February 6th lectureEmbryology Biology 441 Spring 2008 Albert Harris
Fertilization; and development of eggs and sperm.Please notice differences in these processes, between different species and also differences and similarities between male and female gamete (sperm and egg) development.Location of fertilization: Sea urchins and many other kinds of animals that live in the ocean just release their sperm and eggs into the water. (Parenthetical note: ordinary people use the word "egg" to refer to every stage from unfertilized oocytes to early embryos, as late as gastrulation, or even later. Every professional embryologist I have ever known has done the same. Perfectionists sometimes object to this usage, and say it's not at egg once it's fertilized. One variation on external fertilization used by some filter-feeding invertebrates is for sperm to be emitted randomly into the water, but for the eggs to be kept in the body and sperm to reach the eggs from the flow of water being pumped through the parent's body. Many Sea Squirts (tunicates) do this, and so do many (or all) species of sponges. When frogs copulate, the male squirts out sperm next to where the female is squirting out egg cells (oocytes). Many fish breed approximately the same way. Researchers can pick up a female in one hand and a male in the other, and squeeze their bodies enough for the eggs and sperm to flow into the same petri dish. I have done this many times. It only works if the animals are "ripe", in the breeding season, and haven't yet mated. In some species of salamanders, the males deposit globs of sperm on the bottom of ponds and the females come a week or two later and deposit eggs in contact with the sperm masses. In all reptiles, birds and mammals (and in many kinds of fish, too) the male inserts sperm into the lower end of the oviduct of the female, and fertilization occurs inside the female's body. Egg white and egg shells are secreted around the early developing embryo by the walls of the oviduct.
Chemotaxis? And related questions.In species that use external fertilization (like sea urchins) the sperm may have some guidance mechanisms to find egg cells (oocytes) of their own species. Many species have selective adhesion proteins, and sperm of many species are be guided by some form of chemotaxis toward oocytes of their own species.Although the textbook doesn't mention it, there are several very different forms of chemotaxis, without much in common except the net effect. For example, (B) cells might compare the local concentrations of chemotactic attractant substances at different points on each cell's surface, and then turn toward whichever direction the concentration is highest. A different category of chemotaxis would be (B) if each cell moves in an approximately straight line, and detects whether the concentration of attractant substance around it is becoming higher or lower, from one moment to the next, with each cell making a random turn in response to local lowering of the attractant concentration. This is a very effective method of concentrating cells where the attractant is most concentrated, and any na•ve observer will think what is happening must be that the cells are actually detecting the direction of the gradient. A third possibility is ( C ) for moving cells to compare the attractant concentration at their front as compared with the attractant concentration at its rear end (or to compare concentrations at any two concentrations along the cell's length), and make random turns more frequently when the concentration nearer the front becomes less than the concentration near the rear. "Trap action" also tends to be interpreted as chemotaxis. (D) If cells stop whenever the attractant concentration becomes high, this will cause them to accumulate at the top of the gradient, and will seem to many observers that chemotaxis must have occurred. Much the same net effect can be achieved if ( E ) cells stop when the attractant concentrations at front and rear are both high, and approximately the same. In addition to "attracting" sperm toward oocytes, and slime mold amoebae toward each other, there are many important developmental phenomena in which crawling or swimming cells accumulate somewhere, perhaps in response to a chemical gradient. Examples include movement of special "Primordial Germ Cells" to the location where the ovaries and testes will develop, as well as movement of nerve axons toward locations of synapse formation, especially fibers of the optic nerve finding map-like connections in the brain. True molecular mechanisms of these phenomena will never be discovered unless researchers are more careful to distinguish between such fundamentally different kinds of "chemotaxis" as those listed above. Few researchers take the trouble. ++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++ Parthenogenesis means development of an egg cell without fertilization. Note the similarity of this word to Parthenon, which was a temple to the Greek goddess Athena, and comes from a Greek word for "Virgin" Several species of fish, salamanders and lizards are "parthenogenic", and just have females who produce eggs that either develop without being fertilized, or in some cases get fertilized by sperm of related species, but then the male pronuclei get destroyed or extruded, and serve only to initiate oocyte development. Aphids and some other insects reproduce mostly by parthenogenesis, but also produce some oocytes that are fertilized. Incidentally, scientists are not quite sure why all animals don't reproduce by parthenogenesis, because if all individuals were females, then twice as many eggs could be produced. Therefore there is an immediate two-fold advantage in reproduction rate for mutations that produce parthenogenesis. The consensus explanation is that, in the longer run, recombination of genes from different parents allows faster evolution of improvements, enough so that sexually-reproducing species eventually drive parthenogenic species to extinction, by evolving improvements more rapidly. This is difficult to test experimentally. Good evidence comes from rare situations where many sexually reproducing species are competing directly with asexually-reproducing species that are closely related to them. If the environment changes, which will be able to adapt and survive? ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ The fast and slow blocks to polyspermy. "Polyspermy" means entry of two or more sperm into the same oocyte. The effect is almost always that the oocyte develops so abnormally that it effectively dies. So there is a strong evolutionary pressure to prevent polyspermy, and many different mechanisms have evolved to keep any more sperm from getting in after the first one. Polyspermy is just as lethal for the sperm as for the eggs, please notice. Eggs of sea urchins produce what amounts to a protective shield, called the fertilization membrane. This is not really a membrane in the sense of a lipid bilayer. It is a layer of special proteins that attach to the inside surface of the vitelline membrane, which is a jelly-like layer that surrounds the oocytes of most species, including humans. But no kind of vertebrate forms a fertilization membrane. Mature oocytes contain about 10,000 to 20,000 special vacuoles in their cytoplasm near the surface. These are called cortical vesicles, and their contents get secreted just after fertilization. This secretion results from fusion of the oocytes plasma membrane with the membranes that form the surfaces of these vesicles. The substances secreted by these vesicles contain enzymes that digest the adhesion molecules by which sperm stick to eggs and egg jellies. The cortical vesicles of sea urchin eggs also contain the proteins that form the fertilization membrane, plus enzymes that cut the connections between the vitelline membrane and the egg surface, and also contain soluble materials that cause an osmotic pressure that lifts and expands the fertilization membrane. Human and other mammal oocytes also contain thousands of cortical vesicles, very similar to those in sea urchin oocytes. Ours contain enzymes that digest the adhesion proteins by which sperm stick to oocytes. Our eggs don't form fertilization membranes. For mammals, the digestion of the adhesion proteins is the main means of preventing polyspermy, and this loss of adhesiveness is called "the zona reaction". Human and mammal embryology has often been studied by different scientists, who invent different names for processes and structures, instead of using the names used by other embryologists. Two examples are "zona pellucida" (= vitelline membrane) and "zona reaction". ************************************************************************ Please notice the three examples of fusion of membranes that occur in fertilization: 1) Fusion to the acrosome membrane to the plasma membrane of the sperm, thereby releasing the enzymes and other materials that had been inside the acrosome. 2) Fusion of the sperm plasma membrane to the plasma membrane of the oocyte. 3) Fusion of the plasma membrane of the oocyte with the membranes of the cortical vesicles, thereby releasing the enzymes and other materials that had been inside the cortical vesicles. ************************************************************************ Mechanisms to prevent polyspermy are divided into two categories, the slow block and the fast block. In the category of slow blocks to polyspermy are the fertilization membrane and enzymes that digest the adhesion proteins by which sperm stick to eggs. The fast block to polyspermy is a propagated wave of electrical depolarization that spreads rapidly (in second) across the plasma membrane of the oocyte. The mechanism of this electrical depolarization is almost the same as the mechanism of nerve impulses, and of the depolarization waves that coordinate muscle contraction. Before fertilization, oocytes have a negative voltage in their cytoplasm relative to outside their plasma membrane. This voltage is about 70 millivolts, and has the same cause as the resting potential of nerves and muscles. This cause is that the plasma membrane is much more permeable to potassium ions than to any other ions, combined with the fact that certain membrane enzymes called "the sodium pump" use energy from ATP to pump potassium ions in and sodium ions out of the cytoplasm. Leaking ions take charge with them, and produce an excess of their own charge at the place toward which they are leaking. That is why leaking potassium ions (which are positive) create a negative charge on the cytoplasmic side of the plasma membrane. This seems paradoxical, because potassium ions are more concentrated inside the cell than outside, and are positively charged, yet are creating a negative charge inside the cell. This paradox is too difficult for the authors of 95% of biology textbooks (even ours?). They can't see how positive ions that are more concentrated inside can be causing a negative charge inside. What you should keep in mind is that the voltage is not caused by absolute numbers of any kind of ion, but by leakage down a concentration gradient; so positive ions create a positive charge on the side toward which they are leaking. Nerve impulses are caused by voltage-gated sodium channels. These are transmembrane proteins that can let sodium ions through, but do not let them through unless the voltage difference between the inside and the outside is reduced to less than 70 millivolts. Any decrease in this voltage at one part of the cell surface will cause opening of these sodium channels, the result of which is that sodium ions will diffuse from high concentration to low concentration, which means into the cell. They carry their positive charge inward, and make the inside voltage less negative. This depolarizes all nearby areas of the plasma membrane, and is a positive feedback cycle that spreads rapidly over the whole surface. Sperm will only fuse with areas of plasma membrane that still have the 70 millivolt difference. Opening of the sodium channels is a rapid way to prevent an oocyte from being entered by any more sperms, after the first. These mechanisms were proven (in part) by using microelectrodes to depolarize oocytes, before any sperm had fused with them, and showing that this prevents any sperm from entering. The reverse experiment was to use electrodes to keep or restore the 70 millivolt difference, and the result is that unlimited numbers of sperm can enter the oocyte. Voltage-sensitive calcium ion channels also open when the oocyte depolarizes (when the 70 millivolt difference decreases), and depolarization also allows calcium to be released from membrane sacs inside oocytes. Certain chemicals can be bought that emit light wherever the calcium concentration rises enough, and these allow one to make time-lapse movies of the wave of increased calcium concentration. One is almost seeing the fast block spread. One effect of the calcium is to allow the cortical vesicles to fuse with the plasma membrane of the cell, thereby secreting the contents of these thousands of special vacuoles. These contents include enzymes that digest the special proteins by which sperm stick to the oocyte's plasma membrane; and that is the most common category of slow block to polyspermy. The slow block to polyspermy has additional mechanisms in oocytes of sea urchins and some other kinds of animals, such as the formation of the fertilization membrane, which was mentioned before.. --------------------------------------------------------------------------------------------------------------------------
A side issue: A little-known fact, which may be important for future medical research, is that almost all the cells of the body have membrane potentials, or 50 or more millivolts negative inside. These voltages have the same cause as in nerves, muscles and oocytes, i.e. a higher concentration of potassium inside each cell, combined with constant leakage of potassium outward. Furthermore, measurements have been published about the fraction of each cell's ATP that gets expended pumping ions through their plasma membranes; and it seems to be more than a third (!). If you put tissue culture cells in voltage gradients (= D.C. electric currents), the cells respond in one of several ways, depending on which differentiated cell type they are. Mesenchymal cells and muscle cells line up perpendicular to the voltage gradient. Nerves extend axons directionally toward the negative electrode, fish epidermal cells crawl rapidly toward the negative electrode, and osteoclasts and macrophages crawl toward the positive electrode. Many scientists have published research papers on these phenomena, which are very easy to repeat and give consistent results. WARNING- DANGER OF ELECTROCUTION! : In order to create small voltage gradients of just a volt per millimeter in tissue cultures, you have to apply a hundred or more volts to the culture chamber and things called "salt bridges". Therefore, although it is easy to replicate experiments on electrically-induced cell alignment, this research is fundamentally much more dangerous that you would expect. So I don't recommend doing it ---------------------------------------------------------------------------------------------------------
Meiotic divisionsMeiosis consists of two special nuclear divisions, with no S period (DNA synthesis) between the two divisions, so that four haploid cells are produced (Haploid= containing just one copy of each chromosome; the haploid chromosome number in humans is 23)The cells that eventually differentiate into sperm cells (=spermatozoa) undergo both meiotic divisions before they differentiate. Their differentiation includes a) shrinkage of the nucleus, and inactivation of the compressed DNA; b) Formation of a flagellum, by which the sperm will swim; and c) breaking away of nearly all of the cytoplasm, with a few mitochondria remaining, wrapped around the base of the flagellum. Mature sperm have been called 'DNA with an outboard motor'! There are several big differences between sperm and egg (oocyte) development. Oocytes grow very big in comparison to ordinary cells. Mammal and sea urchin eggs are about 100 micrometers (microns) in diameter, but that means their volume is about a thousand times as big as an ordinary 10 micron-diameter cell. Frog, salamander and fish oocytes are millimeters in diameter (1 millimeter = one thousand microns), so that their volumes are millions of times as big as ordinary cells. The yolk of a birds egg is a single oocyte, that has been fertilized & started development before the egg is laid. And for bird and reptile oocytes, the diameters are in the centimeter range. Oocytes of all species become packed with food materials, called "yolk", mostly in the form of millions of small granules each a few micrometers in diameter. Billions of copies of messenger RNA are also stored in oocytes, and also billions of messenger RNAs. The oocyte nuclei have to work very hard to produce all these RNAs, and swell very big. Oocyte nuclei get so big that early embryologists guessed they must be some kind of vacuole, or something, and called them the germinal vesicle. They didn't realize this is their nucleus (gigantic and over-worked!). Therefore, students of embryology were burdened with yet another vocabulary word. A big difference between sperm and egg cell development is that egg cells wait until very late in their differentiation before undergoing meiosis. This is so that those other 3 sets of chromosome DNA (that are going to be discarded in the polar bodies!) can work hard producing RNA transcripts. One result is that early stages of embryos make proteins coded for by all the genes of the mother animal, including any different alleles that were discarded in the polar bodies. Another difference is that meiotic divisions of oocytes are very unequal; instead of dividing the cell into two equal-sized cells, each meiosis produces a small polar body. These contain complete sets of chromosomes, but hardly any cytoplasm. The place on the oocyte surface where meiosis occurred is, by definition, called the animal pole. This is also the area with the least yolk and the smallest yolk granules. And the extreme opposite side of the oocyte (and embryo) is called the vegetal pole. There is always more yolk, relative to cytoplasm, nearer the vegetal pole. People are beginning to think that these poles are defined by the yolk distribution (as the textbook suggests) but really (or originally) the definition of the animal pole is where the polar bodies formed. Eggs of most species don't finish meiosis until after they have been fertilized. This is true of humans, and most other mammals. Our oocytes wait at the metaphase stage of the second meiotic division. Therefore, our embryos are effectively triploid for an hour or so after fertilization. Until completion of the second meiotic division there are three sets of chromosomes in the fertilized egg. Sea urchins are one of the only kinds of animals in which both meiotic divisions have already been completed at the time of fertilizations. Foxes and dogs are the only mammals in which neither meiotic division has occurred yet when their oocytes are fertilized.
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