Eugene D. Albrecht

Expression of estrogen receptors α and β in the baboon fetal ovary

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

Effect of estrogen on dehydroepiandrosterone formation by baboon fetal adrenal cells in vitro

The present study determined if estrogen modulates the responsivity of the adrenal gland of the baboon fetus to tropic hormones such as adrenocorticotropic hormone and prolactin. Adrenal glands were obtained from seven baboon fetuses at midgestation (days 100 to 105). Adrenal cells were dispersed in medium 199 with 0.2% collagenase for 10 minutes at 37 degrees C. Approximately 10(5) cells/4.0 ml of medium 199 were incubated for 24 hours at 37 degrees C with 10 nmol of adrenocorticotropic hormone or 10 nmol of ovine prolactin in the presence or absence of 10(-5) or 10(-6) mol/L of estradiol. The major steroid formed and secreted into the medium was dehydroepiandrosterone. Mean +/- standard error basal formation of dehydroepiandrosterone was 176 +/- 64 ng/10(5) cells/24 hours. Dehydroepiandrosterone formation was increased (p less than 0.05) 3.5-fold and five-fold by adrenocorticotropic hormone and prolactin, respectively. Estradiol at 10(-5) mol/L prevented the response in dehydroepiandrosterone obtained with adrenocorticotropic hormone alone. Estradiol alone had no effect on dehydroepiandrosterone. The results suggest that estrogen modulates the regulatory effects of adrenocorticotropic hormone on dehydroepiandrosterone formation by the adrenal gland of the baboon fetus.

Comparison of cortisol-cortisone interconversion in vitro by the human and baboon placenta

The kinetics of 11 beta-hydroxysteroid dehydrogenase (11HSD) catalyzing the interconversion of cortisol (F) and cortisone (E) were compared in vitro following incubation of homogenates of human (N = 7) and baboon (N = 2) placenta. In both species, enzyme activity catalyzing the conversion of F to E was associated with the membrane fraction of the cell, was greater in the presence of NAD+ than NADP+, was of similar concentration within the placenta, and exhibited a similar Km for F. Moreover, there was no conversion of E to F in either the baboon or human placenta indicating that in both species, term placenta lacks the 11HSD enzyme catalyzing the reduction of the 11-oxo group of corticosteroids. Significantly, the conversion of F to E by both the baboon and human placenta was inhibited when progesterone was added to the reaction mixture at concentrations equimolar to the substrate. We conclude that 11HSD enzyme kinetics in term baboon placental homogenates are similar to those measured in human term placenta. Moreover, progesterone may be a physiologic regulator of 11HSD in both the human and baboon placenta. Collectively, our findings support the use of the baboon as a model for studies of the regulation of placental corticosteroid metabolism during human pregnancy.

The utilization of placental substrates for cortisol synthesis by the baboon fetus near term

Abstract We have determined the proportion of the in vivo cortisol (F) secretion rate (SRF) derived from circulating pregnenolone (P5) and progesterone (P4) in 9 baboon ( Papio papio ) neonates, 5 (4 ♂, 1 ♀) delivered prior to the onset of labor by cesarean section (CS, 164–179 days) and 4 (2 ♂ , 2 ♀) delivered spontaneously at term (SD, 164–179 days; term = 184 days). The metabolic clearance rate (MCR; 1/d/kg) and production rates (PR; mg/d/kg) of P4 and P5 and transfer constants (p) for the reactions P5 → P4 14 P5 → F, and P4 → F, were determined by continuous infusion of [1,2- 3 H] P5 and [4- 14 C] P4. In CS animals the MCR-P5 (41.4 ± 5.6; X ± SE) was lower than the MCR-P4 (74.0 ± 8.5; P We conclude that endogenous adrenal substrates and not placental substrates are the primary precursors utilized for fetal adrenal F production in late baboon gestation.

The effects of estrogen on cortisol metabolism in female baboons

We examined cortisol (F) dynamics in female baboons (Papio anubis) treated with diethylstilbestrol (DES) or estradiol (E2) and compared values with those previously measured in nonpregnant and pregnant animals. Five regularly menstruating baboons (12-18 kg, BW) were administered 5 mg DES daily via fruit or 0.5 mg E2/0.1ml oil sc for 30 days. Blood samples, obtained before and after treatment, were assayed for serum F concentrations and serum cortisol binding capacity (CBC). The metabolic clearance (MCR) and production rate (PR) of F and the catabolism of i.v. administered [3H] F were examined 25 and 30 days after initiation of estrogen treatment. Compared with values in nonpregnant baboons, F metabolism in estrogen treated animals is significantly altered and is characterized by increased formation of unconjugated metabolites, decreased glucuronylation, increased excretion of unconjugated F, cortisone, and highly polar metabolites, and increased CBC. These changes induced by estrogen are similar to those observed in intact pregnant baboons and permit the suggestion that the pattern of F metabolism and the level of CBC in baboon pregnancy are the result of elevated estrogen production. However, estrogen also caused a significant decrease in the MCR and PR of F, parameters which, by contrast, are similar in intact pregnant and nonpregnant baboons. These findings indicate that while estrogen also influences the rate of F clearance and F production, these effects of estrogen are not apparent during pregnancy. Collectively, these findings allow the suggestion that estrogen is a major factor which alters F metabolism and increases serum CBC in baboon gestation. However, additional factors are operative in primate pregnancy which maintain PR and MCR of F at levels similar to those of nonpregnant baboons.

Secretion and Metabolism of Steroids in Primate Mammals During Pregnancy

The principal steroid hormones produced by placenta and fetal adrenal gland during primate pregnancy are estrogen, progesterone, and the glucocorticosteroids. These hormones are critical to preparation of the uterus for implantation early in gestation, the maintenance of pregnancy thereafter, and the development of a healthy newborn at term (reviewed in 1,2). For example, estrogen has a central integrative role in regulating key steps in the biosynthetic pathway for the formation of progesterone, a hormone essential to quiescence of the uterus and the maintenance of mammalian pregnancy. Progesterone, in turn, may have a role in immunosuppression to protect the developing conceptus from rejection by the maternal immune system. Estrogen and progesterone act in conjunction with prolactin, placental lactogen, and other hormones in development and preparation of the mammary gland for lactation. Estrogen also may stimulate utero-placental blood flow to provide nutrients and oxygen to the placenta and fetus for growth and development. Estrogen has been implicated in the cascade of events that initiate the process of labor. Finally, cortisol produced by the fetal adrenal gland has a critical role in the maturation of the lung and other organ systems in the developing fetus.

The effects of cortisone on the interconversion of cortisol and cortisone in the baboon

In the baboon fetus, the conversion of cortisol (F) to cortisone (E) [80%] exceeds the reverse reaction [15%]. Since the fetus is exposed to high quantities of E throughout most of pregnancy, we determined whether F to E interconversion is altered following acute changes in serum E. Adult female baboons (N = 3) were sedated with ketamine, constantly infused for 180 min via an antecubital vein with 15 microCi[14C]E and 15 microCi[3H]F, and saphenous vein blood samples obtained at 70, 80 and 90 min. At 90 min, an infusion of E (166 micrograms/min) was initiated and blood samples obtained at 160-180 min. This protocol was repeated in the same animals treated 24 and 3 h prior to infusion with 3 mg betamethasone. Metabolic clearance (MCR), transfer constants (%) and serum levels (microgram/dl) of F and E were determined. E increased (P less than 0.05; paired t) MCR-E, and serum F and E levels in control and betamethasone-treated baboons. E also decreased %F to E in betamethasone-treated but not control animals. These findings suggest that acute changes in serum E alter MCR-E, do not influence the conversion of E to F and may decrease the conversion of F to E. Therefore, we suggest that the high conversion of F to E in the baboon fetus is probably not the result of elevated concentrations of E.