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We have recently shown that there was a developmental increase in placental trophoblast vascular endothelial growth/permeability factor (VEG/PF) expression and vascularization that closely paralleled maternal serum estrogen levels during advancing baboon gestation. The present study determined whether estrogen regulates these important aspects of primate development. VEG/PF mRNA levels were determined by competitive RT-PCR in isolated villous placental cells, and placental vascularization was assessed by image analysis. Placentas were obtained on d 60 of gestation (length of gestation is 184 d) from baboons in which estrogen levels on d 25-59 were increased by daily administration of aromatizable androstenedione or decreased by aromatase inhibitor CGS 20267. Androstenedione treatment increased maternal serum estradiol levels 3-fold (P < 0.01) and placental villous cytotrophoblast VEG/PF mRNA level to a value (mean +/- se, 26,836 +/- 5,625 attomoles/microg total RNA) 2.5-fold greater (P < 0.05) than that in untreated animals (11,645 +/- 1,746 attomoles/microg RNA). In contrast, administration of CGS 20267 decreased serum estradiol (P < 0.01) and placental cytotrophoblast mRNA (2,912 +/- 693 attomoles/microg RNA; P < 0.05) levels by 75%, effects prevented by concomitant administration of CGS 20267 and estradiol. VEG/PF mRNA levels in inner villous cells were unaltered. Coinciding with the increase in placental VEG/PF expression, the percent vascularized area (3.46 +/- 0.23) and vessel density (493 +/- 34 vessels/mm(2)) of the villous placenta in untreated baboons on d 60 were increased (P < 0.01) in baboons in which estrogen levels were elevated by androstenedione treatment (6.54 +/- 0.56 and 743 +/- 27 vessels/mm(2), respectively). It is concluded that estrogen has an important role in stimulating trophoblast VEG/PF expression and consequently villous placental angiogenesis to promote fetal growth and development in early primate pregnancy.

Estrogen stimulates morphological and functional (i.e. steroidogenesis) differentiation of the primate placental trophoblast, and with advancing gestation there is an increase in estrogen and placental chorionic somatomammotropin (CS) mRNA and protein levels. To examine whether CS formation is regulated by estrogen, placental villous trophoblast CS was determined in baboons in which estradiol levels in uterine vein were increased 2- to 3-fold (P0.01) on d 60 of pregnancy (term = 184 d) by administration of aromatizable androstenedione on d 30-59 or estradiol benzoate on d 45-59 of gestation. Androstenedione and estradiol treatment resulted in a 75% decrease (P0.01) in placental whole villous CS-3 mRNA and CS protein levels, determined by Northern and Western blot analysis, on d 60, and a corresponding decrease in syncytiotrophoblast CS protein and maternal serum CS levels. In contrast, placental villous Delta(5)-3beta-hydroxysteroid dehydrogenase, 11 beta-hydroxysteroid dehydrogenase-2, and P-450 aromatase protein levels were unaltered by androstenedione or estradiol treatment. Collectively, these results suggest that, in elevated levels, estrogen suppressed CS formation by villous syncytiotrophoblast during the first one third of primate pregnancy. Therefore, estrogen has very different and specific actions on steroid and peptide hormone biosynthesis within the placental trophoblast, which we propose are important in regulating placental function and promoting fetal-placental development in the primate.

We recently showed that endometrial vascular endothelial growth/permeability factor (VEG/PF) mRNA expression was decreased by ovariectomy of baboons and restored by chronic administration of estrogen. However, it remains to be determined whether this effect of estrogen reflects genomic up-regulation of VEG/PF and leads to an increase in microvascular permeability, an early physiological event in angiogenesis. Therefore, we determined the temporal expression of VEG/PF mRNA in glandular epithelial and stromal cells isolated by laser capture microdissection from and width of microvascular paracellular clefts that regulate vessel permeability in the endometrium of ovariectomized baboons after acute estradiol and/or progesterone administration.Endometrial VEG/PF mRNA levels were increased in five of five animals within 2 h of estradiol administration and remained elevated at 4 and 6 h. The net increase in glandular epithelial (7.31 ± 2.72 attomol/fmol 18S ribosomal rRNA) and stromal (3.13 ± 0.36) cell VEG/PF mRNA levels after estradiol administration was over 8-fold (P < 0.05) and 2.6-fold (P < 0.01) greater, respectively, than after vehicle (0.90 ± 0.30, glands and 1.20 ± 0.33, stroma). In contrast, endometrial VEG/PF mRNA expression was unaltered by progesterone. After estradiol treatment, endometrial paracellular cleft width was increased (P < 0.01) from a mean (±se) of 71.6 ± 4.6 nm at 0 h to 101.1 ± 6.4 nm at 6 h, whereas vehicle or progesterone had no effect. We suggest that estrogen has a major role in regulating VEG/PF synthesis and early events in angiogenesis in the primate endometrium.

Throughout gestation, the primate fetal adrenal gland is comprised of the fetal zone, which expresses the P-450 17α-hydroxylase-C17,20 lyase (P-450c17) enzyme that catalyzes the synthesis of C19 steroids used for placental estrogen production. The development of the transitional zone comprised of cortical cells that express the P-450c17 and the 3β-hydroxysteroid dehydrogenase-isomerase (3βHSD) enzymes for cortisol production, and the definitive zone, which expresses 3βHSD, but not P-450c17, for mineralocorticoid synthesis, does not occur until relatively late in gestation. Although ACTH is considered essential to fetal adrenal growth and function, the role that ACTH has in the development of the transitional and definitive zones, is less clear. To answer this question, the width of these zones was determined by immunocytochemical expression of P-450c17 and/or 3βHSD in fetal adrenal glands obtained on day 100 (mid) of gestation (term = day 184) from baboons in which ACTH was administered to the fetus on days 95–99 of gestation or on day 165 (late) of gestation from baboons in which fetal ACTH was suppressed by treatment of the mother and fetus with betamethasone on days 150–164 of gestation. At midgestation, the fetal adrenal was comprised almost exclusively of fetal zone cells and a small definitive zone (38 ± 2 μm in width), but was essentially devoid of a transitional zone (7 ± 2 μm). Treatment with ACTH enhanced (P < 0.05) the width of the transitional zone (67 ± 4 μm), but not the size of the definitive zone (10 ± 4 μm). In late gestation, the width of the definitive zone, although 2-fold greater than that on day 100, was smaller (P < 0.05) than that of the transitional zone (120 ± 15 μm), which greatly exceeded that at midgestation. Treatment with betamethasone in late gestation eliminated the transitional zone, but had no effect on the size of the definitive zone (120 ± 8 μm). These findings indicate that the development of the baboon fetal adrenal transitional zone late in gestation is dependent on fetal pituitary ACTH. In contrast, the ontogenesis of the definitive zone at midgestation and its growth in late gestation occur in the relative absence of ACTH.

In the present study, human trophoblast cells were studied in culture to determine the effect of estrogen on low-density lipoprotein (LDL) uptake and progesterone formation. Cytotrophoblasts were obtained from term human placentas and incubated for 72 h with 10% FBS to stimulate formation of syncytia. Syncytiotrophoblasts were then incubated for an additional 48 h with estradiol and/or LDL protein. Estradiol plus LDL stimulated progesterone to a level that was 133% greater (P < 0.05) than control (314 +/- 69 ng/mg protein) or LDL alone, suggesting that estrogen stimulated progesterone formation via an increase in LDL uptake and utilization. To examine this possibility, trophoblast cells were cultured with estradiol for 48 h as above, then incubated for 12 h with 6-200 micrograms/mL [125I]LDL. Mean (+/- SE) specific uptake of [125I]LDL (ng/mg cell protein) was approximately 50% greater (P < 0.01) with estradiol (638 +/- 23) than with vehicle (429 +/- 54). Scatchard analysis demonstrated that the dissociation constant for LDL uptake was similar in the presence (2.9 +/- 0.4 x 10(-6)M) and absence (2.8 +/- 0.6 x 10(-8)M) of estradiol, indicating that estrogen increased LDL receptor number without affecting affinity. LDL uptake was increased (P < 0.05) by incubating trophoblast with as little as 0.10 ng/mL estradiol (approximately 10(-9) M). We conclude that estrogen regulates placental trophoblast cell uptake of LDL and, thus, the availability of cholesterol for progesterone bio-synthesis during human pregnancy.