Fertilization: Beginning a new organism

• Fertilization is the process whereby the sperm and the egg (gametes) fuse to form a zygote

• The zygote is a single celled organism and marks the beginning of a new life

• The genome of the zygote is derived from both parents

Fertilization cont’d

• It accomplishes two main objectives:1)Sex-the combining of genes derived from both

parents2)Reproduction-the creation of a new organism• Fertilization therefore initiates those reactions

in the egg cytoplasm that permit development to proceed among other things

Conception

• Generally consists of four major events:1)Contact and recognition between the egg and the

sperm (ensures they are of the same species)2)Regulation of sperm entry into the egg-only one

sperm nucleus can unite with the egg nucleus3)Fusion of the genetic material of sperm and egg4)Activation of egg metabolism to start

development

Fertilization cont’d

• The egg activates the sperm metabolism essential for fertilization

• The sperm reciprocates by activating the egg metabolism needed for onset of development

Structure of the sperm

• Each sperm cell consists of:• a haploid nucleus, • a propulsion system to move the nucleus, • and a sac of enzymes that enables the nucleus

to enter the egg • During sperm maturation, the haploid nucleus

becomes tightly compressed and its DNA becomes tightly compressed

Sperm structure cont’d

• The acrosomal vesicle (acrosome) lies infront of the compressed haploid nucleus

• The acrosome is derived from the Golgi apparatus and contains enzymes that digest proteins and complex sugars

• These enzymes are used to digest the outer covering of the egg

• During early stages of fertilization in some species, actin proteins are used to extend a finger-like acrosomal process from the sperm

Sperm structure cont’d

• Sperms travel by whipping their flagella• The major motor portion of the flagellum is the

axoneme• The axoneme is formed by microtubules

emanating from the centriole at the base of the sperm nucleus

• Cross section of axoneme reveals that it is made of two central microtubules surrounded by a row of nine doublet microtubules (9+2 arrangement)

• The force for sperm propulsion is provided by a protein called dyenin, attached to the microtubules

• Dyenin is a an ATPase, an enzyme that can hydrolyze ATP converting the released energy into mechanical energy to propel the sperm

• The ATP needed to propel the sperm comes from the mitochondria in the middle piece

• Individuals lacking Dyenin suffer from the genetic syndrome known as Kartagener triad

Kartagener triad symptoms

• The ciliated cells of Kartagener Syndrome individuals are immotile

• Kartagener syndrome males are sterile,• Individuals are susceptible to bronchial

infection• 50% chance of having the heart on the right

side of the body

Sperm maturation

• The sperm released during ejaculation can move but cannot bind and fertilize an egg

• The final stage of sperm maturation is cumulatively referred to as capacitation does not occur until the sperm has been inside the female reproductive tract for a certain period

The egg

• All materials necessary for the beginning of growth and development must be stored in the mature egg

• Unlike the sperm, the egg has retained most of its cytoplasm as it matures

• The mature egg (Oocyte) conserves most of the material it has and actively acquires more as it reaches meiosis, the stage in which it is fertilized

Some cytoplasmic contents• Proteins-the early embryonic cells accumulate yolk

proteins in the egg that are required to provide energy and amino acids

• Many of these proteins are made elsewhere (e.g. liver, fat bodies) and travel through the maternal blood to the oocyte

• Ribosomes and tRNA-these are needed to make proteins. In many species, there is usually a burst of protein synthesis soon after fertilization. This accomplished by ribosomes and tRNA which exists in the egg

The egg continued

mRNA-the oocytes also accumulates mRNA that encode proteins for the early stages of development.

These mRNAs remain repressed until after fertilization• Morphogenic factors-molecules that direct the

differentiation of cells into certain cell types are present in the egg. These include paracrine factors and transcription factors. In many species they are localized in different regions of the egg and are segregated into different parts of the egg during cleavage

The egg cont’d

• Protective chemicals-many eggs contain UV light filters, antibodies, distasteful chemicals and DNA repair enzymes that protect them from sunlight, other damages and predators

Egg cont’d

• In some species such as the sea urchins, the egg pronucleus is already haploid at the time of fertilization

• In some mammals and some worms, the egg nucleus is diploid until the sperm entry and completion of meiosis

• In these species, the egg meiosis will take place while the sperm nuclear material (sperm pronucleus) is travelling towards what will become the female pronucleus

The egg cont’d

• The egg cell membrane must be capable of fusing with sperm cell membrane and must regulate certain ions during fertilization

• Outside the cell membrane is an extra cellular envelope that forms a fibrous mat involved in sperm-egg recognition

• In vertebrates, this envelope is called vitelline envelope containing glycoproteins

• The vitelline envelope is responsible for species-specific binding of sperm

The egg cont’d

• In mammals, the vitelline envelope is a separate and thick extracellular matrix called zona pellucida

• The mammalian egg is also surrounded by a layer of cells called cumulus originating from the ovary (follicle cells that used to nurture the developing egg)

• Mammalian sperm has to get past these cells to fertilize the egg

• The innermost layer of cumulus cells immediately adjacent to the zona pellucida is the corona radiata

• Just beneath the cell membrane is a thin shell of gel-like cytoplasm called the cortex

The egg cont’d

• The cytoplasm in the cortex region contains high concentrations of actin molecules

• During fertilization the actin molecules polymerize to form microfilaments

• Microfilaments are necessary for cell division and also help the egg surface to form microvilli (finger-like projections)

• The microvilli aid in sperm entry into the egg

The egg cont’d• The cortex also contains cortical granules (golgi-derived

structures)• The cortical granules contain proteolytic enzymes and are

homologous to the acrosomal vesicle of the sperm• Cortical granules contain adhesive glycoproteins,

mucopolysaccharides, and hyalin proteins• The enzymes and the mucopolysaccharides prevent polyspermy• The hyalin protein and adhesive glycoproteins surround the

early embryo and provide support for the blastomeres formed during cleavage

• Some eggs also contain a layer of egg jelly outside the vitelline envelope used to attract or activate sperm

Recognition of egg and spermInteraction of egg and sperm generally proceeds by

five basic steps:• Chemoattraction of the sperm to the egg by

soluble molecules secreted by the egg• Exocytosis of the acrosomal vesicle to release its

enzymes• Binding of sperm to the extracellular envelope

(vitelline layer or zona pellucida) of the egg• Passage of sperm through the extracellular

matrix• Fusion of the egg and sperm

Recognition of sperm and egg cont’d

• After the five steps have been accomplished, the haploid sperm nuclei and the egg nuclei fuses and the reactions that initiate development begins

Sperm attraction• Organisms that lay their eggs in water have multiple

problems including; • How would sperms and eggs meet in a dilute

environment? • How come sperms do not fertilize eggs of another

species?• Sperms are attracted to the eggs of their species by

chemotaxis-following a gradient of a chemical released by the egg

• The eggs also regulate the timing of the release of the sperm

Sperm attraction• Sea Urchin spems move as a result of changes in

pH• In the testes, the pH is about 7.2, but in the water

the pH rises to 7.6• This change in pH activates dyenin ATPase• Dyenin ATPases hydrolyzes ATP thereby releasing

energy• The flagella uses this energy to wave and the

sperm swims vigorously • Sperm direction in echinoderms is provided by a

small peptide (14 amino acids) called resact

Acrosome reaction

• A second interaction between sperm and egg is the acrosome reaction

• In most marine invertebrates, the acrosome reaction has two components:

1)Fusion of the acrosomal vesicle with the sperm cell membrane

2)Extension of the acrosomal process• The acrosomal reaction in sea urchins is

initiated by contact of the sperm with the egg jelly (Fig 7.11)

Acrosome reaction cont’d• The contact between sperm and egg jelly causes the

exocytosis of the sperm’s acrosomal vesicle and release of proteolytic enzymes

• The proteolytic enzymes digest a path through the jelly coat to the egg surface

• The acrosomal process adheres to the vitelline envelope and tethers the sperm to the egg

• In sea urchin, the acrosome reaction is initiated by interaction of sperm cell membrane with a specific complex sugar in the egg jelly

• These polysaccharides bind to specific receptors on the sperm cell membrane directly above the acrosome vesicle

Species-specific recognition

• The first species-specific recognition event is achieved by sperm’s contact with the egg

• In sea urchin, another species-specific recognition occurs once the sea urchin sperm penetrates the egg jelly and the acrosomal process of the sperm contacts the surface of the egg (Fig 7.8)

• The protein mediating this reaction in sea urchins is called bindin

Fusion of the egg and sperm cell membrane

• Sperm-egg fusion causes a polymerization of actin molecules in the egg forming a fertilization cone (Fig. 7.15)

• The actin from the sperm and the egg forms a bridge through which the sperm nucleus passes through

Prevention of polyspermy• The entrance of multiple sperms (polyspermy) in most

organisms leads to disastrous consequences• In sea urchin fertilization by two sperms leads to a

triploid nucleus (3 sets of chromosome)• In sea urchins this is achieved in two ways:• A fast reaction caused by electric change in the egg

cell membrane• Slower reaction caused by exocytosis of the cortical

granules • These two reactions were delineated by the famous

African American embryologist, E. E. Just in early 1900s.

The fast reaction to polyspermy

• The cytoplasm of the oocyte has less Na+ than the outside environment

• K+ are more inside the oocyte cytoplasm than the outside environment– This condition is maintained across the cell membrane

by preventing Na+ from entering the cell and prevents K+ from leaking out of the cell

• This maintains the electrode potential difference between the inside of the egg and the ouside at -70 mV

• This potential difference is called resting membrane potential

Fast reaction to polyspermy cont’d

• Immediately (1-3 sec), the first sperm binds the egg cell membrane, the resting membrane potential becomes positive (+20) due to influx of some Na+ into the egg

• Sperms can only fuse with membranes having a resting potential, so no more sperm can fuse with the oocyte when the potential difference is +20

• The positive potential is not permanent, so polyspermy can still occur after the resting potential is restored if the sperms bound to the vitelline envelope are not removed

Slow reaction to polyspermy (cortical granule reaction)

• The removal of the sperms bound to the vitelline membrane is accomplished by the cortical granule reaction (slower reaction)

• The cortical granule reaction is caused by an increase in Ca2+ inside the egg cytoplasm immediately after fertilization

• It takes place a minute after the first successful sperm-egg fusion

• This slow block to polyspermy reaction is found in many animal species including mammals

Cortical granule cont’d

• Upon sperm entry, the cortical granules fuse with the egg cell membrane releasing proteolytic enzymes into the space between the cell membrane and the fibrous mat of the vitelline envelope proteins

• One of these proteins is trypsin-like protease called cortical granule serine protease

• This enzyme dissolves the protein posts that connect the vitelline envelope proteins to the cell membrane

Cortical granule reaction

• This enzyme also clips the bindin receptors and any sperm attached to them

• The components of the cortical granules fuse with the vitelline envelope to form a fertilization envelope

• The fertilization envelope is elevated from the cell membrane by mucopolysaccharides released by the cortical granules

Cortical granule reaction cont’d

• The mucopolysaccharides cause water to rush in the space between the cell membrane and the fertilization envelope causing it to expand and move away from the cell membrane

• The cortical granules releases a peroxidase enzyme that hardens the fertilization envelope

• The fertilization envelope begins to form at the point of contact with the egg and expands around the egg

• Another set of cortical granule proteins including hyalin form a coating around the egg called hyaline layer

• The egg extends microvilli whose tips attach to the hyaline layer and provide support to the blastomeres during cleavage (Fig. 7.19)

Mammalian fertilization

• It is difficult to study the interactions between the mammalian sperm and the egg:

1)Fertilization in these species takes place inside the oviducts of the female

2)We do not know the components of the various natural environments inside the female reproductive system that the sperm must encounter on its way to the egg

Mammalian fertilization cont’d

• Mammalian fertilization takes place in a region of the oviduct called ampulla

• Both the sperm and the egg uses a combination of biochemical interactions and a small amount of physical propulsion to get to the ampulla

• A newly released oocyte is surrounded by a matrix containing cumulus cells-the ovarian follicle cells that were nursing the developing oocyte

Mammalian fertilization cont’d

• If the cumulus-containing matrix is removed or altered, the oviduct does not pick up the oocyte-cumulus complex and the complex is unable to adhere or enter the oviduct

• Once picked up, the oocyte-cumulus complex uses a combination of ciliary beating and muscle contractions to transport the complex to the ampulla for fertilization

Mammalian fertilization • It is believed that sperm motility plays a minor

role in getting the sperm to the oviduct from the vagina

• However, sperm motility is required for mouse sperm to travel through the cervical mucosa and for the encounter with the egg

• Within 30 min of deposition in the vagina, sperms of mice, guinea pigs, cows, and humans, are found in the oviduct (pretty fast for sperm)

• Sperms appear to be transported to the oviduct by the muscular activity of the uterus

Factors affecting sperm motility

• Uterine muscle contractions are critical in getting the sperm into oviduct

• The region of the oviduct before the ampulla may slow down the sperm motility

• Sperm motility becomes hyperactivated once in the vicinity of the oocyte

• Sperm direction cues are provided by temperature gradients in the oviduct and chemicals in the oocytes or cumulus

Factors affecting sperm motility

• The trek from the vagina to the ampulla region of the oviduct enables the sperm to mature (competent) so that it can fertilize the egg once the two meet

• Newly ejaculated mammalian sperms are unable to undergo acrosome reaction until they have been in the female reproductive tract for some time

• The physiological changes that the sperm undergoes in order to become competent to fertilize the eggs are called capacitation

Mammalian fertilization cont’d

• Mammalian sperm contacts the egg at an angle and not at the tip as in sea urchins

• Mammalian gamete fusion is thought to depend on interaction between a sperm protein (izumo) and integrin-associated protein called CD9

• Polyspermy in mammals is a problem as well • Cortical granule reaction does not form a

fertilization membrane• The released enzymes modify zona pellucida so

that it can no longer bind sperm

Mammalian fertilization cont’d• Once the egg cell membrane and sperm cell

membrane fuse, the sperm pronuclei (haploid), mitochondria and its DNA, centriole and a small amount of cytoplasm enters the egg cytoplasm

• The sperm mitochondria and their DNA are degraded in the egg cytoplasm

• Therefore, all the mitochondria of the new individual come from its mother

• In the meantime, the egg pronucleus, completes meiosis which was arrested at metaphase of meiosis II producing a haploid pronucleus

Mammalian fertilization cont’d• The sperm that enters the egg has a haploid

pronucleus• Unlike the sea urchin sperm that enters the egg

perpendicularly, the mammalian sperm enters the egg at an angle (tangentially)

• The sperm fuses with numerous microvilli on the surface of the egg

• The DNA of the sperm nucleus is bound by basic proteins called protamines that are tightly compacted through disulfide bonds

• Glutathione in the egg cytoplasm reduces the disulfide bonds and uncoils the chromatin of sperm

Mammalian fertilization cont’d

• The sperm pronucleus fuses with the egg pronucleus forming a zygote (diploid)

• This marks the beginning of life that goes through rapid cell division (cleavage), gastrulation, organogenesis, metamorphosis (in some organisms), growth & maturation, reproduction, and finally death