Progesterone receptor isoform expression in response to in utero growth restriction in the fetal guinea pig brain.

Intra-uterine growth restriction (IUGR) is a significant in utero complication that can have profound effects on brain development including reduced myelination and deficits that can continue into adulthood. Progesterone increases oligodendrocyte proliferation and myelin expression, an action that may depend on the expression of progesterone receptor (PR) isoforms A (PRA) and B (PRB). The objective of this study was to determine the effect of IUGR on PR isoform expression in the brain of male and female fetuses and whether effects were associated with a reduction in myelination. We used a guinea pig model that involves selective reduction in maternal perfusion to the placenta at midgestation (35 days, term 70 days). This resulted in a significant reduction in body weight with marked sparing of brain weight. PRA, PRB and myelin basic protein (MBP) expression were measured in the brains of male and female growth-restricted and control fetuses at late gestation. MBP, as a measure of myelination, was found to decrease in association with IUGR in the CA1 hippocampal region with no change observed in the cortical white matter. There was a marked increase in PRA, PRB and total PR expression in the IUGR fetal brain. Control female fetuses demonstrated significantly higher PRA:PRB ratios than males; however, this sex difference was abolished with IUGR. These data suggest the central nervous system effects of clinical use of progesterone augmentation therapy in late pregnancy should be carefully evaluated. The overall upregulation of PR isoforms in association with IUGR suggests increased progesterone action and a possible neuroprotective mechanism.

Inhibition of neurosteroid synthesis increases asphyxia-induced brain injury in the late gestation fetal sheep.

Allopregnanolone (AP) is a potent GABAergic agonist that suppresses CNS activity, seizure threshold, and excitotoxicity in the adult brain. AP is present in the fetal sheep brain and increases rapidly after asphyxial insult due to increased 5alpha-reductase type-2 (5alphaR-2) expression. The aim of this study was to use finasteride to suppress fetal neurosteroid synthesis, and then determine the effect on brain injury, particularly in the hippocampus, of asphyxia induced in utero by brief occlusion of the umbilical cord. Catheters and an inflatable umbilical cord cuff were implanted in fetal sheep at approximately 125 days gestation. Five days later the fetuses received either finasteride (20 mg/kg/h) or vehicle (40% hydroxypropyl-beta-cyclodextrin) for 2 h. The umbilical cord was occluded (UCO) for 5 min at 30 min after starting the infusion. The fetal brain was obtained 24 h later for examination of activated caspase-3 expression as an index of apoptosis, and to measure AP content. Finasteride treatment alone significantly reduced AP content and increased the number of caspase-3 positive cells in the hippocampus, cerebellum, and the subcallosal bundle, indicating that AP modulates the normal rate of apoptosis in the developing brain. UCO in vehicle and finasteride-treated fetuses produced a similar, marked decrease in O2 saturation (5.8+/-0.6%), but after finasteride treatment UCO caused a significantly greater increase in the number of caspase-3 positive cells in the hippocampal cornu ammonis 3 (CA3) (57.3+/-1.6%) compared with the vehicle-treated fetuses. Thus, 5alpha-reduced steroids such as AP may be protective in reducing cell death following acute fetal asphyxia. Perturbation of normal fetal neurosteroid levels in late gestation (e.g. due to preterm birth, or maternal synthetic steroid treatment to induce fetal lung maturation) could adversely affect brain development and increase its vulnerability to injury.

Effects of combined IUGR and prenatal stress on the development of the hippocampus in a fetal guinea pig model.

Intrauterine growth restriction (IUGR) and maternal stress during pregnancy are two compromises that negatively impact neurodevelopment and increase the risk of developing later life neuropsychiatric disorders such as schizophrenia, depression and behavioural disorders. Neurosteroids, particularly allopregnanolone, are important in protecting the developing brain and promoting many essential neurodevelopmental processes. Individually, IUGR and prenatal stress (PS) reduce myelination and neurogenesis within affected fetal brains, however less information is available on the combined effects of these two disorders on the term fetal brain. This study aimed to investigate how IUGR and PS impairs the neurosteroid pathway when combined using a guinea pig model, and how these then disrupt the neurodevelopment of the fetus. Uterine artery blood flow restriction was performed at GA30-35 to induce growth restriction, whilst PS was induced by exposure of the dam to a strobe light during gestation commencing GA40 and repeated every 5 days. Exposure in this model caused reductions in hippocampal CA1 MBP immunostaining of male fetuses in both IUGR alone and IUGR+PS paradigms but only by IUGR in the subcortical white mater, compared with control males. Plasma allopregnanolone was reduced by both stressors irrespective of sex, whereas GFAP or MAP2 expression were not affected by either stressor. Female neurodevelopment, as assessed by these markers, was unimpeded by these compromises. The addition of prenatal stress did not further compound these deficits.

Effect of postnatal progesterone therapy following preterm birth on neurosteroid concentrations and cerebellar myelination in guinea pigs.

Allopregnanolone protects the fetal brain and promotes normal development including myelination. Preterm birth results in the early separation of the infant from the placenta and consequently a decline in blood and brain allopregnanolone concentrations. Progesterone therapy may increase allopregnanolone and lead to improved oligodendrocyte maturation. The objectives of this study were to examine the efficacy of progesterone replacement in augmenting allopregnanolone concentrations during the postnatal period and to assess the effect on cerebellar myelination - a region with significant postnatal development. Preterm guinea pig neonates delivered at 62 days of gestation by caesarean section received daily s.c. injections of vehicle (2-Hydroxypropyl-ß-cyclodextrin) or progesterone (16 mg/kg) for 8 days until term-equivalent age (TEA). Term delivered controls (PND1) received vehicle. Neonatal condition/wellbeing was scored, and salivary progesterone was sampled over the postnatal period. Brain and plasma allopregnanolone concentrations were measured by radioimmunoassay; cortisol and progesterone concentrations were determined by enzyme immunoassay; and myelin basic protein (MBP), proteolipid protein (PLP), oligodendroctye transcription factor 2 (OLIG2) and platelet-derived growth factor receptor-α (PDGFRα) were quantified by immunohistochemistry and western blot. Brain allopregnanolone concentrations were increased in progesterone-treated neonates. Plasma progesterone and cortisol concentrations were elevated in progesterone-treated male neonates. Progesterone treatment decreased MBP and PLP in lobule X of the cerebellum and total cerebellar OLIG2 and PDGFRα in males but not females at TEA compared with term animals. We conclude that progesterone treatment increases brain allopregnanolone concentrations, but also increases cortisol levels in males, which may disrupt developmental processes. Consideration should be given to the use of non-metabolizable neurosteroid agonists.

Preterm birth affects GABAA receptor subunit mRNA levels during the foetal-to-neonatal transition in guinea pigs.

Modulation of gamma-aminobutyric acid A (GABAA) receptor signalling by the neurosteroid allopregnanolone has a major role in late gestation neurodevelopment. The objective of this study was to characterize the mRNA levels of GABAA receptor subunits (α4, α5, α6 and δ) that are key to neurosteroid binding in the brain, following preterm birth. Myelination, measured by the myelin basic protein immunostaining, was used to assess maturity of the preterm brains. Foetal guinea pig brains were obtained at 62 days' gestational age (GA, preterm) or at term (69 days). Neonates were delivered by caesarean section, at 62 days GA and term, and maintained until tissue collection at 24 h of age. Subunit mRNA levels were quantified by RT-PCR in the hippocampus and cerebellum of foetal and neonatal brains. Levels of the α6 and δ subunits were markedly lower in the cerebellum of preterm guinea pigs compared with term animals. Importantly, there was an increase in mRNA levels of these subunits during the foetal-to-neonatal transition at term, which was not seen following preterm birth. Myelination was lower in preterm neonatal brains, consistent with marked immaturity. Salivary cortisol concentrations, measured by EIA, were also higher for the preterm neonates, suggesting greater stress. We conclude that there is an adaptive increase in the levels of mRNA of the key GABAA receptor subunits involved in neurosteroid action after term birth, which may compensate for declining allopregnanolone levels. The lower levels of these subunits in preterm neonates may heighten the adverse effect of the premature decline in neurosteroid exposure.

Control of oxytocin secretion by ovine corpora lutea: effects of arachidonic acid, phospholipases, and prostaglandins.

The involvement of arachidonic acid and arachidonic acid metabolites in the control of oxytocin secretion by ovine corpus luteum was investigated, using slices of luteal tissue incubated in vitro. Oxytocin was secreted at steady rates by luteal slices, during 60-min incubations (315.0 +/- 45.3 pg/mg.h). The secretion of oxytocin was stimulated by arachidonic acid, phospholipase A2 (PLA2), and phospholipase C (PLC) in a dose-dependent manner. The highest doses of arachidonic acid, PLA2, and PLC used stimulated oxytocin secretion by 145.8 +/- 23.0% (P less than 0.01; n = 6), 331.5 +/- 42.4% (P less than 0.02; n = 4), and 955.5 +/- 278.6% (P less than 0.01; n = 4), respectively. Oxytocin secretion by luteal slices was not affected by either prostaglandin F2 alpha (PGF2 alpha) or PGE2 over a concentration range from 3-3000 nM. Furthermore, inhibitors of the cyclo-oxygenase pathway of arachidonic acid metabolism did not consistently affect arachidonic acid and PLA2-stimulated oxytocin secretion. Nordihydroguaiaretic acid, which inhibits 5-lipoxygenase, however, totally abolished arachidonic acid- and reduced PLA2-stimulated oxytocin secretion. The presence of CoCl2 in the incubation medium also significantly reduced basal and PLA2- and PLC-stimulated oxytocin secretion [P less than 0.05 (n = 5), P less than 0.05 (n = 5), and P less than 0.01 (n = 6), respectively]. We have shown that oxytocin secretion from slices of ovine corpus luteum incubated in vitro is stimulated by exogenous and endogenously released arachidonic acid. The data show that PGF2 alpha and PGE2 do not have a role in luteal oxytocin secretion in vitro and PG formation does not appear to be involved in the stimulation of oxytocin secretion elicited by arachidonic acid or PLA2. Arachidonic acid may have its effect via the lipoxygenase pathway.

Glucocorticoids stimulate the expression of prostaglandin endoperoxide H synthase-2 in amnion cells.

Corticosteroids increase the production of prostaglandin E2 (PGE2) and the activity of prostaglandin endoperoxide H synthase (PGHS) in cultured amnion cells, although they inhibit prostanoid biosynthesis in numerous other cell types. This suggests that glucocorticoids control the level of PGHS in amnion cells by a hitherto unexplored, positive regulatory mechanism. We have tested the possibility that corticosteroids act by stimulating the expression of messenger RNAs (mRNAs) encoding one or both isoforms of PGHS. Ribonuclease protection assays were used to determine the levels of PGHS-1 and -2 mRNAs and, for reference, gamma-actin mRNA levels in confluent primary cultures of human amnion cells. In untreated cultures, PGHS-1 and -2 mRNA levels were low, often not reaching the level of detection. Dexamethasone (DEX) treatment for 4 h resulted in a measurable level of PGHS-2 mRNA, which increased further 10-fold and 20-fold after incubation with the glucocorticoid for 8 h and 16 h, respectively. The stimulation was dependent on DEX concentration, and was concomitant with an increase in the capacity of the cells to metabolize arachidonic acid to PGE2. PGHS-1 mRNA levels remained low in DEX-treated cells, while the gamma-actin message level showed no change. Estradiol and progesterone had no influence on PGHS-2 mRNA expression, but cortisol increased the PGHS-2 mRNA abundance. The glucocorticoid antagonist RU486 blocked the effect of DEX. Conditioned media of DEX-treated cells did not contain steroid-induced factor(s) stimulating PGE2 production. Inhibition of protein synthesis by cycloheximide potentiated the effect of DEX, and raised the abundance of PGHS-1, PGHS-2, and gamma-actin mRNAs in untreated cells. DEX did not affect the stability of the PGHS-2 mRNA. These results show that glucocorticoids promote PGE2 synthesis by amnion cells by stimulating the expression of PGHS-2 mRNA in a receptor-dependent, selective, and immediate fashion.

Estrogen action in the reproductive tract of rhesus monkeys during antiprogestin treatment.

We previously reported that RU486 can reverse progesterone (P)-induced suppression of the estrogen receptor (ER) in the uterus of pregnant rhesus monkeys, but we did not determine whether estrogen (E) could act through its receptor in the presence of P and RU486. To pursue this question, we treated spayed rhesus monkeys with various hormonal regimens and evaluated the effects of E in the oviduct and endometrium, with and without RU486 treatment, on ER and progestin receptor (PR) levels, morphology, apoptosis, and degree of proliferation. The latter was assessed immunocytochemically with a monoclonal antibody (Ki-67) against a nuclear antigen present only in proliferating cells. Animals were treated for 2 weeks with 17 beta-estradiol (E2) and then for 2 weeks with E2 plus P to produce a regressed oviduct and a secretory endometrium. The animals were then treated for 2 more weeks with four different treatments, as follows: I) E2, P, and vehicle; II) E2 and vehicle; III) E2, P, and RU486; and IV) E2 and RU486 (n = 4 each). In group I, menstruation did not occur, and the endometrium exhibited stromal cell enlargement, extensive development of the spiral arteries, few Ki-67-positive cells, and low levels of ER and PR. Oviducts in group I remained regressed, Ki-67-positive epithelial cells were few, and levels of ER and PR were low. In contrast, in groups II, III, and IV, the oviducts had responded to E2 and were fully ciliated and secretory, with elevated levels of ER and nuclear PR. All animals in these three groups menstruated and then regenerated their endometrium. The regenerated endometria expressed elevations in ER, nuclear PR, and epithelial Ki-67 index. However, the endometria of RU486-treated monkeys in groups III and IV had significantly more epithelial cell death by apoptosis, increased stromal cell compaction, and fewer Ki-67-positive stromal cells than in the E2 controls (group II). In group IV, RU486 caused a significant decrease in endometrial weight. Thus, RU486 blocked P action and allowed E2 to act in a normal fashion in the oviduct and endometrium on several end points, but it also had antiproliferative effects that opposed E2 action, especially in the endometrium.

Prostaglandin endoperoxide-H synthase-1 and -2 messenger ribonucleic acid levels in human amnion with spontaneous labor onset.

Increased prostaglandin (PG) production within the uterine compartment has a pivotal role in the processes leading to labor onset in women. Two PG endoperoxide-H synthase (PGHS) isoenzymes have been identified in a number of cell types. PGHS-1 is constitutively expressed in most cases, whereas PGHS-2 expression is rapidly induced by several agonists. The aims of this study were to determine the levels of PGHS-1 and PGHS-2 expression before and after spontaneous labor (SL) onset in the amnion and to assess the contribution of PGHS-1 and PGHS-2 to enzyme activity. We established and validated ribonuclease protection assays to quantify PGHS-1 and PGHS-2 messenger ribonucleic acid (mRNA) levels in the amnion. PGHS enzyme activity was measured with an established assay. The antisense RNA probes used in the protection assays were generated using human PGHS-1 and PGHS-2 complementary DNAs. These probes specifically detected the 2.8-kilobase mRNA of PGHS-1 and the 4.8-kilobase mRNA of PGHS-2 in amnion RNA samples on Northern blots. We measured mRNA levels in amnion from patients after SL at term and from patients not in labor undergoing elective cesarean section (CS) at term. PGHS-2 mRNA levels were markedly higher after SL compared to levels in CS amnion [5.18 +/- 1.08 (n = 16) and 2.27 +/- 0.50 (n = 15), densitometric units, respectively; P < 0.02], whereas there was no difference in PGHS-1 mRNA levels after labor compared with CS samples. PGHS-2 mRNA levels were also positively correlated with PGHS enzyme activity in 4 separate assays with a total of 25 patients (r = 0.65-0.88; P < 0.05). There was no correlation between PGHS-1 mRNA levels and enzyme activity. We conclude that PGHS-2 mRNA is present in human amnion; its levels are elevated after SL onset, and they are correlated with enzyme activity. The stimulation of PGHS activity at labor onset probably involves increased expression of PGHS-2. The expression of PGHS-1 does not change in association with labor in human amnion.

Steroid hormone receptors in the adrenal glands of fetal and adult rhesus monkeys.

Sex steroid hormone receptors have been identified in the adrenal glands of rodents and may have a role in adrenal function. The highly estrogenic environment during pregnancy has been proposed to influence steroidogenesis by the fetal zone of the primate fetal adrenal gland. In order to determine whether these effects involve receptor-mediated mechanisms, we have examined the concentration and distribution of estrogen receptor (ER), androgen receptor, and progesterone receptor (PR) in the adrenal glands of fetal, immature, and adult rhesus monkeys. Monoclonal antibodies were used for immunocytochemistry (ICC), and in a gradient shift assay, for determination of receptor distribution and concentrations, respectively. There was no difference between the concentrations of ER in the adrenal glands from male and female adult animals (12.4 +/- 2.2, n = 3 and 13.2 +/- 2.0 fmol/mg DNA, n = 7; respectively); however, the concentration of ER in the fetal adrenal glands was markedly lower than in the adults (combined adult 12.7 +/- 1.6, n = 10, and fetal 0.9 +/- 0.4 fmol/mg DNA, n = 7; P less than 0.01). The concentration of ER in the adrenal glands of immature animals was also lower compared to adult animals (6.1 +/- 1.6, n = 6, P less than 0.05). In the adult, ICC revealed that staining for ER was restricted to the cell nucleus and was most dense in the zone fasciculata, with lesser staining in the zona glomerulosa and zona reticularis, and with no detectable staining in the medulla. ER staining was virtually absent in the fetal zone which comprises the major portion of the fetal gland; however, some staining was observed in the narrow definitive zone. The distribution of androgen receptor was similar to that of ER, whereas there was no detectable staining for PR in the adrenals of either adult or fetal animals. We conclude: 1) that the lower concentration of ER in fetal adrenal glands is due to the absence of ER in the fetal zone; 2) the lack of ER and PR in the fetal zone suggests that estrogens and progestins do not influence the growth or function of the fetal zone by receptor-mediated mechanisms; 3) estrogens and androgens may influence the function of the adult adrenal cortex.

Comparison of plasma oxytocin and catecholamine concentrations with uterine activity in pregnant rhesus monkeys.

Pregnant rhesus monkeys exhibit diurnal changes in uterine activity (UA), with episodes of increased UA during the early hours of darkness. The estrogenic environment during late pregnancy serves a permissive role in the maintenance of nocturnal UA episodes and may involve myometrial interactions with oxytocin (OT) and/or alpha-adrenergic stimuli. In the present study we have used chronically catheterized pregnant rhesus monkeys to measure diurnal changes in maternal plasma OT, epinephrine, norepinephrine, and dopamine. We also determined the effects of infusing an OT antagonist (ORF 22164) and the alpha-adrenergic antagonist phentolamine on nocturnal UA episodes. Animals were exposed to a 16-h light, 8-h dark photo-period, with the hours of darkness between 2300-0700 h. Maternal plasma samples were collected at 3-h intervals for 36 h and analyzed by RIA for OT and by high performance liquid chromatography for catecholamines. Plasma OT was correlated with UA in animals that displayed nocturnal UA episodes (r = 0.76; P less than 0.01). Maximal OT concentrations occurred at 2400 h in these animals; plasma OT was higher during the hours of darkness compared to levels during the light phase (10.4 +/- 1.9 and 3.0 +/- 0.3 pmol/L, respectively; n = 4). Some animals did not display nocturnal episodes of increased UA and showed no increase in OT concentrations during the hours of darkness. Maternal plasma catecholamine concentrations were not correlated with nocturnal UA and were maximal during the light phase. Nocturnal UA was abolished within 30 min of infusion of the OT antagonist, but phentolamine infusions had no effect on nocturnal UA. We conclude that 1) changes in maternal plasma catecholamine concentrations are not involved in the generation of nocturnal UA; 2) the presence of episodes of increased UA at night results from increased maternal plasma OT concentrations; and 3) the absence of nocturnal UA in some animals can be explained by a reduced level of OT secretion.

Prostaglandin endoperoxide-H synthase (PGHS) activity and immunoreactive PGHS-1 and PGHS-2 levels in human amnion throughout gestation, at term, and during labor.

Prostaglandins (PGs) are of primary importance in the initiation and maintenance of labor in women. A major intrauterine source of prostaglandins is the amnion, which synthesizes increased amounts of PGE2 at term labor. Because PG endoperoxide-H synthase (PGHS) catalyzes the rate-limiting step of PG synthesis from arachidonic acid, we investigated the changes in amniotic PGHS specific activity during gestation and at term and preterm labor. Also, we determined the level of immunoreactive PGHS protein in the amnion to evaluate the mechanisms by which PGHS activity may be regulated. PGHS specific activity, measured at the amount of PGE2 produced by amnion microsomes under optimal conditions, was 18.2 +/- 3.7 pg PGE2/micrograms protein.min (mean +/- SE; n = 19) at term (37-42 weeks gestation) before the spontaneous onset of labor. PGHS specific activity was significantly higher after spontaneous term labor (38.9 +/- 6.0 pg PGE2/micrograms protein.min; n = 19; P < 0.05). Amnion samples from preterm (< 36 weeks gestation) nonlaboring patients contained low levels of PGHS specific activity (5.9 +/- 1.8 pg PGE2/micrograms protein.min; n = 9), which increased significantly with spontaneous preterm labor (28.3 +/- 6.8 pg PGE2/micrograms protein.min; n = 10; P < 0.05). Longitudinal analysis of the data showed that PGHS specific activity was low in the first and second trimesters of gestation, but increased dramatically before labor onset at term. We detected PGHS protein in all microsomal samples, with an antiovine PGHS antibody recognizing both PGHS-1 and -2 isoforms of the enzyme. However, there was no correlation between PGHS specific activity and the amount of immunoreactive PGHS protein. Using an antibody specific for PGHS-2, we detected immunoreactive protein in only 9 of the 25 tissues examined and found no correlation between PGHS specific activity and the amount of PGHS-2 protein. These results suggest that 1) PGHS specific activity in the amnion increases sharply before the onset of labor at term; 2) further increases in specific activity occur during term and preterm labor; and 3) the specific activity of PGHS in the amnion is not related directly to the amount of immunoreactive enzyme protein.

Effects of pregnanolone on behavioural parameters and the responses to GABA(A) receptor antagonists in the late gestation fetal sheep.

Placental progesterone metabolites may suppress fetal behaviour by interacting with GABA(A) receptors. In an initial study, the effect of 5beta-pregnan-3alpha-ol-20-one (pregnanolone) given as a bolus (2.5 and 5.0 mg) or infused at a rate of 25 mg/h was investigated in unanaesthetized, catheterized fetal sheep, 127-135 days gestation. The incidence of fetal breathing movements (FBM) and behavioural arousal activity, defined as nuchal muscle electromyographic (EMG) activity during low voltage electrocortical (LV ECoG) activity were suppressed by pregnanolone administered as a bolus, while the pregnanolone infusion produced a significant decrease in arousal and EOG activity, and an increase in the presence of HV ECoG. The effect of pregnanolone on fetal behaviour and arousal induced by the GABA(A) antagonist picrotoxin was also investigated. Picrotoxin was given as a bolus (approximately 300 microg/kg) and pregnanolone was subsequently administered as a bolus (5.0 mg), and behavioural parameters were recorded and analysed. The incidence of arousal and FBM were 1.1 +/- 1.6 min/10 min and 2.5 +/- 2.3 min/10 min, respectively, before picrotoxin treatment and increased during the 10-20 and 20-30 min epochs after picrotoxin treatment (arousal: 5.0 +/- 2.2 and 6.5 +/- 3.6 min/10 min, respectively, n = 6, P < 0.05; FBM: 7.3 +/- 3.2 and 9.3 +/- 1.2 min/10 min, respectively, n = 6, P < 0.05). The picrotoxin-induced increases in arousal and FBM were significantly suppressed (n = 6, P < 0.05) by pregnanolone treatment to 1.6 +/- 1.5 min/10 min and 4.6 +/- 2.3 min/10 min, respectively. We conclude that; (i) the GABA(A) active steroid pregnanolone suppresses basal and picrotoxin-induced fetal arousal and FBM; and (ii) steroid sensitive GABA(A) receptors may regulate fetal behaviour and breathing.

Characterisation of GABA(A) receptors in fetal, neonatal and adult ovine brain: region and age related changes and the effects of allopregnanolone.

Progesterone metabolites acting via GABA(A) receptors suppress central nervous system (CNS) activity. The aim of the present study was to examine binding characteristics of GABA(A) receptors in fetal, newborn and adult sheep brains using [(35)S]TBPS, and to determine the effects of allopregnanolone on this binding. Receptor affinity (K(D)) and density (B(MAX)) in the brainstem were not different in fetal, newborn (1-2 days old) and adult brains. In the hypothalamus K(D) and B(MAX) increased significantly in the fetus between 85 and 128 days gestation, and were then similar to postnatal and adult values. In the frontal cortex K(D) and B(MAX) increased progressively between 85 days and term ( approximately 147 days gestation), and were then not different from postnatal and adult values. The K(i) values for the GABA(A) receptor antagonist picrotoxin was similar at all ages. Allopregnanolone inhibited [(35)S]TBPS binding in the presence of 5 microM GABA, but enhanced binding in the absence of GABA. These results show that (i), functional GABA(A) receptors are present in the fetal brain from at least 85 days gestation; (ii), 3alpha-pregnane steroids modify receptor affinity in the late gestation fetal brain; and (iii) there are region-specific changes in GABA(A) receptor binding parameters. Steroid modulation of the GABA(A) receptor in the fetal brain is likely to influence fetal CNS activity in late gestation.

Regulation of oxytocin secretion by the ovine corpus luteum: effect of activators of protein kinase C.

The effect of protein kinase C activation and dibutyryl cyclic AMP on oxytocin secretion by ovine luteal tissue slices was investigated. Several putative regulators of luteal oxytocin secretion were also examined. Oxytocin was secreted by luteal tissue slices at a basal rate of 234.4 +/- 32.8 pmol/g per h (n = 24) during 60-min incubations. Activators of protein kinase C: phorbol 12, 13-dibutyrate (n = 8), phorbol 12-myristate, 13-acetate (n = 4) and 1,2-didecanoylglycerol (n = 5), caused a dose-dependent stimulation of oxytocin secretion in the presence of a calcium ionophore (A23187; 0.2 mumol/l). Phospholipase C (PLC; 50-250 units/l) also caused a dose-dependent stimulation of oxytocin secretion by luteal slices. Phospholipase C-stimulated oxytocin secretion was potentiated by the addition of an inhibitor of diacylglycerol kinase (R59 022; n = 4). These data suggest that the activation of protein kinase C has a role in the stimulation of luteal oxytocin secretion. The results are also consistent with the involvement of protein kinase C in PLC-stimulated oxytocin secretion. The cyclic AMP second messenger system does not appear to be involved in the control of oxytocin secretion by the corpus luteum.

Effect of alteration of maternal plasma progesterone concentrations on fetal behavioural state during late gestation.

Placental progesterone synthesis exposes the fetus to high levels of progesterone and progesterone metabolites during late gestation which may influence fetal behaviour. To determine the role of maternal progesterone synthesis in the control of fetal arousal state and fetal breathing movements (FBM), the effect of raising and lowering maternal progesterone concentrations was examined in chronically catheterised fetal sheep. Fetal and maternal vascular catheters, fetal tracheal and amniotic fluid catheters as well as electrodes for recording fetal electrocortical (ECoG), electro-ocular (EOG) and nuchal muscle electromyographic (EMG) activity were implanted between 118 and 122 days gestational age (GA). Progesterone, 100 mg, administered twice daily i.m. for 3 days (130-133 days GA) resulted in a marked elevation in maternal plasma progesterone concentrations (370 +/- 121%, n = 5, P < 0.05), but had no effect on fetal plasma concentrations. Fetal EOG episodes and the duration of fetal behavioural arousal were significantly suppressed throughout the progesterone treatment period (74.4-81.1% and 58-65% respectively, P < 0.05, n = 5). Four ewes received Trilostane (25 mg i.v.), a 3 beta-hydroxysteroid dehydrogenase inhibitor, between 136 and 140 days GA. Maternal and fetal progesterone concentrations were significantly lowered by 60 min after treatment (19.8 +/- 8.0% and 39.5 +/- 24.3% respectively, P < 0.05). The incidence of fetal EOG activity increased from a pretreatment level of 26.8 +/- 1.5 min/h to 30.3 +/- 2.8 min/h at 1-6 h and to 35.0 +/- 1.7 min/h (P < 0.05) during the 7-12 h after Trilostane treatment. The duration of FBM episodes was significantly higher at 1-6 h and 7-12 h after Trilostane treatment (19.5 +/- 3.0 and 23.6 +/- 5.5 min/h respectively, P < 0.05) compared with pretreatment levels (11.2 +/- 1.2 min/h). We conclude that increasing maternal progesterone levels suppresses fetal EOG activity and behavioural arousal, whereas reducing maternal progesterone synthesis leads to an elevation of EOG activity and FBM.

Changes in the expression of prostaglandin E and F synthases at induced and spontaneous labour onset in the sheep.

The differential production of prostaglandin (PG) F(2 alpha) and PGE(2) within the uterine compartment may play a role in controlling myometrial contraction. We hypothesized that the enzymes downstream of PG endoperoxide synthase-2 (PGHS-2) determine the ratio of PGF(2 alpha) and PGE(2) in the utero-ovarian vein plasma and the time of normal and preterm labour onset. The aim of this study was to simultaneously determine the expression of PGF and PGE synthases (PGFS and PGES) in gestational tissues at spontaneous and induced-preterm labour in sheep. Myometrial, endometrial and placental tissue were obtained from ewes in dexamethasone-induced preterm labour, age-matched control ewes, and ewes in spontaneous term labour for analysis of mRNA expression by real-time PCR. PGFS mRNA expression was significantly increased following dexamethasone-induced and spontaneous labour onset in placentome (P<0.01) but was unchanged in the myometrium and endometrium. In contrast, PGES mRNA expression remained unchanged or decreased. PGHS-2 mRNA expression was increased in all tissues examined in both dexamethasone-induced and spontaneous labour (P<0.001). Plasma PGE(2) and PGF(2 alpha) concentrations rose in both dexamethasone-induced and spontaneous labour with the ratio of PGF(2 alpha):PGE(2) increased with labour onset (P<0.05). These results are consistent with the hypothesis that the increased expression, of PGFS is responsible for the increased PGF(2 alpha):PGE(2) ratio and this, together with increased PGHS-2 expression, accounts for myometrial activity at labour onset. The findings point to PGFS expression as a key factor in regulating the uterotonic process in the sheep.

Effect of a neuroactive steroid infused into the cerebral ventricles of fetal sheep in utero using small infusion volumes.

Placental progesterone metabolites may influence fetal behaviour during late gestation. We have investigated the effect of the neuroactive metabolite, pregnanolone, on the sleep/awake behaviour of fetal sheep. These studies can only be effectively performed in unanesthetized animals with chronically implanted leads and catheters. The relatively large internal volumes of these catheters raises problems for cerebroventriclear infusions. Therefore, we developed a device with negligible deadspace ( approximately 10 microl) consisting of a ventricular cannula attached to a small cap which allowed the instillation of microl volumes of neuroactive steroids into a cerebral ventricle of fetal sheep in utero. Two catheters attached to the cap allow it to be filled in a push-pull configuration. The smaller internal diameter of the ventricular cannular prevents the solution entering the ventricle until the outflow catheter is occluded and solution is forced through the probe. Using this device, the infusion of 5beta-pregnane-3alpha-ol-20-one (pregnanolone, 1 mg), in 35% hydroxypropyl-beta-cyclodextrin, resulted in a marked suppression of fetal behaviour parameters indicative of arousal, while vehicle alone did not affect these parameters. We conclude that neuroactive steroids can be infused into the cerebroventricular system of chronically catheterised fetal sheep in small volumes and that pregnanolone may act directly to influence fetal behaviour.

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.

Effect of pregnane steroids on electrocortical activity and somatosensory evoked potentials in fetal sheep.

The effect of infusing the neuroactive steroids pregnanolone and iso-pregnanolone on somatosensory evoked potentials (SEP) and electrocortical (ECoG) activity was studied in unanaesthetised fetal sheep, 130-135 days gestation. Intravenous infusion of pregnanolone (6 mg/kg per h) significantly increased the proportion of high voltage ECoG (56.1+/-4.8% vs. control 43.5+/-3.2%, P < 0.05), and decreased low voltage ECoG (43.9+/-4.8% vs. control 56.6+/-3.2%, P < 0.05). Pregnanolone treatment decreased the amplitude of the N25 peak of the SEP (89.9+/-2.8% of control, P < 0.05) evoked following stimulation of the skin of the upper lip. In contrast, iso-pregnanolone treatment had no effect on ECoG activities, or on the amplitude and latency of peaks in the SEP. We conclude that 3alpha-hydroxy pregnane steroids are active at GABA(A) receptors in fetal sheep and can modulate sleep/wake activity before birth.