Understanding Sex Differences

Across the animal kingdom, most biological males and females are sexually dimorphic. That means they differ in physical and behavioral characteristics due to their biological sex. These differences can occur through the influence of sex hormones like estrogen and testosterone. These hormones influence the brain through receptors on neurons, which are the cells in your brain that carry information. Think of receptors as antennas that neurons use to receive nearby information. These receptors--estrogen receptor alpha (ER⍺) for estrogen, and androgen receptor for testosterone--can have a lot of unique impacts on neurons and the brain. 

Receptors can change how neurons respond to certain informational signals. These receptors are found in different amounts between males and females. Understanding how these receptors impact neurons can help us to uncover how neurons differ between males and females. Since neurons and their functions are so important for the brain, this type of study can inform us on how to treat certain brain diseases that affect males and females at different rates. For example, autism spectrum disorder and Parkinson’s disease impact more males than females while multiple sclerosis and Alzheimer’s disease impacts more females than males. 

Neuroscientists around the world have looked for populations of neurons with different levels of these sex hormone receptors, estrogen and testosterone receptors, between male and female mice. They first worked to identify different brain regions that are involved in behaviors that are different between males and females, such as mating behavior and aggression. They identified brain regions like the bed nucleus of the stria terminalis (BNST), medial amygdala (MeA), preoptic hypothalamus (POA), and ventromedial hypothalamus ventrolateralis (VMHvl) and found that they are heavily involved in mating and aggression behaviors. These brain regions are part of the limbic system, which influences important internal states like hunger, thirst, sleep, and arousal. This led us to believe that studying the level of receptors, such as ER⍺, in these brain regions could tell us how sex hormones, like estrogen which works through ER⍺, could influence behaviors that differ between males and females. 

We first wanted to know what genes make up the neurons that have estrogen receptors, such as ER⍺. To do this, we looked at RNA. RNA is a special molecule that allows our genes to turn into proteins which are essential for our body’s functions. RNA also enables scientists to figure out what genes and proteins are present in a certain location. RNA sequencing is a special technique that can be used to analyze the RNA from neurons that have a special protein of interest. We used RNA sequencing to look at neurons that have estrogen receptor alpha (ER⍺) to see how these neurons are different between male and female mice. 

When comparing biological males and females, it is important to consider fluctuating sex hormones, as these hormones may impact behavior and the levels of genes and proteins in the brain. Like in the human menstrual cycle, female mice have a cycle of fluctuations in the hormones estrogen and progesterone. There is an estrus stage where estrogen and progesterone rise at certain times, and there is a diestrus stage where both of these hormones are low. To control what hormonal state the females were in, we removed their ovaries and supplied them with estrogen and progesterone in specific ways to mimic their cycle. On the other hand, male mice do not have fluctuating hormones that need to be controlled for. We then performed RNA sequencing on estrus and diestrus females mice as well as male mice to see which genes are found in ER⍺ neurons of the limbic brain regions. 

In each brain region, we found between 29 and 649 genes that were present at different levels between estrus females and males, or between diestrus females and males. This wide range in gene number is due to some limbic brain regions being more different between sexes than others. We found some genes related to autism spectrum disorder are present at higher levels in males than in females. Since autism spectrum disorder impacts males and females differently -- affecting 4 males for every 1 female-- understanding how these male-specific genes impact behaviors may lead to insights in autism research.

We next looked to see if there were genes in limbic brain regions that were present at different levels between estrus and diestrus females. We found between 92 and 301 genes present at different levels between estrus and diestrus females. We saw that some genes were found in the brains of estrus females but not diestrus females, and vice versa. Since estrogen and progesterone levels are different during estrus and diestrus, this finding might allow us to look at how genes change with the presence or absence of estrogen and progesterone. This may be relevant to understanding changes in the brain that occur with menopause, specifically the arrival of hot flashes and changes in sex drive. Further investigation of how these genes change with estrogen and progesterone could lead to more targeted therapies to treat menopause symptoms. 

We next calculated how many genes were present differently across brain regions. We specifically looked at brain regions with high levels of estrogen and testosterone receptors. A substantial number of these genes were different between males and estrus and diestrus females in a brain region known as the bed nucleus of the stria terminalis (BNST). On the other hand, a brain region known as the ventromedial hypothalamus ventrolateralis (VMHvl) had more genes that were different between estrus and diestrus females than between males and females. This suggests that the effects of biological sex or estrus cycle hormones occur in specific brain regions. 

We next looked at the genes that individual neurons have in order to uncover the relationships between a neuron’s genetic makeup and the neuron’s function. We found neurons in the BNST that have a male-specific gene called Tac1. This gene is responsible for mating and aggression. In females, we found neurons in the VMHv1 that have a gene called Cckar. This gene is responsible for mating. 

The brain regions we looked at are also connected to other regions. What is interesting is that, during a female's estrus cycle, these connections strengthen and weaken throughout the cycle. Mating behavior also changes throughout the estrus cycle. It is possible that these neurons, since they have sex hormone receptors and genes related to mating behavior, could be involved. Maybe these neurons are the link between the estrus cycle and mating behavior. Though we need further research to confirm this, these findings point to the information that can be gathered from identifying the genetic makeup of neurons involved in important behaviors such as mating. 

The goal of this work was to define neuron populations in the brain that are different between males and females. We also wanted to see if estrogen receptor neuron populations have different genes depending on whether estrogen is present. In doing this, it is important to consider a few drawbacks. First, controlling the estrus cycle by removing the ovaries also removes other hormones that the ovaries release during the estrus cycle. These other hormones may influence the brain and behavior, so results seen in mice without ovaries may not reflect what happens in mice with a natural estrus cycle. Second, ER⍺ is found in brain regions outside the limbic system so this study does not capture all the neurons that could be influenced by estrogen. Nevertheless, these findings of sex and estrogen-dependent differences in the brain have important implications for understanding diseases that show differences between sexes. 

We can take this work further by asking whether ER⍺-containing neurons in the limbic system alter the genes they have when sex hormones are switched or completely absent. For example, what changes in the brain when testosterone is given to a biological female, or estrogen is given to a biological male? Understanding these changes will enable medicine to optimize hormone replacement therapies for the transgender and intersex communities. This study also benefits the cisgender community, as animal studies and clinical trials historically excluded females due to the inconvenience of accounting for the estrus cycle. This led to the production of treatments that worked for males but not females. With an understanding of what sex differences exist in the brain and how estrogen impacts neurons, researchers can now find ways to accommodate females, both human and animal, into clinical trials. This will increase the number of treatments that work for both males and females. 

Written By: Adarsh Tantry

Academic Editor: Neurobiologist 

Non-Academic Editor: Corporate Lawyer

Original Paper

• Title: A functional cellular framework for sex and estrous cycle-dependent gene expression and behavior

• Journal: Cell 

• Date Published: 17 February 2022

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