Fertilization and gamete formation, Biology tutorial


Fertilization is a process whereby two sex cells (gametes) fuse together to form new individual with genetic potentials derived from both parents. Fertilization achieves two separate ends: sex (combining of genes derived from two parents) and reproduction (creation of new organisms). Therefore, first function of fertilization is to pass on genes from parent to offspring, and second is to start in egg cytoplasm those reactions which allow development to proceed.

 Structure of gametes:

Complex dialogue exists between egg and sperm. Egg activates sperm metabolism which is necessary for fertilization, and sperm reciprocates by activating egg metabolism required for onset of development.

Structure of the Sperm:

Mature sperm, known officially as spermatozoa, have a morphology which most people over age of ten would identify immediately. Nucleus is contained inside head, which, for most mammals, has the flattened, oval shape. During spermiogenesis, haploid sperm cell develops the tail or flagellum, and all of its mitochondria become aligned in the helix around first part of the tail, forming midpiece. The complete cell is, certainly, enveloped by the plasma membrane.

The other structure in mature sperm which plays a vital role in fertilization is acrosome. The acrosome is, in essence, a gigantic lysosome which forms around anterior portion of nucleus. It is bounded by the membrane which is considered to have two faces - inner acrosomal membrane faces nucleus, while the outer acrosomal membrane is in close contact with plasma membrane.

The ruffled appearance of plasma membrane is the artifact of fixation. Acrosome is dark band of material between plasma membrane and nucleus - inner and outer acrosomal membranes are not noticeably visible at this magnification.

Structure of the Egg:

Most mammals ovulate the egg which has matured in secondary oocyte; it is always secondary oocyte which is fertilized. Secondary oocyte is generated along with first polar body due to first meiotic division. Both of these cells are encased in the thick glycoprotein shell known as the zona pellucida. Genetically, the secondary oocyte which arrives in oviduct is in metaphase of second meiotic division. Metaphase plate is situated inside oocyte immediately below first polar body.

Final structural feature of the egg which acts as critical function during fertilization is the set of cortical granules. During oogenesis, oocyte develops thousands of small membrane-bound granules which accumulate in cortical cytoplasm, just beneath the plasma membrane.

Recognition of Egg and Sperm:

Interaction of sperm and egg usually proceeds according to 5 basic steps

1. Chemoattraction of sperm to the egg by soluble molecules secreted by egg.

2. The exocytosis of acrosomal vesicle to release the enzymes.

3. Binding of sperm to extracellular envelope (vitelline layer or zona pellucida) of egg.

4. Passing of sperm by this extracellular envelope.

5. Fusion of egg and sperm cell plasma membranes.

Sperm attraction: Action at a distance:

Species-specific sperm attraction has been documented in several species, comprising cnidarians, molluscs, echinoderms, and urochordates. In several species, sperm are attracted toward eggs of their species by chemotaxis, i.e., by following the gradient of the chemical secreted by egg. Developing oocytes at different stages in their maturation were fixed on microscope slides, and sperm were released at the certain distance from eggs. Though, after second meiotic division was completed and eggs were ready to be fertilized, sperm migrated toward them. Therefore, these oocytes control not only the kind of sperm they attract, but also the time at which they attract them.

Acrosomal Reaction in Sea-urchin:

A second interaction between sperm and egg is acrosomal reaction. In most marine invertebrates, acrosomal reaction has 2 components: fusion of the acrosomal vesicle with sperm plasma membrane (an exocytosis which results in release of the contents of acrosomal vesicle) and extension of acrosomal process. Acrosomal reaction in sea urchins is started by contact of sperm with egg jelly. Contact with egg jelly causes exocytosis of sperm's acrosomal vesicle and release of proteolytic enzymes which can digest path through jelly coat to egg surface.


Gamete binding and recognition in mammals:

ZP3: the sperm-binding protein of the mouse zona pellucida:

The zona pellucida in mammals plays the role analogous to that of vitelline envelope in invertebrates. This glycoprotein matrix that is synthesized and secreted by growing oocyte, plays two main roles during fertilization: it binds sperm, and it starts acrosomal reaction after sperm is bound. Binding of sperm to zona is comparatively, but not absolutely, species-specific. Binding of mouse sperm to mouse zona pellucida can be inhibited by first incubating sperm with zona glycoproteins. Bleil and Wassarman isolated an 83-kDa glycoprotein, ZP3, from mouse zona which was the active competitor for binding in this inhibition assay. Other two zona glycoproteins they found, ZP1 and ZP2, failed to struggle for sperm binding. Furthermore, they found that radio-labeled ZP3 bound to heads of mouse sperm with intact acrosomes. Therefore, ZP3 is specific glycoprotein in mouse zona pellucida to which sperm bind. ZP3 also starts acrosomal reaction after sperm have bound to it.

Induction of mammalian acrosomal reaction by ZP3:

Unlike the sea urchin acrosomal reaction, the acrosomal reaction in mammals takes place only after sperm has bound to zona pellucida. Mouse sperm acrosomal reaction is induced by cross linking of ZP3 with receptors for it on sperm membrane. This cross linking opens calcium channels to increase concentration of calcium in sperm. Mechanism by which ZP3 induces opening of calcium channels and subsequent exocytosis of acrosome remains controversial, but it may engage receptor's activating the cation channel (for sodium, potassium, or calcium), that would change resting potential of the sperm plasma membrane. Calcium channels in membrane would be sensitive to the change in membrane potential, permitting calcium to enter sperm.

Secondary binding of sperm to the zona pellucida:

During acrosomal reaction, anterior portion of the sperm plasma membrane is shed from the sperm. This region is where ZP3-binding proteins are situated, and yet sperm should still remain bound to zona to lyse the path through it. In mice, it seems that secondary binding to zona is achieved by proteins in inner acrosomal membrane which bind specially to ZP2. While acrosome-intact sperm will not bind to ZP2, acrosome-reacted sperm will. Furthermore, antibodies against ZP2 glycoprotein won't prevent binding of acrosome-intact sperm to the zona, but will inhibit attachment of acrosome-reacted sperm. Structure of zona comprises of repeating units of ZP3 and ZP2, infrequently cross linked by ZP1. It seems that acrosome-reacted sperm transfer their binding from ZP3 to adjacent ZP2 molecules.

Action at the Distance: Mammalian Gametes:

It is very tricky to study interactions which might be taking place between mammalian gametes before sperm-egg contact. One obvious reason for this is that mammalian fertilization takes place inside oviducts of female. While it is comparatively easy to imitate situations surrounding sea urchin fertilization, we don't yet know components of the different natural environments which mammalian sperm encounter as they travel to egg. The second reason for complexity is that sperm population ejaculated in female is possibly very heterogeneous, having spermatozoa at different phases of maturation. As fewer than 1 in 10,000 sperm get close to egg, it is hard to examine those molecules which might allow sperm to swim toward egg and become activated. There is great deal of controversy concerning mechanisms underlying translocation of mammalian sperm to oviduct, the chance that egg may be attracting the sperm by chemotaxis, and capacitation and hyperactivation reactions which appear essential for some species' sperm to bind with egg.

Translocation and Capacitation:

Reproductive tract of female mammals plays the very active role in mammalian fertilization process. While sperm motility is needed for mouse sperm to encounter egg once it is in oviduct, sperm motility is possibly the minor factor in getting sperm in oviduct in first place. Sperm are discovered in oviducts of mice, guinea pigs, hamsters, cows, and humans inside 30 minutes of sperm deposition in vagina, a time "too short to have been attained by even the most Olympian sperm relying on their own flagellar power". Rather, sperm seem to be transported to oviduct by muscular activity of uterus.

Hyperactivation and chemotaxis:

Different regions of female reproductive tract may secrete different, regionally particular molecules. These factors may influence sperm motility and capacitation. In addition to increasing activity of sperm, soluble factors in oviduct may also give directional component of sperm movement. There has been speculation which the ovum (or, more probable, ovarian follicle in which it developed) may secrete chemotactic substances which attract sperm toward egg during last phases of sperm migration tested this hypothesis using follicular fluid from human follicles whose eggs were being utilized for in vitro fertilization. Performing the experiment like the one described earlier with sea urchins, they microinjected the drop of follicular fluid in the larger drop of sperm suspension. Microinjection of other solutions didn't have this effect. These studies didn't rule out chance that effect was because of a general stimulation of sperm movement or metabolism. Though, these investigations uncovered the fascinating correlation: fluid from only about half the follicles tested illustrated a chemotactic effect, and in nearly every case, egg was fertilizable if, and only if, fluid illustrated chemotactic ability (P < 0.0001).

Fusion of the genetic Material:

Fusion of genetic material in sea urchins:

In sea urchins, sperm nucleus goes into egg perpendicular to egg surface. After fusion of sperm and egg plasma membranes, the sperm nucleus and its centriole divide from mitochondria and flagellum. Mitochondria and flagellum disintegrate inside egg, so very few, if any, sperm-derived mitochondria are found in developing or adult organisms. In mice, it is evaluated that only 1 out of every 10,000 mitochondria is sperm-derived. Therefore, though each gamete contributes the haploid genome to zygote, mitochondrial genome is transmitted mainly by maternal parent. Egg nucleus, once it is haploid, is known as female pronucleus. Once inside egg, the sperm nucleus decondenses to form male pronucleus. Sperm nucleus goes through dramatic transformation. Nuclear envelope vesiculates in small packets, thus exposing compact sperm chromatin to egg cytoplasm. Proteins holding sperm chromatin in its condensed, inactive state are exchanged for other proteins derived from egg cytoplasm. This exchange allows decondensation of sperm chromatin. In sea urchins, decondensation seems to be started by phosphorylation of two sperm-specific histones which bind tightly to DNA. This procedures starts when sperm comes in contact with glycoprotein in egg jelly which elevates level of cAMP-dependent protein kinase activity.

Fusion of genetic material in mammals:

In mammals, procedure of pronuclear migration takes approx 12 hours, compared with less than 1 hour in sea urchin. Mammalian sperm enters almost tangentially to surface of egg rather than approaching it perpendicularly, and it fuses with many microvilli. Mammalian sperm nucleus also breaks down as its chromatin decondenses and is then reconstructed by coalescing vesicles. DNA of sperm nucleus is bound by basic proteins known as protamines, and these nuclear proteins are tightly compacted through disulfide bonds. In egg cytoplasm, glutathione decreases these disulfide bonds and permits uncoiling of sperm chromatin. Mammalian male pronucleus enlarges while oocyte nucleus completes its second meiotic division. Centrosome (new centriole) accompanying male pronucleus generates its asters (largely from proteins stored in the oocyte) and contacts female pronucleus.

Rearrangement of Egg Cytoplasm:

Fertilization can start radical displacements of egg's cytoplasmic materials. While these cytoplasmic movements are not obvious in mammalian or sea urchin eggs, there are numerous species in which these rearrangements of oocyte cytoplasm are vital for cell differentiation later in development. Such cytoplasmic movements are also simple to see in amphibian eggs. In frogs, single sperm can enter anywhere on animal hemisphere of egg; when it does, it changes cytoplasmic pattern of egg.

Initially, the egg is radially symmetrical about the animal-vegetal axis. After sperm entry, however, the cortical (outer) cytoplasm shifts approx 30° toward point of sperm entry, relative to inner cytoplasm. In some frogs (like Rana), a region of the egg which was formerly covered by dark cortical cytoplasm of animal hemisphere is now exposed. This underlying cytoplasm, situated near equator on side opposite the point of sperm entry, has diffuse pigment granules and thus seems gray.

Preparation for cleavage:

Increase in intracellular free calcium ions which activates DNA and protein synthesis also sets in motion apparatus for cell division. Mechanisms by which cleavage is started possibly vary among species, depending on phase of meiosis at which fertilization takes place. Though, in all species studied, the rhythm of cell divisions is handled by synthesis and degradation of the protein known as cyclin.

Cleavage has the special relationship to egg regions established by cytoplasmic movements described above. In tunicate embryos, first cleavage bisects egg, with its established cytoplasmic pattern, in mirror-image duplicates. From that phase on, every division on one side of cleavage furrow has the mirror-image division on opposite side. Likewise, gray crescent is bisected by first cleavage furrow in amphibian eggs. Therefore, position of first cleavage is not random, but tends to be specified by point of sperm entry and subsequent rotation of egg cytoplasm. Coordination of cleavage plane and cytoplasmic rearrangements is probably mediated through microtubules of sperm aster.

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