Research Symposium: Sexual Differentiation of Physiological Functions

Transcription

1 Research Symposium: Sexual Differentiation of Physiological Functions

2 128 Sexual Differentiation of Physiological Functions J. Physiol. (2001). 531.P Molecular and genetic approaches to sexual differentiation of brain and behaviour Donald Pfaff Department of Neurobiology &- Behaviour, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA Proving causal connections between individual genes and specific mammalian brain functions will be difficult for several reasons (Pfaff et a!. 1999). Nevertheless, for a simple sex-differentiated mating behaviour whose neural and genetic determinants have been thoroughly analysed ('Drive', 1999), we have demonstrated an absolute dependence on the classical oestrogen receptor (ER) gene (Ogawa, 1996). Notably, the phenotype of the ER knockout mouse depends on the gender in which the ER gene is knocked out. The dogma for explaining sexual differentiation of the brain depends exclusively upon testosterone circulating in the genetic male. Endocrine pharmacological evidence, as well as antisense DNA evidence from our laboratory (McCarthy, 1993), indicate that testosterone must be aromatized to oestrogen in the CNS for full masculinization and defeminization to occur. Because not all recent results, particularly with songbirds, can be explained by this mechanism, a new look at the dogma is justified. First, Keverne has provided evidence for genetic imprinting in the CNS. Alleles contributed from the maternal and paternal genomes are expressed differently in the forebrain and probably influence forebrain development in a differential manner. Secondly, we have in situ hybridization evidence that a particular Mullerian inhibiting substance (MIS) receptor is expressed in the developing mouse brain. Therefore, as well as testosterone, gene products associated with the development of the gonadal ridge may playa direct role in sexual differentiation of the brain. McCarthy, 1\1.1\1., Schlenker, E. & Pfaff, DW. (1993). Endocrinology 133, Ogawa, S., Taylor, J., Lubahn, D.B., Korach, K.S. & Pfaff, DW. (1996). Neuroendocrinology 64, Pfaff, D. (1999). Drive. The MIT Press, Cambridge, MA, USA. Pfaff, D., Berrettini, W., Joh, T. & Maxson, S. (1 999). Genetic Influence on Neural and Behavioral Function. CRe Press, Boca Raton, FL, USA. Sex differences in the human brain D.F. Swaab, W.C.J. Chung, F. Kruijver and M.A. Hofman Netherlands Institute for Brain Research, Meibergdreef 33, 1105 AZ Amsterdam, The Netherlands Several hypothalamic structures differentiate in a sexually dimorphic manner. Examples of structural sex differences have been reported in the sexually dimorphic nucleus of the preoptic area, interstitial nuclei of the hypothalamus (INAH), bed nucleus of the stria terminalis (BST) and the anterior commissure (Swaab & Hofman, 1995). Moreover, a functional sex difference was found recently that is not reflected in differences in neuronal numbers, but only in neuronal activity. In the supraoptic nucleus, the vasopressin neurons are more active in males than in females, explaining the sex difference in plasma vasopressin levels (Ishunina et al. 1999). In addition, structural dimorphism related to sexual orientation (i.e. homosexuality and heterosexuality) has been described in the suprachiasmatic nucleus and INAH-3 (Swaab & Hofman, 1990; LeVay, 1991). A different remarkable observation has been made in transsexuals, who have the strong feeling, often from childhood onwards, of having been born the wrong sex. The central nucleus of the BST (BSTc), which plays an essential role in rodent sexual behaviour, was found to be about half the size in women compared with men. In men the size or number of somatostatin neurons of the BSTc did not vary a{)cording to heterosexual or homosexual orientation. However, in maleto-female transsexuals we found a female-sized BSTc. In one female-to-male transsexual the opposite was observed (Zhou et al. 1995; Kruijver et al. 2000). The size of the BSTc in transsexuals appeared thus to agree with their gender identity, and not with their genetic or biological sex. The observation that strong changes in sex hormone levels in adulthood did not change the size or cell number of the BSTc strongly suggests that the female-sized BSTc observed in genetically male transsexuals arose during development. These data suggest that there are complex neuronal networks that are the basis of our sexual orientation and gender identity, i.e. the feeling of being male or female. The sex differences develop between 5 years of age and adulthood, depending on the area that is studied. Currently sex differences are mapped in the adult human brain for oestrogen receptor a and /1, and for the androgen receptor. They all reveal clear sex differences in various hypothalamic and adjacent areas. The degree to which these sex differences are determined by circulating sex hormones is currently being investigated (Fernandez-Guasti et al. 2000; Ishunina et a!. 2000). Brain material was obtained from the Ketherlands Brain Bank (co-ordinator Dr R. Ravid). Fernandez-Guasti, A.,' Kruijver, F.P.M., Fodor, 1\L & Swaab, D.F. (2000). J. Camp. Neural. 425, Ishunina, T.A., Kruijver, F.P.1\f., Balesar, R. & Swaab, D.F. (2000). J. Clin. Endoerinol. Metabo/. 85, Ishunina, T.A., Salehi, A., Hofman, :\LA. & Swaab, D.F. (1999). J. Neuroendocrinol. 11, Kruijver, F.P.1\1., Zhou, J.1\., Pool, CW., Hofman, l\1.a., Gooren, L.J.G. & Swaab, D.F. (2000). J. Clin. Endoerinol. 11etabol. 85, LeVay, S. (1991). Science 253, Swaab, D.F. & Hofman, M.A. (1990). Brain Res. 537, Swaab, D.F. & Hofman, M.A. (1995). Trends Neurosei. 18,

3 J Physiol. (2001). 531.P Sexual Differentiation of Physiological Functions 13S Zhou, J.N., Hofman, M.A., OOOl'en, L.J.O. & Swaab, D.F. (1995). Nature 378, Sexual differentiation of mating partner preferences in a carnivore, the ferret: the role of olfaction M.J. Baum Department of Biology, Boston University, Boston, MA, USA One of the earliest indications that the mammalian nervous system is sexually differentiated stemmed from the observation of Phoenix and co-workers (1959) that the capacity of female guinea-pigs to show lordosis behaviour in response to ovarian steroids was permanently reduced by the transplacental administration of testosterone. Subsequent research using several rodent species showed that the male is typically less responsive than the female to the lordosisinducing effects of oestradiol and progesterone, presumably as a result of the perinatal actions of testosterone secreted by the developing testes. Studies using a range of mammalian species including rodents (e.g. rat), carnivores (e.g. ferret) and non-human primates (e.g. rhesus monkey) have shown that male-typical patterns of sexual motivation, including the preference to approach and mate with a sexually receptive female, are also organized by the perinatal actions of testosterone. In male ferrets the organization of male-typical heterosexual partner preference has been linked to the differentiation of a sexuallv dimorphic nucleus in the dorsal-medial preoptic area/a;terior hypothalamus (mpoa/ AH). This male nucleus of the mpoa/ AH develops in response to the fetal action of oestradiol formed via the neural aromatization of testosterone secreted by the testes. Bilateral excitotoxic lesions centred in the mpoa/ AH caused adult male ferrets to prefer to approach and interact with other sexually active male conspecifics instead of with oestrous females in T-maze tests. Female ferrets which received similar lesions continued to prefer to approach and interact with sexually active males. Homosexual partner preference among male ferrets with bilateral mpoa/ AH lesions persisted even when distal stimuli (olfactory, visual and auditory) as opposed to physical interactions with stimulus animals were provided. These results raise the possibility that neurons in the male's mpoa/ AH somehow promote sexual motivation by integrating information about the smell or physical appearance of conspecifics in a male-typical manner. By contrast, females which lack this nucleus (or males with lesions of the mpoa/ AH) may integrate distal signals from conspecifics in a female-typical fashion, which leads them to prefer to approach and mate with a male. Using nuclear Fos protein immunoreactivity (IR) as a marker of neuronal activation, we found that exposure to odours emitted from bedding soiled by either an oestrous female or a stud male augmented the number of Fos-IR granule cells in the main olfactory bulb (MOB) equally in male and female ferrets. No Fos responses to soiled bedding were seen in the accessory olfactory bulbs (AOB) of either sex, suggesting that the main as opposed to the accessory olfactory system detects socially relevant odours in this species. The MOB conveys information about socially relevant odours to the limbic system and hypothalamus via a polysynaptic circuit which includes the anterior-cortical and medial amygdaloid nuclei, the bed nucleus of the stria terminalis, medial preoptic area (mpoa) and ventromedial nucleus of the hypothalamus. In response to odours from oestrous females or stud males, significant increments in neuronal Fos-IR occurred in the female's, but not the male's mpoa. This suggests that odours from con specifics may play different roles in male and female ferrets in controlling sexual attraction and/or coital pel'formance. Using a Y -maze, we assessed the contribution of con specific odours, either alone or in combination with visual and auditory signals or physical interaction, to heterosexual partner selection by gonadectomized, steroid-treated male and female ferrets. Ferrets' nares were either bilaterally occluded using dental impression material or were sham-occluded. Independent behavioural and histological evidence showed that subjects were profoundly anosmic after bilateral nares occlusion and remained this way for many weeks of behavioural testing. Prior to coital experience, shamoccluded females and males preferred to approach odour only or odour + visual + auditory cues from opposite-sex conspecifics, whereas nares-occluded ferrets showed no such preference. All ferrets subsequently mated successfully in tests of sexual behaviour conducted in a small chamber. When re-tested in the Y-maze, sham-occluded males and females again preferred to approach odour only or odour + visual + auditory cues from opposite-sex conspecifics, whereas nares-occluded ferrets showed no such preference even in tests when a brief physical interaction with tethered stimulus ferrets was allowed after each trial. The only sex difference in ferrets' behavioural responses to being anosmic was seen when subjects were allowed to interact physically with stimulus ferrets after each trial: the latency of nares-occluded females to approach either the male or female stimulus ferrets was notably shorter than in previous trials that were run under 'odour only' or 'ordour + visual + auditory' cue conditions, even though females approached the two types of stimulus on an equal percentage of trials. In contrast, nares-occluded males continued to have long latencies to approach both the opposite- and same-sex stimulus ferrets, even when physical interaction was allowed. This suggests that peripheral anosmia disrupted the motivation to approach any social stimulus more profoundly in male than in female ferrets. This work was supported by NIH grant HD21094.

4 148 Sexual Differentiation of Physiological Functions J. Physioi. (2001). 531.P Influences of sexual steroids on neurosecretory oxytocin neurones D.A. Poulain, J.-M. Israel and D.T. Theodosis IN8ER}'1 U378, Universite Victor Segalen Bordeaux 2, Bordeaux, France Both sex steroids and the neurohormone oxytocin (OT) are important in reproductive physiology. As seen from numerous molecular, biochemical and physiological data, there is a great degree of interplay between the two, although detailed mechanisms are still obscure. Two aspects of the influence of sex steroids on the OT neurosecretory system will be discussed here, one concerning the anatomical plasticity of the OT system during parturition and lactation, the other, the electrophysiological behaviour of OT neurons at different phases of the reproductive cycle. The magnocellular OT system constitutes a striking model of morphological plasticity associated with physiological functions (Theodosis & Poulain, 1993). At the end of parturition, glial coverage of OT cells diminishes, leaving large segments of neuronal surface membrane directly juxtaposed. At the same time, there is proliferation of synapses containing glutamate, GABA or noradrenaline. This neuronal-glial and synaptic remodelling persists throughout lactation and the system reverts to its initial state after weaning. Besides peripherally, OT is released within the magnocellular nuclei, essentially via a somatodendritic release dependent on oestrogen (Pow & Morris, 1989). This central OT modulates the activity of OT neurons. It is also a potent agent for inducing plasticity since its intracerebral infusion in virgin rats provokes the same changes as those observed at the end of parturition (Theodosis et af. 1986), an effect which requires the concomitant action of sexual steroids. The morphological changes occur rapidly, within a few hours, as shown by our recent observations of acute hypothalamic slices from preparturient rats treated with OT and oestradiol. Sexual steroids also modify electrophysiological properties of OT neurons. Their activity during lactation consists of brief bursts of action potentials occuring synchronously in the whole population and resulting in release of OT into the blood and milk ejection. However spectacular, this electrophysiological behaviour is not a strictly fixed neuroendocrine mechanism. It is gradually programmed at the end of parturition and is modified throughout lactation. At the end of gestation, a sharp, transient fall in progesterone coincides with a sharp rise in oestrogen. Thereafter, oestradiol levels, low at the beginning of lactation, rise progressively, whereas progesterone levels decline. At late pregnancy, OT neurons exhibit a GABA A receptor subunit composition that confers sensitivity to the progesterone metabolite aiiopregnanalone (Brussard et al. 1999). As the concentration of progesterone falls, neuronal sensitivity to GABA also decreases. OT itself attenuates the efficacy of GABA but this effect is blocked by aiiopregnanalone. The fall of progesterone at term may therefore precipitate a state of positive feedback disinhibition which favours the expression of bursting in OT cells. Changes in steroid levels at term and throughout lactation may contribute to the modulation of the milk ejection reflex itself since ovariectomy and/or treatment with ovarian steroids alter milk ejection frequency and the facilitatory influence of OT itself on the reflex. We recently showed that oestrogens can influence OT neurones directly during lactation (Israel & Poulain, 2000). As seen from recordings of acute hypothalamic slices, the basal electrical properties of OT neurones differ in the course of lactation, so that at the end of lactation, there is an increased spike duration, decreased HAP amplitude and disappearance of spike frequency adaptation. Administration of 17 f3 -oestradiol restores all these properties to their values at the beginning of lactation. In parallel, sensitivity of OT neurons to glutamate diminishes at the end of lactation but is restored by application of 17f3-oestradiol. Oestradiol is rapidly washed out, so that adding 17 p -oestradiol to the preparation at late lactation merely restores oestrogen levels to those observed in vivo. This suggests, therefore, that oestradiol contributes to maintain normal firing characteristics and sensitivity to glutamate at late lactation, in the face of a declining excitability of the OT system and a diminishing frequency of suckling by the growing young. Brussarcl A.B., De\Oay, P., Leytig-Vermeulen, J.L. & Kits, K.S. (1999). J. Physioi. 516, Israel, J.-M. & Poulain, D.A. (2000). J. Physioi. 524, Pow, D.V. & Morris, J.F. (1989). Neuroscience 32, Theodosis, D.T., Montagnese, C., Rodriguez, F., Vincent, J.D. & Poulain, D.A. (1986). Nature 322, Theoclosis, D.T. & Poulain, D.A. (1993). Neuroscience 57, The role of sexual steroids in the regulation of neuroendocrine function Mary L. Forsling Neuroendocrine Laboratories, GKT School of Medicine, Guy's Campus, London Bridge, London 8E1 1 UL, UK The neurohypophysial system represents a useful model for the study of a neurosecretory system as the cell bodies are easily identified and their responses including electrophysiological activity are well characterised. Furthermore, the peptides released from the nerve terminals are directly secreted into the systemic circulation, so that the activity of the neurons can be directly monitored in the human. Studies on gender differences in osmotic and volume regulation of vasopressin release indicate a role for gonadal

5 J. Physioi. (2001). 531.P Sexual Differentiation of Physiological Functions 158 steroids in the release of vasopressin. Reproductive status also affects the regulation of vasopressin release, the change being most marked during pregnancy. A series of studies have been carried out in the rat on the effect of ovariectomy on the basal and stimulated release of both oxytocin and vasopressin in the rat and the effect of oestrogen replacement. In addition to hormone release, immediateearly gene expression, manifest as Fos protein expression, and mrna in the supraoptic nucleus (SON) have been determined. While the increase in oxytocin concentrations during hypertonic saline infusion was unaffected by ovariectomy and oestrogen replacement, vasopressin release was reduced on ovariectomy. The response was increased although not restored to normal by oestrogen administration. The profiles of Fos protein expression in the supraoptic nucleus reflected these changes, indicating that modulation occurs at a hypothalamic level. In contrast, both the oxytocin and vasopressin response to hypovolaemia were reduced on ovariectomy, an effect largely reversed by oestradiol administration. Again the changes in circulating hormone concentrations were reflected in Fos protein expression in the SON. Ovariectomy also reduced the oxytocin and vasopressin mrna in the SON in normally hydrated animals and attenuated the increase seen on dehydration with oestradiol reversing the change. Oestrogen receptors have been demonstrated in the neurohypophysial system, but the steroid could also act on pathways projecting to the SON. Gonadal steroids affect not only neurohypophysial hormone release in response to altered hydrational status, but also the increase seen during the hours of sleep. Neurohypophysial hormone concentrations in the male rat increase during the hours of daylight, falling again during the night. This pattern is markedly affected in female rats by the stage of the oestrous cycle. In men the increase in hormone concentrations occurs during the hours of darkness, a time when melatonin concentrations are elevated, but in women the 24 h profile additionally is related to reproductive status. The increase in neurohypophysial hormone release during the period of rest appears to depend in part on melatonin, with physiological concentrations of melatonin being inhibitory in the rat and stimulatory in man. Higher concentrations are stimulatory in the rat and inhibitory in men, both basal and stimulated release being affected A similar response is seen in women during the follicular phase, but during the luteal phase stimulation is seen with both physiological and pharmawlogical doses. This potentially enhanced release of neurohypophysial hormones during the night could be of physiological importance during pregnancy and parturition when the enhanced vasopressin release would contribute to the increased fluid retention and the nocturnal oxytocin increase to parturition.