Research Update #2

We finally got LH results back from UVA.  And none of them are unreadable!  Yay.  No matter what they are, they have got to be better than the results we’ve been getting for the past year.

Please recall that we were testing to see if we could get an LH response from our mice (at all) in the absence of estrogen and testosterone implants.  We were trying to see if an LH difference still existed between the nonresponsive and responsive lines in both males and females, quantify LH levels without steroid feedback, and see if the duration of elapsed time between GnRH injection and blood collection made a difference.

A quick summary: there was not a significant difference between NR and R mice in either males or females.  Hopefully this is due to the small sample size (n=2 for the females and n=3 for the males), but it’s possible the steroid negative feedback is an important component of the LH difference.    (A quick word about “significant” differences: the word significant has statistical meaning in science.  It means that statistical tests designed to account for sample size and the effect of variables, among other things, have been run on the data.  A significant difference means these tests have determined the difference (or lack thereof) you observed in your data is due to the effect of your manipulated variables, and not due to chance.)  There was also not a significant effect of elapsed time on LH level, which was most surprising to me.  The success of LH responses did confirm that the UVA assay works on our mice, that we can actually achieve an LH response, and that forgoing hormone implants might be a viable option in the future.

Two very useful numbers that we got back were the LH values in pooled male and female blood.  In females, resting LH levels are around 0.78 ng/mL, and in males resting LH values are around 0.33.  These are helpful because they provide a baseline.  Now, we will be better able to tell when a GnRH injection has an effect or not.  On that note; the GnRH injection of 25 ng we gave the females did not appear to significantly raise their LH levels beyond the females that we ovariectomized but did not inject as controls.  This is problematic for obvious reasons; the whole point is that we need the mice to be responding to the GnRH.  One of my hypotheses to explain this is that 25 ng is too high of a dose.  I have been reading a lot about GnRH agonists and antagonists (for reasons I will explain in a hot second) and there is a good body of evidence suggesting that agonists eventually desensitize the pituitary GnRH receptors.  Put simply, it’s not the dose but the timing that matters in these pulsatile hormones.  (Quickly: an agonist is a molecule that mimics another by binding to the same receptor and eliciting a similar physiological response.  An antagonist is a molecule that binds somewhere else on the same receptor, causing it to change in conformation and essentially shut down the ability of the original molecule to perform its function.)

Some thoughts that I’ve hammered out while processing these results:

  1. There are obvious differences between males and females that need to be taken more seriously and approached more systematically.
  2. Previous work in staining GnRH neurons in the brain found that NR mice have 50% more pituitary GnRH receptors than R mice do.  This work was done in males but I think our lab should replicate it in females.
  3. Similarly, the 2010 LH paper that established a difference in LH levels between NR and R mice was done in females.  I think we should replicate it in males.
  4. Perhaps we have exhausted the method of injecting GnRH to elicit a response and it is time to give up and try another approach.
    1. In the event this is not true, I think we should give it one more go with no more than a 10 ng dose, implants with a purposefully less-than-optimal hormone dose, and 20 minutes between injection and collection (20 minutes had a high LH level with the least amount of variation).
    2. In the event this is true: I want to look into GnRH antagonists.  I think if we blocked pituitary GnRH receptors (some, not all of them) we might be better able to examine pituitary sensitivity directly.  (Hence the reading I’ve been doing.)

Stay tuned!