Soon after the organizational hypothesis was published, researchers reported a paradoxical finding: when newborn female rats were treated with estrogens, they failed to show lordosis behavior in adulthood (Feder and Whalen, 1965). Researchers were very puzzled to find that neonatal treatment with a very small dose of estradiol, regarded at that time as a female hormone, could permanently masculinize these behaviors. The results were especially strange because during development, all rat fetuses are exposed to high levels of estrogens that originate in the mother and cross the placenta. If estrogens masculinize the developing brain, why aren’t all females masculinized by maternal estrogens?

A closer look at the synthesis of steroid hormones reveals the answer. Testosterone and estradiol molecules are very closely related in structure. In fact, testosterone is the precursor for the manufacture of estradiol in the ovary. In a single chemical reaction, called aromatization, the enzyme aromatase converts testosterone to estradiol (and other androgens to other estrogens). The ovaries normally contain a great deal of aromatase, and the brain was found to have high levels of aromatase as well. From this evidence arose the aromatization hypothesis, which suggests that testicular androgens enter the brain and are converted there into estrogens, and that these estrogens are what masculinize the developing rodent nervous system.

Why, then, aren’t the brains of females masculinized by maternal estrogens? A blood protein called alpha-fetoprotein binds estrogens and prevents them from entering the brain (Bakker et al., 2006). Although both male and female fetuses produce alpha-fetoprotein, this protein does not bind testosterone. The male rat’s brain is masculinized when testosterone from his testes is conveyed by the bloodstream (unimpeded by alpha-fetoprotein) to his brain, where it is aromatized to an estrogen within individual neurons. This newly synthesized estrogen binds to local estrogen receptors, and the steroid-receptor complex regulates gene expression to cause the brain to develop in a masculine fashion. If no androgens are present, there can be no estrogenic action in the brain (because alpha-fetoprotein has blocked estrogens of peripheral origin), so the fetus develops in a feminine fashion. A lack of aromatase seems to play a role in the unusual sexual differentiation of the spotted hyena, as described in A Step Further 8.6.

You may wonder why, if alpha-fetoprotein binds up estrogens, injecting rat pups with estrogen affects lordosis. The answer is that estrogen injections quickly flood the bloodstream with hormone molecules, saturating the alpha-fetoprotein, allowing many estrogen molecules to enter the brain.

The aromatization hypothesis was soon shown to be fully applicable to masculine copulatory behavior in rats. If a male rat was castrated at birth, it grew up to have a small penis and show few intromissions, even when given replacement testosterone in adulthood. If a male was castrated at birth and given the androgen dihydrotestosterone (DHT), which cannot be aromatized into an estrogen, it grew up to have a penis of normal size but still showed few or no intromissions when given testosterone. On the other hand, males castrated and treated with estrogens as newborns were able to achieve intromissions regularly when treated with androgens as adults, despite having very small penises (no larger than in untreated castrated males). Thus, it is hormonal masculinization of the brain, not the genitalia, that organizes male rat copulatory behavior.

In primates, including humans, aromatization does not seem to play an important role in masculinization of the nervous system (Grumbach and Auchus, 1999). The human brain produces significant quantities of aromatase, but men who have mutations in the aromatase gene—and are thus unable to produce aromatase—nonetheless have masculine gender identities and sexual development. Estrogen resistance caused by mutations in the gene encoding the alpha estrogen receptor (one of the two known isoforms of estrogen receptors) similarly does not affect the development of masculine gender behavior in men. Finally, recall that people with AIS display feminine behavior, even though they produce lots of testosterone and have functional estrogen receptors. The details of the masculinization of the primate nervous system remain to be worked out, but hormonal masculinization must be accomplished through the androgen receptor rather than the estrogen receptor. Whichever specific steroid receptor is involved, many vertebrate species display distinct sex differences in the brain.

References

Bakker, J., De Mees, C., Douhard, Q., Balthazart, J., et al. (2006). Alpha-fetoprotein protects the developing female mouse brain from masculinization and defeminization by estrogens. Nature Neuroscience 9: 220–226.

Feder, H. H., and Whalen, R. E. (1965). Feminine behavior in neonatally castrated and estrogen-treated male rats. Science 147: 306–307.

Grumbach, M. M., and Auchus, R. J. (1999). Estrogen: Consequences and implications of human mutations in synthesis and action. Journal of Clinical Endocrinology and Metabolism 84: 4677–4694.