Sexual differentiation of the nervous system: where the action is.

Almost everyone would agree that gonadal steroid hormones cause sex differences in the nervous system. Testosterone, acting via androgenic or estrogenic metabolites, alters the form and function of the developing brain. What is not so clear, however, is exactly where androgens and estrogens act to cause neural sex differences. Consider the dilemma: most brain regions are composed of multiple cell types, several of which may express androgen or estrogen receptors. Each region receives input from dozens (if not hundreds) of sites and in turn projects to dozens more; many of these afferent and efferent cell groups are also likely to express the relevant receptors. Thus, testosterone may cause a given sex difference by acting at the sexually dimorphic cells themselves, at afferents or target cells or even at neighboring glial cells. Given this complexity, the problem of identifying the crucial site(s) of hormone action becomes almost insurmountable. Ideally what you would like is to examine the effects on sexual differentiation of expressing the relevant steroid receptor in only a single location. This is in effect what Niel et al. (1) have done by taking advantage of a simple, sexually dimorphic neuromuscular system that has been a focus of research in sexual differentiation for almost 30 years. The spinal nucleus of the bulbocavernosus (SNB) is a cluster of motoneurons in the lumbar spinal cord that innervates striated muscles in the perineum (2). Males of many mammalian species have more of these neurons than do females, and the target muscles of SNB neurons are absent or vestigial in females (reviewed in Ref. 3). A substantial amount of previous work set the stage for the current report. It is known, for example, that SNB motoneurons and perineal muscles initially form in female rats and mice but then degenerate around the time of birth (4,5,6). The SNB system is permanently masculinized in females treated with testosterone perinatally and is completely female-like in Tfm males with an inactivating mutation of the androgen receptor gene (7,8,9). Thus, testosterone acts via androgen receptors to prevent the degeneration of SNB motoneurons and their target muscles in males. But androgen receptors where? The original paper by Breedlove and Arnold (2) that reported the sex difference in the SNB was exciting, in part, because androgen receptors were found in SNB motoneurons of adult rats. However, SNB cells do not express the receptors during the perinatal period when androgens can save the system (10,11). In addition, Breedlove and colleagues eliminated the brain as a necessary site of hormone action by showing that testosterone could rescue SNB motoneurons in spinally transected newborn female rats (12). As evidence accumulated, it appeared that in fact androgens act at the perineal muscles: the muscles express androgen receptors during the critical period for sexual differentiation of this system (10,13), and they can be rescued by testosterone even if the entire lumbosacral spinal cord (and thus neural input) is removed (14). In addition, the masculinizing effect of testosterone on SNB motoneuron number can be blocked by antiandrogen administered to the muscle at a dose that is ineffective when given systemically (15). More evidence for testosterone’s site of action came from work with female carriers of the Tfm mutation. These animals have the wild-type androgen receptor on one X chromosome and the inactive Tfm receptor on the other X. Due to the process of random X inactivation [in which one X chromosome in each cell is silenced during early development (16)], about half of the cells in Tfm carrier females are androgen sensitive, whereas the others are not. Freeman et al. (17) found that androgens could rescue SNB motoneurons irrespective of whether they expressed the functional androgen receptor. This confirmed that the SNB cells are not the site of hormone action for masculinization of this system. Because the perineal muscles in the Tfm carrier females contained at least some wild-type androgen receptor, the muscles were identified as the likely site of hormone action (17) and the question seemed resolved, more or less. Niel et al. (1), however, now report on a new animal model designed to critically test the hypothesis that hormone action solely at androgen receptors in perineal muscle fibers is sufficient to masculinize the SNB. To do this, they crossed female rats carrying the Tfm mutation with transgenic males expressing the human androgen receptor gene driven by a skeletal muscle-specific promoter. Some of the male offspring of this cross should have the nonfunctional Tfm androgen receptor in all tissues except for striated muscle fibers, in which both the Tfm and the wild-type human androgen receptor would be expressed. The authors first validate the model by confirming that muscle fibers, and only muscle fibers, express the wild-type human androgen receptor in Tfm males bearing the transgene. They then compare the SNB neuromuscular system in wild-type males, wild-type females, Tfm males, and Tfm males expressing the androgen receptor transgene. Contrary to expectation, both perineal muscle size and SNB motoneuron number were completely feminine in the Tfm males, regardless of whether the muscle fibers had the functional androgen receptor transgene (Fig. 1​1).). This suggests that expression of androgen receptors in muscle fibers is not sufficient to rescue the SNB system. The authors considered the possibility that the transgenic males might not produce enough androgens to masculinize the SNB. If so, then the negative finding could be dismissed on the basis of lack of androgen stimulation. This was ruled out, however, by treating rats with testosterone perinatally. Whereas the testosterone treatment completely masculinized the SNB of wild-type females, it had no effect on the SNB system of males expressing functional androgen receptors only in muscle fibers (1). The work is doubly satisfying in that it uses a fresh approach to address a long-standing question and comes up with a surprising answer. Figure 1 SNB motoneurons are shown innervating striated perineal muscles that attach to the base of the penis. The survival of this neuromuscular system is dependent on androgens. Left, In the study by Niel et al. (1), the expression of functional androgen receptors ... As with any study, there are a few caveats. The authors show functionality of the human androgen receptor by demonstrating that testosterone increases myoglobin expression in a leg muscle of adults bearing the transgene. Tfm animals expressing the human androgen receptor in muscle fibers also have increased overall lean body mass (1), consistent with transgene functionality. It is not known, however, whether the transgenic receptor is completely normal in terms of its ability to regulate gene transcription, in particular within the perineal muscles during early development. Indeed, it would be difficult to assess this because the crucial gene targets for testosterone action in the perineal muscles are not known. Overall, however, the findings are convincing and force a reevaluation of previous work that seemed to point to muscle fibers as the site of hormone action in masculinization of SNB neuron number. It remains possible that another cell type within the perineal muscles is the critical site of action. For example, both satellite cells and fibroblasts in muscle tissue express androgen receptors (18). The perineal region also contains adipocytes, endothelial cells, epithelial cells, and Schwann cells. Although these might seem like odd places to look for the source of neural sex differences, each must now be considered. It remains possible, of course, that androgens act at multiple cell types, including perineal muscle fibers, to masculinize the SNB system, but one of these sites is not likely to be the SNB motoneurons, as described above. Regardless of how the story ends, the new findings should give us all pause. Even in this simple neuromuscular system, identifying the site of hormone action has turned out to be anything but simple. Work on the site of hormone action in the brain has largely been limited to describing where particular hormone receptors are expressed. The study by Niel et al. (1) suggests that we may be in for some surprises when hypotheses about sites of hormone action are put to the test.