ASCI 341-Sperm Transport, Capacitation, and Fertilization

Movement of Sperm

1-immediate transport

(retrograde loss from backflow) plugs to prevent backflow

phagocytosis-neutrophils

entrance into cervix/uterus, sperm need to get on mucus into cervical crypts/cervix

2 cervix

“privileged pathway”

removal of nonmotile sperm

removal of tail abnormalities

3 uterus

CAPACITATION initiated

phagocytosis

4 Oviduct

capacitation completed

hyperactive motility  in UTJ (to contact oocyte)

5 Fertilization

acrosome reaction

spermatozoa penetrates oocyte

male and female pronuclei form

Relative neutrophil numbers

at insemination really high, trail off over 48 hours

two types of sperm transport

rapid- reach oocyte within a few minutes

non viable

Sustained- sperm are transported to oviducts in a trickle-like effect from reservoirs in cervix + UTJ\

-viable

Retrograde loss

putting semen further up in uterine horn to reduce retrograde loss (no difference in percent of sperm recovered)

if it cervix deposition there is DRAMATIC loss

AI fact

60% of sperm deposited in cow uterus via AI are lost to the exterior of the tract within 12 hours after deposition by retrograde loss (transport)

Comparing rabbit treatments

control 16% fertilization

treatment with agonists of smooth muscle contractions

phenylephrine or ergonovine

fertilization 52% and 63%

Ovary treatments

No seminal plasma on one side, ligate other uterine horn with seminal plasma + ovulation of egg 11 hours earlier

specific factor fund by people in Germany

2 types of mucus in cervix

sinlomucin= low viscosity—produced by cells of cervical crypts = “priviledge pathway”

sperm can move through this

Sulfomucin= high viscosity

apical areas of surface epithelium= sperm CANNOT move through this

Sperm movement

Moving through epididymis = complement of surface molecules (proteins, carbs, provide surface) obtain with maturation + seminal plasma

After mixing with seminal plasma

will be ejaculated (also coated with specific proteins from seminal plasma) + female tract

As they enter the female tract, proteins are removed, opens recognition site so that sperm can interact with the zone of pellucida

Capacitation of sperm

a biochemical change to sperm that allows the true acrosome reaction to occur (occurs as sperm move through reprotract to be allowed to bind to oocyte)

removal of glycoproteins coating the sperm head

in uterus + oviduct

Rabbits

Follicular phase

uterus 10 hrs

oviduct 13hrs

BOTH = 6 hours, reduces time to capacitate with coordination between uterus and oviduct

sperm can capacitate during luteal phase, it just takes a long time (24 hrs)

Time to capiciate

cow 3-6 hrs

pigs 3 hrs

ewe 1-2 hrs

Decapacitation factors

glycoproteins from epididymis and seminal plasma which coat the sperm

Seminal plasma contains factors which inhibit sperm from fertilizing the egg

oviductal and uterine fluid remove coat allow capacitation

Flow of effort in capacitation

hyperactive motility (to initiate sperm oocyte contact)

binding to zone of pellucida

acrosomal reaction

penetration of zone of pellucida

Sperm-oocyte membrane fusion

Sperm engulfed

Decondensation of sperm nucleus

Formation of male pronucleus

Hyperactive motility

occurs in the oviduct-sperm change from a linear progressive movement to a frenzied dancing motion that isn’t linear and localized in a small area

facilitates the sperm oocyte contact

Zone of pellucida 3 glycoproteins

zona protein 1 and 2-structural protein maintain integrity of the zone of pellucida

zona protein 3-like receptor for hormone, binds to proteins on sperm plasma membrane

sperm binding to zone of pelluicda requires 10,000-50,000 ZP-3

two binding sites on sperm plasma membrane

zona binding region (ZBR)-actually binds to sperm on ZP3 site

acrosome reaction promoting region (ARPR)

binding between this region and ZP3 activates signal transduction cascade (second message system) initiate acrosome reaction

coordination of events

sperm transport with estrogen production

if breed too early, the sperm will die

if oocyte aging also bad for fertile life of sperm

can increase polyspermy

Ovulation

copulation closer to ovulation cause pregnancy (best case scenario is still only 33%)

process

perivitelline space, sperm fuses with vetilline membrane and iniate meiosis 2

post acrosomal region of sperm attaches to vitelline membrane for first polar body

oocyte phagocytosis to uptake sperm

pronuclei aof male/female forms

syngamy-fusion of pronucli to create diploid zygote

no nuclear membrane formed until duplication of chromosome/cell division

1st CLEAVAGE DIVISION
hours after sperm penetration

12-14 sow

16-21 in ewe

20-24 in cow

Vesiculation

multiple fusion sites between plasma membrane and outer acrosomal membrane of sperm results in the formation of membrane vesicles

tiny pores created for acrosomal enzymes to escape

two purposes of acrosome reaction

enables sperm to penetrate zone of pellucida

exposes equatorial segment so that it can later fuse with the plasma membrane of the oocyte

penetration of the zone of pellucida only takes a few minutes

cortical reaction and cortical granules

during 1st and second meiosis

small dense coritcal granules move to periphery of the oocyte cytoplasm

cortical granules contain:

mucopolysaccharides

proteases

plasminogen activator

acid phosphatase

oxidase

Two blocks to polyspermy

zona block (reaction): zona pellucida changes biochemically so that sperm can’t penetrate it SLOW

vitelline block: FAST prevents additional sperm from binding to the oocyte plasma membrane

zona block versus vitelline block

zona block: sheep, dog, mouse

vitelline block: rabbit, pig

Order

hyperactive motility

binding to zona pellucida

acrosomal reaction

penetration of zona

sperm oocyte fusion

sperm engulfed

decondensation of sperm nucleus

formation of male pronucleus

connection between egg and sperm

zp3 on outside of zone of pellucida

zpr and arpr then acrosome contents

then sperm nucleus

acrosome reaction

binding of zp3 to arpr and zpr

fusion sites between outermembrane and plasma membrane,

causes fusion bubble sites (vesicles)

leads to vesiculation

creates tiny pores to let enzymes out of acrosome

eventually vesicles sloughed from head, now just inner acrosomeal membrane + nuclear membrane

along equatorial segment——reveal fusion proteins to allow sperm to interact with the plasma membrane

sperm past zone of pellucida

perivitelline space

microvilli from oocyte (cortical granules after first and second meiosis) around peripheral of cytoplasm

sperm on bed of microvilli directed b y fusion protein equatorial with plasma membrane of oocyte

becomes engulfed

sperm head and tail cytoplasm

cortical granules fuse with plasma, exoctyosis, contents of cortical granules enter perivitelline space

^cortical reaction

changes in cytoplasm of oocyte and zone of pellucida

do not want ploidy problems

(vitelline = plasma membrane of ooctye) fast block as want to prevent sperm if also in perivitelline space

nuclear membrane disappear, inactivated DNA are unwinding to form male pronucleus

changes to block polyspermy

sperm attach to vetilline membrane

lose sperm receptors for zona pellucida

zona hardening like forcefield to prevent digestion

make vitelline membrane altered so less penetrable

mouse have like none on the zone of pellucida

other species are caked

Process based off of diagram

plasma membrane of sperm binds to zp3

zpr + arpr, acrosome contents, slightly bigger whole of digestion

use tail to help penetrate

sperm enter perivitelline space, lay of bed, fusion of equatorial segment, induces: extrusion of second polar body (1n)

cortical granules undergo exocytosis for zona block and vitelline block to polyspermy

zona-take away zp3

sperm + tail engulfed into cytoplasm, nuclear membrane break down

things that decrease effectiveness of blocks of polyspermy

age of ova (need to time insemination, older is worse)

heating ova, increases metabolism and lifespan of egg

fever/environment

excessive sperm at site of fertilization

AIJ, cervix, uterus, UTJ (blocks for polysperm)

need to not overrun them

Fertile lifespan of sperm + egg

sperm longer than oocyte

6-8 hours in horse oocyte, sperm 72 hr-120 hrs

coordination of events

breed female early to allow capacitation, but not too early, breed late enough so the fertile life span match

but not vulnerable to polyspermy