W3006     Fall 2003                   Physiology           Mini-exam #1        

1.  “Ovariectomy” refers to removal of the ovaries.  Why were the mice ovariectomized in this assay?  (10)

 To remove the source of their own (endogenous) estrogen production, so they would have a consistent baseline to observe the effect of administering exogenous estrogen.

2.  The authors call this as a bioassay, but the experimental procedure they describe differs in some ways from the method we discussed.  Let’s say that you wanted to do a bioassay for estrogen, using female mice, and based on the known response of the uterus to estrogen. 

A.  Explain how you would get the data you need to draw a dose-response curve. (30)

 1. Take a bunch of mice and remove their ovaries.
 2. Inject each mouse with a different dose of estrogen. 
-2 if you said "ovarian extract".  For this study, it's clear that they've identified & isolated estrogen.
 3. Wait a few days for estrogen to have its effect, then remove the uteri from the mice and weigh them.
 4. Repeat this with several mice at each dose, for a more accurate dose-response curve.

You lost 10 points for saying "measure the response" without indicating what response you'd measure.  

  B.   Draw below the dose-response curve you would expect to make.  Label the x and y axes. (20)

    weight of uterus


C.  Suggest a reason why the authors did not make this kind of dose-response curve.  (10)

 They looked at only two points, one without estrogen, and one with estrogen.  They assumed that the dose of estrogen that they gave would have caused maximal uterine growth, in the plateau region indicated by A.  But without using more doses, we can't be certain that the dose they gave would have been this effective in these mice, and the dose they gave may have been anywhere between B and A.  So why didn't they make a proper dose-response curve?  Scientists sometimes do this sort of pilot study, or "quick and dirty" study, to save time and money and animal lives.  If the herbs did have a greater effect on the uterus, then the researchers probably would have produced a standard curve to determine exactly how much of an estrogenic effect they had.  

Some answers we took off for:  (-5) If you said "They just wanted to compare to the untreated mice".  Not exactly.  They wanted to see if the herbs acted like estrogen or not.  Without knowing the way estrogen affects the system, they would have no way of interpreting their results.   (-3) If you said, "They were interested in the response to herbs, not to estrogen", that is correct, but to determine whether the response to herbs was similar to the response to estrogen, they need to produce a standard curve to show just how the uterus responds to estrogen.   (-10) if you said "because other hormones may have influenced uterine weight".  That is true, but they wanted to see what effect estrogen has, and whether the herbs act like estrogen.  (-4) if you said "the authors relied on a dose-response curve that they had previously made."  That would not be good laboratory practice, because the dose-response curve is showing us how these particular animals respond at this particular time with this particular technician doing the injections with this particular batch of  hormone.  So the curve should be made at the same time and with the same animals as used in the test with herbs. 

3.  The authors also did an in vitro bioassay, using breast cancer cells that are estrogen-dependent.  Adding estrogen caused an increase in cell proliferation.  At high doses, dong quai and ginseng also caused cell proliferation, but licorice and black cohosh did not.    These results seem to suggest that dong quai and ginseng have estrogenic activity in one assay and not in another.  Suggest a way to resolve this seeming contradiction.  (10)

The authors tested to see if these herbs act like estrogen, using two tests:  An in vivo bioassay in mice, and an in vitro bioassay using breast cancer cells.  Since both systems are estrogen-sensitive, you'd expect that if herbs act like estrogen then the herbs would show the same response in both systems.  But they don't.  The dong quai and ginseng show an estrogen-like response in the in vitro assay, but not in the in vivo assay.   How could we explain this?   One possibility:  Estrogen stimulates cell growth and the herbs stimulate cell growth, but they do so via different pathways, so the herbs may have shown a similar response in the breast cell assay, but still not be working in the same way as estrogen.  Another possibility:  Perhaps the herbs were acting like estrogen in the breast cell assay, and did stimulate the estrogen-receptor in these cells.  But perhaps the estrogen-receptor in uterine cells is different.  There are, in fact, two different estrogen receptors that have been identified, and so it's possible that the herbs would stimulate one but not the other.  You lost credit if you said they should do a third assay, because that still doesn't explain the contradictory results.  We don't decide scientific questions democratically by taking a vote as to how many assays come out positive! 

4.  What advantage does the ELISA have over the RIA?  (10)  

Doesn't use radioactivity, which could contaminate the environment, be dangerous to health of lab workers.  Cheaper.

Some of you said that ELISA doesn't use animals.  Actually, neither of these are done in vivo, so animals not used like in bioassay.  I did mention use of an animal to make the antibodies, in the early RIAs, but today antibodies can be made in culture:  http://www.accessexcellence.org/AB/GG/monoclonal.html

5.  Rank these in order from largest =1  to smallest =5 contribution to the water found in a single human body:  (10)

__3__  ECF    ___5_  plasma      ___4_  ISF     __2__  ICF    _1__  total body water      See  Lecture 1, Slide 21   If you mixed up two of these, you lost 4 pts.