DEVBIOL 3 Fertilization

Fertilization

Union of sex cells; begins the formation of a new organism.

Types of fertilization

External fertilization (ex vivo) and internal fertilization (in vivo).

External fertilization (ex vivo)

Characteristic of aquatic vertebrates such as fish and amphibians.

Challenge of external fertilization

Releasing thousands of eggs in a single spawning to ensure species survival.

Spawning

Release of eggs into the aquatic environment.

Species-specific attraction challenge

Ensuring attraction between sex cells of the same species.

Solution to attraction challenge

Presence of chemoattractant released by the jelly coat of the oocyte.

Chemoattractant

Responsible for species-specific sperm attraction and activation.

Internal fertilization (in vivo)

Characteristic of avians and mammals.

Insemination

Deposition of sperm cells into the female reproductive tract.

Fertilization site (internal)

Female reproductive tract, specifically in the oviduct (uterine or fallopian tube).

Major events in fertilization

1. Contact and recognition between sperm and oocyte; 2. Regulation of sperm entry into the oocyte (prevention of polyspermy); 3. Fusion of the genetic materials of sperm and oocyte (pronuclear fusion or amphimixis); 4. Activation of the oocyte metabolism to start development (early and late metabolic responses).

Fertilization process (sea urchin fertilization)

Based on studies by the Hertwig brothers (Oscar and Richard) using sea urchins as model organisms.

Outer layer of egg cell

Vitelline envelope and jelly coat.

Function of jelly coat

Secretes chemoattractant for species-specific recognition of sex cells in the aquatic environment.

Contact and recognition between sperm and oocyte

Primary egg components: egg plasma membrane, vitelline layer, and jelly coat.

Contact step

Sperm head comes into contact with the jelly coat; acrosomal cap breaks down releasing hydrolytic enzymes that digest the jelly coat.

Acrosomal reaction

Formation of acrosomal processes (fibrillar protein) to further penetrate the vitelline layer.

Vitelline layer

Contains the species-specific receptor for the sperm.

Sea urchin fertilization (handwritten notes)

1.) Sperm contacts jelly coat, acrosome cap release enzymes penetrate PM; 2.) Acrosomal processes (fibrillar protein); 3.) PM of sperm and egg fuse, fertilization cone forms; 4.) Sperm head moves into cytoplasm → cortical reaction; 5.) Release of cortical granules into perivitelline space.

Contact and fusion of sperm and egg membrane

Forms the fertilization cone.

Entry of sperm nucleus

Movement of sperm head into the cytoplasm of the oocyte causing cortical reaction.

Cortical reaction definition

Bursting of cortical granules releasing their chemical contents into the perivitelline space.

Perivitelline space

Space between the plasma membrane of the oocyte and the vitelline layer.

Fertilization envelope

Formed when the vitelline layer is lifted off from the plasma membrane.

Cortical reaction (details)

Formation of fertilization cone and fusion of plasma membranes of the sex cells; cortical reaction releases contents of cortical granules (proteases, mucopolysaccharides, peroxidases).

Proteases

Enzymes that break molecular bonds between the vitelline envelope and the plasma membrane.

Mucopolysaccharides

Carbohydrates that create osmotic gradients causing water to rush between the vitelline envelope and the plasma membrane.

Example of mucopolysaccharides

Like tapioca pearls that become bloated after being soaked in water for a long time.

Function of mucopolysaccharides

Separates the vitelline membrane from the plasma membrane.

Peroxidases

Enzymes that harden the fertilization membrane by cross-linking tyrosine residues of adjacent proteins.

Result of cortical granule breakdown

Formation of the fertilization membrane.

Regulation of sperm entry into the oocyte

Involves plasma membrane fusion and prevention of polyspermy.

Plasma membrane fusion

Sperm acts on receptors on the plasma membrane of the oocyte.

Oocyte activation

The previously inactive oocyte becomes activated through membrane depolarization.

First block to polyspermy

Electrical in nature; fast block to polyspermy.

Purpose of first block

Prevents entry of more than one sperm into the oocyte.

Need for backup block

Fast block may not be sufficient, requiring a slower second block to polyspermy.

Fusion of plasma membranes (first block mechanism)

Sperm activates sodium channels in the oocyte membrane causing sodium influx.

Membrane potential at rest

Ranges from -50 to -70 mV, more negative inside the cell.

Membrane potential change during activation

Sodium and calcium influx causes reversal of potential from -70 mV to +10 mV (fertilization potential).

Result of first block

Inside of the cell becomes more positive than the outside.

Second block to polyspermy

Chemical in nature; slow block to polyspermy.

Mechanism of second block

Fusion of plasma membranes activates phospholipase C in the egg cell plasma membrane.

Products of phospholipase C activation

Inositol trisphosphate (IP3) and diacylglycerol (DAG).

Function of IP3

Activates release of calcium ions from calcium reserves in the endoplasmic reticulum (ER).

Function of calcium increase

Triggers cortical reaction or breakdown of cortical granules releasing proteases, peroxidases, and mucopolysaccharides.

Result of calcium-triggered cortical reaction

Formation of the fertilization cone and establishment of slow block to polyspermy.

Early metabolic responses

Both the first and second blocks represent early metabolic activation of the oocyte.

Handwritten notes: early metabolic reactions

Fusion of plasma membrane → sodium channels open → Na⁺ influx; resting cell more negative inside; Na⁺ and Ca²⁺ influx → fast reversal of potential; backup slow block triggered by calcium release; cortical granules burst into perivitelline space; bonds between plasma membrane and vitelline envelope break; osmotic gradient pushes water; peroxidases harden fertilization membrane.

Fusion of the genetic materials of male and female pronuclei

Fusion of male and female genetic materials forming the zygote nucleus (amphimixis).

Mammalian oocyte structure

Surrounded by zona pellucida and corona radiata.

Depolarization in mammals

Sperm penetration causes depolarization of egg plasma membrane (first block to polyspermy).

Onset of cortical reaction (mammals)

Leads to hardening of zona pellucida similar to vitelline membrane hardening in sea urchins.

Male sperm head in cytoplasm

Compact nucleus enters oocyte cytoplasm and undergoes decondensation and nuclear breakdown.

Formation of male pronucleus

Occurs after decondensation and replacement of protamines by histones.

Metaphase II arrest

Lifted off after sperm entry and second polar body is released.

Pronuclear fusion (amphimixis)

Male and female pronuclei meet to form the zygote nucleus.

Completion of fertilization

Polar bodies are visible; meiosis II completed forming functional oocyte (zygote nucleus).

Metabolic activation of the oocyte

Based on studies using sea urchins; involves early and late responses of the fertilized oocyte.

Early response

Fast block to polyspermy and cortical reaction.

Fusion of plasma membranes

Activates phospholipase C in the oocyte membrane.

Possible enzyme activation

Tyrosine kinase C may also be stimulated in the oocyte plasma membrane (uncertain).

Phospholipase C activation result

Production of inositol trisphosphate (IP3) and diacylglycerol (DAG).

Function of IP3

Triggers release of calcium ions from internal stores.

Effect of calcium release

Activates calcium-dependent kinases like NAD⁺ kinase that phosphorylates NAD⁺ to NADP⁺.

Function of NADP⁺

Serves as a coenzyme for lipid synthesis.

Purpose of lipid synthesis

Prepares fertilized oocytes for generation of plasma membranes of new cells during cleavage and embryogenesis.

Cortical exocytosis

Sets up the slow block to polyspermy.

Hyaline layer formation

Occurs after cortical granule release.

Activation of other kinases

Stimulates protein synthesis, DNA replication, and cytoplasmic movements of morphogenetic material.

Function of DAG

Activates protein kinase C.

Protein kinase C effects

Activates Na⁺/H⁺ exchange increasing intracellular pH.

Effect of increased pH

Stimulates protein synthesis, DNA replication, and cytoplasmic movements.

Phosphorylation targets of protein kinase C

Proteins involved in DNA replication and cell cycle regulation (cyclins, CDKs, histones).

Hyaline layer

Formed in aquatic mammals but absent in some terrestrial mammals.

Fertilization significance

Acts as a wake-up call for the oocyte to enter fast-paced activity (DNA replication, protein synthesis, chromosomal and regulatory protein formation).

Activated oocyte

Fully prepared for cleavage and embryonic development.

Transport of gametes and fertilization in mammals

Occurs within the female reproductive tract associated with the ovary.

Ostium

Mouth or opening of the fallopian tube surrounded by finger-like fimbriae.

Oocyte capture during ovulation

Oocyte released and captured by fimbriae to travel down to the uterus.

Oocyte travel time

Takes 4-5 days from oviduct to uterus; around 7-8 days to reach uterine vicinity.

Site of fertilization in mammals

Ampulla of the fallopian tube.

Oocyte fate

Whether fertilized or not, it travels down to the uterus.

Sperm transport in the female reproductive tract

Process following insemination.

Insemination

Deposition of sperm into female reproductive tract.

Common site of insemination in mammals

Upper vaginal canal.

Site of insemination in rodents

Uterus.

Sperm journey

From vaginal canal → cervix → muscular uterus → uterotubal junction → ampulla of fallopian tube.

Sperm motility in fallopian tube

Hyperactivated motility allows sperm to penetrate the oocyte.

Barriers during sperm transport

Natural vaginal acidity, thick cervical mucus, wide uterus, utero-tubal junction.

Vaginal acidity

Normally pH 3.5 (acidic); semen buffers to about pH 7.2.

Cervical mucus consistency

Becomes watery due to hormonal changes during ovulation aiding sperm motility.

Uterus as barrier

Wide muscular space—like sperm crossing the Pacific Ocean due to size difference.

Utero-tubal junction

Narrow passage (more constricted in rodents than common mammals).

Ampulla function

Final site of fertilization and location of blocks to polyspermy.

Capacitation

Conditioning period for sperm during passage through the female reproductive tract.

Purpose of capacitation

Removal of glycoprotein coat and seminal proteins covering acrosome cap.

Species variation in capacitation duration

Mouse - 1 hour; Rabbit - 6 hours; Human - 5-8 hours.

In vitro fertilization capacitation

Sperm are incubated in capacitating medium to remove glycoprotein coat covering acrosome.