LABORATORY #3 FERTILIZATION, SEA URCHIN PREPARATION AND MATERIALS PER GROUP OF FOUR STUDENTS:two sea urchins, syringe, 2 small beakers (50 ml), filtered sea water, ph 5,7,8 sea water, CA++Mg++free sea water, hand centrifuge, container of ice.

THIS INVESTIGATION REQUIRES: SLIDES and coverslips ANIMALS:sea urchins (Echinodermata) UTENSILS:50 ml beakers, syringe and needle for injection of sea urchins, centrifuge tubes, pipettes, petri plates.

PRELAB; LOOK UP BINDIN, VITELLINE MEMBRANE, SPERM MOTILITY, ACROSOME REACTION, CORTICAL REACTION.

OBJECTIVES: to see sperm movement, attachment to egg, lifting away of fertilization membrane from surface of egg, first cleavage and to investigate mechanisms involved.

REQUIRED TASKS: fertilize eggs, observe fertilization membrane lifting off the surface of egg.

OBSERVE EFFECTS OF PH, TEMPERATURE, CA++ CONTENT ON FERTILIZATION. Students to work in pairs and turn in completed lab sheet at the end of the lab

PROCEDURES: In this section you will determine the relationship between eggs and sperm and correct environmental conditions required for normal fertilization, fertilization membrane formation, and first cleavage LABEL ALL DISHES OF EGG FERTILIZED WITH THE TIME OF SPERM ADDITION, AND ANY VARIABLE CONDITION. MAKE SURE YOU HAVE AN UNTREATED CONTROL WHICH HAS NO SPERM ADDED, IF THAT IS REQUIRED IN YOUR EXPERIMENT.

1.inject two sea urchins with 1 cc of .55 M KCl and then give them a good shake and wait to see what sex they are: the gametes come out five gonopores at the aboral surface (opposite the oral surface where the mouth is) if the gametes are white, it is sperm and should be shed by turning the male upside down in a petri dish placed on ice; if the gametes are yellow, it is a female and the eggs should be shed into filtered sea water (pH 8) by inverting them over a beaker full of sea water. The beaker should be of smaller diameter than the sea urchin so that it won't fall in. Keep injecting until you get both sexes, after appropriate waits to see what the next one is. You can trade with other groups, also.

ALWAYS KEEP THE EGGS AT ROOM TEMPERATURE AND THE SPERM ON ICE This means use room temperature sea water in all egg treatments and at fertilization. If it is cold, warm it by setting it in a pan of warm water. Give another 1 cc injection when egg spawning slows down, then leave the female to complete the spawning for about ten more minutes. In the meantime, design your experiments and label your dishes.

2. After spawning is complete pour off the sea water into another beaker, so you don't lose your eggs by mistake.

Removal of jelly: Add 25 ml ph5 sea water to the eggs from the the beaker, and allow it to stand for three minutes before adding .5 ml 1 M tris pH 8 to raise the pH rapidly. (the pH 5 treatment removes the egg jelly, but prolonged treatment leads to damage to the eggs.) Now we must remove the dissolved egg jelly by washing the eggs. SAVING THE FIRST SUPERNATE, LABEL IT JELLY, so pour it off into a tube labeled jelly and wash the eggs with sea water pH 8, 2 times, suspending the eggs each time as above.

3. Now we have our eggs prepared for the experiment.

EACH GROUP OF 2 STUDENTS Sperm are much more delicate than the eggs, so they must be kept undiluted in ice (the seminal fluid has substances in it to keep the sperm inactive and the ice also helps to do that.) They must be diluted prior to their addition to the eggs, or we get polyspermy and abnormal cleavage. Add one drop of concentrated sperm to 10 cc of sea water in a graduated cylinder, stir to disperse evenly. This diluted sperm suspension will only last for 20 minutes, so do it right before you need it, and keep it in the ice bath. Prepare a slide with a drop of eggs, have it set up on a microscope prior to sperm addition adjusting the light the eggs on the slide easily visible. Add a drop of the sperm, slam the cover slip on the slide and place under the low power objective and observe fertilization. If you don't get fertilization try it again with another batch of sperm from someone who found good fertilization. Then proceed to fertilize your aliquots of eggs. 4. Fertilization: ADD 0.1 ML DILUTED SPERM TO 10 ML OF SEA WATER CONTAINING .1 ML concentrated EGGS. Observe, using dissection microscope and dark field (obtained by putting mirror on its side until field appears black and eggs appear white) or compound microscope. The fertilization membrane is formed by a lifting away of the vitelline membrane, and an addition of material to it from the ruptured and released contents of the cortical granules. Look at the number of sperm around one egg, record for a few eggs. Observe and record the sperm shape and size compared to the egg, movement of the flagellum.

For polyspermy, get one dish of eggs with about 100 sperm per egg. Check the effect on cleavage, as compared to the control. 5. Test the effect of pH on fertilization. Fertilize a DISH of eggs in the same manner, but use pH 5 sea water in one dish, pH 7 in another. Record the % fertilization membranes formed after 1 min, 5 min. What is your control?

IF YOU ARE GOING TO CULTURE THE EMBRYOS TO LATER STAGES, DECANT OFF THE SPERM LADEN SEA WATER ATER THEY SETTLE, AND POUR A SHALLOW LAYER INTO PYREX BAKING DISHES AND INCUBATE THEM AT 15 DEGREES C FOR ANYTHING PAST THE 2 CELL STAGE OR THE CLEAVAGES WILL NOT BE SYNCHRONOUS. IT TAKES ABOUT 1 1/2 HRS TO GET TO THE FIRST CLEAVAGE. KEEP CHECKING TO SEE IF YOU CAN SEE THE MITOTIC APPARATUS DEVELOPING.

EFFECT OF TEMPERATURE ON FERTILIZATION. Fertilize a dish of eggs that has been sitting on ice for ten minutes. Compare the percent fertilization after and 5 min. with the control. .UL EFFECT OF CA++ ON FERTILIZATION. Fertilize a dish of eggs using Ca++Mg++free sea water instead of regular sea water. Compare the results with the control.

ANSWER SHEET FOR LABORATORY #1 NAME_____________________________

What is an independent variable?

What is the dependent variable in all the experiments?

Why is the lifting of the fertilization membrane a good indication that a sperm has penetrated the egg?

What is the effect of ph on fertilization?

What is the effect of mixing egg jelly with a sperm suspension?

What is the effect of adding egg jelly to a dish of eggs before fertilization?

What causes this?

WHY IS CA++ important for fertilization?

What is the effect of ‑SH reagents on the fertilization membrane?

What is the effect of polyspermy?