The Female Reproductive Tract
Meet The Gametes
Ovulation
The Oocyte Cumulus Complex
The Zona Pellucida
Pick-up of the Oocyte Cumulus Complex by the Oviduct
Fertilization of the Oocyte
The Cortical Granule Reaction
Pronuclear Formation, Cleavage, and Implantation
THE FEMALE REPRODUCTIVE TRACT:

The female reproductive tract consists of the ovaries, oviduct, and a uterus, as shown for humans in the figures below. The ovaries produce both hormones and the female gametes or oocytes. The oviduct is the site of fertilization and preimplantation development, and the uterus is the organ where the embryo implants and develops until birth.


FIGURE: A schematic diagram of a female reproductive tract (one side) showing the ovary, oviduct, and uterus. Notice that the oviduct is divided into three anatomical regions, the infundibulum, ampulla, and isthmus.


FIGURE: A human reproductive tract.
Meet the Gametes:

Fertilization in mammals involves two gametes, the sperm, which are made by males in the testes, and the oocytes, which are made by females in the ovaries.

Sperm are specialized streamlined cells produced in the testes during the process of spermatogenesis. In humans it takes about 64 days to make a mature sperm. The figure below on the left shows a human testis that has been cut open revealing its many seminiferous tubules where sperm are produced. Mature sperm are haploid cells that contain one set of chromosomes in their nucleus. Mature sperm are also characterized by a flagellum for motility and a secretory vesicle, the acrosome, at their leading edge.


FIGURE: Human testis that has been cut open. Inside are numerous seminiferous tubules in which sperm are produced. The areas between tubules contain Leydig cells that produce male hormones.


FIGURE: A histological section through a seminiferous tubule in a testis. The letters denote cells in various stages of spermatogenesis. For example, "a" is a spermatogonium and "b" is a primary spermatocyte. As sperm mature, they move closer to the lumen of the tubule.
When mature sperm leave the testis, they move into the epididymis and vas deferens where they are stored until they are needed for fertilization. Sperm were once thought to contain a preformed miniature human, as shown in the homunculus below. However, we now know that mammalian sperm have a head containing a nucleus with DNA and an acrosome or secretory vesicle that is lost during fertilization. The head attaches to a flagellum that provides motility for penetration of the coats surrounding the oocyte. The basic structural features of sperm - nucleus, acrosome and flagellum - are present in all mammalian sperm. However, sperm from different species have very distinct morphologies and expert spermatologists can identify a species by looking at its sperm. Several examples of mammalian sperm are shown below.


FIGURE: Low and high magnifications of guinea pig sperm. Guinea pig sperm are stacked in rouleaux when they leave the testes as shown in the low magnification micrograph (left). The guinea pig acrosome (AC) is very large and can be seen directly with light microscopy (middle). The shape of the acrosomal cap is even better seen in the higher resolution scanning electron micrograph (right).


FIGURE: Examples of hamster sperm showing the head and flagellum. The acrosome has begun lifting off the two sperm in the lower part of the figure. The shape of the hamster and guinea pig sperm heads are quite different.


FIGURE: Histochemical reaction performed on guinea pig testes to reveal the location of DPP-II. The reaction product (golden color) is localized to the acrosomes of the sperm (LDiCarlantonio and Talbot, 1988).


FIGURE: Within the acrosome itself, the DPP II reaction product is confined to a compartment called the dorsal bulge. The reaction product appears as small black grains (arrow). Within the dorsal bulge, there are also spherical zones that lack reaction product (asterisk).
The Oocyte: Oocytes are produced in the ovarian follicles during oogenesis. Like sperm, they are haploid cells when mature and carry a single set of chromosomes. While human sperm can be made in about 64 days, human oocytes require many years to complete oogenesis. As oocytes mature, their follicle also matures and becomes very large and filled with fluid. During oogenesis, the oocytes secrete an extracellular matrix, the zona pellucida, that is made up of three glycoproteins, ZP1, ZP2, and ZP3.


FIGURE: Germinal vesicle stage oocyte from a mouse. This oocyte is arrested in meiosis I. A large nucleus is present near the center of the oocyte. The arrow points to the surface of the oocyte, beyond which is the zona pellucida. Prior to ovulation, the oocyte will resume meiosis and proceed as far as metaphase II of meiosis before it is fertilized. It will not actually complete meiosis until after it is fertilized.


FIGURE: A maturing follicle with its oocyte (at tip of finger). The oocyte is surrounded by its zona pellucida (pink staining ring) that is in turn surrounded by numerous small cells that will become cumulus cells. The follicle has accumulated fluid (sunflower) in its antrum.
In response to luteinizing hormone (LH) from the anterior pituitary gland, the cumulus cells surrounding the oocyte secrete an extracellular matrix that is rich in hyaluronan (see figure below). Hyaluronan is a large negatively charged polymer that draws fluid into the space between the cumulus cells, thereby causing the cumulus cells to expand or push apart from each other. The oocyte, zona pellucida, cumulus cells and cumulus extracellular matrix together form the oocyte cumulus complex (OCC).


FIGURE: Hyaluronan is a large polymer of glucuronic acid and N-acetylglucosamine. This repeating disaccharide unit can form huge molecules that are even larger than some bacteria. GAG = glycosaminoglycan.


FIGURE: Part of an oocyte cumulus complex. The oocyte is the large central cell surrounded by numerous small cumulus cells. The matrix between the cumulus cells can not be seen in this figure.


OVULATION:

Oocytes, which develop in ovarian follicles, are surrounded by numerous cumulus cells that are held together by a sticky extracellular matrix. The thick, multilayered wall of the follicle retains the oocyte cumulus complex in the fluid-filled antrum of the follicle. Before an oocyte can be fertilized, it must be ovulated from an ovarian follicle and picked-up by the infundibulum of the ovary. Ovulation presents an interesting, complex biological problem - how can an oocyte escape from the enclosed follicle which has a wall made up of a number of cell layers and extracellular matrix. For ovulation to occur, enzymes first weaken the wall of the follicle, then smooth muscle cells in the base of follicles contract. This contraction pushes the oocyte cumulus complex up against the weakened apex and eventually causes it to rupture, thereby, squeezing the oocyte cumulus complex out through the rupture site. The figures below show how the shape of the follicle changes when muscles in its base contract and how a V-shaped constriction forms in its base during contraction. The oocyte cumulus complex is pushed towards and through the enzymatically weakened apex by this contraction (Martin et al,. 1981).


FIGURE: Electron micrograph showing a muscle cell from the base of a hamster ovarian follicle. Organelles are concentrated at the poles of the nucleus and the remaining cytoplasm is packed with actin and myosin for contraction.


FIGURE: External views of hamster follicles showing how the profile of the follicle changes as the smooth muscle cells at the base of the follicle contract. As the muscle cells contract, the follicle becomes taller and narrower. This pushes the cumulus mass towards the rupture site at the apex, thereby helping to thin the apex.


FIGURE: Scanning electron micrograph showing a bird's eye view of a rupture site forming in a hamster follicle. The apex of the follicle is already torn and blood cells and fluid (arrow) are leaking through the tear.


FIGURE: Histological sections showing mature hamster follicles undergoing ovulation. The arrowheads show the base of the follicle as it constricts due to contraction of smooth muscle cells. A V-shaped constriction forms in the base due to this contraction. The arrows show the apex of the follicle which weakens and eventually ruptures as the oocyte cumulus complex is pressed against it by muscle cell contraction.


FIGURE: Scanning electron micrographs showing bird's eye views of the base of follicles as the muscle in the follicle wall contracts. The contour of the base, which is initially smooth (not shown) becomes folded by contraction. The arrow points to a fold in the base of one follicle.


Topical treatment of follicles with drugs that inhibit smooth muscle cell contraction, such as verapamil, prevents formation of the V-shaped constriction and inhibits ovulation (Martin et al., 1981b). If a rupture site is artificially poked in a follicle wall before smooth muscles in the base contract, the oocyte cumulus complex can escape from the follicle; however, a long time is required for this (Talbot and Chacon, 1982). Apparently escape under these conditions occurs because fluid outside of the follicle is drawn into the cumulus matrix, which acts like a sponge, and eventually the accumulated fluid pushes the cumulus mass out of the follicle through the artificial rupture site. During natural ovulation, however, the pushing is done by muscle contraction at the base of the follicle.

Pressure has been measured in hamster follicles before and during ovulation. During muscle contraction, the pressure within the antrum does not increase and in fact may decrease slightly because the rupture site is leaky and apparently oozes fluid and cells at the time the muscles contract (Schroeder and Talbot, 1982). Thus pressure does not increase prior to ovulation, as was once thought, but instead pressure is actually bled off the follicle through the leaky rupture site.

The oocyte cumulus complex forms a solid structure that the contracting follicle can press against the apex. By having the oocyte embedded in the larger oocyte cumulus complex, the opportunity for successful ovulation increases. If the oocyte were surrounded only by a zona pellucida, it might easily become trapped within the follicle and fail to escape during ovulation.
THE OOCYTE CUMULUS COMPLEX:

Ovulation results in the escape of the oocyte cumulus complex from the follicle. This complex is ovulated into the peritoneal cavity in humans or bursal cavity in rodents. The oocyte cumulus complex contains a centrally located oocyte surrounded by its zona pellucida. Outside of the zona are layers of tightly packed cells called the corona radiata, and outside of the corona are loosely packed cumulus cells. The space between the corona and cumulus cells is filled with an extracellular matrix synthesized and released by the cells prior to ovulation. The figure below (left) shows an ovulated oocyte cumulus complex, as it would appear shortly after ovulation. The structure of a hamster oocyte cumulus complex is shown in greater detail in the scanning electron micrograph below (right).


FIGURE: An ovulated oocyte cumulus complex. An oocyte (O) is at the center of the complex. The zona pellucida (ZP) is not visible in this micrograph, but would be just beyond the tip of the ZP arrow. Cells just outside the zona are densely packed forming the corona radiata (CR). Cumulus cells are widely separated from each other in the cumulus layer (CL).


FIGURE: Scanning electron micrograph of a hamster oocyte cumulus complex showing surface of the zona pellucida and corona radiata cells. Some corona cells attach directly to the surface of the zona. The matrix that is normally present between cells is not shown in this figure.
The space in between cumulus cells contains an extracellular matrix rich in hyaluronan that is covalently linked to inter-alpha-trypsin inhibitor, creating a stable matrix that holds the oocyte cumulus complex together. If the complex is treated with the enzyme hyaluronidase, which degrades hyaluronan, the cumulus cells will fall away from the rest of the complex.


FIGURE: Micrograph of an oocyte cumulus complex treated with India ink to demonstrate the extracellular matrix (black) that outlines the white cumulus cells. Ink particles can diffuse in between cells and therefore stain the matrix. Ink, however,can not penetrate the cumulus cell membranes, so the cells appear white.


FIGURE: Confocal micrograph showing a hamster oocyte cumulus complex labeled with an antibody to inter-alpha-trypsin inhibitor. The antibody (green) has labeled the extracellular matrix between the cumulus cells.


FIGURE: Scanning electron micrograph of a hamster oocyte cumulus complex. Cumulus cells (green) and matrix (gray) are shown. Small blood clots (red) also often appear in oocyte cumulus complexes.


At the ultrastructural level, the cumulus matrix is comprised of granules that are sensitive to trypsin and filaments that are sensitive to hyaluronidase (Talbot and DiCarlantonio, 1984c).


FIGURE: Cumulus matrix showing granules (arrowhead) and filaments that are preserved by fixation in presence of ruthenium red.


FIGURE: Another transmission electron micrograph showing the granules and filaments in the cumulus matrix. Some granules (arrowheads) attach to the surface of a cumulus cell.


FIGURE: Cumulus matrix after treatment with trypsin. Filaments are present while granules (arrowheads) are reduced in size and number.


FIGURE: The cumulus matrix after treatment with hyaluronidase. Filaments are absent but granules (arrowheads) remain.
When the matrix is digested with the enzyme hyaluronidase to remove cumulus cells, the oocyte and zona pellucida are readily seen, as shown in the figure below left. However, it is the oocyte cumulus complex that is actually ovulated and travels into the oviduct where fertilization occurs. The granule-filament matrix that is present between cumulus cells also extends into the pores of the zona pellucida and the perivitelline space, as shown below to the right (Talbot and DiCarlantonio, 1984; Dandekar et al., 1992).


FIGURE: A cumulus cell free oocyte surrounded by a zona pellucida as it would appear just prior to fertilization. One polar body has been produced by completion of the first meiotic division. The perivitelline space (arrow) is large near the polar body.


FIGURE: Scanning electron micrograph of a hamster zona pellucida showing its porous nature. Matrix and corona cell processes extend into these pores in vivo.


FIGURE: The granule and filament matrix (yellow) shown above in the cumulus layer is also present in the perivitelline space (purple) of unfertilized oocytes. ZP = zona pellucida, O = oocyte, yellow = granule-filament matrix, g = granule, arrowhead = filaments.


THE ZONA PELLUCIDA:

Mammalian oocytes are surrounded by an extracellular matrix, the zona pellucida, as shown above to the left. Other labs have demonstrated that the mouse zona is made of three proteins which are called ZP1, ZP2, and ZP3. These proteins are made and secreted by the oocyte during oogenesis. ZP2 and ZP3 are thought to form heterodimeric filaments that are cross bridged by ZP1. Dr. Paul Wasserman's lab first showed that ZP3 also binds to sperm and induces the acrosome reaction. Dr. Jurrien Dean’s lab has created knock out mice lacking each of the zona proteins. Interestingly, the ZP1 knockouts still had zonas, although they were thinner than in the wild animals (Rankin et al., 1999). The ZP1 knockouts were also fertile indicating that the zona can exist without ZP1 and that additional factors must stabilize the ZP2-ZP3 filaments. The oocytes from ZP3 knockouts lacked zonas and were not fertile, illustrating the importance of this extracellular matrix in reproduction.


FIGURE: Scanning electron micrographs comparing zonas from ZP1 knockouts (nulls) and normal females. In the knockouts, the zona is very thin and the surface of the oocyte can be seen through pores in the zona (arrow C). Also the zona creeps up around the cumulus cells in the knockouts (B),but not in the normal females (E).
PICK-UP OF THE OOCYTE CUMULUS COMPEX BY THE OVIDUCT:

The oocyte cumulus complex is ovulated into the peritoneal cavity or bursal cavity and must be picked up by cilia on the exterior of the oviduct and carried through the ostium into the ampulla of the oviduct where fertilization occurs. Oocyte cumulus complex pick-up is important as it positions the oocyte in the correct place for fertilization.


FIGURE: Scanning electron micrograph showing the opening or ostium of a hamster oviduct. The ostium is normally closed but becomes opened like this as oocyte cumulus complexes pass through it.
Pick-up of the oocyte cumulus complex depends on two processes – adhesion of the complex to the oviductal cilia and beating of the cilia which moves the complex into the oviduct (Talbot et al., 1999). If small particles such as Lycopodium spores are placed on the infundibulum, the spores, because they are small and light weight, will be carried into the oviduct by the current created above the beating cilia. This can be seen in the movie below. The spores actually tumble and roll over the surface of the infundibulum before entering the ostium.


FIGURE: Explanted hamster infundibulum with spores on its surface. (Click on Image) to see a movie of the spores being carried by ciliary currents over the surface of this infundibulum. This video was made in vitro. Watch carefully and you can see cilia beating on the edge of the infundibulum.


FIGURE: Scanning electron micrograph showing the epithelium on the exterior surface of a hamster infundibulum. Most of the cells are ciliated (arrows), while some cells are secretory and have short microvilli (right side of figure). The ciliated cells beat in the direction of the ostium (see figure above).
In contrast to spores, oocyte cumulus complexes are large structures with considerable mass. They are too large to be carried in the ciliary current. For successful pick-up, the oocyte cumulus complex must first adhere to the surface of the infundibulum. The beating cilia then provide the motor for pulling the adherent complex toward the opening of the oviduct. As the complex moves over the surface of the infundibulum, adhesion at the trailing end of the complex is disrupted so that forward movement toward the ostium is possible. The movie below shows an oocyte cumulus complex (stained blue) moving over the surface of a hamster infundibulum.


FIGURE: Infundibulum with oocyte cumulus complex (blue) attached. Please Click on Image to load the movie showing an oocyte cumulus complex moving over the surface of an infundibulum.


FIGURE: Scanning electron micrograph showing an oocyte cumulus complex (blue) entering the ostium of the oviduct.
The extracellular matrix between the cumulus cells adheres to the "crown" at the tips of the cilia, as shown in the electron micrograph below (Lam et al., 2000). This adhesion is strong enough to keep the oocyte cumulus complex attached to the infundibulum, yet weak enough to allow the beating cilia to move the complex toward the ostium.

As the oocyte cumulus complex travels over the surface of the infundibulum, some of its extracellular matrix apparently tears and remains bound to the tips of the cilia as shown in the scanning electron micrograph below.


FIGURE: Transmission electron micrograph showing the cumulus matrix adhering to the tips of oviductal cilia. The tip of each cilium has a "crown" to which the matrix adheres (see high magnification views on right side of figure).


FIGURE: Scanning electron micrograph showing cumulus matrix (yellow) that has remained stuck to the tips of cilia (purple) after an oocyte cumulus complex traveled over the infundibulum.
If the extracellular matrix is not present between cumulus cells, the oocyte cumulus complex can not adhere to the oviduct and pick-up does not occur (Lam et al., 2000). This is illustrated in the figure below.


FIGURE: Only expanded oocyte cumulus complexes with extracellular matrix are picked up by oviduct. (C) The oocyte cumulus complex (blue) was not expanded and hence did not have extracellular matrix and did not move into the oviduct even after several hours. (F) The expanded oocyte cumulus complex in this preparation moved over the infundibulum and into the oviduct in about 10 seconds. It can be seen here entering the ostium of the oviduct.
Factors that interfere with proper adhesion of the complex to the infundibulum preclude pick-up of the oocyte cumulus complex by the oviduct. For example, if adhesion is increased by incubating either the infundibulum or the oocyte cumulus complex in the lectin WGA, the complex and infundibulum adhere so tightly that the cilia are not able to move the complex to the ostium (Lam et al., 2000).


FIGURE: Effect of WGA treatment on adhesion and pick-up rate. (A) shows that untreated infundibula and treated OCCs (UI-TOCC), or treated infundibula and untreated OCCs (TI-UOCC), or treated infundibula and treated OCCs (TI-TOCC) all adhere control. (B) Shows that treatment of the infundibulum or of both the infundibulum and OCC stops OCC pick-up. (C) Shows that the above inhibition of pick-up rate is not due to an effect on ciliary beating, and therefore can be attributed to the increase in adhesion observed in panel.
Conversely, if the cumulus matrix is crushed so that it is no longer sticky enough, the oocyte cumulus complex will not bind tightly to the cilia and will fall off during pick-up, as illustrated in the movie below.


FIGURE: Click on image to see a movie showing that this OCC, which has a crushed matrix, does not adhere to the infundibulum strongly enough to be picked up by ciliary beating.
Pick-up can also be inhibited by exposing the infundibulum or the oocyte cumulus complex to chemicals that are present in cigarette smoke (Knoll and Talbot, 1998), suggesting that environmental factors can affect this process and could therefore affect pregnancy. More information on this topic can be found at this website on our research on smoking page.

It is interesting that in hamsters the oocyte cumulus complex is actually too wide in diameter to fit through the ostium. The ostium also is normally not wide open, so it has to be “opened” by the first oocyte cumulus complex to go through. To gain entrance to the oviduct, the large oocyte cumulus complex undergoes “churning” when it reaches the ostium. Cilia provide the motive force for churning which compresses the extracellular matrix of the cumulus layer, thereby making the diameter of the complex smaller so that it can fit through the ostium and enter the oviduct. The movie below illustrates the process of churning.


FIGURE: Bird's eye view of an oocyte cumulus complex entering hamster oviduct. The cumulus mass is too wide to fit through the ostium, so it “churns” which compresses the matrix and makes it smaller. It can eventually fit through the ostium and enter the oviduct. Click on image to view a moving showing churning of an oocyte cumulus complex.
FERTILIZATION OF THE OOCYTE:

Penetration of the Cumulus: After passing through the ostium, the oocyte cumulus complex is quickly moved into the ampulla of the oviduct where fertilization occurs. The actual number of sperm that reach the site of fertilization is small, about 10-1000 in most mammals. Sperm must first penetrate the cumulus matrix, which requires that they first undergo capacitation. During capacitation, the surface of the sperm changes enabling it to pass through the cumulus layer. Sperm that are uncapacitated get stuck in the cumulus and do not reach the zona (Corselli and Talbot, 1987). It used to be thought that sperm released the enzyme hyaluronidase from their acrosome as they penetrated the cumulus. While hyaluronidase on the sperm surface may facilitate penetration of the cumulus, it is not required since sperm from knockout mice lacking this enzyme are able to fertilize oocytes (Baba et al., 2002, J Biol Chem 277: 30310) and sperm from non-mammalian species that do not have hyaluronidase in their acrosomes are able to penetrate to the zona (Talbot et al., 1985).


FIGURE: Electron micrograph showing a sea urchin sperm that has penetrated the cumulus layer and bound to the zona pellucida. Sea urchin sperm do not have hyaluronidase. Frog sperm and the alga Chlamydomonas are also able to penetrate the cumulus layer without hyaluronidase. Chlamydomonas has two flagella at its front end and swims using the "breast stroke," further suggesting that the cumulus matrix is relatively easy for motile cells to penetrate.
Sperm Binding to the Zona Pellucida: After penetrating the cumulus layer, sperm bind to the zona pellucida by their head. Initially, this binding is weak as sperm may bind and detach and rebind repeatedly. Sperm bind to ZP3, one of the three zona proteins, but it is not yet clear what molecule(s) on the sperm surface binds to ZP3.


FIGURE: Light micrograph showing hamster sperm bound to the zona pellucida of a hamster oocyte undergoing in vitro fertilization. When oocytes are fertilized in vivo, fewer sperm bind to the zona.
The Acrosome Reaction: Dr. Paul Wasserman’s lab first showed that after sperm reach the zona pellucida and bind to ZP3, they undergo an acrosome reaction, apparently induced by ZP3. The acrosome reaction occurs when the plasma membrane of the sperm undergoes multiple vesiculations with the underlying acrosomal membrane. As first shown by Dr. Claudio Barros et al. The acrosome reaction causes the release of hydrolytic enzymes, including the protease acrosin, whose functions in fertilization still remain uncertain.


FIGURE: The guinea pig sperm acrosome reaction as viewed with the light microscope. Guinea pig sperm have a large acrosome (AC) that can easily be seen with light microscopy. The sperm on the left is unreacted and the acrosome is clearly seen. The middle sperm has completed an acrosome reaction and the nucleus remains. The sperm on the right of the panel is in the process of undergoing an acrosome reaction. The acrosome appears crenulated.


FIGURE: The hamster sperm acrosome reaction. Hamster sperm have a smaller acrosome than guinea pig sperm but it is nevertheless large enough to see with light microscopy. (Near right) Light micrograph showing hamster sperm before (two at bottom), during (upper left), and after (upper right) completing an acrosome reaction. A remnant of the acrosome can be seen on the sperm in the upper left of the figure.


FIGURE: Electron micrograph showing a sperm in the process of acrosome reacting and a second sperm that has already lost its acrosome. N = nucleus, a = remnant of the acrosome, p = perforatorium at the leading edge of a reactedsperm.
A remnant of the acrosome, sometimes called the acrosomal ghost, consisting of the vesiculated membranes and residual insoluble contents of the acrosome remains attached to the outer surface of the zona pellucida as shown below. Sperm must free themselves from the acrosomal ghost by swimming through it before they can penetrate the zona pellucida.


FIGURE: An acrosomal “ghost” left on the surface of the zona pellucida (ZP). The ghost consists of hybrid vesicles (arrow) formed from multiple fusions of the plasma and outer acrosomal membranes and insoluble material (arrowheads).


FIGURE: Scanning electron micrograph of human sperm bound to an oocyte. Sperm bind to the numerous microvilli on the oocyte"s surface.


FIGURE: Human sperm that have fused with zona free hamster oocytes. Oocyte microvilli (m) have fused with the sperm’s plasma membrane over the postacrosomal region of the sperm head. The arrowhead points to the apparent initial site of fusion. Sperm and oocyte cytoplasm are continuous in this region. The arrow points to an oocyte microvillus that appears to have wrapped around the sperm and begun fusion.


FIGURE: A sperm has fused with an oocyte and the sperm nucleus is already partially decondensed, as shown at the tip of the arrow.
THE CORTICAL REACTION:

Prior to fertilization, mammalian oocytes have about 5,000 cortical granules beneath their plasma membrane. These granules contain a number of proteins that may include proteinases and peroxidase (Hoodbhoy and Talbot, 1994). In response to fertilization, the oocyte releases its cortical granules by exocytosis. This event is called the cortical reaction. The contents of the cortical granules are thought to diffuse into the zona pellucida, where they modify the zona so that additional sperm cannot penetrate, and indeed sperm do not bind well to the zona once fertilization has occurred. This change in the zona induced by the cortical granules exudate is termed the zona reaction, and it protects the oocyte from polyspermy, which in mammals is lethal. The oocytes below have been labeled with an antibody produced in Dr. Pat Calarco's lab (ABl2) that binds specifically to cortical granules and shows their location before and after fertilization.


FIGURE: An optical section through an unfertilized hamster oocyte showing numerous cortical granules beneath the plasma membrane.


FIGURE: An optical section through a 2-cell stage hamster preimplantation embryo labeled to show the cortical granule exudate after fertilization. Much of the exudate remains in the perivitelline space. The zona is faint blue.
The cortical granule material can be seen in electron micrographs of oocytes fixed in the presence of ruthenium red (Dandekar and Talbot, 1992). As the exudate is released from granules, it can be followed ultrastructurally. The cortical granule exudate appears granular after its release and much of the exudate remains in the perivitelline space where it forms a new extracellular layer around fertilized oocytes. This layer is termed the cortical granule envelope, and it is retained until the blastocyst hatches from its zona pellucida. Some evidence indicates that additional envelope protein is synthesized during preimplantation development (Hoodbhoy et al., 2001). The cortical granule envelope has been found in various different species including hamster, mice and humans (Dandekar and Talbot, 1992; Dandekar et al., 1995), but it is best demonstrated in the opossum (Dandekar et al., 1995). Examples of cortical granule envelopes are shown below.


FIGURE: Transmission electron micrograph of a fertilized opossum oocyte with the cortical granule envelope indicated by the white bar. The envelope is not present around unfertilized oocytes. ZP = zona pellucida; O = oocyte.


FIGURE: Transmission electron micrograph showing the cortical granule envelope forming around a eutherian oocyte. After fertilization, the cortical granule exudate is released into the perivitelline space (PVS) where it forms the cortical granule envelope which remains until blastocyst hatching.
remnant of the acrosome, sometimes The ABL2 antigen(s) appears to be a component of the cortical granule envelope as it is found in the perivitelline space of fertilized or artificially activated hamster oocytes (Hoodbhoy et al., 2001). The identity of the ABL2 antigen is not yet known, but it shows immunological similarity to sea urchin hyaline (Hoodbhoy et al., 2000).


FIGURE: Plate showing the distribution of the ABl2 antigen after fertilization. Labeled material is present on the surface of blastomeres and in the perivitelline space until the blastocyst hatches from the zona. (A) Fertilized oocyte. (C-D) 2 cell stage. (E-F) 8 cell stage. (G-H) blastocyst stage.
The function of the cortical granule envelope is not yet known; however, antibodies to one cortical granule protein (the ABL2 antigen) when injected into the oviducts of hamsters, retard preimplantation cleavage divisions (Hoodbhoy et al., 2001). A similar retardation in cleavage rate is found when antibodies to sea urchin hyaline are used in place of anti-ABL2 (Hoodbhoy et al., 2000). Preimmune antibodies or function blocking antibodies to b1 integrin, when injected similarly, have no effect on cleavage. These data suggest that the ABL2 antigen, which remains in the perivitelline space and on the oocyte's surface after its release, is involved in regulating cleavage of preimplantation embryos and support the novel idea that the cortical reaction could signal the fertilized oocyte to begin and sustain cleavage divisions.


FIGURE: Preimmune IgG or anti-ABL2 was injected into oviducts of hamsters on day 2 after fertilization, then 5-8 cell embryos were counted on day 3. A dose dependent inhibition in cleavage rate was observed following anti-ABL2 treatment. Similar data were obtained with an antibody to sea urchin hyaline. Interestingly, removal of the sea urchin hyaline layer also retards growth.
PRONUCLER FORMATION, CLEAVAGE, AND IMPLANTATION:

After fertilization, the oocyte completes its second meiotic division and a male and female pronucleus form (see figure below). The fertilized oocyte begins cleaving within 18-24 hours after fertilization. The cleavage divisions take place as it travels through the ampulla and isthmus of the oviduct. When it reaches the uterus, the preimplantation embryo is in the blastocyst stage. In the uterus, it hatches from the zona pellucida and then begins implanting in the wall of the endometrium of the uterus where it will continue its post-implantation development. The length of time devoted to post-implantation development varies. It can be as short as 15 days in hamsters or 9 months in humans.


FIGURE: Mission accomplished! A fertilized oocyte with two pronuclei that have begun to fuse. This zygote will have the potential to undergo cleavage divisions in the oviduct, implant in the uterus, and develop into a new individual.
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