Cheryl Rosenfeld

Seminal work

How unruly data led MU scientists to discover a new microbiome
By Roger Meissen | MU Bond Life Sciences Center

seminal vesicles 3_11_16.jpg

This seminal vesicle contains a newly-discovered microbiome in mice. Some of its bacteria, like P. acnes, could lead to higher occurrences of prostate cancer. | contributed by Cheryl Rosenfeld

It’s a strange place to call home, but seminal fluid offers the perfect environment for particular types of bacteria.

Researchers at MU’s Bond Life Sciences Center recently identified new bacteria that thrive here.

Cheryl Rosenfeld1.jpg “It’s a new microbiome that hasn’t been looked at before,” said Cheryl Rosenfeld, a Bond LSC investigator and corresponding author on the study. “Resident bacteria can help us or be harmful, but one we found called P. acnes is a very important from the standpoint of men. It can cause chronic prostatitis that results in prostate cancer. We’re speculating that the seminal vesicles could be a reservoir for this bacteria and when it spreads it can cause disease.”

Experiments published in Scientific Reportsa journal published by Nature — indicate these bacteria may start disease leading to prostate cancer in mice and could pass from father to offspring.


A place to call home

From the gut to the skin and everywhere in between, bacterial colonies can both help and hurt the animals or humans they live in.

Seminal fluid offers an attractive microbiome — a niche environment where specific bacteria flourish and impact their hosts. Not only is this component of semen chockfull of sugars that bacteria eat, it offers a warm, protected atmosphere.

“Imagine a pond where bacteria live — it’s wet it’s warm and there’s food there — that’s what this is, except it’s inside your body,” said Rosenfeld. “Depending on where they live, these bacteria can influence our cells, produce hormones that replicate our own hormones, but can also consume our sugars and metabolize them or even cause disease.”

Rosenfeld’s team wasn’t trying to find the perfect vacation spot for a family of bacteria. They initially wanted to know what bacteria in seminal fluid might mean for offspring of the mice they studied.

“We were looking at the epigenetic effects — the impact the father has on the offspring’s disease risk — but what we saw in the data led us to focus more on the effects this bacterium, P. acnes, has on the male itself,” Rosenfeld said. “We were thinking more about effect on offspring and female reproduction — we weren’t even considering the effect the bacteria that live in this fluid could have on the male — but this could be one of the more fascinating findings.”

But, how do you figure out what might live in this unique ecosystem and whether it’s harmful?

First, her team found a way to extract seminal fluid without contamination from potential bacteria in the urinary tract.

“We gowned up just like for surgery and we had to extract the fluid directly from the seminal vesicles to avoid contamination,” said Angela Javurek, primary author on the study and recent MU graduate. “You only have a certain amount of time to collect the fluid because it hardens like glue.”

Once they obtained these samples, they turned to a DNA approach, sequencing it using MU’s DNA Core.

They compared it to bacteria in fecal samples of the same mice to see if bacteria in seminal fluid were unique. They also compared samples from normal mice and ones where estrogen receptor genes were removed.


The difference in the data

It sounds daunting to sort and compare millions of DNA sequences, right? But, the right approach can make all the difference.

“A lot of it looks pretty boring, but bioinformatics allow us to decipher large amounts of data that can otherwise be almost incomprehensible,” said Scott Givan, the associate director of the Informatics Research Core Facility (IRCF) that specializes in complicated analysis of data. “Here we compared seminal fluid bacterial DNA samples to publicly available databases that come from other large experiments and found a few sequences that no one else has discovered or at least characterized, so we’re in completely new territory.”

The seminal microbiome continued to stand out when compared to mouse poop, revealing 593 unique bacteria.

One of the most important was P. acnes, a bacteria known to cause chronic prostatitis that can lead to prostate cancer in man and mouse. It was abundant in the seminal fluid, and even more so when estrogen receptor genes were present.

“We’re essentially doing a lot of counting, especially across treatments to see if particular bacteria species are more common than others,” said Bill Spollen, a lead bioinformatics analyst at the IRCF. “The premise is that the more abundant a species is, the more often we’ll see its DNA sequence and we can start making some inferences to how it could be influencing its environment.”

Although this discovery excites Rosenfeld, much is unknown about how this new microbiome might affect males and their offspring.

“We do have this bacteria that can affect the male mouse’s health, that of his partner and his offspring,” Rosenfeld said. “But we’ve been studying microbiology for a long time and we still find bacteria within our own bodies that nobody has seen before. That blows my mind.”

The study, “Discovery of a Novel Seminal Fluid Microbiome and Influence of Estrogen Receptor Alpha Genetic Status,” recently was published in Scientific Reports, a journal published by Nature.

 

BPA overrides temperature to decide turtle sex

The environmental build-up of bisphenol A (BPA) can result in a life-changing shift for aquatic animals.

For painted turtles, exposure to this chemical can disrupt sexual differentiation,, according to new research in the  General and Comparative Endocrinology.

Scientists at the University of Missouri have teamed up to show how low levels of certain endocrine disruptors like BPA can cause males to possess female gonadal structures in newly-hatched turtles. This collaboration between MU, Westminster College, the U.S. Geological Survey (USGS) and the Saint Louis Zoo exposed turtle eggs to levels of BPA similar to those currently found in the environment.

“It’s important because this is one of the first times we’ve seen low doses of BPA causing disorganization or reorganization of the male gonad to resemble females,” said Dawn Holliday, adjunct assistant professor of pathology & anatomical sciences at MU’s School of Medicine and assistant professor of biology at Westminster College. “We’re not sure what this means in terms of population-level effects, but certainly it can cause some reproductive dysfunction for turtles.”

Painted turtle eggs were brought from a hatchery in Louisiana, candled to ensure embryo viability and then incubated at male-permissive temperatures in a bed of vermiculite. Those exposed to BPA developed deformities to testes that held female characteristics.  Photo by Roger Meissen | © 2015 - MU Bond Life Sciences Center

Painted turtle eggs were brought from a hatchery in Louisiana, candled to ensure embryo viability and then incubated at male-permissive temperatures in a bed of vermiculite. Those exposed to BPA developed deformities to testes that held female characteristics.
Photo by Roger Meissen | © 2015 – MU Bond Life Sciences Center

Endocrine disruptors leach into rivers and streams and concern scientists because of potential effects on animals and humans. While BPA is used as a hardening agent in plastics, it also is used to line cans and in manufacturing where more than 15 billion tons are produced each year.

In the case of painted turtles, these chemicals have potential to alter sex ratios, which are normally regulated by temperature during incubation. Eggs exposed to cooler temperatures normally produce males and those hatched at warmer temperatures produce females.

Turtle eggs incubated at cooler temperatures result in male hatchlings while warmer temperatures cause females. Researchers are measuring the temperature and weight of this turtle. Photo by Roger Meissen | © 2015 - MU Bond Life Sciences Center

Turtle eggs incubated at cooler temperatures result in male hatchlings while warmer temperatures cause females. Researchers are measuring the temperature and weight of this turtle. Photo by Roger Meissen | © 2015 – MU Bond Life Sciences Center

In this experiment, turtle eggs were incubated at temperatures known to rear males and dosed with low, medium and high levels of BPA. BPA-exposed turtles were compared to hatchlings not exposed to chemicals as well as a group exposed to high levels of ethinyl estradiol — an endocrine disruptor found in birth control — at the USGS Columbia Environmental Research Center.

These doses resulted in turtle sex organs that should have been male , but abnormally contained female gonadal elements. The low dose represented BPA concentrations found in fields where turtles can nest while the mid and high doses approximate BPA levels near contaminated sites like landfills.

“We exposed the eggs for a limited amount of time right when they were most vulnerable to the effects,” said Cheryl Rosenfeld, a researcher at MU’s Bond Life Sciences Center and an associate professor of biomedical sciences in the College of Veterinary Medicine. “We found that we got partial feminization in more than 30 percent of turtle eggs exposed to BPA despite being incubated at male-permissive temperatures.”

Dawn Holliday (left), Caitlin Jandegian and Cheryl Rosenfeld examine turtle gonadal tissue to determine if BPA affected proper sexual development. Photo by Roger Meissen | © 2015 - MU Bond Life Sciences Center

Dawn Holliday (left), Caitlin Jandegian and Cheryl Rosenfeld examine turtle gonadal tissue to determine if BPA affected proper sexual development. Photo by Roger Meissen | © 2015 – MU Bond Life Sciences Center

These results give the team a look into what real-world exposure levels might mean in the wild and a starting point for comparison.

“Turtles are the most endangered vertebrate taxa and they have all sorts of conservation issues coming at them from people harvesting them to disease, and endocrine disruptors are another potentially big whammy they have against their conservation status,” said Sharon Deem, director of the Saint Louis Zoo’s Institute for Conservation Medicine. “This research is a stepping stone, and we are hoping we can apply these results to populations of turtles throughout the state and use these results as a marker to look at endocrine disruptors in the wild.”

Future studies plan to look at the underlying mechanisms behind sexual disruption and will extend the study to animals including fish and mammals. Rosenfeld’s laboratory is in the process of examining how early exposure of turtles to endocrine disruptors might affect cognitive behaviors, including spatial navigation ability.

Fred vom Saal, Curators Professor of Biological Sciences in the College of Arts and Science at MU, Don Tillitt, an adjunct professor of biological sciences at MU and a research toxicologist with the USGS, Ramji Bhandari, an assistant research professor of biological sciences and a visiting scientist with the USGS at MU and Caitlin Jandegian, a senior research technician at MU, all collaborated on the study.

Candling helps determine whether the painted turtle embryo is viable for the experiment. Photo by Roger Meissen | © 2015 - MU Bond Life Sciences Center

Candling helps determine whether the painted turtle embryo is viable for the experiment. Photo by Roger Meissen | © 2015 – MU Bond Life Sciences Center

Funding was provided by Mizzou Advantage, an MU initiative that fosters interdisciplinary collaboration among faculty, staff, students and external partners to solve real-world problems in four areas of strength identified at the University of Missouri. These areas include Food for the Future, Sustainable Energy, Media for the Future and One Health/One Medicine.