Beginnings and Kinks

I feel that I should begin with a better explanation of what exactly my project is, before this post becomes a confusing, angry rant.  It might turn into a little bit of a rant (fair warning) but hopefully it will also make a little bit of sense.

GnRH stands for gonadotropin-releasing hormone.  It is the “master regulator” of puberty and reproduction.  The hormones that control and regulate sexual maturity and fertility and all that good stuff travel along a path of organs and glands known as the hypothalamic-pituitary-gonadal axis (known hereafter as the HPG axis).  GnRH originates in the hypothalamus and signals to your anterior pituitary gland to release luteinizing hormone (LH) and follicle-stimulating hormone (FSH).  These two hormones travel to your testes or ovaries, depending on your gender of course, and stimulate gametogenesis (the production of sperm and eggs) and production of sex steroid hormones testosterone, estrogen and progesterone.  Both testosterone and estrogen are then involved in a negative feedback loop in which they travel up to the brain and inhibit the release of GnRH.  Estrogen is also part of a positive feedback cycle in females only that leads to successive LH surges which ultimately culminate in ovulation.  Of course I am leaving many details out, but this is the gist of it.

In Dr. Heideman’s Lab of Evolutionary Physiology, we work with a unique mouse model.  Our mice are unique because they’re not lab mice per se.  Dr. Heideman caught a parent population in 1995 in the College Woods, and his lab has raised subsequent generations under laboratory conditions ever since.  This is important because genetically designed “lab mice” are homogenous.  That is, they are basically the same in their behavior, mechanisms and genetic make-up. Our mice, on the other hand, are of the species Peromyscus leucopus, and theyvary from individual to individual just like humans do.  This individual variation is key to the overall research goals of our lab.  Most importantly, the mice vary in their sexual response to a winter, short day photoperiod.  During winter in the wild, some mice in the population suppress reproduction while some continue to reproduce despite the decrease in day length.  This trait, response to winter photoperiod, is genetic.  We have dubbed the mice that continue to reproduce the nonresponsive line (NR) and their counterparts that shut down reproduction the responsive line (R).

Our lab has found that the NR line of mice have more LH than R mice do.  The goal of the GnRH Challenge is to determine whether this difference is due to an inherent difference in number of GnRH neurons between lines or a difference in pituitary sensitivity to the hormone between lines.  The basic procedure is to inject mice from both lines with the same dose of GnRH.  If they turn out to have the same LH levels after the injection, the observed difference is probably due to neuron number.  If NR mice still have more LH than R mice, the difference is probably due to pituitary sensitivity.  Understanding this detail of the pathway will allow us to better understand the pieces required for fertility and in the future might contribute to treatment of infertility in humans.

(Side note: The above paragraph is an important one in terms of “the process of science.”  I stated the purpose of what we are doing, its importance and “real life” relevance, and then laid out our hypothesis and predictions.  These steps are universal in the world of research.)

Seems simple, right?  Inject hormone, measure LH in blood.  Evidently not that simple, because the experiment has been in its pilot stages for more than a year at this point.  Last summer, we figured out that we were administering too much testosterone as a control.  Too much steroid negative feedback was occurring, and an LH pulse could not be generated.  There have been a plethora of other issues besides that, but the main problem is that we can’t seem to produce readable levels of LH in the blood of any mice, regardless of line or GnRH dose.  Even without an injection of GnRH, there should be some LH in the blood.  This past spring semester, we decided to try a new chemical form of the hormone from a different company than the one we had been ordering.  One of our group members met up with Dr. Coleman, a biochemistry guy.  He advised us that we may have been using a molecular form that was not quite as effective at binding to GnRH receptors as another form might be.  Full of hope, we embarked on a new pilot testing three forms and three different doses of GnRH.

The title of this post stems from the fact that this pilot failed just like all the others.  Of 26 blood samples sent off for LH assay, only 6 were returned with readable LH levels in no discernible order or informative pattern.  We felt like we were at a dead end.  We can’t seem to figure out what we’re doing wrong, methods used in the past don’t seem to be working for us and our patience is running out.  So, we have decided to try one more thing and if this does not work, we will most likely give up on this project (DUN DUN DUN).  Normally, we surgically remove the gonads of our mice and implant testosterone and estrogen replacements to control variable amounts of these hormones between individuals.  These implants are relatively simple, but they seem to be giving us issues although we are not sure why.  This time around, we are not going to use hormone implants.  The effort to control variable hormone levels should still be achieved, these levels will just be zero.  As a result, LH should be extra high and this can only work to our advantage at this point.  Secondly, we decided to play around with the time that elapses between GnRH injection and blood collection.  In that past we have waited 30-45 minutes, but we have read some literature that used as little as 10-15 minutes, and so we have considered that it’s possible we are missing the LH surge we’re trying to induce.  If these measures don’t work (knock on wood), I don’t know what will.

We’ve sent off several serum samples for LH assay after carrying out this last-ditch procedure and we should get the results within a week or so.  Keep your fingers crossed for me, and I’ll keep you updated!