Fertilization

Front 1

a. How long after ejaculation is the sperm viable?
a. After the egg has been ovulated, how long is it receptive to sperm?

Back 1

b. About 72hrs
b. Between 15-48hrs

Front 2

There is a window of time that intercourse must happen for egg and sperm to be viable:

Back 2

Lasts between 72 hours before ovulation, and no later than 24 hours after ovulation (but usually 15 hours). Best time is at ovulation.

Front 3

Sperm’s Journey

Back 3

Produced in the seminiferous tubule -> rete testis -> efferent ductile -> epididymus -> vas deferens -> ejaculatory duct -> urethra -> exported into female tract

Front 4

How do sperm move from Sertoli cells -> Rete testis ?

Back 4

Aided by reabsorption of water. Pulls sperm from the sertoli cells into rete testis. Sertoli cells also release luminal fluid that push sperm into the rete testis.

Front 5

How do sperm move from Efferent ductules -> Epididymus ?

Back 5

Cilia movement in the efferent ductules causes movement in one direction. Smooth muscles of epididymus contract in waves through peristalsis like contractions and propel sperm forward.

Front 6

How do sperm move from Epididymus -> Vas deferens -> Ejaculatory duct -> Urethra ?

Back 6

Also use powerful peristaltic contractions.

Front 7

Divisions of the Epididymus:

Back 7

1. Head (caput)
a. Contains efferent ducts
2. Body (corpus)
3. Tail (cauda)

Front 8

Days to go from the head to tail of the epididymus:

Back 8

Takes between 6-12 days

Front 9

Changes to sperm between the head and tail of the epididymus:

Back 9

1. Sperm is concentrated
2. Change in shape and size of the acrosome
3. Sperm acquire motility
4. Spermatozoa is coated in glycoproteins
5. Change in lipid composition of the plasma membrane makes it more stable
6. Change in metabolism
a. Changes from fatty acid oxidation -> increased dependence on external sources, specifically fructose, for glucose oxidation as a means to obtain ATP

Front 10

Cells that line the epididymus also secrete nutrients for the sperm:

Back 10

1. Carnitine
Transports long fatty acid chains into the mitochondria for energy production
2. Glycerophosphorylcholine
3. Glycoproteins
Coat the head of the sperm
4. Fructose

Front 11

Sperm maturation in the epididymus is dependent on __ presence.

Back 11

androgen

Front 12

Androgens reach the epididymus through the __, bound on __ secreted by the __.

Back 12

lymphatic system, ABP, Sertoli cells

Front 13

Semen is composed of:

Back 13

1. Spermatozoa
2. Seminal fluid
a. 60% seminal plasma
b. 30% prostate secretion
c. 10% bulbourethral secretion

Front 14

The __ lie behind the bladder. The __ is below, and then the __. As sperm pass through the duct system, these glands secrete their substances into it.

Back 14

seminal vesicles, prostate gland, bulbourethral gland

Front 15

Contributions of seminal vesicles:

Back 15

1. Water
2. Fructose
3. Fibrinogen
a. Liquid form of protein involved in blood clotting
4. Ascorbic acid and citric acid
a. Serve as nutrients
5. Prostaglandins

Front 16

Contributions of the prostate:

Back 16

1. Water
2. Bicarbonate
3. Fibrinogenase
4. Fibrinolytic enzyme
5. Acid phosphatase

Front 17

Contributions of the bulbourethral gland:

Back 17

1. Water
2. Phosphate and bicarbonate
3. Mucus
a. For lubrication

Front 18

Semen is released in 3 stages:

Back 18

1. Excitement phase
2. Orgasmic phase
Prostate and the seminal vesicles are stimulated to secrete their portion of semen
3. Resolution phase

Front 19

Ejaculation involves __ of __ surrounding the urethra.

Back 19

rhythmic contractions, smooth muscle

Front 20

Sperm must swim about __ inches through the female reproductive tract.

Back 20

6

Front 21

Millions of sperm are deposited by ejaculation, but many are lost because of death, improper direction, swimming in circles, etc. Only __ sperm will make it to the uterus.

Back 21

100

Front 22

After 1min in the vagina, the sperm becomes thicker due to action of __—converts __ into __. Coagulation occurs to prevent loss of sperm from the vagina. Semen then liquefies by the action of __, allowing the sperm to start swimming into the female tract.

Back 22

fibrinogenase, fibrinogen, fibrin, fibrinolytic enzyme

Front 23

The vagina has a pH of __. The semen have __ reagents like __ and __ that neutralize this pH to __. The acidity of the vagina prevents the sperm from swimming, but once these reagents increase the pH, the sperm becomes capable of swimming.

Back 23

4.2~5.7, buffering, bicarbonate, phosphate, 7.2

Front 24

It takes a minimum of __ hours for a sperm to swim through the vagina and cause fertilization.

Back 24

3

Front 25

When the sperm enters the cervix:

Back 25

Some sperm get stuck in crevasses of the cervix. The cervix also has mucus and thin fibers. At the time of ovulation, the mucus becomes clear and fibers become thin, so it’s easier to swim through the cervix. After ovulation, mucus becomes thicker, which makes penetration more difficult for sperm. The space is quite narrow, so the sperm form a line to pass through.

Front 26

3 phases of movement from cervix to uterus:

Back 26

1. Initial rapid phase
2. Colonization phase
Sperm finds a place in the crypts of the cervix. They can survive for a couple of hours nourished by the cervical mucus
3. Prolonged phase of transport
Some uterine contraction facilitates movement. The rate of swimming is about 3mm/min, so at that speed we cannot account for the distance the sperm travel in a short period of time. Dead sperm reach the oviduct, and dead sperm cannot swim. Inert particles also reach the oviduct in less than 15 minutes. It is also thought that stimulation of the cervix promotes release of oxytocin, causing rhythmic uterine contractions.

Front 27

Sperm can only survive in the uterus for a short time. Their presence causes a surge of __ into the uterus that aim to destroy them.

Back 27

white blood cells

Front 28

Passage of egg from ovary to uterus:

Back 28

1. Ovary to fallopian tube
Antral fluid, cilia, smooth muscle
2. Passage into oviduct occurs in three phases:
a. Fimbria to the ampulla-isthmic
b. Ampulla-isthmic to junction
c. Enters uterus

Front 29

After ovulation, the egg has not finished with Meiosis II. Has a zona pellucida surrounded with some granulosa cells and a corona radiata. The sperm must pass through the __ and the __, then the __ to fuse with the membrane of the ovum.

Back 29

corona radiata, zona pellucida, pre-vitelline space

Front 30

Capacitation

Back 30

Glycoproteins from the head are stripped off. Sperm switches from undulation to hyperactive motility (a whip-like motion).
Changes in surface of plasma membrane: these increase responsiveness of the sperm to signals encountered near the egg. Cholesterol is been removed from the bilayer, destabilizing it.
There is an increase in permeability to:
Calcium: helps hypermotility
Bicarbonate
Adenyl cyclase is activated, which leads to phosphorylation of tyrosine residues of proteins that will activate tyrosine kinase
All this occurs while sperm is in the oviduct, but sperm has not yet encountered the egg

Front 31

Activation (of the acrosome)

Back 31

a. Occurs once the sperm encounters the egg
b. The acrosomal sac breaks open
c. Portions of the plasma membrane fuse with portions of the acrosomal membrane, forming pores through which acrosomal enzymes are secreted
d. Other proteins found in the inner portion of the membrane are exposed
e. Timing is very slow because if it happens too quickly, the sperm can only digest a small section of the membrane
f. Contains 2 most notorious enzymes in the head of the sperm:
i. Hyaluronidase: released first; digests hyaluronic acid, which is the component of the matrix that holds the granulosa cells of the corona radiata together
ii. Acrosin: trypsin-like enzyme; protease that helps digest the zona pellucida; released a little later than the first
g. Potentially caused by following stimuli:
i. Progesterone of cumulus cells could make the acrosome more permeable to Ca2+, which can cause the release of hyaluronidase enzyme
ii. Presence of a glycoprotein in the zona pellucid called Zona Pellucida protein 3 (ZP3).

Front 32

Zona pellucida proteins:

Back 32

1. ZP1:
2. ZP2: Inner receptors bind to ZP2 and maintain the contact of the sperm with the egg.
3. ZP3: First ZP3 receptors exposed by the bursting of the acrosomal sac bind here

Front 33

Steps of fusion:

Back 33

1. Membrane recognition: may involve protein-protein or protein-carbohydrate mediated binding of 2 membranes. Ensure target specificity.
2. Membrane apposition: only the equatorial region of the sperm head engages in binding to proteins of the oocyte.
a. Oocyte has 2 regions of the plasma membrane:
i. Microvillar free region: overlay the meiotic spindle
ii. Microvillar rich portion: majority of the membrane; involved in fusion
b. When the proteins of the sperm plasma membranes bind to those of the oocyte, a conformational change occurs and causes the protein of the sperm to fall back on itself in a hinge movement, bringing the membrane of the sperm and the oocyte a lot closer together.
3. Lipid mixing
a. Lipids of plasma membranes of sperm and egg causes the cytoplasm of the sperm to be continuous with that of the oocyte.
b. The whole head is brought in; the neck and tail are separated.
c. Contributes DNA and a centriole (important for spindle formation)
d. The egg lacks a centriole due to uneven developme

Front 34

Name 3 proteins likely involved in fusion:

Back 34

1. CD9
a. Has small loop EC1 and large loop EC2
b. Study 1: If CD9 is knocked out, the egg is infertile. However, the egg goes through normal oogenesis, the animal produces mature oocytes in the same amount as the wild type. This indicates that something is wrong with fertilization.
c. Study 2: If the egg is incubated with a protein that specifically binds the large loop EC2, and then exposed to sperm, fusion is inhibited.
d. Study 3: When you take a CD9-deficient oocyte and inject it with messenger RNA that encodes the wild-type CD9, 55% of those oocytes are rescued and able to fuse.
e. Study 4: When you take a CD9-deficient oocyte and inject it with messenger RNA that encodes for mutations in the EC2 large loop, they show that there is no effect.
f. Studies suggest that CD9 must be present for fertilization.
2. GPI (glycosyl phosphatidyl inositol)
a. Lack of gene leads to inhibition of sperm fusion
3. Fertilin
a. Alpha and beta: mutations or knockout causes either
i. Sperm having difficulty travelling th

Front 35

What happens once fusion occurs?

Back 35

1. Increase in egg calcium levels
2. Increased nuclear condensation—forms a membrane around the pronucleus
3. Egg destroys everything else

Front 36

Sperm binds to __ and then __ receptors. Once it binds to the plasma membrane:

Back 36

ZP3, ZP2
1. Cortical reaction
2. Union of genes
3. Secretion of enzymes necessary for embryogenesis

Front 37

Cortical granules contain a series of enzymes:

Back 37

1. B-hexosamidase B: digests or destroys oligosaccharide molecule that is attached to ZP3. Since the oligosaccharide is the recognition site; no more sperms can bond.
2. Another enzyme causes the destruction of ZP2. Sperm that are halfway through cannot bind to ZP2, so even those that had the acrosomal reaction cannot pass.
3. Another enzyme cross links proteins of the zona pellucida to harden it. Makes sperm get stuck.

Front 38

Completion of meiosis involves:

Back 38

• Before meiosis, maturation promoting factor (MPF) stabilizes the spindle. Cytostatic factor (CSF) stabilizes MPF. This is how metaphase is arrested.
o MPF is made of Cyclin B and cyclin-dependent kinase 1 (Cdk1)
• Increase in calcium concentration when sperm fuses with the egg.
• Increasing calcium concentration activates a calcium-dependent enzyme. This enzyme destroys CSFand cyclin B.
• Cyclin B dissociates from cdk1.
• Meiotic spindle is now unstable and can finish meiosis II.
• Second polar body is extruded and now the pronucleus of the egg can fuse with the pronucleus of the male.

Front 39

Models of how spermatozoa cause increased calcium:

Back 39

Receptor hypothesis: The sperm has some kind of antigen that is recognized by receptors found in the egg plasma membrane. These could be associated with tyrosine kinase or g-coupled receptors, which have the second messenger inositol triphosphate that increases calcium.
Cytoplasmic factor hypothesis: could be a sperm factor present in the sperm cytosol that when released, binds to a receptor in the ER that activates calcium channels to release Ca2+ into the cytosol.

Front 40

Describe the zygote after fusion.

Back 40

Membrane disintegrates, DNA merges, new membrane forms.
Oocyte is now referred to as a zygote.
Zona pellucida surrounds it, 2 polar bodies are found in the pre-vitelline space.

Front 41

Multiple embryos:

Back 41

Monozygotic: come from one zygote; caused by separation of a 2-cell zygote into two separate zygotes. They share the same genetic material and the same placenta.
Dizygotic: come from two zygotes; caused by two eggs fertilized by two different sperm; have different sites of implantation and each has its own placenta.

Front 42

Errors in fertilization

Back 42

• Polyspermy: two or more sperm deposit genetic material into the egg
• Polygyny: failure of the oocyte to extrude the second polar body; leaves an extra set of female chromosomes
• Gynogenesis: pronuclei cannot fuse; embryo only contains mother chromosomes but zygote only has half the chromosomes necessary
• Aging egg: the older the egg, the more errors can occur
o During meiosis: nondisjunction can lead to trisomy
o Polar body took an extra chromosome
o Turner’s syndrome: XO chromosome

Front 43

In vitro fertilization

Back 43

1. Female goes through hormonal treatment to stimulate multiple follicles to develop simultaneously. From that cohort of follicles, we try to get all (more than 5) to reach the dominant stage.
2. Sperm sample is collected
3. Put in petri dish and check for fertilization to occur.
4. Check for embryos
5. Implanted into the uterus at the blastocyst stage: too early and they will not implant. Usually more than one egg is implanted.
6. Female also treated with estrogen and progesterone to make sure the endometrium is ready for implantation.