Search

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.

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 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.

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.

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.

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.

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.

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.

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

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.

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.

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.

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.

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

In the present study, we determined whether expression of the messenger ribonucleic acids (mRNAs) for insulin-like growth factor-II (IGF-II), and its principal IGF type-1 receptor and IGF-binding protein-2 (IGFBP-2), as well as basic fibroblast growth factor (bFGF), was developmentally regulated in the baboon fetal adrenal gland. In the second phase of this study, fetal pituitary ACTH was suppressed by the administration of betamethasone to determine the possible effect on the mRNA levels for those factors, i.e. IGF-II and IGFBP-2, shown to be expressed at high levels in the adrenal late in fetal development. Adrenals were obtained from fetuses delivered via Cesarean section on days 60 (early), 100 (mid), and 165 (late) of gestation (term=184 days) from untreated baboons and on day 165 from baboons in which betamethasone was administered to the fetus, or to fetus and mother, every other day between days 150 and 164 of gestation. Although the mRNA levels of IGF-II in the fetal adrenal were similar at early, mid and late gestation, IGF type-1 receptor mRNA levels were approximately 2- to 3-fold greater (P

This review summarizes the experimental evidence supporting the concept that oestrogen has a central integrative role in modulating the communication that occurs between the placenta and the fetus which results in primate fetal–placental development. Thus oestrogen, acting within placental trophoblasts, regulates the functional differentiation of syncytiotrophoblasts, manifested as an upregulation of key components of the progesterone biosynthetic pathway and the 11β-hydroxysteroid dehydrogenase (11β-HSD)-1 and -2 enzymes controlling cortisol–cortisone interconversion. The increase in 11β-HSD expression results in the switch in the qualitative and quantitative patterns of transplacental corticosteroid metabolism that induces maturation of the primate fetal hypothalamic pituitary adrenocortical axis. The studies outlined in this review, therefore, provide new insight into the role that oestrogen plays during the course of primate pregnancy and demonstrate that an oestrogen-dependent signalling system exists in utero that coordinates the placental and fetal dialogue critical to development of the placenta and endocrine systems underlying neonatal self-sufficiency.

In the baboon, estrogen regulated 11beta-hydroxysteroid dehydrogenase (11beta-HSD) catalyzed metabolism of cortisol and cortisone by the placenta is an important component in the sequence of events regulating the fetal pituitary-adrenocortical axis. The present study was designed to isolate and sequence the promoter region of the baboon 11beta-HSD-2 gene and to produce constructs of this gene and the 1.7 kb fragment of 5'-flanking region of baboon 11beta-HSD-1 isolated previously in order to determine whether the promoters of these two genes were activated in human placental JEG-3 cells and whether expression could be modulated by estradiol. The 11beta-HSD-2 genomic DNA was isolated from a baboon kidney genomic library using a human 11beta-HSD-2 cDNA as a probe. The sequence of a 1.2 kb fragment of the 5'-flanking region showed extensive homology with that published by others for human 11beta-HSD-2, particularly in exon 1 (>95%) and in the proximal promoter (>90%). Primer extension confirmed that the baboon 11beta-HSD-2 gene has multiple transcriptional start sites which are preceded by a GC box. To determine promoter activity of 11beta-HSD-2 and -1, the 5'-flanking regions of these genes were subcloned into luciferase reporter pGL3 vectors, transiently transfected into human placental JEG-3 cells, and then incubated for 16-18 h in the presence or absence of 10-8 M 17beta-estradiol or 17alpha-estradiol. To augment the low level of estrogen receptor (ER) in JEG cells, promoter activity studies were also performed in JEG cells co-transfected with an expression vector containing the human ER cDNA. The promoters of both 11beta-HSD-1 and -2 were activated following transient transfection into JEG-3 cells although basal activity of 11beta-HSD-2 (87+/-21 RLU/microg protein) always exceeded (P

We have shown that oestrogen has a central integrative role in regulating key components of the progesterone biosynthetic and corticosteroid metabolic pathways within syncytiotrophoblasts that govern placental function and maturation of the fetal pituitary-adrenocortical axis. Studies utilizing classic binding procedures and RNAse protection have demonstrated that human placental villous tissue exhibits specific high affinity oestrogen binding and expresses the mRNA for the oestrogen receptor. However, it is not known whether the oestrogen receptor is expressed specifically in syncytiotrophoblasts. Therefore, the present study determined whether the oestrogen receptor protein was detectable by immunocytochemistry in cultured human syncytiotrophoblast maintained in a low oestrogen/progestin environment. Cytotrophoblasts were isolated from human term placentae by trypsin dispersion and Percoll gradient centrifugation and cultured for 5, 7 or 10 days. Incubation of syncytiotrophoblast with 5-10 micrograms/ml of the anti-oestrogen receptor rat monoclonal antibody D-75, which is specific for the primate oestrogen receptor, resulted in identification of the oestrogen receptor in the nuclei of these cells. In contrast, there was no reactivity of the trophoblasts to either rat IgG or an irrelevant rat monoclonal antibody IgG2a against mouse common leukocyte antigen T200. Collectively, these findings indicate that oestrogen receptor is expressed in the nuclei of human placental syncytiotrophoblasts and support the suggestion that the syncytiotrophoblast is an oestrogen-responsive tissue.

Using the baboon as a model for studies of human reproductive biology, we previously showed that placental estrogen regulates fetal ovarian follicle development. In this study, offspring of baboons untreated or treated in utero with the aromatase inhibitor letrozole (estradiol reduced >95%) or letrozole and estradiol were reared to adulthood to determine whether estrogen programming of the fetal ovary impacted puberty and reproduction in adulthood. All offspring exhibited normal growth and blood pressure/chemistries. Puberty onset in untreated baboons (43.2 ± 1.4 mo) was delayed (P < 0.01) in animals of letrozole-treated mothers (49.0 ± 1.2 mo) and normal in offspring of mothers treated with letrozole and estradiol (42.7 ± 0.8 mo). During the first 2 yr postmenarche, menstrual cycles in estrogen-suppressed animals (43.2 ± 1.3 days) were longer (P < 0.05) than in untreated baboons (38.3 ± 0.5 days) or those treated with letrozole and estrogen (39.6 ± 0.8 days). Moreover, in estrogen-suppressed offspring, serum levels of estradiol were lower and follicle-stimulating hormone greater (P < 0.05) in the follicular and luteal phases, and the elevation in luteal-phase progesterone extended (P < 0.02). Thus, puberty onset was delayed and menstrual cycles prolonged and associated with altered serum hormone levels in baboon offspring that developed in an intrauterine environment in which estradiol levels were suppressed. Because puberty and follicle development, as shown previously, were normal in baboons treated in utero with letrozole and estradiol, we propose that fetal ovarian development and timely onset of puberty in the primate is programmed by fetal exposure to placental estrogen.

At midgestation in the baboon, 11beta-hydroxysteroid dehydrogenase (11beta-HSD) catalysed interconversion of cortisol (F) and cortisone (E) during transuterine passage favors the formation of F. Because the site(s) of oxidation/reduction of F and E are not clear, the present study compared F-E interconversion in placenta, decidua and chorion in vitro. In addition, because the reduction of E to F is catalysed only by the 11beta-HSD-1, we also determined whether the mRNA for this enzyme was expressed in baboon placenta, including placental syncytiotrophoblast cells; the site of fetal-maternal exchange. Placentas were obtained on day 100 of gestation (term = day 184) and villous tissue, decidua and chorion isolated, minced in HBSS and incubated (300-400 mg) in duplicate for 0.1-24 h in Medium 199 containing 10% fetal bovine serum and [3H]F and [14C]E. Radiolabelled F and E were purified from incubates and the percentage conversion of F to E and E to F was calculated. In decidua, mean (+/- SE; n = 3) conversion of E to F (69 +/- 2%) was greater than oxidation of F to E (26 +/- 2%). Conversion of E to F in placenta (50 +/- 1%) and chorion (39 +/- 9%) was also extensive and greater than or equal to that for the oxidation of F to E (39 +/- 4% and 32 +/- 4%, respectively). The apparent ratio of 11beta-HSD reductive/oxidative activity was maintained when respective tissues from four baboons were incubated for 18 h with or without 4.4 microM excess radioinert substrate. Expression of 11beta-HSD-1 mRNA was determined by Northern blot by hybridization of poly (A) + -enriched RNA from tissues obtained at midgestation with [32P]labelled human 11beta-HSD-1 cDNA. This cDNA hybridized to a single mRNA species of 1.6 kb in decidua, whole placental villous tissue and syncytiotrophoblast cells, but not to RNA isolated from fetal baboon kidney. The results of the present study demonstrate that at midgestation in the baboon, 11beta-HSD activity in intact placenta and decidua in vitro favored the formation of F from E, presumably catalysed by the 11beta-HSD-1 enzyme protein, the mRNA for which is present in placental syncytiotrophoblasts. Moreover, based on overall mass and extensive vascularity, we suggest that the net formation of F from E during transuterine passage in vivo at this stage of gestation results from the 11beta-HSD-1 in the syncytiotrophoblast cells of the baboon placenta.

The present study determined whether the elevation in oestrogen, which occurs with advancing baboon pregnancy, is associated with a developmental increase in expression of the placental enzymes catalysing progesterone synthesis. The mRNA levels for P-450 cholesterol side-chain cleavage (P-450scc), adrenodoxin, and delta 5-3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta-HSD) were assessed by Northern blot analysis in placental syncytiotrophoblasts isolated from baboons in early (days 58-65), mid- (days 97-113) and late (days 161-175), gestation (term = 184 days). Placental villous tissue was dispersed and subjected to 50 per cent Percoll density gradient centrifugation to obtain primarily syncytiotrophoblasts. Mean (+/- S.E.) P-450scc mRNA level, expressed as a ratio of beta-actin in the syncytiotrophoblast-rich fraction, progressively increased with advancing pregnancy to a level in late gestation (1.81 +/- 0.28 arbitrary units) that was approximately sixfold (P0.01) greater than in early gestation (0.31 +/- 0.08) and approximately twofold greater (P0.05) than in mid-gestation (0.97 +/- 0.24). In contrast adrenodoxin mRNA expression was similar at early (0.97), mid- (1.14 +/- 0.12) and late (1.16 +/- 0.13) gestation. Syncytiotrophoblast 3 beta-HSD mRNA levels also remained constant in early (1.69), mid- (1.89 +/- 0.41) and late (1.34 +/- 0.41) gestation. On the basis of these findings, we propose that villous syncytiotrophoblasts undergo functional/biochemical differentiation, resulting in a coordinated upregulation of specific components of the steroid biosynthetic pathway required for progesterone biosynthesis during the course of primate pregnancy.

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)

Expression of the molecules that modulate the synthesis and action of estrogen in, or reflect function of, Sertoli cells was determined in the fetal testis of baboons in which estrogen levels were suppressed in the second half of gestation to determine whether this may account for the previously reported alteration in fetal testis germ cell development. P-450 aromatase, estrogen receptor (ER) β, and α-inhibin protein assessed by immunocytochemistry was abundantly expressed in Sertoli cells of the fetal baboon testis, but unaltered in baboons in which estrogen levels were suppressed by letrozole administration. Moreover, P-450 aromatase and ERα and β mRNA levels, assessed by real-time RT-PCR, were similar in germ/Sertoli cells and interstitial cells isolated from the fetal testis of untreated and letrozole-treated baboons. These results indicate that expression of the proteins that modulate the formation and action of estrogen in, and function of, Sertoli cells is not responsible for the changes in germ cell development in the fetal testis of estrogen-deprived baboons.

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 (P0.01) within 4 hr of estradiol administration. In contrast, TSP-1 mRNA levels decreased four- to fivefold (P0.01) in endometrial glandular epithelial and stromal cells 4-6 hr after estradiol, whereas Ang-2 and Tie-2 expression was unaltered. Immunostaining for Ang-1 increased, TSP-1 decreased, and Ang-2 and Tie-2 were unaltered in the endometrium during the secretory compared with the proliferative phase of the cycle. Endometrial Ang-1 protein expression, quantified by PLA, increased 10-fold (P0.05) between the early proliferative and late proliferative/mid-secretory phases of the menstrual cycle in association with the rise in estrogen. In summary, estrogen induced a rapid, divergent, and cell-specific change in expression of angiostimulatory and angioinhibitory growth factors in the endometrium of the nonhuman primate.

The present study determined the placental and whole-body metabolism of androstenedione originating in the maternal and fetal compartments of the pregnant baboon at mid (day 100; n = 4) and late (day 165; n = 3) gestation (term = day 184) in untreated animals and at midgestation in animals (n = 3) treated with pellets (50 mg) of androstenedione inserted at 8-day intervals in the mother between days 70 and 100 of gestation. Baboons were anesthetized with ketamine-halothane-nitrous oxide, blood samples obtained from maternal, uterine, fetal and umbilical vessels during constant infusion of [3H] or [14C]androstenedione via the fetal or maternal circulation, respectively, and radiolabeled precursor/products in plasma purified by HPLC. The metabolic clearance rate (MCR; 1/day/kg body wt) of androstenedione in the mother was similar at mid (81 ± 6) and late (69 ± 12) gestation and was unaltered by treatment with androstenedione (92 ± 17). Fetal MCR of androstenedione was 3-fold greater (P < 0.05) than in the mother and was similar in the three treatment groups. In the maternal compartment, the conversion ratio of androstenedione to estradiol (range 26–37%) exceeded (P < 0.05) that to testosterone (range 15–19%) which exceeded (P < 0.05) that to estrone (range 7–14%), a pattern unaffected by stage of gestation or treatment with androstenedione in vivo. Similar results were observed in the fetal compartment although values for each conversion were always 3–4-fold lower (P < 0.05) than in the maternal compartment. Regardless of stage of gestation or treatment with androstenedione, [14C]estradiol in the uterine vein (95 ± 15 cpm/ml) exceeded (P < 0.05) that in the unbilical vein (3 ± 1) indicative of preferential secretion of estradiol to the maternal compartment. In contrast, the concentration of [14C]estrone in uterine (15 ± 4) and umbilical (18 ± 4) vessels were similar indicating that estrone was secreted equally into the mother and fetus. Similar observations were noted for respective values for [3H]estrogens derived from fetal [3H]androstenedione. Placental extraction of fetal androstenedione (range 86–93%) exceeded (P < 0.05) that for androstenedione originating in the mother (range 44–54%) and neither were affected by stage of gestation or treatment with androstenedione in vivo. Less than 1% of fetal [3H]androstenedione reached the maternal circulation unaltered, presumably due to placental catabolism. Similarly, the concentration of maternally-derived [14C]androstenedione present in fetal plasma (

Although estrogen regulates important aspects of maternal cardiovascular physiology, the role of estrogen on uteroplacental and fetal blood flow is incompletely understood. This study tested the hypothesis that chronically suppressing endogenous estrogen production during the second half of baboon pregnancy alters uterine and fetal blood flow dynamics assessed by ultrasonography. Pregnant baboons were untreated or treated daily with the aromatase inhibitor letrozole or letrozole plus estradiol on days 100– 160 of gestation (term = 184 days). Blood flow dynamics were determined by Doppler ultrasonography on day 60 and longitudinally between days 110 and 160 of gestation. Letrozole decreased maternal serum estradiol and estrone concentrations by 95% ( P < 0.001). Fetal growth biometrical parameters increased ( P < 0.001) between days 110 and 160 of gestation and were similar in untreated and letrozole-treated animals. Uterine, umbilical, and fetal middle cerebral artery pulsatility index and resistance index declined ( P < 0.01) by 30–50% and uterine artery volume flow increased sixfold ( P < 0.001) between days 60 and 160, but values were similar in untreated, letrozole-treated, and letrozole plus estradiol-treated baboons. Thus uterine and fetal artery blood flow indexes, uterine artery volume flow, and fetal growth were maintained at normal levels despite chronic estrogen suppression in the second half of baboon pregnancy. This suggests that elevated levels of endogenous estrogen are not required to maintain low impedance blood flow within the uteroplacental vascular bed during the second half of nonhuman primate pregnancy.

The foundation for development of the male reproduction system occurs in utero, but relatively little is known about the regulation of primate fetal testis maturation. Our laboratories have shown that estrogen regulates key aspects of the physiology of pregnancy and fetal development. Therefore, in the present study, we characterized and quantified germ cells and Sertoli cells in the fetal baboon testis in late normal gestation (i.e., Day 165; term is 184 days) and in baboons administered the aromatase inhibitor letrozole throughout the second half of gestation to assess the impact of endogenous estrogen on fetal testis development. In untreated baboons, the seminiferous cords were comprised of undifferentiated (i.e., type A) spermatogonia classified by their morphology as dark (Ad) or pale (Ap), gonocytes (precursors of type A spermatogonia), unidentified cells (UI), and Sertoli cells. In letrozole-treated baboons, serum estradiol levels were decreased by 95%. The number per milligram of fetal testis (x10(4)) of Ad spermatogonia (0.42 +/- 0.11) was 45% lower (P = 0.03), and that of gonocytes (0.58 +/- 0.06) and UI (0.45 +/- 0.12) was twofold greater (P0.01 and P = 0.06, respectively), than in untreated baboons. Moreover, in the seminiferous cords of estrogen-deprived baboons, the basement membrane appeared fragmented, the germ cells and Sertoli cells appeared disorganized, and vacuoles were present. We conclude that endogenous estrogen promotes fetal testis development and that the changes in the germ cell population in the estrogen-deprived baboon fetus may impair spermatogenesis and fertility in adulthood.

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 mm(2)) were increased (P0.01) between mid (6 +/- 1 ng/ml; 15.8 +/- 2.4) and late (257 +/- 28 ng/ml; 57.3 +/- 5.1) gestation, reduced (P0.01) in late-gestation fetuses in which estradiol was suppressed (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 (P0.01) between mid (48.8 +/- 9.5) and late (17.4 +/- 3.2) gestation, remained elevated (P0.01) 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 showed that estrogen regulates baboon fetal ovarian follicle development and oocyte integrity. Because iron incorporated into cells by the transferrin receptor is essential for cell/nuclear function, we determined whether fetal oocyte expression of transferrin receptor and the nuclear protein Ki67 were developmentally regulated by estrogen and associated with DNA integrity/fragmentation. Transferrin-receptor expression was minimal at midgestation and abundant in late gestation and localized to the cytoplasm and surface of oocytes of primordial follicles. Expression of transferrin receptor, however, was negligible in oocytes in fetuses in which serum estradiol-17beta levels were suppressed (95%) by daily maternal treatment between mid- and late gestation with the aromatase inhibitor letrozole and partially restored by treatment with letrozole and estradiol benzoate. Ki67 was localized to pregranulosa and germ cells at midgestation and throughout the oocyte nucleus in late gestation in estrogen-replete fetuses. In contrast, in estrogen-suppressed fetuses, Ki67 was localized to a limited number of foci around the oocyte nucleus. Apoptosis detected in pregranulosa and germ cells at midgestation was not observed in late gestation in estrogen-replete/-suppressed fetuses. We conclude that estrogen regulates fetal oocyte transferrin-receptor expression and that inhibition of receptor development is associated with alterations in Ki67 expression by the oocyte but not apoptosis. Collectively, these results and our previous studies further define the essential role of estrogen in regulating development of follicles comprised of healthy oocytes by the baboon fetal ovary.