Steroidome and metabolome analysis in gilt saliva to identify potential biomarkers of boar effect receptivity.

Optimal management of gilt reproduction requires oestrus synchronization. Hormonal treatments are used for this purpose, but there is a growing demand for non-hormonal alternatives, especially in organic farms. The boar effect is an important alternative opportunity to induce and synchronize oestrus without hormones. Before puberty, gilts exhibit a 'waiting period' during which boar exposure could induce and synchronize the first ovulation. We searched for salivary biomarkers of this period of boar effect receptivity to improve detection of the gilts to stimulate with the perspective of enhancing the efficacy of the boar effect. Saliva samples were collected from 30 Large-White×Landrace crossbred gilts between 140 and 175 days of age. Gilts were exposed twice a day to a boar and subjected to oestrus detection from 150 to 175 days of age. Among the 30 gilts, 10 were detected in oestrus 4 to 7 days after the first introduction of the boar and were considered receptive to the boar effect, 14 were detected in oestrus more than 8 days after first boar contact, and six did not show oestrus and were considered non-receptive. Saliva samples from six receptive and six non-receptive gilts were analyzed for steroidome and for metabolome using gas chromatography coupled to tandem mass spectrometry and 1H nuclear magnetic resonance spectroscopy, respectively. Four saliva samples per gilt were analyzed: 25 days and 11 days before boar introduction, the day of boar introduction, 3 days later for receptive gilts or 7 days later for non-receptive gilts. Twenty-nine steroids and 31 metabolites were detected in gilt saliva. Salivary concentrations of six steroids and three metabolites were significantly different between receptive and non-receptive gilts: progesterone and glycolate 25 days before boar introduction, 3α5ß20α- and 3ß5α20ß-hexahydroprogesterone, dehydroepiandrosterone, androstenediol, succinate, and butyrate 11 days before boar introduction, and 3ß5α-tetrahydroprogesterone on the day of boar introduction. Thus, nine potential salivary biomarkers of boar effect receptivity were identified in our experimental conditions. Further studies with higher numbers of gilts and salivary sampling points are necessary to ascertain their reliability.

Sex differences in brain mitochondrial metabolism: influence of endogenous steroids and stroke.

Steroids are neuroprotective and a growing body of evidence indicates that mitochondria are a potential target of their effects. The mitochondria are the site of cellular energy synthesis, regulate oxidative stress and play a key role in cell death after brain injury and neurodegenerative diseases. After providing a summary of the literature on the general functions of mitochondria and the effects of sex steroid administrations on mitochondrial metabolism, we summarise and discuss our recent findings concerning sex differences in brain mitochondrial function under physiological and pathological conditions. To analyse the influence of endogenous sex steroids, the oxidative phosphorylation system, mitochondrial oxidative stress and brain steroid levels were compared between male and female mice, either intact or gonadectomised. The results obtained show that females have higher a mitochondrial respiration and lower oxidative stress compared to males and also that these differences were suppressed by ovariectomy but not orchidectomy. We have also shown that the decrease in brain mitochondrial respiration induced by ischaemia/reperfusion is different according to sex. In both sexes, treatment with progesterone reduced the ischaemia/reperfusion-induced mitochondrial alterations. Our findings indicate sex differences in brain mitochondrial function under physiological conditions, as well as after stroke, and identify mitochondria as a target of the neuroprotective properties of progesterone. Thus, it is necessary to investigate sex specificity in brain physiopathological mechanisms, especially when mitochondria impairment is involved.

Progesterone and allopregnanolone in the central nervous system: response to injury and implication for neuroprotection.

Progesterone is a well-known steroid hormone, synthesized by ovaries and placenta in females, and by adrenal glands in both males and females. Several tissues are targets of progesterone and the nervous system is a major one. Progesterone is also locally synthesized by the nervous system and qualifies, therefore, as a neurosteroid. In addition, the nervous system has the capacity to bio-convert progesterone into its active metabolite allopregnanolone. The enzymes required for progesterone and allopregnanolone synthesis are widely distributed in brain and spinal cord. Increased local biosynthesis of pregnenolone, progesterone and 5α-dihydroprogesterone may be a part of an endogenous neuroprotective mechanism in response to nervous system injuries. Progesterone and allopregnanolone neuroprotective effects have been widely recognized. Multiple receptors or associated proteins may contribute to the progesterone effects: classical nuclear receptors (PR), membrane progesterone receptor component 1 (PGRMC1), membrane progesterone receptors (mPR), and γ-aminobutyric acid type A (GABAA) receptors after conversion to allopregnanolone. In this review, we will succinctly describe progesterone and allopregnanolone biosynthetic pathways and enzyme distribution in brain and spinal cord. Then, we will summarize our work on progesterone receptor distribution and cellular expression in brain and spinal cord; neurosteroid stimulation after nervous system injuries (spinal cord injury, traumatic brain injury, and stroke); and on progesterone and allopregnanolone neuroprotective effects in different experimental models including stroke and spinal cord injury. We will discuss in detail the neuroprotective effects of progesterone on the nervous system via PR, and of allopregnanolone via its modulation of GABAA receptors.

Revisiting the roles of progesterone and allopregnanolone in the nervous system: resurgence of the progesterone receptors.

Progesterone is commonly considered as a female reproductive hormone and is well-known for its role in pregnancy. It is less well appreciated that progesterone and its metabolite allopregnanolone are also male hormones, as they are produced in both sexes by the adrenal glands. In addition, they are synthesized within the nervous system. Progesterone and allopregnanolone are associated with adaptation to stress, and increased production of progesterone within the brain may be part of the response of neural cells to injury. Progesterone receptors (PR) are widely distributed throughout the brain, but their study has been mainly limited to the hypothalamus and reproductive functions, and the extra-hypothalamic receptors have been neglected. This lack of information about brain functions of PR is unexpected, as the protective and trophic effects of progesterone are much investigated, and as the therapeutic potential of progesterone as a neuroprotective and promyelinating agent is currently being assessed in clinical trials. The little attention devoted to the brain functions of PR may relate to the widely accepted assumption that non-reproductive actions of progesterone may be mainly mediated by allopregnanolone, which does not bind to PR, but acts as a potent positive modulator of γ-aminobutyric acid type A (GABA(A) receptors. The aim of this review is to critically discuss effects of progesterone on the nervous system via PR, and of allopregnanolone via its modulation of GABA(A) receptors, with main focus on the brain.

Steroid profiling in brain and plasma of male and pseudopregnant female rats after traumatic brain injury: analysis by gas chromatography/mass spectrometry.

Steroids in brain arise from the peripheral endocrine glands and local synthesis. In traumatic brain injury (TBI), the endogenous circulating hormones at the time of injury are important for neuroprotection. In particular, pseudopregnant females recover better than males from TBI. We investigated the effect of pseudopregnancy and TBI on steroid levels in plasma and in three brain regions (within, adjacent, and distal to the lesion site), 6 and 24 h after prefrontal cortex injury. The following steroids were analyzed by gas chromatography/mass spectrometry: pregnenolone, progesterone, 5alpha-dihydroprogesterone, 3alpha,5alpha-tetrahydroprogesterone, 3beta,5alpha-tetrahydroprogesterone, dehydroepiandrosterone, Delta(4)-androstenedione, testosterone, 5alpha-dihydrotestosterone, 3alpha,5alpha-tetrahydrotestosterone, 3beta,5alpha-tetrahydrotestosterone, and 17beta-estradiol. Corticosterone was assayed in plasma to account for stress in the rats. We found different steroid profiles in brain and plasma of male and pseudopregnant female rats and specific profile changes after TBI. In sham-operated pseudopregnant females, much higher levels of progesterone, 5alpha-dihydroprogesterone, 3alpha,5alpha-tetrahydroprogesterone, and 3beta,5alpha-tetrahydroprogesterone were measured in both brain and plasma, compared with sham-operated males. Plasma levels of corticosterone were high in all groups, indicating that the surgeries induced acute stress. Six hours after TBI, the levels of pregnenolone, progesterone, and 5alpha-dihydroprogesterone increased, and those of testosterone decreased in male brain, whereas levels of 5alpha-dihydroprogesterone and 3beta,5alpha-tetrahydroprogesterone increased in brain of pseudopregnant female rats. Plasma levels of 5alpha-dihydroprogesterone did not change after TBI, suggesting a local activation of the 5alpha-reduction pathway of progesterone in both male and pseudopregnant female brain. The significant increase in neurosteroid levels in the male brain after TBI is consistent with their role in neuroprotection. In pseudopregnant females, high levels of circulating progestagens may provide protection against TBI.

Injury elicited increase in spinal cord neurosteroid content analyzed by gas chromatography mass spectrometry.

The effects of spinal cord injury (SCI), combined with castration and adrenalectomy, and of progesterone (PROG) treatment on neurosteroid levels and steroidogenic enzyme expression were investigated in the adult male rat spinal cord (SC). Steroid levels were quantified by gas chromatography/mass spectrometry in SC and plasma, and mRNAs of enzymes by quantitative real-time RT-PCR. The levels of pregnenolone (PREG), PROG, 5alpha-dihydroprogesterone, 3alpha,5alpha-tetrahydroprogesterone increased in SC 75 h after transection without significant increase in the plasma. After combined adrenalectomy and gonadectomy, significant levels of PREG and PROG remained in the SC, suggesting their local biosynthesis. In the SC of adrenalectomized and gonadectomized rats, there was an increase of PREG 24 h after SCI, followed at 75 h by a concomitant increase in its direct metabolite, PROG. These observations are consistent with a sequential increase of PREG biosynthesis and its conversion to PROG within the SC in response to injury. However, no significant change in P450-side chain cleavage and 3beta-hydroxysteroid dehydrogenase/Delta5-Delta4 isomerase mRNA levels was observed after SCI. Systemic PROG treatment after SCI, resulted in a very large increase in PROG, 5alpha-dihydroprogesterone, and 3alpha,5alpha-tetrahydroprogesterone in both plasma and SC. Furthermore, high levels of 3beta,5alpha-tetrahydroprogesterone were detected in SC, whereas their plasma levels remained barely detectable. Because the ratio of reduced metabolites to PROG was 65-times higher in SC than in the plasma, it appears likely that reduced metabolites mainly originated from local biosynthesis. Our results strongly suggest an important role for locally biosynthesized neurosteroids in the response of the SC to injury.

Steroid hormones and neurosteroids in normal and pathological aging of the nervous system.

Without medical progress, dementing diseases such as Alzheimer's disease will become one of the main causes of disability. Preventing or delaying them has thus become a real challenge for biomedical research. Steroids offer interesting therapeutical opportunities for promoting successful aging because of their pleiotropic effects in the nervous system: they regulate main neurotransmitter systems, promote the viability of neurons, play an important role in myelination and influence cognitive processes, in particular learning and memory. Preclinical research has provided evidence that the normally aging nervous system maintains some capacity for regeneration and that age-dependent changes in the nervous system and cognitive dysfunctions can be reversed to some extent by the administration of steroids. The aging nervous system also remains sensitive to the neuroprotective effects of steroids. In contrast to the large number of studies documenting beneficial effects of steroids on the nervous system in young and aged animals, the results from hormone replacement studies in the elderly are so far not conclusive. There is also little information concerning changes of steroid levels in the aging human brain. As steroids present in nervous tissues originate from the endocrine glands (steroid hormones) and from local synthesis (neurosteroids), changes in blood levels of steroids with age do not necessarily reflect changes in their brain levels. There is indeed strong evidence that neurosteroids are also synthesized in human brain and peripheral nerves. The development of a very sensitive and precise method for the analysis of steroids by gas chromatography/mass spectrometry (GC/MS) offers new possibilities for the study of neurosteroids. The concentrations of a range of neurosteroids have recently been measured in various brain regions of aged Alzheimer's disease patients and aged non-demented controls by GC/MS, providing reference values. In Alzheimer's patients, there was a general trend toward lower levels of neurosteroids in different brain regions, and neurosteroid levels were negatively correlated with two biochemical markers of Alzheimer's disease, the phosphorylated tau protein and the beta-amyloid peptides. The metabolism of dehydroepiandrosterone has also been analyzed for the first time in the aging brain from Alzheimer patients and non-demented controls. The conversion of dehydroepiandrosterone to Delta5-androstene-3beta,17beta-diol and to 7alpha-OH-dehydroepiandrosterone occurred in frontal cortex, hippocampus, amygdala, cerebellum and striatum of both Alzheimer's patients and controls. The formation of these metabolites within distinct brain regions negatively correlated with the density of beta-amyloid deposits.

Characterization and regulation of the 3beta-hydroxysteroid dehydrogenase isomerase enzyme in the rat sciatic nerve.

In the peripheral nervous system, progesterone (PROG) has a stimulatory effect on myelination. It could be derived from local synthesis, as Schwann cells in culture express the 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and convert pregnenolone (PREG) to PROG. Although 3beta-HSD mRNA can be detected by RT-PCR in peripheral nerves, the activity of the enzyme has so far not been demonstrated and characterized in nerve tissue. In this study, we show that homogenates prepared from rat sciatic nerves contain a functional 3beta-HSD enzyme and we have analysed its kinetic properties and its regulation by steroids. The activity of 3beta-HSD in homogenates was evaluated using 3H-labelled PREG as a substrate and NAD+ as a cofactor, the levels of steroids formed were calculated either by extrapolating the relationship between tritiated peaks obtained by TLC to the initial amount of PREG, or by gas chromatography/mass spectrometry determination. A rapid increase in PROG formation was found between 0 and 50 min of incubation and no further significant changes were observed between 1 and 4 h. The calculated Km value (1.06 +/- 0.19 microm) was close to the values described for the 3beta-HSD type-I and type-IV isoforms. Trilostane, a competitive inhibitor of the 3beta-HSD caused a potent inhibition of the rate of conversion of PREG to PROG (IC50 = 4.06 +/- 2.58 microm). When the effects of different steroids were tested, both oestradiol and PROG significantly inhibited the conversion of PREG to PROG.

3 Beta-hydroxysteroid dehydrogenase expression in rat spinal cord.

In adult male rats, 3beta-hydroxysteroid dehydrogenase/delta5-delta4-isomerase (3beta-HSD) expressing cells were identified in the spinal cord from the cervical to the sacral segments. An in situ hybridization study, using an oligonucleotide common to the four known isoforms of rat 3beta-HSD, revealed its mRNA in gray matter. Measurements of optical densities in autoradiograms showed the following regional distribution: dorsal horn (layers I-III) > central canal (layer X) > or = ventral horn (layers VIII-IX) > ventral funiculus = lateral funiculus. At the cellular level, the number of grains was higher on the large motoneurons than on small neurons of the dorsal horn, but the grain density per cell was similar. Further evidence for the expression of 3beta-HSD in the spinal cord was obtained by western blot analysis, which revealed an immunoreactive protein of approximately 45 kDa in the dorsal and ventral parts of the spinal cord. Castration and adrenalectomy did not influence the expression of 3beta-HSD mRNA and protein. Gas chromatography/mass spectrometry measurements showed higher levels of pregnenolone and progesterone in the spinal cord than in the plasma. After castration and adrenalectomy, their levels remained elevated in the spinal cord, suggesting that these neurosteroids may be synthesized locally. The wide distribution of 3beta-HSD, and the high levels of pregnenolone and progesterone in the spinal cord even after castration and adrenalectomy, strongly suggest a potential endogenous production of progesterone and an important signalling function of this steroid in the spinal cord.

Validation of an analytical procedure to measure trace amounts of neurosteroids in brain tissue by gas chromatography-mass spectrometry.

A selective and extremely sensitive procedure has been developed and optimized, using high-performance liquid chromatography (HPLC), specific derivatization and gas chromatography-mass spectrometry (GC-MS), to simultaneously quantify very small amounts of different neurosteroids from rat brain. Unconjugated and sulfated steroids in brain extracts were separated by solid-phase extraction. The unconjugated fraction was further purified by HPLC, the steroids being collected in a single fraction, and the sulfated fraction was solvolyzed. All steroids were derivatized with heptafluorobutyric acid anhydride and analyzed by GC-MS (electron impact ionization) using selected-ion monitoring. High sensitivity and accuracy were obtained for all steroids. The detection limits were 1 pg for pregnenolone (PREG), dehydroepiandrosterone (DHEA) and their sulfate esters PREG-S and DHEA-S, 2 pg for progesterone (PROG) and 5 pg for 3alpha,5alpha-tetrahydroprogesterone (3alpha,5alpha-THP). In a pilot study on a rat brain, the concentrations of PREG-S and DHEA-S were 8.26+/-0.80 and 2.47+/-0.27 ng/g, respectively. Those of PREG, DHEA and PROG were 4.17+/-0.22, 0.45+/-0.02 and 1.95+/-0.10 ng/g, respectively. Good linearity and accuracy were observed for each steroid. The methodology validated here, allows femtomoles of neurosteroids, including the sulfates, found in small brain samples (at least equal to 10 mg) to be quantified simultaneously.