Material below is adapted from the SfN Short Course session How to Study the Origins of Sex Differences in Brain and Behavior, by Margaret M. McCarthy. Short Courses are daylong scientific trainings on emerging neuroscience topics and research techniques held the day before the start of SfN’s annual meeting.
Scientists have known for nearly 60 years that the hormones produced by the endocrine system influence fetal brain development and subsequent adult behavior. Yet only now are researchers beginning to gain a greater understanding of how neuroscience and those hormones interact in both male and female animals.
Sex is largely determined by an organism’s chromosomes. Mammals use an XY chromosome system in which biological females have two X chromosomes and biological males have both an X and Y, which carries the Sry gene that directs the development of the testes, the male gonads or sex glands. The testes arise early during fetal development and quickly start producing endocrine hormones that influence the growth and development of other sex organs and the masculinization or sexual differentiation of the brain.
Brain development happens in stages, and the brain is sensitive to hormones at particular times. The male brain is unique in that there is a period during which it must be exposed to male sex hormones. In contrast, the female brain has a sensitive period right after birth during which exposure to male sex hormones can masculinize the brain and generate characteristics similar to those observed in biological males. This ability to nudge females toward having more masculinized brains is a useful tool for researchers who want to understand sex differences, but they must reconcile the difference in age between males, whose brains receive these signals in utero, and females, who receive them after birth and thus are several days older.
Scientists also know that estradiol, which was traditionally considered a female hormone, actually works as a masculinizing hormone in the brain. Testosterone exerts at least some of its effects after neurons convert it to estradiol: When researchers block estrogen activity in newborn males, they also block masculinization of the brain. Testosterone and estrogen are each processed differently by neurons in different parts of the brain, and the two hormones can act either independently or together to control different developmental outcomes for the central nervous system.
Two tools are available for studying the influence of hormones on sexual differentiation of the brain: genetic mouse models and steroid hormone administration. One example of a genetic tool is a mouse that lacks the hormone that converts testosterone to estradiol. The problem with using a genetic model, however, is that if a gene is inactivated early in development, it can be difficult to distinguish its early effects from later ones. Steroids, on the other hand, while easy to administer as early as the newborn stage, can be difficult to dose and may need cofactors and receptors to function properly.
To manipulate sex differences in the brain and behavior, scientists must know whether they are dealing with male or female animals. It is possible to distinguish the sexes in mice and rats early by observation of the distance between the anus and the urethra, or by abdominal dissection to ascertain the presence of testes. Researchers also use some genotyping strategies to determine sex early on.
It is possible to add in extra steroid hormones as well as to block an animal’s natural steroid hormones. Adding in extra hormones is generally done by subcutaneous injection into newborns. Researchers pinpoint specific hormonal effects by using appropriate controls, such as a testosterone analog that cannot be converted to estradiol.
Because hormonal effects start in utero and continue after birth, blocking hormones to affect sex differences in the brain can be difficult. In this case, scientists can use chemical tools that either block the estrogen receptor or disrupt the hormone that converts testosterone to estradiol. Blocking testosterone synthesis or receptors is more difficult because fewer reliable tools are available.
Masculinization of the brain is a more straightforward target for investigation because it is an active process that relies on the presence of the testes and testosterone. Brain feminization is the default that happens without testes, but it, too, is an active process, only less well understood. Researchers have attempted to understand feminization in the brain by studying defeminization — primarily by removing brain circuitry specific to female sexual behavior.
So far, this discussion has focused on internal factors — genes and hormones — that affect sexual differentiation in the central nervous system, but the environment also plays a role. For instance, mouse mothers treat male and female offspring differently. They retrieve lost male pups more quickly, which likely leads to less stress for the pups, and perform more urogenital licking, which encourages motor neuron growth in the penis and enhances the pups’ reproductive function as adults. Stress that the mother mouse experiences during pregnancy also affects the brains of male pups in ways that they may even be pass to their offspring, without affecting those of female pups.