Whereas the evidence of estrogen-mediated sexual differentiation is irrefutable, the strength of this evidence may have inadvertantly prejudiced researchers against looking to other factors in the differentiation process. Consideration of the role of progesterone, as well as additional non-steroidal ovarian secretions, may provide additional insights into mechanisms of sexual differentiation. Further consideration of non-ovarian factors, such as the potential role of genes involved in peripheral sexual differentiation and the influence of changes in afferent input, would be equally valuable in elucidating the mechanisms of sexual differentiation.
2. Fitch and Denenberg (1995) point out that it has previously been elucidated by Sodersten that while it is clear there is some role for the ovary in female differentiation, "the nature and mechanisms of action of these ovarian secretions remains to be determined...". The emphasis is frequently on the notion that all ovarian secretions must be steroidal, and in general the focus is on estrogen. However, there are also numerous growth factors synthesized by the ovary in the adult, including vascular endothelial growth factor (VEG-F), basic fibroblast growth factor (b-FGF), epidermal growth factor (EGF) and insulin-like growth factor (IGF), to name a few. The potential for these growth factors to be synthesized by the neonatal ovary seems likely. Borrowing from an idea suggested to me by Dr. Barney Schlinger of UCLA, it's possible the developing gonads produce and secrete sufficient quantities of some growth factors that they can influence the developing brain. The lack of a mature blood-brain-barrier would allow ready access to the brain of even low levels of circulating growth factors. Additional factors that are produced in high quantities by the ovary include oxytocin, prostaglandins and inhibin. To the best of my knowledge there have been no investigations of potential influences of these ovarian secretions on the developing brain. Evidence in adult canaries reveals a role for non-estrogenic ovarian influences on the process of neurogenesis in sexually dimorphic brain regions (Hidalgo et al., 1995) and the extension of this mechanism into the developmental period seems likely.
3. An additional ovarian factor that is often not discussed in the context of sexual differentiation of the brain is progesterone. As pointed out by Fitch and Denenberg, there is some precedent for an action of progesterone in the developing brain as there is an effect on cortical thickness (Pappa, Diamond and Johnson, 1979), but these experiments did not involve progesterone treatment until day 40 of life. However, although circulating levels of progesterone are very low during the neonatal period, there is evidence for an earlier action of progesterone as demonstrated by an effect of administration of antiserum to progesterone on the day of birth (reviewed in Hendricks, 1992). It would appear there are at least four possible modes of action of progesterone in the sexual differentiation process. First is the classic genomic effect of progesterone on an estrogen-induced progesterone receptor. Differential exposure to estrogen in male and female brains could also lay the ground work for differential progesterone action. Second, progesterone often has antagonistic effects on estrogen, through mechanisms that are not entirely understood and likely involve both genomic and non-genomic steroid actions. Third, low levels of progesterone also act as an anti-androgen through an action at the androgen receptor. Differential interference with androgen binding by varying levels of progesterone between males and females has obvious implications for sexual differentiation. And fourth, it's possible that peripherally produced progesterone is metabolized in the brain into a neuroactive steroid that acts at the GABA-A receptor. These neurosteroids can have either agonist or antagonistic effects on the GABA-A receptor and may exert local influence on neuronal excitability (reviewed in McCarthy, 1995). There is increasing evidence for an important role of GABAergic transmission in the developing brain and steroid modulation of this transmitter could have differentiating effects.
4. Aside from ovarian factors, the evidence for additional variables influencing sexual differentiation is becoming increasingly tantalizing. Work by Reisert and Pilgram (1991) has demonstrated a genomic differentiation of monoaminergic neurotransmission that is independent of any steroid influences. The potential for genes involved in peripheral sexual differentiation, such as Mullerian Inhibiting Substance (MIS) and the Sex-determining Region (SRY) gene that determines development of the testis, have also been suggested as possible mediators of brain differentiation (McCarthy 1994). Reports of investigations into both these factors seems imminent.
5. An additional source of variation in the sexual differentiation of the brain is found in the data of Denenberg et al. (1991) regarding the influence of handling on androgens effects on corpus callosum thickness in females. The authors interpret these findings as "suggesting a synergy between the presence of testosterone and the effects of handling on adrenal corticosteroids". However, the possibility of the handling itself, independent of any adrenal steroid release, having a synergistic action with androgens also exists. In other words, there is a role for afferent input in the determination of brain sexual differences and handling may unmask the potential of afferent input by activating or inhibiting certain neural systems. The idea of afferent input as an important mediator in the process of sexual differentiation was well elucidated in a thoughtful review by Beyer and Feder (1987). The recent re-affirmation that neurotransmitters can directly influence and activate steroid receptors (Mani et al., 1994) further indicates the significance of changes in afferent input in hormonally-mediated sexual differentiation.
6. We are currently investigating the role of differences in one form of afferent input by manipulating a peripheral stimulus. It is well known that the anogenital licking performed by the dam on her pups is not only crucial for their survival, it is also important to the establishment of normal sexual behavior in adulthood (see for review Ward, 1992). This maternal behavior is not uniformly performed on all pups, rather the mother spends considerably more time anogenital licking male pups compared to females. If females are treated with testosterone neonatally, the dam will increase the frequency of anogenital licking of the female. We have been mimicking the anogenital licking (with a stiff bristle paint brush) in male and female pups and then looking for activation of neurons in specific brain areas as evidenced by induction of the immediate early gene product, c-fos. Induction of this protein is a reliable indicator of neuronal activation. We are finding a two-fold induction of c-fos protein in several brain areas of stimulated animals compared to unstimulated controls (McCarthy, Jacobs and Besmer, unpublished data). We take this as evidence for how differential afferent input might help to establish sex differences in neural structure and are currently engaged in determining if testosterone alters the profile of c-fos induction after stimulation.
7. In conclusion, while the evidence for estrogen playing an active role in the feminization of the brain is strong, there should also be consideration of additional, and perhaps non-steroidal, factors. Assessment of the level of ovarian functioning in the neonate remains an active area of investigation and students of the brain are well advised to remain informed of its status.
Beyer, C. and Feder, H.H. (1987) Sex steroids and afferent input: Their roles in brain sexual differentiation. Ann. Rev. Physiol. 49:349-364.
Denenberg, V.H., Fitch, R.H., Schrott, L.M., Cowell, P.E. and Waters, N.S. (1991) Corpus callosum interactive effects of testosterone and handling in the rat. Behavioral Neuroscience 105: 562-566.
Fitch, R.H. and Denenberg, V.H. (1995) A Role for Ovarian Hormones in Sexual Differentiation of the Brain. PSYCOLOQUY 6(5) sex-brain.1.fitch.
Hendricks, S.E. (1992) Role of estrogens and progestins in the development of female sexual behavior potential. In: Handbook of Behavioral Neurobiology, vol. 11: Sexual Differentiation, A.A. Gerall, J. Moltz, and I.L. Ward (Eds.) Plenum Press, New York. p. 129-155.
Hidalgo, A., Barami, K., Iversen, K. and Goldman, S.A. (1995) Estrogens and non-estrogenic ovarian influences combine to promote the recruitment and decrease the turnover of new neurons in the adult female canary brain. J. Neurobiol. 27: 470-487.
Mani, S.K., Allen, J.M.C., Clark, J.H., Blaustein, J.D. and O'Malley, B.W. (1994) Convergent pathways for steroid hormone- and neurotransmitter-induced rat sexual behavior. Science 265: 1246-1249.
McCarthy, M.M. (1994) Molecular Aspects of Sexual Differentiation of the Rodent Brain. Psychoneuroendocrinology 19:415-427.
McCarthy, M.M. (1995) Functional significance of steroid modulation of GABAergic neurotransmission: Analysis at the behavioral, cellular and molecular levels. Horm. Behav. 29:131-140.
Pappas, C.T.E., Diamond, M.C. and Johnson, R.E. (1979) Morphological changes in the cerebral cortex of rats with altered levels of ovarian hormones. Behavioral and Neural Biology 26: 298-310.
Reisert, I. and Pilgram, C. (1991) Sexual differentiation of monoaminergic neurons - genetic or epigenetic? Trends Neurosci. 14: 468-473.
Rucklidge, J. (1995) Incorporating estrogen's masculinizing role. PSYCOLOQUY 6(18).sex-brain.2.rucklidge.
Ward, I.L. (1992) Sexual behavior: The product of perinatal hormonal and prepubertal social factors. In: Handbook of Behavioral Neurobiology, vol. 11: Sexual Differentiation, A.A. Gerall, J. Moltz, and I.L. Ward (Eds.) Plenum Press, New York. p. 129-155.