Sex-specific activation of cell death signalling pathways in cerebellar granule neurons exposed to oxygen glucose deprivation followed by reoxygenation

Neuronal death pathways following hypoxia–ischaemia are sexually dimorphic, but the underlying mechanisms are unclear. We examined cell death mechanisms during OGD (oxygen-glucose deprivation) followed by Reox (reoxygenation) in segregated male (XY) and female (XX) mouse primary CGNs (cerebellar granule neurons) that are WT (wild-type) or Parp-1 [poly(ADP-ribose) polymerase 1] KO (knockout). Exposure of CGNs to OGD (1.5 h)/Reox (7 h) caused cell death in XY and XX neurons, but cell death during Reox was greater in XX neurons. ATP levels were significantly lower after OGD/Reox in WT-XX neurons than in XY neurons; this difference was eliminated in Parp-1 KO-XX neurons. AIF (apoptosis-inducing factor) was released from mitochondria and translocated to the nucleus by 1 h exclusively in WT-XY neurons. In contrast, there was a release of Cyt C (cytochrome C) from mitochondria in WT-XX and Parp-1 KO neurons of both sexes; delayed activation of caspase 3 was observed in the same three groups. Thus deletion of Parp-1 shunted cell death towards caspase 3-dependent apoptosis. Delayed activation of caspase 8 was also observed in all groups after OGD/Reox, but was much greater in XX neurons, and caspase 8 translocated to the nucleus in XX neurons only. Caspase 8 activation may contribute to increased XX neuronal death during Reox, via caspase 3 activation. Thus, OGD/Reox induces death of XY neurons via a PARP-1-AIF-dependent mechanism, but blockade of PARP-1-AIF pathway shifts neuronal death towards a caspase-dependent mechanism. In XX neurons, OGD/Reox caused prolonged depletion of ATP and delayed activation of caspase 8 and caspase 3, culminating in greater cell death during Reox.     

Sex chromosomes and brain gender

In birds and mammals, differences in development between the sexes arise from the differential actions of genes that are encoded on the sex chromosomes. These genes are differentially represented in the cells of males and females, and have been selected for sex-specific roles. The brain is a sexually dimorphic organ and is also shaped by sex-specific selection pressures. Genes on the sex chromosomes probably determine the gender (sexually dimorphic phenotype) of the brain in two ways: by acting on the gonads to induce sex differences in levels of gonadal secretions that have sex-specific effects on the brain, and by acting in the brain itself to differentiate XX and XY brain cells.     

Genetic study of the production of sexually dimorphic cuticular hydrocarbons in relation with the sex-determination gene transformer in Drosophila melanogaster

In Drosophila melanogaster, the main cuticular hydrocarbons (HCs) are some of the pheromones involved in mate discrimination. These are sexually dimorphic in both their occurrence and their effects. The production of predominant HCs has been measured in male and female progeny of 220 PGa14 lines mated with the feminising UAS-transformer transgenic strain. In 45 lines, XY flies were substantially or totally feminised for their HCs. Surprisingly, XX flies of 14 strains were partially masculinised. Several of the PGa14 enhancer-trap variants screened here seem to interact with sex determination mechanisms involved in the control of sexually dimorphic characters. We also found a good relationship between the degree of HC transformation and GAL4 expression in oenocytes. The fat body was also involved in the switch of sexually dimorphic cuticular hydrocarbons but its effect was different between the sexes.     

Dissociable Effects of Sry and Sex Chromosome Complement on Activity,Feeding and Anxiety-Related Behaviours in Mice

Whilst gonadal hormones can substantially influence sexual differentiation of the brain, recent findings have suggested that sex-linked genes may also directly influence neurodevelopment. Here we used the well-established murine ‘four core genotype’ (FCG) model on a gonadally-intact, outbred genetic background to characterise the contribution of Sry-dependent effects (i.e. those arising from the expression of the Y-linked Sry gene in the brain, or from hormonal sequelae of gonadal Sry expression) and direct effects of sex-linked genes other than Sry (‘sex chromosome complement’ effects) to sexually dimorphic mouse behavioural phenotypes. Over a 24 hour period, XX and XY gonadally female mice (lacking Sry) exhibited greater horizontal locomotor activity and reduced food consumption per unit bodyweight than XX and XY gonadally male mice (possessing Sry); in two behavioural tests (the elevated plus and zero mazes) XX and XY gonadally female mice showed evidence for increased anxiety-related behaviours relative to XX and XY gonadally male mice. Exploratory correlational analyses indicated that these Sry-dependent effects could not be simply explained by brain expression of the gene, nor by circulating testosterone levels. We also noted a sex chromosome complement effect on food (but not water) consumption whereby XY mice consumed more over a 24hr period than XX mice, and a sex chromosome complement effect in a third test of anxiety-related behaviour, the light-dark box. The present data suggest that: i) the male-specific factor Sry may influence activity and feeding behaviours in mice, and ii) dissociable feeding and anxiety-related murine phenotypes may be differentially modulated by Sry and by other sex-linked genes. Our results may have relevance for understanding the molecular underpinnings of sexually dimorphic behavioural phenotypes in healthy men and women, and in individuals with abnormal sex chromosome constitutions.     

Sex differences in juvenile mouse social behavior are influenced by sex chromosomes and social context

Play behavior in juvenile primates, rats and other species is sexually dimorphic, with males showing more play than females. In mice, sex differences in juvenile play have only been examined in out-bred CD-1 mice. In this strain, contrary to other animals, male mice display less play soliciting than females. Using an established same-sex dyadic interaction test, we examined play in in-bred C57BL/6J (B6) 21-day-old mice. When paired with non-siblings, males tended to be more social than females, spending more time exploring the test cage. Females displayed significantly more anogenital sniffing and solicited play more frequently than did males. To determine if the origin of the sex difference was sex chromosome genes or gonadal sex, next we used the four core genotype mouse. We found significant interactions between gonadal sex and genotype for several behaviors. Finally, we asked if sibling pairs (as compared to non-siblings) would display qualitatively or quantitatively different behavior. In fact, XX females paired with a sibling were more social and less exploratory or investigative, whereas XY males exhibited less investigative and play soliciting behaviors in tests with siblings. Many neurobehavioral disorders, like autism spectrum disorder (ASD), are sexually dimorphic in incidence and patients interact less than normal with other children. Our results suggest that sex chromosome genes interact with gonadal hormones to shape the development of juvenile social behavior, and that social context can drastically alter sex differences. These data may have relevance for understanding the etiology of sexually dimorphic disorders such as ASD.     

Sexually and Developmentally Dimorphic Grooming: A Comparative Survey of the Ungulata

Grooming is effective in removing fitness‐compromising ectoparasites, such as ticks. Tick‐removal grooming is regulated both by a central control mechanism (programmed grooming model) and by cutaneous stimulation from tick bites (stimulus‐driven model). The programmed grooming model predicts that organismic factors that impact the cost‐benefit ratio of grooming (e.g., sex and body size) will influence the rate of grooming. The ‘vigilance principle’ predicts that breeding males of sexually dimorphic species will groom less than females to maintain high levels of vigilance for rival males and oestrus females. The intraspecific body size principle predicts that juveniles will engage in more frequent grooming than larger‐bodied adults to compensate for the higher cost of tick infestation for smaller animals. To examine the generalizability of these predictions, we surveyed the grooming rate of 53 species/subspecies of ungulates (primarily Bovidae and Cervidae) in tick‐free zoological parks in which stimulus‐driven grooming was controlled for. Matched‐pair analysis supported both predictions. Males delivered fewer oral grooming episodes per hour than females in 85% of sexually dimorphic species (n = 40), but only 56% of monomorphic species (n = 11) exhibited sexually dimorphic grooming, which was not different from random. Juvenile oral episodes per hour exceeded that of adult females in 74% of surveyed species (n = 36). As predicted by the body size principle, the grooming rate of juveniles declined as juveniles grew larger and less vulnerable to tick infestation. In separate analyses of Bovidae and Cervidae to control for common ancestry, both families supported sexually dimorphic grooming, but developmentally dimorphic grooming was supported only for Bovidae. Our results indicate that sexually dimorphic grooming is widespread in the ungulate species surveyed, suggesting that programmed grooming evolved at least as early as the common ancestor to the Artiodactyla.     

Neuroeffectors for vocalization in Xenopus laevis: hormonal regulation of sexual dimorphism

South African clawed frogs use sex-specific vocalizations during courtship. In the male, vocalizations are under the control of gonadal androgen. Though females have moderate levels of circulating androgen, they do not give male-typical mate calls. Both muscles of the vocal organ and neurons of the central nervous system (CNS) vocal pathway are sexually dimorphic and androgen-sensitive. Recent studies suggest that the failure of androgen to masculinize adult females results from a male-specific, androgen-regulated developmental program. At metamorphosis the larynx is sexually monomorphic and feminine in morphology, muscle fiber number and androgen receptor content. During the next six months, under the influence of increasing androgen titers and high receptor levels, myoblasts proliferate in the male and muscle fibers increase at an average rate of 100/day. Females have much lower hormone levels, receptor values decline and they display no net addition of fibers. At metamorphosis, both males and females have approximately 4000 muscle fibers. By adulthood, males have eight times the female fiber number. In the CNS, adult laryngeal motor neurons are more numerous with larger somata and dendritic trees in males than in females. Certain connections of neurons in the vocal pathway are also less robust in females. Unlike the periphery, motor neuron number does not appear to be established by androgen-induced proliferation. Our current hypothesis is that androgen acts at the level of laryngeal muscle to produce more muscle fibers and thus provide more target for motor neurons in the male. This process could regulate cell number by ontogenetic cell death. In the CNS, androgen-target neurons become capable of accumulating hormone shortly before metamorphosis. Androgen receptor in laryngeal motor neurons may permit the dendritic growth characteristic of males by increasing sensitivity to afferent stimuli. Such a process could account for the observed differences in CNS vocal "circuitry" in X. laevis and thus behavioral differences between the sexes.     

Role of DNA-dependent protein kinase in neuronal survival

DNA-dependent protein kinase (DNA-PK) is a DNA repair enzyme composed of a DNA-binding component called Ku70/80 and a catalytic subunit called DNA-PKcs. Many investigators have utilized DNA-PKcs-deficient cells and cell lines derived from severe combined immunodeficiency (scid) mice to study DNA repair and apoptosis. However, little is known about the CNS of these mice. This study was carried out using primary neuronal cultures derived from the cerebral hemispheres of new-born wild-type and scid mice to investigate the effects of loss of DNA-PK function on neuronal maturation and survival. Purified neuronal cultures developed comparably in terms of neurite formation and expression of neuronal markers, but scid cultures showed a significant increase in the percentage of dying cells. Furthermore, when apoptosis was induced by staurosporine, scid neurons died more rapidly and in higher numbers. Apoptotic scid neurons exhibited nuclear condensation, DNA fragmentation and caspase-3 activation, but treatment with the general caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-(O-methyl) fluoromethyl ketone did not prevent staurosporine-induced apoptosis. We conclude that a DNA-PK deficiency in cultured scid neurons may cause an accumulation of DNA damage and increased susceptibility to caspase-independent forms of programmed cell death.     

In vitro and in vivo neuroprotective effect and mechanisms of glabridin, a major active isoflavan from Glycyrrhiza glabra (licorice)

Stroke is a life-threatening disease characterized by rapidly developing clinical signs of focal or global disturbance of cerebral function due to cerebral ischemia. A number of flavonoids have been shown to attenuate the cerebral injuries in stroked animal models. Glabridin, a major flavonoid of Glycyrrhiza glabra (licorice), possesses multiple pharmacological activities. This study aimed to investigate whether glabridin modulated the cerebral injuries induced by middle cerebral artery occlusion (MCAO) in rats and staurosporine-induced damage in cultured rat cortical neurons and the possible mechanisms involved. Our study showed that glabridin at 25mg/kg by intraperitoneal injection, but not at 5mg/kg, significantly decreased the focal infarct volume, cerebral histological damage and apoptosis in MCAO rats compared to sham-operated rats. Glabridin significantly attenuated the level of brain malonyldialdehyde (MDA) in MCAO rats, while it elevated the level of two endogenous antioxidants in the brain, i.e. superoxide dismutase (SOD) and reduced glutathione (GSH). Co-treatment with glabridin significantly inhibited the staurosporine-induced cytotoxicity and apoptosis of cultured rat cortical neurons in a concentration-dependent manner. Consistently, glabridin significantly reduced the DNA laddering caused by staurosporine in a concentration-dependent manner. Glabridin also suppressed the elevated Bax protein and caspase-3 proenzyme and decreased bcl-2 induced by staurosporine in cultured rat cortical neurons, facilitating cell survival. Glabridin also inhibited superoxide production in cultured cortical neurons exposed to staurosporine. These findings indicated that glabridin had a neuroprotective effect via modulation of multiple pathways associated with apoptosis. Further studies are warranted to further investigate the biochemical mechanisms for the protective effect of glabridin on neurons and the evidence for clinical use of licorice in the management of cerebral ischemia.     

Two regions within the proximal steroidogenic factor 1 promoter drive somatic cell-specific activity in developing gonads of the female mouse

Targets of steroidogenic factor 1 (SF1; also known as NR5A1 and AD4BP) have been identified within cells at every level of the hypothalamic-pituitary-gonadal and -adrenal axes, revealing SF1 to be a master regulator of major endocrine systems. Mouse embryos express SF1 in the genital ridge until Embryonic Day 13.5 (E13.5). Thereafter, expression persists in the male and is substantially lower in the female gonad until birth. We hypothesize that the sexually dimorphic expression of Sf1 during gonadogenesis is mediated by sex-specific regulation of its promoter. To investigate dimorphic regulation within the fetal gonad, we developed an experimental strategy using transient transfection of E13.5 gonad explant cultures and evaluated various Sf1 promoter constructs for sexually dimorphic DNA elements. The proximal Sf1 promoter correctly targeted reporter activity to SF1-expressing cells in both XY and XX gonads. Stepwise deletion of sequences from the Sf1 promoter revealed two regions that affected regulation within female gonads. Mutation of both sequences together did not cause further disruption of reporter activity, suggesting the two sites might work in concert to promote activity in female somatic cells. Results from gel mobility shift assays and fetal gonad-chromatin immunoprecipitation showed that TCFAP2 binds to one of the two female-specific sites within the proximal promoter of Sf1. Together, we show that transient transfection experiments performed within developing testes and ovaries are a powerful tool to uncover elements within the Sf1 promoter that contribute to sex-specific expression.     

Seabream GnRH immunoreactivity in brain and pituitary of XX and XY Nile tilapia, Oreochromis niloticus during early development

Seabream gonadotropin-releasing hormone (sbGnRH)-the chief preoptic area-hypothalamus (POA-H) form of GnRH in tilapia is involved in sexual maturation. In this study, we investigated the qualitative changes in ontogeny of sbGnRH immunoreactivity (ir-), between sexes to understand its impending role during sex differentiation. For this, the differences in immunocytochemical localization of sbGnRH in genetically male (XY) and female (XX) fish were studied from 1 day after hatching (dah), through the critical period of sex differentiation (7-21 dah) to 40 dah and mature Nile tilapia. Specific antisera against sbGnRH were used for immunolocalization. SbGnRH ir- neurons were observed in POA-H as early as 5 and 15 dah in XY fish and XX fish, respectively. Higher ir- was detected in the POA-H of XY tilapia compared with XX population till 10 dah. There was a qualitative drop in sbGnRH ir- neurons/cell bodies in POA-H around 20 dah till 30 dah in XY population compared with other durations. SbGnRH ir- cells were detected in pituitary of XX fish by 15 dah and in XY fish around 10 dah but seemed to drop down by 20 dah in XY whereas it continued to remain steady in XX fish. The sbGnRH ir- in XY fish showed a rise from 35 dah and thence till 40 dah. This study revealed subtle differences in POA-H and pituitary sbGnRH ir- during early development between genetic male and female fish with possible implications in sex differentiation.     

Antioxidant and bioenergetic coupling between neurons and astrocytes

Oxidative and nitrosative stress underlie the pathogenesis of a broad range of human diseases, in particular neurodegenerative disorders. Within the brain, neurons are the cells most vulnerable to excess reactive oxygen and nitrogen species; their survival relies on the antioxidant protection promoted by neighbouring astrocytes. However, neurons are also intrinsically equipped with a biochemical mechanism that links glucose metabolism to antioxidant defence. Neurons actively metabolize glucose through the pentose phosphate pathway, which maintains the antioxidant glutathione in its reduced state, hence exerting neuroprotection. This process is tightly controlled by a key glycolysis-promoting enzyme and is dependent on an appropriate supply of energy substrates from astrocytes. Thus brain bioenergetic and antioxidant defence is coupled between neurons and astrocytes. A better understanding of the regulation of this intercellular coupling should be important for identifying novel targets for future therapeutic interventions.     

Sex-specific apoptosis regulates sexual dimorphism in the Drosophila embryonic gonad

Sexually dimorphic development of the gonad is essential for germ cell development and sexual reproduction. We have found that the Drosophila embryonic gonad is already sexually dimorphic at the time of initial gonad formation. Male-specific somatic gonadal precursors (msSGPs) contribute only to the testis and express a Drosophila homolog of Sox9 (Sox100B), a gene essential for testis formation in humans. The msSGPs are specified in both males and females, but are only recruited into the developing testis. In females, these cells are eliminated via programmed cell death dependent on the sex determination regulatory gene doublesex. Our work furthers the hypotheses that a conserved pathway controls gonad sexual dimorphism in diverse species and that sex-specific cell recruitment and programmed cell death are common mechanisms for creating sexual dimorphism.     

The sex-determination genes fruitless and doublesex specify a neural substrate required for courtship song

Courtship song is a critical component of male courtship behavior in Drosophila, making the female more receptive to copulation and communicating species-specific information [1-6]. Sex mosaic studies have shown that the sex of certain regions of the central nervous system (CNS) is critical to song production [7]. Our examination of one of these regions, the mesothoracic ganglion (Msg), revealed the coexpression of two sex-determination genes, fruitless (fru) and doublesex (dsx). Because both genes are involved in creating a sexually dimorphic CNS [8, 9] and are necessary for song production [10-13], we investigated the individual contributions of fru and dsx to the specification of a male CNS and song production. We show a novel requirement for dsx in specifying a sexually dimorphic population of fru-expressing neurons in the Msg. Moreover, by using females constitutively expressing the male-specific isoforms of fru (Fru(M)), we show a critical requirement for the male isoform of dsx (Dsx(M)), alongside Fru(M), in the specification of courtship song. Therefore, although Fru(M) expression is sufficient for the performance of many male-specific behaviors [14], we have shown that without Dsx(M), the determination of a male-specific CNS and thus a full complement of male behaviors are not realized.     

FGF9 promotes survival of germ cells in the fetal testis

In addition to its role in somatic cell development in the testis, our data have revealed a role for Fgf9 in XY germ cell survival. In Fgf9-null mice, germ cells in the XY gonad decline in numbers after 11.5 days post coitum (dpc), while germ cell numbers in XX gonads are unaffected. We present evidence that germ cells resident in the XY gonad become dependent on FGF9 signaling between 10.5 dpc and 11.5 dpc, and that FGF9 directly promotes XY gonocyte survival after 11.5 dpc, independently from Sertoli cell differentiation. Furthermore, XY Fgf9-null gonads undergo true male-to-female sex reversal as they initiate but fail to maintain the male pathway and subsequently express markers of ovarian differentiation (Fst and Bmp2). By 14.5 dpc, these gonads contain germ cells that enter meiosis synchronously with ovarian gonocytes. FGF9 is necessary for 11.5 dpc XY gonocyte survival and is the earliest reported factor with a sex-specific role in regulating germ cell survival.     

Role of cell death in the formation of sexual dimorphism in the Drosophila central nervous system

Currently, sex differences in behavior are believed to result from sexually dimorphic neural circuits in the central nervous system (CNS). Drosophila melanogaster is a common model organism for studying the relationship between brain structure, behavior, and genes. Recent studies of sex‐specific reproductive behaviors in D. melanogaster have addressed the contribution of sexual differences in the CNS to the control of sex‐specific behaviors and the development of sexual dimorphism. For example, sexually dimorphic regions of the CNS are involved in the initiation of male courtship behavior, the generation of the courtship song, and the induction of male‐specific muscles in D. melanogaster. In this review, I discuss recent findings about the contribution of cell death to the formation of sexually dimorphic neural circuitry and the regulation of sex‐specific cell death by two sex determination factors, Fruitless and Doublesex, in Drosophila.