Biology: Fertilization

• Fertilization is the process where sperm and oocyte fuse to create a new zygote.

Results of Fertilization

• A new organism is produced through fertilization.
• The diploid chromosome number (2n) is achieved when haploid gametes (n) combine.
• Fertilization stabilizes chromosome number across different species.
• Homologous recombination during fertilization causes species variation.
• The embryo's sex is determined during fertilization.
• Natural selection occurs.

Place and Timing

• Fertilization takes place in the ampulla, located in the fallopian tube, after a mature oocyte is released from the ovary.
• Approximately 200 out of 300,000,000 ejaculated sperm reach the fertilization site.
• Viability of sperm is ~72 hours.
• Fertilization potential of sperm is ~48 hours
• Viability of human oocytes is ~12-24 hours

Fertilization Stages

• Sperm and oocyte binding
• Capacitation
• Hyperactivation
• Corona radiata and sperm interaction
• Penetration of zona pellucida (first binding step with ZP3, acrosomal reaction, and second binding step with ZP2)
• Penetration with oolemma
• Oocyte activation (Zona reaction, completion of II. Meiosis and the externalization of the II. Polar body (PB), Pronucleus formation)
• Cell division (DNA replication, Homologous chromosome pairing, Oocyte meiotic spindle activation, The first cell division

Sperm and Oocyte Binding

• Sperm travels through the vagina and fallopian tubes, to reach the cells that surround the oocyte.
• Binding involves mechanical, enzymatic, and physiological processes.
• Sperm motility aids in the mechanical aspect.
• Acrosomal enzymes (e.g., hyaluronidase) and tubal mucosal enzymes contribute enzymatically.
• Interaction relies on sperm receptors on both the oocyte and sperm.

Capacitation

• Sperm conditioning in the female tract is a process of capacitation.
• Capacitation lasts about 7 hours.
• Capacitation removes glycoproteins and seminal plasma proteins from the sperm's acrosomal region.
• Only capacitated sperm undergo the acrosome reaction and fertilize the oocyte.
• Capacitation involves a number of biochemical and biophysical cellular changes.
• Capacitation results in remodelling of the cell surface architecture, intracellular pH changes, altered motility patterns, and initiation of complex signal transduction pathways.
• Capacitation happens when sperm enters the female genital tract (mucosal secretions).
• Adenyl cyclase activity increases the permeability of ions in the membrane.
• Metabolic changes occur, and energy consumption increases by glycolytic activation.
• Intracellular ionic shifts occur, with an intake of Ca++ changing ion concentration..
• The sperm membrane changes as cholesterol is removed and fluidity is increased

Hyperactivation

• Asymmetry in flagellum motility increases after capacitation
• Progressive motility decreases and trashes, helix and starpin motility is seen.
• Hyperactivation is a pattern of motility needed for sperm to penetrate through the cumulus cell layer and the zona pellucida to reach the oocyte's inner membrane.
• Hyperactivation causes high amplitude and curvature.
• The velocity is high, but the path is not linear.
• Hyperactivation occurs after three hours and diminishes after 6-24 hours in vitro.

Corona Radiata and Sperm Interaction

• The corona radiata interacts with sperm

Penetration of Zona Pellucida

• The space between the oocyte and the first layer of granulosa cells contains an extracellular material called the zona pellucida.
• Three glycoproteins secreted by the oocyte make up the zona pellucida.
• Sperm receptors in the zona pellucida are critical for binding specific proteins and activating acrosomal activation on the sperm surface.
• The zona pellucida is composed of three glycoproteins, ZP1, ZP2 and ZP3.
• The oocyte produces 3 glycoproteins while growing.
• ZP2 and ZP3 assemble into long filaments
• ZP1 is cross-linked to filaments for a three-dimensional network
• ZP3 mediates Sperm-Specific Egg Binding
• ZP2 mediates subsequent sperm binding
• ZP1 cross-links ZP2 and ZP3 as protein meshwork

The First Binding Step with ZP3

• In the first binding step, the sperm plasma membrane binds with ZP3
• Sperm receptors interact with zona glycoproteins (ZP3?) and activates signaling mechanisms leading to acrosome reaction

Acrosome Reaction

• The sperm head is capped by an organelle called the acrosome.
• Trypsin-like protein (digesting enzyme) and hyaluronidase digests hyaluronic acid.
• Contact with the zona pellucida triggers an acrosome reaction in the sperm head.
• Acrosomal reaction causes enzymes like hyaluronidase and acrosin to be released from the acrosomal cap.

Acrosomal Enzymes include

• α-L fucosidase
• Acrosin
• Arylsulphatase
• Hyaluronidase
• Phosphatase phospholipase-c
• Neuroaminidase
• Esterase
• Collegenase
• Acid proteinase
• Cathepsin-D

The Second Binding Step with ZP2

• Lysis of the zona pellucida (ZP2) and plasma membrane occurs by acrosomal digestive enzymes.

Penetration of Zona Pellucida (Summary)

• Signal molecules recognize ZP3
• Binding of ZP3 relies on extracellular Ca.
• The acrosome reacts and acrosomal membrane receptors (Acrosin) begin secondary binding with ZP2.
• Penetration into the inner regions happens around 1 hour later and the sperm cell crosses the zona.

Penetration with Oolemma

• Sperm-oocyte membrane fusion occurs

Ca Release and Oocyte Activation

• Initial microvilli contact between oolemma and sperm leads to membrane fusion.
• Oocyte subcortical region's actin and myosin filaments aid incorporation.

Zona (Cortical) Reaction

• The oocyte's initial response to incoming sperm is a cortical reaction.
• To prevent polyspermy, cortical granules are secreted into the perivitelline space.
• This is a fast polyspermy blockage
• The cortical granules creates a barrier (specialized secretory vesicles) and are found within the egg's cortex (region beneath plasma membrane).
• Existing extracellular matrix is modified to become impenetrable to sperm.
• Sperm binding activity of ZP3 is lost.
• Occurs via cortical granule enzymes (N-Acetyl glycosaminidase)
• These enzymes blocks Gal-Tase binding sites.
• Biochemical composition of the ZP changes.

Mechanism

• The sperm's penetration of the zona pellucida causes a change, the zona reaction, which makes it impermeable to other sperms.
• Cortical reaction happens after binding

Incorporation

• This involves sperm nucleus, sperm mitochondria, centrosome (lies across the nucleus in the neck region), a piece of the tail, and perinuclear organelles
• Oolemma, Cell membrane of the spermatozoon, Kinocilium Nucleus (compact) of the spermatozoon, and Centrosome of the spermatozoa are released into the oocyte cytoplasm

Metabolic Activation of Oocyte

• Cortical reaction and second polar body extrusion indicate an activated oocyte.
• Fertilization starts oocyte activation and initiates second meiosis.
• The 2nd polar body (PB) is extruded.
• Activation factors (SAOAF, etc.) are carried by sperm and transduced via receptors.

Pronucleus Formation

• The genetic material in the nucleus reorganizes and forms a pronucleus.
• Haploid genome (N) is carried in the pronucleus
• Key fertilization markers are the formation of two pronuclei (from mother and father's genetic material) and two polar bodies.
• The oocyte becomes a zygote.
• As the pronuclei form and grow, replication is initiated.

Female Pronucleus Formation

• Mature oocyte's maternal chromosomes (22 + X) condense to form the female pronucleus.
• The oocyte's chromosomes in Anaphase II scatter into the cytoplasm
• Vesicles begin to gather around the scattering chromosomes
• The vesicles combine to create the female pronucleus membrane.
• The female pronucleus is near the first polar body

Male Pronucleus Formation

• The sperm nucleus membrane dissolves.
• Sperm chromatin is dissolved in the oocyte's cytoplasm (decondensation).
• The male pronucleus membrane reforms as the male pronucleus.
• In immature oocytes, the sperm membrane does not dissolve since the dissolving factor requires oocyte activation.
• The nucleus swells.

Pronucleus Migration

• Both pronuclei move to the zygote's center around the 20th hour following insemination.
• Sperm aster accompanies this process.
• Sperm's centrioles use microtubules as a motor,
• The actins (cell skeleton microfilaments) help the movement of the PN migration.

Pronucleus Combining

• The pronucleus membrane dissolves, and the two haploid genomes (n) unite to form a diploid (2n) zygote.
• The zygote is at the 1 cell stage (1C).
• Both sets of chromosomes come together in the two cell-stage blastomeres

First Cleavage

• Maternal pronuclei and parental loose their cell membranes.
• The chromosomes form two nuclei (23 in each) combine to form a diploid (i.e., 46 chromosome in the zygote

Clinic Correlation

• Sperm and oocyte maturation are key factors for fertilization
• Key oocyte elements include the nucleus, cytoplasm, membrane, and zona pellucida (ZP).
• Key sperm aspects include the nucleus, cytoplasm, and developmental stage
• Oocyte cytoplasmic characteristics (homogeneity, no granulation, size, shape, and polar body morphology) and motility, concentration, morphology, and DNA integrity of sperm are important elements of cell quality.
• Genetic factors is a clinic correlation in fertilization

Oocyte Defects

• Maturation problems
• Granulated cytoplasm
• Cytoplasmic vacuol
• Refractil body
• Oocyte size
• Oocyte shape
• PB anomalies
• Zona anomalies
• Empty zona
• Binovular oocyte
• Degenerated oocyte
• Genetic problems

Sperm Defects

• Strong correlation between semen parameters and fertilization exists
• Semen factors include concentration, motility, morphology, ROS level, & DNA fragmentation level.
• Sperm maturation
• Genetic problems

Fertilization Anomalies

• Anomalies include One gamete activation (1PN) in which one gamete is activated and one pronucleus is formed.
• Other gametes are sometimes activated or not activated.
• Triploidy (3 PN) can occur
• During no fertilization, 70% of the oocytes fertilize and No PN is observed in the remaining

Unfertilization

• Activation problems of the gametes
• Maturation problems
• Molecular problems
• Genetic problems
• Microtubule defects
• Unknown reasons

What to do

• ART (Assisted reproductive techniques) can be used.