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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).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.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).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 ObstetricsGynecology published by John WileySons Ltd on behalf of International Society of Ultrasound in Obstetrics and Gynecology.

Uterine spiral artery remodeling (UAR) is essential for placental perfusion and fetal development. A defect in UAR underpins placental ischemia disorders, e.g., preeclampsia, that result in maternal systemic vascular endothelial dysfunction and hypertension. We have established a model of impaired UAR by prematurely elevating maternal serum estradiol levels during the first trimester of baboon pregnancy. However, it is unknown whether this experimental paradigm is associated with maternal vascular endothelial dysfunction. Therefore, in the present study baboons were administered estradiol on days 25–59 of gestation to suppress UAR and maternal vascular function determined on day 165 (term = 184 days) peripherally and in skeletal muscle, which accounts for over 40% of body mass and 25% of resting systemic vascular resistance. Maternal serum sFlt-1 levels were 2.5-fold higher (P < 0.05), and skeletal muscle arteriolar endothelial nitric oxide synthase (eNOS) protein expression and luminal area, and skeletal muscle capillary density were 30-50% lower (P < 0.05) in UAR suppressed baboons. Coinciding with these changes in eNOS expression, luminal area, and capillary density, maternal brachial artery flow-mediated dilation and volume flow were 70% and 55% lower (P < 0.05), respectively, and mean arterial blood pressure 29% higher (P < 0.01) in UAR defective baboons. In summary, maternal vascular function was disrupted in a baboon model of impaired UAR. These results highlight the translational impact of this primate model and relevance to adverse conditions of human pregnancy underpinned by improper uterine artery transformation. NEW & NOTEWORTHY Maternal vascular dysfunction is a hallmark of abnormal human pregnancy, particularly early-onset preeclampsia, elicited by impaired UAR. The present study makes the novel discovery that maternal systemic vascular dysfunction was induced in a baboon experimental model of impaired UAR. This study highlights the translational relevance of this nonhuman primate model to adverse conditions of human pregnancy underpinned by defective UAR.

We have shown that normal weight offspring born to estrogen-deprived baboons exhibited insulin resistance, although liver and adipose function and insulin receptor and glucose transporter expression were unaltered. The blood microvessels have an important role in insulin action by delivering insulin and glucose to target cells. Although little is known about the regulation of microvessel development during fetal life, estrogen promotes capillary proliferation and vascular function in the adult. Therefore, we tested the hypothesis that estrogen promotes fetal microvessel development and thus vascular function and insulin sensitivity in offspring. Capillary/myofiber ratio was decreased 75% (P

Extravillous trophoblast remodeling of the uterine spiral arteries is essential for promoting blood flow to the placenta and fetal development, but little is known about the regulation of this process. A defect in spiral artery remodeling underpins adverse conditions of human pregnancy, notably early-onset preeclampsia and fetal growth restriction, which result in maternal and fetal morbidity and mortality. Many in vitro studies have been conducted to determine the ability of growth and other factors to stimulate trophoblast cells to migrate across a synthetic membrane. Clinical studies have investigated whether the maternal levels of various factors are altered during abnormal human pregnancy. Animal models have been established to assess the ability of various factors to recapitulate the pathophysiological symptoms of preeclampsia. This review analyzes the results of the in vitro, clinical, and animal studies and describes a nonhuman primate experimental paradigm of defective uterine artery remodeling to study the regulation of vessel remodeling.

Uterine spiral artery remodeling (SAR) is essential for promoting placental perfusion and fetal development. A defect in SAR results in placental ischemia and increase in placental expression and serum levels of the soluble fms-like tyrosine kinase-1 (sFlt-1) receptor that binds to and suppresses vascular endothelial growth factor (VEGF) bioavailability, thereby leading to maternal vascular dysfunction. We have established a nonhuman primate model of impaired SAR and maternal vascular dysfunction by prematurely elevating estradiol levels in early baboon pregnancy. However, it is unknown whether this primate model of defective SAR involves an increase in placental expression of sFlt-1, which may suppress VEGF bioavailability and thus SAR in the first trimester. Therefore, to establish the role of sFlt-1 in early pregnancy, SAR was quantified in baboons treated on days 25 through 59 of gestation (term = 184 days) with estradiol or with the sFlt-1 gene targeted selectively to the placental basal plate by ultrasound-mediated/microbubble-facilitated gene delivery technology. Placental basal plate sFlt-1 protein expression was 2-fold higher (P 25 µm in diameter was 72% and 63% lower (P

In the field of protein biology, immunology-based techniques are continuously evolving for the detection and quantification of individual protein levels, protein-protein interaction, and protein modifications in cells and tissues. The proximity ligation assay (PLA), a method of detection that combines immunologic and PCR-based approaches, was developed to overcome some of the drawbacks that are inherent with other detection methods. The PLA allows for very sensitive and discretely quantifiable measures of unmodified, native protein levels and protein-protein interaction/modification complexes in situ in both fixed tissues and cultured cells. We describe herein the PLA method and its applicability to quantify the effects of estrogen on expression of angioregulatory factors, e.g., endothelial nitric oxide synthase (eNOS) in the vasculature, vascular endothelial growth factor (VEGF) in the placenta, and melanocortin 2 receptor (MC2R)/accessory protein (MRAP) in the fetal adrenal of the nonhuman primate.

Using our nonhuman primate baboon model, we showed that offspring born to mothers deprived of estrogen during the second half of gestation exhibited insulin resistance and a deficit in first phase insulin release. Although insulin resistance was not due to an impairment of fetal or offspring growth, nor to an alteration in adipose or hepatic sensitivity to insulin, skeletal muscle microvacularization critical for delivery of nutrients/insulin was significantly reduced in fetuses and offspring deprived of estrogen in utero. Skeletal muscle myofiber maturation occurs in utero and estrogen modulates myofiber growth in adults. Therefore, the current study determined whether fetal skeletal muscle development was altered in baboons in which estradiol levels were suppressed/restored during the second half of gestation by maternal treatment with letrozole ± estradiol benzoate. In estrogen-suppressed animals, fetal skeletal muscle fascicles were structurally less organized, smaller, and comprised of slow type I and fast type II fibers, the size, but not the number of which were smaller than in untreated baboons. Moreover, the proportion of non-muscle fiber tissue was greater and that of muscle fibers lower in estrogen-deprived fetuses. Thus, the maintenance of fetal body weight in estrogen-deprived animals was maintained at the expense of muscle fibers and likely reflected increased deposition of non-muscle proteins. Importantly, fetal skeletal muscle development, including fascicle organization, myofiber size and composition was normal in baboons treated with letrozole and estradiol benzoate. Collectively, these and our previous findings support our proposal that exposure of the fetus to estrogen is important for fetal skeletal muscle development and glucose homeostasis in adulthood.

Uterine spiral artery remodeling is essential for placental perfusion and fetal growth and, when impaired, results in placental ischemia and pregnancy complications, e.g., fetal growth restriction, preeclampsia, premature birth. Despite the high incidence of adverse pregnancies, current treatment options are limited. Accordingly, research has shifted to the development of gene therapy technologies that provide targeted delivery of “payloads” to the placenta while limiting maternal and fetal exposure. This review describes the current strategies, including placental targeting peptide-bound liposomes, nanoparticle or adenovirus constructs decorated with specific peptide sequences and placental gene promoters delivered via maternal IV injection, directly into the placenta or the uterine artery, as well as noninvasive site-selective targeting of regulating genes conjugated with microbubbles via contrast-enhanced ultrasound. The review also provides a perspective on the effectiveness of these technologies in various animal models and their practicability and potential use for targeted placental delivery of therapeutics and genes in adverse human pregnancies affected by placental dysfunction.

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.

Placental extravillous trophoblast remodeling of the uterine spiral arteries is important for promoting blood flow to the placenta and fetal development. Heparin-binding EGF-like growth factor (HB-EGF), an EGF family member, stimulates differentiation and invasive capacity of extravillous trophoblasts in vitro. Trophoblast expression and maternal levels of HB-EGF are reduced at term in women with preeclampsia, but it is uncertain whether HB-EGF is downregulated earlier when it may contribute to placental insufficiency. A nonhuman primate model has been established in which trophoblast remodeling of the uterine spiral arteries is suppressed by shifting the rise in estrogen from the second to the first trimester of baboon pregnancy. In the present study, we used this model to determine if placental HB-EGF is altered by prematurely elevating estrogen early in baboon gestation. Uterine spiral artery remodeling and placental expression of HB-EGF and other EGF family members were assessed on day 60 of gestation in baboons treated with estradiol (E2) daily between days 25 and 59 of gestation (term = 184 days). The percentages of spiral artery remodeling were 90, 84 and 70% lower (P 100 µm diameter in E2-treated compared with untreated baboons. HB-EGF protein quantified by immunocytochemical staining/image analysis was decreased three-fold (P 2-treated versus untreated baboons, while amphiregulin (AREG) and EGF expression was unaltered. Therefore, we propose that HB-EGF modulates the estrogen-sensitive remodeling of the uterine spiral arteries by the extravillous trophoblast in early baboon pregnancy.

We recently demonstrated that offspring delivered to baboons deprived of estrogen during the second half of gestation exhibited insulin resistance prior to onset of puberty. Because gonadal hormones have a profound effect on insulin action and secretion in adults, we determined whether insulin resistance is retained after initiation of gonadal secretion of testosterone and estradiol. Glucose tolerance tests were performed in postpubertal baboon offspring of untreated and letrozole-treated animals (serum estradiol reduced >95 %). Basal fasting levels of insulin (P < 0.05) and peak 1 min and 1 + 3 + 5 min levels of glucose after glucose tolerance tests challenge (P < 0.03) were greater in offspring delivered to letrozole-treated, estrogen-deprived baboons than untreated animals. Moreover, the value for the HOMA-IR, an accepted index of insulin resistance, was 2-fold greater (P < 0.05) in offspring delivered to baboons treated with letrozole than in untreated animals. Collectively these results support the proposal that estrogen normally has an important role in programming mechanisms in utero within the developing fetus that lead to insulin sensitivity after birth.

This study tested the hypothesis that estrogen programs mechanisms within the primate fetus that promote insulin sensitivity and glucose homeostasis in offspring. Glucose tolerance tests were performed longitudinally in prepubertal offspring of baboons untreated or treated on days 100 to 165/175 of gestation (term is 184 days) with the aromatase inhibitor letrozole, which decreased fetal estradiol levels by 95%. Basal plasma insulin levels were over two-fold greater in offspring delivered to letrozole-treated than untreated animals. Moreover, the peak 1min, average of the 1, 3, and 5min, and area under the curve blood glucose and plasma insulin levels after an i.v. bolus of glucose were greater (PPin utero than in untreated animals and partially or completely restored in letrozole plus estradiol-treated baboons. The value for the homeostasis model assessment of insulin resistance was 2.5-fold greater (PPin utero indicates that pancreatic beta cells had the capacity to secrete insulin, but that peripheral glucose uptake and/or metabolism were impaired, indicative of insulin resistance and glucose intolerance. We propose that estrogen normally programs mechanisms in utero within the developing primate fetus that lead to insulin sensitivity, normal glucose tolerance, and the capacity to metabolize glucose after birth.

We recently demonstrated that offspring delivered to baboons deprived of estrogen during the second half of gestation exhibited insulin resistance. Therefore, because skeletal muscle accounts for80% of insulin dependent glucose disposal, we suggested that estrogenThe fasting serum levels of adiponectin, leptin, glucose, and analytes of liver function as well as the basal levels of serum FFA were similar in offspring of estrogen replete/suppressed baboons. Moreover, the normal glucose-induced decline in serum FFA levels measured in untreated offspring was also measured in offspring of letrozole-treated baboons. Fetal serum levels of adiponectin and leptin in late gestation also were similar and expression of nitrotyrosine negligible in fetal liver and adipose of untreated and letrozole-treated animals.These results indicate that offspring of letrozole-treated baboons have normal adipose and liver function and do not exhibit adipose insulin resistance. Therefore, we suggest that the insulin resistance observed in estrogen-deprived offspring primarily reflects a decline in insulin-stimulated glucose clearance by skeletal muscle and which supports our original suggestion that estrogen

This article will summarize the factors controlling the ontogeny of and production of hormones by the fetal hypothalamus, pituitary, thyroid, gonads, adrenal, parathyroid, and pancreas; the integration and coordination of fetal endocrine gland function with maternal and placental hormonogenesis and placental transport of maternal nutrients; and the role of the fetal hormones on maintenance of homeostasis, growth and the maturation of fetal organs systems important for physiologic function in adulthood.

We have shown that fetal adrenal fetal zone (FZ) volume and serum dehydroepiandrosterone sulfate (DHAS) levels were increased, whereas definitive and transitional zone (DZ/TZ) volume was unaltered, in baboons in which estrogen levels were suppressed by the administration of the aromatase inhibitor letrozole. The interaction of the melanocortin 2 receptor (MC2R) with its accessory protein (MRAP) is essential for trafficking MC2R to the adrenal cell surface for binding to ACTH. The present study determined whether the estrogen-dependent regulation of fetal adrenocortical development is mediated by ACTH and/or expression/interaction of MC2R and MRAP. Fetal pituitary proopiomelanocortin mRNA and plasma ACTH levels and fetal adrenal MC2R-MRAP interaction were assessed in baboons in which estrogen was suppressed/restored by letrozole/letrozole plus estradiol administration during the second half of gestation. Although fetal pituitary proopiomelanocortin and plasma ACTH levels and fetal adrenal MC2R and MRAP protein levels were unaltered, MC2R-MRAP interaction was 2-fold greater (P < .05) in the DZ/TZ in letrozole-treated baboons than in untreated animals and restored by letrozole plus estradiol treatment. We propose that the increasing levels of estradiol with advancing pregnancy suppress interaction of MC2R with MRAP, thereby diminishing MC2R movement to the cell membrane in the DZ/TZ. This would be expected to reduce progenitor cell proliferation in the DZ and migration to the FZ, thereby restraining FZ growth and DHAS production to maintain fetal adrenal DHAS and placental estradiol levels in a physiological range late in gestation.

We showed that the volume of the fetal zone of the fetal adrenal gland and serum dehydroepiandrosterone sulfate (DHAS) levels at term were increased in baboons in which estradiol levels were suppressed by treatment with aromatase inhibitor 4,4-[1,2,3-triazol-1yl-methylene] bis-benzonitrite (letrozole). The fetal zone remodels postnatally into the reticular zone and DHAS production, and serum levels decline with age. Therefore, we determined whether the trajectory of reticular zone DHAS secretion and response to ACTH were altered in offspring deprived of estrogen in utero. Female offspring were delivered to baboons untreated or treated daily throughout the second half of gestation with letrozole (estradiol reduced >95%) or letrozole plus estradiol and cortisol and DHAS determined in blood samples obtained bimonthly between 4 and 125 months and after iv bolus of ACTH. The slope/rate of decline in serum DHAS with advancing age was greater (P < .01) in letrozole-treated (−0.54 ± 0.005) than untreated (−0.32 ± 0.003) baboons and partially restored by letrozole-estradiol (−0.43 ± 0.004). Serum cortisol was similar and relatively constant in all offspring. Moreover, in letrozole-treated offspring, serum DHAS at 61–66, 67–95, and 96–125 months were lower (P < .05), and cortisol to DHAS ratio was greater (P < .05) than in untreated offspring. ACTH at high level increased cortisol and DHAS in untreated baboons and cortisol but not DHAS in letrozole-treated offspring. We propose that postnatal development of the primate adrenal cortex, including the decline in reticular zone DHAS production, response to ACTH and maintenance of cortisol to DHAS ratio with advancing age is modulated by exposure of the fetal adrenal to estradiol.

In the field of protein biology, immunology-based techniques have been evolving for detection and quantification of protein levels, protein-protein interaction, and protein modifications in cells and tissues. The proximity ligation assay (PLA), a method of detection that combines immunologic and PCR-based approaches, was developed to overcome some of the drawbacks that are inherent to other detection methods. The PLA allows for very sensitive and discretely quantifiable measures of unmodified, native protein levels, and protein-protein interaction/modification complexes in situ in both fixed tissues and cultured cells. We describe herein the PLA method and its applicability to quantify the effects of estrogen on expression of angioregulatory factors, e.g., angiopoietin-1 (Ang-1) in the endometrium, vascular endothelial growth factor (VEGF) in the placenta, and melanocortin 2 receptor (MC2R)/accessory protein (MRAP) in the fetal adrenal of the nonhuman primate.

The present study was conducted to determine the impact of suppressing trophoblast remodeling of the uterine spiral arteries by prematurely elevating estrogen levels in the first trimester of baboon pregnancy on uterine and umbilical blood flow dynamics. Uteroplacental blood flow was assessed by Doppler ultrasonography after acute administration of saline (basal state) and serotonin on days 60, 100, and 160 of gestation (term: 184 days) to baboons in which uterine spiral artery remodeling had been suppressed by the administration of estradiol on days 25–59 of gestation. Maternal blood pressure in the basal state was increased ( P < 0.01), and uterine artery diastolic notching and the umbilical artery pulsatility index and systolic-to-diastolic ratio, reflecting downstream flow impedance, were increased ( P < 0.01) after serotonin administration on day 160, but not earlier, in baboons treated with estradiol in early gestation. These changes in uteroplacental flow dynamics in serotonin-infused, estradiol-treated animals were accompanied by a decrease ( P < 0.05) in uterine and umbilical artery volume flow and fetal bradycardia. The results of this study show that suppression of uterine artery remodeling by advancing the rise in estrogen from the second trimester to the first trimester disrupted uteroplacental blood flow dynamics and fetal homeostasis after vasochallenge late in 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.

Estrogen has an important role in the reconstruction of a new vascular network in the endometrium during each menstrual cycle; however, the underlying mechanisms are incompletely understood. Angiopoietin-1 (Ang-1) promotes vessel assembly, whereas Ang-2 and thrombospondin-1 (TSP-1) cause vessel breakdown. To determine the potential effect of estrogen on the expression of these angioregulatory factors in the endometrium, Ang-1, Ang-2, TSP-1, and Tie-2 receptor mRNA levels were assessed by real-time reverse transcriptase polymerase chain reaction in glandular epithelial and stromal cells isolated from the endometrium of ovariectomized baboons treated acutely with estradiol. Corresponding protein expression was assessed by immunocytochemistry and the proximity ligation assay (PLA) during advancing stages of the baboon menstrual cycle. Serum estradiol levels in ovariectomized baboons were 400 pg/ml within 4–6 hr of estradiol treatment. Ang-1 mRNA levels in glandular epithelial cells increased threefold (P

During human and nonhuman primate pregnancy, an extensive blood vessel network is established within the villous placenta to support fetal growth and follicles develop within the fetal ovary to provide a pool of oocytes for reproductive function in adulthood. These two important developmental events occur in association with a progressive increase in placental estrogen production and levels. This review will describe the developmental processes required for placental vascularization and fetal follicular maturation and recent studies which show that estrogen has an important role in regulating these events.

We previously showed that fetal adrenal fetal zone growth was increased and the number of follicles in the fetal ovary reduced in baboons in which estradiol was suppressed by treatment with the aromatase inhibitor letrozole between mid and late gestation periods. Because adrenal/ovarian development was restored in animals treated with letrozole and estradiol, and both tissues express estrogen receptor, we proposed that estrogen regulates fetal adrenal/ovary development via a direct action. However, because prolactin can modulate fetal adrenal and adult pituitary/ovarian function, the current study determined whether estrogen action involved estradiol-regulated changes in fetal prolactin/luteinizing hormone (LH) expression. Fetal prolactin levels and the number of prolactin-positive fetal pituitary cells (per 0.37 mm2) were increased (P 95%) by letrozole (61 ± 11 ng/ml; 19.3 ± 2.0), and minimally but not significantly increased by letrozole and estradiol (99 ± 11 ng/ml; 32.7 ± 5.2). In contrast, the number of LH-positive fetal pituitary cells decreased (P < 0.01) between mid (48.8 ± 9.5) and late (17.4 ± 3.2) gestation, remained elevated (P < 0.01) in estrogen-suppressed animals (56.6 ± 5.1), and was partially but not significantly decreased by letrozole-estradiol (28.8 ± 5.2). We conclude that estrogen regulates fetal pituitary prolactin and LH expression and fetal prolactin levels. However, because prolactin and LH expressions in estrogen-suppressed fetuses were inversely related to previously demonstrated changes in adrenal/ovarian development, we propose that estrogen regulates the fetal ovary and adrenal gland directly and not via action on the fetal pituitary gland.

We previously demonstrated that the number and height of oocyte microvilli were reduced in baboon fetuses deprived of estrogen in utero and restored to normal in animals supplemented with estradiol. Phosphorylated ezrin and Na+/H+ exchange regulatory factor 1 (NHERF, now termed SLC9A3R1) link f-actin bundles to the membrane, whereas alpha-actinin cross-links f-actin to form microvilli. Therefore, we determined whether these proteins were expressed in oocytes of the fetal baboon ovary and whether expression and/or localization were altered between mid and late gestation in association with an increase in estrogen and in late gestation in animals in which estrogen was suppressed (>95%) or restored by treatment with an aromatase inhibitor with or without estradiol. Expression of alpha-actinin was low at mid gestation, increased on the surface of oocytes of primordial follicles in late gestation, and was negligible in the ovaries of estrogen-suppressed fetuses and normal in animals treated with estro...

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.

In fetal ovaries of estrogen-suppressed baboons, we have previously shown that follicle numbers were 50% lower than in estrogen-replete animals and contained oocytes with a reduced number of microvilli. In the baboon fetal ovary, although estrogen receptor (ER)alpha and beta have been detected by immunocytochemistry in granulosa cells, it is not known whether oocytes express ER. Because the actions of estrogen are mediated by interaction with cell-specific receptors, the current study determined whether ERalpha/beta mRNA were expressed in oocytes of baboon fetal ovaries obtained on day 165 (term = day 184) of gestation. Oocyte nuclei and cytoplasm from primordial follicles were isolated by laser capture microdissection and ERalpha, ERbeta, GATA-4 (granulosa cell specific marker) mRNAs, and 18S rRNA determined by RT-PCR and products verified by sequencing. ERbeta mRNA was expressed in oocytes of 5 of 5 fetuses. In contrast, fetal oocytes did not express ERalpha mRNA. Although 18S rRNA was expressed in all oocytes, GATA-4 mRNA was not detected in oocytes and only detected in granulosa cells confirming purity of oocytes sampled. We conclude that oocytes of the fetal baboon ovary express ERbeta mRNA, thereby providing a mechanism by which estrogen regulates oocyte function, e.g. microvillus development.

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 (P0.05) 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 (P0.05) 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.

Although the human and the nonhuman primate fetal adrenal glands undergo a highly unique pattern of cortical zone-specific intrauterine growth and development, studies of the regulatory components of the cell cycle responsible for this growth have not been conducted. Therefore, the present study determined expression of the cell cycle regulators, cyclin D1 and cyclin E, and their cyclin-dependent kinases, Cdk2, Cdk4, and Cdk6, and Ki67 a marker of cell proliferation within the baboon fetal adrenal cortex during advancing stages of gestation. Fetal adrenal glands were obtained on days 60 (early), 100 (mid), and 160–170 (late) of gestation (term = 184 days). Mean ( ± s.e.) cyclin D1 mRNA levels, determined by RT-PCR and expressed relative to 18S rRNA, were similar at early (0.85 ± 0.09) and mid (1.04 ± 0.08) gestation, then decreased (P < 0.001, ANOVA) approximately 50% by late gestation (0.57 ± 0.04). Cyclin E mRNA levels were also similar at early (2.03 ± 0.07) and mid (1.63 ± 0.31) gestation, and decreased by 70% (P < 0.001) in late gestation (0.53 ± 0.09). Coinciding with the decrease in cyclin D1 and cyclin E, the percentage of Ki67 positive cells in the definitive zone decreased twofold (P < 0.01) between mid (28.2 ± 3.6) and late (13.8 ± 1.7) gestation. The cyclin D1 and cyclin E proteins, determined by immunocytochemistry, were expressed at high levels in the definitive zone of baboon fetal adrenal gland, where they decreased between mid- and late gestation. In contrast, immunocytochemical expression of the functionally important steroidogenic enzyme Delta; 5-3β-hydroxysteroid dehydrogenase (3β-HSD) became abundant in the definitive and transitional zones with advancing pregnancy. However, fetal adrenal Cdk2, Cdk4, and Cdk6 mRNA levels and protein immunoexpression were similar in the baboon fetal adrenal at early-, mid-, and late gestation. In summary, expression of cyclin D1, cyclin E, and Ki67 decreased, while 3β-HSD expression increased, in the fetal adrenal cortex, particularly in the definitive zone, between mid- and late-baboon gestation. We propose that a developmental decline in cellular proliferation permits functional differentiation of fetal adrenal cortical cells, leading to increased production of steroid hormones important for placental estrogen synthesis and maturation of organ systems within the developing fetus.

We recently showed an increase in vascular endothelial growth factor (VEGF), decrease in angiopoietin-1 (Ang-1) and unaltered Ang-2 expression by the villous placenta with advancing baboon pregnancy. Moreover, placental VEGF expression was increased by estrogen in early pregnancy. In the present study, we determined whether placental Ang-1 and Ang-2 are regulated by estrogen. Ang-1 and Ang-2 mRNA and protein were determined by RT-PCR and immunocytochemistry in the placenta of baboons on Day 60 of gestation (term is 184 days) after administration of estrogen precursor androstenedione on Days 25-59 or on Day 54 after acute estradiol administration. Chronic androstenedione treatment increased serum estradiol levels three-fold (P0.001) and decreased (P0.05) villous cytotrophoblast Ang-1 mRNA to a level (0.36 +/- 0.08 relative to 18S rRNA) that was one-third of that in untreated animals (0.98 +/- 0.26). Within 2 hr of estradiol administration, cytotrophoblast Ang-1 mRNA was decreased to a level (0.24 +/- 0.05) one-fifth (P0.05) of that in untreated animals (1.14 +/- 0.23). However, Ang-2 mRNA levels were unaltered. Ang-1, Ang-2 and estrogen receptors alpha and beta protein were localized within villous cytotrophoblasts providing a mechanism for estrogen action at this site. In summary, estrogen increased VEGF, decreased Ang-1, and had no effect on Ang-2 expression within placental cytotrophoblasts during early baboon pregnancy. We propose that the estrogen-dependent differential regulation of these angioregulatory factors underpins the unique pattern of neovascularization established within the villous placenta during primate pregnancy.

In the field of protein biology, immunology-based techniques have been evolving for detection and quantification of protein levels, protein-protein interaction, and protein modifications in cells and tissues. The proximity ligation assay (PLA), a method of detection that combines immunologic and PCR-based approaches, was developed to overcome some of the drawbacks that are inherent to other detection methods. The PLA allows for very sensitive and discretely quantifiable measures of unmodified, native protein levels, and protein-protein interaction/modification complexes in situ in both fixed tissues and cultured cells. We describe herein the PLA method and its applicability to quantify the effects of estrogen on expression of angioregulatory factors, e.g., angiopoietin-1 (Ang-1) in the endometrium, vascular endothelial growth factor (VEGF) in the placenta, and melanocortin 2 receptor (MC2R)/accessory protein (MRAP) in the fetal adrenal of the nonhuman primate.

The human and nonhuman primate placenta produces estradiol and progesterone and peptide hormones that function independently and as components of paracrine, autocrine, and endocrine control systems essential for maternal–fetal homeostasis and successful pregnancy. This chapter summarizes the developmental pattern of expression, action, and regulation of these placental hormones. For example, chorionic gonadotropin promotes luteal progesterone synthesis and thus pregnancy maintenance, while placental growth factors regulate placental and fetal vasculogenesis, and uterine artery remodeling essential for placental perfusion and delivery of substrates to the fetus. Placental lactogen, insulin-like growth factor, and adipokines coordinate maternal metabolic processes essential for fetal growth. Placental estrogen and neuropeptides, including corticotrophin-releasing hormone, participate in the timing of delivery, regulate uterine blood flow and contractility, and regulate cortisol metabolism for timely maturation of the fetal pituitary–adrenocortical axis. Dysregulation of the placental hormones results in pregnancy complications, including fetal growth restriction and preeclampsia, that predispose offspring to diseases (e.g., diabetes and hypertension) in adulthood.

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.

The present study determined whether estrogen plays a role in regulating invasion and remodeling of the uterine spiral arteries by extravillous trophoblasts during early baboon pregnancy. The level of trophoblast invasion of spiral arteries was assessed on day 60 of gestation (term is 184 days) in baboons untreated or treated on days 25-59 with estradiol or aromatizable androstenedione. The administration of estradiol or androstenedione increased (P0.01) maternal serum estradiol levels approximately 3-fold above normal. The mean+/-SE percentage of spiral arteries/arterioles invaded by extravillous cytotrophoblasts in estradiol-treated baboons for vessels with diameters of 26-50 microm (0.0+/-0.0), 51-100 microm (1.2+/-0.7) and100 microm (13.2+/-5.5) was 100%, 90%, and 75% lower (P0.001), respectively, than in untreated baboons (2.4+/-1.2%; 11.0+/-5.5%, and 54.5+/-8.5%, respectively). Similar results were obtained with androstenedione treatment. However, the distribution of uterine spiral arteries grouped by diameter or number of arteries per basal plate area, i.e. microvessel density, were similar in untreated and estrogen-treated baboons. We suggest, therefore, that the low levels of estrogen exhibited during early primate pregnancy are required to permit normal progression of trophoblast vascular invasion and that the surge in estrogen which occurs during the second-third of normal pregnancy has a physiological role in suppressing further arterial trophoblast invasion. Consequently, we propose that the estrogen-dependent restraint of spiral artery invasion/remodeling ensures optimal blood flow dynamics across the uteroplacental vascular bed to promote normal fetal growth and development.

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.

Vascular endothelial growth/permeability factor (VEG/PF) has an established role in angiogenesis, however, the regulation of placental VEG/PF expression during primate pregnancy is incompletely understood. A temporal study was conducted in baboons to determine the effect of acute administration of estradiol on the expression of VEG/PF by cells of the villous placenta. VEG/PF mRNA levels were determined by reverse transcription-polymerase chain reaction in isolated placental cell fractions of baboons after acute i.v. and i.m. administration of estradiol. Within 2 h of estradiol treatment, VEG/PF mRNA (attomoles/ micrograms total RNA) increased within villous cytotrophoblasts to a level (mean ± SEM, 12 612 ± 2419) that was almost 2-fold greater (P < 0.05) than in untreated controls (6810 ± 1368). Cytotrophoblast VEG/PF mRNA levels remained elevated (P < 0.01) 6 h after estradiol treatment (15 006 ± 506), but were not different from controls 18 h after estradiol administration. VEG/ PF mRNA levels i...

Background: Alterations in sex steroids during pregnancy are associated with the development and exacerbation of reactive lesions involving the gingiva. Currently, few experimental animal models similar to humans are available to examine regulatory pathways involving sex steroids and the periodontium. Methods: In the present study, we used the baboon as a novel experimental model for the study of the regulatory actions of estrogen on the periodontium during pregnancy. Pregnant baboons (N = 5) were administered the potent, highly specific aromatase inhibitor CGS 20267 (2 mg/day subcutaneously) daily on days 60 through 165 of gestation (term = 184). Untreated females (N = 10) and females (5) concomitantly administered aromatase inhibitor and estradiol benzoate (2.0 mg/day each subcutaneously) served as controls. Gingival biopsies were taken between days 145 and 165 of gestation. Results: Administration of CGS 20267 in all females suppressed maternal serum concentrations of estradiol by 95% and induced the development of an exuberant papillomatous enlargement of the gingiva by gestational day 110, with the most prominent development involving the labial aspects of the anterior sextants. None of the untreated pregnant controls or females concomitantly administered aromatase inhibitor and estradiol benzoate developed gingival overgrowth. Thus, estradiol alone prevented the onset of gingival overgrowth induced by estrogen suppression. In all baboons, discontinuation of the aromatase inhibitor at time of cesarean section resulted in spontaneous regression and resolution of the papillomatous hyperplasia within 4 to 6 weeks. Clinically, the gingival papillary overgrowth was erythematous and edematous, with a propensity toward spontaneous subgingival hemorrhage. Histologically, the biopsy specimens demonstrated hyperplasia of the epithelium typified by mild hyperkeratosis, acanthosis, and elongation and isolated anastamoses of rete ridges. Subjacent to the intact epithelium was a loose connective tissue stroma with isolated areas of inflammatory cell infiltrate. Special stains verified the presence of isolated bacterial biofilms; however, no evidence of fungal filaments was present. Histological features suggestive of viral infection were notably absent in the epithelium. No evidence of viral particles or capsids was identified using transmission electron microscopy. Reverse transcription polymerase chain reaction analysis, using a panel of degenerate primers, was negative for papilloma family viruses. Conclusions: These results are consistent with a significant role for estrogen during primate pregnancy in the regulation of cellular proliferation and differentiation within the gingiva. The baboon represents an important experimental model for studying the regulatory actions of estrogen on the periodontium during pregnancy. J Periodontol 2004;75:693-701.

Although studies in transgenic mice suggest that estrogen is important for development of the testis, very little is known about the potential role of estrogen in maturation of the primate fetal testis. Therefore, as a first step to determine whether estrogen regulates maturation of the fetal primate testis, we used immunocytochemistry to determine estrogen receptor (ER) alpha and beta expression in the fetal baboon testis. Second, we established methods to quantify ERbeta mRNA levels by competitive reverse transcription-polymerase chain reaction in Sertoli cells isolated by laser capture microdissection (LCM) from the fetal baboon testis. ERbeta protein expression was abundant in the nuclei of Sertoli, peritubular, and interstitial cells in baboon fetuses at mid (Day 100) and late (Day 165) gestation (term is 184 days). ERbeta mRNA level was 0.03 attomole/femtomole 18S rRNA in Sertoli cell nuclei and associated cytoplasm isolated by LCM. ERalpha was expressed in low level in seminiferous tubules and in moderate level in peritubular cells on Day 165. Germ cells expressed very little ERalpha or ERbeta protein, whereas the baboon fetal epididymis exhibited extensive ERalpha and ERbeta immunostaining at mid- and late gestation. In contrast to the robust expression of ERbeta, androgen receptor protein was not demonstrable within the cells of the seminiferous cords but was abundantly expressed in epididymal epithelial cells of the fetal baboon. In summary, the results of this study show that the fetal baboon testis and epididymis expressed the ERalpha and ERbeta, and we suggest that our nonhuman primate baboon model can be used to study the potential role of estrogen on maturation of the fetal testis.

We recently demonstrated that the reduction in the number of primordial follicles in ovaries of near-term baboon fetuses deprived of estrogen in utero was associated with increased expression of alpha-inhibin, but not activin betaA and betaB or the activin receptors. Therefore, we proposed that estrogen regulates fetal ovarian follicular development by controlling the intraovarian inhibin:activin ratio. As a prelude to conducting experiments to test this hypothesis, in the current study we determined whether the primate fetal ovary expressed Smads 2/3 and 4 and whether expression of these activin-signaling proteins was altered in fetal ovaries of baboons in which estrogen production was suppressed. Western blot analyses demonstrated that the 59 kDa Smad 2, 54 kDa Smad 3, and 64 kDa Smad 4 proteins were expressed in fetal ovaries of untreated baboons at both mid and late gestation and that the level of expression was not significantly altered in late gestation by in vivo treatment with CGS 20267 or CGS 20267 and estrogen. Immunocytochemistry localized Smads 2/3 and 4 to cytoplasm of oocytes and pregranulosa cells at midgestation and oocytes and granulosa cells of primordial follicles in late gestation. Smad 4 was also detected in granulosa cell nuclei in late gestation, and nuclear expression appeared to be decreased in fetal ovaries of baboons deprived of estrogen. The site of localization of Smads correlated with localization of the activin receptors IA and IIB, which we previously showed were abundantly expressed in oocytes and (pre)granulosa cells at both mid and late gestation and unaltered by estrogen deprivation. In summary, the results of the current study are the first to show that the intracellular signaling molecules required to transduce an activin signal are expressed in the baboon fetal ovary and that expression was not altered by estrogen deprivation in utero. These findings, coupled with our previous observations showing that estrogen deprivation reduced follicle numbers and upregulated/induced expression of inhibin but not activin or the activin receptors, lend further support to the hypothesis that estrogen regulates fetal ovarian folliculogenesis by controlling the intraovarian activin:inhibin ratio.

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.

Although estrogen plays a central integrative role in regulating key aspects of placental and fetal endocrine development in the primate, our understanding of the regulation of maturation of the fetal liver is incomplete. In adults, estrogen modulates several aspects of hepatic function. Therefore, the current study determined whether fetal hepatic gene expression development was modulated by estrogen. mRNA differential display was used to identify genes whose expression was altered in fetal livers obtained on d 165 of gestation (term=d 184) from baboons that were untreated or treated on d 60–164 with the aromatase inhibitor CGS 20267 (2 mg/d; sc), which suppressed estrogen levels in the fetus by > 95% (p

The ovarian steroid hormones, estrogen and progesterone, have important roles in establishing the new vascular bed within the endometrium during each menstrual cycle; however, little is known about the mechanisms underlying this process. We recently showed that mRNA and protein levels for the angiogenic factor vascular endothelial growth/permeability factor (VEG/PF) in endometrial glandular epithelial and stromal cells of baboons were decreased to very low levels by ovariectomy, and we proposed that the levels of estrogen and progesterone exhibited during the menstrual cycle regulate endometrial VEG/PF expression in the primate. To test this hypothesis, VEG/PF mRNA levels were determined by reverse transcription-polymerase chain reaction in glandular epithelial and stromal cells isolated by laser-capture microdissection from, and VEG/PF protein was determined by immunocytochemistry in the endometrium of baboons after ovariectomy and chronic administration of estradiol and progesterone in levels d...

In the adult ovary, pituitary FSH via interaction with its receptor (FSHR) is required for follicular maturation and granulosa cell development. In humans and nonhuman primates, the pool of follicles available for adult ovarian function is established in utero. However, our understanding of the ontogeny and developmental regulation of FSHR in the ovary of the primate fetus is incomplete. Our goal was to determine whether the baboon fetal ovary expresses the full-length FSHR mRNA transcript and whether levels are developmentally regulated. Fetal ovaries were obtained at mid (Day 100) and late (Day 165) gestation (term = Day 184) from untreated baboons and on Day 165 from baboons in which fetal estrogen levels were either decreased by >95% by treatment with the aromatase inhibitor CGS 20267 or restored to 30% of normal by treatment with CGS 20267 plus estradiol benzoate administered s.c. to the mother on Days 100–164. The full-length 2088-base pair FSHR mRNA transcript was expressed in ovaries of a...

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.

The human endometrium develops new capillaries from existing microvessels, i.e. angiogenesis, which then undergo maturation and remodeling, i.e. investment of microvessels with periendothelial mural cells, into a new vascular network during each menstrual cycle. Improper vascularization of the endometrium may cause implantation failure and infertility. Estrogen and progesterone have pivotal roles in establishing this vascular bed, but the cellular sites and mechanisms of action of these steroid hormones are incompletely understood. Vascular endothelial growth factor (VEGF), angiopoietin-1, and angiopoietin-2 and their receptors are expressed in the human and nonhuman primate endometrium and interact to control vascular development and remodeling. VEGF synthesis within and neovascularization of the endometrium seem to be sustained and ongoing processes designed to progressively promote growth and development of the endometrium with advancing stages of the menstrual cycle. However, estrogen rapidly upregulates VEGF expression by endometrial glandular epithelial and stromal cells in vivo in the nonhuman primate and in vitro in the human. Reports of the effects of progesterone on endometrial VEGF formation, however, are inconsistent and may reflect regulatory actions on particular isoforms of VEGF. In addition, estrogen has effects on vascular endothelial and smooth muscle cells, which may be direct or mediated by VEGF. Very little is known, however, about the steroid hormone regulation of other angiostimulatory and angioinhibitory factors, e.g. angiopoietin-1 and -2, in the endometrium. Moreover, the role of steroid hormones acting directly, or indirectly via VEGF and other angiogenic factors, on expression of integrins, cell adhesion and other molecules required for cell-cell and cell-extracellular matrix interactions important for angiogenesis in the human and nonhuman primate endometrium is largely unknown. Finally, further study is needed of cell-specific responsivity and function in the human endometrium with respect to steroid hormone regulation of angiogenesis.

Ovarian function in adult human and nonhuman primates is dependent on events that take place during fetal development, including the envelopment of oocytes by granulosa (i.e., folliculogenesis). However, our understanding of fetal ovarian folliculogenesis is incomplete. During baboon pregnancy, placental production and secretion of estradiol into the fetus increases with advancing gestation, and the fetal ovary expresses estrogen receptors a and b in mesenchymal-epithelial cells (i.e., pregranulosa) as early as midgestation. Therefore, the current study determined whether estrogen regulates fetal ovarian follicular development. Pregnant baboons were untreated or treated with the aromatase inhibitor CGS 20267, or with CGS 20267 plus estradiol benzoate administered s.c. to the mother on Days 100‐164 (term 5 Day 184). On Day 165, baboon fetuses were delivered by cesarean section and the number of total follicles and interfollicular nests consisting of oocytes and mesenchymal-epithelial cells in areas (0.33 mm2) of the outer and inner cortices of each fetal ovary were quantified using image analysis. Maternal and umbilical serum estradiol levels were decreased by .95% with CGS 20267. Treatment with CGS 20267 and estrogen restored maternal estradiol to normal and fetal estradiol to 30% of normal. Although fetal ovarian weight was unaltered, the mean number of follicles 6 SEM/0.33 mm2 in the inner (59.0 6 1.7) and outer (95.3 6 2.4) cortical regions of fetal ovaries in untreated animals was 35%‐50% lower (P , 0.01) in estrogen-depleted baboons (25.9 6 1.4, inner cortex; 62.5 6 2.7, outer cortex) and was restored to normal by treatment with CGS 20267 and estrogen. In contrast, the number of interfollicular nests was 2-fold greater (P , 0.01) in fetal ovaries of estrogen-suppressed animals, a change that was prevented by treatment with estrogen. In summary, fetal ovarian follicular development was significantly altered in baboons in which estrogen was depleted during the second half of gestation and restored to normal by estradiol. We propose that estrogen plays an integral role in regulating, and perhaps programming, primate fetal ovarian development. estradiol, follicle, oocyte development, ovary, pregnancy

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 adult mammals, estrogen regulates ovarian function, and estrogen receptor (ER) is expressed in granulosa cells of antral follicles of the adult baboon ovary. Because the foundation of adult ovarian function is established in utero, the present study determined whether ERalpha and/or ERbeta were expressed in fetal ovaries obtained on Days 100 (n = 3) and 165-181 (n = 5) of baboon gestation (term = Day 184). On Day 100, ERalpha protein was detected by immunocytochemistry in surface epithelium and mesenchymal-epithelial cells but not oocytes in germ cell cords. ERbeta protein was also detected by immunocytochemistry on Day 100 of gestation and was abundantly expressed in mesenchymal-epithelial cells in germ cell cords, lightly expressed in the germ cells, but was not detected in the surface epithelium. On Days 165-180 of gestation, ERalpha expression was still intense in the surface epithelium, in mesenchymal-epithelial cells throughout the cortex, and in nests of cells between follicles. ERalpha expression was lighter in granulosa cells and was not observed in all granulosa cells, particularly in follicles close to the cortex. In contrast, ERbeta expression was most intense in granulosa cells, especially in flattened granulosa cells, was weaker in mesenchymal-epithelial cells and nests of cells between follicles, and was absent in the surface epithelium. Using an antibody to the carboxy terminal of human ERbeta, ERbeta protein was also detected by Western immunoblot with molecular sizes of 55 and 63 kDa on Day 100 and primarily 55 kDa on Day 180. The mRNAs for ERalpha and ERbeta were also detected by Northern blot analysis in the baboon fetal ovary. These results are the first to establish that the ERalpha and ERbeta mRNAs and proteins are expressed and exhibit changes in localization in the primate fetal ovary between mid and late gestation. Because placental estrogen production and secretion into the baboon fetus increases markedly during advancing pregnancy, we propose that estrogen plays an integral role in programming fetal ovarian development in the primate.

We have previously shown that estrogen selectively suppresses growth of the fetal zone of the baboon fetal adrenal cortex, which produces the C19-steroid precursors, eg, dehydroepiandrosterone sulfate, which are aromatized to estrogen within the placenta. In the present study, we determined whether fetal adrenal expression of cell cycle regulators are altered by estrogen and thus provide a mechanism by which estrogen regulates fetal adrenocortical development. Cyclin D1 mRNA levels in the whole fetal adrenal were increased 50% (P < .05), and the number of cells in the fetal adrenal definitive zone expressing cyclin D1 protein was increased 2.5-fold (P < .05), whereas the total number of cells in the fetal zone and fetal serum dehydroepiandrosterone sulfate levels were elevated 2-fold (P < .05) near term in baboons in which fetal serum estradiol levels were decreased by 95% (P < .05) after maternal administration of the aromatase inhibitor letrozole and restored to normal by concomitant administration of letrozole plus estradiol throughout second half of gestation. However, fetal adrenocortical expression of cyclin D2, the cyclin-dependent kinase (Cdk)-2, Cdk4, and Cdk6, and Cdk regulatory proteins p27Kip1 and p57Kip2 were not changed by letrozole or letrozole plus estradiol administration. We suggest that estrogen controls the growth of the fetal zone of the fetal adrenal by down-regulating cyclin D1 expression and thus proliferation of progenitor cells within the definitive zone that migrate to the fetal zone. We propose that estrogen restrains growth and function of the fetal zone via cyclin D1 to maintain estrogen levels in a physiological range during primate pregnancy.