LABORATORY # BIOLOGY 441

TITLE : PREGNANCY TEST

Preparation and materials: per group of four students UCG SLIDE‑TEST for chorionic gonadotropin.

This investigation requires: slides and stirring sticks, lamp, pregnant and control urine.

Prelab; carefully read the experiment and read about antigens and antibodies in an elementary textbook. Look up multivalent antibody and agglutination.

Objectives: to learn to use an immunochemical test for chorionic gonadotropin, and to distinguish a positive pregnancy test, showing CG presence in the urine.

Required tasks; prepare a labelled slide for each of the four urine samples. Shake the antigen sample, well. Make sure reagents are room temperature before use. Hold them in your hand until they are. No more than 4 tests should be set up at any one time. Hypothesis to be tested; pregnant urine contains chorionic gonadotropin whereas non‑pregnant urine does not. Students to work in pairs and each turn in completed lab sheet at the end of the lab.

Procedures: 1. Place one drop of urine on labeled slide. 2. Add one drop of antiserum reagent to the drop of urine. Be careful not to contaminate the pipette by touching the urine with its tip. 3. Add one drop of shaken antigen reagent and mix together with the flat part of a new stirrer (don't use it over without washing it with soap. 4. Rock gently for 2 minutes and observe with a gooseneck lamp right above it at the end of that time. Check for agglutination. The beads are coated with HCG. We are adding urine which is another possible source of antigen. When there is no antigen in the urine, what would you expect to happen when you add the antigen and stir? What will happen in the case where there is antigen (hormone) in the urine that can compete with the antigen on the beads?

A positive test does not agglutinate. A negative test agglutinates by the two minutes, and you will see alternating areas of clear and opaque appearance. This is because the latex beads are coated with antigen, and can be agglutinated by the antibody when the two are mixed. However, in the presence of an outside source of the antigen, the antibody reacts with that instead of with the beads, so there is inhibition of agglutination. In a positive pregnancy test the urine contains the chorionic gonadotropin to react with the antibody to interfere with its agglutination of the antigen on the beads human chorionic gonadotropin appears in the human circulation in concentrations which can usually by detected by 30 days after the last period. This test can work as soon as five days after the day of the missed menses. If the test is negative, and there is still no menstrual period, the test can be tried again after one week. Just as the name suggests, the hormone is made by the chorion of the placenta, and normally has as its function the maintainance of the corpus luteum in the mother's ovary, taking the place of the pituitary gonadotropin. First‑urination of the morning is used for the collection, since the hormone is more concentrated in the urine at that time. Caution should be used when there is a possibility of menapause, since there is also an elevation of the hormone from the pituitary at that time which could lead to a false positive.

In this section you will determine the relationship between the variables of the experiment are 1) amount of chorionic gonadotropin the dependent variable is the amount of aggutination of the latex beads make sure that you vary only one condition at a time. Collect data: record it in either a table, a drawing or a graph on a separate page.

ENDOCRINE TISSUES AND THEIR TARGET ORGANS

We are going to study the structure of the pituitary and of some reproductive organs affected by pituitary hormones. We have already studied ovaries and testes, and we have seen gametogenesis which is regulated by pituitary gonadotropins, so integrate that knowledge with this exercise.

Materials: slides of pituitary, vagina, uterus in various stages of hormone stimulation, prostate, mammary glands, placenta

PITUITARY

Examine the slide of the pituitary. Notice that there are distinctly different staining regions: The darkly staining anterior pituitary, or pars distalis; the pars intermedia next to the cleft of Rathke's pouch; and the posterior pituitary or pars nervosa.

Anterior pituitary (Adenohypophysis): There are acidophilic cells which stain quite darkly just in front of the cleft which is the remnant of Rathke's pouch from which the anterior pituitary is derived. There are many blood vessels since there is a portal system bringing blood directly from the hypothalamus to the anterior pitutiary, with the releasing factors made in the hypothalamus. The releasing factors will cause the anterior pituitary cells to secrete their gonadotropins or other trophic hormones which will control release of hormones by many other endocrine glands in the body (thyroid, pancreas, gonads, for example.)

Intermediate pituitary: This tissue lies between the anterior and posterior pituitary and makes a melanocyte stimulating hormone in some animals.

Posterior pituitary (neurohypophysis): Made from the infundibulum of the forebrain (diencephalon), and containing many nerve fibers since this tissue will release hormones at this region made in the hypothalamus, but stored in the cells here, much as neurotranmitters are stored at the ends of nerves a long distance from their cell body, and can be released due to stimulation. This is the region where oxytocin (for milk ejection) and vasopressin or antidiuretic hormone are released.

A stimulus to the nipple from suckling causes neural stimulus to brain which then causes oxytocin release.

UTERUS

Compare slides of different times of the menstrual cycle of normal uterus (see the difference between the proliferative and secretory phases), uterus from someone taking the pill, pregnant uterus regarding the following:

a. the thickness of the wall (you have a pie shaped sector cut from one side of the uterus on each slide, and the whole thickness of the wall is there.) Measure the thickness with a ruler. Prepare a table comparing the thicknesses.

b. the amount of glandular tissue (lots of deep grooves in the surface of the endometrium).

c. the relative amount of endometrium and myometrium. The myometrium has many muscle layers.

VAGINA

Find the lumenal side. Count the layers of cells in the lumenal epithelium (there may be 20-40 layers.) What kinds of muscle can you see? There should be layers of both smooth and striated muscle, since there are both voluntary and involuntary contractions of the vagina.

Compare the epithelium with the epithelium of the cervex and uterus. Notice how the cervex transitions between the vagina and uterus.

MAMMARY GLAND

Compare the ducts and glandular tissue in normal and lactating gland. How can you tell the difference?

PLACENTA

Examine the preparation of the PIG fetus with fetal membranes: measure the length of the whole placenta. Can you see the folds on its surface which interdigitate with folds of the uterus)? There are no villi on pig chorion which correspond to those on the disc of the human placenta. The connection with the mother is much less intimate, so there is little bleeding at birth of pigs. In order to see these folds, use the dissection microscope. Examine the fetal pig in the jar which has an injected placenta, with arteries in red and veins in blue. Compare it to placentas of sheep, cat, other mammals shown mounted in jars and to the human placentas we have seen. Next, cut open the fetal membranes (the chorion vesicle is outermost) carefully so you can keep them, BEING CAREFUL NOT TO CUT A SECOND MEMBRANE INSIDE, then examine the connection of the umbilical cord with the membranes and see if you can tell the difference between the allantois, (which is fused with the chorion and is used to store urine in pig development, and to bring the circulation to the uterine lining to get oxygen and release wastes) and the amnion, which contains the fluid which protects the embryo from drying and mechanical injury, and the outer chorion, and yolk sac.

Look at the slide of human placenta villi. The round or irregularly shaped structures are sections of the villi. Each villus after the fourth month has:

1. a blood vessel in it which will probably contain blood cells.

2. Around the blood vessel is connective tissue which contains a few cells and a lot of extracellular material which is fibers and matrix material.

3. Around the connective tissue is the syncytiotrophoblast (syntrophoblast) which separates the mother's blood from the rest of the villus, since the mother's blood is in the

4. intervillous space or lacuna between the villi.

MAKE DRAWINGS LABELING THESE STRUCTURES.

Questions:

1. The placental villi contain blood vessels containing:

a. fetal blood

b. maternal blood

2. The chorionic villi are bathed on the outside in the intervillus sinusoids or lacuna by blood from:

a. the embryo

b. uterine arterioles

NAME__________________________

ENDOCRINE AND UG

1. The difference in vagina as compared to uterus is, vagina:

a. has a thicker wall

b. has more glands

c. has striated muscle

d. has a more stratified (layered) epithelium

e. more than one of the above

3. The part of the uterus which is shed during menstruation and after birth is the:

a. entire endometrium

b. myometrium

c. outer layer of endometrium (the stratum functionalis), down to basal layer

d. connective tissue

e. basalis blood vessels

4. A lactating mammary gland can be distinguished from a non-secretory one by:

a. the amount of connective tissue

b. the number of fat cells

c. the size of the alveoli and ducts

d. the amount of hormone the mammary gland produces

5. The pars distalis or anterior pituitary has compared to the pars nervosa or posterior pituitary:

a. more different kinds of cells staining uniquely

b. fewer cell types since fewer hormones are released from it

c. more neurons entering it from the hypothalamus

d. fewer capillaries

SHORT ANSWER

1. What is the difference in the uterine wall in the first and last half of the menstrual cycle?

2. What is the difference in the uterine wall in the first half of the menstrual cycle with and without the pill?

3.How can you tell the lumenal side of the vagina?