Fertilization: The Beginning of Development in

a New Organism

Lange

BIOL 370 – Developmental Biology

Topic #4

Fertilization: The process where the gametes fuse together to create a new instance of an organism whose genome is derived from two parents.

There are four major events in this process:

1.Contact and recognition between gametes (species recognition is a key aspect of this process).

2.Regulation of the merging of gametes (typically regulation of the sperm into the egg and the inhibition of additional sperm from entering).

3.Fusion of the genetic material of the gametes.

4.Activation of metabolism in the fertilized product to start development.

Sperm – the “male” gamete which typically is much smaller than the ova.

First discovered by Anton van Leeuwenhoek in the 1670s, but mistakenly suggested that sperm were actually parasites in the semen.

The human infant preformed in the sperm, as depicted by Nicolas Hartsoeker (1694)

Hartsoeker proposed that there would be found within sperm a homuncular body and drew this image of what he hoped would be discovered with improvements in microscopy.

It was not until the 1876 that Oscar Hertwig and Herman Fol both demonstrated that sperm were definitively entering the egg.

Oscar Hertwig Herman Fol

Modification of a germ cell to form a mammalian sperm – SPERM ANATOMY

General overview of sperm formation in mammals.

Modification of a germ cell to form a mammalian sperm (Part 1)

• Acrosomal vesicle (acrosome) – a structure derived from the Golgi apparatus that associates with the highly condensed nucleus. The acrosome produces enzymes and is a secretory vesicle. The use of these enzymes is to digest through the outer coverings of the egg during fertilization. This vesicle will form the acrosomal cap that is associated with capacitation of sperm.

• Notice how the flagellum develops from the centriole.

Modification of a germ cell to form a mammalian sperm (Part 2)

• Sperm head – the acrosome and the nucleus

• Flagellum – the tail-like projection

Motile apparatus of the sperm (Part 1)

• Axoneme – the flagellar structures that are formed from microtubules originating at the centriole within the midpiece.

Motile apparatus of the sperm (Part 2)

• Tubulin – the major protein constituent of the microtubules

• Dynein – the protein that is going to hydrolyze ATP (it is actually an ATPase) to release energy.

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Modification of a germ cell to form a mammalian sperm (Part 3)

• Capacitation – the final stages of sperm maturation. In most mammals this occurs OUTSIDE of the testes and is typically within the vaginal/uterine environment of the female. This occurs. The uterus aids in the steps of capacitation by secreting variety of chemicals that react with the outer aspects of the sperm.

• Non-mammalian spermatozoa do not undergo capacitation step and are ready to fertilize an oocyte immediately after release from the male.

• When capacitation occurs the sperm must still undergo the acrosome reaction.

Structure of the sea urchin egg at fertilization

Be sure to notice difference in sperm size and egg size.

•Oocyte – a developing egg that CANNOT yet be fertilized

•Ovum (or egg) – female gamete capable of being fertilized

Eggs of different species can be at different developmental stages of meiosis prior to fertilization.

Other important aspects about the structure of the egg:

•Cytoplasm content in the egg is roughly 10,000 X that of sperm in a typical sea urchin egg (similar differences are seen in many other species).

•Cytoplasm of the egg contains nutritive proteins, ribosomes, tRNA, mRNA, protective factors (such as filters, repair enzymes, distasteful chemicals, antibiotics), and morphogenetic factors (to regulate development).

Stages of egg maturation at the time of sperm entry in different animal species

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Figure 27.19

Sea urchin egg cell surface

Vitelline envelope – an extracellular matrix that forms a “fibrous” mat around the egg. It is used typically in sperm-egg recognition

Egg jelly – seen in some species, resides outside the vitelline envelope, but when present it serves to attract and activate (capacitate) sperm

(Egg) Cortex – a thin layer of cytoplasm along the interior of the cell membrane of most eggs that is more dense than most other cytoplasm. The cortex ** is especially rich in actin that helps in the formation of microfilaments and microvilli.

Cortical granules – homologus structures to the acrosomal vesicles in sperm.

Mammalian eggs immediately before fertilization

In mammals we see a zona pellucida & corona radiata – a thick matrix (z.p.) and layer of cells (c.r.) that mammalian sperm cells need to pass through prior to fertilization.

In this image, the zona pellucida is the thick, white band and the corona radiata (and its cells) surrounds the zona pellucida.

Summary of events leading to the fusion of egg and sperm cell membranes in the sea urchin and the mouse

Sperm chemotaxis in the sea urchin Arbacia punctulata

Chemotaxis –

the phenomenon whereby somatic cells, bacteria, and other single-cell or multicellular organisms direct their movements according to certain chemicals in their environment. In fertilization, the chemicals are positively

chemotaxic.

Model for chemotactic peptides in sea urchin sperm

• Resact is a small peptide chemical that stimulates a cell membrane protein on the head of the sperm.

• The resulting opening of calcium channels causes an influx of calcium into the sperm cell.

• This leads to swimming behavior in the sperm.

Note that this image depicts the SPERM CELL.

Species-specific induction of the acrosome reaction by sulfated polysaccharides characterizing the egg jelly coats of three species of sea urchins

Acrosomal reaction - the reaction that occurs in the acrosome of the sperm as it approaches the egg. The acrosome is a cap-like structure over the anterior half of the sperm's head.

In this example, different egg jelly layer polysaccharides initiate this reaction in a species specific fashion.

Acrosome reaction in sea urchin sperm

In sea urchins, the protein BINDIN is seen at the base of globular cluster of acrosomal enzymes. During the acrosomal reaction, the formation of the acrosomal process occurs and the bindin is important in the recognition of the differences in egg jelly.

(Look at Vacquier and Moy, 1977)

Species-specific binding of acrosomal process to egg surface in sea urchins

Scanning electron micrographs of the entry of sperm into sea urchin eggs

Following the acrosome reaction, the sperm enters the egg causing actin to polymerize and the egg forms a fertilization cone (the homologous process for the egg).

• Monospermy – the normal condition of fertilization that results in the formation of a diploid cell.

• Polyspermy – the enterance of more than one sperm into the ovum. In animals and most organisms this leads to abnormal development and typically fatal consequences for the cell or early stage embryo.

Theodor Boveri – demonstrated through experimentation the problems associated with polyspermy in 1902.

Ova combat the risks of polyspermy in ways that have tremendous similarities to how neurons function:

“Fast Block” to polyspermy – “electrical” membrane potential of the egg changes immediately after sperm entry. The this change is due to the closing of sodium channels. The change can be similar to that of a neuron stimulated and activating an action potential. The change in this fast block is from the resting potential of -70 mV to a new potential of +20 mV.

Membrane potential of sea urchin eggs before and after fertilization

Notice how in the sea urchin, we can see the “fast block” occurs very quickly following addition of sperm.

Formation of the fertilization envelope and removal of excess sperm

“Slow Block” to polyspermy – more technically called the “CORTICAL GRANULE REACTION”.

•In this process, cortical granules that line the inner layer of the egg’s cell membrane fuse with the membrane. •The release of their contents via exocytosis will put a layer of an enzyme between the cell membrane and the vitelline envelope.•The enzyme is called cortical granule serine protease, and it helps in breaking protein bonds and is part of the formation of the fertilization envelope.

Cortical granule exocytosis

This “slow block” mechanism develops the fertilization envelope.

Aberrant development in a dispermic sea urchin egg

This diagram shows how the rare occurrence of polyspermy results in developmental defects that prevent in virtually all cases, the survival of the organism.

Wave of Ca2+ release across a sea urchin egg during fertilization

The cortical granule reaction is initiated by a wave of calcium ions that begins where the single sperm has entered the egg. This calcium wave flowing across the egg takes on average about 30 seconds.

Endoplasmic reticulum surrounding cortical granules in sea urchin eggs

In most (but not all) organisms, the calcium used in this process is stored in the ER associated with the cortical granules.

Postulated pathway of egg activation in the sea urchin

Variations in the process of fertilization related specifically to internal fertilization occurring in mammals:

•Occurs in the oviducts, making it difficult to study•Heterogeneity of sperm development•The potential of MULTIPLE mechanisms of sperm acrosome reaction in mammals

Fertilization in mammals typically occurs in the ampulla of the oviduct.

DES – Endocrine Disrupting Prescription Medication Used in the 1950s & 1960s

Diethylstilbestrol (DES) is a synthetic nonsteroidal estrogen first synthesized in 1938. •It is classified as an endocrine disruptor.

Human exposure to DES occurred through diverse sources:•dietary ingestion from supplemented cattle feed •medical treatment for certain conditions (breast and prostate cancers) •from about 1950 to 1971, DES was given to pregnant women in the mistaken belief it would reduce the risk of pregnancy complications and losses.

DES

Sperm Transport in Mammals:

•Ejaculation by the male will deposit sperm into the vaginal orifice

•Timing from release by the male and reaching of the ampulla in humans is roughly around 30 minutes in many instances of fertilization. Sperm are not able to navigate that distance by flagellar swimming in that short a time frame.

•Muscular activity of the uterus is critical for sperm movement from vagina to ampulla.

****See Baylis & Baker, 1993

Capacitiation of sperm:

•Newly ejaculated sperm are not able to undergo the acrosome reaction (and hence cannot fertilize the egg)

•Time must be spent in the female reproductive tract where physiological changes occur called CAPACITATION.

• Sperm cell membrane is denuded of cholesterol• This removal leads to changes in position of lipids in the

membrane surface (called “lipid rafts”) that surround specific receptor proteins.

• These receptor proteins (on the “lipid rafts”) then migrate to the anterior head of the sperm

• These proteins are associated with the acrosome reaction and binding to the zona pellucida.

Hypothetical model for mammalian sperm capacitation

SEM (artificially colored) showing bull sperm as it adheres to the membranes of epithelial cells in the oviduct of a cow prior to entering the ampulla

Hyperactivation – following capacitation, the swimming rate of sperm increases dramatically

Chemotaxis “Part A” – capacitation and hyperactivation may permit the sperm to sense a chemical gradient within the oviduct

Thermotaxis – capacitiation allows sperm to be more sensitive to temperature gradients. Studies have suggested that capacitated sperm can sense differences in temperature as small as 0.014 degrees Celcius. Sperm have been shown to be positively thermotaxic (meaning they will migrate toward warmer temperatures).

Chemotaxis “Part B” – in the ampulla, it appears that progesterone is a chemical mediator that attracts sperm.

Acrosome reaction in hamster sperm

Entry of sperm into a golden hamster egg

End.