Category: Honors College

This project is the culmination of my work over the past six months studying how Danazol impacts the embryonic development of zebrafish. Although most widely used in the treatment of endometriosis, adenomyosis, and fibrocystic breast disease for over 40 years—Danazol has an extensive array of biologic effects as well as diverse clinical applications in the management of countless other diseases and disorders.

My interest in Danazol, however, is due to its role as an inhibitor of adrenal steroidogenesis— the process by which steroid hormones (such as aldosterone, progesterone, testosterone, and cortisol to name a few) are synthesized by the adrenals and the gonads.

In the adult, human model: Danazol suppresses this system through the competitive inhibition of specific enzymes expressed by the adrenal glands. Likewise, once in the bloodstream, Danazol binds avidly to the sex-hormone binding globulin—displacing estradiol and testosterone. The net effect is a significant increase in the amount of testosterone in the body and a decrease in cortisol and other steroid hormones.

My question with this project was: “how do increased levels of testosterone, or the endocrine-disrupting activity of Danazol more generally, affect normal embryonic development?”. Unlike in adults, where these hormonal disruptions are actually therapeutic, I predicted that exposure to Danazol would actually induce a model of the disorders that arise to due to abnormal adrenal ontology and function.

Specifically—because Danazol is a synthetic androgen, or hormone that regulates the development and maintenance of male sex characteristics—I predicted that exposure to the drug would result in virilizing, otherwise known as masculinizing, effects: phenotypes consistent with the intersex condition congenital adrenal hyperplasia (CAH). However, given that Danazol has never before been tested in zebrafish or another live developmental model at this stage— I didn’t have any prior work to ground these preliminary assumptions in.

Over the spring semester, as I conducted pilot studies to identify a non-lethal dosing regimen of Danazol, what I saw turned my hypothesis right on its head. Instead of observing virilizing effects, or phenotypes consistent with CAH—I saw dramatic bradycardia (or a decreased basal heart rate) along with other cardiac deformities, including pericardial and yolk sack edema and blood pooling.

The goal of this project moving forward, and over the summer term, was to repeat the same drug trials, but over a statistically significant sample size—allowing me to quantitatively describe the effects of the drug treatment, specifically the reduction in heart rate.

To conduct my experiment, embryos were obtained from the zebrafish colony housed in the Animal Facility at the Krasnow Center for Advanced Study. Once acquired, any coagulated (or dead) embryos were removed and those that remained were then sorted into four petri dishes such that each contained 75 healthy embryos.

The interrenal gland, the zebrafish organ equivalent to the mammalian adrenal glands, begins to form between 24 to 30-hours-post fertilization (abbreviated to hpf)—and the steroidogenic enzymes bound by Danazol begin to be transcribed at 24 hpf. As a result, I chose to treat the embryos by exposing them to Danazol (solubilized in 5 microliters of DMSO) between 24 to 26 hpf.

Every trial consisted of a control group (egg medium with 5 microliters of DMSO) and three treatment groups (at 0.2, 0.4, and 0.6 micrograms/mL of Danazol). A starting sample size of 300 embryos were used for each trial—and I completed 3 trials over the course of the summer.

Beginning at 72 hpf, one at a time, I recorded a 15-second video of every surviving embryo in all four groups from a lateral view. I then counted the heart rate for each embryo in every video.

Here we can see the results from each of the three trials, which suggests that Danazol does not decrease heart rate in a linear, dose-dependent manner. Heart rate data was assessed using a one-way ANOVA with a post-hoc Tukey-Kramer test for multiple comparisons. This allowed me to determine if there was a statistically significant difference between the test groups, and if so, which.

For all three trials, there was found to be a statistically significant decrease in heart rate between the control group and all three treatment groups—and, with the exception of trial 1, there was no statistically significant difference in heart rate between the three treatment groups. The significant difference between the 0.2 ug/mL and 0.4 ug/mL groups in trial 1 is likely due to experimental error and not attributable to the effects of the drug.

These findings demonstrate that in addition to widely reported accounts of virilization, exposure to androgens during embryonic development poses significant risk for cardiovascular pathology. Future work in this regard may seek to identify Danazol’s mechanism of action in interfering with heart organogenesis by recording data at the 30 and 48-hpf mark to study errors in heart looping and chamber ballooning or directly measuring the levels of steroid hormones in circulation with ELISA assays.

Finally, I would like to thank my mentor Dr. Valerie Olmo—thank you for your constant support and guidance, both with this project and over the course of my senior year. I would also like to thank Dr. Karen Lee and the rest of the OSCAR team for funding my work and giving me this opportunity. And finally—I would like to thank Nathan Ridings for his filming/audio/and editing expertise. Thank you for listening.

So, a lot of our cats here are permanent residents. So, this means that a lot of them have come in before the age of 6 months and because of that, their mothers were not able to teach them the proper skills to actually survive in the wild. So, some skills including what prey to hunt, such as duikers, steenboks, springboks, kudus and oryx’s as well as what predators to avoid: lions, snakes and hyenas. And so, a lot of them have actually also been useful for research and understanding more about the species themselves and how to conserve them the best.

So here on our Namibian campus, we have 29 cheetahs and on Somaliland’s facility we have 86 cheetahs. So a lot of these cheetahs have come in at very different ages, so these siblings came to us at the age of four weeks. Others have come in at 5 days, 10 days, so very young. A lot of the cheetahs that have come in were rescued with different scenarios, so some farmers actually might have killed the mothers, some farmers might have captured them and in other cases people have kept the cheetahs as pets as well. So, like I mentioned before, having them here has been very useful for research and also their conservation.

Here are three of our cheetah cubs at CCF who arrived when they were 3 weeks old. Speaking of cheetah cubs for my research project, I have been working with Dr. Laurie marker, and other CCF staff members as well as my mentor Dr. David Luther where I am investigating the comparison of Somaliland and Namibian cheetah cubs growth rates. From data collected back from 2006 until most recently 2022, I have been gathering all this information on a new excel spreadsheet from individuals between the ages 0-6 months.

Other than spending time on my research project, I have been part of the cheetah team working to take care of the cheetahs by feeding them, giving their meds if needed, cleaning and managing their enclosures.

Moving on to the results for the project so far. I have created some graphs as well as produced some statistical analysis of the data through RStudio.

This first graph demonstrates the average weights of Somaliland and Namibian Cheetah cubs from the age of Day 1-14. As you can see there is a linear relationship between the weights and days of the cubs. The R-squared value is around 0.59 indicating a moderate goodness of fit of the model. We notice the Somaliland average weights are less than the Namibian weights. The second graph demonstrates the average weights of Somaliland and Namibian Cheetah cubs from the ages of Week 3-26. The R-squared value of 0.91 demonstrates a very strong linear relationship. Once again, the Namibian weights are larger than the Somaliland weights on average.

The third graph is a boxplot displaying the first two weeks of age. We notice the Namibian data appears to have more range and a higher average weight. The last graph is a boxplot displaying weeks 3-26 of age. We notice the Namibian data appears again to have more range and a higher average weight; however, it appears less robust than the previous graph. Running these last two graphs’ data through R, we found no significance in terms of their weights and comparing them between the two locations. Meaning the p-value was greater than 0.05.

Last but not least, the sum of the weights for all weeks did not appear to have significance with meat and milk consumption, yet the location of Somaliland was significant indicating their cubs are less likely to survive, and the males in general appear to have higher growth rates than females (indicating greater chance of survival).

We still have many questions to answer, such as how much the cheetah cubs grow in general, what month they grow the most and how exactly does diet affect their growth, especially relating between the two locations of cheetahs.

Thank you so much for watching and for everyone who has helped me on my project so far! Bye!