Lecture 6 - Fertilisation

Question 1

Human spermatozoa structure

Answer 1

• Acrosome - contains proteolytic & other enzymes
• Nucleus - haploid paternal DNA, 23 chromosomes
• Mitochondria densely packed in the midpiece - energy source
• Flagella - means of motility

Question 2

Deposition of semen: how much semen reaches the egg for fertilisation, why do so many sperms not make it, and what does the semen do to aid survival?

Answer 2

~10² (from the average of 10⁸ deposited - 99% lost)

Different environmental conditions - the migration through female reproductive track is related to sperm selection (cryptic female choice) and only the fittest sperm can reach oviduct and participate in fertilization

Semen coagulates, becoming gelatinous (enzymes released from the prostate act on a fibrinogen-like substance to create a fibrin-like gel), this gel retains sperm and may buffer them against the acidic cervical fluid (pH 5.7)

Question 3

Internal/external cervical os: what do they mean and what does os stand for?

Answer 3

Internal cervical os - opening between the uterus and cervic
External cervical os - opening between the cervix and vagina

Ostium

Question 4

Sperm colonise cervical canal and crypts

Answer 4

Within one minute of mating sperm can be found in cervical crypts
Can survive here up to 24h
Crypts may act as a sperm reservoir
Possibly low resistance mucus channels

Question 5

Fertilisation: where does it occur?

Answer 5

Ampulla of fallopian tube

Question 6

Movement of sperm to the fallopian tube: how does it work, does it become immobilised at any point, what proteins interact with sperm, andcan sperms live if immobilised?

Answer 6

• By their own motility (mediated by movement of the flagellum through dynein-mediated microtubule sliding).
• Possibly aided by uterine cilia-driven currents or uterine muscular movements
• The contractions of the oviduct promote sperm progression toward the ampulla (mainly travel through isthmus)

Sperm become immotile at isthmus of oviduct binding oviductal epithelium (can act as a maternally controlled reservoir of sperm)
Proteins required for successful passing utero-tubal junction: pAce, Calr3,Clgn, Pdilt, Pmis2, Rnase10, disintegrin, ADAM3

Sperm can wait in the epithelium for several days for ovulation to occur while maintaining their ability to fertilize the egg

Question 7

Hyperactive sperm: what is it and what is it caused by?

Answer 7

The curvature increases in amplitude on one side of the flagellum only and produces an asymmetrical beat that might help release the sperm from the oviductal reservoir - sperm in this state are said to be hyperactivated

• Bicarbonate Ion from female reproductive track?
• Sperm activation may occur after removing inhibitory factors, such as surface‑attached glycoproteins, seminal plasma proteins and the depletion of membrane cholesterol
• We don’t yet have a conclusive answer

Question 8

Bicarbonate ion: what is it and what is its potential role in fertility?

Answer 8

HCO₃⁻

• Essential for sperm motility, capacitation, hyperactivation, and acrosome reaction
• Important for fertility, and a defective HCO₃⁻ transporting mechanism may cause female infertility
• Plays a critical role in the entire process of reproduction, including fertilization, spermatogenesis, and early embryo development

Question 9

CatSper: what is it, when is it activated, what is it regulated by, and what is an example of a pathway it is involved in?

Answer 9

Cation channel of Sperm - a sperm specific pH–dependent Ca²⁺ channel

Constitutively active but stimulated directly by progesterone to induce hyperactivation and asymmetric (directed) motility

• Progesterone
• Cyclic nucleotides (e.g., cAMP, cGMP)
• Zona pellucida (ZP) glycoproteins
• Bovine serum albumin (BSA)

Transports the Ca²⁺ involved in the regulation of the cAMP-PKA-dependent pathway required for sperm capacitation

Question 10

When does the acrosome reaction occur?

Answer 10

Before the sperm contacts the ZP

Question 11

Sperm penetration: what must sperm penetrate and what factors do sperm use to penetrate?

Answer 11

Has to penetrate between cells of cumulus - eggs are not released as discrete cells:
* Surrounded by a glycoprotein matrix called the zona pellucida (ZP)
* COC (cumulus-oocyte complex) - hyaluronic acid–rich jelly like structure containing somatic cumulus and the oocyte

• Hyaluronidases, including SPAM1 and HYAL5, are displayed on the surface of sperm, and their activity presumably eases the sperm’s passage through the cumulus matrix (digesting hyaluronic acid)
• Hyperactivated motility
• Glycosylphophatidylinositol (GPI) anchored proteins (hyaluronidase PH-20)

Question 12

Zona pellucida binding and penetration: are digestive enzymes required for it?

Answer 12

Surprisingly none of the digestive enzymes are necessary

Question 13

Polyspermy: what is it, is it possible, and is it harmful?

Answer 13

When multiple sperms enter an egg

Yes, but many mechanisms exist to prevent it:
* Cortical granules inside the egg which release enzymes that change the structure of the zona pellucida and make it impermeable to sperm fertilisation
* Ovastacin - An oolemma protein that cleaves the ZP2 protein in the zona pellucida, preventing other sperm from binding to the egg
* Zinc ions - The release of zinc ions during fertilization hardens the zona pellucida by modifying its microarchitecture

Question 14

Acrosome reaction: what is it, why is it necessary, and what causes it to occur?

Answer 14

The fusion of the sperm plasma membrane and the outer acrosome membrane - excluding the equatorial segment (this allows for sperm membrane fusion with oolemma)

Different mechanisms could induce acrosome reaction, which would depend on the status of the egg:
* A tight cumulus mass surrounding freshly ovulated eggs could induce acrosome exocytosis before sperm penetration through the zona matrix * Conversely, a more dispersed cumulus mass, would fail to induce acrosome exocytosis and would therefore be dependent on zona penetration
* Moreover, the most recent studies have indicated that the majority of sperm have undergone acrosome exocytosis before encountering the cumulus mass

Therefore acrosome exocytosis of the fertilizing sperm might be occurring during sperm migration through the female oviduct and the acrosome-reacted sperm cross the cumulus before binding to the zona

Question 15

Acrosome reaction mechanism

Answer 15

Phospholipase C (PLC) cleaves phosphatidylinositol 4,5-bisphosphate (PIP₂) to Inositol trisphosphate (IP₃) which releases internal Ca²⁺ from calcium stores by binding Ca²⁺ ion channels

Results in release of acrosome proteins - acrosin, hyaluronidase, etc (active proteases)
Causes the reloalisation of IZUMO1 - may be one of the main functional roles

Question 16

Zona pellucida: what is it, what is it formed from, what are the proteins present, and what does it do?

Answer 16

The extracellular coat surrounding all mammalian eggs that can be penetrated by capacitated sperms

Formed from a sponge-like mesh consisting of interwoven microfilaments, The ZP proteins establish non-covalent interactions with other ZP proteins to form a three-dimensional zona matrix

Three or four glycoproteins: ZP1, ZP2/ZPA, ZP3/ZPC and ZP4/ZPB (ZP1/2/3/4 in humans)

• Mediates species-specific gamete recognition
• Prevents polyspermy - maybe due to the cleavage of ZP2, disrupting the native conformation of this putative sperm binding site
• Protects the preimplantation embryo from being resorbed into the oviduct epithelial lining

Question 17

Can sperm bind and penetrate zona pellucidas from different species?

Answer 17

Zona proteins are fairly specific and sperm from different species usually do not bind or, if binding occurs, do not penetrate the ZP to reach the egg

Question 18

ZP protein mutations

Answer 18

• ZP1 mutations results in a misshapen, thinner and more fragile zona structure and result in female subfertility
• ZP2 or ZP3 mutation more severe : homozygous null females form a very thin zona (Zp2Null) or do not form a zona at all (Zp3Null), which results in defective follicle formation, scarcity of ovulated eggs (that also lack the zona) and ultimately in female infertility

Question 19

Sperm penetration though the zona pellucida: what is it facilitated by and what type of proteases may be involved?

Answer 19

Motility, protease and glycosidase activity work together

Probably include both those attached to the inner acrosomal membrane and in the released acrosomal contents

Question 20

Sperm-oocyte membrane binding: what happens, what proteins are involved specifically, and what conditions are necessary for the fusion event?

Answer 20

Equatorial segment (tip) of a sperm membrane binds to oolemma away from MII chromosomes

Binding may involve the same proteins which drive fusion of sperm and oocyte membrane and is a part of the second species-specific gamete recognition process

Sperm - IZUMO1 and SPACA6
Oolemma - CD9 and CD81 (gamete adhesion), JUNO (oolemma receptor for IZUMO1)

Extracellular (but not intracellular) calcium was quickly demonstrated to be required for the fusion event, as were controlled pH and temperature

Question 21

Sperm factors required in sperm-oocyte fusion: what are some of them and what do they stand for?

Answer 21

• Sperm acrosome membrane-associated protein 6 (SPACA6)
• Transmembrane protein 95 (TMEM95)
• Fertilization influencing membrane protein (FIMP)
• Sperm-oocute fusion-required 1 (SOF1)
• DC-STAMP domain-containing protein 1/2 (DCST1 and DCST2)

Mutation in all of these proteins result in the lack of gametes fusion

Question 22

IZUMO1: where is it both pre- and post-acrosome reaction?

Answer 22

IZUMO1 is not detectable on the plasma membrane initially

IZUMO1 localises to the equatorial segment, mediating gamete adhesion

Question 23

GPI(APs): what are they, what are some examples, and what do they do?

Answer 23

Glycosylphosphatidylinositol (GPI)- anchored protein

Folate receptor 4 (FOLR4/IZUMO1R/JUNO) - facilitates gamete fusion

Hyaluronidase PH-20 - sperm penetration

Question 24

Sperm oocyte fusion: what are some fusion proteins and what is the process?

Answer 24

• JUNO-IZUMO1 - may lead to accumulation of CD9 in the adhesion area
• CD-9 and CD-81 may interact with tetraspanin partners on oocyte membrane in microvilli

Upon gamete adhesion, a closer apposition of the membranes establishes mixing of lipid bilayers, followed by the formation of fusion pores, which generates cytoplasmic continuity and gamete fusion

Question 25

Consequences of fertilisation

Answer 25

• Reconstruction of normal diploid chromosome complement (total 46) 44 autosomes + XX or XY
• Resumption of meiosis and completion of meiosis II
• Introduction of the sperm centriole - forms spindle poles in subsequent development
• Block to polyspermy
• Reconstruction of the multicellular state (cleavage asymmetric -> symmetric)
• Initiation of the developmental program of the embryo
• Determination of the sex of the embryo

Question 26

Egg activation: what ion is presumed to have an effect in egg activation, how does the process work, and where is it sourced from?

Answer 26

Calcium ions

• Sperm triggers a a series of oscillations in intracellular free Ca²⁺
• Calcium transients start within a few minutes of sperm–egg interaction

This can be monitored by using a Ca²⁺ sensitive dye such as fura2 which fluoresces on binding Ca²⁺

Endoplasmic reticulum

Question 27

Calcium: how long do its transients occur for, what does calcium do, what protein does it associate with, and is it regenerative or induced once?

Answer 27

Transients continue until pronuclear formation (sperm and oocyte nucleus)

Requirement for cortical granule extrusion, resumption of meiosis and pronuclear formation latter 2 events also dependent on CAM kinase II

Ca²⁺/calmodulin-dependent protein kinase

Regenerative - shown by injection of calcium into the oocyte which itself generates a calcium wave, but the egg is more sensitive to injection of calcium after fertilisation suggesting that sperm egg binding leads to cytoplasmic events which increase sensitivity to calcium induced calcium release

Question 28

Ca²⁺ oscillations

Answer 28

Phospholipase C zeta (PLCζ) mimics the calcium transients observed after IVF or sperm extract injection

• Sperm-receptor interaction
• Entry of sperm factor after fusion

During egg Ca²⁺ oscillations mediated by sperm factor PLC, the IP3 receptor releases Ca²⁺ from the Ca²⁺ stores

Question 29

Possible mechanisms of calcium oscillations

Answer 29

Three main possibilities:
* Sperm interacts with a receptor - ligand binding activates proteins (e.g. a tyrosine kinase) which in turn leads to activation of phospholipase Cγ, PLC gamma hydrolyses PIP2 into IP3 and DAG, IP3 can bind to the ER and trigger calcium release
* Sperm GPCR activation may result in associated trimeric G-protein activation - leading to phospholipaseCbeta (PLC beta)
* Sperm factor - fusion of sperm and egg plasma membranes results in the release of a molecule present in the sperm cytosol into the oocyte

Question 30

Polyspermy: why is it required, how is it blocked, and is there a way to remove supernumerary sperm?

Answer 30

Polyspermic polyploid embryos are not viable

Increased intracellular Ca²⁺ induces exocytosis of cortical granules before meiosis II continues - proteases including ovastacin cleaves ZP-2 preventing additional binding and zinc may affect sperm motility and/or sperm-egg interaction

No way to remove supernumerary sperm - must be prevented from penetrating the plasma membrane

Question 31

Cortical granules: what are they, how many are there, and where are they located?

Answer 31

Membrane-bound vesicles derived from the Golgi apparatus that contain biochemical matrix remodelling apparatus

There are thousands of granules in the egg cortex

The enzyme rich granule contents are extruded into the space between the plasma membrane and the zona pellucida

Question 32

Zona reaction

Answer 32

The hardening process of preventing polyspermy