Fertilization

Question 1

In ruminants, primates, rodents and cats, Upon intromission and ejaculation, semen is deposited into…

Answer 1

The Vagina

Question 2

In pigs and equids, Upon intromission and ejaculation, semen is deposited into….

Answer 2

The cervix to into the uterus

Question 3

In dogs, Upon intromission and ejaculation, semen is deposited into…

Answer 3

The uterus

Question 4

Retrograde transport of semen

Answer 4

60% lost in cattle 12 hours post-insemination

Gel fraction reduces in some species (pig, horse, primates, rodents)

forms a visible vaginal “plug” in rodents

Question 5

Major barrier to sperm transport in the female tract

Answer 5

Cervix (except in pigs and dogs)

Question 6

Cervical sperm transport description

Answer 6

Rapid phase:
Injection to uterus by ejaculatory force (especially mares)

Slow phase:
Sperm swim upgrade through “channels” in cervical crypts to avoid retrograde mucous flow

Question 7

In the slow phase of cervical transport sperm swim through…

Answer 7

“channels” in cervical crypts to avoid retrograde mucous flow

Question 8

In the uterus sperm is transported anterograde towards the…

Answer 8

Utero-tubule junction by peristaltic contraction (estrogen induced)

Question 9

The uterus has peristaltic contractions induced by ________ that move sperm anterograde towards the UTJ

Answer 9

Estrogen

Question 10

In the female tract the _____-________ _________ acts as a reservoir for sperm

Answer 10

Utero-tubule junction
Sperm accumulate and bind tot he epithelium in the folds and are released slowly to the oviduct

Question 11

Sperm Capacitation definition

Answer 11

physiological changes that sperm undergo in the female tract to gain the capacity to undergo the acrosome reaction (AR) (gaining the capacity to fertilize)

Capacitation normally occurs in the female tract (uterus, uterine tube) but can also be induced in vitro

Question 12

Capacitation of sperm normally occurs in the….

Answer 12

Female tract (uterus, uterine tube) but can also be induced in vitro

Question 13

Sperm undergo the acrosome reaction when…

Answer 13

Triggered by binding to the zona pellucida (oocyte is present)

Question 14

Fertilizing capacity of an ejaculate can be stretched out over many hours, thereby increasing the odds of fertilization since….

Answer 14

It will not undergo acrosome reaction unless bound to zona pellucida of oocyte

(Once sperm are capacitated and/or undergo AR, their lifespan is dramatically shortened)

Question 15

Requirement for ____________ stretches out the fertilizing lifespan of an ejaculate

Answer 15

Capacitation

Question 16

Sperm Changes in Capacitation

Answer 16

Removal of surface factors (DF) (seminal plasma):
-ASF, Caltrin, CRISP-1
-Changes surface charge
-Unmasking of binding sites

Binding of surface factors
-Modulators of AC/cAMP: Calcitonin, FPP, adenosine, angiotensin II
-Modulators of Ca++ influx: progesterone, GABA
-Adhesion molecules: SPAM1 from female tract, via clusterin

Ability to undergo membrane fusion event (acrosome reaction):
-Membrane cholesterol efflux
-Increased plasma membrane fluidity and movement of some surface protein (e.g., dopamine receptors)

Motility changes (faster!!!!)
-Progressive motility
-Hypermotility (aka hyperactivation)

Metabolic changes
-Influx of Ca++
-Increased metabolism

Question 17

Acrosome reaction steps

Answer 17

Sperm binds to zona pellucida via ligand-receptor interaction (ZP3)

Receptor on sperm triggers calcium influx
Intracellular [Ca++] (rises F-actin -> G-actin)

Ca++ induces membrane fusion
-Outer acrosomal and sperm cell membrane fuse
-Resulting pores permit escape of soluble acrosomal contents
-Exposure of inner acrosomal membrane, which binds to ZP2

Question 18

Zona penetration by sperm

Answer 18

Hyaluronidase digests hyaluronic acid secreted by cumulus cells

Acrosin digests a pathway through zona pellucida (ZP2-3 matrix, ZP1 x-links)

Hyperactivated motility provides motile force

Question 19

Sperm penetration depends on…

Answer 19

Acrosin-digested pathway

Hyperactivated motility to propel sperm through pathway

Question 20

Sperm-egg binding

Answer 20

Entire sperm enters perivitelline space

Motility diminishes or ceases

Sperm binds to oocyte cell membrane
-Binding domain restricted to post-acrosomal region of sperm

Sperm ligand (fertilin, PH-30) binds to oocyte surface receptor (integrin?)
-Binding event triggers surface depolarization event
-Momentary opening of calcium channels causes massive increase in oocyte [Ca++]
-Cortical granule exocytosis (Block to polyspermy)
-Sperm-egg fusion (PLC) and endocytosis
-Sperm nucleus decondensation
-Resumption of oocyte meiosis

Question 21

How the oocyte blocks polyspermy

Answer 21

Cortical reaction
Cortical granule exocytosis once sperm enters into the oocyte Perivitelline space and binds to oocyte surface receptor

Cortical granule exocytosis release a variety of proteases (e.g., plasminogen activator)
Protease diffuse across PVS
Proteases alter zona structure (zona reaction), rendering zona resistant to acrosin digestion
Results in block to polyspermy

Question 22

Sperm-egg binding: the seminal fertilization event

Answer 22

1. Sperm-egg binding
2. Cytoplasmic [Ca++]
   a. Cortical Reaction (exocytosis)
   b. Fast Block to polyspermy
   c. Sperm endocytosis
   d. Sperm nucleus decondensation
   e. Resumption/completion of oocyte meiosis

Question 23

The Cortical reaction leads to a release of a variety of…

Answer 23

proteases (e.g., plasminogen activator)
Protease diffuse across Perivitelline space
(PVS)

Proteases alter zona structure (zona reaction), rendering zona resistant to acrosin digestion

Results in block to polyspermy

Question 24

Sperm endocytosis into the oocyte

Answer 24

Increase in cytoplasmic [Ca++]

Sperm and oocyte membranes fuse (endocytosis)

Decondensing sperm nucleus (1N)

Completion of oocyte Meiosis II
(extrusion of PBII)

Question 25

Resumption/completion of oocyte meiosis happens when....

Answer 25

Sperm endocytosis into the oocyte with increased cytoplasmic calcium (Egg has already at metaphase and has extruded polar body and is ready to accept more DNA) Sperm nucleus then decondenses and completion of oocyte meiosis II is complete and 2nd polar body is extruded- HAPLOID male and female pronuclei

Question 26

First zygotic cell cycle

Answer 26

After Meiosis II is complete there is formation of haploid male pronucleus (1N) and female pronucleus (1N)

Question 27

Sperm head decondensation

Answer 27

When the sperm head first enters the ooplasm, it immediately begins to swell The dense chromatin “unpacks”, involving reduction of protamine disulfide bonds Protamines are replaced by histones Haploid genome becomes organized into chromosomes

Question 28

The enlarging sperm head is called the...

Answer 28

Male pronucleus As it enlarges, it becomes surrounded by a pronuclear envelope/membrane and microtubules It moves towards the center of the ovum. It is also busy duplicating its chromosomes so that by the time it reaches the center, it is ready for the metaphase of mitosis.

Question 29

As the male pronucleus enlarges it becomes surrounded by...

Answer 29

a pronuclear envelope/membrane and microtubules It moves towards the center of the ovum. It is also busy duplicating its chromosomes so that by the time it reaches the center, it is ready for the metaphase of mitosis.

Question 30

In the oocyte, immediately upon extrusion of the second polar body, the remaining haploid genome forms...

Answer 30

a female pronucleus It also forms a pronuclear envelope, duplicates its chromosomes, and moves towards the center of the fertilized ovum.

Question 31

In some species the female pronucleus is typically _______ than the male pronucleus.

Answer 31

Smaller

Question 32

Syngamy process

Answer 32

When the 2 pronuclei reach the center, the pronuclear envelopes disappear and the two genomes become one. The chromosomes with duplicated chromatids line up on the metaphase plate for the first mitosis (first cleavage).

Question 33

First zygotic cell cycle

Answer 33

Interphase:haploid male and female pronuclei replicate chromosomes Prophase: chromosomes condense; nuclear membrane breaks down Metaphase: syngamy occurs as maternal and paternal chromosomes align together on metaphase plate

Question 34

Cell stages of developing embryo

Answer 34

2, 4, 6, 12 At some point between 8 – 16 cells (depending on species), the cells produce a cell adhesion molecule on their surfaces, compaction process to create the MORULA

Question 35

Morula

Answer 35

At some point between 8 – 16 cells (depending on species), the cells produce a cell adhesion molecule on their surfaces Uvomorulin, an E-cadhedrin Forms tight junctions between adjacent cells, so that individual cell boundaries become difficult to see (morula becomes compacted; process is called “COMPACTION”.) Some cells are now located in the interior of the ball of cells Early morula- some cell boundaries still visible Late morula- boundaries between cells not visible Becomes blastocyst once fluid accumulates in cavities to form blastocoel

Question 36

Blastocyst description

Answer 36

multiple small fluid-filled cavities are formed. These coalesce into a single cavity – the blastocoel, marks the transition from morula to blastocyst The cells on the inside form the Inner Cell Mass (ICM) which will eventually give rise to the embryo. The cells forming the periphery will eventually form the embryonic side of placental tissues (trophoblast). Blastocyst is still surrounded by zona pellucida

Question 37

Blastocyst growth

Answer 37

ICM and trophoblast cells continue to multiply Largest increase in blastocyst volume comes from accumulation of blastocoel fluid This causes outward pressure on zona pellucida which stretches and thins This pressure-caused thinning, together with proteases in the endometrium, eventually causes a zona rupture, through which the blastocyst escapes (hatches)