Experimental approaches for the study of oxytocin and vasopressin gene expression in the central nervous system.

Intron-specific probes measure heteronuclear RNA (hnRNA) levels and thus approximate the transcription rates of genes, in part because of the rapid turnover of this intermediate form of RNA in the cell nucleus. Previously, we used oxytocin (Oxt)- and vasopressin (Avp)- intron-specific riboprobes to measure changes in Oxt and Avp hnRNA levels in the supraoptic nucleus (SON) by quantitative in situ hybridization (ISH) after various classical physiological perturbations, including acute and chronic salt loading, and lactation. In the present experiments, we used a novel experimental model to study the neurotransmitter regulation of Oxt and Avp gene expression in the rat SON in vivo. Bilateral cannulae connected via tubing to Alzet osmotic mini-pumps were positioned over the SON. In every experiment, one SON was infused with PBS and served as the control SON in each animal, and the contralateral SON received infusions of various neurotransmitter agonists and antagonists. Using this approach, we found that Avp but not Oxt gene expression increased after acute (2-5h) combined excitatory amino acid agonist and GABA antagonist treatment, similar to what we found after an acute hyperosmotic stimulus. Since both OXT and AVP are known to be comparably and robustly secreted in response to acute osmotic stimuli in vivo and glutamate agonists in vitro, our results indicate a dissociation between OXT secretion and Oxt gene transcription in vivo.

Neuronal nitric oxide synthase and calbindin delineate sex differences in the developing hypothalamus and preoptic area.

Throughout the hypothalamus there are several regions known to contain sex differences in specific cellular, neurochemical, or cell grouping characteristics. The current study examined the potential origin of sex differences in calbindin expression in the preoptic area and hypothalamus as related to sources of nitric oxide. Specific cell populations were defined by immunoreactive (ir) calbindin and neuronal nitric oxide synthase (nNOS) in the preoptic area/anterior hypothalamus (POA/AH), anteroventral periventricular nucleus (AVPv), and ventromedial nucleus of the hypothalamus (VMN). The POA/AH of adult mice was characterized by a striking sex difference in the distribution of cells with ir-calbindin. Examination of the POA/AH of androgen receptor deficient Tfm mice suggests that this pattern was in part androgen receptor dependent, since Tfm males had reduced ir-calbindin compared with wild-type males and more similar to wild-type females. At P0 ir-calbindin was more prevalent than in adulthood, with males having significantly more ir-calbindin and nNOS than have females. Cells that contained either ir-calbindin or ir-nNOS in the POA/AH were in adjacent cell groups, suggesting that NO derived from the enzymatic activity of nNOS may influence the development of ir-calbindin cells. In the region of AVPv, at P0, there was a sex difference with males having more ir-nNOS fibers than have females while ir-calbindin was not detected. In the VMN, at P0, ir-nNOS was greater in females than in males, with no significant difference in ir-calbindin. We suggest that NO as an effector molecule and calbindin as a molecular biomarker illuminate key aspects of sexual differentiation in the developing mouse brain.

Studies of oxytocin and vasopressin gene expression in the rat hypothalamus using exon- and intron-specific probes.

To develop a comprehensive approach for the study of oxytocin (OT) and vasopressin (VP) gene expression in the rat hypothalamus, we first developed an intronic riboprobe to measure OT heteronuclear RNA (hnRNA) levels by in situ hybridization histochemistry (ISHH). Using this 84-bp riboprobe, directed against intron 2 of the OT gene, we demonstrate strong and specific signals in neurons confined to the supraoptic (SON) and paraventricular (PVN) nuclei of the rat hypothalamus. We used this new intronic OT probe, together with other well-established intronic and exonic OT and VP probes, to reevaluate OT and VP gene expression in the hypothalamus under two classical physiological conditions, acute osmotic stimulation, and lactation. We found that magnocellular neurons in 7- to 8-day lactating female rats exhibit increased OT but not VP hnRNA. Since VP mRNA is increased during lactation, this suggests that decreased VP mRNA degradation during lactation may be responsible for this change. In contrast, whereas there was the expected large increase in VP hnRNA after acute salt loading, there was no change in OT hnRNA, suggesting that acute hyperosmotic stimuli produce increased VP but not OT gene transcription. Hence, the use of both exon- and intron-specific probes, which distinguish the changes in hnRNA and mRNA levels, respectively, can provide insight into the relative roles of transcription and mRNA degradation processes in changes in gene expression evoked by physiological stimuli.

Of mice and missing data: what we know (and need to learn) about male sexual behavior.

With recent advances in molecular genetics, the popularity of mice as subjects for behavioral neuroscience is increasing at an exponential rate. Unfortunately, the existing body of knowledge on sexual behavior in male mice is not large and many basic gaps exist. The assumption that what is true of rats is also true of mice is a dangerous one that can misdirect and, in the worst case, impede progress. We summarize the current knowledge about the sexual behavior of male mice, with an emphasis on hormonal bases of these behaviors. Behavioral differences between strains, developmental actions of steroids, activational actions of steroids given peripherally and in the brain, and data generated in various receptor knockout and related mice are discussed. In addition, suggestions are made for the standardization of experimental protocols used in investigations of the sexual physiology and behavior of male mice in order to facilitate between-experiment and between-laboratory comparisons and to expedite the growth of knowledge in this area.

Lack of functional estrogen receptor beta gene disrupts pubertal male sexual behavior.

The estrogen receptor-beta (ERbeta) mediates estrogen action in the female gonads, reproductive tract, and central nervous system. In addition, in rats and mice, gonadotropin-releasing hormone (GnRH-I) neurons coexpress ERbeta. Here we asked if ERbeta plays a role in the onset of puberty and in hypothalamic-pituitary-gonadal (HPG) axis function in male mice. We examined mating behavior, testosterone concentrations, steroid negative feedback on gonadotropins, and GnRH-I function in male ERbeta knockout (ERbetaKO) and wild-type (WT) mice. Peripubertal ERbetaKO males displayed their first ejaculation at a significantly older age than WT littermates. Castrated, adult ERbetaKO mice had significantly higher plasma luteinizing hormone (LH) than WT counterparts. Estradiol (E2) treatment reduced LH and follicle stimulating hormone (FSH) concentrations to an equivalent degree in castrates of both genotypes. In three different measures of the adult GnRH-I system, no genotypic differences were observed. These data show that ERbeta plays an important role in the timing of male sexual behavior at puberty, but does not appear to be involved in adult HPG axis functioning. Furthermore, our data suggest that a primary role of ERbeta may be to regulate ejaculatory behavior.

Aggression in male mice lacking functional estrogen receptor alpha.

Estrogen receptor alpha knockout (ERaKO) male mice fail to display sexual behavior. The authors hypothesized that ERalphaKOs require higher testosterone (T) concentrations than wild-type (WT) males to exhibit copulatory behavior. Increasing T stimulated sexual behavior and preference for females in WT males but failed to do so in ERalphaKOs. However, T did induce female-directed aggression in ERaKOs. In aggression tests, WT residents selectively attacked T-treated male intruders. ERaKO residents attacked female, T-treated male, and estrogen-treated male intruders equally. Increased access to olfactory cues prior to direct contact reduced overall aggression in ERalphaKO versus WT males but did not cause ERalphaKOs to differentially attack male and female opponents. Results suggest that ERalpha is essential for normal social behavior, perhaps via processing of chemoinvestigatory cues, which are required to discriminate males from females.

Estrogen receptor alpha influences socially motivated behaviors.

Male mice lacking estrogen receptor alpha (ERalphaKO) show reduced social behaviors. We hypothesized that this might be due to either socially elicited or generalized anxiety. Male ERalphaKOs and wild type (WT) mice were given a series of behavioral tests: elevated plus maze, T-maze, and social recognition. Each test included a social dimension by exposing males to ovariectomized (OVX) females. In addition plasma concentrations of corticosterone were measured, and open field activity was assessed. In the elevated plus maze, WT males exposed to an OVX female 1 min prior to the test were more anxious than WT controls. ERalphaKO males showed anxiety in this test whether or not they were preexposed to a female. In the T-maze, WT males increased exploration of a novel arm when it contained an OVX female. The presence or absence of a female in a novel arm did not affect behavior of ERalphaKO males. In social recognition tests, ERalphaKO males spent less time than WT littermates investigating an OVX female that was repeatedly introduced into their home cage. On the final trial, when a novel female was introduced, WT males increased their chemo-investigation but ERalphaKOs did not. Plasma corticosterone levels were lower in ERalphaKO than in WT males when plasma was taken directly after a brief (control) cage disturbance. In the open field WT and ERalphaKO males behaved essentially the same. Taken together, the results of these experiments suggest the ERalphaKO males avoid contact with other conspecifics, perhaps due to an inability to be aroused by social cues.

Roles of estrogen receptor alpha and androgen receptor in the regulation of neuronal nitric oxide synthase.

In brain and peripheral tissues, steroid hormones regulate nitric oxide synthase (nNOS). We asked whether estrogen receptor-alpha (ERalpha) and/or androgen receptor (AR) regulated nNOS immunoreactivity in mouse brain. First, we quantified cells singly labeled for nNOS immunoreactivity or labeled dually with ERalpha-immunoreactive (-ir) or AR-ir cells in the nucleus accumbens (Acb), preoptic area (POA), bed nucleus of the stria terminalis (BNST), posterior dorsal and posterior ventral regions of the medial amygdala (MePD and MePV, respectively), and paraventricular nucleus (PVN). The POA and MePD contained the greatest number of double-labeled cells. More nNOS-ir cells were colabeled with ERalpha immunoreactivity compared with AR immunoreactivity. Next, by using a double mutant mouse in which males lacked functional ERalpha, AR, or both, we investigated the roles of these steroid receptors in nNOS-ir cell numbers and immunoreactive area staining under testosterone (T) and estradiol (E2) conditions. Our data show that functional ERalpha is correlated with more nNOS-ir cells under T conditions and more immunoreactive area staining in the POA under both T and E2 conditions. However, ERalpha decreases nNOS-ir cell number in the BNST under E2 treatment. In summary, the data suggest that AR has organizational actions on nNOS-ir cell numbers in the MePV, that interactions between ERalpha and AR genes occur in PVN, and that sex differences in nNOS-ir area staining are limited to the POA. Thus, we show that ERalpha and AR interact to regulate nNOS in male and female brain in a site-specific manner.

Estrogen receptor-alpha (ERalpha), but not ERbeta, modulates estrogen stimulation of the ERalpha-truncated variant, TERP-1.

Estrogens regulate pituitary gene expression through two nuclear receptors (ERs), ERalpha and ERbeta. Rodent pituitary also expresses high levels of the pituitary-specific ERalpha isoform, truncated ER product-1 (TERP-1), which modulates the response of both ER forms to 17beta-estradiol (E2). Under physiological conditions, E2 stimulates TERP-1 expression from an ERalpha intronic promoter containing several potential binding sites for ERs. To evaluate the role of intact ER proteins on TERP-1 expression, we measured basal expression and steroid stimulation of TERP-1 in wild-type (WT) mice and mice in which either the ERalpha (ERalphaKO) or the ERbeta (ERbetaKO) gene was disrupted. TERP-1 mRNA expression was assessed by semiquantitative RT-PCR, and protein expression was evaluated by immunoblots. Both TERP-1 mRNA and protein were expressed in pituitaries from castrate WT, ERalphaKO, and ERbetaKO male and female mice. E2 stimulated TERP-1 mRNA expression in WT and ERbetaKO mice of both sexes, but had no effect on TERP-1 mRNA in either male or female ERalphaKO mice. Testosterone treatment also stimulated TERP-1 in WT, ERalphaKO, and ERbetaKO male mice. We conclude that ERalpha is critical for E2 stimulation, but not basal expression, of the TERP promoter, and that testosterone may act through the androgen receptor to stimulate the TERP-1 promoter in males.

A model system for study of sex chromosome effects on sexually dimorphic neural and behavioral traits.

We tested the hypothesis that genes encoded on the sex chromosomes play a direct role in sexual differentiation of brain and behavior. We used mice in which the testis-determining gene (Sry) was moved from the Y chromosome to an autosome (by deletion of Sry from the Y and subsequent insertion of an Sry transgene onto an autosome), so that the determination of testis development occurred independently of the complement of X or Y chromosomes. We compared XX and XY mice with ovaries (females) and XX and XY mice with testes (males). These comparisons allowed us to assess the effect of sex chromosome complement (XX vs XY) independent of gonadal status (testes vs ovaries) on sexually dimorphic neural and behavioral phenotypes. The phenotypes included measures of male copulatory behavior, social exploration behavior, and sexually dimorphic neuroanatomical structures in the septum, hypothalamus, and lumbar spinal cord. Most of the sexually dimorphic phenotypes correlated with the presence of ovaries or testes and therefore reflect the hormonal output of the gonads. We found, however, that both male and female mice with XY sex chromosomes were more masculine than XX mice in the density of vasopressin-immunoreactive fibers in the lateral septum. Moreover, two male groups differing only in the form of their Sry gene showed differences in behavior. The results show that sex chromosome genes contribute directly to the development of a sex difference in the brain.

Oestrogen's masculine side: mediation of mating in male mice.

This review focuses on the role of oestrogen in male sexual behaviour using oestrogen receptor alpha and beta knockout (ERalphaKO and ERbetaKO) mouse models. ERbetaKO mice are capable of mating and producing offspring, whereas ERalphaKO mice are unable to do either. When ERalphaKO males are treated with testosterone or dihydrotestosterone (DHT), < 50% display mounting behaviour, few intromit and none ejaculate. However, concurrent treatment with testosterone and a dopamine agonist instates masculine sexual behaviour in both male and female ERalphaKO mice. Dopamine content in the preoptic area and associated regions is not affected by oestrogen receptor alpha gene disruption. However, expression of neuronal nitric oxide synthase immunoreactivity is severely reduced in ERalphaKO males compared with wild-type males. These findings, together with studies conducted in aromatase knockout mice, are at odds with the dogma that oestrogen is required during development for expression of male sexual behaviour in adults. However, they do support a role for oestrogens, mediated by oestrogen receptor alpha, in regulation and production of neuronal nitric oxide synthase, which in turn may control dopamine agonist release. As has been shown in male rats, in mice dopamine agonist release is likely to be an essential component of the neural pathway that mediates male sexual behaviour.