Lecture notes for Wednesday, April 11, 2018 The lecture began with some comments on stents used to open blocked arteries. This was prompted by a recent article in the New York Times.    Fertilization Sperm meets egg; sperm fuses with egg; egg refuses any other sperm. How does it do this? If an egg ("oocyte" = egg cell) gets fertilized by 2 sperm, that will make the embryo triploid, and therefore infertile or dead. "Polyspermy" means fertilization by more than one sperm. Many mechanisms have evolved to 'lock the doors' as soon as one sperm had managed to fertilize the egg: "Blocks to polyspermy" The "fast block" to polyspermy uses a propagated decrease in electrical voltage across the plasma membrane of the oocyte. This depolarization is closely equivalent to nerve action potentials! (and presumably evolved before nerves! Protozoa also use it.) Before being fertilized, the voltage is about 70 millivolts more positive outside the cell than inside = 70 mv more negative inside than out! This voltage is caused by leakage of potassium ions (+ charge) from high concentrations inside the cell to low outside. NOTICE THE PARADOX! The positive charge outside is being caused by higher concentrations of (positively-charged) potassium ions inside the cell. Their leakage outward creates this voltage. (This is like the "resting potential" of nerve cells, muscle cells, and 99% of biologists don't know, but also almost all other cell types) When the oocyte is fertilized, then ion channels open in its plasma membrane that let sodium ions leak in (& also calcium ions). The membrane voltage depolarizes, as a result. The wave of depolarization propagates across the egg surface, in only a minute or so. (much slower than a nerve impulse) The oocyte membrane (somehow!) won't fuse with the sperm membrane after it has depolarized. Just under the plasma membrane of oocytes are thousands of vesicles, each with its own membrane around it. The increased calcium concentration causes these to fuse with the plasma membrane, releasing the content of the ~ 20,000 "Cortical vesicles" The contents of these vesicles causes slow blocks to polyspermy Different kinds of slow blocks to polyspermy: a) Enzymes in the cortical vesicles digest away adhesion molecules on the oocyte surface, that are needed for sperm to stick to eggs. Mammals have only this kind of slow block to polyspermy; it is called "the zona reaction" (but you don't need to remember that). Phenomena in humans and mammals tend to be called by special names even when the same phenomena also occur in lower animals. For example, the mammal vitelline membrane is called the zona pellucida. b) In sea urchins, and amphibians, enzymes in the cortical granules digest adhesion molecules that stick the vitelline membrane to the plasma membrane of the oocyte. This allows the jelly coat to lift away from the egg cell's surface (tending to push sperm away, too) Video of sea urchin fertilization This detachment of the oocyte surface from the jelly coat also allows frog eggs to rotate, so the heavier (yolkier) white side rotates downward. In a not-yet-fertilized batch of frog eggs, you can see many of the eggs still have their white sides upward, randomly. And if you flip the mass of eggs over, the cells can't rotate. You can really impress parents, friends and future in-laws with the ability to distinguish fertilized from unfertilized eggs that frogs lay in their swimming pool or bird-bath! c) In sea urchins, a special protein in the cortical granules precipitates onto the inner surface of the vitelline membrane, to form a sperm-proof layer called "the fertilization membrane" Notice differences in the location of fertilization (in different kinds of animals) (external & internal etc.) Sea urchins: simply release sperm and oocytes into sea water Sea squirts: release sperm into sea water; but hold oocyte in body;      (sperm get sucked in during filter-feeding) Frogs, most salamanders, & most teleost fish: male and female come together, female releases eggs into the water, and then the male releases sperm onto the eggs. Some salamanders: Males come to ponds about a week before females, and deposit gooey mucus blobs ("spermatophores"). Then, on the next rainy night, the females come to the pond and pick up the spermatophores with their rear ends and lay eggs past them. Many fish, & all reptiles birds and mammals: Male inserts sperm into the lower end of the female oviduct, and sperm cells meet the oocytes Which kinds of differentiated cells have "resting potentials"? = Negative voltage inside their plasma membranes, caused by > 20 times higher concentrations of potassium ions inside their plasma membranes compared with the potassium concentration in the surrounding water, combined with their plasma membranes being permeable to potassium ions. Outward leakage of about a millionth of this difference in potassium concentration creates a positive voltage outside, and this voltage pulls back any more potassium ions from leaking out. Action potentials are positive feed-back "voltage-gated" leakage of sodium and calcium ions into cells. 1) Nerve cells use resting potentials and acting potentials to transmit signals, by stimulating secretion of synaptic vesicles. 2) Muscle cells use resting potentials and acting potentials to synchronize contraction. 3) Heart muscle cells use resting potentials and acting potentials to time contractions, and to cause this spontaneous contraction to occur in waves. 4) Oocytes use resting potentials and acting potentials as their fast block to polyspermy. Notice the irony that depolarization increases fusion of cortical granules to the plasma membrane, but inhibits the fusion of sperm plasma membranes to the oocyte plasma membrane! 5) Some pigment cells in the skins of zebra fish use resting potentials and acting potentials to stimulate and inhibit each other in ways that (somehow I do not yet understand) produce alternating white & black horizontal lines. 6) Paramecia and other ciliate protozoa use action potentials (= quick depolarizations) to cause temporary increases in calcium ion concentrations in their cytoplasm, to reverse direction of cilia power strokes. By this means, paramecia back up when they bump into an obstacle. [and remember the video of ciliate chemotaxis? Do you think they must depolarize whenever an attractant concentration depolarizes?] Maybe depolarization also can cause reversal of direction of other kinds of cell locomotion? For example, contact inhibition of crawling by tissue cells could result from cell-cell touching causing one or both of them to depolarize, combined with depolarization stimulating cells to crawl in an opposite direction. 7) Venus fly traps use changes in resting potentials to control decreases in osmotic pressure, which is how they close their trap-like leaves. 8) Maybe many other cell types also use resting potentials and acting potentials to control many different kinds of changes in cell properties (that need to occur faster than diffusion). 9) ??? If the sodium pumps of most body cells continually use as much as 20-40% of their ATP energy, doesn't that strongly imply that their resting potentials have some major use? 10) ??? Please invent some possible uses? Voltages are suspected of helping regeneration. What kinds of evidence would suggest that a given cell type used resting and/or action potentials? (How to test whether cells are controlling some process using changes in voltage differences between the cytoplasm and outside their plasma membrane? A) Create a ~100 millivolt voltage gradient in the tissue culture medium surrounding cells. Observe whether cells' behaviors change in any way. B) Raise potassium concentrations in the cells' surrounding water. C) Poison cells' sodium pumps. D) Ionophore chemicals (VERY poisonous, even if you touch a tiny drop!) that allow calcium (or certain other ions) to leak through membranes E) Use microelectrodes inserted into cells to force increases or decreases of trans-membrane voltages; and see if the cells do something consistent in reverse. F) Use fluorescent dyes to look for spatial and/or time changes in calcium or other ion concentrations in cytoplasm. video of cells in tissue culture responding to an electric field   The paper handed around in class (optional reading, not required): Harris et al. 1990 paper on galvanotaxis. ========================================================== Some evidence supports the idea that electric fields may control bone formation and osteoporosis. In that case, the voltage has been assumed to be caused by piezoelectricity. [Many crystals (quartz, sucrose, but not calcium phosphate) create electrical voltages when compressed, stretched or twisted.] These voltages can be many volts, but are very brief, with tiny current. Applying an external voltage to a piezoelectric crystal will cause it to elongate or contract. Sonar pingers work that way; also sonicators and quartz watches. Pierre Curie figured out these relations between symmetries of locations of charged ions, voltage generation by changing crystal's shapes & sizes, etc. This probably was the first example of "Curie's Principle. ****************************************************************** Many orthopedic researchers have confirmed that voltages are generated (temporarily) by compressing bone, cartilage, and collagen. They say this must be piezoelectricity. I think it is really caused by electroosmosis. (Are they unfair to regard me as pedantic?) If we get a voltage by squeezing tissues, who cares whether this is caused by crystal symmetry or by dissolved positive counter-ions. What are your thoughts? This might lead to a cure for osteoporosis. ******************************************************************

 

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