This exam has 6 pages and a total of 50 points.
You will have 90 min. to complete it.

Make sure your name is on all pages.
You will receive 1 bonus point for doing this!!

Answer all short answer questions in the space provided.

Rolling of a sheet of tissue that causes internalization of original surface material.

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B. Syngamy:

In fertilized mammalian oocytes, the sperm and oocytepronuclei lie side by side but
their DNA does not mix until first mitosis.

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C. ICSI:

Intracytoplasmic sperm injection; sperm is directly injected into an oocyte and the
oocyte is activated by aspirating oocyte cytoplasm into a pipette and then
reinjecting it into the oocyte.

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D. Capacitation:

Process by which mammalian sperm are activated in the female reproductive tract.

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E. Blastocoel:

Hollow, fluid-filled cavity created at the blastula stage as a result of cleavage.

2. The regulation ofreproduction in humans involves regulating hormones before fertilization as well as after fertilization.

A. Complete the following table, which refers to the approximate levels (low or high) of several hormones involved in reproduction at various times in the human reproductive cycle. Provide your reasoning in each case.

Hormone/ Factor	Stage in Cycle	Expected Hormone Level	Reasoning
human chorionic gonadotropin	Immediately following release of oocyte from mature follicle	Low	hCG is produced by zygote after fertilization
luteinizing hormone	Immediately prior to release of oocyte from mature follicle	High	Surge of LH and FSH precedes follicle rupture, so we expect LH levels to be high.
progesterone	Three days after ovulation in the absence of fertilizing sperm	Low	Corpus luteum degenerates in the absence of zygote’s hCG, resulting in low levels of progesterone

B. Briefly describe how the drug RU486 acts, and why it is effective as an abortifacient. Provide molecular details where appropriate. (2 points).

RU486 binds to progesterone receptors in cells to the uterus. However, once bound by the drug, the receptors are not capable of carrying out signaling in the nucleus, and the result is that the uterine lining is sloughed off, resulting in prevention of implantation and the loss of the embryo.

3. You believe a novel sperm surface protein (which you name protein W) is required for binding to the zona pellucida during mammalian fertilization.

A. You use a variety of techniques to study protein W's function. For each piece of information you learned about protein W, state what technique you used to obtain the information. (3 points)

Discovery	Major Technique Used To Obtain Information
Lack of protein W gene function in mice results in sperm that cannot bind to the zona	Targeted mutation ("knockout")
Protein W mRNA is not produced in any other tissue in mice expect in secondary spermatocytes and spermatids in the male testis	in situ hybridization (Northern blot also accepted)
In the occasional mouse sperm from a normal male that cannot bind to the zona well, protein W is in an abnormal location	Immunostaining
You isolate a DNA fragment from humans that corresponds to a portion of the coding region for protein W	RT-PCR or screen a cDNA library
You discover to your surprise that the nematode C. elegans has a gene similar to the protein W gene	Look up sequence in database (screen genomic library also accepted)
You wish to block the function of the human protein W to test its function in fertilization	Use blocking antibodies

B. Based on what you already know, what protein would you predict protein W interacts with in the zona?

Since ZP3 is the protein in the zona to which sperm bind, it’s likely ZP3.

C. You assay sperm from male mice that lack a functional protein W gene. As expected, you discover that their acrosomal vesicles are intact, even when they are placed in contact with large quantities of zona pellucida proteins. Why did you expect this? Clearly state your reasoning (1 point)

Binding to ZP3 initiates the acrosome reaction, but since sperm lacking protein W function can’t bind the zona, they would be expected not to acrosome react.

4. The ability of sperm to bind to and fuse with eggs is a key event in fertilization, and crucial for blocking polyspermy. Please complete the following table, which asks whether or not sperm will bind to eggs under a variety of circumstances. Indicate your reasoning in the space provided:

Type of Sperm	Type of Egg	Fertilization Successful (Yes/No)?	Reasoning
Sea urchin sperm exposed to egg jelly	Normal sea urchin eggs from a different species	No	Fertilization is species-specific in sea urchins (via bindin)
Untreated sea urchin sperm	sea urchin egg treated with calcium ionophore	No	Ionophore induces slow block, so fertilization can't occur
Human sperm directly removed from the epididymis	human oocyte with intact zona pellucida	No	Sperm aren't capacitated
Sea urchin sperm	sea urchin egg held at -70 mV by a "voltage clamp"	Yes	This is the normal resting potential, so egg should be fertilizable.

5. Answer the following questions based on your knowledge of cytostatic factor's effects on cell division.

A. If purified c-mos were injected into one cell of a two-cell embryo immediately after first cleavage, what would you predict would happen at the time of the next division? Clearly state your reasoning. (2 points)

c-mos results in stabilization of active MPF; injected cell will make it to metaphase, but will arrest there. Other cell will cleave normally.

B. Frog eggs can be artificially activated in the absence of sperm by pricking them with a needle. What would you expect would happen to levels of cytostatic factor in such an artificially activated egg? Clearly state your reasoning. (2 points)

c-mos (CSF) levels would drop as they would following fertilization.

6. Complete the following table referring to cleavage patterns in various animals.

Animal	Yolk Distribution	Completeness of Cleavage	Orientation of Cleavage
Ostrich	extremely telolecithal	meroblastic	radial (discoidal)
Snail Gastropod Mollusc	isolecithal	holoblastic	spiral
Human	isolecithal	holoblastic	rotational
Sea cucumber Echinoderm	isolecithal	holoblastic	radial

7. By altering the external ionic environment or the properties of some eggs or oocytes, it is possible to study the role of signal transduction in the blocks to polyspermy and egg activation. Please answer the following questions regarding these ionic events. In all cases, briefly explain your reasoning.

A. If eggs are placed in sea water made mildly basic with ammonia in the absence of sperm, what effects would you expect on the treated eggs? (2 points).

Increase in pH stimulates protein synthesis.

However, it is not stimulated as much as in normally fertilized eggs.

B. A frog oocyte is injected with mRNA for serotonin receptors and allowed to undergo maturation. The oocyte is then treated with serotonin. What effects do you expect? (2 points)

This stimulates G protein mediated signal transduction, leading to IP3 production and calcium release. This activates the egg.

C. A frog oocyte is injected with a calcium chelator compound (a compound that binds to calcium ions, preventing them from moving freely within the cytoplasm) and subsequently fertilized. What effects do you expect? (2 points)

Without calcium wave, egg activation would be blocked or significantly delayed.

8. Circle the appropriate response (T = True, F = False) for each of the following statements, which refer to mammalian embryos (6 points):

T	In frog cloning experiments, the likelihood of producing a viable tadpole from a donor nucleus placed into an enucleated oocyte decreases the older the animal is from which the donor nuclei are obtained..
F	The inner cell mass cells of the mammalian blastocyst predominantly form the extraembryonic membranes of the later fetus.
F	In chicken embryos, some epiblast cells involute to form the hypoblast, which forms the floor of the embryo.
T	Unlike protostomes, the first opening of the gut formed in echinoderms such as sea urchins forms the anus.
T	Like European Union countries, the United States prohibits federal funding of human cloning experiments.
F	Cortical granules in sea urchin eggs are crucial in the fast block to polyspermy, because they contain components that help to produce the fertilization envelope.
T	Unlike mammalian sperm, sea urchin sperm produce a long acrosomal process containing bundles of actin.
F	Treatment of fertilized frog eggs with nocodazole prevents rotation of subcortical cytoplasm, preventing ventral structures from forming.
F	Pronuclear migration would likely be inhibited by cytochalasins, since it is an actin-dependent process.
F	"Pre-embryo" biopsy is still difficult, because current methods only allow genetic identification of homozygous embryos.
T	The archenteron of the sea urchin embryo is a convenient system in which to study invagination of a cell sheet.
F	Fate mapping indicates that extensive cell movement occurs in embryos prior to gastrulation.

9. The developmental potential of cells in the early mammalian embryo has been examined in several ways.

A. Describe one way that primate embryonic stem cells have been produced (2 points)

"Immunosurgery" was used to remove the trophoblast. The remaining ICM cells were cultured, and the resulting cells and their progeny behave like stem cells.

OR

Gonadal ridges of fetuses were isolated and the primordial germ cells were cultured. They also seem to behave like stem cells.

B. Describe at least two experiments that demonstrate that cells in the pre-compacted mammalian embryo are totipotent (2 points)

(1) Make allophenic mice by combining multiple embryos; result is a normal embryo.

(2) Remove one cell as late as uncompacted 8-cell, and you still get a normal embryo.

 

10. Many processes in early development require proper cell adhesion. Match the appropriate responses on the right to the processes on the left. Note: There may be more than one correct answer for each event listed on the left. (3 points)

Adhesion of frog blastula cells	A	A. Requires cadherin-mediated adhesion
Compaction of mouse embryos	A	B. Requires integrin-based adhesion
Binding of acrosome-reacted sperm and oocyte in mammals	B	C. A & B
"Sorting out' of amphibian embryonic cells	A	D. None of the above
Binding of sea urchin sperm to sea urchin eggs	D	_

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