is altered Acrosome is altered

Increased intracellular Ca2+ levels & pHIncreased intracellular Ca2+ levels & pH

Tyrosine phosphorylation: Possibly Tyrosine phosphorylation: Possibly modulated by ROS (0modulated by ROS (022

--; H; H22OO2; 2; progesterone progesterone stimulates both ROS production and stimulates both ROS production and capacitation); cAMP; Ca2+; PKA; PKC, capacitation); cAMP; Ca2+; PKA; PKC, extracellular signal-regulated kinaseextracellular signal-regulated kinase ((ERKs), phosphatases.ERKs), phosphatases.

NOT FULLY REVERSIBLENOT FULLY REVERSIBLE

Signaling Cascade of CapacitationSignaling Cascade of Capacitation

1.1. Bicarbonate influx via Na/HCO Bicarbonate influx via Na/HCO transporter or via CO2 diffusiontransporter or via CO2 diffusion

2.2. Activation of sperm adenyl cyclaseActivation of sperm adenyl cyclase

3.3. cAMP activates PKA concomitantly cAMP activates PKA concomitantly with cholesterol efflux (depleted by with cholesterol efflux (depleted by BSA)BSA)

4.4. PKa induces plasma membrane PKa induces plasma membrane bilayer distribution (lateral bilayer distribution (lateral redistribution of seminolipid and redistribution of seminolipid and cholesterol)cholesterol)

5.5. Membrane hyperpolarization opens Membrane hyperpolarization opens Ca-channelsCa-channels

6.6. Ca-influx increases tyrosine Ca-influx increases tyrosine phosphorylationphosphorylation

HyperactivationHyperactivation

Increase in the flagellar bend amplitude and asymmetry, Increase in the flagellar bend amplitude and asymmetry, lateral head displacement, velocity observed in lateral head displacement, velocity observed in spermatozoa from oviductal ampulla.spermatozoa from oviductal ampulla.

Enhances release from sperm reservoir, progressive Enhances release from sperm reservoir, progressive motility and sperm penetration of cumulus ECM.motility and sperm penetration of cumulus ECM.

Is accompanied by CaIs accompanied by Ca2+2+ oscillations in sperm head and oscillations in sperm head and midpiece, regulated by cAMP (cyclic-nucleotide gated midpiece, regulated by cAMP (cyclic-nucleotide gated ion channel modulation) and calmodulin (Caion channel modulation) and calmodulin (Ca2+2+ binding binding protein).protein).

Results in tyrosine phosphorylation of AKAP82 and Results in tyrosine phosphorylation of AKAP82 and other proteins.other proteins.

REVERSIBLEREVERSIBLE

CatSper1-4CatSper1-4 Mutant Mice Mutant Mice

InfertileInfertile

MotileMotile

No hyperactivationNo hyperactivation

No penetrationNo penetration

ICatSper = voltage-gated Ca2+ ion-ICatSper = voltage-gated Ca2+ ion-channelchannel

Required for calcium entry in the flagellum Required for calcium entry in the flagellum

Formation of Membrane RaftsFormation of Membrane Rafts

Cholesterol is removed/redistributedCholesterol is removed/redistributedGPI-anchored peripheral membrane proteins cluster togetherGPI-anchored peripheral membrane proteins cluster togetherLipid ordered microdomain (membrane raft) is formedLipid ordered microdomain (membrane raft) is formedCholesterol and saturated fatty acids are enriched in raftCholesterol and saturated fatty acids are enriched in raftFreely diffusable transmembrane proteins are excluded from raftFreely diffusable transmembrane proteins are excluded from raft

From Gadella & Visconti, 2006; In: From Gadella & Visconti, 2006; In: The Sperm Cell,The Sperm Cell, Cambridge University Press Cambridge University Press

Oocyte TransportOocyte Transport

Capture by ovarian Capture by ovarian bursa: facilitated by bursa: facilitated by expanded cumulus, expanded cumulus, fimbria and fimbria and mesosalphinx mesosalphinx contractions. contractions.

Oocyte Transport is Facilitated By:Oocyte Transport is Facilitated By:

Oviductal muscle contractions Oviductal muscle contractions (Low amplitude & high (Low amplitude & high frequency during estrus)frequency during estrus)

Cilia* (back-and-forth Cilia* (back-and-forth movement). movement).

* Female Kartagener’s syndrome/immotile * Female Kartagener’s syndrome/immotile cilia patients and some smokers are cilia patients and some smokers are infertileinfertile

Oocyte Transport Cont’d.Oocyte Transport Cont’d.

•Diameter of oviductal lumen matches the size of ovulatory Diameter of oviductal lumen matches the size of ovulatory product product (no cumulus in marsupials; Bedford, 1996)(no cumulus in marsupials; Bedford, 1996)

•Isthmus is narrow to minimize the likelihood of a miss.Isthmus is narrow to minimize the likelihood of a miss.

Sperm-Oocyte RecognitionSperm-Oocyte Recognition

Components of the olfactory signaling pathways are present in spermatozoa (G-Components of the olfactory signaling pathways are present in spermatozoa (G-protein-coupled receptors, cAMP-gate ion channel, IP3).protein-coupled receptors, cAMP-gate ion channel, IP3).

N-formyl-Met-Leu-Phe (fMLP), progesterone and atrial natriuretic peptide (ANP) often N-formyl-Met-Leu-Phe (fMLP), progesterone and atrial natriuretic peptide (ANP) often debated but none provendebated but none proven

Spermatozoa acquire chemotactic responsiveness during capacitationSpermatozoa acquire chemotactic responsiveness during capacitation

Exposure to cumulus cells alters sperm motility Exposure to cumulus cells alters sperm motility

SMIF, sperm motility initiation factor in fish; SPERACT and RESACT peptides in sea SMIF, sperm motility initiation factor in fish; SPERACT and RESACT peptides in sea urchin; no known homologues in mammals.urchin; no known homologues in mammals.

• Chemotaxis: Chemo-attractants may Chemotaxis: Chemo-attractants may be present in follicular fluid be present in follicular fluid

that accompanies the eggs that accompanies the eggs

• Thermotaxis: 2Thermotaxis: 2ººC difference between C difference between isthmus and ampullaisthmus and ampulla

A model for Mammalian Gamete A model for Mammalian Gamete Transport (Suarez, 2002).Transport (Suarez, 2002).

1. Deposition of spermatozoa & sperm-protective seminal plasma1. Deposition of spermatozoa & sperm-protective seminal plasma

2. Acquisition of motility & rapid swimming 2. Acquisition of motility & rapid swimming through cervixthrough cervix

3. Sperm movement through uterine cavity 3. Sperm movement through uterine cavity assisted by muscle contractionsassisted by muscle contractions

4. Slowing of the sperm in uterotubal junction or 4. Slowing of the sperm in uterotubal junction or isthmusisthmus

5. Binding to oviductal epithelium (sperm reservoir)5. Binding to oviductal epithelium (sperm reservoir)

6. Capacitation, hyperactivation and sperm 6. Capacitation, hyperactivation and sperm release induced by ovulationrelease induced by ovulation

7. Oocyte picked by cilia on fimbria, moved down ampula by cilia and contractions.7. Oocyte picked by cilia on fimbria, moved down ampula by cilia and contractions.

8. Fertilization occurs in ampullary-isthmic junction8. Fertilization occurs in ampullary-isthmic junction

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