Expert Insight: New insights into reproductive dysfunction with advanced spatial genomics

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Dr. Lique Coolen, Professor, Department of Biological Sciences, Associate Dean, College Arts and Sciences, and Dr. Aleisha Moore, Assistant Professor, Department of Biological Sciences, Kent State University

In this SelectScience webinar, now available on demand, Dr. Lique Coolen and Dr. Aleisha Moore, Kent State University, discuss how they have integrated powerful RNAscope in situ hybridization technology into their research to study the mechanisms underlying spinal cord injury and neuroendocrine dysfunction.

In part one of this session, Moore presents work in her laboratory identifying changes in neuronal circuits controlling fertility that may lead to polycystic ovarian syndrome. This is followed by a talk from Coolen, who provides an overview of studies in her lab that examine the impact of spinal cord injury on the spinal reflex generator for sexual function in the lumbar spinal cord in male rats.

Think you’d benefit but missed the live event? Register now to watch the webinar at a time that suits you and read on to find highlights from the live Q&A session.

Q: Do hormone changes associated with PCOS have any other health ramifications besides PCOS itself, and how might these particular cells play into these health issues?

AM: There have been quite a few epidemiological studies looking at testosterone concentrations in the blood of hormonal PCOS, and they've been able to correlate this highly with instances of obesity, hyperinsulinemia, and insulin resistance, as well as other diseases such as cardiovascular disease and mood disorders. There's a lot of work going on to decipher the mechanisms that lead to this high androgen causing these other symptoms that are seen in PCOS women. With KNDy cells, we are also looking to see whether they're also involved in nonreproductive effects in PCOS. For instance, the KNDy neurons have been hypothesized to provide a site through which the metabolic factors can influence reproduction. Therefore, they may provide a site in PCOS in which factors such as insulin resistance may be causing changes in the brain that then impact reproduction.

Q: You show that GnRH neuron activity is higher in PCOS mice. Has anyone looked at whether KNDy neuron activity is also elevated?

AM: Yes, GnRH neurons have been shown using the in vitro cytolytic physiology to have higher activity and that correlates with the increase in LH pulse frequency. But to date, although we are seeing many anatomical changes, we haven't been able to look at whether or not the KNDy cells have higher activity. You may have seen that in my HiPlex assay, I added FOS RNA probe to see if we had higher activity in the cell just by looking at the FOS. However, FOS clears so quickly we were unable to detect any differences. We're going to need to use a different technique that allows us to visualize the KNDy neurons across longer periods of time to see whether or not they also show an increase in activity, which may then drive a high GnRH neuron activity and a high LH output.

Q: You showed changes that may affect the gesture and negative feedback within KNDy neurons and also presynaptic populations. How will you know which changes are sufficient to drive the PCOS symptoms?

AM: Using the mouse rather than other animal models, I'm able to use different transgenic technologies to specifically target the KNDy cells, and then specifically target the GABAergic or glutamatergic cell in different regions. I can then use function manipulations such as optogenetics, chemogenetics, and others to be able to tell which of these components, which of the changes is leading to the high LH pulse frequency or if these changes are contributing to other symptoms in the PCOS disease.

Q: You showed us the use of RNAscoped visualized G protein-coupled receptor, GRPR. Why did you not choose to use immunolabeling as that would've shown you the localization of the receptor protein?

LC: Antibodies for G protein-coupled receptors are extremely difficult to work with. They're not always sensitive and validation steps are very critical for using those antibodies. For the receptors in this system that we are interested in, it has been very difficult to find reliable, validated antibodies. We were also having trouble with visualizing olfactory expression through receptors. We therefore chose to use RNAscope as a very reliable and sensitive method to first get a sense of where receptors were expressed within the system. However, it is important that we next follow this up with additional immunofluorescence studies. In some of our findings, we found RNA in cell bodies where we didn’t expect the receptor to be expressed. That means we have to follow up with immunofluorescence to detect whether the receptors are expressed postsynaptically on the soma or presynaptically in axon terminals. We also need to do some other physiological studies to follow up on the exact location of these receptors. RNAscope is a great, very sensitive technique. However, there's never one technique that will give you all of the answers to your questions, a combination of approaches will always be necessary.

Q: You showed experiments focused on GRP, but you also saw changes in guanine mRNA. Have you done similar experiments for guanine as for GRP with similar results?

LC: Those experiments are currently undergoing. We have done some pharmacology experiments and the preliminary analysis is looking very promising. We have not yet conducted any RNAscope experiments to visualize the receptor expression and that is exactly what my lab is currently working on.

How did you prepare your tissues for using RNAscope?

LC: For all the experiments that I showed today, we use the tissues that were perfused using paraformaldehyde following the protocol that ACD Bio-Techne provides for a preparation using the v2 assay. This is an assay that requires the use of TSA-conjugated constructs, so it's focused on paraformaldehyde perfusion, and tissues are then postfixed overnight and then go through a radiative series of sucrose. 

Can CellProfiler detect changes in the level of expression or is it a binary count of particles?

AM: The CellProfiler software is taking advantage of the fact that with RNAscope, a single present particle is equivalent to a single RNA. Therefore, by counting the number of these present particles, you can get an idea of the expression levels within a cell. This is why with CellProfiler, not only do we detect the number of cells which express any number of these particles, but also then the number of these particles within the cell. You may see these differences in the intensity of the fluorescence, but the number of probes that are binding does not necessarily mean differences between your groups. The number of cells and the number of particles is your best way to be able to detect changes in gene expression.

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