Jonathan J. Hirst

Progesterone receptor localization and isoforms in myometrium, decidua, and fetal membranes from rhesus macaques: evidence for functional progesterone withdrawal at parturition.

Objective: It is not known whether withdrawal of progesterone (P) action is a prerequisite for parturition in women or in nonhuman primates because concentrations of circulating progesterone or progesterone receptors (PR) in myometrium and decidua do not decrease before delivery. To examine this potentially important regulatory mechanism, we determined PR isoforms, PR localization, and mRNA in myometrium, decidua, and fetal membranes from rhesus monkeys during pregnancy and in spontaneous labor at term. Methods: Gestational tissues were obtained midpregnancy (day 80-100), late pregnancy (day 130-145), and during spontaneous labor at term (day 161-167). Samples of rhesus monkey myometrium, decidua, chorion-decidua, and amnion were collected and analyzed for total nuclear and cytosolic PR by competitive binding assay. Progesterone receptor isoforms were identified and quantified by Western blot analysis, and PR mRNA was determined by a specific ribonuclease protection assay. Nuclear PR was localized by immunohistochemistry with monoclonal anti-PR (JZB39) after microwave stabilization. Results: Myometrium and decidua showed no change in total PR during pregnancy and labor. Nuclear PR was not detected in fetal membranes by binding assay but was localized in amnion epithelial and mesenchymal cells and in chorion laeve cytotrophoblasts by immunohistochemistry. Staining for PR was substantially less by serial antibody dilution in fetal membranes than in decidua. Message for PR was confirmed in all tissues analyzed. A significant (P < .05) shift in the ratio of PR isoforms (from PR-B dominance at midpregnancy to PR-A dominance in labor) was observed in myometrium but not in decidua. Both PR-A and PR-B isoforms and PR nuclear staining were nearly undetectable in amnion obtained during labor. Conclusion: A shift to PR-A dominance in myometrium at term together with a loss of PR in fetal membranes provides evidence for a functional progesterone withdrawal mechanism, which may facilitate the initiation of parturition in primates.

Cerebrovascular Responses in the Fetal Sheep Brain to Low-Dose Endotoxin

Clinical and experimental evidence indicate that infection in pregnancy is associated with fetal brain damage. However, the inflammatory processes that compromise the fetal brain are not fully understood. In this study, we used a single, low dose of lipopolysaccharide (LPS, 0.1 μg/kg i.v.) to provoke an acute-phase response in unanesthetized fetal sheep in utero. COX-2 mRNA was increased in the cortex and cerebellum at 24 and 48 h after LPS, and immunoreactive COX-2 protein was increased in perivascular cells throughout gray and white matter at 24 h after LPS administration. Plasma albumin was observed in the parenchyma of the brain in cortex, thalamus, hypothalamus, corpus callosum, fornix, hippocampus, midbrain, subcallosal bundle, and cerebellar Purkinje cells. Large, rounded, lectin-positive cells with the appearance of macrophages were observed around blood vessels in subventricular white matter. These results indicate that blood-brain barrier permeability is increased in the fetal brain after exposure to endotoxin and suggests that cytotoxic and pro-inflammatory substances could pass from the circulation into the brain after peripheral inflammatory stimulation.

Changes in 5α-pregnane steroids and neurosteroidogenic enzyme expression in the perinatal sheep

Pregnane steroids have sedative and neuroprotective effects on the brain as a result of interactions with the steroid-binding site of the GABAA receptor. To determine whether the fetal brain is able to synthesize pregnane steroids de novo from cholesterol, we measured the expression of cytochrome P450 side-chain cleavage (P450scc) and 5α-reductase type II (5αRII) enzymes in fetal sheep from 72 to 144 d gestation (term ≈147 d) and in newborn lambs at 3 and 19-26 d of age. Both P450scc and 5αRII expression was detectable by 90 d gestation in the major regions of the brain and also in the adrenal glands. Expression increased with advancing gestation and was either maintained at fetal levels or increased further after birth. In contrast, the relatively high content (200-400 pmol/g) of allopregnanolone (5α-pregnan-3α-ol-20-one), a major sedative 5α-pregnane steroid, present throughout the brain from 90 d gestation to term, was reduced significantly (<50 pmol/g) immediately after birth. These results suggest that although the perinatal brain has the enzymes potentially to synthesize pregnane steroids de novo from cholesterol, either the placenta is a major source of these steroids to the brain or other factors associated with intrauterine life may be responsible for high levels of allopregnanolone production in the fetal brain until birth.

Role of neurosteroids in regulating cell death and proliferation in the late gestation fetal brain

The neurosteroid allopregnanolone (AP) is a GABAergic agonist that suppresses central nervous system (CNS) activity in the adult brain, and by reducing excitotoxicity is considered to be neuroprotective. A role for neurosteroids in the developing brain, particularly in late gestation, is still debated. The aim of this study was to investigate effects on proliferation and cell death in the brain of late gestation fetal sheep after inhibition of AP synthesis using finasteride, a 5alpha-reductase type 2 (5alpha-R2) inhibitor. Catheters were implanted in fetal sheep at approximately 125 days of gestation. At 3-4 days postsurgery, fetuses received infusions of either finasteride (20 mg/kg/h; n=5), the AP analogue alfaxalone (5 mg/kg/h; n=5), or finasteride and alfaxalone together (n=5). Brains were obtained at 24 h after infusion to determine cell death (apoptotic or necrotic) and cell proliferation in the hippocampus and cerebellum, areas known to be susceptible to excitotoxic damage. Finasteride treatment significantly increased apoptosis (activated caspase-3 expression) in hippocampal CA3 and CA1, and cerebellar molecular and granular layers, an effect abolished by co-infusion of alfaxalone and finasteride. Double-label immunohistochemistry showed that both neurons and astrocytes were caspase-3 positive. Finasteride treatment also increased the number of dead (pyknotic) cells in the hippocampus and cerebellum (Purkinje cells), but not when finasteride+alfaxalone was infused. Cell proliferation (Ki-67-immunoreactivity) increased after finasteride treatment; double-labeling showed the majority of Ki-67-positive cells were astrocytes. Thus, steroids such as AP appear to influence the constitutive rate of apoptosis and proliferation in the hippocampus and cerebellum of the fetal brain, and suggest an important role for neurosteroids in the development of the brain.

Fetal Responses to Maternal and Intra-Amniotic Lipopolysaccharide Administration in Sheep

Abstract A link between intrauterine infection and premature labor is widely accepted, yet the fetal inflammatory responses to such infections are not well understood. Our aim was to use a sheep model in which an inflammatory state was induced by lipopolysaccharide (LPS) administration during pregnancy to the maternal systemic, intra-amniotic or extra-amniotic compartments. Fetal and maternal blood gases and uterine electromyographic activity along with fetal and maternal circulating concentrations of prostaglandins PGE2 and PGFM, cortisol, and interleukin-6 were determined. Maternal systemic LPS treatment resulted in mild maternal hypoxemia, a rise in temperature, greater fetal hypoxemia, and a marked rise in fetal cortisol and PGE2 concentrations that persisted for 48 h. Intra-amniotic administration of LPS at doses higher than those used systemically caused an increase in fetal cortisol and PGE2 concentrations as well as a rise in uterine activity, but these were lesser in magnitude. Extra-amniotic LPS administration caused no overt fetal or maternal inflammatory responses. We conclude that maternal LPS treatment markedly elevated fetal cortisol and PGE2 concentrations. This may be a potential protective mechanism that aids the fetus in the event of premature delivery. The attenuated fetal response to intra-amniotic LPS treatment, despite the much higher dose used, may support a role for the amniotic fluid in protecting the fetus from endotoxin exposure during pregnancy.

Increased allopregnanolone levels in the fetal sheep brain following umbilical cord occlusion

Allopregnanolone (AP) is a potent modulator of the GABAA receptor. Brain AP concentrations increase in response to stress, which is thought to provide neuroprotection by reducing excitation in the adult brain. Umbilical cord occlusion (UCO) causes hypoxia and asphyxia in the fetus, which are major risk factors associated with poor neurological outcome for the neonate, and may lead to adverse sequelae such as cerebral palsy. The aims of this study were as follows: (i) to determine the effect of 10 min UCO on AP concentrations in the extracellular fluid of the fetal brain using microdialysis, and (ii) to compare the content of the steroidogenic enzymes P450scc and 5α‐reductase type II (5αRII) with brain and CSF neurosteroid concentrations. UCO caused fetal asphyxia, hypertension, bradycardia and respiratory acidosis, which returned to normal levels after 1–2 h. AP concentrations in dialysate samples from probes implanted in grey and white matter of the parietal cortex were significantly increased 1 h after UCO from control levels of 10.4 ± 0.4 and 12.4 ± 0.3 to 26.0 ± 5.1 and 27.6 ± 6.4 nmol l−1, respectively (P < 0.05), before returning to pre‐occlusion levels by 3–4 h after UCO. When fetal brains were collected 1 h after a 10 min UCO, the relative increases of AP and pregnenolone content in the parietal cortex were similar to the increase observed in the extracellular (dialysate) fluid. AP, but not pregnenolone, was increased in CSF at this time. P450scc and 5αRII enzyme expression was significantly increased in the cerebral cortex in the UCO fetuses compared to control fetuses. These results suggest that the fetal brain is capable of transiently increasing neurosteroid production in response to asphyxia. The action of the increased neurosteroid content at GABAA receptors may serve to diminish the increased excitation due to excitotoxic amino acid release, and provide short‐term protection to brain cells during such stress.

Allopregnanolone in the brain: Protecting pregnancy and birth outcomes

A successful pregnancy requires multiple adaptations in the mothers brain that serve to optimise foetal growth and development, protect the foetus from adverse prenatal programming and prevent premature delivery of the young. Pregnancy hormones induce, organise and maintain many of these adaptations. Steroid hormones play a critical role and of particular importance is the progesterone metabolite and neurosteroid, allopregnanolone. Allopregnanolone is produced in increasing amounts during pregnancy both in the periphery and in the maternal and foetal brain. This review critically examines a role for allopregnanolone in both the maternal and foetal brain during pregnancy and development in protecting pregnancy and birth outcomes, with particular emphasis on its role in relation to stress exposure at this time. Late pregnancy is associated with suppressed stress responses. Thus, we begin by considering what is known about the central mechanisms in the maternal brain, induced by allopregnanolone, that protect the foetus(es) from exposure to harmful levels of maternal glucocorticoids as a result of stress during pregnancy. Next we discuss the central mechanisms that prevent premature secretion of oxytocin and consider a role for allopregnanolone in minimising the risk of preterm birth. Allopregnanolone also plays a key role in the foetal brain, where it promotes development and is neuroprotective. Hence we review the evidence about disruption to neurosteroid production in pregnancy, through prenatal stress or other insults, and the immediate and long-term adverse consequences for the offspring. Finally we address whether progesterone or allopregnanolone treatment can rescue some of these deficits in the offspring.

Plasma oxytocin and nocturnal uterine activity: Maternal but not fetal concentrations increase progressively during late pregnancy and delivery in rhesus monkeys*

OBJECTIVE The purpose of the study was to determine whether rising maternal or fetal plasma oxytocin concentrations could be responsible for the increasing levels of nocturnal uterine activity on the nights preceding delivery. STUDY DESIGN Chronically catheterized pregnant rhesus monkeys were exposed to a 16-hour light, 8-hour dark photoperiod (dark 11 PM to 7 AM). Uterine activity and maternal arterial plasma oxytocin concentrations were measured concurrently at weekly intervals in late gestation, on the night preceding term delivery (158 to 167 days, n = 4), and during delivery (149 to 170 days, n = 6). Fetal carotid arterial plasma oxytocin levels were measured during episodes of nocturnal uterine activity in six animals. The effect of oxytocin infusions into the fetus (30 to 480 ng/kg/hr) on uterine activity and on maternal and fetal plasma oxytocin levels was also determined (n = 3). RESULTS Maximal nocturnal oxytocin concentrations in the maternal plasma rose progressively during late gestation from 9.9 +/- 3.5 pg/ml at 130 to 139 days to 28.7 +/- 9.8 pg/ml on the night preceding term delivery (p < 0.005); a significant increase in nocturnal uterine activity accompanied this rise (p < 0.001). Maternal oxytocin concentrations were elevated during labor and increased further at delivery (62.5 +/- 5.5 pg/ml, p < 0.05). There was no increase in fetal plasma oxytocin during nocturnal uterine activity (3.1 +/- 0.2 pg/ml) or during labor. Fetal oxytocin infusions raised fetal plasma oxytocin concentrations sixtyfold but had no effect on maternal plasma oxytocin concentrations or on uterine activity. CONCLUSIONS Elevated maternal plasma oxytocin concentrations are responsible, at least in part, for the increasing magnitude of nocturnal uterine activity episodes as term approaches and for the elevated uterine activity before delivery at night. Fetal plasma oxytocin does not contribute to nocturnal uterine activity or to maternal plasma oxytocin concentrations.

Neurosteroids in the fetus and neonate: Potential protective role in compromised pregnancies

Complications during pregnancy and birth asphyxia lead to brain injury, with devastating consequences for the neonate. In this paper we present evidence that the steroid environment during pregnancy and at birth aids in protecting the fetus and neonate from asphyxia-induced injury. Earlier studies show that the placental progesterone production has a role in the synthesis and release of neuroactive steroids or their precursors into the fetal circulation. Placental precursor support leads to remarkably high concentrations of allopregnanolone in the fetal brain and to a dramatic decline with the loss of the placenta at birth. These elevated concentrations influence the distinct behavioral states displayed by the late gestation fetus and exert a suppressive effect that maintains sleep-like behavioral states that are present for much of fetal life. This suppression reduces CNS excitability and suppresses excitotoxicity. With the availability of adequate precursors, mechanisms within the fetal brain ultimately control neurosteroid levels. These mechanisms respond to episodes of acute hypoxia by increasing expression of 5alpha-reductase and P450scc enzymes and allopregnanolone synthesis in the brain. This allopregnanolone response, and potentially that of other neurosteroids including 5alpha-tetrahydrodeoxycorticosterone (TH-DOC), reduces hippocampal cell death following acute asphyxia and suggests that stimulation of neurosteroid production may protect the fetal brain. Importantly, inhibition of neurosteroid synthesis in the fetal brain increases the basal cell death suggesting a role in controlling developmental processes late in gestation. Synthesis of neurosteroid precursors in the fetal adrenal such as deoxycorticosterone (DOC), and their conversion to active neurosteroids in the fetal brain may also have a role in neuroprotection. This suggests that the adrenal glands provide precursor DOC for neurosteroid synthesis after birth and this may lead to a switch from allopregnanolone alone to neuroprotection mediated by allopregnanolone and TH-DOC.

Sex-dependent effect of a low neurosteroid environment and intrauterine growth restriction on foetal guinea pig brain development

Progesterone and its neuroactive metabolite, allopregnanolone, are present in high concentrations during pregnancy, but drop significantly following birth. Allopregnanolone influences foetal arousal and enhances cognitive and behavioural recovery following traumatic brain injury. Inhibition of allopregnanolone synthesis increases cell death in foetal animal brains with experimental hypoxia. We hypothesised that complications during pregnancy, such as early or preterm loss of placental steroids and intrauterine growth restriction (IUGR), would disrupt the foetal neurosteroid system, contributing to poor neurodevelopmental outcomes. This study aimed to investigate the effects of chronic inhibition of allopregnanolone synthesis before term and IUGR on developmental processes in the foetal brain. Guinea pig foetuses were experimentally growth restricted at mid-gestation and treated with finasteride, an inhibitor of allopregnanolone synthesis. Finasteride treatment reduced foetal brain allopregnanolone concentrations by up to 75% and was associated with a reduction in myelin basic protein (MBP) (P = 0.001) and an increase in glial fibrillary acidic protein expression in the subcortical white matter brain region (P < 0.001). IUGR resulted in decreased MBP expression (P < 0.01) and was associated with a reduction in the expression of steroidogenic enzyme 5α-reductase (5αR) type 2 in the foetal brain (P = 0.061). Brain levels of 5αR1 were higher in male foetuses (P = 0.008). Both IUGR and reduced foetal brain concentrations of allopregnanolone were associated with altered expression of myelination and glial cell markers within the developing foetal brain. The potential role of neurosteroids in protecting and regulating neurodevelopmental processes in the foetal brain may provide new directions for treatment of neurodevelopmental disorders in infants who are exposed to perinatal insults and pathologies.