B-flow/spatiotemporal image correlation M-mode: novel ultrasound method that detects decrease in spiral artery luminal diameter in first trimester in primate model of impaired spiral artery remodeling.

OBJECTIVE: To determine if B-flow/spatiotemporal image correlation (STIC) M-mode ultrasonography detects a decrease in spiral artery luminal diameter and volume flow during the first trimester in a non-human primate model of impaired spiral artery remodeling (SAR). METHODS: Pregnant baboons were treated daily with estradiol benzoate on days 25-59 of the first trimester (term, 184 days), or remained untreated. On day 60 of gestation, spiral artery luminal diameter (in seven untreated and 12 estradiol-treated baboons) and volume flow (in four untreated and eight estradiol-treated baboons) were quantified by B-flow/STIC M-mode ultrasonography. In addition, in 15 untreated and 18 estradiol-treated baboons, the percent of spiral arteries remodeled by extravillous trophoblasts was quantified ex vivo by immunohistochemical image analysis on placental basal plate tissue collected via Cesarean section on day 60. Findings were compared between treated and untreated animals. The correlation between spiral artery luminal diameter and percent of SAR was assessed in three untreated and six estradiol-treated baboons which underwent both B-flow/STIC M-mode ultrasound and quantification of SAR. RESULTS: The proportion of spiral arteries greater than 50 µm in diameter remodeled by extravillous trophoblasts was 70% lower in estradiol-treated baboons than in untreated animals (P = 0.000001). Spiral artery luminal diameter in systole and diastole, as quantified by B-flow/STIC M-mode in the first trimester of pregnancy, was 31% (P = 0.014) and 50% (P = 0.005) lower, respectively, and volume flow was 85% lower (P = 0.014), in SAR-suppressed baboons compared with untreated animals. There was a significant correlation between spiral artery luminal diameter as quantified by B-flow/STIC M-mode ultrasonography and the percent of SAR (P < 0.05). CONCLUSION: B-flow/STIC M-mode ultrasonography provides a novel real-time non-invasive method to detect a decrease in uterine spiral artery luminal diameter and volume flow during the cardiac cycle, reflecting decreased distensibility of the vessel wall, in the first trimester in a non-human primate model of defective SAR. © 2021 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of International Society of Ultrasound in Obstetrics and Gynecology.

Differential expression of placental villous angiopoietin-1 and -2 during early, mid and late baboon pregnancy.

Although vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang-1) and Ang-2 have important roles in angiogenesis, very little is known about the regulation of these factors in the villous placenta during human pregnancy. In the present study, to investigate whether placental expression of Ang-1, Ang-2 and VEGF was altered in a cell-specific manner with advancing baboon gestation, the mRNA levels of these growth factors were determined by RT-PCR in cells isolated by Percoll gradient centrifugation from and protein localization assessed by immunocytochemistry in the villous placenta at early (day 60), mid (day 100) and late (day 170, term is 184 days) baboon gestation. Mean (+/-SE) Ang-1 mRNA levels, relative to 18S rRNA, in villous syncytiotrophoblast (3.92+/-0.68) and cytotrophoblast (1.31+/-0.31) cell fractions were highest on day 60 of gestation, then decreased by approximately 2.5-fold (P<0.05) to 1.39+/-0.29 and 0.49+/-0.07, respectively, on day 170. Moreover, Ang-1 mRNA levels in the villous stromal cells and Ang-2 mRNA levels in all placental villous cell fractions were similar on days 60, 100, and 170 of gestation. In contrast to Ang-1 and Ang-2, placental villous cytotrophoblast VEGF mRNA levels were increased 2.94-fold (P<0.05) between mid (0.67+/-0.15) and late (1.97+/-0.49) gestation. A corresponding decrease in Ang-1, absence of change in Ang-2, and increase in VEGF protein immunocytochemical expression were exhibited in placental trophoblast with advancing baboon pregnancy. Ang-1/Ang-2 and the angiopoietin Tie-2 receptor were expressed in vascular endothelial cells of the villous placenta, indicating that these blood vessel cells are a major site of ligand-receptor interaction for angiogenesis during primate pregnancy. We conclude that there is a cell-specific differential change in placental villous trophoblast expression of VEGF, Ang-1, and Ang-2 which we propose is important in regulating angiogenesis in the villous placenta during primate pregnancy.

Developmental regulation of placental insulin-like growth factor (IGF)-II and IGF-binding protein-1 and -2 messenger RNA expression during primate pregnancy.

The present study was conducted to determine the developmental expression of placental insulin-like growth factor (IGF)-II, IGF-binding protein (IGFBP)-1 and -2, and IGF-II receptor mRNA expression during baboon pregnancy and whether estrogen, the levels of which increase with advancing pregnancy, regulates placental trophoblast IGF-II mRNA expression. Levels of the IGF-II 6.1-kilobase (kb) and 4.9-kb mRNA transcripts determined by Northern blot analysis progressively increased three- to fourfold in placental syncytiotrophoblast and whole-villous tissue between early (Day 60), mid (Day 100), and late (Day 170) baboon gestation (term = 184 days). In contrast, syncytiotrophoblast IGFBP-1 and -2 mRNA levels decreased, and IGF-II receptor mRNA expression remained relatively constant, with advancing baboon pregnancy. Placental cytotrophoblast IGF-II mRNA levels determined by competitive reverse transcription-polymerase chain reaction on Day 54 of gestation were increased (P < 0.05) almost twofold at 18 h after acute administration of estradiol to baboons, whereas long-term estrogen treatment had no effect. We propose that these changes in trophoblast IGF expression would provide a mechanism for enhancing net bioavailability and bioreactivity of IGF-II locally to promote the growth and development of the placenta and, consequently, of the fetus during primate pregnancy.

Developmental regulation of vascular endothelial growth/permeability factor messenger ribonucleic acid levels in and vascularization of the villous placenta during baboon pregnancy.

Vascular endothelial growth/permeability factor (VEG/PF) has an important role in angiogenesis; however, very little is known about the developmental regulation of VEG/PF and the vascular system within the placenta during human pregnancy. In the present study, therefore, a developmental approach was used in the baboon to determine the placental source of VEG/PF and its fms-like tyrosine kinase (flt-1) and kinase-insert domain containing (KDR/flk-1) receptors, and whether the rise in estrogen with advancing pregnancy was associated with a corresponding increase in placental VEG/PF expression and vascularization. VEG/PF messenger RNA (mRNA) levels were determined by competitive RT-PCR in villous cell fractions isolated by Percoll gradient centrifugation from placentas obtained on days 45 and 54 (very early), 60 (early), 100 (mid), and 165-170 (late) of baboon pregnancy (term = 184 days). Maternal peripheral serum estradiol increased from very low concentrations early in gestation (0.15-0.20 ng/ml) to an early surge of over 2.5 ng/ml on days 60-85, and peak levels of 4-6 ng/ml late in baboon pregnancy. VEG/PF mRNA was expressed in low level in the syncytiotrophoblast (<2,000 attomol/microgram total RNA), and values in this fraction did not change significantly with advancing gestation. VEG/PF mRNA expression was slightly greater in the inner villous core cell fraction; however, levels decreased (P < 0.05) between early and late gestation. Cytotrophoblasts were a major source of VEG/PF mRNA and levels increased (P < 0.01) from 3,631 +/- 844 attomol/microgram total RNA on day 45 to 25,807 +/- 5,873 attomol/microgram total RNA on day 170. VEG/PF protein expression determined by immunocytochemistry was abundant in cytotrophoblasts and lower in the syncytiotrophoblast and inner villous core cells. The flt-1 and KDR/flk-1 receptors were expressed in the vascular endothelial cells of the baboon villous placenta. The percentage of villous placenta occupied by blood vessels and the number of vessels/mm(2) villous tissue, determined by image analysis, progressively increased (P < 0.001; r = 0.97) from 3.4 +/- 0.2% and 447 +/- 29, respectively, on day 54 to 15.9 +/- 0.9% and 1,375 +/- 71, respectively, on day 170. In summary, the present study shows that villous cytotrophoblasts were a major source of VEG/PF mRNA and protein in the baboon villous placenta, and that cytotrophoblast VEG/PF mRNA levels and vascularization of the villous placenta closely paralleled the increase in estradiol concentrations of advancing pregnancy. These results are consistent with the concept that estrogen has an important role in establishing the new vascular system within the developing placenta during primate pregnancy and that VEG/PF mediates this process.

Developmental regulation of morphological differentiation of placental villous trophoblast in the baboon.

The present study determined whether morphological differentiation of placental villous cytotrophoblasts into syncytiotrophoblast during primate pregnancy was developmentally regulated and whether oestrogen has a role in this process. Placental volumetric composition of the cytotrophoblast and syncytiotrophoblast was determined by the test-point counting method on days 45-54, 60, 100, and 170 of gestation (term=184 days) in untreated baboons, on day 60 after placental oestrogen production was prematurely elevated by administration of aromatizable androstenedione or oestradiol, and on day 170 after oestrogen production was suppressed by administration of aromatase inhibitor CGS 20267. Cytotrophoblast and syncytiotrophoblast volumes and oestrogen levels increased (P< 0.01) with advancing gestation. Due to the rise in syncytiotrophoblast volume (12-fold) exceeded that of the cytotrophoblast (threefold), the mean (sem) ratio of syncytiotrophoblast and cytotrophoblast volumes increased (P< 0.001) from 3.4 (0.5) ml on day 60 to 12.1 (2.8) ml on day 170. Androstenedione administration elevated serum oestradiol levels threefold (P< 0.01) and increased the ratio of syncytiotrophoblast: cytotrophoblast volumes on day 60 by 50 per cent (P< 0.03) to that normally observed on day 100. However, the ratio of trophoblast volumes was unaltered in oestradiol-treated and CGS 20267-treated baboons. It is concluded that there is a developmental increase in morphological differentiation of the placental villous trophoblast during primate pregnancy and that androstenedione potentially via its conversion to oestrogen has a role in this process.

Identification and developmental expression of the estrogen receptor alpha and beta in the baboon fetal adrenal gland.

We have previously shown that estrogen regulates the development and function of the fetal and definitive/transitional zones of the primate fetal adrenal gland. Thus, during baboon pregnancy estrogen acts directly on the fetal zone to suppress ACTH-stimulated dehydroepiandrosterone (DHA) formation, potentially to modulate C19-steroid production and consequently placental estrogen synthesis. It is proposed that this action of estrogen is mediated by the estrogen receptor. Therefore, in the present study a developmental approach was used to determine whether the messenger RNA (mRNA) and protein for the estrogen receptor were expressed in the fetal and definitive/transitional zones ofthe baboon fetal adrenal gland at mid (day 100) and late (day 170) gestation (term = 184 days). Estrogen receptor alpha mRNA levels, determined by competitive RT-PCR, were approximately 7-fold greater (P < 0.02) in the fetal adrenal of late (187.8+/-40.3 attomoles/microg RNA) compared with mid (27.4+/-5.4 attomoles/microg RNA) gestation. Moreover, estrogen receptor alpha mRNA expression, determined by quantitative in situ hybridization, was approximately 2.5-fold greater (P < 0.05) in the definitive/transitional zones (21.6+/-0.5 silver grains/0.025 mm2) than in the fetal zone (8.3+/-1.5 grains/0.025 mm2) late in gestation. The mRNA for the beta-isoform of the estrogen receptor was also expressed in the baboon fetal adrenal cortex. There was a gradient of immunocytochemical staining for the estrogen receptor alpha and beta proteins, with extensive immunoreactivity for both isoforms in the definitive zone and lower staining in the transitional zone and the fetal zone. In summary, the results of the present study show that estrogen receptor alpha and beta were expressed in the fetal and definitive/transitional zones of the baboon fetal adrenal cortex at mid and late gestation. The presence of the estrogen receptor provides a mechanism for mediating the action of estrogen in modulating ACTH-dependent and cortical zone-specific development and function of the primate fetal adrenal gland.

Decline in adrenocorticotropin receptor messenger ribonucleic acid expression in the baboon fetal adrenocortical zone in the second half of pregnancy.

We have previously shown a decrease in fetal zone-specific ACTH-stimulable dehydroepiandrosterone formation and an increase in definitive zone-specific cortisol biosynthesis in the baboon fetal adrenal gland in the second half of gestation. Therefore, the fetal and definitive zones seem to develop a divergence in functional capacity with advancing gestation. We have proposed, therefore, that there is a selective decrease in ACTH receptor expression and thus tropic responsivity to ACTH within the fetal zone in the second half of primate pregnancy. The present study examined this possibility and whether corresponding changes occurred in the developmental expression of major components required for steroidogenesis. ACTH receptor messenger RNA (mRNA) levels, determined by in situ hybridization, in the fetal zone of the baboon fetal adrenal were approximately 2-fold greater (P < 0.05) at mid (i.e. day 100) than at late (i.e. day 170) gestation and 3-fold greater (P < 0.01) in the definitive zone than in the fetal zone in late gestation (term = 184 days). Both ACTH receptor and low density lipoprotein receptor mRNA levels, determined by Northern blot in the whole fetal adrenal, also decreased (P < 0.001) by approximately 50%, whereas the mRNA levels for the definitive zone-specific delta5-3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD) enzyme required for cortisol biosynthesis increased over 13-fold (P < 0.001) between mid and late gestation. In contrast, mRNA expression of the steroidogenic enzymes P-450 cholesterol side-chain cleavage and 17alpha-hydroxylase/17-20 lyase were unchanged throughout gestation. We conclude that the decrease in ACTH receptor mRNA expression and ACTH-stimulable dehydroepiandrosterone formation in the second half of gestation reflect a decline in functional capacity of the fetal zone, whereas the increase in 3beta-HSD mRNA expression and cortisol production results from the ACTH receptor-mediated development and enhanced functional capacity of the definitive zone.

Estrogen regulation of placental P-450 cholesterol side-chain cleavage enzyme messenger ribonucleic acid levels and activity during baboon pregnancy.

The present study was conducted to determine whether estrogen regulates the P-450 cholesterol side-chain cleavage (P-450scc) enzyme component of the progesterone biosynthetic pathway in the placenta during the second half of baboon pregnancy. Placental estrogen formation was suppressed by removing the fetus, i.e. fetectomy, and thus fetal adrenal C19-steroid estrogen precursors, on day 100 of baboon gestation (term = 184 days). P-450scc activity and messenger ribonucleic acid (mRNA) levels were then determined in placentas obtained on day 160 after fetectomy alone and after fetectomy and s.c. administration of the estrogen precursor and rostenedione or estradiol benzoate. Placentas were maintained in situ after fetectomy, and placental villi were comprised of syncytiotrophoblasts that seemed morphologically normal, based on their histology and immunocytochemical expression of pregnancy-specific-beta 1-glycoprotein. In untreated baboons, peripheral serum estradiol increased with advancing gestation, and mean (+/-SE) concentrations were 1.22 +/- 0.05 ng/ml on days 101-160 of gestation. After fetectomy serum estradiol concentrations decreased to 24% (P < 0.01) of normal. Androstenedione or estradiol administration after fetectomy increased serum estradiol levels to values that were 57% (P < 0.01) of, or 90% (P < 0.001) greater than intact controls, respectively,. Placental P-450scc specific activity, determined on a mitochondrial-enriched fraction of villous tissue, was 281.1 +/- 15.0 pmol pregnenolone plus progesterone formed per mg mitochondrial protein in untreated control baboons. Fetectomy resulted in a 52% decrease (P < 0.001) in placental P-450scc activity. Administration of androstenedione or estradiol after fetectomy increased P-450scc activity to values that were not significantly different from control. P-450scc mRNA levels were quantified by competitive RT-PCR. P-450scc mRNA levels in placental villous tissue of fetectomized baboons was 38% lower (P < 0.01) than that in the intact controls (110.9 +/- 5.9 attomoles/microgram RNA). The administration of androstenedione after fetectomy restored P-450scc mRNA to a level that was not different from the untreated controls. The results of this study show that there was close association between the levels of estrogen and the specific activity of and the mRNA levels for placental P-450scc in the second half of baboon pregnancy. Therefore, we propose that the P-450scc enzyme that catalyzes the conversion of substrate cholesterol to pregnenolone is regulated, for the most part, by estrogen in the primate placenta.

Estrogen stimulation of P450 cholesterol side-chain cleavage activity in cultures of human placental syncytiotrophoblasts.

The present study determined whether estrogen has a role in regulating the P450 cholesterol side-chain cleavage enzyme (P450scc) and/or de novo/deesterification cholesterol pathways involved in progesterone biosynthesis within human syncytiotrophoblasts. Human placental syncytiotrophoblasts were cultured for 48 h with estradiol, and P450scc activity was determined by the formation of progesterone from 25-hydroxycholesterol. Estradiol at 10(-7) or 10(-6) M and 25-hydroxycholesterol increased mean (+/- SE) progesterone production by syncytiotrophoblasts (ng/0.5 x 10(6) cells) to a value (19.2 +/- 1.1) that was 104% (p < 0.001) higher than that of the untreated controls (9.4 +/- 0.8) and 52% higher (p < 0.001) than with 25-hydroxycholesterol alone (12.6 +/- 0.9). The stimulation of progesterone secretion apparently was not the result of a change in progesterone metabolism to its principal metabolite, because 20alpha-hydroxypregn-4-en-3-one represented a minor secretory component (0.7-1.7 ng/0.5 x 10(6) cells) under these conditions, and levels were not substantially altered by estrogen. In contrast to the stimulatory effect of estradiol on P450scc activity, estrogen did not alter either the P450scc mRNA levels or the activities of 3-hydroxy-3-methylglutaryl coenzyme-A reductase and cholesterol ester hydrolase-rate-limiting enzymes for the de novo and deesterification pathways, respectively, for cholesterol formation in syncytiotrophoblasts in culture. Collectively, these results indicate that estrogen regulates the P450scc component of the progesterone biosynthetic pathway, which we suggest signals functional/biochemical differentiation of syncytiotrophoblasts during primate pregnancy.

Developmental increase in expression of the messenger ribonucleic acid and protein levels of 11beta-hydroxysteroid dehydrogenase types 1 and 2 in the baboon placenta.

Cortisol-cortisone interconversion is catalyzed by the NADP/NADPH-dependent oxido-reductase, 11beta-hydroxysteroid dehydrogenase-1 (11betaHSD-1) and the NAD-dependent oxidase, 11betaHSD-2. Because of the importance of placental corticosteroid metabolism in dictating the amount of cortisol arriving in the fetus to regulate fetal pituitary-adrenocortical function, the present study determined whether there was a developmental change in the expression of 11betaHSD-1 and/or -2 in placental syncytiotrophoblast, the site of maternal:fetal exchange. A syncytiotrophoblast-enriched (>95%) cell fraction was isolated from baboon placentas obtained at early (day 60), mid (day 100), and late (day 165) gestation (term = day 184), and 11betaHSD-1 and -2 messenger RNA (mRNA) and protein levels were determined by Northern and Western blots. The levels (mean +/- SE) of the single 1.6-kilobase (kb) mRNA for 11betaHSD-1, expressed as a ratio to beta-actin, increased (P < 0.05) between early (0.36 +/- 0.16; n = 4) and mid (0.95 +/- 0.21; n = 11) gestation and further increased (P < 0.05) by late gestation (1.82 +/- 0.29; n = 13). Similarly, the levels of the single 1.9-kb mRNA for 11betaHSD-2 in late gestation (2.46 +/- 0.35; n = 8) were greater (P < 0.05) than respective values at mid (1.36 +/- 0.22; n = 8) and early (0.64; n = 2) gestation. The levels of 11betaHSD-1 (arbitrary densitometric units), detected as a dominant band of 34 kDa, were greater (P < 0.05) in late gestation (2.6 +/- 0.2; n = 4) than at early (1.2 +/- 0.1; n = 4) or mid (1.9 +/- 0.3; n = 4) gestation. In contrast, 11betaHSD-2 was not detected by Western blot in syncytiotrophoblast isolated by collagenase dispersion. However, immunocytochemistry revealed that 11betaHSD-2 was present in and localized to the syncytiotrophoblast layer of the baboon placenta and that expression in late gestation (n = 4) appeared to exceed that in placentas of early (n = 4) and mid (n = 4) gestation. These results indicate that both 11betaHSD-1 and 11betaHSD-2 were expressed in syncytiotrophoblasts of the baboon placenta and that the mRNA and protein levels of these two 11betaHSD enzymes increased with advancing gestation. However, because 11betaHSD-2 was not detected in syncytiotrophoblast isolated by collagenase dispersion, we suggest that the 11betaHSD-1 and -2 reside in different membrane fractions of the syncytiotrophoblast. Consequently, the estrogen-regulated change in transplacental cortisol metabolism with advancing gestation may result in a developmental change in the expression and location of the two 11betaHSD enzymes controlling cortisol-cortisone metabolism and transfer into the fetus, resulting in activation of the fetal pituitary adrenocortical system.

Developmental expression of placental trophoblast P-450 cholesterol side-chain cleavage, adrenodoxin and delta 5-3 beta-hydroxysteroid dehydrogenase/isomerase messenger ribonucleic acids during baboon pregnancy.

The present study determined whether the elevation in oestrogen, which occurs with advancing baboon pregnancy, is associated with a developmental increase in expression of the placental enzymes catalysing progesterone synthesis. The mRNA levels for P-450 cholesterol side-chain cleavage (P-450scc), adrenodoxin, and delta 5-3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta-HSD) were assessed by Northern blot analysis in placental syncytiotrophoblasts isolated from baboons in early (days 58-65), mid- (days 97-113) and late (days 161-175), gestation (term = 184 days). Placental villous tissue was dispersed and subjected to 50 per cent Percoll density gradient centrifugation to obtain primarily syncytiotrophoblasts. Mean (+/- S.E.) P-450scc mRNA level, expressed as a ratio of beta-actin in the syncytiotrophoblast-rich fraction, progressively increased with advancing pregnancy to a level in late gestation (1.81 +/- 0.28 arbitrary units) that was approximately sixfold (P < 0.01) greater than in early gestation (0.31 +/- 0.08) and approximately twofold greater (P < 0.05) than in mid-gestation (0.97 +/- 0.24). In contrast adrenodoxin mRNA expression was similar at early (0.97), mid- (1.14 +/- 0.12) and late (1.16 +/- 0.13) gestation. Syncytiotrophoblast 3 beta-HSD mRNA levels also remained constant in early (1.69), mid- (1.89 +/- 0.41) and late (1.34 +/- 0.41) gestation. On the basis of these findings, we propose that villous syncytiotrophoblasts undergo functional/biochemical differentiation, resulting in a coordinated upregulation of specific components of the steroid biosynthetic pathway required for progesterone biosynthesis during the course of primate pregnancy.

Biphasic developmental expression of adrenocorticotropin receptor messenger ribonucleic acid levels in the baboon fetal adrenal gland.

We have previously shown an estrogen-dependent developmental regulation of placental oxidation of cortisol to cortisone that results in enhanced fetal pituitary ACTH production and the induction of steroidogenic enzymes in and de novo cortisol production by the fetal adrenal in the second half of baboon pregnancy. However, it is not known whether the receptor for ACTH is simultaneously generated at this time in development to provide a mechanism for mediating the tropic action of ACTH on steroidogenesis in the primate fetal adrenal gland. Therefore, in the present study we determined the levels of ACTH receptor messenger RNA (mRNA) and correlated ACTH receptor expression with appearance of the mRNA for delta5-3beta-hydroxysteroid dehydrogenase/isomerase (3betaHSD), the enzyme protein that signals functional maturation of the definitive cortical zone in the baboon fetal adrenal. A baboon ACTH receptor complementary DNA was cloned and hybridized with polyadenylated RNA isolated from baboon (Papio anubis) fetal adrenals obtained in early (days 58-64; RNA from seven baboon fetuses pooled to yield three samples), mid-(days 99-103; RNA from five baboons pooled to yield four samples), and late (days 165-168; RNA of four individual baboon fetuses) gestation (term = 184 days). Expression of the primary 3.4-kilobase ACTH receptor mRNA transcript, determined by Northern blot and expressed as a ratio of beta-actin mRNA, was minimal early in gestation (mean +/- SE, 0.11 +/- 0.05 arbitrary densitometric units). However, fetal adrenal ACTH receptor mRNA levels increased (P < 0.001, by ANOVA) approximately 13-fold to 1.41 +/- 0.16 at midgestation, then declined by 70% (P < 0.001) to 0.41 +/- 0.10 in late gestation. To determine whether the decrease in ACTH receptor expression by the fetal adrenal in the second half of pregnancy reflected programmed cell death, the integrity of genomic DNA was assessed by 32P-labeled DNA gel electrophoresis and in situ DNA end labeling. Because DNA oligonucleosomes and apoptotic DNA strand breaks characteristic of apoptosis were absent in the adrenal glands of fetal baboons, the decline in ACTH receptor mRNA levels in the fetal adrenal did not seem to reflect programmed cell death. Expression of the single 2.0-kilobase mRNA transcript for 3betaHSD, an enzyme localized specifically in the definitive zone of the fetal adrenal, was minimal in early (0.01 +/- 0.00 arbitrary units) and mid- (0.10 +/- 0.01) gestation. However, 3betaHSD mRNA levels were markedly increased late in gestation to a value (1.38 =/- 0.34) approximately 13-fold greater (P < 0.001) than that in midgestation. These findings indicate that there was a biphasic monomodal developmental expression of the ACTH receptor in the baboon fetal adrenal, which contrasted with the progressive increase in adrenal weight, 3betaHSD expression, and de novo cortisol production previously determined. Because the fetal adrenal is comprised mainly of the fetal cortical zone throughout gestation, the decrease in ACTH receptor expression between mid- and late gestation seems to occur primarily in the latter zone and may signal a selective decline in tropic responsivity of and delta5-C19-steroid, e.g. dehydroepiandrosterone, biosynthesis within the baboon fetal adrenal gland.

Developmental increase in low density lipoprotein receptor messenger ribonucleic acid levels in placental syncytiotrophoblasts during baboon pregnancy.

We have previously shown that there was an estrogen-regulated developmental increase in low density lipoprotein (LDL) uptake by placental syncytiotrophoblasts during baboon pregnancy. To determine whether this reflected enhanced expression of the LDL receptor, the levels of LDL receptor messenger RNA (mRNA) were determined by Northern blot and reverse transcription-polymerase chain reaction in placental tissue obtained from baboons (Papio anubis) in early (days 58-64; pooled to yield 5 samples), mid- (days 97-110; pooled to yield 12 samples), and late (days 161-175; pooled to yield 15 samples) gestation (term = 184 days). Whole villous tissue and a trophoblast cell fraction isolated by 50% Percoll gradient centrifugation were analyzed. The latter cell fraction was equally comprised predominantly of syncytiotrophoblasts at early, mid-, and late gestation as determined by extensive immunocytochemical reactivity with antisera to syncytiotrophoblast-specific peptides. Tissues were extracted with guanidine isothiocyanate and 5 micrograms polyadenylated-enriched RNA hybridized to a 32P-labeled human LDL receptor complementary DNA (cDNA). A major 6.2-kilobase LDL receptor mRNA transcript was expressed in syncytiotrophoblasts and whole villous tissue, as determined by Northern blot. In the syncytiotrophoblast-rich cell fraction, LDL receptor mRNA levels, analyzed by Northern blot and autoradiodensitometry and expressed as a ratio of beta-actin, were similarly low in early (0.66 +/- 0.12 arbitrary units) and mid- (1.15 +/- 0.23) gestation, then increased to a level in late gestation (2.71 +/- 0.33) that was over 4- and 2-fold greater (P < 0.01) than that in early or midgestation, respectively. In contrast, in whole villous tissue, LDL receptor and beta-actin mRNA levels exhibited no consistent change or decreased slightly with advancing pregnancy, so that when corrected for beta-actin, LDL receptor mRNA levels were similar in early (1.53 +/- 0.33), mid- (1.44 +/- 0.16), and late (2.32 +/- 0.29) gestation. The unchanged levels of LDL receptor mRNA in whole placental villous tissue with advancing primate gestation may reflect potential villous tissue with advancing primate gestation may reflect potential confounding effects that nontrophoblast, e.g. vascular, components of the developing placenta may have on assessing trophoblast endocrine function in whole villous tissue. Amplification of trophoblast RNA by reverse transcription-polymerase chain reaction with LDL receptor primers generated a single cDNA product of approximately 258 base pairs.(ABSTRACT TRUNCATED AT 400 WORDS)

Interconversion of cortisol and cortisone in baboon trophoblast and decidua cells in culture.

In baboons, transplacental cortisol (F)/cortisone (E) metabolism changes from reduction (E to F) at midgestation to oxidation (F to E) near term when estrogen becomes elevated. Indeed, estrogen regulates the placental microsomal 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) enzyme catalyzing F oxidation. However, regulation of 11 beta-HSD-reductase (E to F) is unknown, because this enzyme is destroyed by microsomal isolation. Therefore, we used cell culture to determine the role of estrogen on placental reduction of E to F and to ascertain whether estrogen regulation of the oxidation of F to E was specific to trophoblast. Placentas were obtained on day 165 (n = 6; term, day 184) and on day 100 of gestation from baboons untreated (n = 8) or treated (n = 6) with 50-mg implants of androstenedione (delta 4A) inserted sc in the mother between days 70 and 100 of gestation to increase placental estrogen production. After removal of fetal membranes, the decidua basalis and trophoblast were separated, rinsed repeatedly in medium-199, minced, and then incubated in trypsin/DNase. Dispersed cells were layered onto a discontinuous Percoll gradient (5-70%), and purified cytotrophoblast (TC; 1.048-1.062 g/ml) and decidua (DC; 1.048-1.062 g/ml) were harvested. After incubation in media containing 10% fetal bovine serum to permit attachment, cells were incubated (24 h) in Dulbecco's modified Eagle's medium containing 10,100, or 500 ng [3H]F or [3H]E. F and E in medium were purified by HPLC and the interconversion of F/E calculated. Equilibrium was achieved by 12 h, and F/E metabolism was proportional to cell number and substrate (10-500 ng) concentration. At substrate concentrations of 500 ng/ml, the reduction of E to F (range, 81-195 ng F produced/24 h) in the DC (0.5 x 10(6) cells) was greater (P less than 0.05) than oxidation of F to E (19-28 ng E/24 h) in all groups. This pattern of metabolism by DC was not affected by time of gestation or treatment with delta 4A. In the TC (2.5 x 10(6) cells), oxidation of F to E always exceeded (P less than 0.05) reduction of E to F. Moreover, the conversion of F to E by TC of day 100 (86 +/- 26 ng E/24 h; mean +/- SE) was increased (P less than 0.05) by delta 4A (195 +/- 35) and greater (P less than 0.05) at day 165 (213 +/- 40). In contrast, TC metabolism of E (21-57 ng F/24 h) was similar in all groups.(ABSTRACT TRUNCATED AT 400 WORDS)

Regulation of progesterone biosynthesis by estrogen during baboon pregnancy: placental mitochondrial cholesterol side-chain cleavage activity in antiestrogen (ethamoxytriphetol, MER-25)-treated baboons.

We determined whether the reduction in placental progesterone (P4) production observed after administration of the antiestrogen ethamoxytriphetol (MER-25) to pregnant baboons was associated with a decline in activity and/or content of the placental mitochondrial cholesterol side-chain cleavage system (P-450scc). Pregnant baboons (Papio anubis) were untreated (n = 9) or administered MER-25 (25 mg/kg BW, orally; n = 6) daily on days 140-170 of gestation (term = 184 days). Placentas were obtained by cesarean section on day 170 of gestation, and P-450scc activity and cytochrome P-450 content were determined on mitochondria-rich fractions. Administration of MER-25 to pregnant baboons resulted in a 40% reduction (P less than 0.01, by Student's t test) in the mean (+/- SE) peripheral serum P4 concentration (6.3 +/- 0.3 ng/ml) compared to that in untreated (10.4 +/- 0.3 ng/ml) baboons. P-450scc activity, as determined by formation of pregnenolone (P5) and P4 during a 30-min incubation (picomoles per mg protein), was 37% lower (P less than 0.01) in placental mitochondria obtained from MER-25-treated baboons (179.8 +/- 25.0) than in that from untreated (285.4 +/- 13.4) baboons. Mitochondrial cytochrome P-450 content, assessed by spectral analysis, was 28% lower (P less than 0.02) in antiestrogen-treated (46.7 +/- 2.1 pmol/mg protein) than in untreated (64.8 +/- 5.2 pmol/mg protein) baboons. The initial (time zero) free cholesterol content (nanomoles per mg protein) of mitochondrial-rich preparations was not significantly different in antiestrogen-treated (189.3 +/- 13.0) and untreated (225.0 +/- 15.1) animals. Collectively, these results suggest that the decline in placental P4 production observed in baboons in response to MER-25 occurs at least in part as a result of a decrease in cytochrome P-450scc activity. The loss in P-450scc activity appears to be an intramitochondrial event and not a result of depletion of the total mitochondrial cholesterol pool. We propose, therefore, that one mechanism by which estrogen may regulate the production of P4 by the placenta during primate pregnancy is via the maintenance of placental mitochondrial cytochrome P-450, the terminal oxidase of cytochrome P-450scc.

Effect of the antiestrogen ethamoxytriphetol (MER-25) on placental low density lipoprotein uptake and degradation in baboons.

The present study determined if the decline in placental progesterone (P4) production that results from administration of the antiestrogen ethamoxytriphetol (MER-25) to pregnant baboons results from a change in placental low density lipoprotein (LDL) uptake and/or degradation. Pregnant baboons (Papio anubis) were untreated (n = 10) or received MER-25 (25 mg/kg BW, orally; n = 10) daily on days 140-170 of gestation (term, 184 days). Placentas were removed by cesarean section on day 170 of gestation, and villous tissue was dispersed with 0.1% collagenase at 37 C for 40 min. Placental cells (10(6)) were incubated in medium 199 (pH 7.2) for 12 h at 37 C with increasing amounts (5-100 micrograms) of [125I]LDL, with or without a 100-fold excess of unlabeled baboon LDL. Mean (+/- SE) peripheral serum P4 concentrations on days 140-170 of gestation were 51% lower (P less than 0.01) in MER-25-treated (5.7 +/- 0.3 ng/ml) than in untreated (11.6 +/- 0.5 ng/ml) baboons. The uptake of LDL was 56% lower (P less than 0.01) in placental cells from antiestrogen-treated (6.3 +/- 1.6 ng/micrograms cell protein) than in those from untreated (14.4 +/- 1.9 ng/micrograms cell protein) baboons. The dissociation constants for placental LDL uptake, as assessed by Scatchard analysis, however, were similar in untreated (0.80 microgram/ml) and MER-25-treated (0.76 microgram/ml) animals. The amount of [125I]LDL concomitantly degraded by cells from baboons that received MER-25 was 54% of that degraded by cells from untreated controls. The relative decline in LDL degradation by cells of antiestrogen-treated baboons was proportionate to the decline in overall LDL uptake. The results indicate, therefore, that antiestrogen treatment decreased the amount of placental LDL uptake, but did not change the affinity for the lipoprotein. We suggest that the decline in placental P4 production elicited in pregnant baboons by antiestrogen results, at least in part, from subnormal LDL uptake. We propose that one of the mechanisms by which estrogen regulates the biosynthesis of P4 by the placenta during baboon pregnancy is by increasing receptor-mediated placental cell uptake of cholesterol in the form of LDL. Estrogen, therefore, may regulate LDL uptake by the placenta and thus the availability of cholesterol for P4 biosynthesis via the LDL pathway.

Insulin provokes a transient activation of phospholipase C in the rat epididymal fat pad.

Insulin is known to increase the de novo synthesis of inositol phospholipids in rat epididymal fat pads. We presently examined the effects of insulin on the hydrolysis of inositol phospholipids in this tissue. Relatively small (30-40%) but significant increases in inositol phosphates (mono-, di-, and tri-) were apparent within 30-60 s of insulin treatment in fat pads (and adipocytes); thereafter, inositol phosphates returned to control levels. These rapid insulin-induced increases in inositol phosphates appeared to be due to phospholipase C-mediated hydrolysis of inositol phospholipids, since there were associated transient decreases in these lipids during 32P pulse-chase experiments. Increases in the synthesis of inositol phospholipids were also apparent within a few minutes of insulin treatment and persisted for at least 2 h. We conclude that, in the rat epididymal fat pad, insulin has two phospholipid effects, viz. a transient activation of phospholipase C, and a persistent increase in de novo phospholipid synthesis.

The de novo phospholipid effect of insulin is associated with increases in diacylglycerol, but not inositol phosphates or cytosolic Ca2+.

We have previously reported that insulin increases the synthesis de novo of phosphatidic acid (PA), phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP), phosphatidylinositol 4,5-bisphosphate (PIP2) and diacylglycerol (DAG) in BC3H-1 myocytes and/or rat adipose tissue. Here we have further characterized these effects of insulin and examined whether there are concomitant changes in inositol phosphate generation and Ca2+ mobilization. We found that insulin provoked very rapid increases in PI content (20% within 15 s in myocytes) and, after a slight lag, PIP and PIP2 content in both BC3H-1 myocytes and rat fat pads (measured by increases in 32P or 3H content after prelabelling phospholipids to constant specific radioactivity by prior incubation with 32Pi or [3H]inositol). Insulin also increased 32Pi incorporation into these phospholipids when 32Pi was added either simultaneously with insulin or 1 h after insulin. Thus, the insulin-induced increase in phospholipid content appeared to be due to an increase in phospholipid synthesis, which was maintained for at least 2 h. Insulin increased DAG content in BC3H-1 myocytes and adipose tissue, but failed to increase the levels of inositol monophosphate (IP), inositol bisphosphate (IP2) or inositol trisphosphate (IP3). The failure to observe an increase in IP3 (a postulated 'second messenger' which mobilizes intracellular Ca2+) was paralleled by a failure to observe an insulin-induced increase in the cytosolic concentration of Ca2+ in BC3H-1 myocytes as measured by Quin 2 fluorescence. Like insulin, the phorbol diester 12-O-tetradecanoylphorbol 13-acetate (TPA) increased the transport of 2-deoxyglucose and aminoisobutyric acid in BC3H-1 myocytes. These effects of insulin and TPA appeared to be independent of extracellular Ca2+. We conclude that the phospholipid synthesis de novo effect of insulin is provoked very rapidly, and is attended by increases in DAG but not IP3 or Ca2+ mobilization. The insulin-induced increase in DAG does not appear to be a consequence of phospholipase C acting upon the expanded PI + PIP + PIP2 pool, but may be derived directly from PA. Our findings suggest the possibility that DAG (through protein kinase C activation) may function as an important intracellular 'messenger' for controlling metabolic processes during insulin action.