A review of fundamental principles for animal models of DOHaD research: an Australian perspective.

Epidemiology formed the basis of 'the Barker hypothesis', the concept of 'developmental programming' and today's discipline of the Developmental Origins of Health and Disease (DOHaD). Animal experimentation provided proof of the underlying concepts, and continues to generate knowledge of underlying mechanisms. Interventions in humans, based on DOHaD principles, will be informed by experiments in animals. As knowledge in this discipline has accumulated, from studies of humans and other animals, the complexity of interactions between genome, environment and epigenetics, has been revealed. The vast nature of programming stimuli and breadth of effects is becoming known. As a result of our accumulating knowledge we now appreciate the impact of many variables that contribute to programmed outcomes. To guide further animal research in this field, the Australia and New Zealand DOHaD society (ANZ DOHaD) Animals Models of DOHaD Research Working Group convened at the 2nd Annual ANZ DOHaD Congress in Melbourne, Australia in April 2015. This review summarizes the contributions of animal research to the understanding of DOHaD, and makes recommendations for the design and conduct of animal experiments to maximize relevance, reproducibility and translation of knowledge into improving health and well-being.

Effects of omega-3 and omega-6 fatty acids on human placental cytokine production.

INTRODUCTION: Dietary supplementation with omega-3 long chain polyunsaturated fatty acids (n-3 PUFAs) may exert benefits in pregnancy through inhibition of placental inflammation. However, studies on the anti-inflammatory effects of n-3 PUFAs in the placenta are lacking. We compared the cytokine responses of human placental explants in vitro after 4 days pre-incubation with either: a) individual n-3 or n-6 PUFAs (20 µM), or b) physiologically relevant combinations of low, medium or high n-3 or n-6 PUFA concentrations. METHODS: Placental cytokine (IL-6 and TNF-α) mRNA expression and protein production were assessed at 4 h and 12 h, respectively. Cytokine and fatty acid concentrations were also measured in placentas delivered at term by women who ingested either low (n = 12) or high (n = 10) amounts of fish/fish oil in the month prior to delivery. RESULTS: Pre-exposure to n-3 PUFAs as individual fatty acids results in reduced placental IL-6 production (P < 0.05), whereas exposure to complex fatty acid mixtures enriched in n-3 PUFAs (high n-3:n-6 ratios) results in a significant stimulation of IL-6 production (P < 0.05). There were no differences in placental n-3 or n-6 PUFA levels between women with either high or low dietary fish oil intake and no differences in cytokine expression. DISCUSSION: In summary, data from our complex lipid explant model and an observational cohort study do not support a role for n3 PUFAs in the suppression of pro-inflammatory cytokine expression in the human placenta. Results from studies of placental tissues exposed to single n-3 and n-6 PUFAs should be interpreted with considerable caution.

The inflammatory state of the rat placenta increases in late gestation and is further enhanced by glucocorticoids in the labyrinth zone.

INTRODUCTION: Inflammation plays central roles in key aspects of successful reproduction: ovulation, implantation and parturition. In this study we characterised the inflammatory profile of the rat placenta in late gestation with and without maternal glucocorticoid (dexamethasone) treatment. METHODS: Placentas (n = 6/group) were collected from untreated (Con) rats at days 16 and 22 (term = day 23) and from dexamethasone-treated (Dex) rats at day 22. mRNA and protein expression was determined for enzymes of prostaglandin synthesis and metabolism (Ptgs-1, Ptgs-2, 15-Pgdh), pro-inflammatory cytokines (Tnf-α, Il-1ß, Il-6), and the macrophage marker Emr-1 in the junctional (JZ) and labyrinth (LZ) zones of the placenta. RESULTS: Tnf-α, Il-1ß and Il-6 mRNAs all increased (2- to 4-fold) in both placental zones between days 16 and 22 (P < 0.01). Ptgs-2 mRNA (30-fold; P < 0.01) and PTGS-2 protein (2.4-fold; P < 0.05) similarly increased in LZ. In contrast, 15-Pdgh expression increased in JZ but decreased in LZ; these changes were accompanied by decreased levels of PGE2 in the JZ and a trend towards increased LZ levels. Dex treatment inhibited fetal and placental growth, but had minimal effects on expression of Ptgs-1, Ptgs-2 or 15-Pdgh. Nevertheless, Dex treatment increased LZ PGE2 levels (5-fold, P < 0.01) at the end of gestation. Dex treatment increased Tnf-α mRNA expression in LZ (40%; P < 0.05), but modestly suppressed cytokine protein expression in JZ. CONCLUSIONS: These data demonstrate that the inflammatory state of the LZ increases near term coincident with the known increase in local glucocorticoid levels. This suggests the classic anti-inflammatory actions of glucocorticoids do not occur in the placental LZ.

Kiss1 and Kiss1r mRNA expression in the rat placenta: changes with gestational age and regulation by glucocorticoids.

INTRODUCTION: Kisspeptin, the neuropeptide product of the KISS1 gene, is synthesized by neurons within the hypothalamus and is critical for fertility. Human placenta also expresses KISS1 and kisspeptin receptor (KISS1R) mRNA within the trophoblast compartment, where it is thought to act as a physiological invasion inhibitor. METHODS: We determined the expression of Kiss1 mRNA in rat placenta and examined the effect of gestational age and feto-placental growth restriction, achieved through excess maternal glucocorticoid exposure. RESULTS: Dexamethasone induced fetal growth restriction at both day 16 and day 22 of gestation, but placental growth restriction only at day 22. Real-time quantitative RT-PCR revealed an increase in Kiss1 and Kiss1r mRNA from day 16-22 in the labyrinth and junctional zones of the rat placenta. Immunolocalization confirmed kisspeptin expression in the placenta and was prominent in trophoblast tissue. Dexamethasone exposure elevated the expression of Kiss1 mRNA in the labyrinth and junctional zones of day 16 placentas. In contrast, Kiss1 mRNA in the labyrinth zone was reduced following dexamethasone-treatment at day 22. Kiss1r expression was increased in both placental zones at day 16 and 22 in response to dexamethasone-treatment. CONCLUSIONS: We confirm the presence of Kiss1 and Kiss1r mRNA in the rat placenta with expression increasing over the final third of pregnancy, suggestive of a role in restricting placental growth. Furthermore, the effects of dexamethasone on placental Kiss1/Kiss1r suggest glucocorticoid-induced placental growth retardation could be mediated, in part, via early stimulation of Kiss1 and the subsequent inhibition of trophoblast proliferation and invasion.

A rhythmic placenta? Circadian variation, clock genes and placental function.

Physiological rhythms entrained by the circadian clock are present in virtually all organs including those of the reproductive system. In mammals, circadian timing is driven by a 'master clock' in the suprachiasmatic nucleus that influences peripheral tissue clocks via endocrine, autonomic and behavioral cues. The molecular clock machinery comprises a network of 'clock' genes, namely Clock, Bmal1, Per1, Per2, Per3, Cry1 and Cry2. These clock genes generate endogenous oscillations that drive rhythmic expression of downstream genes and thus physiological and behavioral processes. Importantly, disturbances in clock gene expression are implicated in a range of pathologies including cancer and obesity. The recent recognition that clock genes are expressed in the placenta, together with observations linking circadian disruption with compromised placental function, suggests that circadian variation may be an important component of the normal placental phenotype. In this review we consider this possibility in the context of maternal circadian physiology in pregnancy. While there is good evidence for rhythmic expression of several genes in the rodent placenta, the conventional transcriptional-translational feedback loops of the clock machinery appear less robust and coordinated. Further study is needed to elucidate the function of the placental clock genes across gestation and among different species, particularly those in which greater circadian development occurs in utero. Such studies will likely provide important insights into placental physiology and pathology.

Oxysterols inhibit differentiation and fusion of term primary trophoblasts by activating liver X receptors.

Oxygenated cholesterol metabolites known as oxysterols display potent biological activities ranging from regulation of lipid homeostasis to cytotoxicity. Oxysterols have previously been shown to inhibit the invasion of first trimester trophoblasts, an effect which involves activation of the nuclear liver X receptors (LXRs). In the present study, we investigated the effects of several oxysterols on syncytialisation (differentiation and fusion) in term placental trophoblasts. Treatment of cultured term primary trophoblast cells with oxysterols [25-hydroxycholesterol, 7-ketocholesterol, 22(R)-hydroxycholesterol] and the synthetic LXR agonist T0901317 at non-toxic doses decreased expression of GCM-1 and HERV-W mRNA and reduced hCG secretion and placental alkaline phosphatase activity, indicative of diminished trophoblast differentiation. Furthermore, treatment with these compounds also decreased cell fusion measured by E-cadherin immunostaining and quantification of syncytialised nuclei. Treatment with an LXR antagonist (geranylgeranyl diphosphate) abrogated the inhibitory effects of oxysterols and T0901317 on trophoblast syncytialisation indicating that these effects are mediated by LXR. These findings suggest that oxysterols impair differentiation and fusion of term trophoblast cells via an LXR-dependent mechanism.

IFPA Meeting 2009 workshops report.

Workshops are an important part of the annual meeting of the International Federation of Placenta Associations (IFPA). At IFPA Meeting 2009 diverse topics were discussed in twelve themed workshops. Topics covered included: immune response to pregnancy; signaling between fetus and placenta; bioactive lipids in placenta; placenta in agricultural species; epigenetics and placentation; trophoblast deportation; glucocorticoids and placental function; endothelium; placental transport; genes and placenta; uteroplacental blood flow and placental stem cells. This report is a full summary of the various topics covered.

Placental and fetal growth retardation following partial progesterone withdrawal in rat pregnancy.

This study investigated placental expression of the two main isoforms of the progesterone receptor and the regulation of placental and fetal growth by progesterone over the final third of rat pregnancy, the period of maximal fetal growth. Expression patterns of mRNAs encoding the two major progesterone receptor isoforms (PR-A and PR-B) were measured by real-time RT-PCR in the two morphologically- and functionally-distinct regions of the placenta, the basal and labyrinth zones, at days 16 and 22 of pregnancy (term=day 23). PR-A and PR-B mRNA expression was extremely low in labyrinth zone on days 16 and 22, close to the limits of detection. In contrast, the basal zone exhibited much higher levels of mRNA expression for both PR-A (>10-fold higher than in labyrinth zone) and PR-B (3-fold higher at day 16). To assess the role of progesterone in placental growth, maternal progesterone was reduced from day 16 by ovariectomy with full estradiol replacement and partial progesterone replacement until day 22. Progesterone reduction lowered fetal (10%), whole placental (24%), basal zone (37%) and labyrinth zone (14%) weights at day 22 compared with sham-controls, whereas fetal and placental weights (both zones) were maintained in ovariectomised rats given full estradiol/progesterone replacement. The effects of progesterone withdrawal were not associated with changes in placental expression of either IGF-II or IGFBP-2, both important players in growth factor regulation of placental growth. Importantly, however, IGF-II expression remained elevated in the labyrinth zone but fell markedly in basal zone ( approximately 7-fold) between days 16 and 22 of normal pregnancy, consistent with the growth patterns of these two placental regions over this period.

The expression of apoptosis related genes in the first trimester human placenta using a short term in vitro model.

Using in vitro model for studying the induction and inhibition of spontaneous apoptosis in human first trimester placental villi, mediated by the free radical scavenger SOD, we have examined the expression of bcl-xL, bax, Caspase-3 and PARP (Poly ADP-ribosyl). An increase in apoptosis was associated with activation of PARP and an increase and activation of Caspase-3. There was no significant change in bcl-x or bax. Therefore bcl-x and bax do not appear to have a significant role in apoptosis in the first trimester in vitro. Cleavage of Caspase-3 rather than transcriptional regulation appears to be the main determinant of Caspase-3 activity in first trimester placental villi.

Developmental changes in plasma leptin and hypothalamic leptin receptor expression in the rat: peripubertal changes and the emergence of sex differences.

Leptin, the peptide hormone product of the ob gene, regulates food intake and energy expenditure at the hypothalamic level via the long-form of the leptin receptor (Ob-Rb). Leptin also plays a key role in determining the onset of puberty, but there is controversy as to whether leptin provides a trigger for puberty or is a permissive signal. Thus, although leptin administration can advance puberty onset in rodents, circulating leptin appears stable across puberty. While these data suggest a permissive role for leptin in rat puberty, it is possible that a change in hypothalamic response to leptin (e.g. via increased Ob-Rb expression) could enhance leptin action and thus trigger puberty without a rise in circulating leptin. In the present study we assessed developmental changes in hypothalamic Ob-Rb mRNA and protein expression in female and male rats from late fetal to postpubertal life. Quantitative RT-PCR showed that Ob-Rb mRNA increased (P<0.05) by around fivefold from fetal to postpubertal life in both females and males. These increases in Ob-Rb mRNA expression were gradual, but did not increase significantly between postnatal day 30 (pre-puberty) and day 51 (post-puberty). By day 51, hypothalamic Ob-Rb mRNA expression was higher (P<0.05) in females relative to males. Hypothalamic Ob-Rb protein showed a comparable developmental pattern (approximate threefold increase from fetal to postpubertal life), although a significant increase (15%; P<0.05) was observed between days 30 and 51 in females. Plasma leptin levels exhibited a dynamic pattern in both male and female rats during the prepubertal period, characterised by a precipitous fall after birth, relative stability to day 5, then a rapid increase to a transient peak on day 12. Plasma leptin then remained unchanged from day 15 in female rats but increased in males after puberty, thus confirming the well-recognised sex difference in adult rat leptin levels. In conclusion, this study shows that developmental increases occur not only in plasma leptin but also in hypothalamic Ob-Rb expression, suggesting that both are likely to influence the timing of puberty onset. Moreover, our data show that sex differences in both hypothalamic Ob-Rb and plasma leptin emerge only after puberty.

Myometrial expression of 11 beta-hydroxysteroid dehydrogenase type 2 in rat pregnancy.

The enzyme 11 beta-hydroxysteroid dehydrogenase type 2 (11 beta-HSD2), which reduces glucocorticoid potency in target cells by metabolism of active glucocorticoids, is expressed in the non-pregnant rat uterus in an oestrogen-dependent manner. Because glucocorticoids appear to facilitate parturition in many species, expression of 11 beta-HSD2 in pregnant myometrium is likely to influence pregnancy maintenance and possibly the onset and progression of labour. The present study therefore examined myometrial 11 beta-HSD2 mRNA, protein and bioactivity across rat pregnancy, with emphasis on the peripartum period. A single 1.9 kb transcript of 11 beta-HSD2 mRNA was evident in myometrium at all stages, with maximal (P<0.05) levels observed at day 16 (term=day 23). Consistent with this pattern of mRNA expression, Western blot analysis showed the presence of a 40 kDa 11 beta-HSD2 protein at all stages, with the maximal immunoreactive signal also observed on day 16. The 11 beta-HSD2 signal was immunolocalized to myometrial smooth muscle cells and endometrial stromal cells. Bioactivity specific to 11 beta-HSD2 was detectable in myometrium at all stages, but in contrast to the patterns of 11 beta-HSD2 mRNA and protein, the V(max) decreased at the beginning of pregnancy and remained stable until term. The apparent K(m) of 11 beta-HSD2 for corticosterone increased from 47 +/- 11 nM in non-pregnant myometrium to 75 +/- 7 nM by day 10 of pregnancy, and remained high until returning to an intermediate level on the day of delivery (60 +/- 8 nM). Progesterone competitively inhibited 11 beta-HSD2 bioactivity (K(i)=1.75 muM) whereas 20 alpha-hydroxypregn-4-en-3-one, the other major progestin present during rat pregnancy, had no such effect. In conclusion, these data suggest that local levels of active glucocorticoid in the myometrium are determined by the net effects of myometrial 11 beta-HSD-1 and -2 expression across pregnancy. Because the previously reported increase in myometrial 11 beta-HSD-1 near term occurs with little change in myometrial 11 beta-HSD2 bioactivity, this is likely to facilitate parturition by increasing local concentrations of active glucocorticoid.

Sex differences in response to steroids in preterm sheep lungs are not explained by glucocorticoid receptor number or binding affinity.

We recently reported that prenatal glucocorticoid therapy is less effective at promoting an improvement in lung function in male than in female sheep. This observation, and the higher incidence of respiratory distress syndrome in human males, suggests that the male fetal lung may be less responsive to glucocorticoids than is the female fetal lung. Since glucocorticoids are known to exert their effects via specific cytoplasmic glucocorticoid receptors (GR), we hypothesized that there may be sexual dimorphism in either the number or binding affinity of lung GR. To test the hypothesis, binding of dexamethasone (a synthetic glucocorticoid, 0.5-40 nM) by cytosolic fractions of male (n = 16) and female (n = 16) fetal sheep lung was measured at 125 days gestation (term = 148 days). Scatchard analysis of dexamethasone binding showed that the total number of GR (Bmax) did not significantly differ between male (346 +/- 42 fmol/mg protein) and female (277 +/- 23 fmol/mg protein) fetuses. The measured binding affinity (Kd) in male fetal lungs (6.85 +/- 0.43 nM) was not significantly different from that in females (8.46 +/- 1.02 nM). In conclusion, this study suggests that sex differences in fetal sheep lung responses to glucocorticoid therapy are not due to differences in the number or binding affinity of lung GR.

Apoptosis in rat placenta is zone-dependent and stimulated by glucocorticoids.

Apoptosis, or physiological cell death, is elevated in the placenta of human pregnancies complicated by fetal growth retardation, suggesting that placental apoptosis may be a key factor in the overall control of feto-placental growth. The present study used DNA internucleosomal fragmentation analysis to characterize apoptosis in the two morphologically and functionally distinct regions of the rat placenta, the basal and labyrinth zones, during the last week of pregnancy (Days 16, 22, and 23). In addition, because glucocorticoids are potent inhibitors of feto-placental growth and can stimulate apoptosis in other tissues, we examined whether dexamethasone treatment in vivo induces placental apoptosis. DNA fragmentation was clearly evident in both placental zones at each stage of pregnancy, with higher levels evident in the basal zone compared with the labyrinth zone on Days 22 and 23. TUNEL analysis, which identifies dying cells in situ, demonstrated positive staining of cells in the basal zone, particularly giant trophoblast cells. Dexamethasone treatment increased DNA fragmentation in the basal zone but not the labyrinth zone. Similarly, maternal treatment with carbenoxolone, which can enhance local concentrations of endogenous glucocorticoid by inhibition of 11 beta-hydroxysteroid dehydrogenase, also increased DNA fragmentation in the basal zone but not in the labyrinth zone. These effects of dexamethasone and carbenoxolone on placental apoptosis were associated with reduced placental and fetal weights. In conclusion, this study shows that apoptosis occurs in both zones of the rat placenta, particularly in the basal zone near term, and is elevated after increased glucocorticoid exposure in vivo. These data support the hypothesis that placental apoptosis is an important player in the regulation of feto-placental growth, and establish the rat as a useful model to study the endocrine control of placental apoptosis.

Increased fetal glucocorticoid exposure delays puberty onset in postnatal life.

The fetal environment is now recognized as a key determinant of the adult phenotype, being linked to development of diseases, including hypertension, as well as the timing of puberty. Such links may be related, in part, to the level of fetal exposure to maternal glucocorticoids in utero, which is normally regulated by placental expression of the enzyme 11beta-hydroxysteroid dehydrogenase (11beta-HSD). The present study examined whether manipulation of fetal glucocorticoid exposure, either directly or indirectly via 11beta-HSD inhibition, influences the subsequent timing of puberty. Administration of dexamethasone acetate at low (LDEX, 0.25 microg/ml drinking water) or high doses (HDEX, 1 microg/ml) or carbenoxolone (CBX, 2 x 10 mg/day, sc; an inhibitor of 11beta-HSD) to pregnant rats from day 13 to term (day 23) reduced offspring birthweight (LDEX: 9%; HDEX: 27%; CBX: 8%) and resulted in a subsequent delay in the onset of puberty in females (control: 41.4 +/- 0.5; LDEX: 44.8 +/- 0.7; HDEX: 48.5 +/- 0.4; CBX: 43.6 +/- 0.5 days). Importantly, the effects of CBX were not observed in the absence of maternal adrenals, indicating that they were mediated by increased fetal exposure to endogenous maternal glucocorticoids. In contrast, maternal treatment with metyrapone (MET; an inhibitor of glucocorticoid synthesis; 500 microg/ml drinking water from day 13) increased birthweight by 5% and advanced puberty onset in male offspring (control: 48.8 +/- 1.0; MET: 45.7 +/- 0.8 days). Changes in the timing of puberty onset were not attributable to changes in either bodyweight at puberty or peripubertal plasma leptin concentrations. Peripubertal plasma LH was also unaffected in animals with delayed puberty but was elevated in male offspring of MET-treated mothers. Collectively, these results demonstrate that fetal glucocorticoid exposure is an important determinant of the timing of puberty onset in postnatal life, and that this effect is operable within the normal physiological range of glucocorticoid concentrations.

Full induction of rat myometrial 11beta-hydroxysteroid dehydrogenase type 1 in late pregnancy is dependent on intrauterine occupancy.

The 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD-1) enzyme catalyses the conversion of the biologically inert glucocorticoid 11-dehydrocorticosterone to active corticosterone (11-oxoreductase activity) in vivo, and it is dramatically up-regulated in uterine myometrium in the days leading up to parturition. 11beta-HSD-1 is likely to enhance local concentrations of glucocorticoid within the myometrium and thus facilitate uterine contractility, but the stimulus for the increase in myometrial 11beta-HSD-1 is unknown. The objective of the present study was to test whether the induction of myometrial 11beta-HSD-1 is dependent on uterine occupancy or systemic hormonal signals of late pregnancy. This involved use of a unilateral pregnancy (ULP) model in which the gravid and nongravid uterine horns are both exposed to the normal systemic hormonal milieu of pregnancy. Western blot analysis showed that the 11beta-HSD-1 signal was only partially induced in the nongravid horn of ULP rats on Day 22 of pregnancy (term: Day 23). Moreover, artificial distension of this nongravid horn had no effect on myometrial 11beta-HSD-1 immunoreactivity or bioactivity at either Day 16 or Day 22 of pregnancy. Removal of fetuses and placentas on Day 18 reduced myometrial 11beta-HSD-1 bioactivity 4 days later, and this effect was not overcome by artificial maintenance of uterine distension. In contrast, after fetectomy at Day 18 (i.e., removal of the fetus but not placenta), myometrial 11beta-HSD-1 bioactivity was largely maintained on Day 22, indicative of placental support for myometrial 11beta-HSD-1 over this period. In conclusion, our data show that full induction of myometrial 11beta-HSD-1 expression and associated 11-oxoreductase bioactivity late in rat pregnancy is dependent upon intrauterine occupancy. Although the hormonal milieu of late pregnancy appears to stimulate myometrial 11beta-HSD-1 marginally, full induction clearly requires an additional stimulus. Manipulations involving fetectomy and artificial uterine distension indicate that the placenta provides at least part of this stimulus, but uterine stretch does not appear to play a role.

Dual function of 11beta-hydroxysteroid dehydrogenase in placenta: modulating placental glucocorticoid passage and local steroid action.

Target cell metabolism of glucocorticoids is now recognized as an important modulator of ligand access to the glucocorticoid receptor (GR). This metabolism occurs via two distinct 11beta-hydroxysteroid dehydrogenase (11beta-HSD) enzymes (types 1 and 2) that catalyze interconversion of active glucocorticoids (cortisol and corticosterone) and their inactive 11-keto products (cortisone and 11-dehydrocorticosterone, respectively). The focus of this review is on the biology of the 11beta-HSD enzymes in the placenta, where they also regulate passage of maternal glucocorticoids to the fetus. The presence of this metabolic barrier at the maternal-fetal interface is potentially crucial to fetal growth and development, since maternal glucocorticoid levels are elevated in pregnancy and since excess glucocorticoid exposure in fetal life has detrimental effects on prenatal growth and increases susceptibility to disease in subsequent adult life. In primates, transplacental glucocorticoid passage also appears to play an important role in the induction of an autonomous fetal hypothalamic-pituitary-adrenal axis near term. Placental 11beta-HSD is also likely to modulate glucocorticoid actions within the placenta, per se, by regulating their access to placental GR. Moreover, because some progesterone effects are exerted via the GR, placental 11beta-HSD may regulate progesterone-glucocorticoid competition for access to this receptor and thereby affect the biological actions of both steroids in the placenta.

Tissue-specific messenger ribonucleic acid expression of 11beta-hydroxysteroid dehydrogenase types 1 and 2 and the glucocorticoid receptor within rat placenta suggests exquisite local control of glucocorticoid action.

Placental 11beta-hydroxysteroid dehydrogenase (11beta-HSD) regulates transplacental passage of maternal glucocorticoids to the fetus and is thus a key determinant of fetal glucocorticoid levels. It has also been proposed that placental 11beta-HSD expression may influence local glucocorticoid actions by regulating access of corticosterone to the glucocorticoid receptor (GR) or mineralocorticoid receptor (MR). Therefore, the present study used a rat model to assess whether the GR or MR are coexpressed with the two forms of 11beta-HSD (types 1 and 2) in the placental labyrinth zone, the major site of maternal-fetal transfer, and in the basal zone, the primary site of placental hormone synthesis. In situ hybridization analysis was used to assess messenger RNA (mRNA) expression for the GR, MR, 11beta-HSD-1, and 11beta-HSD-2 in the two placental zones on days 16, 19 and 22 of pregnancy (term = day 23). Whereas expression of the GR appeared relatively unchanged in both zones at these three stages of pregnancy, that of 11beta-HSD-1 clearly increased in the labyrinth zone but fell in basal zone, whereas the opposite pattern of expression was observed for 11beta-HSD-2. MR expression was not detected at any stage. The pattern of placental 11beta-HSD-2 mRNA expression over days 16, 19, and 22 of pregnancy was paralleled by changes in 11beta-HSD-2-specific bioactivity, but despite clear expression of 11beta-HSD-1 mRNA, no bioactivity attributable to this enzyme was measurable in either placental zone. To assess the role of fetal adrenal maturation on these changes in 11beta-HSD, two experimental models, maternal adrenalectomy and fetectomy, were employed. Maternal adrenalectomy on day 13 advanced maturation of the fetal adrenal cortex but had no effect on 11beta-HSD-2 bioactivity in either of the placental zones at day 19. Placental 11beta-HSD-2 bioactivity on day 22 was also unaffected by fetectomy 3 or 6 days earlier. In conclusion, the consistent expression of the GR in the two placental zones late in pregnancy suggests that concomitant and marked changes in 11beta-HSD-1 and 11beta-HSD-2 expression could have a major influence on glucocorticoid action in the placenta at this time. Moreover, the changes in 11beta-HSD expression appear to be unrelated to development of the fetal adrenal cortex and are likely to reduce the placental glucocorticoid barrier near the end of pregnancy.

Immunolocalization of 11beta-hydroxysteroid dehydrogenase types 1 and 2 in rat uterus: variation across the estrous cycle and regulation by estrogen and progesterone.

Glucocorticoid hormone action in several target tissues is dependent not only on the expression of the glucocorticoid receptor, but also on that of the 11beta-hydroxysteroid dehydrogenase (11betaHSD) enzymes, 11betaHSD-1 and -2. In the uterus, glucocorticoids can exert inhibitory effects on a range of important functions, particularly in relation to the effects of estrogen. Therefore, the present study examined immunolocalization of the two 11betaHSD enzymes in the rat uterus at each stage of the estrous cycle and after ovariectomy with or without estrogen/progesterone replacement. In cycling rats 11betaHSD-1 was localized to luminal and glandular epithelial cells and to eosinophils in both the endometrial stroma and myometrium. In contrast, 11betaHSD-2 immunostaining was localized to endometrial stromal cells and myometrial cells, with no staining evident in epithelial cells or eosinophils. Immunostaining for both enzymes was cycle dependent, being maximal at proestrus and minimal at diestrus. Western blot analysis of whole uterus at proestrus showed the presence of 34- and 40-kDa immunoreactive species for 11betaHSD-1 and -2, respectively. These immunoreactive signals were almost abolished by ovariectomy, but this effect was reversed for both enzymes by estrogen replacement with or without progesterone. These effects of ovariectomy and steroid replacement were confirmed by immunocytochemical analysis, with the exception that progesterone appeared to enhance the stimulatory effects of estrogen on 11betaHSD-2 specifically within the endometrial stroma. In conclusion, these results establish the presence of both 11betaHSD-1 and -2 in the nonpregnant rat uterus and show distinct distributions for the two enzymes and cyclic variation related to positive regulation by ovarian steroids. The physiological implications of these patterns of 11betaHSD expression will ultimately depend on the reaction direction for each enzyme, but 11betaHSD-2 is likely to limit disruptive effects of glucocorticoids on the endometrial stroma, and 11betaHSD-1 may then serve to selectively reactivate glucocorticoids in epithelial cells.

Circadian variation in basal plasma corticosterone and adrenocorticotropin in the rat: sexual dimorphism and changes across the estrous cycle.

Sexual dimorphism in the rat hypothalamic-pituitary-adrenal axis was investigated by determination of plasma corticosterone and immunoreactive (I-) ACTH in males and in females at each stage of the estrous cycle. A serial blood-sampling technique enabled assessment of covariation of the two hormones across the full circadian range of their concentrations within individual animals. Distinct diurnal rhythms in plasma corticosterone were evident in all rats, and the degree and timing of this rhythmicity, determined by cosinor analyses, did not vary with gender or cycle stage. There were, however, marked differences in absolute levels of corticosterone across the estrous cycle, with the average daily concentration (mesor) increasing progressively from a minimum at estrus (129 +/- 11 ng/ml) to a maximum 3 days later at proestrus (246 +/- 14 ng/ml). The mesor corticosterone value in male rats (102 +/- 21 ng/ml) was not different from that in estrous females, but was lower than that in females at all other stages of the cycle. In contrast, no gender- or cycle-related differences were detected in absolute levels of I-ACTH, although distinct diurnal rhythms, synchronous with those for corticosterone, were evident in all groups. Accordingly, a strong and positive within-rat relationship between plasma corticosterone and I-ACTH was observed in all groups, but there was a clear shift in the nature of this relationship across the estrous cycle, such that the slope (i.e. concentration of plasma corticosterone per unit concentration of I-ACTH) was minimal in males and estrous females and maximal in proestrous females. In conclusion, this study shows that the extent of sexual dimorphism in resting plasma corticosterone levels is dependent on estrous cycle stage, being absent at estrus and maximal at proestrus. Moreover, this variation in plasma corticosterone was not accompanied by corresponding changes in plasma I-ACTH, suggestive of cycle-related changes in responsiveness of the adrenal cortex to trophic stimulation.