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Extravillous trophoblast (EVT) uterine artery remodeling (UAR) promotes placental blood flow, but UAR regulation is unproven. Elevating estradiol (E(2)) in early baboon pregnancy suppressed UAR and EVT vascular endothelial growth factor (VEGF) expression, but this did not prove that VEGF mediated this process. Therefore, our primate model of prematurely elevating E(2) and contrast-enhanced ultrasound cavitation of microbubble (MB) carriers was used to deliver VEGF DNA to the placental basal plate (PBP) to establish the role of VEGF in UAR. Baboons were treated on days 25 to 59 of gestation (term, 184 days) with E(2) alone or with E(2) plus VEGF DNA-conjugated MBs briefly infused via a maternal peripheral vein on days 25, 35, 45, and 55. At each of these times an ultrasound beam was directed to the PBP to collapse the MBs and release VEGF DNA. VEGF DNA-labeled MBs per contrast agent was localized in the PBP but not the fetus. Remodeling of uterine arteries >25 µm in diameter on day 60 was 75% lower (P < 0.001) in E(2)-treated (7% ± 2%) than in untreated baboons (30% ± 4%) and was restored to normal by E(2)/VEGF. VEGF protein levels (signals/nuclear area) within the PBP were twofold lower (P < 0.01) in E(2)-treated (4.2 ± 0.9) than in untreated (9.8 ± 2.8) baboons and restored to normal by E(2)/VEGF (11.9 ± 1.6), substantiating VEGF transfection. Thus, VEGF gene delivery selectively to the PBP prevented the decrease in UAR elicited by prematurely elevating E(2) levels, establishing the role of VEGF in regulating UAR in vivo during primate pregnancy.

We previously showed that advancing the increase in estradiol levels from the second to the first third of baboon pregnancy suppressed placental extravillous trophoblast (EVT) invasion and remodeling of the uterine spiral arteries. Cell culture studies show that vascular endothelial cell growth factor (VEGF) plays a central role in regulating EVT migration and remodeling of the uterine spiral arteries by increasing the expression/action of certain integrins that control extracellular matrix remodeling. To test the hypothesis that the estradiol-induced reduction in vessel remodeling in baboons is associated with an alteration in VEGF and integrin expression, extravillous placental VEGF and integrin expression was determined on d 60 of gestation (term is 184 d) in baboons in which uterine artery transformation was suppressed by maternal estradiol administration on d 25–59. EVT uterine spiral artery invasion was 5-fold lower (P < 0.01), and VEGF protein expression, quantified by in situ proximity ligation assay, was 50% lower (P < 0.05) in the placenta anchoring villi of estradiol-treated than in untreated baboons. α1β1 and α5β1 mRNA levels in cells isolated by laser capture microdissection from the anchoring villi and cytotrophoblastic shell of estradiol-treated baboons were over 2-fold (P < 0.01) and 40% (P < 0.05) lower, respectively, than in untreated animals. In contrast, placental extravillous αvβ3 mRNA expression was unaltered by estradiol treatment. In summary, extravillous placental expression of VEGF and α1β1 and α5β1 integrins was decreased in a cell- and integrin-specific manner in baboons in which EVT invasion and remodeling of the uterine spiral arteries were suppressed by prematurely elevating estradiol levels in early pregnancy. We propose that estrogen normally controls the extent to which the uterine arteries are transformed by placental EVT in primate pregnancy by regulating expression of VEGF and particular integrin extracellular remodeling molecules that mediate this process.

To assess whether there is a link between estrogen, vascular endothelial growth factor (VEGF), and early aspects of uterine angiogenesis, an acute temporal study was conducted in which ovariectomized baboons were pretreated with VEGF Trap, which sequesters endogenous VEGF, and administered estradiol at time 0 h. Serum estradiol levels approximated 500 pg/ml 4–6 h after estradiol administration. VEGF mRNA levels in endometrial glandular epithelial and stromal cells were increased to values 6 h after estradiol that were 3.74 ± 0.99-fold (mean ± se) and 5.70 ± 1.60-fold greater (P < 0.05), respectively, than at 0 h. Microvessel interendothelial cell tight junctions, which control paracellular permeability, were present in the endometrium at time 0 h, but not evident 6 h after estradiol administration. Thus, microvessel paracellular cleft width increased (P < 0.01, ANOVA) from 5.03 ± 0.22 nm at 0 h to 7.27 ± 0.48 nm 6 h after estrogen. In contrast, tight junctions remained intact, and paracellular cleft widths were unaltered in estradiol/VEGF Trap and vehicle-treated animals. Endometrial microvessel endothelial cell mitosis, i.e. percent Ki67+/Ki67− immunolabeled endothelial cells, increased (P < 0.05) from 2.9 ± 0.3% at 0 h to 21.4 ± 7.0% 6 h after estrogen treatment but was unchanged in estradiol/VEGF Trap and vehicle-treated animals. In summary, the estrogen-induced disruption of endometrial microvessel endothelial tight junctions and increase in endothelial cell proliferation were prevented by VEGF Trap. Therefore, we propose that VEGF mediates the estrogen-induced increase in microvessel permeability and endothelial cell proliferation as early steps in angiogenesis in the primate endometrium.

Although Na+/H+ exchange is important to maintenance of pH and volume and thus placental-fetal homeostasis, regulation of the Na+/H+ exchange system is incompletely understood. We previously showed that Na+/H+ exchanger (NHE)1 and -3 and their regulatory factors NHERF1 and -2 were expressed in human and nonhuman primate placenta. Our laboratories have also shown that estrogen regulates key aspects of primate placental function and development including the 11beta-hydroxysteroid dehydrogenase enzymes controlling cortisol metabolism. Therefore, it is possible that localization and/or expression of components of the syncytiotrophoblast NHE system are also estrogen dependent. As a first step in testing this possibility, the current study compared the immunocytochemical localization and level of NHE1, NHE3, NHERF1, and NHERF2 in baboon placentas obtained at mid- (d 100) and late gestation (d 165; term = d 184). NHE3 and NHERF2 were abundantly expressed at midgestation and localized to the cytoplasm and juxtanuclear compartment but were not detected in the microvillus membrane. By late gestation, NHE3 and NHERF2 expression were markedly reduced in the juxtanuclear compartment but not the cytoplasm. NHERF2 was also abundantly expressed in fetal vascular endothelium in which levels, as assessed by immunoblot exhibited a 3-fold developmental increase. In contrast, levels of NHE1 and NHERF1, which were abundantly expressed in and localized almost exclusively to syncytiotrophoblast microvillus membrane, were similar at mid- and late gestation. We conclude that the subcellular distribution and levels of key components of the Na+/H+ system in the baboon syncytiotrophoblast are developmentally regulated.

In the present study, we determined whether endogenous estrogen, the levels of which increase with advancing pregnancy, regulates growth and development of the baboon fetal adrenal cortex. Fetal adrenal glands were obtained at mid- (d 100) and late (d 165, term is 184 d) gestation from untreated baboons and on d 165 from animals in which endogenous estrogen production was suppressed by administration of aromatase inhibitor CGS 20267 between d 100 and 165. Volumes of the respective cortical zones were determined by zone-specific immunocytochemical staining of steroidogenic enzymes and image analysis. Fetal adrenal weight and volume increased (P0.01) 3-fold between mid- and late gestation and an additional 70% (P0.01) by administration of CGS 20267, which decreased (P0.001) fetal serum estradiol levels by more than 95%. Mean +/- se volume (x10(-10) mum(3)) of the fetal cortical zone increased from 3.45 +/- 0.28 at midgestation to 6.64 +/- 0.69 at late gestation in untreated baboons and to 12.55 +/- 0.99 (P0.01) in baboons in which estrogen production was suppressed by CGS 20267 administration. The levels of umbilical artery serum dehydroepiandrosterone sulfate, which is secreted primarily by the fetal zone, were increased almost 3-fold (P0.01) by administration of CGS 20267. Concomitant administration of CGS 20267 and estradiol returned fetal cortical zone volume and serum dehydroepiandrosterone sulfate levels to normal. In contrast to the effect of estrogen deprivation on the fetal zone, the volumes of the definitive and transitional zones in untreated baboons late in gestation (3.18 +/- 0.63 and 2.62 +/- 0.43, respectively) and levels of fetal serum cortisol, a steroid secreted from the transitional zone, were not altered by estrogen suppression. The changes in fetal zone growth were not associated with alterations in fetal pituitary proopiomelanocortin mRNA levels. We propose that estrogen acts directly on the fetal adrenal cortex to selectively repress the morphological and functional development of the fetal zone, potentially as a feedback system to maintain physiological secretion of estrogen precursors and thus placental estrogen production to promote normal primate fetal and placental development.

We recently showed that the number of primordial follicles was reduced by 50% in ovaries of near-term fetal baboons deprived of estrogen in utero and restored to normal in animals supplemented with estrogen. Oocytes are avascular and rely on surrounding granulosa cells for nutrients, a process facilitated by microvilli on the oocyte surface. However, our understanding of oocyte microvillus development in the primate fetal ovary is incomplete. Thus, we determined whether estrogen regulates formation of oocyte microvilli in utero. Fetal ovaries were obtained on d 165 gestation (term = d 184) from baboons untreated (n = 3) or treated on d 100-165 with aromatase inhibitor CGS 20267 (estrogen suppressed by 95%; n = 5) or CGS 20267 and estradiol (n = 4). Follicles with intact (homogeneous cytoplasm) or nonintact (cytoplasm vacuolated) oocytes were quantified and the number/height of oocyte microvilli determined by electron microscopy. In untreated baboons, the mean (+/-se) number of follicles/0.08 mm(2) with an intact oocyte (11.5 +/- 0.5) was decreased (P < 0.05) by 70% in fetal ovaries of estrogen-suppressed baboons (3.4 +/- 0.2) and restored (P < 0.05) by CGS 20267 and estradiol (11.2 +/- 1.2). In estrogen-deprived fetuses, the number of microvilli/intact oocyte (23 +/- 3) was 56% lower (P < 0.01) than normal (52 +/- 5) and restored by CGS 20267 and estrogen (62 +/- 4). Moreover, in intact oocytes of estrogen-suppressed baboons, height (nm) of microvilli (105 +/- 11) was 54-62% lower (P < 0.01) than in intact oocytes of fetal ovaries of untreated (228 +/- 13) or estrogen-treated (274 +/- 17) baboons. In estrogen-replete baboons, the number of microvilli in intact oocytes was 2-fold greater (P < 0.01) than in nonintact oocytes. However, in estrogen-deprived baboons, no microvilli were detected in nonintact oocytes and the number of microvilli in intact oocytes was similar to that in nonintact oocytes of untreated fetuses. We conclude that development of microvilli in oocytes of primordial follicles in the primate fetal ovary is regulated by estrogen. Collectively, these results and those of our previous studies indicate that estrogen regulates fetal ovarian folliculogenesis and development of follicles with oocytes composed of microvilli critical for nutrient uptake and presumably long-term survival.

We have previously shown that estrogen plays a central integrative role in regulating key aspects of fetal-placental development and that inhibition of estrogen production during the second half of baboon pregnancy suppressed fetal adrenal function. Because maturation of the fetal lung is dependent on cortisol of fetal adrenal origin, the current study determined whether lung development and expression of surfactant proteins (SPs) A and B were altered at term in estrogen-deprived baboons. Fetal lungs were obtained on d 100, 165, and 175 of gestation (term = d 184) from untreated baboons and on d 165 from animals treated daily during the second half of pregnancy either with the aromatase inhibitor CGS 20267 alone or with CGS 20267 and estradiol benzoate. Umbilical venous estradiol levels were suppressed by more than 95% by CGS 20267 and elevated by CGS 20267 and estrogen. Although umbilical serum cortisol levels were also suppressed by 35% by CGS 20267, cortisol levels in the fetal lung of estrogen-suppressed baboons were similar to values in untreated animals. Immunocytochemistry demonstrated that CGS 20267 treatment did not alter fetal lung expression of the 11β-hydroxysteroid dehydrogenase enzyme-1 enzyme catalyzing reduction of cortisone to cortisol. However, immunocytochemical expression of the 11β-hydroxysteroid dehydrogenase enzyme-2 catalyzing oxidation of cortisol to cortisone appeared lower in lungs of estrogen-deprived fetuses and restored to normal by CGS 20267 and estrogen. SP-A levels in fetal lungs of untreated baboons were increased 16- to 20-fold between d 100 and d 165–175 of gestation in untreated baboons and baboons treated with CGS 20267 or CGS 20267 and estrogen. Similarly, SP-B levels in fetal lungs of untreated baboons were increased 10-fold between d 100 and d 165–175 of gestation in untreated baboons and baboons treated with CGS 20267 or CGS 20267 and estrogen. Moreover, in estrogen-suppressed baboons, as in untreated animals, the fetal lung continued to grow and exhibited normal alveolarization on histology. We conclude that development of the primate fetal lung can occur in utero in baboons in which fetal serum cortisol levels have been suppressed by the relative absence of estrogen perhaps because of the ability of the lung to coordinate local production of cortisol.

Vascular endothelial growth/permeability factor (VEG/PF) has a crucial role in angiogenesis, and neovascularization is essential in preparing the uterine endometrium for implantation. However, the regulation of VEG/PF synthesis by particular cell types of the endometrium during the human menstrual cycle is not well understood. Therefore, in the present study the baboon was used as a nonhuman primate to determine the role of the ovary in vivo in endometrial VEG/PF expression. VEG/PF mRNA levels were quantified by competitive RT-PCR in whole uterine endometrium and in glandular epithelial and stromal cells isolated from the endometrium by laser capture microdissection of baboons during the normal menstrual cycle and after ovariectomy, which decreased serum estradiol and progesterone to undetectable levels. Mean (±se) levels (attomoles per micrograms of total RNA) of the 323-bp VEG/PF mRNA product, which reflected collective expression of all VEG/PF isoforms, in whole endometrium were 785 and 727 ± 158 during the mid and late follicular phases, respectively, and 1108 ± 320 during the midcycle surge in serum estradiol. VEG/PF mRNA levels then declined briefly before increasing to 1029 ± 365 attomoles/μg RNA during the late luteal phase of the menstrual cycle. VEG/PF mRNA levels (attomoles per femtomole of 18S rRNA) were similar in glandular epithelial (2.27 ± 1.11) and stromal (2.54 ± 0.70) cells at the midcycle estradiol peak and the midluteal phase of the menstrual cycle (2.34 ± 1.30 and 1.49 ± 0.53, respectively). Immunocytochemical expression of VEG/PF protein was abundant in glandular and luminal epithelium, stroma, and vascular endothelium. Endometrial vessel density and percent vascularized area, determined by morphometric image analysis, were similar during the various stages of the baboon menstrual cycle. After ovariectomy, VEG/PF mRNA levels (attomoles per femtomole of 18S rRNA) in the endometrial glands (0.52 ± 0.21) and stroma (0.22 ± 0.11) were decreased to values that were approximately 20% and 10% (P < 0.05), respectively, of those in intact baboons during the midcycle estrogen surge. Moreover, there was relatively little VEG/PF protein immunostaining in the endometrial glands, stroma, and vascular endothelium after ovariectomy.In summary, VEG/PF mRNA and protein expression in glandular epithelial and stromal cells were markedly suppressed after ovariectomy, indicating that synthesis of this angiogenic factor in these endometrial cells is dependent upon a product(s) secreted by the ovary. Moreover, endometrial VEG/PF expression remained relatively constant and thus was available as a component of the angiogenic system throughout the menstrual cycle, presumably to progressively promote vascular reconstruction of the endometrium.

In polarized epithelial cells of several organ systems, e.g. the kidney, a family of Na(+)/H(+) exchangers (e.g. Na(+)/H(+) exchanger-1 and -3) and their regulatory proteins, Na(+)/H(+) exchanger regulatory factor and Na(+)/H(+) exchanger-3 kinase A regulatory protein play a major role in regulating Na(+)/H(+) exchange integral to cellular homeostasis. Because the primate placenta regulates exchange of Na(+) and H(+) between the mother and fetus critical to fetal-placental homeostasis, the current study determined whether Na(+)/H(+) exchanger-1 and -3 were compartmentalized and associated with expression of Na(+)/H(+) exchanger regulatory factor and Na(+)/H(+) exchanger-3 kinase A regulatory protein in baboon and human syncytiotrophoblast. Using RT-PCR, single 413-bp Na(+)/H(+) exchanger-1 and 190-bp Na(+)/H(+) exchanger-3 products were expressed by baboon and human syncytiotrophoblasts. The 104-kDa Na(+)/H(+) exchanger-1 protein was detected by Western blot in microvillus membranes and to a much lesser extent in the basal membranes of the baboon and human syncytiotrophoblasts. In contrast, the 85-kDa Na(+)/H(+) exchanger-3 protein was detected primarily in membranes contiguous with the basal membranes of the syncytiotrophoblast of both species. Differential localization of Na(+)/H(+) exchanger-1 and -3 was confirmed by immunocytochemistry. The Na(+)/H(+) exchanger-3 regulatory protein, Na(+)/H(+) exchanger-3 kinase A regulatory protein, resided almost exclusively in the basal membranes, whereas Na(+)/H(+) exchanger regulatory factor was localized primarily to the microvillus membranes in the baboon and human syncytiotrophoblast. Collectively, these results are the first to show that the baboon and human term placental syncytiotrophoblast expressed the mRNAs and proteins for Na(+)/H(+) exchanger-1 and -3 and their regulatory factors and that Na(+)/H(+) exchanger-1 and Na(+)/H(+) exchanger regulatory factor resided primarily in the microvillus membranes, whereas Na(+)/H(+) exchanger-3 and Na(+)/H(+) exchanger-3 kinase A regulatory protein were localized to membranes contiguous with the basal membranes and to the basal membranes, respectively. We conclude that a complete Na(+)/H(+) exchange system is present in the baboon and human term placental syncytiotrophoblast and suggest that the primate placenta exhibits polarity with respect to the capacity for regulation of Na(+)/H(+) exchange between the placenta and the maternal and fetal circulations.

We previously showed that the messenger RNA and protein levels of the 11ss-hydroxysteroid dehydrogenase (11betaHSD) enzymes catalyzing glucocorticoid reduction (11betaHSD-1) and oxidation (11betaHSD-2) increased with advancing baboon gestation and concluded that the estrogen-regulated change in placental cortisol metabolism from reduction at midgestation to oxidation near term is not simply the result of a change in the relative concentrations of these two enzymes. Therefore, in the current study we determined whether 11betaHSD-1 and -2 are located in different regions of the baboon and human syncytiotrophoblast and whether there is a developmental change in their localization with advancing baboon gestation. Western blot analyses, immunofluorescence, and electron microscopic immunocytochemistry indicated that 11betaHSD-1 expression was abundant in microvillus membranes (MVM) juxta the maternal circulation, and their levels are significantly lower, but detectable, in more internal regions of the syncytiotrophoblast, including membranes contiguous with the basal membrane (BM(m)) facing the fetal vasculature in both the human and baboon. In contrast, in both species 11betaHSD-2 expression was limited in the MVM and extensive throughout the remainder of the syncytiotrophoblast, including the BM(m). In the baboon, the relative mean (+/-SE) concentrations (arbitrary densitometric units per microgram protein) of 11betaHSD-1 in the MVM were similar at mid (i.e. day 100; 38,859 +/- 3,484; n = 3) and late (i.e. day 180; 43,561 +/- 1,784; n = 3) gestation (term = day 184) and exceeded (P < 0.01) respective values for 11betaHSD-2 by approximately 16-fold. In contrast, levels of 11betaHSD-1 in the BM(m) declined (P < 0.05) by approximately 50% between mid (7,099 +/- 758) and late (4,013 +/- 738) gestation, whereas levels of 11betaHSD-2 in this fraction increased. Thus, the ratio of 11betaHSD-2 to 11betaHSD-1 in the BM(m) at midgestation (1.22 +/- 0.10) was increased (P < 0.05) 2-fold in late gestation (2.66 +/- 0.05). Collectively, these findings indicate that the 11betaHSD-1 and -2 enzymes are localized to different membrane fractions of the baboon and human placental syncytiotrophoblast. Moreover, we propose that the developmental increase in the ratio of 11betaHSD-2 to 11betaHSD-1 in membranes facing fetal blood near term is consistent with and perhaps the subcellular mechanism responsible for the previously demonstrated switch in transplacental glucocorticoid metabolism from reduction at midgestation to oxidation late in gestation and appears to be responsible for the activation/maturation of the fetal pituitary-adrenocortical axis.

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 of the baboon fetal adrenal gland at mid (day 100) and late (day 170) gestation (term = 184 days). Estrogen receptor α 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/μg RNA) compared with mid (27.4 ± 5.4 attomoles/μg RNA) gestation. M...

In the present study, the baboon was used as a model to investigate the effects of steroid hormones on vascular endothelial growth factor (VEG/PF) and aromatase expression and on proliferation of the normal mammary gland. Immunocytochemistry revealed that both aromatase and VEG/PF were expressed in the epithelial cells of the terminal ductal lobular units. Mammary tissue biopsies were obtained from female baboons during the follicular and luteal phases of the menstrual cycle, 4 weeks after ovariectomy (OVX), and after 2 weeks of treatment with estradiol benzoate (E2B; 500 μg/day, im). Although there was little apparent difference in aromatase messenger ribonucleic acid (mRNA) in tissue from follicular and luteal phases or after ovariectomy, aromatase mRNA was decreased in tissue from ovariectomized (OVX) animals treated for 2 weeks with E2B. Furthermore, aromatase activity in tissue from these animals was markedly reduced compared to activity in tissue from the OVX animals before treatment (P < 0.001). In...

Although the human placenta at term exhibits high levels of phospholipase A2 (PLA2) enzyme activity, our understanding of the ontogenesis, regulation, and specific roles of placental phospholipases A2 remains relatively incomplete. Using the baboon as the experimental model, the present study determined whether the levels of the messenger ribonucleic acid (mRNA) for the cytosolic (cPLA2) and/or type IIa, nonpancreatic secretory (sPLA2) enzymes are developmentally regulated and modulated by glucocorticoid treatment. Total RNA was extracted from whole villous placenta obtained on days 60 (early; n = 3), 100 (mid; n = 3), and 165 (late; n = 4) of gestation (term = day 184) from untreated baboons and on day 100 (n = 4) after maternal administration on days 60–99 of betamethasone (3 mg/day). The complementary DNA to cPLA2 recognized a single 2.9-kb mRNA transcript in both baboon and human placenta. Mean (±se) levels of cPLA2 mRNA, expressed, in arbitrary units as a ratio to that of β-actin, were similar at ear...

Although fetal pituitary ACTH is important to fetal adrenal growth and steroidogenesis in the second half of primate pregnancy, its role in adrenal development and function has not been established in vivo in the first half of gestation. In the present study, therefore, baboons were treated at midgestation with betamethasone to determine the effect of fetal pituitary ACTH on fetal adrenal growth, development, and ACTH receptor and P-450 enzyme messenger ribonucleic acid (mRNA) levels. The administration of betamethasone to baboon mothers on days 60–99 of gestation (term = 184 days) decreased fetal pituitary POMC mRNA levels by 54% (P < 0.01) and fetal serum ACTH levels to undetectable values (P < 0.05). The decline in ACTH was associated with decreases in fetal adrenal weight (P < 0.001), cortical cell size (P < 0.05), appearance of apoptosis and cellular disorganization, and a loss of immunocytochemically demonstrable definitive zone-specificΔ 5-3β-hydroxysteroid dehydrogenase expression. The concomitant...

The objective of the present study was to determine whether, in addition to the onset of chorionic somatomammotropin (CS) production previously shown to result from the morphological differentiation of cytotrophoblasts into syncytiotrophoblasts, there is a further developmental increase in the capacity of syncytiotrophoblasts to produce CS with advancing stages of baboon pregnancy. Placentas were obtained from baboons in early (days 48–62), mid (days 97–110), and late (days 161–175) gestation (term = 184 days), and CS messenger ribonucleic acid (mRNA) and protein levels were determined in a syncytiotrophoblast-rich cell fraction isolated by Percoll gradient centrifugation. CS mRNA levels in syncytiotrophoblasts, expressed as a ratio of β-actin, exhibited a progressive increase from early (0.04 ± 0.04 relative arbitrary units) to mid (2.37 ± 0.33; P < 0.001) to late (3.66 ± 0.39; P < 0.05) gestation. Levels of the 22-kDa CS protein were very low on days 48–55 (0.83 ± 0.09 arbitrary units), increased 10-fol...

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 fe...

We have proposed that estrogen, via regulation of placental metabolism of maternal cortisol, regulates the baboon fetal hypothalamic-pituitary-adrenal axis and the timing of the onset of de novo cortisol production. In support of this hypothesis, we demonstrated that the ontogenesis of fetal adrenal steroidogenic enzymes near term could be induced at midgestation by maternal estrogen treatment. In the present study, we determined whether maturation of the fetal adrenal near term and at midgestation after estrogen treatment reflects enhanced expression of the messenger RNA (mRNA) for the ACTH precursor molecule POMC. Fetal pituitaries were obtained on day 100 (n = 7) and day 165 (n = 5) of gestation (term = day 184) from untreated baboons and on day 100 after maternal treatment with estradiolbenzoate (sc; days 70-100; n = 6). Sections were fixed in paraformaldehyde and hybridized with saturating concentrations of an antisense (or sense) oligodeoxynucleotide complementary to bases 297-326 of human POMC mRNA that was 3' end-labeled with [35S]dATP. After stringent washes, sections were placed against Kodak X-Omat film (Eastman Kodak, Rochester, NY) and then dipped in Kodak NTB-2, developed, and counterstained. POMC mRNA (antisense minus sense) was quantified by densitometry and image analysis of silver grains. Specificity of labeling was documented by selective distribution of grains over a dispersed population of cells in sections of anterior pituitary hybridized with antisense, the relative absence of grains in sections incubated with sense, and the absence of grains in neurohypophyseal sections incubated with antisense. Moreover, silver grains were not visible when sections were pretreated with excess radioinert probe. The mean (+/- SE) maternal serum estradiol concentration in baboons treated with estradiol benzoate at midgestation (2.9 +/- 0.4 ng/ml) was greater (P0.05) than that in untreated baboons on day 100 (1.0 +/- 0.3) but not significantly different from that in late gestation (1.9 +/- 0.3). In umbilical serum, estradiol concentrations were greater (P0.05) at term (3.7 +/- 0.9) than at midgestation (0.7 +/- 0.2) but, unlike maternal values, were not significantly increased at midgestation after treatment of the mother with estradiol (1.1 +/- 0.2). Based on densitometric analysis, mean (+/- SE) pituitary POMC mRNA (absorbance units) was greater (P0.05) in baboon fetuses at term (0.57 +/- 0.05) than at midgestation (0.28 +/- 0.03) and increased (P0.05) on day 100 (0.43 +/- 0.04) in estrogen-treated animals. Similar results were obtained when data were analyzed as the number of silver grains/0.025 mm2.(ABSTRACT TRUNCATED AT 400 WORDS)

We have previously demonstrated that the estrogen-regulated change in transuteroplacental metabolism of cortisol (F) and cortisone (E) from preferential reduction (E to F) at midgestation to oxidation (F to E) near term results in activation of the hypothalamic-pituitary-adrenal axis of the baboon and the ontogenesis of rate-limiting steroidogenic enzymes, culminating in de novo F secretion. It is well established that transcription of messages activated by peptide-mediated binding to membrane receptors can occur via cAMP-dependent protein kinase-A (PKA) and/or phospholipid/calcium-dependent protein kinase-C (PKC). The present study was designed to determine whether basal levels of PKA and PKC in the fetal adrenal are developmentally regulated during baboon gestation and, thus, could provide the mechanism(s) by which activation of the fetal adrenal near term is mediated. Fetal adrenal glands were obtained on day 100 (n = 8) and day 165 (n = 6) of gestation (term = day 184) from untreated baboons and on day 100 after treatment of the mother with estradiol benzoate, injected sc between days 70-100 to increase estrogen production. PKA activity (picomoles of 32P incorporated into kemptide per min/mg protein) was determined by incubation of adrenal cytosol (12,000 x g; 0.3-30 micrograms protein) in reaction mixtures containing 0.25 mM ATP, 1 x 10(6) dpm [lambda-32P]ATP, and 3 micrograms kemptide in the presence or absence of 0.02 mM cAMP. PKC activity (picomoles of 32P incorporated into histone IIIS per min/mg protein) was determined in cytosol (105,000 x g) and detergent-solubilized membrane fractions after incubation with 0.02 mM ATP, 50 micrograms histone IIIS, and 1 x 10(6) dpm [lambda-32P]ATP in the presence or absence of calcium and phospholipids. Mean (+/- SE) maternal serum estradiol concentrations (nanograms per ml) were 3-fold greater (P < 0.05) at term (1.9 +/- 0.3) than at midgestation and increased (P < 0.05) after treatment with estradiol. PKA activity was greater at term (3965 +/- 546) than at midgestation (2130 +/- 240) and increased (P < 0.05) 2-fold after treatment with estrogen (3525 +/- 416) at midgestation. PKC activity was always 3- to 4-fold lower than that of PKA and was similar in the cytosol and membrane fractions of the cell. In contrast to PKA, cytosolic PKC activity was similar at mid (265 +/- 98)-and late (353 +/- 99) gestation and was not altered by treatment with estradiol (223 +/- 27).(ABSTRACT TRUNCATED AT 400 WORDS)

The present study was designed to determine whether the baboon fetal pituitary at midgestation was responsive in utero to a bolus injection of CRH. On day 100 of gestation (term = day 184), baboons were anesthetized with halothane/nitrous oxide, the fetus was exteriorized, and a cannula was inserted into a fetal carotid artery. Five minutes later (experimental time zero), a fetal carotid blood sample was obtained, and saline (0.5 ml) with (n = 6) or without (n = 3) ovine CRH (100 ng estimated to equal 500 ng/kg BW) was then infused via the fetal carotid over a 3-min period. Fetal blood samples were taken 5, 15, 30, 45, and 60 min post-CRH/saline treatment and assayed for ACTH. Mean (+/- SE) pretreatment fetal plasma ACTH concentrations were similar in animals that subsequently received saline (26 +/- 3 pg/ml) or CRH (29 +/- 6 pg/ml). Fetal plasma ACTH remained constant after the infusion of saline. In contrast, CRH increased (P less than 0.05) fetal plasma ACTH within 5 min in six of six baboons to a value (58 +/- 12 pg/ml) that exceeded (P less than 0.05) the zero time value and the respective mean value (27 +/- 5 pg/ml) in saline-treated fetuses. Fetal plasma ACTH concentrations continued to rise in four of six baboons 15 min after CRH injection to a level (68 +/- 15 pg/ml) which exceeded that in saline controls (27 +/- 2 pg/ml). In fetuses treated with CRH, overall mean fetal plasma ACTH concentrations from 0-60 min increased at a rate (1.47 pg/min) greater (P less than 0.05) than that in fetuses injected with saline (0.07 pg/min). In contrast to the effects of intracarotid CRH injection, fetal plasma ACTH was not increased after the infusion of 100 ng CRH into a fetal antecubital vein of three additional animals. Collectively, these findings indicate that intracarotid injection of a bolus of CRH into the baboon fetus rapidly increased fetal plasma ACTH concentrations. Moreover, the site of action of CRH was presumably the fetal pituitary. Therefore, we suggest that the baboon fetal hypothalamic-pituitary axis at midgestation has the capacity to secrete ACTH in response to a challenge of CRH.

We have recently demonstrated that treatment of pregnant baboons with androstenedione (delta 4 A) at midgestation to increase estrogen production induced a pattern of placental cortisol (F) metabolism which was similar to that at term and resulted in de novo F production by the fetus, presumably by activation of the fetal hypothalamic-pituitary-adrenocortical axis. The present study was designed to examine the subcellular events in the fetal adrenal that were apparently stimulated by estrogen-induced alterations in transplacental corticosteroid metabolism. Therefore, we determined the effects of estrogen treatment at midgestation and removal of estrogen action near term on the specific activity of the rate-limiting enzymes delta 5-3 beta-hydroxysteroid dehydrogenase (3 beta HSD) and 17-hydroxylase-17,20-lyase (17 alpha-OHase). Fetal adrenals were obtained on day 100 (n = 11) or day 165 (n = 11) of gestation (term = day 184) from untreated animals, on day 100 from animals receiving delta 4 A daily between days 70-100 (n = 9) to increase placental estrogen production, and on day 165 from baboons treated daily between days 130-164 with antiestrogen ethamoxytriphetol (MER-25; n = 7). The activity of 17 alpha-OHase was determined by incubating adrenal microsomes (105,000 x g) with [3H] progesterone, NAD+, and NADH in phosphate buffer. The radiolabeled products 17-hydroxyprogesterone, delta 4 A, and testosterone were purified, and enzyme activity expressed as picograms of product per min/mg tissue. The activity of 3 beta HSD was determined by incubating adrenal microsomes with [3H]pregnenolone and NAD+ in phosphate buffer. The radiolabeled progesterone product was purified, and enzyme activity was expressed as nanograms per min/mg tissue. Treatment with delta 4 A increased estrogen concentration at midgestation 3-fold to levels comparable to those measured near term. Although fetal adrenal weight was greater at term than at midgestation (p less than 0.05), weight was not increased by delta 4 A treatment. The specific activity (mean +/- SE) of fetal adrenal 17 alpha-OHase at midgestation (181 +/- 29) was increased (P less than 0.05) 3-fold by treatment with delta 4 A to levels (591 +/- 105) comparable to those in adrenal microsomes prepared from untreated animals near term (816 +/- 130). Enzyme activity in adrenals of MER-25-treated baboons was 40%, but not significantly lower than that in term controls.(ABSTRACT TRUNCATED AT 400 WORDS)

High levels of maternal estrogens are likely to gain access to the fetal brain, yet little is known regarding the role of the steroid hormone 17beta-estradiol in neuronal differentiation and maturation of primate neurons. Previous research documented the presence of estrogen receptors during development in the hippocampus and cortex of the primate brain, but the functional significance of steroid exposure has not been widely investigated. Using both an in vitro preparation of primary hippocampal and frontal cortex neurons and Western blot analysis of fetal hippocampal and frontal cortex tissue, we documented the effects of in utero and acute in vitro exposure to 17beta-estradiol on the development of neuronal responsiveness to the amino acid transmitters gamma-aminobutyric acid (GABA) and glutamate in fetal baboon, Papio anubis, hippocampal, and cortical neurons. We found that in utero 17beta-estradiol exposure enhanced the excitatory action of the GABAergic system on immature cortical and hippocampal neurons, as manifest by increases in intracellular calcium after transient muscimol application and changes in the relevant ion cotransporters. Acute exposure to 17beta-estradiol in vitro had limited effect on GABAergic responses in cultured hippocampal and frontal cortex neurons. Moreover, there was limited effect of both prolonged in utero and acute estradiol on the response to glutamatergic system activation, consistent with previous findings in the rat. Along with documenting a prominent role for 17beta-estradiol in maturation of the GABAergic system, these findings increase our understanding of neuronal differentiation and maturation in the fetal primate brain.

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)

We have previously shown that the change in transuteroplacental cortisol (F)-cortisone (E) metabolism in vivo from preferential reduction (E to F) at midgestation to oxidation by term (F to E) does not occur in baboons in which the production or action of estrogen have been blocked. Moreover, because the administration of androstenedione (Δ4A) to baboons increased estradiol (E2) production at midgestation and induced a pattern of F-E metabolism similar to that at term, we suggested that estrogen regulates placental F-E interconversion. The present study was designed to ascertain whether estrogen regulates the activity of the placental 11β-hydroxysteroid dehydrogenase (11βHSD) enzyme catalyzing the oxidation of F to E. Placentas were obtained on day 100 (n = 10) and day 165 (n = 10) of gestation (term = day 184) from untreated baboons, on day 100 from animals (n = 7) treated with Δ4A between days 70-100 of gestation, and on day 165 from animals in which placental estrogen was decreased by fetectomy (n = 5)...

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.

During rat pregnancy the placenta appears to provide androgens, particularly androstenedione (delta 4A) as a source of precursor for estradiol (E2) formation by the ovary. The present study determined if the ovary and, specifically, estrogen have roles in regulating placental delta 4A production during the second half of rat pregnancy. Pregnant rats were ovariectomized (OVX) on day 9 of gestation, treated daily with 4 mg progesterone (P4) to maintain pregnancy, and received a Silastic capsule containing either E2 or oil on day 10 of gestation only. Placental steroidogenesis was then determined in vitro on day 14 by the ability of this tissue to convert [3H]pregnenolone ([3H]P5) substrate to the intermediate [3H]P4 and product [3H]delta 4A. Mean (+/- SE) placental formation of delta 4A from P5 increased (P less than 0.01) from 6.3 +/- 0.5% in control animals to 10.7 +/- 1.6% in OVX P4-treated rats. In contrast, placentae from OVX animals treated with P4 and E2 exhibited a decline in delta 4A formation (2.5 +/- 0.3%) compared to that in both control (P less than 0.01) and OVX P4-treated (P less than 0.001) animals. The amount of P5 converted to P4 and thus not further metabolized to delta 4A decreased (P less than 0.05) from 64.0 +/- 3.1% in control animals to 49.0 +/- 4.7% in OVX rats treated with P4 alone. However, OVX animals treated with P4 and E2 exhibited an increase in placental conversion of P5 to P4 (80.3 +/- 4.7%) compared to values in both control (P less than 0.05) and OVX P4-treated (P less than 0.001) animals. The peripheral serum concentrations of delta 4A and testosterone (T) were 3- and 2-fold greater (P less than 0.01), respectively, in OVX P4-treated animals compared to concentrations in the untreated controls. Rats that had been OVX and treated with both P4 and E2 had peripheral serum delta 4A and T concentrations that were approximately 10-15% (P less than 0.001) of the concentrations in OVX P4-treated animals. In conclusion, ovariectomy or ovariectomy and estrogen supplementation resulted in comparable alterations in the formation of delta 4A within the placenta and the concentrations of this steroid in the peripheral circulation. Thus, ovariectomy caused an elevation in and ovariectomy with E2 treatment caused a decline in both placental delta 4A formation and peripheral serum androgen concentrations. We suggest, therefore, that ovarian estrogen may feed back to inhibit placental delta 4A and T production, thereby regulating its substrate availability during the second half of rat pregnancy.

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.

The present study determined whether the reduction in serum progesterone (P4) concentrations which follows the administration of the antiestrogen ethamoxytriphetol [1-(rho-2-diethylaminoethoxyphenyl)1-phenyl-2-rho-methoxyphenyl ethanol (MER-25)] to pregnant baboons reflects a decline in placental and/or luteal function. Maternal saphenous venous blood was collected at 1- to 4-day intervals between day 70 of gestation and term in pregnant baboons. Four females received no other treatment, and eight females received MER-25 (15 mg/kg BW, orally) daily between day 130 of gestation and term. Four of the MER-25-treated baboons received no other treatment, and four had the corpus luteum of pregnancy surgically excised between days 104 and 118 of gestation. Serum P4 concentrations in the untreated baboons fluctuated, but no significant progressive rise or fall in P4 occurred. Administration of antiestrogen to intact pregnant baboons resulted in a 50% decline (P less than 0.001) in serum P4 concentrations from mean pretreatment values of 7.0-25.1 to 4.2-10.8 ng/ml thereafter. Although removal of the corpus luteum alone had no effect on serum P4, administration of MER-25 to luteectomized females resulted in an 80% decrease (P less than 0.001) in serum P4 concentrations from pretreatment means of 10.6-16.6 to 2.5-3.2 ng/ml thereafter. The results indicate that most or all of the P4 that remained in the peripheral circulation after MER-25 administration to intact pregnant baboons originated from the ovary, primarily the corpus luteum. Thus, the major site of action of antiestrogen in reducing P4 production during baboon pregnancy is on the placenta.

The metabolic clearance rate (MCR) of progesterone was examined in the baboon to determine whether the increased serum progesterone concentrations during gestation result from increased production or decreased clearance. MCR was determined by constant infusion of [3H]progesterone in 6 non-pregnant baboons and 13 pregnant animals, between 58 and 175 days gestation. Progesterone MCR (mean+/-SE) was significantly greater in pregnant (1316+/-105 1/day, p less than 0.025; 87.3 +/- 6.2 1/day/kg, p less than 0.01) than in non-pregnant animals (893 +/- 75 1/day; 53.1 +/- 3.5 1/day/kg). There was no correlation between progesterone MCR and gestational age. Mean (+/- SE) progesterone MCR was similar during the follicular (938+/-119 1/day; 58.8+/-3.8 1/day/kg) and luteal (802+/-78 1/day; 52.4+/-6.8 1/day/kg) phases of the menstrual cycle. Serum progesterone concentrations (mean +/- SE, determined by radioimmunoassay, were 11.3+/-1.2 ng/ml in pregnant animals and 5.9+/-2.2 ng/ml during the luteal phase of the menstrual cycle. The mean (+/- SE) calculated progesterone production rates (MCR X serum progesterone concentration) were 4.57+/-1.74 mg/day during the luteal phase of the menstrual cycle and 15.26+/-2.48 mg/day during the last two-thirds of gestation. Thus, increased serum progesterone concentrations in pregnant baboons reflect enhanced production rather than decreased clearance. This is analogous to humans, but contrasts with rhesus monkeys, in which production is not increased during pregnancy. The elevated baboon progesterone MCR, despite increased serum corticosteroid binding capacity, indicates that other factors such as changes in maternal metabolism may also be important in regulating progesterone clearance.

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.

The conversions of [3H]pregnenolone (P5) to [3H]progesterone (P4) and [3H]20 alpha-hydroxypregn-4-en-3-one (20-OHP4) were determined in vitro in ovaries of 3 (young)- and 11 (aged)-month-old rats throughout pregnancy. Aged females exhibited an increased incidence of embryonic-fetal resorption beginning at midgestation. The mean +/- SE numbers of live and dead fetuses in aged females between day 11 of gestation and term were 8.6 +/- 0.5 and 2.3 +/- 0.3, respectively, compared to 12.0 live fetuses in young females. In young animals, ovarian conversion of P5 to P4 approximated 30% on days 1-3 of gestation, increased (P less than 0.001) to a plateau of about 45% on days 6-11, then declined to 23.5% by day 15. In aged females, ovarian formation of P4 from P5 showed no significant progressive rise or fall throughout pregnancy. The increment in P4 formation observed on days 6-11 in young rats, therefore, did not occur in aged females, and the percent conversion of P5 to P4 was 40% lower (P less than 0.05 to P less than 0.005) in aged rats during this interval. Ovarian P4 formation was similarly low in aged females that did or did not exhibit embryonic death on days 9-11. From day 12 of gestation until term, the percent conversion of P5 to P4 was similar in all young and aged animals. Conversion of P5 to 20-OHP4 in young rats increased (P less than 0.005) steadily to a peak of 21.9 +/- 5.8% (mean +/- SE) at midgestation, then declined thereafter. 20-OHP4 formation was about 50% greater in aged than in young females on days 2-6, but values were similar thereafter. The mean ratio of conversion of P5 to P4 to that of P5 to 20-OHP4, therefore, was 50% lower (P less than 0.025) in aged (1.8 +/- 0.2) than in young (3.3 +/- 0.4) rats between days 6 and 11 of gestation. The subnormal amount of P4 recovered after ovarian incubation in aged females apparently reflected subnormal P4 formation and/or enhanced P4 catabolism to steroid metabolites. The present results, therefore, demonstrate intrinsic changes in ovarian function during aged rat pregnancy. The onset of fetal demise in aged females, however, does not appear to be related to this alteration in ovarian steroidogenesis.

We determined the role of the fetus and estrogen on transuteroplacental cortisol (F)-cortisone (E) metabolism in the baboon (Papio anubis). The interconversion of F-E at mid-gestation (day 100; term = day 184) was compared with that in animals near term (day 170) in which the fetus, but not the placenta, was removed (fetectomy) on day 100 and that in baboons treated daily between days 140 and 170 of gestation with the antiestrogen ethamoxytriphetol [1-(p-diethylamino-ethoxyphenol)1-phenyl-2-p-methoxyphenolethan ol (MER-25)]. In fetectomized animals at term, transuteroplacental conversion of E to F (30%) exceeded (P less than 0.05) that of the reverse reaction (7%). This pattern of metabolism was significantly different from that measured in intact pregnant animals at term, in which oxidation of F to E (28%) exceeded reduction of E to F (4%). In contrast, placental metabolism in fetectomized baboons at term was similar to that in pregnant animals at mid-gestation, in which conversion of F to E (20%) was lower (P less than 0.05) than reduction of E to F (39%). Treatment of intact pregnant baboons with MER-25 also resulted in a pattern of F-E metabolism across the placenta at term which was similar to that measured at midgestation but different from that in untreated baboons at term. Collectively, our findings show that the striking alteration in F-E interconversion from reduction (E to F) at midgestation to oxidation (F to E) by term, as measured across the placenta in vivo during the second half of baboon pregnancy, does not occur in animals lacking a fetus or in intact baboons in which the action of estrogen was inhibited. Therefore, we suggest that the fetus and/or the hormones of pregnancy that are dependent upon the fetus (i.e. estrogen) regulate transuteroplacental corticosteroid metabolism.

The present study was designed to characterize the dynamics of progestin metabolism peripherally and across the uterus during normal baboon pregnancy and to determine whether the decline in placental progesterone (P4) production which results from administration of the antiestrogen ethamoxytriphetol (MER-25) to baboons reflects a decrease in conversion of pregnenolone (P5) to P4 and thus delta 5-3 beta-hydroxysteroid dehydrogenase activity. To examine this possibility, the conversion of [3H]P5 to [3H]P4 was determined by the constant infusion method on day 100 (midgestation) and day 175 (near term) of gestation in baboons that received MER-25 (25 mg/day X kg BW, po) on days 95-100 or 140-175 (term = 184 days). Baboons were sedated with ketamine HCl, then received a constant iv infusion of [3H]P5 (1.0 mu Ci/0.388 ml X min) and [14C]P4 (0.2 mu Ci/0.388 ml X min for 110 min. Radiolabeled progestins were purified from blood samples withdrawn from saphenous, uterine, and umbilical vessels, and the MCR of P4 and P5, uterine extraction of P5, and transfer constants (rho) for the peripheral, transuterofetoplacental, and transuteroplacental conversion of P5 to P4 were determined. The formation of P4 from P5 by incubates of placental cells obtained on day 175 from untreated and MER-25-treated baboons was also assessed. During normal baboon pregnancy the mean (+/- SE) % P5 extracted (i.e. metabolized) by the uterus was 31.0 +/- 3.3 at midgestation and 45.7 +/- 5.6 late in gestation. Peripheral and transuterofetoplacental rho values of P5 to P4 in untreated baboons were 6.9 +/- 1.8% and 37.3 +/- 7.9%, respectively, at midgestation and 6.1 +/- 0.6% and 46.8 +/- 10.1%, respectively, near term. The transuteroplacental rho of P5 to P4 was only slightly lower than the transuterofetoplacental values, indicating minimal conversion of P5 to P4 by the fetus. The peripheral contribution of P5 production to the total production rate of P4 at term in baboons was 1%. The contribution of uteroplacental conversion of P5 to P4 to the total conversion of P5 to P4 at midgestation was estimated to be 22%. MER-25 caused a 53% decline (P less than 0.01) in serum P4 concentrations from a mean (+/- SE) of 12.5 +/- 2.4 ng/ml during the pretreatment period to 5.4 +/- 0.3 ng/ml between days 140 and 175 of gestation.(ABSTRACT TRUNCATED AT 400 WORDS)

The present study employed the dehydroepiandrosterone (DHA) loading test to determine if the placenta in baboons remains functional following fetectomy. A bolus iv injection of 100 mg DHA was given to 4 baboons prior to and at various intervals following fetectomy at midgestation. Blood samples were withdrawn over a 4-h period following DHA and assayed for estradiol (E2). Placentas were maintained in situ following removal of the fetus. Serum E2 reached peak concentrations 30-60 min after injection of DHA prior to and after fetectomy. Although initial serum E2 concentrations were very low after fetectomy, the mean +/- SE net increase (delta) in peak serum E2 concentrations following the DHA bolus were similar before (3.92 +/- 0.71 ng/ml) and after (3.72 +/- 0.64 ng/ml) fetectomy. Based on the capacity for aromatization, therefore, the placenta remains equally competent and functional in baboons despite chronic absence of the fetus.