Sex differences in the brain expression of steroidogenic molecules under basal conditions and after gonadectomy

The brain is a steroidogenic tissue. It expresses key molecules involved in the synthesis and metabolism of neuroactive steroids, such as steroidogenic acute regulatory protein (StAR), translocator protein 18 kDa (TSPO), cytochrome P450 cholesterol side‐chain cleavage enzyme (P450scc), 3β‐hydroxysteroid dehydrogenases (3β‐HSD), 5α‐reductases (5α‐R) and 3α‐hydroxysteroid oxidoreductases (3α‐HSOR). Previous studies have shown that the levels of brain steroids are different in male and female rats under basal conditions and after gonadectomy. In the present study, we assessed gene expression of key neurosteroidogenic molecules in the cerebral cortex and cerebellum of gonadally intact and gonadectomised adult male and female rats. In the cerebellum, the basal mRNA levels of StAR and 3α‐HSOR were significantly higher in females than in males. By contrast, the mRNA levels of TSPO and 5α‐R were significantly higher in males. In the cerebral cortex, all neurosteroidogenic molecules analysed showed similar mRNA levels in males and females. Gonadectomy increased the expression of 5α‐R in the brain of both sexes, although it affected the brain expression of StAR, TSPO, P450scc and 3α‐HSOR in females only and with regional differences. Although protein levels were not investigated in the present study, our findings indicate that mRNA expression of steroidogenic molecules in the adult rat brain is sexually dimorphic and presents regional specificity, both under basal conditions and after gonadectomy. Thus, local steroidogenesis may contribute to the reported sex and regional differences in the levels of brain neuroactive steroids and may be involved in the generation of sex differences in the adult brain function.     

Neuroactive steroids and peripheral myelin proteins

The present review summarizes observations obtained in our laboratories which underline the importance of neuroactive steroids (i.e., progesterone (PROG), dihydroprogesterone (5α-DH PROG), tetrahydroprogesterone (3α, 5α-TH PROG), testosterone (T), dihydrotestosterone (DHT) and 5α-androstan-3α,17β-diol (3α-diol)) in the control of the gene expression of myelin proteins (i.e. glycoprotein Po (Po) and the peripheral myelin protein 22 (PMP22)) in the peripheral nervous system. Utilizing different in vivo (aged and adult male rats) and in vitro (Schwann cell cultures) experimental models, we have observed that neuroactive steroids are able to stimulate the mRNA levels of Po and PMP22. The effects of these neuroactive steroids, which are able to interact with classical (progesterone receptor, PR, and androgen receptor, AR) and non-classical (GABA_A receptor) steroid receptors is further supported by our demonstration in sciatic nerve and/or Schwann cells of the presence of these receptors. On the basis of the observations obtained in the Schwann cells cultures, we suggest that the stimulatory effect of neuroactive steroids on Po is acting through PR, while that on PMP22 needs the GABA_A receptor. The present findings might be of importance for the utilization of specific receptor ligands as new therapeutical approaches for the rebuilding of the peripheral myelin, particularly in those situations in which the synthesis of Po and PMP22 is altered (i.e. demyelinating diseases like Charcot–Marie–Tooth type 1A and type 1B, hereditary neuropathy with liability to pressure palsies and the Déjérine–Sottas syndrome, aging, and after peripheral injury).     

Purkinje cell loss is due to a direct action of the weaver gene in Purkinje cells: evidence from chimeric mice

Within the cerebellum of the adult homozygous weaver mutant mouse there is an approximate 50% reduction in the number of vermal Purkinje cells. It is not known if this deficit is due to a primary action of the weaver gene or if the cell loss is due to a secondary effect of the weaver gene. We examined this question using chimeric mice, produced by fusing C57BL/6 homozygous or heterozygous weaver embryos (high beta-glucuronidase activity, Gusb) with C3HAw wild-type embryos (low beta-glucuronidase activity, Gush). Chimeric cerebella were stained for beta-glucuronidase activity and counts were made of the number of wv/- (Gusb) and +/+ (Gush) Purkinje cells. If the weaver gene acts intrinsically in the Purkinje cells, then the number of genetically wv/- and not +/+ Purkinje cells should be decreased. Alternatively, if the Purkinje cells are extrinsically affected by the weaver gene, then both wv/- and +/+ should be equally reduced. In this study, using comparative measures of chimerism and Purkinje cell numbers, only weaver Purkinje cells were reduced, while the +/+ Purkinje cells were unaffected in the chimera. These results indicate that the decrease in Purkinje cell number seen in the wv/wv and wv/+ cerebellum is a direct effect of the weaver gene. In concordance with previous work, the disorganization of the Purkinje cells in the cerebellum, however, results from an indirect effect of the weaver gene.     

Repetitive behavior and increased activity in mice with Purkinje cell loss: a model for understanding the role of cerebellar pathology in autism

Repetitive behaviors and hyperactivity are common features of developmental disorders, including autism. Neuropathology of the cerebellum is also a frequent occurrence in autism and other developmental disorders. Recent studies have indicated that cerebellar pathology may play a causal role in the generation of repetitive and hyperactive behaviors. In this study, we examined the relationship between cerebellar pathology and these behaviors in a mouse model of Purkinje cell loss. Specifically, we made aggregation chimeras between Lc/+ mutant embryos and +/+ embryos. Lc/+ mice lose 100% of their Purkinje cells postnatally due to a cell‐intrinsic gain‐of‐function mutation. Through our histological examination, we demonstrated that Lc/+?+/+ chimeric mice have Purkinje cells ranging from zero to normal numbers. Our analysis of these chimeric cerebella confirmed previous studies on Purkinje cell lineage. The results of both open‐field activity and hole‐board exploration testing indicated negative relationships between Purkinje cell number and measures of activity and investigatory nose‐poking. Additionally, in a progressive‐ratio operant paradigm, we found that Lc/+ mice lever‐pressed significantly less than +/+ controls, which led to significantly lower breakpoints in this group. In contrast, chimeric mice lever‐pressed significantly more than controls and this repetitive lever‐pressing behavior was significantly and negatively correlated with total Purkinje cell numbers. Although the performance of Lc/+ mice is probably related to their motor deficits, the significant relationships between Purkinje cell number and repetitive lever‐pressing behavior as well as open‐field activity measures provide support for a role of cerebellar pathology in generating repetitive behavior and increased activity in chimeric mice.     

Sex differences in steroid levels and steroidogenesis in the nervous system: Physiopathological role

The nervous system, in addition to be a target for steroid hormones, is the source of a variety of neuroactive steroids, which are synthesized and metabolized by neurons and glial cells. Recent evidence indicates that the expression of neurosteroidogenic proteins and enzymes and the levels of neuroactive steroids are different in the nervous system of males and females. We here summarized the state of the art of neuroactive steroids, particularly taking in consideration sex differences occurring in the synthesis and levels of these molecules. In addition, we discuss the consequences of sex differences in neurosteroidogenesis for the function of the nervous system under healthy and pathological conditions and the implications of neuroactive steroids and neurosteroidogenesis for the development of sex-specific therapeutic interventions.     

Peripheral nerves: a target for the action of neuroactive steroids

Peripheral nervous system possesses both classical and non-classical steroid receptors and consequently may represent a target for the action of neuroactive steroids. The present review summarizes the state of art of this intriguing field of research reporting data which indicate that neuroactive steroids, like for instance progesterone, dihydroprogesterone, tetrahydroprogesterone, dihydrotestosterone and 3α-diol, stimulate the expression of two important proteins of the myelin of peripheral nerves, the glycoprotein P0 (P0) and the peripheral myelin protein 22 (PMP22). Interestingly, the mechanisms by which neuroactive steroids exert their effects involve classical steroid receptors, like for instance progesterone and androgen receptors, in case of P0 and non-classical steroid receptors, like GABA_A receptor, in case of PMP22. Moreover, neuroactive steroids not only control the expression of these specific myelin proteins, but also influence the morphology of myelin sheaths and axons suggesting that these molecules may represent an interesting new therapeutic approach to maintain peripheral nerve integrity during neurodegenerative events.     

The male bias in the number of Purkinje cells and the size of the murine cerebellum may require Müllerian inhibiting substance/anti-Müllerian hormone

There is a male bias in the size of the cerebellum, with males, on average, having more Purkinje cells than females. The critical periods in cerebellum development occur when the immature testes secrete Müllerian inhibiting substance (MIS; synonym anti-Müllerian hormone) but only trace levels of testosterone. This suggests that the male bias in the cerebellum is generated by a different mechanism to the testosterone-sensitive reproductive nuclei. Consistent with this, in the present study, we report that Purkinje cells and other cerebella neurones express receptors for MIS, and that MIS(-/-) male mice have female-like numbers of Purkinje cells and a female-like size to other parts of their cerebellum. The size of the cell bodies of Purkinje cells was also dimorphic, although only a minority of this was a result of MIS. This suggests that MIS induces the initial male bias in the cerebellum, which is then refined by pubescent testosterone and/or other sex-specific factors.     

Modifications of Neuroactive Steroid Levels in an Experimental Model of Nigrostriatal Degeneration: Potential Relevance to the Pathophysiology of Parkinson’s Disease

An important link between neuroactive steroids and neurodegenerative disorders has recently been suggested. Indeed, in several neurodegenerative experimental models the levels of neuroactive steroids are affected and their administration exerts neuroprotective effects. However, scarce information has so far been obtained on the neuroactive steroid levels present in Parkinson’s disease. To this aim, using an experimental model of loss of nigrostriatal dopaminergic neurons obtained by stereotaxic injection of the neurotoxin 6-hydroxydopamine (6-OHDA), we evaluated by liquid chromatography tandem mass spectrometry the levels of several neuroactive steroids in the striatum and cerebral cortex of 6-OHDA-lesioned male rats. Among the neuroactive steroid levels assessed (i.e., pregnenolone, progesterone, dihydroprogesterone, tetrahydroprogesterone, isopregnanolone, testosterone, dihydrotestosterone, 3α-diol, dehydroepiandrosterone, 17α-estradiol, and 17β-estradiol), we observed a significant decrease of pregnenolone in the striatum. A similar effect was also observed on the levels of dihydroprogesterone present in this cerebral area and also in the cerebral cortex. Interestingly, an increase of isopregnanolone also occurred in the striatum and in the cerebral cortex. Altogether, these results suggesting that progesterone metabolism is affected in an experimental model of Parkinson’s disease further highlight the link between neuroactive steroids and the neurodegenerative diseases.     

Changes in CCK-4 induced panic after treatment with the GABA-reuptake inhibitor tiagabine are associated with an increase in 3α,5α-tetrahydrodeoxycorticosterone concentrations

There is evidence that gamma-amino-butyric acid type A (GABA_A)-receptor modulating neuroactive steroids play a role in the pathophysiology of panic disorder. Antidepressant treatment has been suggested to stabilize the concentrations of neuroactive steroids. In this pilot study we investigated neuroactive steroid concentrations during GABAergic treatment, which might represent an alternative anxiolytic pharmacotherapeutic strategy. Neuroactive steroid concentrations were determined in 10 healthy subjects treated with tiagabine. To evaluate the anxiolytic effects of tiagabine a cholecystokinine-tetrapeptide (CCK-4) challenge was performed before and after treatment. Treatment with tiagabine led to a significant increase in 3α,5α-tetrahydrodeoxycorticosterone (3α,5α-THDOC) from 0.49 to 1.42 nmol/l (Z = −2.80, p = .005), which was significantly correlated with a decrease of panic symptoms in the CCK-4 challenge. Thus, it might be hypothesized that the anxiolytic effects of GABAergic treatment might in part be mediated by their influence on 3α,5α-THDOC concentrations.     

Localization of p450scc and 5alpha-reductase type-2 in the cerebellum of fetal and newborn sheep

Prenatally, neuroactive steroids that modulate GABAergic activity may be synthesized de novo within the fetal brain. We have examined changes in immunoreactivity staining for the steroidogenic enzymes cholesterol P450 side-chain cleavage (P450scc), and 5alpha-reductase type-2 in the cerebellum of late gestation (130-145 days gestation) fetal sheep and newborn lambs (1-4 weeks of age). Both enzymes were predominantly localized in the Purkinje cell body and dendrites of the fetal and newborn cerebellum, with weaker immunoreactivity in a few cells of the inner granular layer. P450scc immunoreactivity was present in Purkinje neurons expressing either of the neuronal microtubule associated proteins MAP1b/5 or MAP2a/b, but was absent from GFAP and HNK-1 positive cells. Soma of Purkinje neurons were also immunopositive for 5alpha-reductase type-2 in the fetuses, but expression decreased to just detectable levels in the 1-2 and 2-4 week old lambs. Both MAP1b/5- and MAP2a/b-positive Purkinje neurons showed 5alpha-reductase type-2 expression in the fetus, whereas the residual 5alpha-reductase staining in the newborn lamb was present only in MAP2a/b-positive Purkinje neurons. Allopregnanolone in the cerebellum decreased from 21.8+/-1.9 ng/g wet weight in fetuses at 140-145 days gestation to 6.7+/-0.5 ng/g in 2-4 week old lambs (P<0. 05). Thus, synthesis of neuroactive steroids from cholesterol is possible in cerebellar neurons in late gestation and persists into neonatal life, 5alpha-reductase type-2 expression is greater in the fetus compared to the neonate, and allopregnanolone concentrations in the cerebellum decrease significantly after birth.