During my junior year as a Biology major at UIC, I began working in the Physiology Department and the laboratory of Dr. Buck Hales. During the last two years pursuing research, I've experienced the excitement of scientific discovery, as well as the frustration of experimental failure. Each new finding in our lab has prompted new questions, new conclusions and new hypotheses. Our scientific process has been hard work, but I have found the lab bench and experimental science to be both challenging and great fun. Research has pushed me to expand my understanding and comprehension of life at the cellular level, providing me with a much deeper appreciation for Biology. Regardless of whether your future career ambitions involve medicine, the allied health professions, or science, I would highly recommend spending some of your undergraduate career doing research. I was once a Pre-Med myself, but after doing lab research, my career ambitions changed. I hope my explanation of our work will share some new knowledge, and perhaps spark an interest in research for some of you.

Introduction and Background

Testosterone is the essential masculinizing steroid hormone found in the male. Testosterone promotes the early and sustained development of male secondary sex characteristics, and it is necessary to promote the normal development of spermatozoon. Thus, testosterone is a key hormone that influences the reproductive capacity and sexual development of the male. Testosterone is produced within a specialized cell type known as the Leydig cell located within the interstitial space of the testes. Closely associated with Leydig cells are macrophages, which are specialized immune system cells that hunt down and engulf bacteria. Upon recognition of bacteria, macrophages become activated and they produce reactive oxygen in the form of hydrogen peroxides and oxidized halogens, compounds which are toxic and kill invading bacteria. The close physical proximity between Leydig cells and macrophages suggests the two cell types might be functionally interacting. The Hales laboratory is interested in examining the functional interactions between Leydig cells and macrophages, and in particular elucidating how the immune system regulates testosterone production.

Testosterone is produced in Leydig cells in a multi-step biosynthetic pathway, which is under the control of the pituitary gonadotropin luetinizing hormone or LH. Biosynthesis of testosterone is outlined in the figure below. Of particular importance is the transfer of cholesterol into the mitochondria, the rate limiting step in Testosterone production. Cholesterol transfer is facilitated by the Steroidogenic Acute Regulatory Protein (StAR). Decreasing the transcription or translation of StAR prevents cholesterol transfer and inhibits testosterone synthesis (1).

Many of our recent experiments have tested the hypothesis that reactive oxygen produced by the immune system inhibits the production of testosterone. As mentioned previously, when macrophages recognize bacteria, they will produce reactive oxygen in the form of hydrogen peroxides to kill the invaders. Thus, if a bacterial infection occurs in the testes, macrophages will produce reactive oxygen to counteract the infection, but this reactive oxygen could also have paracrine effects on the closely neighboring Leydig cells. Reactive oxygen is also produced constantly within all cells as a by-product of metabolism and enzymatic chemical reactions. Reactive oxygen build-up in cells can interfere with cellular processes, leading to abnormal function. Cells have sophisticated antioxidant defense systems within them that prevent reactive oxygen build-up. Two examples of this are Vitamins A and C, which quench free radicals and reactive oxygen within cells. The aging process in all living organisms is associated with an increased build-up of free radical reactive oxygen, which overwhelms the cellular antioxidants. Thus, aging leads to a build-up of reactive oxygen leading to inefficient cellular processes and even cell death.

The Experiments

We hypothesized that during an immune response or aging, reactive oxygen might affect Leydig cells and inhibit testosterone. To test this, I did several in-vitro tissue culture experiments using mouse Leydig cells. We grew the cells in culture and then treated them with increasing concentrations of the reactive oxygen species hydrogen peroxide (H2O2), a scenario mimicking what might happen in vivo during an infection and macrophage activation. After the incubation with H2O2, treatment medias were removed and saved for later analysis, and cells were lysed and protein solublized in a buffer. We analyzed the treatment medias for hormone concentration by radioimmunoassay (RIA) to see if testosterone production was affected by H2O2. Protein from the cells was analyzed by western blot to determine if reactive oxygen affected any of the enzymes that make testosterone. I also examined hydrogen peroxide treated cells under a light microscope equipped with fluorescence filters. We incubated the cells with reactive oxygen then added a dye to the cells which measures the electrochemical gradient in the mitochondria. We also performed a northern blot analysis to examine the effects of H2O2 on mRNA expression of the enzymes which make testosterone.

Our Findings

Our experiments found that 100uM hydrogen peroxide treatment significantly inhibited testosterone production in Leydig cells, and 250uM profoundly reduced testosterone production by approx. 50%. What could cause this decrease? Our western blot data showed that StAR protein expression was also greatly reduced after the reactive oxygen treatment. StAR protein levels were decreased by nearly 80% after 250uM hydrogen peroxide treatment. We found that StAR mRNA levels were not effected by the reactive oxygen treatments. Since StAR mRNA levels remained unchanged, but protein levels were reduced, this indicated that hydrogen peroxide inhibits StAR post-transcriptionally. The light microscope experiments showed that Leydig cell mitochondria were disrupted by the reactive oxygen. Their electrochemical gradient, a necessary component for cholesterol transfer and testosterone production, was severely depolarized.

These collected findings showed that reactive oxygen inhibits testosterone production in Leydig cells by disrupting their mitochondria and by decreasing StAR protein expression (2, 3). Since StAR protein levels were reduced, less cholesterol was transferred into the mitochondria, resulting in reduced testosterone production. So, yes, reactive oxygen does affect Leydig cells as we hypothesized! This inhibition of testosterone production by reactive oxygen likely occurs in disorders such as infections, reperfusion injury, alcohol toxicity, and aging. Decreased testosterone production in these disorders can cause a reduced reproductive capacity and reduced virility in the male.

Meetings and the Undergraduate Research Symposium

I have been fortunate to have the opportunity to present our findings at several local and national meetings within the last two years. Last July, I traveled with some of our lab to Madison, Wisconsin for the annual Society for the Study of Reproduction meeting, which was a fantastic experience. Being at a large scientific meeting was a new experience for me and a bit intimidating, but seeing the numerous reproductive science findings, including cutting edge stem cell and cloning work, excited and motivated me. This last April, I presented our findings at the 2nd Annual Undergraduate Research Symposium at UIC. The organizers for the symposium did a top-notch job, and I thoroughly enjoyed the experience of sharing my research with other UIC students. I really encourage all undergraduates to at least attend if not participate in this campus research meeting next spring. My future plans involve an oral presentation at the Center for the Study of Reproduction meeting at Northwestern University. I plan to continue my research in the Hales lab while pursuing a PhD in Physiology.