Laboratory Exercise for October 23—November 20, 2007

Radioimmunoassay of Rat Ovarian Extracts for Progesterone & Prostaglandin E₂

In this experiment, along with injecting hormones and anti-ovulatory agents into the rats and removing their ovaries last week, this week you will be homogenizing the ovaries, centrifuging the homogenates to obtain a simple aqueous extract, performing a radioimmunoassay (for progesterone and prostaglandin E₂) on the extract, and estimating the amount of blood in the extracts.

WORK SCHEDULES FOR CLASS ON TUESDAY, November 07, 2006

Time, Treatment, and Student Assignments

See Schedule #1

I. Experimental Animals:

The animals are sexually immature Wistar rats (25-days-old) that were induced to ovulate by first treating them with a single subcutaneous injection of 10 I.U. of equine chorionic gonadotropin (eCG) to cause maturation (i.e., folliculogenesis) of the ovarian follicles (I did this injection for you). After 48 hours, the animals were given 10 I.U. of human chorionic gonadotropin (hCG) as a substitute for LH to induce the ovulatory process. Animals treated with this hormone protocol will normally begin ovulating 12 hours after receiving hCG. At 3 hours after hCG, one group of animals was given Indomethacin (INDO), a very potent anti-inflammatory agent that inhibits cyclooxygenase (and prostaglandin synthesis) and is also an inhibitor of ovulation. We are also going to pretend that another group of animals was given Epostane (EPO), an inhibitor of progesterone synthesis and ovulation, at 3 hours after hCG administration. You will determine the effect of these two drugs on ovarian progesterone and prostaglandin synthesis during ovulation.

II. Numbering the Animals:

The animals were numbered as follows:

#0-1 to 0-6 = 0-hours after hCG

#4-1 to 4-6 = 4-hours after hCG

#8-1 to 8-6 = 8-hours after hCG

#8I-1 to 8I-6 = 8-hours after hCG (this group received 1 mg of indomethacin at 3 hours after hCG)

#8E-1 to 8E-6 = 8-hours after hCG (this group received 5 mg of epostane at 3 hours after hCG)

#12-1 to 12-6 = 12-hours after hCG

#24-1 to 24-6 = 24-hours after hCG

NOTE: Epostane (EPO) is an effective inhibitor of ovulation, and exerts its anti-ovulatory action by inhibiting 3b-hydroxysteroid dehydrogenase, the enzyme that converts pregnenolone into progesterone.

NOTE: Indomethacin (INDO) is a very potent anti-inflammatory agent that reportedly inhibits ovulation by inhibiting prostaglandin synthase (cyclooxygenase), which converts arachidonic acid into prostaglandins.

NOTE: Dr. Espey has previously determined the ovulation rates following administration of these agents by counting the number of ova in the oviducts, and you will receive these data at a later date.

III. Initial Ovarian Extraction: (to be performed the second week in lab)

1. The rats are killed by CO₂ euthanasia at 0, 4, 8, 12 and 24 hours after hCG was given.

2. The ovaries are stored frozen in acetate buffer until there is adequate time to extract and assay them.

NOTE: See Schedule #2 for work on November 13, 2007.

3. The ovaries are thawed and homogenized in 1.0 ml of acetate buffer (pH 4.5) for 30 sec.

4. The homogenate is centrifuged at 5,000 rpm for 10 minutes, and the supernatant fluid is collected (by decantation) into a fresh tube.

NOTE: This supernatant fluid is a simple aqueous extract of the ovary that can be used to assay progesterone, prostaglandins and other metabolites in the ovary.

5. 500 ml of the extract will be diluted with 2,500 ml of deionized water and vortexed.

NOTE: ~ 2,000 ml of this solution will be used later to estimate the change in ovarian blood volume by spectrophotometry.)

NOTE: Now, you are ready to perform progesterone and prostaglandin RIAs on 100 ml aliquots of the diluted extract that you prepared in the previous step. Be aware that you will be performing each RIA in "duplicate", i.e., you will need two RIA tubes for each ovarian extract. Be sure that these tubes are properly numbered with the correct animal number. You should mark the first tube in each duplicate with an "a" and the second tube with a "b", i.e., 8-1a, 8-1b, etc., (with the "8" indicating 8 hours after hCG injection, and the "1" indicating the animal number in this experimental group).

IV. Radioimmunoassay (RIA) Procedure for Progesterone (P₄):

1. Pipet 100 ml of diluted extract from one rat into each of two marked tubes.

2. Pipet 1000 ml of radioactively labeled [¹²⁵I]-P₄ (i.e., tracer) into each of your assay tubes.

(NOTE: To pipet radioisotopes, you must have on a lab coat, safety glasses, and a pair of disposable gloves.) Although the amount of radioactivity you will be working with is quite low, we will follow all of the normal precautions while working with radioactive materials. By following these simple precautions, it is quite safe to handle these materials.)

3. Incubate these tubes for exactly 40 minutes at 37ºC (see Mrs. Garcia for location of incubator).

(NOTE: While these tubes are incubating, proceed to the prostaglandin RIA that is described in Section "V", below. When you have finished this pipeting, proceed to Section "VI" to estimate by spectrophotometry the relative amount of blood in each of the ovarian extracts.)

4. After the 40-minute incubation in #3, above, decant the supernatant fluid out of the tube and store the tubes in the refrigerator.

5. Next week, we will read the results on a gammacounter.

V. Radioimmunoassay (RIA) Procedure for Prostaglandin E (PGE):

1. Pipet 100 ml of diluted extract from one rat into each of two marked tubes.

2. Pipet 100 ml of radioactively labeled [¹²⁵I]-PGE₂ (i.e., tracer) into each of your assay tubes.

3. Pipet 100 ml of prostaglandin E₂ antiserum into each assay tube and vortex for 3 seconds.

4. Place the tubes in a rack marked "prostaglandin" overnight at 4°C.

5. Tomorrow morning, your kindly professor will:

a) add 1 mL of precipitating reagent to each tube

b) incubate the tubes in the refrigerator for 30 minutes

c) centrifuge the tubes at 2000 x g for 30 minutes

d) decant the supernantant fluid from each tube

e) store all the tubes until they can be counted on the gamma counter next week

VI. Estimating Ovarian Blood Volume

1. Take the remaining ovarian extract (about 2000 ml) and put it in a spectrophotometer cuvette.

2. Prepare a "blank" cuvette by pipetting 2000 ml of distilled water in it. (Mark it with a "B".)

3. Take your cuvettes to the sectrophotometer in the outer room of Dr. Espey’s laboratory, where someone will show you how to operate the spectrophotometer.

4. First, perform a wavelength scan to determine the absorbance peak for hemoglobin.

5. Then, set the spectrophotometer at this wavelength and read the absorbance of your samples.

6. Finally, print and provide your absorbance values to the professor before you leave for the day.