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.

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.

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.

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.

The hypothalamic-pituitary-adrenal axis in rat pregnancy and lactation: circadian variation and interrelationship of plasma adrenocorticotropin and corticosterone.

The circadian variation in immunoreactive (I-) ACTH and corticosterone was studied at several stages throughout rat pregnancy and compared with those before pregnancy and during lactation. Serial blood samples were obtained from chronically cannulated, conscious rats at 2- to 3-h intervals beginning at 0800 h at diestrus of the cycle; on days 2, 6, 10, 14, 18, and 22 of pregnancy (term = day 23); and on day 4 of lactation. Plasma I-ACTH and corticosterone were determined in all samples, and indices of their circadian variation (acrophase, mesor, and amplitude) were derived by cosinor analyses within each rat. A circadian variation in corticosterone was clearly evident in all groups, with individual cosinor r2 values being consistently high. Plasma I-ACTH also exhibited distinct circadian variation up to day 14 of pregnancy, but the cosinor r2 value then fell (P < 0.05, by analysis of variance) and remained low during lactation. Mesor levels (midpoint of the derived circadian range) of I-ACTH fell (P < 0.05) by 34% to 20.6 +/- 2.4 pg/ml by day 2 of pregnancy; corticosterone also fell (P < 0.05) by 34% to 141 +/- 27 ng/ml, and both changes were due primarily to reductions in peak levels. I-ACTH mesors then remained effectively unchanged for the remainder of pregnancy, but mesor corticosterone increased progressively after day 10 to reach a maximum of 286 +/- 28 ng/ml by day 22, and this rise was due to elevations in both trough and peak corticosterone levels. Further changes occurred after parturition, with mesor I-ACTH increasing more than 2-fold between day 22 of pregnancy and day 4 of lactation, whereas mesor corticosterone declined by more than half over the same period. Plasma corticosterone and I-ACTH were positively associated within rats in all groups (P < 0.01), but the common slope of this association increased considerably after midgestation, indicative of higher corticosterone levels for a given concentration of I-ACTH. In summary, this study identifies marked changes in the hypothalamic-pituitary-adrenal axis during rat pregnancy and lactation. Although a circadian variation in corticosterone was observed throughout, that in plasma I-ACTH was evident early in pregnancy but declined after midgestation. Absolute concentrations of plasma I-ACTH and corticosterone fell in parallel early in pregnancy, but corticosterone subsequently increased without any change in I-ACTH.(ABSTRACT TRUNCATED AT 400 WORDS)

11beta-Hydroxysteroid dehydrogenase type 2 in the rat corpus luteum: induction of messenger ribonucleic acid expression and bioactivity coincident with luteal regression.

The corpus luteum (CL) is the major source of progesterone during rat pregnancy, and its regression precedes and is essential for parturition. Recent studies show that luteal regression in the rat can be blocked by the administration of synthetic glucocorticoids, yet endogenous glucocorticoids are maximal at the time of normal luteal regression in pregnancy. This suggests that endogenous glucocorticoid may be inactivated locally within the CL, presumably via the enzyme 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD-2), which is known to regulate glucocorticoid access to receptors in other target tissues. This possibility was examined in the present study by measurement of 11beta-HSD-2 messenger RNA (mRNA) expression and bioactivity in rat CL over the second half of pregnancy, thus covering periods of maximal and minimal progesterone secretion. 11beta-HSD-2 bioactivity was measured in luteal homogenates obtained from rats on days 11, 16, 19, and 22 of pregnancy (term = day 23). Bioactivity was measurable in CL at each stage of pregnancy, with an apparent Km for corticosterone of approximately 100 nM. Enzyme activity was lowest on day 11 (maximum velocity, 1.0 +/- 0.6 pmol/min x mg protein), increased more than 5-fold by day 16 (6.2 +/- 0.5), then increased by an additional 4-fold by day 19 (24.3 +/- 4.3), and this high level of activity was maintained to day 22 (26.5 +/- 5.2). In kidney, the apparent Km for corticosterone was lower than that in CL, but remained unchanged throughout pregnancy (overall mean, 28.9 +/- 1.9 nM) as did the maximum velocity (overall mean, 25.4 +/- 1.3 pmol/min x mg protein). Consistent with the pattern of bioactivity in CL, mRNA for 11beta-HSD-2 was not detectable in CL by Northern analysis on either day 11 or day 16, but was clearly evident on days 19 and 22. In situ hybridization also revealed a substantial up-regulation of 11beta-HSD-2 expression specifically within the CL on days 19 and 22, whereas glucocorticoid receptor mRNA expression was consistent across all stages. In contrast, there was no detectable mRNA expression in CL for either 11beta-HSD-1 or the mineralocorticoid receptor at any stage. These data show that a marked induction of 11beta-HSD-2 mRNA expression and bioactivity occurs within the CL late in rat pregnancy and thus suggest that local inactivation of endogenous glucocorticoids facilitates luteal regression.

The effects of adrenocorticotropin and prolactin on adrenal dehydroepiandrosterone secretion in the baboon fetus.

The present study was designed to determine whether PRL, in addition to ACTH, stimulates adrenal secretion of dehydroepiandrosterone (DHA) in vivo at midgestation in the baboon fetus (Papio anubis). On day 100 of gestation (term = day 184), fetuses were exteriorized, and a constant infusion of saline (0.1 ml/min) was initiated via a fetal femoral vein. Forty minutes later, a bolus injection of 30 nmol ACTH/ml saline (n = 5), 40 nmol ovine PRL/ml saline (n = 4), or 1 ml saline (n = 5) was administered via the fetal femoral venous catheter. ACTH (0.15 nmol/min.0.1 ml saline), PRL (0.20 nmol/min.0.1 ml saline), or saline (0.1 ml/min) was then infused for an additional 25 min. Blood samples were obtained from the contralateral fetal femoral vein and the maternal saphenous vein immediately before and after peptide infusion and from the umbilical vein and artery at the end of the infusion. Fetal serum DHA concentrations (range, 9-11 micrograms/100 ml) were significantly increased (P less than 0.05) by PRL and ACTH, but not by saline. In contrast, fetal concentrations of cortisol (15-20 micrograms/100 ml) and DHA sulfate (DHAS; 13-18 micrograms/100 ml) were not altered by infusion of test substances into the fetus. The maternal concentrations of F (49-61 micrograms/100 ml) and DHAS (19-22 micrograms/100 ml) exceeded (P less than 0.05) respective values in the fetus, whereas DHA concentrations (2-3 micrograms/100 ml) in the mother were lower (P less than 0.05) than those in fetal serum. The serum concentrations of DHA, DHAS, and cortisol in the mother were not altered by PRL or ACTH. Regardless of the treatment, concentrations of DHA and DHAS in umbilical vein were lower (P less than 0.05) than those in the umbilical artery. These findings indicate that PRL as well as ACTH are effective in vivo in stimulating serum DHA concentrations in fetal baboons at midgestation. The greater concentration of DHA in umbilical artery vs. umbilical vein as well as the lack of response in maternal DHA concentrations indicate that the site of action of PRL and ACTH is the fetal adrenal. Therefore, we conclude that at midgestation, there is the potential for multifactorial regulation of baboon fetal adrenal androgen production and that PRL, in addition to ACTH, can function as a fetal adrenocorticotropic factor in vivo.

Metabolism of cortisol and cortisone in the baboon fetus at midgestation.

The metabolism of cortisol (F) and cortisone (E) in the fetal circulation is likely to influence the availability/biological potency of these corticosteroids and hence maturation of fetal organ systems. Therefore, we determined the MCR, production, peripheral interconversion, and placental extraction of F and E in the baboon fetus at midgestation. Radiolabeled F and E were infused into a femoral vein of fetuses (n = 7; 4 female, 3 male) exteriorized on day 100 of gestation (term = day 184). The MCR of F in the fetus (5.6 +/- 0.8 1/day) was lower (P less than 0.01) than that of E (13.1 +/- 2.2 1/day). Placental extraction of F (72.8 +/- 5.3%) and E (87.8 +/- 2.4%) were extensive indicating that the placenta contributes to fetal F/E MCR. Although the serum concentration (micrograms per dl) of F (20 +/- 2) exceeded (P less than 0.01) that of E (12 +/- 1), the calculated production rate (milligrams per day) of F (1.09 +/- 0.12) was not significantly different from that of E (1.55 +/- 0.27). The transfer constant for fetal conversion of F to E (29.0 +/- 6.0%) exceeded (P less than 0.01) that for reduction of E to F (1.8 +/- 0.4%). Therefore, the proportion of total F production derived from circulating E was only 2.2%, whereas the proportion of E derived from circulating F was 26.7%. These findings demonstrate that at midgestation the baboon fetus has minimal capacity for peripheral conversion of biologically inactive E to biologically active F, whereas the reverse conversion (F to E) is substantial.

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.

Activation of the baboon fetal hypothalamic-pituitary-adrenocortical axis at midgestation by estrogen-induced changes in placental corticosteroid metabolism.

We have hypothesized that the change in placental cortisol (F)-cortisone (E) metabolism induced by estrogen late in gestation is important to activation of the baboon fetal hypothalamic-pituitary-adrenocortical axis, culminating in the ontogenesis of de novo F secretion by the fetal adrenal. The present study tested this hypothesis in vivo by comparing the proportion of F in the fetus derived via maternal and fetal production on day 100 (n = 7; term = day 184) and day 165 (n = 4) in untreated baboons and on day 100 in baboons (n = 9) in which 50-mg pellets of androstenedione were implanted sc in the mother in increasing numbers (i.e. two on day 70, four on day 78, six on day 86, and eight on day 94) to increase placental estrogen production. Maternal, uterine, and umbilical venous samples were collected during constant maternal infusion (120 min) of [3H]F/[14C]E, endogenous and radiolabeled F/E content was determined, and corticosteroid dynamics were quantified. The MCR and peripheral interconversion of F and E as well as the production rate of F were unaltered in the mother. However, at midgestation, androstenedione increased (P less than 0.05) estrogen by 62% and altered transuterofeto placental F-E metabolism from preferential reduction of E (%F----E = 17 +/- 4; %E----F = 27 +/- 7) to preferential oxidation of F (%F----E = 26 +/- 2; %E----F = 5 +/- 2), a pattern similar to that at term (%F----E = 32 +/- 6; %E----F = 9 +/- 4). In untreated baboons, on day 100 none of the F in the fetus was due to fetal production, whereas by day 165, 49 +/- 6% was of fetal origin. In animals treated with androstenedione at midgestation, 22 +/- 4% of fetal F was derived de novo within the fetus. Thus, production of F by the fetus was negligible on day 100, increased near term in association with an increase in transplacental oxidation of F to E, and was induced at midgestation in baboons in which placental F-E metabolism was altered by an increase in estrogen production. These findings provide indirect evidence that supports our hypothesis that the change in placental F-E metabolism induced by increasing estrogen late in pregnancy results in activation of the fetal hypothalamic-pituitary-adrenocortical axis and, thus, ontogenesis of fetal F production near term.

Effect of estrogen on pituitary peptide-induced dehydroepiandrosterone secretion in the baboon fetus at midgestation.

We have previously shown that ACTH and PRL stimulate baboon fetal adrenal dehydroepiandrosterone (DHA) production both in vitro and in vivo and that estrogen diminishes the responsivity of the adrenal to tropic peptides in vitro. In the present study we determined the effects of increasing placental estrogen production by the administration of androstenedione at midgestation on DHA production by the baboon fetus in vivo. Pregnant baboons were untreated (n = 8) or treated (n = 9) with increasing numbers of androstenedione implants inserted in the mother at 8-day intervals between days 70-100 of gestation (term = day 184). On day 100, the fetuses were exteriorized, and a constant infusion of saline (0.1 ml/min) was initiated via a catheter inserted into a femoral vein of the fetus. At 40 min, a bolus injection of either 30 nmol ACTH or 40 nmol ovine PRL was administered to fetuses. ACTH or PRL (0.2 nmol/min.0.1 ml saline) were then infused for an additional 25 min. The concentrations of serum estradiol (E2) in the uterine vein (20.2 +/- 1.5 ng/ml; mean +/- SE) and estrone (E1) in umbilical vein (11.9 +/- 3.1 ng/ml) of androstenedione-treated baboons were 2-fold greater (P less than 0.05) than respective values in untreated baboons. Baseline concentrations of DHA in the femoral vein of the fetus were similar in all treatment groups (overall mean, 120 +/- 20 ng/ml) and greater (P less than 0.05) than values (27 +/- 3) in the mother. In untreated control baboons, basal DHA concentrations in the fetus were increased (P less than 0.05) by 69 +/- 17% and 94 +/- 29% after fetal injection of ACTH (n = 4) or PRL (n = 4), respectively. In contrast, neither PRL (n = 5) nor ACTH (n = 4) had any effect on serum DHA when injected into androstenedione-treated baboons. Regardless of treatment, injection of ACTH or PRL into the fetus had no effect on DHA concentrations in the mother. Collectively, these findings indicate that the ability of the fetal adrenal to increase DHA production in response to an acute infusion of ACTH or PRL was abolished in baboons in which placental estrogen production was increased prematurely at midgestation. Therefore, we suggest that during the second half of gestation in the baboon a regulatory system may exist in utero, in which there is feedback control of the placental product estrogen on the formation of the fetal adrenal precursor DHA.

Induction of myometrial 11beta-hydroxysteroid dehydrogenase type 1 messenger ribonucleic acid and protein expression late in rat pregnancy.

Myometrial function in pregnancy is regulated by a range of hormonal stimuli, including glucocorticoids, particularly in the period leading up to parturition. Glucocorticoid hormone action is dependent not only on expression of glucocorticoid receptor (GR) within target cells, but also on local expression of the enzyme 11beta-hydroxysteroid dehydrogenase (11betaHSD). Therefore, this study examined changes in myometrial 11betaHSD bioactivity and expression (messenger RNA and protein) of the 11betaHSD-1 isoform and whether 11betaHSD-1 and GR are colocalized to myometrial cells. Myometrial 11-oxoreductase activity (conversion of [3H]11-dehydrocorticosterone to [3H]corticosterone) was only just detectable (<6%) at the postestrus stage of the cycle and on days 5 and 10 of pregnancy, but then increased markedly by day 16 (45 +/- 2%). This activity increased further to maximal levels on day 22 of pregnancy (55 +/- 3%) and remained high on day 23 (term; 34 +/- 3%) before decreasing by 24 h postpartum (9 +/- 2%). High 11beta-dehydrogenase activity was evident before (87 +/- 1%) and during the first half (day 5,91 +/- 1%; day 10, 88 +/- 2%) of pregnancy, was lower on days 16 (55 +/- 2%), 22 (39 +/- 3%), and 23 (58 +/- 1%), then returned to prepregnancy levels 24 h postpartum (86 +/- 1%). The marked induction of 11-oxoreductase activity late in pregnancy was strongly and positively correlated with both 11betaHSD-1 messenger RNA expression (by Northern analysis) and protein (by Western analysis; r = 0.96 and 0.98, respectively; P < 0.001). Moreover, 11betaHSD-1 and GR immunoreactivity were colocalized to the smooth muscle cells of the myometrium and the uterine epithelium late in pregnancy. Collectively, these data demonstrate that a marked induction of 11betaHSD-1 expression occurs in the rat myometrium near term, and this is associated with increased 11-oxoreductase bioactivity. As GR is coexpressed in the myometrium, we suggest that the induction 11betaHSD-1 serves to enhance local glucocorticoid actions and thus facilitate parturition.

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.

The regulation of transplacental cortisol-cortisone metabolism by estrogen in pregnant baboons.

The present study tests the hypothesis that estrogen regulates the alteration in baboon placental cortisol (F)/cortisone (E) interconversion from preferential reduction (E----F) at midgestation to oxidation (F----E) near term. Five pregnant baboons (Papio anubis) received increasing numbers of 50-mg implants of androstenedione inserted sc at 8-day intervals between days 70 and 100 of gestation (term = day 184) to elevate the production of estrogen. Five animals served as controls at midgestation and received implants containing no steroid, while four baboons were studied near term between days 164-170 of gestation. All animals were bled from a maternal saphenous vein at 2-day intervals, and the serum was assayed for estradiol. On days 100 or 170 of gestation, transuterofetoplacental corticosteroid dynamics were determined by the constant infusion method. Baboons were anesthetized with ketamine and halothane-nitrous oxide and a constant infusion of [3H]F/[14C]E initiated via a maternal saphenous vein. At 60 min, animals were laparotomized and at 70, 80, and 90 min, blood samples were obtained from right and left uterine veins and from a maternal saphenous vein. At 95 min, an incision was made in the uterus, and blood samples were obtained from the umbilical vein and artery. Radiolabeled F and E were extracted from serum and purified by paper chromatography. Maternal serum E2 concentrations (nanograms per ml; mean +/- SE) were greater (P less than 0.01) between days 94 and 100 of gestation in androstenedione-treated baboons (2.4 +/- 0.3) than in untreated animals at midgestation (0.7 +/- 0.2), but lower than those near term (4.5 +/- 1.0). On day 100 of gestation, conversion of E to F across the uterus in control animals (30%) was similar to that of the reverse reaction (23%). In androstenedione-treated baboons at midgestation the conversion of E----F (8%) was lower (P less than 0.05) than the oxidation of F----E (27%) and not different from that in untreated baboons at term (E----F = 13%; F----E = 28%). The dominance of transuterofetoplacental conversion of F----E over the conversion of E----F in term and in androstenedione-treated animals at midgestation was maintained when transfer constants were corrected for fetal metabolic contributions. We conclude that the increase in placental estrogen production induced by androstenedione administration at midgestation alters the pattern of transuterofetoplacental F-E metabolism, supporting the hypothesis that estrogen regulates placental corticosteroid metabolism.

11 beta-Hydroxysteroid dehydrogenase in the rat placenta: developmental changes and the effects of altered glucocorticoid exposure.

The enzyme 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) catalyses the interconversion of corticosterone, the major glucocorticoid of the rat, and the biologically-inactive 11-dehydrocorticosterone. In the placenta, 11 beta-HSD is thought to regulate glucocorticoid transport between maternal and fetal compartments, and may also affect the local action of glucocorticoids. The present study assessed whether 11 beta-dehydrogenase (corticosterone to 11-dehydrocorticosterone) and 11-oxoreductase (11-dehydrocorticosterone to corticosterone) activities are both present in rat placenta, and whether these activities change with advancing pregnancy. Enzyme activity was estimated on days 16, 19 and 22 of pregnancy (term = day 23) in placental fragments incubated for 6 h with either [3H]corticosterone or [3H]11-dehydrocorticosterone. The percentage conversion of these substrates to [3H]11-dehydrocorticosterone and [3H]corticosterone, respectively, were determined at the end of the incubation. Both 11-oxoreductase and 11 beta-dehydrogenase activities were clearly evident in placental tissue fragments, and while 11-oxoreductase activity declined with advancing pregnancy (P < 0.01), 11 beta-dehydrogenase activity increased (P < 0.01). Thus, 11-oxoreductase exceeded (P < 0.05) 11 beta-dehydrogenase at day 16, but thereafter activities were similar. These changes do not appear to be glucocorticoid-induced, since pretreatment of rats with either metyrapone or dexamethasone acetate from day 15 of pregnancy did not affect placental 11 beta-HSD on day 22.(ABSTRACT TRUNCATED AT 250 WORDS)

Release of bioactive ACTH by perifused human placenta at early and late gestation.

This study assessed whether bioactive ACTH is released by the human placenta during perifusion in vitro at early and late gestation. Human placental villous fragments from early (8-12 weeks) and late (38-40 weeks) gestation were perifused at a constant rate for 6.5 h. To assess ACTH-like bioactivity released by this tissue, the perifusion effluent was redirected through adjacent chambers containing freshly dispersed adrenocortical cells obtained from adult rats. Baseline secretion of corticosterone by these adrenocortical cells averaged 95 +/- 26 (S.E.M.) fmol/min, and this increased at least fivefold (P < 0.01, two-way ANOVA) in response to placental effluent at early and late gestation. The magnitude of this increase, expressed as a percentage of the maximal response to a subsequent stimulus with ACTH(1-24), was similar for placentas obtained at early (41 +/- 12% of maximal response) and late (42 +/- 17%) gestation. Immunoreactive (I)-ACTH was readily detectable in placental effluent from all preparations (5.5 +/- 2.3 fmol/min per g tissue), and there was no apparent difference with stage of gestation. To determine whether all of the ACTH-like bioactivity released by the placenta was attributable to I-ACTH, a second series of placental/adrenal perfusions was conducted. In these, I-ACTH was selectively removed from placental effluent by immunoneutralization, and the residual bioactivity measured. Immunoneutralization involved preincubation of placental effluent with ACTH antiserum (1:100), and preincubation with normal rabbit serum (NRS) served as a control.(ABSTRACT TRUNCATED AT 250 WORDS)

Production rate, metabolic clearance rate and blood concentration of progesterone in conscious and anaesthetized pregnant rats.

Both production rate and metabolic clearance rate (MCR) of progesterone may vary rapidly and so effect short-term changes in blood concentration of the hormone. Here, a constant infusion and sampling technique was used to estimate these three characteristics of progesterone metabolism in seven conscious and ten anaesthetized rats on day 16 of pregnancy. After steady state was achieved, four samples were collected during a 1-h period from each rat. Mean values for production rate and MCR of progesterone in conscious rats were 14.0 +/- 1.4 mumol/day and 63.2 +/- 6.2 litres/day respectively. Both values were substantially reduced in anaesthetized rats (8.6 +/- 0.8 mumol/day and 39.4 +/- 3.4 litres/day respectively) and so blood concentration was unchanged. The production rate was positively related to the total mass of luteal tissue (common correlation coefficient, r = 0.61, P less than 0.05). There were no consistent changes in the three characteristics with time but variation within rats was high. The estimated coefficients of variation for production rate, MCR and blood concentration within rats were 26, 18 and 17% in conscious and 27, 20 and 23% in anaesthetized rats respectively. Short-term changes in production rate and MCR generally were in the same direction (P less than 0.05). This reduced variation in blood concentration which would otherwise have occurred if production rate and MCR were unrelated. The pregnant rat is clearly capable of rapid shifts in production rate, MCR and blood concentration of progesterone and the positive relationship between production rate and MCR has a homeostatic effect on blood concentration.

Production rate, metabolic clearance rate and uterine extraction of corticosterone during rat pregnancy.

This study examined changes in the blood concentration of corticosterone with the onset and progression of pregnancy in the rat. To identify the source of variation in blood corticosterone, the metabolic clearance rate (MCR) and production rate of corticosterone were also determined. Measurements were made in conscious rats (n = 4-7 per group) in the morning of dioestrus and days 5, 10, 16 and 22 of pregnancy (term = day 23). Corticosterone levels were 713 +/- 38 nmol/l (mean +/- S.E.M.) in non-pregnant rats, remained unchanged to day 10 of pregnancy, then increased to 1036 +/- 52 nmol/l by day 16 and remained high at day 22. The production rate of corticosterone appeared to increase during pregnancy from 25.6 +/- 1.7 mumol/day on day 10 to reach 36.3 +/- 3.3 mol/day on day 22, but this did not reach statistical significance (one-way ANOVA). The MCR of corticosterone was similar among all groups (overall mean 34.6 +/- 2.5 l/day), although a slight but non-significant fall was apparent at day 16. When account was taken of changes in maternal weight, the MCR decreased progressively from 139 +/- 10 1/day per kg before pregnancy to reach a minimum of 88 +/- 7 1/day per kg on day 16. Transuterine extraction of corticosterone on day 22 of pregnancy was 19.2 +/- 3.1% and so, based on this and estimates of uterine blood flow, the uterus must account for around 15% of corticosterone clearance at this time. Because this uterine contribution is effectively additional clearance, it is likely that without it the MCR of corticosterone would have fallen during pregnancy.(ABSTRACT TRUNCATED AT 250 WORDS)

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.