Fertilization

Sperm motility score

Represents the percentage of sperm that are actively swimming in a forward direction

How long does it take for sperm to reach the egg in the oviduct?

Species specific

Average = 15 minutes

Wh

Cows/rabbits = few minutes

Mouse, guinea pig, sow, rat = 15 minutes

Hamster = 2-60 minutes

Dog/ewe = few minutes to hours

Woman = 5 minutes - 68 minutes

What is the fertile life of sperm after ejaculation?

Species specific

Range: 1.5-6 days

Rabbit: 1.5 days

Cattle/sheep: 1-2 days

Horse: 4-5 days

Human: 5-6 days

What is the fertile life of an egg after ovulation

Species specific

Range: 8-24 hours

Rabbit: 8 hours

Sheep: 15-24 hours

Cattle/horse/human: 24 hours

What direction is the egg traveling in?

Down the oviduct

What happens in the infundibulum?

Cilia-oocyte cumulous complex "pick up" the egg + granulosa cells

Lots of cilia to help move the egg

What is the site of fertilization?

The ampulla

Oocyte cumulous cell (OCC)

Ova + the granulosa cells that surround the oocyte in the ovary

Specific adhesion between cilia and OCC matrix

OCC is compacted by the cilia in the infundibulum - churning

Needs to be compacted or the OCC won't fit in the oviduct

Species variation in sperm count/ejaculate

Boars/bulls/rams: 10 billion

Rodents: 50-60 million

Humans: 200-500 million

What sperm count is considered infertile in humans?

Anything below 200 million/ejaculate

How many sperm meet the egg in the ampulla?

Under 100

Only some of those become able to fertilize an egg, and even fewer are actually successful

99.9%+ don't get there

In what species is sperm deposited vaginally?

Cows

Sheep

Rabbit

Rodents

Primates

Dogs

Cats

In what species is sperm deposited in the cervix?

Pigs

Horses

Camelids

Physical nature of the ejaculate - pigs/horses

Final fraction highly viscous - "rice pudding"

Gelatinous - blocks other animals from mating (forms plug for boars)

Physical nature of the ejaculate - rodents

Coagulating protein in semen forms vaginal plug

Good mating marker

Physical nature of the ejaculate - pigs/camelids

Seminal fluid can induce ovulation

Physical nature of the ejaculate - pH

In humans and bulls

Seminal fluid: neutral pH (6.7-7.4), allows for maximum sperm movement

Neutralizes vaginal acidity (pH 4) - hydrogen ions inhibit sperm movement

Phagocytosis process

Neutrophils (leukocytes or WBCs) are recruited during estrus to line vagina

Scavenge any dead sperm, sperm that aren't moving well, bacteria, viruses, etc and eat them

First line responders to infections - attacks "foreign" proteins

Huge spike in neutrophils immediately following insemination from the uterine mucosa into the uterine lumen

Phagocytosis importance

Sperm loss/leukocytes prevent tract infection

What controls contractility of the reproductive tract?

Estradiol

Progesterone

Oxytocin

PGF2a & PGE1

Estradiol effect on contractility

High at insemination

Increases contractility

Progesterone effect on contractility

Not present during estrus

Blocks contractility

Oxytocin effect on contractility

Released at coitus

Increases contractility

Prostaglandins effect on contractility

Present in seminal plasma

Increase contractility

Effect of smooth muscle stimulants of fertility

Increases fertilization due to increased contractility

Done with tests on rabbits:

Saline = 16% fertilization

Phenylephrine = 52%

Ergonovine = 63%

What are the types of cervical mucus?

Sulfomucin

Sialomucin

Sulfomucin

High viscosity = sticky

Apical - sits on top of ridges

Barrier to sperm due to stickiness - if sperm aren't swimming well, they get stuck

Sialomucin

Low viscosity

In basal areas of cervical crypts - valleys

Sperm that move well can get into the basal area and move much more easily

"Privileged pathways"

Regions where it is easy for sperm to move through cervix into vagina

Through sialomucin in basal layers of cervix

Also acts as storage - allowing for slow release of sperm from a reservoir

Artificial insemination - what locations are best for sperm depositing?

Uterine body/uterine horn

Cervix is a big barrier to insemination - sperm deposited in the cervix is much more likely to flow back out of the vagina

AI techniques

Intracervical insemination - within the cervix (sows)

Transcervical insemination - bypass the cervix and inject directly into the uterine horns (cows)

Factors that inhibit sperm

Low pH

Mucins

Neutrophils

Retrograde loss

Sperm-sperm competitionF

Factors that protect sperm

Seminal fluid (pH, ovulation triggers (prostaglandins))

Smooth muscle stimulants

Sheer numbers

Chemotaxis

Production of a signal that attracts sperm

Resact

Sea urchin peptide (14aa) emitted by egg

Sperm can detect the concentration gradient in the water to find the egg

Increases sperm motility

Chemotaxis in mammals

Sperm attracted to follicular fluid, eggs, and cumulous complexes

We don't know what it is that attracts the sperm

Depends on capacitation

Thermotaxis

Sperm can orient in the thermal gradient and move up the temperature gradient

Human oviduct:

Isthmus = 37 C

Ampulla = 39 C

Sperm become hyperactive at increased temperatures

Capacitation

Series of changes that render sperm competent to fertilize the egg

Sperm mature at the epididymis but require an extra step to properly dock at the egg

Not species specific

Reversible (add back seminal plasma and sperm will be decapacitated)

Capacitation steps

Epididymal:

Surface proteins, CHO added

Ejaculated:

Seminal plasma coats surface proteins to prevent them from being activated

Capacitated:

Seminal plasma coating + some surface proteins removed by the female tract (pH and fluid of tract)

Why is capacitation important?

Holds sperm in check - prevents sperm from trying to fertilize random cells

As seminal plasma is removed, sperm gain ability to penetrate oocyte

Capacitation to fertilization sequence of events

Hyperactive motility

Binding to ZP

Acrosomal reaction

Penetration of ZP

Sperm-oocyte membrane fusion

Sperm engulfed

Decondensation of sperm nucleus

Formation of male pronucleus

Sperm hyperactive motility

Sperm become hyperactive in the oviduct

Motility patterns change from linear to more frenzied

Big sweeping motion helps chemotaxis and facilitates sperm/oocyte contact

What controls sperm hyperactive motility?

Controlled molecular reaction

CatSper protein - opens Ca2+ channels = increased motility

Sperm-egg recognition

ZP = Mucopolysaccharide shell = extracellular matrix of the egg

ZP is vital for fertility, blocks polyspermy, and ensures species specific sperm-egg binding

Polyspermy

Fertilization of oocyte by more than one sperm

Results in embryo death

ZP proteins

ZP1, ZP2, ZP3

ZP1 & ZP2

Structural proteins - integrity

Change when block to polyspermy happens

ZP3

Receptor for spermatozoa - what sperm recognize

Acrosomal reaction

Enzymes in acrosomal cap are activated

Proacrosin = inactive, high affinity for ZP

Acrosin = active, hydrolyzes (weakens) ZP locally

Steps leading up to acrosome reaction

Sperm plasma membrane contains 2 receptor like regions - Zona Binding Region (ZBR) and Acrosome Reaction Promoting Region (ARPR)

Both bind to ZP3

ZBR secures sperm to ZP3

ARPR initiates release of acrosomal enzymes

Parts of sperm head before acrosome reaction

Nucleus

Inner acrosomal membrane (IAM)

Acrosomal contents

Outer acrosomal membrane (OAM)

Plasma membrane covers everything

Also post nuclear cap - covers the bottom half of the nucleus below the acrosome

What occurs during acrosome reaction

Plasma membrane fuses with the OAM --> vesiculation

Basically forms a bunch of bubbles similar to chainmail - acrosomal contents leak through

What happens after the acrosome reaction?

Oocyte plasma membrane fuses with equatorial segment (brought on by fusion protein)

Acrosomal contents and the plasma membrane bubbles disconnect - the sperm is now the nucleus, with the IAM on the top and the post nuclear cap/plasma membrane on the bottom

Equatorial segment

Middle part of the sperm head at the very bottom of the acrosome

Fold in the plasma membrane

Has equatorial fusion proteins

Order of events of acrosome reaction

Sperm binds to ZP

Acrosome reaction begins

Penetration of ZP

Sperm oocyte fusion

Cortical reaction

Cortical reaction

Little vesicles full of enzymes sit right under the surface of the oocyte plasma membrane

Once the sperm fuses with the oocyte, exocytosis of cortical granules harden the ZP = block to polyspermy

Fusion events

Complete penetration

Fusion begins

Nucleus breaks apart

Where does fusion occur?

Perivitelline space - between the ZP and the oocyte plasma membrane

Before membrane fusion

Microvilli on oocyte grab at sperm

During membrane fusion

Oocyte plasma membrane fuses with equatorial segment of sperm - the sperm plasma membrane becomes part of the oocyte plasma membrane

Cortical granules are released into the perivitelline space

After membrane fusion

Oocyte plasma membrane finishes engulfing sperm

Cortical granules all released

Sperm nucleus decondenses - DNA unwinds (disulfide x-links reduced) so it can interact with the egg (egg nucleus also decondenses)

Sperm mitochondria degrade

What are the types of blocks to polyspermy

Zona block

Vitelline block

Zona block

Cortical reaction creates barrier at ZP - ZP changes to block polyspermy

All species

Vitelline block

Cortical reaction can alter oocyte membrane - changes fusion proteins on egg so they can't be recognized

Only some species

Syngamy

Moment of fertilization

Fusion of male and female pronuclei - haploid genomes mix together to form a diploid

Zygote formed

Twins

Identical = monozygotic

Fertilized egg --> 2 cell stage --> single zygote divides in half

Fraternal = disygotic

2 fertilized eggs --> each becomes their own zygote

Fast block vs slow block to polyspermy

Non mammals:

Fast block

Electrical

Mammals:

Slow block

Cortical granules

Fertile period

Can get pregnant up to 5 days before ovulation, but the closer to ovulation copulation is, the higher the likelihood of pregnancy

Sequence of events overview

1. Immediate transport

- Retrograde loss

- Phagocytosis

- Entrance into cervix/uterus

2. Cervix

- Privileged pathways

- Removal of non-motile sperm

- Removal of some abnormalities

- Cervical crypts

3. Uterus

- Capacitation initiated

- Phagocytosis

4. Oviduct

- Capacitation completed

- Hyperactive motility

5. Fertilization

- Acrosome reaction

- Spermatozoon penetrates oocyte

- Male and female pronuclei form

What controls contractility of the reproductive tract?

Estradiol

Oxytocin