Jeffery S. Babischkin

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.

Identification and Developmental Expression of the Estrogen Receptor α and β in the Baboon Fetal Adrenal Gland1

We have previously shown that estrogen regulates the development and function of the fetal and definitive/transitional zones of the primate fetal adrenal gland. Thus, during baboon pregnancy estrogen acts directly on the fetal zone to suppress ACTH-stimulated dehydroepiandrosterone (DHA) formation, potentially to modulate C19-steroid production and consequently placental estrogen synthesis. It is proposed that this action of estrogen is mediated by the estrogen receptor. Therefore, in the present study a developmental approach was used to determine whether the messenger RNA (mRNA) and protein for the estrogen receptor were expressed in the fetal and definitive/transitional zones of the baboon fetal adrenal gland at mid (day 100) and late (day 170) gestation (term = 184 days). Estrogen receptor α mRNA levels, determined by competitive RT-PCR, were approximately 7-fold greater (P < 0.02) in the fetal adrenal of late (187.8 ± 40.3 attomoles/μg RNA) compared with mid (27.4 ± 5.4 attomoles/μg RNA) gestation. M...

Developmental regulation of placental insulin-like growth factor (IGF)-II and IGF-binding protein-1 and -2 messenger RNA expression during primate pregnancy.

Abstract The present study was conducted to determine the developmental expression of placental insulin-like growth factor (IGF)-II, IGF-binding protein (IGFBP)-1 and -2, and IGF-II receptor mRNA expression during baboon pregnancy and whether estrogen, the levels of which increase with advancing pregnancy, regulates placental trophoblast IGF-II mRNA expression. Levels of the IGF-II 6.1-kilobase (kb) and 4.9-kb mRNA transcripts determined by Northern blot analysis progressively increased three- to fourfold in placental syncytiotrophoblast and whole-villous tissue between early (Day 60), mid (Day 100), and late (Day 170) baboon gestation (term = 184 days). In contrast, syncytiotrophoblast IGFBP-1 and -2 mRNA levels decreased, and IGF-II receptor mRNA expression remained relatively constant, with advancing baboon pregnancy. Placental cytotrophoblast IGF-II mRNA levels determined by competitive reverse transcription-polymerase chain reaction on Day 54 of gestation were increased (P < 0.05) almost twofold at 18 h after acute administration of estradiol to baboons, whereas long-term estrogen treatment had no effect. We propose that these changes in trophoblast IGF expression would provide a mechanism for enhancing net bioavailability and bioreactivity of IGF-II locally to promote the growth and development of the placenta and, consequently, of the fetus during primate pregnancy.

Developmental expression of placental trophoblast P-450 cholesterolside-chain cleavage, adrenodoxin and Δ5-3β-Hydroxysteroid dehydrogenase/isomerase messenger ribonucleic acids during baboon pregnancy

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 (P < 0.01) greater than in early gestation (0.31 +/- 0.08) and approximately twofold greater (P < 0.05) 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.

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.

Prematurely Elevating Estradiol in Early Baboon Pregnancy Suppresses Uterine Artery Remodeling and Expression of Extravillous Placental Vascular Endothelial Growth Factor and α1β1 and α5β1 Integrins

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.

Divergent regulation of angiopoietin-1 and -2, Tie-2, and thrombospondin-1 expression by estrogen in the baboon endometrium.

Estrogen has an important role in the reconstruction of a new vascular network in the endometrium during each menstrual cycle; however, the underlying mechanisms are incompletely understood. Angiopoietin‐1 (Ang‐1) promotes vessel assembly, whereas Ang‐2 and thrombospondin‐1 (TSP‐1) cause vessel breakdown. To determine the potential effect of estrogen on the expression of these angioregulatory factors in the endometrium, Ang‐1, Ang‐2, TSP‐1, and Tie‐2 receptor mRNA levels were assessed by real‐time reverse transcriptase polymerase chain reaction in glandular epithelial and stromal cells isolated from the endometrium of ovariectomized baboons treated acutely with estradiol. Corresponding protein expression was assessed by immunocytochemistry and the proximity ligation assay (PLA) during advancing stages of the baboon menstrual cycle. Serum estradiol levels in ovariectomized baboons were 400 pg/ml within 4–6 hr of estradiol treatment. Ang‐1 mRNA levels in glandular epithelial cells increased threefold (P < 0.01) within 4 hr of estradiol administration. In contrast, TSP‐1 mRNA levels decreased four‐ to fivefold (P < 0.01) 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 (P < 0.05) 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. Mol. Reprod. Dev. 77: 430–438, 2010.

Expression of P-450 aromatase, estrogen receptor α and β, and α-inhibin in the fetal baboon testis after estrogen suppression during the second half of gestation

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.

Assessment of Protein Expression by Proximity Ligation Assay in the Nonhuman Primate Endometrium, Placenta, and Fetal Adrenal in Response to Estrogen

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.