FERTILIZATION

FERTILIZATION

2.       FERTILIZATION

Both egg and sperm acquire structural specializations for fertilization. Eggs are non-motile, surrounded by protective egg coverings. These serve to recognize the sperm specifically and prevent fertilization by more than one sperm and prevent polyspermy. The mammalian egg has a zona pellucida layer around the plasma membrane. After the fertilization sperms are highly motile cells consisting of a nucleus, mitochondria to provide energy and a flagellum for movement. The anterior end of the sperm is highly specialized which aids in penetration of the egg. Sperms are typically designed to activate the egg and to deliver their nuclei into the egg cytoplasm.

Basic requirements of fertilization

Fertilization requires a fluid medium in most of the animals. It may be seawater in marine forms, fresh water in freshwater forms and body fluid in viviparous animals. To increase the probability of fertilization, the number of sperms must exceed the number of eggs. Moreover, the life span of gametes is limited; therefore fertilization must take place within a short duration of time. Eggs that are shed in the water like that of most invertebrates, fishes and amphibians, have shorter life span while those fertilized within the body of the female, generally have a longer life span.

Site of fertilization

Fertilization may be either external or internal. In external fertilization, the gametes are discharged in the aquatic medium and the fertilization occurs outside the body of both male and female parents, as in most invertebrates and some vertebrates (fish and frog). The aquatic medium for external fertilization may be either seawater or fresh water. When fertilization occurs inside the body of the female parent, it is internal fertilization, as in Drosophila, birds and mammals.

Mechanism of fertilization

Fertilization is a process of gamete fusion that is most important to develop a new organism. The process concerned with the fusion of two haploid gametes of the opposite sex like in animals it is concerned with the fusion of sperm with the ovum. The process of fertilization in animals is classified into two categories, on the basis of site of occurrence.

2.1.         External fertilization :

The strategy of fertilization in the open medium rather than inside a parental body organ. In this, both parents are required to release numerous eggs or sperms in the medium to increase the chances of meeting and fertilization. It is characteristically found in lower animals habitats in the aquatic ecosystems like sea urchin coral, hydra, salmon, cichlids, amphibians.

The mechanism of external fertilization in a model organism sea urchin (Arabcia punctulata). During external fertilization, the main problem concerned with the meeting of dispersed gametes to only their species specificity. To resolve this problem many species evolved a way of chemotaxis (chemoattraction), through which the sperms got attracted by eggs on the release of a species-specific chemoattractant.

2.1.1.     Chemotactic movement :

The eggs produced by Arabacia punctulata are surrounded by a jelly coat. The egg release. The egg the resact is chemotactic peptide molecule of 14 amino acid. The receptor for resact is only found in the membrane of species-specific sperms. The resact bind at that GPCR receptor, a signally cascade initiates inside the cytoplasm that leads to the transient influx of Ca+2. This regulates the flagellar beats and swimming trajectory by activating dynein ATPase and mitochondrial ATP synthase.

Due to this sperm reaches its competent egg and make contact with their outer coating jelly coat. This contact again activates the second signalling pathway, that leads to the release of acrosomal content of sperm. The sperm acrosome has a variety of enzymes that can digest both sugars and proteins and make a path through the jelly to the egg surface.

This event is termed as acrosomal reaction thought to be initiated by a polysaccharide present in egg jelly. This fucose-containing ligand acts on sperm to allow calcium influx leading to exocytosis of acrosomal vesical. These ligands are also species-specific.

2.1.2.     Acrosomal reaction

The acrosome lies at the front end of the sperm cell just below the cell membrane. It is derived from the Golgi body and, because of its content of digestive enzymes, is now considered to be a specialized lysosome-related organelle.

The enzymes contained within the acrosome are designed to penetrate the egg coats during fertilization. Here's a list of those that have been characterized in humans.

Protein Digestion

Acid Proteinase: a general protease that hydrolyzes protein at acidic pH

Collagenase: a protease that digests collagen

Acrosin: A special human acrosomal protease that digests zona pellucida proteins may also mediate sperm-zona pellucida binding. However, mouse null acrosin mutants can still penetrate the zona indicating that acrosin may not be critical in the mouse.

Serine Proteases: Found in mice but not yet in humans.

Carbohydrate Digestion

Beta-glucuronidase: An enzyme that removes Beta-glucuronic acid residues from carbohydrates

Hyaluronidase: An enzyme that digests carbohydrates containing hyaluronic acid residues

Neuraminidase: An enzyme that cleaves neuraminic (sialic) acid residues from carbohydrates

Lipid Digestion

Phospholipase C: A lipase that hydrolyzes phosphoinositol phospholipids, in the egg membrane

Human Acrosin

Acrosomal serine protease

Bound to the inner surface of the acrosomal membrane

Digests pathway through the zona pellucida which is primarily made up of three proteins: ZP1, ZP2, ZP3

In sea urchins, acrosome reaction occurs when direct binding of sperm head with egg outer surface takes place. After acrosomal reaction acrosomal content released causes influx of Na+ and efflux of H+.

2.1.3.     Acrosome filament formation :

Efflux of H+ increases the pH of sperm. Increase in pH causes dissociation of the profilactin complex (actin and profiling) and release of actin monomers which get polymerize to form the acrosomal filament. This process is called as Acrosomal filament formation. The acrosomal reaction occurs synchronously with the polymerization of globular actin in sperm, leads to the extension of the acrosomal process with a membrane protein, binding.

The binding protein present on acrosome filament interacts with the plasma membrane of sperm. This allows the fusion of the sperm and egg plasma membranes. It provides the first set of species-specific recognition events provided by the sperm's contact with an egg's jelly coat sperm penetrated the jelly and the acrosomal process of the sperm contacts the surface of the egg provided another critical species-specific binding event. The acrosomal protein bindin mediating this recognition, the bindin protein of sea urchins present on the acrosomal process and its receptor present on the egg plasma membrane. The bindin acts as a ligand on the vitelline membrane of egg and binds to a specific receptor EBR1.

2.1.4.     Fusion :

The adhesion of sperm on egg causes the extension of several microvilli that forms the fertilization cone. Then the plasma membranes of egg and span join together and the sperm nucleus passes through the cytoplasmic bridge formed. The fusion process is mediated by “fusogenic” proteins actively and allow the entry of the sperm nucleus, mitochondria, centriole and flagellum. The organism that has another strategy of fertilization inside the female body also resembles the molecular mechanism exhibited in case of external fertilization.

2.2.         Internal fertilization

It is also a popular strategy evolved in higher animals in which generally male gametes are transferred into the female body by the process of copulation. The animals which exhibit internal fertilization are known as ovoviviparous and viviparous animals.

During the copulation, the semen is ejaculated in the cervix of the female reproductive tract. But even the mature sperms are not able to fertilize the ovum without capacitation.

Capacitation is the second last step of sperm activation or maturation. Semen gets diluted into the vagina. The sperm undergoes several changes. The membrane becomes more fluid to increase the Ca+2 ion permeable by removal of surface antigens of sperm, like steroids and non covalently bound seminal glycoproteins and thus Ca+2 levels increase intercellularly. Beat frequency of tail is increased. This increase in sperm mobility. Sperm is become more accessible to bind to the egg due to the loss of these proteins. By albumin proteins in the female reproductive tract cause removal of cholesterol. The removal of cholesterol exposed the site of sperm to egg binding.

Fast event

When sperms come in contact with the uterine isotonic medium containing HCO3– and Ca+2, they get inside via Na+/HCO3 Cotransporter (NBC) and a sperm-specific Ca+2 channel respectively. The influx of bicarbonate and calcium ion increases the phosphokinase A (PKA) activity by stimulating the kind of adenylate cyclase.

The events occur after the long period of incubation of sperm in such condition. Sperms became able to fertilize an egg due to the increase in their motility.

Protein kinase A (PKA) activate the protein tyrosine kinases as well as causes inactivation of protein phosphatases. The kinases phosphorylate the proteins that are essential for capacitation. As a result efflux of K+ occurs which cause hyperpolarization of the sperm membrane. The speed of sperm movement is 1-3 mm per second that is too much slow to reach the egg. This time is reduced by a strong uterine contraction that pulls the sperms and transported it to the fallopian tube. The uterine muscle contraction is due to a chemical, prostaglandin present in the seminal fluid. The seminal fluid without sperms is withdrawn out via cervix and vagina after the few minutes of intercourse (copulation). The hyperactivated sperm is attracted towards egg in the fallopian tube because of a known chemoattractant relatively bind to the zone pellucida of an egg.

2.2.2.     Zone Interaction :

Sperm-egg binding is not so simple in humans case. The complicating factor is the thick zona pellucida is that factor that prevents sperm from binding close to the egg plasma membrane. Zona pellucida is found outside the plasma membrane, composed of glycoproteins that are synthesized and secreted by growing oocyte. The zona pellucida is structurally a bilayer of glycoproteins ZP1, ZP2 and ZP3, where the ZP2 and ZP3 are crosses linked by ZP-1 and their orientation is alternative. The ZP-3 glycoprotein is specific for binding of sperm and acts as a ligand for the O-linked glycans receptors of the sperm.

These glycans bind specifically at the Ser332/Ser334 residue of ZP-3 peptide at C-terminal, and later they are named as GalT (galactosyl transferases) receptors. Zona pellucida glycoproteins of ZP-3 (ZPGP III) is the sperm receptor. O-linked oligosaccharide of ZP III acts as a receptor and bind to galactosyl transferase, the receptor of sperm.

This binding causes the repolarization of the sperm membrane from the resting potential of – 60 mV by the transient opening of a Ca+2 ion channel. This binding also causes activation of IP3 signalling mediated by PLC pathway leads to Ca+2 high availability in sperm cytoplasm. The Ca+2 inside the cytoplasm causes exocytosis of the vesicle. The acrosomal reaction followed by the actin filament polymerisation with the help of Ca+2 and ATP.

2.2.3.     Acrosome reaction :

Acrosome reaction takes place by irreversible binding of the sperm to the egg, in turn, acrosomal reaction triggers by the zona pellucida. The outer plasma membrane of the acrosome fuses at multiple sites with the plasma membrane of oocyte and acrosome’s contents are released, acrosome’s contents contain two important components, acrosin (a serine protease) and N-acetylglucosaminidase. A hole gets bores into the zona pellucida by acrosin B digestive enzyme. As a result, sperm can reach the egg itself. N-acetylglucosaminidase hydrolyzes the O-linked oligosaccharides in ZP Glycoprotein III that bind to galactosyltransferase present on sperm. This allows the sperm to detach. A new surface, the inner acrosomal membrane is exposed on the sperm plasma membrane. The inner acrosomal membrane is containing new binding sites for ZP Glycoprotein II of an egg. Thus, the acrosomal activated sperm digest the ZP3 via their proteolytic and hyaluronidases and interact with lateral ZP2 molecules. Thus the acrosome intact spem interact with ZP III of egg and acrosome activated sperm interact with ZP-2 of an egg. This is known as secondary binding. The actin polymerization leads to the formation of acrosome filament.

This tangential entry of sperm facilitates the binding of fertilin ligands to the receptors residing on the egg plasma membrane. Then both membranes of gametes fuses and the content of sperm are released in ooplasm like in the case of sea urchin.

2.2.4.     The fast block of polyspermy

Block of Polyspermy: Fast block of polyspermy just after the fusion. It lasts up to 60 second due to the change in membrane potential.

Sea urchin: The resting potential of the egg membrane is   70 mV and shifts to about + 20 mV due to the influx of Na+ ion present in seawater.

Mammals: In mammalian ovum, the fast block of polyspermy is similar to sea urchin but here the Na+ ions or other cations come from fallopian tube environment and causes the fast block of polyspermy.

2.2.6.     Slow Block of polyspermy

The fast block of polyspermy remains up to 60 seconds then the membrane potential is restored to original resting potential by other ion exchange. The binding of sperm’s ligands to the receptors of egg plasma membrane induces a signalling cascade, that causes the exocytosis of cortical granules.

The cortical granules also contain the mucosal polysaccharides that absorb water and form a mechanical viscous coat around the fertilized egg. This fertilization envelop is known as the hyaline layer. This layer causes a permanent block of polyspermy after few minutes of fertilization.

The immediate events take place after fertilization that prevents polyspermy. Fertilization of the egg by more than one sperm called polyspermy. Polyspermy is resulting in a zygote that contains greater than a diploid amount of DNA, which in turn cause the early arrest of development as well as embryonic defects.

The cortical granule reaction causes the release of two important enzymes into the perivitelline space:

1.     Ovoperoxidase:

In sea urchins, the egg extracellular membrane contains tyrosine residues which get crosslinked by ovoperoxidase. This makes extracellular matrix more tough and insoluble (analogous to the tanning of leather) and now this act as a physical barrier, which prevents other sperm from fertilizing the egg. In mammals, ovoperoxidase does not catalyze tyrosine cross-linking to the point of insolubility. In mammals, its major effect is thought to be as a spermicial agent.

2.      Hydrolase :

A specific hydrolase degrades O-linked oligosaccharides on zona pellucida glycoprotein III (ZPGP III). Now the zona pellucida become incapable of binding with additional sperm, thus preventing polyspermy. Initiation of development of the new zygote also included in egg activation, upregulation of protein synthesis and other metabolic processes also takes place, to provide for the developing embryo.

The beating of the tail stops immediately as the sperm fuses with the egg. Through elongation and fusion of the egg’s microvilli sperm is drawn into the egg. As a result, incorporation of the sperm nucleus and other organelles are taken place into the egg cytoplasm. After that series of changes occur within the sperm nucleus, like chromatin decondensation takes place along with the formation of a new nuclear envelope, as a result, the formation of a male pronucleus takes place. Migration of the male pronucleus to the center of the cell done with the guidance of microtubules and at the center of the cell, male pronucleus fuses with the female pronucleus to reconstitute a diploid nucleus. During early cleavage stages of the embryo, other sperm organelles (e.g. mitochondria) also persist and it is speculated that they may play a part in development. The fertilized eggs are then metabolically activated for the further development process.

2.3.         Types of egg

According to the concentration of yolk in an egg

  1. Alecithal: no yolk
  2. Microlecithal: less or little yolk
  3. Mesolecithal: more yolk
  4. Macrolecithal: large concentration of yolk

According to the distribution of yolk in an egg

Isolecithal: uniform distribution of yolk

Telolecithal: yolk in the large amount present at the vegetal pole

Discolecithal: cytoplasm at one end of the egg

Centrolecithal: yolk present at center

Type of division

Holoblastic is a complete division. It if found in alecithal egg, discolecithal egg, mesolecithal eggs.

Meroblastic is called as incomplete division: It is found in lecithal egg and centrolecithal egg.  

2.4.         Type of cleavage according to the position of the cleavage furrow

Meridional: cleavage furrow passes vertically from the center, thus both blastomeres receive an equal amount of animal and vegetal pole.

Vertical: cleavage furrow passes vertically parallel to the meridional, thus the unequal distribution of animal and vegetal pole between blastomere.

Equitorial: cleavage furrow passes horizontally from the center, thus one blastomere receives vegetal pole and other receives animal pole.

Horizontal: cleavage furrow passes horizontally parallel to the equatorial, thus one blastomere receive a whole animal and some part of vegetal and other blastomere receive only some part of vegetal.

2.5.         Type of cleavage with respect to the metaphasic plate

2.5.1.     Radial cleavage: When cleavage furrow passes from the plane of the metaphasic plate or right angle of the plane of metaphasic plate.

2.5.2.     Spiral cleavage: when cleavage furrow passes from an angle to the metaphysic plate rather than right angle.

2.5.3.     Rotational cleavage: It is the type of random cleavage, cleavage furrow passes from any direction. In this cleavage, second cleavage onwards, the pattern of blastomere cleavage is different.

2.5.4.     Bilateral cleavage: In this type of cleavage, blastomeres produce by the first cleavage divided in the same pattern and this same pattern of the division followed by each blastomere pair produces by cleavage and thus blastomere produces the mirror image of each other.

2.5.5.     Superficial cleavage: cleavage occurs superficial due to center located yolk. In this type of cleavage, karyokinesis occurs without cytokinesis. In this type of cleavage pole cells are formed at the posterior pole. The pole cell becomes primordial germ cells and gives rise to gonads.


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