Graham W. Aberdeen

The role of estrogen in the maintenance of primate pregnancy

OBJECTIVEnThe aim of this study was to determine the role of estrogen in pregnancy maintenance in baboons by suppressing estrogen synthesis through administration of a highly specific nonsteroidal aromatase inhibitor, CGS 20267.nnnSTUDY DESIGNnCGS 20267 was administered subcutaneously at maximal dosages of 2.0 mg/d to pregnant baboons (n = 24) daily beginning on either day 30 (n = 8), day 60 (n = 8), or day 100 (n = 8) of gestation (normal length of gestation is 184 days) until animals miscarried or were delivered abdominally on days 160 through 168 of gestation. CGS 20267 and estradiol (n = 9), each at maximal dosages of 2 mg/d, were administered at the same intervals of gestation. Twenty baboons served as untreated control animals. Serum estradiol and progesterone levels were determined by radioimmunoassay from serum samples obtained at 1- to 3-day intervals from a maternal peripheral vein.nnnRESULTSnWithin 1 to 3 days of the initiation of CGS 20267 administration, maternal serum estradiol concentration decreased to and remained at a level that was substantially lower (mean +/- SE, 0. 096 +/- 0.003 ng/mL) than in the untreated control animals throughout gestation (0.35-4.0 ng/mL; P <.001). Although pregnancy was maintained in 19 of the 20 untreated control baboons (95%), only 12 of the 24 animals that received CGS 20267 (50%) maintained pregnancy. In contrast, all the baboons treated concomitantly with estradiol and CGS 20267 (9/9) maintained pregnancy. Thus estradiol alone prevented the high rate of miscarriage induced by the antiestrogenic agent CGS 20267. Serum progesterone concentrations were not significantly different throughout the experimental period between the CGS 20267-treated baboons that maintained pregnancy (12. 9 +/- 1.4 ng/mL) and those that miscarried (13.6 +/- 1.6 ng/mL) and were not lower in antiestrogen-treated baboons than in untreated control baboons (10.6 +/- 0.8 ng/mL).nnnCONCLUSIONnEstrogen, acting directly, indirectly, or both through a factor or factors other than the level of progesterone, plays a critically important physiologic role in the maintenance of primate pregnancy.

Analysis of apoptosis and expression of bcl-2 gene family members in the human and baboon ovary

Recent data support a role for apoptosis, under tight regulatory control by bcl-2, oxidative stress response, tumor suppressor, and CASP gene family members, in mediating granulosa cell demise during follicular atresia in the rodent and avian ovary. Herein we evaluated the occurrence of apoptosis in the human and baboon ovary relative to follicular health status, and analyzed expression of several cell death genes in these tissues. In situlocalization of DNA strand breaks in fixed human and baboon ovarian tissue sections indicated that apoptosis was essentially restricted to granulosa cells of atretic antral follicles. Biochemical analysis of DNA oligonucleosomes in individual follicles isolated from baboon ovaries during the ovulatory phase revealed the presence of apoptotic DNA fragments in subordinate but not dominant follicles, thus substantiating the in situ labeling studies. Messenger RNA transcripts encoded by the bax death susceptibility gene, the bcl-xlong survival gene, the bcl-xshort pro-apoptosis gene, the p53 tumor suppressor gene, and two members of the CASP gene family (CASP-2/Ich-1, CASP-3/CPP32), were detected by Northern blot analysis of total RNA prepared either from human ovaries or from Percoll-purified granulosa-lutein cells obtained from patients undergoing assisted reproductive technologies. Lastly, immunohistochemical localization of the BAX death-susceptibility protein in the human ovary revealed abundant expression in granulosa cells of early atretic follicles, whereas BAX protein was extremely low or non-detectable in healthy or grossly-atretic follicles. We conclude that apoptosis occurs during, and is probably responsible for, folicular atresia in the human and baboon ovary. Moreover, apoptosis in the human ovary is likely controlled by altered expression of the same cohort of cell death regulatory factors recently implicated as primary determinants of apoptosis induction or suppression in the rodent ovary.

Effect of Estrogen on Vascular Endothelial Growth/Permeability Factor Expression by Glandular Epithelial and Stromal Cells in the Baboon Endometrium

Abstract 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 designed to replicate the hormonal profiles that are characteristic of the proliferative and secretory phases of the menstrual cycle. Administration of estradiol to ovariectomized baboons in levels that replicated the late-proliferative phase of the menstrual cycle (209 ± 40 pg/ml serum) increased/restored VEG/PF mRNA to levels in the glands (5.57 ± 1.53 amol/fmol 18S rRNA, P < 0.01) and stroma (2.61 ± 1.57 amol/fmol 18S rRNA, P < 0.02) that were approximately 10-fold greater than those observed after ovariectomy alone (0.52 ± 0.21 and 0.22 ± 0.11 amol/fmol 18S rRNA, respectively) and were similar to those previously shown in intact baboons. Concomitant administration of estradiol and progesterone to ovariectomized baboons in levels that replicated the midsecretory phase of the menstrual cycle (44 ± 15 pg/ml serum and 9.8 ± 2.2 ng/ml serum, respectively) resulted in glandular epithelial (3.65 ± 1.42 amol/fmol 18S rRNA) and stromal (1.25 ± 0.77 amol/fmol 18S rRNA) VEG/PF mRNA levels that were not significantly different from those exhibited after ovariectomy or ovariectomy and estradiol treatment. Comparable results were obtained for VEG/PF mRNA expression in whole-endometrial tissue, although the relative 2-fold increase (P < 0.03) in VEG/PF mRNA levels induced by estrogen in mixed endometrial cells of ovariectomized baboons appeared to be less marked than that in isolated glandular epithelial and stromal cells. After ovariectomy, endometrial width (0.98 ± 0.09 mm) was approximately one-third of that in intact baboons (3.58 ± 0.32 mm), and endometrial VEG/PF protein expression was low. Estradiol restored endometrial width (3.00 ± 0.12 mm, P < 0.01) and VEG/PF protein expression to normal. In summary, estrogen has a significant role in regulating and maintaining VEG/PF expression by glandular epithelial and stromal cells of the endometrium during the menstrual cycle.

Ask1 gene deletion blocks maternal diabetes-induced endoplasmic reticulum stress in the developing embryo by disrupting the unfolded protein response signalosome

Apoptosis signal–regulating kinase 1 (ASK1) is activated by various stresses. The link between ASK1 activation and endoplasmic reticulum (ER) stress, two causal events in diabetic embryopathy, has not been determined. We sought to investigate whether ASK1 is involved in the unfolded protein response (UPR) that leads to ER stress. Deleting Ask1 abrogated diabetes-induced UPR by suppressing phosphorylation of inositol-requiring enzyme 1α (IRE1α), and double-stranded RNA-activated protein kinase (PKR)-like ER kinase (PERK) blocked the mitochondrial translocation of proapoptotic Bcl-2 members and ER stress. ASK1 participated in the IRE1α signalosome, and removing ASK1 abrogated the proapoptotic kinase activity of IRE1α. Ask1 deletion suppressed diabetes-induced IRE1α endoriboneclease activities, which led to X-box binding protein 1 mRNA cleavage, an ER stress marker, decreased expression of microRNAs, and increased expression of a miR-17 target, thioredoxin-interacting protein (Txnip), a thioredoxin binding protein, which enhanced ASK1 activation by disrupting the thioredoxin-ASK1 complexes. ASK1 is essential for the assembly and function of the IRE1α signalosome, which forms a positive feedback loop with ASK1 through Txnip. ASK1 knockdown in C17.2 neural stem cells diminished high glucose– or tunicamycin-induced IRE1α activation, which further supports our hypothesis that ASK1 plays a causal role in diabetes-induced ER stress and apoptosis.

Vascular Endothelial Growth Factor Mediates the Estrogen-Induced Breakdown of Tight Junctions between and Increase in Proliferation of Microvessel Endothelial Cells in the Baboon Endometrium

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.

Placental growth factor in the first trimester: relationship with maternal factors and placental Doppler studies

Placental growth factor (PlGF) is a potent angiogenic factor that impacts on early placental vascular development. It was our aim to clarify relationships between PlGF and first‐trimester maternal/placental factors that are related to placental development.

Expression of Estrogen Receptors α and β in the Fetal Baboon Testisand Epididymis

Abstract 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) α and β expression in the fetal baboon testis. Second, we established methods to quantify ERβ mRNA levels by competitive reverse transcription-polymerase chain reaction in Sertoli cells isolated by laser capture microdissection (LCM) from the fetal baboon testis. ERβ 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). ERβ mRNA level was 0.03 attomole/femtomole 18S rRNA in Sertoli cell nuclei and associated cytoplasm isolated by LCM. ERα was expressed in low level in seminiferous tubules and in moderate level in peritubular cells on Day 165. Germ cells expressed very little ERα or ERβ protein, whereas the baboon fetal epididymis exhibited extensive ERα and ERβ immunostaining at mid- and late gestation. In contrast to the robust expression of ERβ, 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 ERα and ERβ, 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.

Suppression of trophoblast uterine spiral artery remodeling by estrogen during baboon pregnancy: impact on uterine and fetal blood flow dynamics.

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.

Uterine and fetal blood flow indexes and fetal growth assessment after chronic estrogen suppression in the second half of baboon pregnancy

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.

Methoxychlor and Its Metabolites Inhibit Growth and Induce Atresia of Baboon Antral Follicles

Methoxychlor (MXC), an organochlorine pesticide, inhibits growth and induces atresia of antral follicles in rodents. MXC metabolites, mono-OH MXC (mono-OH) and bis-OH MXC (HPTE), are thought to be more toxic than the parent compound. Although studies have examined the effects of MXC in rodents, few studies have evaluated the effects of MXC in primates. Therefore, the present study tested the hypothesis that MXC, mono-OH, and HPTE inhibit growth and induce atresia of baboon antral follicles. To test this hypothesis, antral follicles were isolated from adult baboon ovaries and cultured with vehicle (dimethylsulfoxide; DMSO), MXC (1–100 μg/ml), mono-OH (0.1–10 μg/ml), or HPTE (0.1–10 μg/ml) for 96 hr. Growth was monitored at 24 hr intervals. After culture, follicles were processed for histological evaluation of atresia. MXC, mono-OH, and HPTE significantly inhibited follicular growth and increased atresia compared to DMSO. Moreover, the adverse effects of MXC and its metabolites on growth and atresia in baboon antral follicles were observed at lower (100-fold) doses than those causing similar effects in rodents. These data suggest that MXC and its metabolites inhibit growth and induce atresia of baboon antral follicles, and that primate follicles are more sensitive to MXC than rodent follicles.