I. Reisert

Transcription of the Y chromosomal gene,Sry, in adult mouse brain

The Y chromosomal gene Sry encodes a putative transcription factor which appears to serve as a master switch initiating testicular development. Here we show that this gene is transcribed in hypothalamus, midbrain, and testis of adult male but not adult female mice. In contrast to its circular transcripts in adult testis, those in brain are linear and may be translated. We propose that Sry exerts a role in the regulation of sex differentiation of the mammalian nervous system.

HP1-β is required for development of the cerebral neocortex and neuromuscular junctions

HP1 proteins are thought to be modulators of chromatin organization in all mammals, yet their exact physiological function remains unknown. In a first attempt to elucidate the function of these proteins in vivo, we disrupted the murine Cbx1 gene, which encodes the HP1-β isotype, and show that the Cbx1−/−-null mutation leads to perinatal lethality. The newborn mice succumbed to acute respiratory failure, whose likely cause is the defective development of neuromuscular junctions within the endplate of the diaphragm. We also observe aberrant cerebral cortex development in Cbx1−/− mutant brains, which have reduced proliferation of neuronal precursors, widespread cell death, and edema. In vitro cultures of neurospheres from Cbx1−/− mutant brains reveal a dramatic genomic instability. Our results demonstrate that HP1 proteins are not functionally redundant and that they are likely to regulate lineage-specific changes in heterochromatin organization.

Developmental profile of Sry transcripts in mouse brain

ABSTRACTTransient activation of the gene Sry in the gonadal ridge during a brief period of embryonic development is believed to function as a key signal for sex determination. However, a number of reports suggest that Sry expression is not as restricted in space and time as one would expect if its role was confined to directing male-specific differentiation in the early gonadal anlage. We have previously reported the occurrence of Sry/SRY transcripts in adult murine and human brain. The present communication is concerned with the study of the ontogenetic time course of Sry transcripts in mouse brain as detected by reverse transcription-polymerase chain reaction (RT-PCR). Particular emphasis was placed on the identification of two different forms of Sry mRNA, which can be linear or circular. To this aim, we used specific RT-PCR strategies to distinguish between both. Sry transcripts were found in male brain tissue of all ontogenetic stages investigated. Circular, presumably untranslatable, transcripts were found in embryonic brains of day 11 through 19. In contrast, postnatal Sry transcripts were linear, and thus translatable, and were found in diencephalon, midbrain, and cortex. The change from one transcript form to the other suggests that expression of the Sry gene in mouse brain is developmentally regulated, presumably by a switch in promoter selection. This supports the notion that Sry expression in brain is biologically significant.

Early sexual differentiation of diencephalic dopaminergic neurons of the rat in vitro

SummaryDevelopment of dopaminergic neurons was investigated in dissociated cell cultures raised from the diencephalon of male and female rat fetuses from days 14 and 17 of gestation. Striking differences in morphology and function of male and female dopaminergic neurons were observed. Outgrowth of tyrosine hydroxylase-immunoreactive processes initially proceeded at a faster rate in female than in male cultures. Morphological differences disappeared in cultures of gestational day 17. Irrespective of the age of the cultures and of the length of cultivation, the uptake capacity for (3H)dopamine per immunoreactive neuron was twice as high in female than in male cultures. Treatment of the cultures with sex steroids did not influence morphology, numbers, or transmitter uptake of tyrosine hydroxylase-immunoreactive neurons. The results suggest that diencephalic dopaminergic systems exhibit a sexual dimorphism that develops unexpectedly early in ontogeny and is independent of the action of gonadal hormones.

Neurotoxicity of Dopamine and Protective Effects of the NMDA Receptor Antagonist AP-5 Differ between Male and Female Dopaminergic Neurons

Neurodevelopmental and neurodegenerative disorders related to dopaminergic transmission typically exhibit a sex-specific prevalence. In order to investigate the underlying cellular mechanisms, primary cultures of dissociated embryonic rat midbrain were subject to a 24 h treatment with dopamine in concentrations between 1 and 1000 microM. Dopamine caused a dose-dependent loss of neurons and reduction of neurites immunoreactive to tyrosine hydroxylase with a LD50 of about 100 microM. Application of D1-like or D2-like receptor agonists instead of dopamine did not induce cell loss. Neither D1-like or D2-like receptor antagonists nor the nitric oxide synthase inhibitor N-nitro-L-arginine were capable of blocking dopamine-induced cell death. When tissue from male and female donors was cultured separately, a twofold sex difference was consistently present: (1) Survival rates of female dopaminergic neurons in the presence of LD50 concentrations of dopamine were about twice those of male neurons. (2) The N-methyl-D-aspartate-receptor antagonist AP-5 was capable of rescuing female but not male dopaminergic neurons from dopamine-induced cell death. It is concluded that dopamine neurotoxicity is not mediated by dopamine receptors and is aggravated by glutamate excitotoxicity but not by nitric oxide. The male-specific vulnerability is the first direct evidence that the prevalence of certain neurodevelopmental or neurodegenerative disorders may have a basis in the biology of the single dopaminergic neuron.

GENOTYPE-DEPENDENT SEX DIFFERENTIATION OF DOPAMINERGIC NEURONS IN PRIMARY CULTURES OF EMBRYONIC MOUSE BRAIN

In order to investigate genetic factors that interfere with hormone-mediated sex differentiation of dopaminergic neurons, we raised sex-specific primary cultures from embryonic day 13 diencephalon (D) or mesencephalon (M) of three different strains of mice, NMRI, CBA/J, and BALBc/J. Part of the cultures were maintained for 6 or 13 days in vitro (DIV) in medium containing 17 beta-estradiol or testosterone. The cultures were analyzed for sex differences in numbers of tyrosine hydroxylase-immunoreactive neurons, endogenous dopamine (DA) levels, and specific uptake of [3H]DA. Previous results obtained with cultures of embryonic Sprague-Dawley rats had shown that these parameters develop sex-specific characteristics in the absence of sex differences in hormone environment. Similar steroid-independent sex differences as they occur in the rat were found in M cultures of NMRI but not in CBA and BALBc mice. Long-term sex steroid treatment did not affect any of the above parameters in any strain. It is concluded that cell-autonomous realization of the genetic sex of dopaminergic neurons depends on the genetic background.

Effects of sex and estrogen on tyrosine hydroxylase mRNA in cultured embryonic rat mesencephalon

In order to elucidate cellular events responsible for sex differentiation of the nigro-striatal system, we studied the influence of estrogen on the expression of tyrosine hydroxylase (TH) in sex-specific dissociated cell cultures of embryonic day 14 rat mesencephalon. Cultures were raised in the absence or presence of 17 beta-estradiol (10(-12) M) and hybridized with a [35S]oligonucleotide specific to TH. Cultured cells and tissues were probed for estrogen receptor (ER) transcripts by hemi-nested PCR. More TH mRNA containing cells were present in control cultures from female than from male donors. Estrogen treatment resulted in an up-regulation of TH expression in male cells only and induced a reversal of the sex difference in TH mRNA levels present in early control cultures. ER message was detectable in hypothalamic and uterine tissues but not in mesencephalic tissue or cultured cells. Estrogen exposure failed to induce ER expression in cultured mesencephalic cells. It is concluded that there are sex differences in TH mRNA expression of developing midbrain dopaminergic neurons which are independent of the steroid environment. Estrogen can up-regulate TH mRNA in a sex-specific fashion by modulating signal transduction mechanisms other than the classical nuclear receptor pathway.

Sex steroids do not alter sex differences in tyrosine hydroxylase activity of dopaminergic neurons in vitro

SummaryIn order to distinguish the effects of genetic sex from those of sex hormones on the sexual differentiation of dopaminergic neurons, catecholamine synthesis was studied in gender-specific cultures of embryonic day-14 rat diencephalon. In addition to embryos from normal dams, embryos were used whose mothers had been treated with the estrogen antagonist tamoxifen or the testosterone antagonist cyproterone acetate on days 12 and 13 of gestation. Cultures from embryos of untreated dams were fed daily with a medium containing 17β-estradiol or testosterone. After 10 days in vitro, cultures were immunostained for tyrosine hydroxylase and the accumulation of dihydroxyphenylalanine (DOPA) was measured in the presence of the DOPA decarboxylase inhibitor NSD 1015. Rates of DOPA synthesis, unlike the numbers of tyrosine hydroxylase-immunoreactive neurons, were markedly higher in female cultures under all experimental conditions. Treatment of dams with antisteroids prior to removal of the embryos had no influence on these results. Treatment of cultures with both steroids decreased DOPA formation in a dose-dependent manner without altering the sex difference. These results suggest that cultured diencephalic dopaminergic neurons develop sex differences in the activity of tyrosine hydroxylase. This sexual dimorphism is initiated independently of the action of gonadal steroid hormones. Sex hormones exert an additional modulatory influence on the activity of the enzyme but do not abolish or reverse sex differences. Therefore, the concept of a purely epigenetic mode of sexual differentiation of the mammalian brain needs to be broadened to incorporate other mechanisms, such as the cell-autonomous fulfillment of a sex-specific genetic program.

Sex differences of hypothalamic prolactin cells develop independently of the presence of sex steroids

There is evidence for a hypothalamic prolactin (PRL) system that expresses sexually dimorphic traits. The aim of this in vitro study is to gain an insight into the process of sexual differentiation of hypothalamic PRL cells. In particular, we wanted to determine whether sexual differentiation of these cells can occur independently of the surge of gonadal testosterone which, in the male rat embryo, takes place at embryonic day (E) 18 and is commonly believed to start the critical period of sexual differentiation of the brain. Gender-specific cell cultures were prepared from E 14 or E 17 rat diencephalon and raised in the absence of gonadal steroids. After 10 days in vitro, numbers of PRL-immunoreactive (IR) cells and PRL levels were quantified by immunocytochemistry and Western blotting, respectively. Numbers of PRL-IR cells and PRL levels were 2-3 times higher in cultures prepared from female than from male embryos of either age. It is concluded that sexual differentiation of hypothalamic PRL cells starts well before the generally acknowledged onset of the critical period and may proceed independently of the action of gonadal testosterone. Besides gonadal steroids, other mechanisms, such as cell-intrinsic realization of a sex-specific genetic program, may be responsible for initiating the development of sexually dimorphic neuronal phenotypes.

Pre- and postnatal development of dopaminergic neuron numbers in the male and female mouse midbrain

Quantitative information about dopaminergic neuron numbers in the mesencephalon is needed to assess the significance of physiological cell death in the regulation of the development of this neural system. Therefore, stereological techniques were applied to determine absolute numbers of mesencephalic neurons immunoreactive to tyrosine hydroxylase during the ontogenetic period between embryonic day (E) 13 and postnatal day (P) 90. Male and female CBA/J mice were examined separately. The most rapid development with a 2.5-fold increase of total counts of immunostained cells per midbrain took place in the prenatal period. Beginning at E21, immunostained cells were counted separately in their three main locations, substantia nigra (SN), ventral tegmental area (VTA), and retrorubral field (RRF). Neuron numbers in RRF and VTA reached adult levels perinatally. In contrast, counts of immunostained cells in SN continued to increase postnatally. The only sign of cell loss was a transient decrease in VTA cell numbers (but not in total numbers of immunostained midbrain neurons) between E21 and P14. There were no statistically significant sex differences in cell numbers at any time point investigated. It is concluded that physiological cell death is not a major factor in the developmental regulation of dopaminergic cell numbers in the mouse midbrain.