Featured Scientist: Rosario Marroquin-Flores (she/her/hers), PhD, 2022, Illinois State University. Currently a Postdoctoral Research Associate in the Department of Biological Sciences at Texas Tech University.
Birthplace: Albuquerque, New Mexico
My Research: I study temperature-dependent sex determination (TSD) in turtles. Turtles that have TSD will become male, or female, based on the temperature they experience as an egg.
Research Goals: I have recently transitioned into a new field of research, Biology Education Research (BER). People who work in BER study how students learn biology in the classroom. I would like to explore new ways of teaching that help students connect their biology content knowledge to real-world problems.
Career Goals: I am interested in teaching and doing research at the university level.
Hobbies: I enjoy camping, backpacking, and spending time with my partner and my pets.
Favorite Thing About Science: I enjoy the autonomy of science. I like that I get to decide what I want to study. I get to ask my own questions and find my own answers. I also like how dynamic it is. Every day I get to do something different. I’m very busy, but I’m never bored!
Scientist Upbringing: I have always had a passion for living things. I used to sit in the grass in my front yard and try to be as still as possible, and just wait for everything around me to come alive. When I was a little girl, those interests manifested in the desire to be a veterinarian. However, when I got into my undergraduate program, I started to do research. My research experiences ignited a deep curiosity that encouraged me to explore living things more deeply.
My Team: I am the first author on this publication, but this has been a collaborative project. My advisors, Dr. Rachel Bowden and Dr. Ryan Paitz, helped me come up with the project, assisted with data analysis, and helped in writing the publication. My wonderful lab mate, Anthony Breitenbach, helped me organize my eggs into treatments, complete dissections, and provided lots of emotional support and very loud jokes.
Organism of Study: I study the red-eared slider turtle (Trachemys scripta)
Field of Study: Eco-physiology
What is Eco-Physiology? Eco-physiology is the study of the relationship between the body and the environment. In my research, I study how temperature affects the way turtles develop in the egg.
Citation: Marroquín-Flores, R.A., Paitz, R.T and Bowden, R.M., 2022. Temperature fluctuations and estrone sulfate affect gene expression via different mechanisms in a species with temperature-dependent sex determination. Journal of Experimental Biology, 225(16), p. jeb244211.
Research At A Glance: Living things are closely connected to the world around them. External conditions like the environment, light, or water availability can affect how animals develop. At the same time, internal conditions like hormones or metabolism, can also affect how animals develop. In my research, we focused on how temperature influences development in a species of turtle. Animals that have temperature-dependent sex determination (TSD) will develop as male or female based on temperature. For our research, we studied the red-eared slider turtle, a species of reptile with TSD. In the red eared slider turtle, warm temperatures of about 31°C will result in females and cool temperatures of about 26°C will result in males. Temperature can lead to male or female development when some genes are turned on and off. In previous studies, researchers have found two genes that are important for regulating male development, Kdm6b and Dmrt1. Kdm6b is a gene that is sensitive to temperature and is responsible for turning on the Dmrt1 gene. Dmrt1 is responsible for activating the male pathway by turning on other genes that help the turtle to develop as male. When turtle eggs are exposed to cool temperatures, Kdm6b and Dmrt1 are turned on, but when eggs are exposed to warm temperatures, they are turned off. While temperature is important, hormones also play an important role in development for the red-eared slider turtle. Mother turtles will leave female hormones, like estrogens, in the eggs when they are laid. Eggs that are laid later in the nesting season have more estrogens and are more likely to develop as female. Previous studies show that estrogen can affect which genes are turned on or off. In this study, we explore how temperature and a specialized form of estrogen, called estrone sulfate, effect genes and development in the red-eared slider turtle.
Highlights: For this research, we placed turtle eggs in a temperature-programmed incubator to mimic the temperature that turtle eggs experience in nature. In the wild, temperatures go up during the day and down at night, so we programmed our incubator to do the same. Our goal was to understand how genes respond to these temperature changes. We started with three experimental treatments. In the first treatment, we initially incubated eggs using cool temperatures, then shifted the eggs to warm temperatures during the window of development that turtles are most sensitive to temperature. This treatment was designed to create females. In the second treatment, we incubated the eggs using cool temperatures and added our hormone, estrone sulfate, to the eggs to create females. In the third treatment, we incubated eggs using only cool temperatures to create males. The full experimental design is pictured below in Figure 1.
We sampled eggs at several points. We sampled during the window of development that turtles are most sensitive to temperature. The time that we sampled was critical because this is when we expected to see a change in which genes were turned on. We also let some of the eggs hatch to make sure that our experimental treatments worked. We wanted to make sure that our warm temperature treatment led to females, that our estrone sulfate treatment led to females, and that our cool temperature treatment led to males.
At the end of the experiment, we found that our treatments were effective. Eggs that were incubated under warm temperatures or treated with estrone sulfate hatched as female and eggs that were incubated under cool temperatures hatched as male. The results for the Kdm6b gene are outlined in Figure 2. Kdm6b responded to temperatures in the way that we expected. As you can see in Figure 2, there was more Kdm6b in the eggs when they were incubated under cool temperatures and less when they were under warm temperatures. However, Kdm6b did not respond to estrone sulfate as we expected. The Kdm6b gene was still turned on in the estrone sulfate treatment. Figure 2 shows Kdm6b expression looked the same between eggs incubated under cool temperatures and those that had estrone sulfate added to the eggs. This was an interesting result because Kdm6b is a male gene, but it was turned on even though the turtles were hatching as female. These findings suggest that Kdm6b responds specifically to cool temperatures.
The results for the Dmrt1 gene are outlined in Figure 3. Dmrt1 responded to temperature the way that we expected. Dmrt1 tends to turn on later during the window of development that turtles are most sensitive to temperature. In Figure 3, there is more Dmrt1 in eggs that are incubated under cool, male temperatures and this increase happens later in development, near our last sampling point. There is also less Dmrt1 in eggs incubated under warm, female temperatures. However, Dmrt1 did not respond to estrone sulfate the way we expected. Interestingly, the amount of Dmrt1 was low in eggs that had estrone sulfate added to them, and even lower than eggs that were incubated under warm temperatures. These results suggest that Dmrt1 responded more strongly to our female hormone than our female temperature. Recall that Kdm6b is responsible for turning on Dmrt1. Our results suggest that estrogens can override the effect of Kdm6b on Dmrt1 and lead to female development.
What My Science Looks Like: The steps involved in our research are pictured in the image below. To sample the eggs, we dissected bipotential gonads from undeveloped turtles. Bipotential gonads are tissues that will later develop into either ovaries or testes. They are located on top of another tissue that will later become the kidney. Even though they have not yet become ovaries or testes, the bipotential gonads will still turn on genes that show if the turtle is on the path towards male or female development. After dissection, we isolate the DNA from the tissue. We can then target our genes of interest using small pieces of DNA, called primers. Primers will only bind to strands of DNA that match the gene of interest. Once this step is done, we use a lab technique called Quantitative polymerase chain reaction (qPCR) which makes many copies of the gene of interest and counts how much of the gene is in a sample. qPCR is accomplished with the help of computers. The samples are prepared using a solution that contains an ingredient that will glow each time a new copy of the gene is made. A computer then will count the number of times the light is emitted to count how many copies of the gene exist in the sample. The computer continues to make copies of the gene until it reaches a certain threshold. If it takes a long time to reach the threshold, that means that there is a smaller amount of the gene in the sample. If it takes a short amount of time to reach the threshold, that means that there is a larger amount of the gene found in the sample. This method is what allowed us to figure out how much of the Kdm6b and Dmrt1 gene was present in the bipotential gonads of turtles in each treatment.
The Big Picture: As a result of climate change, the environment is becoming unpredictable, and the earth is becoming warmer. We are expected to experience heat waves that are longer lasting and of a higher intensity. For many animals, development and survival are closely tied to the environment. That is why it’s crucial to understand how external conditions, like temperature, and internal conditions, like hormone signaling, can affect development. I used the red-eared slider turtle because it has a common form of sex determination. While our research focused on the red eared slider turtle, our findings can be also applied to other reptiles, including sea turtles, which are critically endangered. Our research is also important because of the type of hormone that we used in our research. Estrogen comes in many forms. One of these forms, estrone sulfate, is often considered an inactive form of estrogen. Our findings counter this theory. Our findings suggest that estrone sulfate was able to affect Dmrt1, an important gene in the male pathway. Estrone sulfate was also able to override the effect of male temperatures to induce female development in turtles. Our research opens the door for many exciting opportunities and work to be done with the hormone, estrone sulfate.
Decoding the Language:
Autonomy: Autonomy is the ability to act on your own values and interests. It is having the power to make your own decisions independently and do the things that are important to you.
Bipotential gonads: Bipotential gonads are tissues that can be found inside an embryo, or an undeveloped animal. The bipotential gonads are still early in development and will go on to become either the ovaries or the testes when the animal finishes developing. In red eared turtles, the bipotential gonads are located on top of a tissue that will later become the kidney. Adult ovaries will produce female hormones, like estrogen, and adult testes will produce male hormones, like testosterone.
Climate change: Climate change are shifts and changes in global weather patterns that happen over a long period of time. It refers to the different increasing global temperatures like drought and rising temperatures that cause heat waves. Humans use of fossil fuels that release greenhouse gasses into the atmosphere, which contributes to climate change.
Data analysis: Data analysis involves examining and comparing data (in this case, the amount of the Kdm6b in turtles) across groups (known as “treatments”) in an experiment. Data analysis is important to draw accurate and meaningful conclusions after an experiment.
Deoxyribonucleic acid (DNA): DNA is a molecule that’s like a blueprint of the body. It contains all the instructions that the body needs to grow, develop, and function.
Doublesex and mab-3 related transcription factor 1 (Dmrt1): Dmrt1 is a gene that is important for male development in many species. It helps male reproductive organs grow. In turtles, Dmrt1 is turned on by Kdm6b. If Dmrt1 is intentionally turned off by researchers, then the turtle will develop as female.
Estrogen: Estrogen is a female hormone that is produced by the ovaries. It can exist in three forms: estradiol, estrone, and estriol. In this study, we looked at the effects of one type of estrogen, estrone-sulfate.
Estrone sulfate: Estrone sulfate is the combination of estrone, a form of estrogen, and a sulfate group. The conjugated form of this estrogen (estrone + a sulfate group) is often considered to be inactive. However, our findings show that estrone sulfate can make changes in the body.
Experimental control: An experimental control is a technique used to minimize, or “control”, unintended effects of a particular treatment. In this research, estrone sulfate was dissolved in ethanol and applied to eggs in the hormone treatment. This means that eggs in the hormone treatment were exposed to both estrone sulfate and ethanol. To ensure that the only differences between eggs in the hormone treatment and those in the other treatments was due to hormones, we applied ethanol to eggs in the other treatment groups as a control.
Gene: A gene contains the Information that will determine your traits and characteristics and it also contains the instructions that your body needs to make proteins.
Hatchling: A hatchling is any animal that was recently born from an egg.
Hormone: A hormone is a chemical messenger that travels through the blood to induce changes in the body. There are many types of hormones. For example, testosterone is a male hormone that is produced by the testes and leads to the development of masculine features, like a beard. Estrogen is a female hormone produced by the ovaries and leads to feminine features. Cortisol is a stress hormone that is released during our fight or flight response.
Histone H3 lysine 27 (H3K27) demethylase (Kdm6b): Kdm6b is a gene that is important for male development in TSD species. When turtles are incubated under cool, male temperatures, the amount of Kdm6b increases. If turtles are incubated under male temperatures, but Kdm6b is intentionally turned off by researchers, then the turtle will develop as female. Kdm6b is important because it responds directly to temperature, and it turns on another gene that is critical for male development.
Primers: Primers are short pieces of DNA that can be specifically designed in the lab to match a small section of a gene. This allows researchers to target the specific gene of interest from a large pool of DNA that contains many genes.
Quantitative polymerase chain reaction (qPCR): qPCR is a laboratory technique that is similar to Polymerase chain reaction (PCR) in that it involves making many copies of DNA. The only difference is that qPCR takes this one step further by counting how much of a specific gene is in a sample. It involves making several copies of a gene until it can be detected by a computer, then counting how much is there.
Temperature-dependent sex determination (TSD): TSD is a special form of sex determination where temperature will determine whether an animal will develop either as a male or female. For some animals, like the red-eared slider turtle, warm temperatures create females and cool temperatures create males. For other animals, it’s the exact opposite. Animals, like the American alligator, have males that are produced at high and low temperatures, but females are produced at intermediate temperatures.
A YouTube video that describes how climate change can affect animal species
A brief article and YouTube video from the University of Wisconsin – Eau Clair on estrogen pollution in water systems
A YouTube video that describes how Kdm6b and Dmrt1 regulate sex determination
A YouTube Video that explains how PCR and qPCR work.
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