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The metabolism of testosterone (T) was examined during the second half of pregnancy in the rat to deter- mine whether utilization of Tfor estradiol (E2) synthesis occurs via conversion of T to androstenedione (A). On Days 11, 16. and 21 of gestation (term = Day 23), rats (n = 7-9/group) were anesthetized and a constant infusion ofHJT was initiated. At 60 mm, blood was obtained from a jugular vein and the ovaries (Days 11, 16, and 21), and piacentae and uterine tissue (Day 16 only) were removed. In a second study performed in rats on Day 16 of gestation (n = 8-JO/group), the ovaries and/or gravid uterus were removed 15 mm after initiation of ? HIT infusion, and blood was taken from a jugular vein 60 mm later. Radiolabeled T and A were purified from serum and tissues by paper chromatography. In a third group of rats (n = 6), jugular vein samples were obtained sequentially on Days 11, 16 and 21 of gestation and serum concentrations of T were measured by radioimmu- n oassay. The metabolic clearance rate of T was Constant during the study period (overall mean = 31 1/day). In con- trast, the serum concentration of T (pg/mi) on Day 16 of gestation (863 ± 108) exceeded (p

The present study was designed to characterize the dynamics of progestin metabolism peripherally and across the uterus during normal baboon pregnancy and to determine whether the decline in placental progesterone (P4) production which results from administration of the antiestrogen ethamoxytriphetol (MER-25) to baboons reflects a decrease in conversion of pregnenolone (P5) to P4 and thus delta 5-3 beta-hydroxysteroid dehydrogenase activity. To examine this possibility, the conversion of [3H]P5 to [3H]P4 was determined by the constant infusion method on day 100 (midgestation) and day 175 (near term) of gestation in baboons that received MER-25 (25 mg/day X kg BW, po) on days 95-100 or 140-175 (term = 184 days). Baboons were sedated with ketamine HCl, then received a constant iv infusion of [3H]P5 (1.0 mu Ci/0.388 ml X min) and [14C]P4 (0.2 mu Ci/0.388 ml X min for 110 min. Radiolabeled progestins were purified from blood samples withdrawn from saphenous, uterine, and umbilical vessels, and the MCR of P4 and P5, uterine extraction of P5, and transfer constants (rho) for the peripheral, transuterofetoplacental, and transuteroplacental conversion of P5 to P4 were determined. The formation of P4 from P5 by incubates of placental cells obtained on day 175 from untreated and MER-25-treated baboons was also assessed. During normal baboon pregnancy the mean (+/- SE) % P5 extracted (i.e. metabolized) by the uterus was 31.0 +/- 3.3 at midgestation and 45.7 +/- 5.6 late in gestation. Peripheral and transuterofetoplacental rho values of P5 to P4 in untreated baboons were 6.9 +/- 1.8% and 37.3 +/- 7.9%, respectively, at midgestation and 6.1 +/- 0.6% and 46.8 +/- 10.1%, respectively, near term. The transuteroplacental rho of P5 to P4 was only slightly lower than the transuterofetoplacental values, indicating minimal conversion of P5 to P4 by the fetus. The peripheral contribution of P5 production to the total production rate of P4 at term in baboons was 1%. The contribution of uteroplacental conversion of P5 to P4 to the total conversion of P5 to P4 at midgestation was estimated to be 22%. MER-25 caused a 53% decline (P less than 0.01) in serum P4 concentrations from a mean (+/- SE) of 12.5 +/- 2.4 ng/ml during the pretreatment period to 5.4 +/- 0.3 ng/ml between days 140 and 175 of gestation.(ABSTRACT TRUNCATED AT 400 WORDS)

To determine the maturational changes leading to adrenocortical self-sufficiency in baboon neonates, fetal adrenal growth and cortisol (F) production, in vitro, from pregnenolone (P5), progesterone (P4) and 11-deoxycortisol (S) were examined at various times during gestation. Adrenal weight of animals delivered prematurely (100-179 days gestation, term = 184) by cesarean section (N = 17) or spontaneously (N = 13) at term (181 ± 4 days, X ± SE), tripled during the second half of gestation (r = 0.82; P

The present study employed the dehydroepiandrosterone (DHA) loading test to determine if the placenta in baboons remains functional following fetectomy. A bolus iv injection of 100 mg DHA was given to 4 baboons prior to and at various intervals following fetectomy at midgestation. Blood samples were withdrawn over a 4-h period following DHA and assayed for estradiol (E2). Placentas were maintained in situ following removal of the fetus. Serum E2 reached peak concentrations 30-60 min after injection of DHA prior to and after fetectomy. Although initial serum E2 concentrations were very low after fetectomy, the mean +/- SE net increase (delta) in peak serum E2 concentrations following the DHA bolus were similar before (3.92 +/- 0.71 ng/ml) and after (3.72 +/- 0.64 ng/ml) fetectomy. Based on the capacity for aromatization, therefore, the placenta remains equally competent and functional in baboons despite chronic absence of the fetus.

A short term incubation of baboon fetal adrenal cells obtained at midgestation and near term was used to determine whether a change in the regulation of androgen formation occurs with advancing gestation. Adrenal glands were removed from baboon (Papio anubis) fetuses on day 100 (mid; n = 7) or day 170 (late; n = 5) of gestation, and cells were dispersed with 0.2% collagenase. Cells (10(5] were incubated at 37 C for 3 h in medium 199 in the presence or absence of 10 nM ACTH, 10 nM ovine PRL, 10 nM ovine GH, 50 nM hCG, or 50 nM human chorionic somatomammotropin. Dehydroepiandrosterone (DHA), DHA sulfate (DHAS), cortisol (F), and androstenedione concentrations were determined in the medium by RIA. At midgestation, ACTH, PRL, and GH elevated (P less than 0.05) DHA (168%, 169%, and 178%, respectively) and DHAS (142%, 210%, and 197%, respectively) formation. Near term, ACTH and PRL retained their ability to stimulate (P less than 0.05) DHA (307% and 220%, respectively), but not DHAS, synthesis. The fetal adrenal at late gestation, however, lost its ability to respond to treatment with GH. hCG and human chorionic somatomammotropin did not stimulate steroidogenesis at either time of gestation. F formation at midgestation was less (P less than 0.05) than that at term and not responsive to ACTH. ACTH stimulated (P less than 0.05) F secretion by 68% in fetal adrenal cells obtained near term. The secretion of androstenedione was not affected by any peptide treatment at either stage of gestation. These data indicate that the responsivity of the baboon fetal adrenal to various pituitary peptides is different at two developmentally distinct stages of gestation. We conclude, therefore, that the regulation of fetal adrenal steroidogenesis changes with advancing gestation.

The metabolism of iv administered 14C-cortisol (F) by mature female baboons was compared with that reported for humans. Approximately 60% of 14C was recovered in urine within 24 hr and 80% within 120 hr after injection. The distribution of urinary 14C was 12.8% unconjugated, 50.3% glucuronoside, and 7.5% sulfate (H2SO4-e,thyl acetate) with 21% unextractable with ethyl acetate. Metabolites were isolated by paper chromatography and purified by crystallization and derivative formation. More than 50% of unconjugated and sulfate fractions were more polar than the cortols with a significant portion of the former even more polar than 6β-hydroxy-F. Unconjugated F plus cortisone [ratio < 1] represented < 2% urinary 14C. Tetrahydro-F (THF) and tetrahydrocortisone (THE) [ratio < 1], constituting 20% of the glucuronoside fraction, were the principal C21 metabolites isolated. Thirty-five percent of the glucuronoside fraction was less polar than cortisone indicating extensive side-chain cleavage. The daily production r...

In the present study, a culture system of human placental cells was established to examine the role of estrogen and androgen in progesterone (P4) formation. Normal human placentae were obtained at term, and cells were dispersed in Hank's Balanced Salt Solution (5 ml/g tissue) containing 0.1% collagenase, 0.1% hyaluronidase, 0.01% deoxyribonuclease, and 1% fetal bovine serum for 2 h at 37 C. Dispersed placental cells (10(6) cells/ml) were placed in medium 199 with modified Earle's salts (pH 7.4) containing 10% fetal bovine serum, 12.5 mM HEPES buffer, 26 mM NaHCO3, and 40 micrograms/ml Gentamycin-SO4 and incubated for 72 h at 37 C and 5% CO2 in air to allow cell attachment. Medium was then changed (time zero), and P4 formation was studied thereafter. Culture of placental cells for 96 h resulted in linear increases in P4 and estradiol (E2) formation, indicating the maintenance of cell viability and steroidogenic function. Mean +/- SE P4 formation at 48 h was 246 +/- 16 pg/micrograms DNA. To assess the role of estrogen on P4 formation, placental cells were incubated for a period of 48 h with various amounts (10(-7)-10(-4)M) of the antiestrogen ethamoxytriphetol (MER-25), the aromatase inhibitor 4-hydroxyandrostenedione (4-OHA), and/or E2. Both MER-25 and 4-OHA resulted in a dose-dependent decline (P less than 0.01) in P4 formation (greater than 80% decline at 10(-4)M MER-25 or 4-OHA). The marked reduction in P4 formation caused by 4-OHA alone was reversed by concomitant addition of E2; however, E2 alone had no effect. To assess the role of androgens on P4 formation, cells were incubated for 48 h with increasing amounts (10(-7)-10(-4)M) of androstenedione, dehydroepiandrosterone (DHA), or dihydrotestosterone. Although the formation of E2 was enhanced by DHA, formation of P4 was not affected by the aromatizable androgens DHA or androstenedione or the nonaromatizable dihydrotestosterone. The decline in P4 formation by human placental cells in culture elicited by MER-25 or 4-OHA supports the hypothesis of a regulatory role for estrogen in placental P4 formation during human pregnancy. The lack of effect of exogenous estrogen suggests that the action of estrogen on P4 formation may be permissive.

The present study determined the source and regulation of 17 alpha-hydroxyprogesterone (17-OHP4) during mid-late baboon pregnancy. Serum 17-OHP4 (ng/ml) in 5 untreated baboons increased from low values at mid-late gestation to a mean (+/- SEM) of 0.49 +/- 0.02 during the final 20 days of gestation. Fetectomy of 5 baboons resulted in serum 17-OHP4 concentrations which declined to and remained at baseline. Serum 17-OHP4 concentrations were 5- to 10-fold greater (P less than 0.001) in the uterine, utero-ovarian, and umbilical veins than peripherally. Apparently the fetal adrenal provides precursors for placental 17-OHP4 formation because the fetal adrenal gland develops delta 5-3 beta-hydroxysteroid dehydrogenase only late in gestation, and because the fetal adrenal and not the placenta has the capacity for 17-hydroxylation. Thus, at mid-late gestation the placenta appears to supply a major, and at term the corpus luteum a minor portion of the total 17-OHP4. Administration of the estrogen antagonist ethamoxytriphetol (MER-25, 15 mg/kg BW) to 4 baboons did not affect 17-OHP4 during mid-late gestation, when the placenta was the only source of 17-OHP4. However, MER-25 resulted in serum 17-OHP4 concentrations (ng/ml) at term which were greater (1.08 +/- 0.10, P less than 0.001) than in untreated baboons (0.49 +/- 0.02). Prior removal of the corpus luteum of pregnancy in 4 animals subsequently given MER-25 prevented this rise in 17-OHP4. This suggests that the marked elevation in 17-OHP4 observed near term after MER-25 administration was of luteal origin and that antiestrogen enhanced 17-OHP4 secretion by the corpus luteum.

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.

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.

The present study tests the hypothesis that estrogen regulates the alteration in baboon placental cortisol (F)/cortisone (E) interconversion from preferential reduction (E----F) at midgestation to oxidation (F----E) near term. Five pregnant baboons (Papio anubis) received increasing numbers of 50-mg implants of androstenedione inserted sc at 8-day intervals between days 70 and 100 of gestation (term = day 184) to elevate the production of estrogen. Five animals served as controls at midgestation and received implants containing no steroid, while four baboons were studied near term between days 164-170 of gestation. All animals were bled from a maternal saphenous vein at 2-day intervals, and the serum was assayed for estradiol. On days 100 or 170 of gestation, transuterofetoplacental corticosteroid dynamics were determined by the constant infusion method. Baboons were anesthetized with ketamine and halothane-nitrous oxide and a constant infusion of [3H]F/[14C]E initiated via a maternal saphenous vein. At 60 min, animals were laparotomized and at 70, 80, and 90 min, blood samples were obtained from right and left uterine veins and from a maternal saphenous vein. At 95 min, an incision was made in the uterus, and blood samples were obtained from the umbilical vein and artery. Radiolabeled F and E were extracted from serum and purified by paper chromatography. Maternal serum E2 concentrations (nanograms per ml; mean +/- SE) were greater (P less than 0.01) between days 94 and 100 of gestation in androstenedione-treated baboons (2.4 +/- 0.3) than in untreated animals at midgestation (0.7 +/- 0.2), but lower than those near term (4.5 +/- 1.0). On day 100 of gestation, conversion of E to F across the uterus in control animals (30%) was similar to that of the reverse reaction (23%). In androstenedione-treated baboons at midgestation the conversion of E----F (8%) was lower (P less than 0.05) than the oxidation of F----E (27%) and not different from that in untreated baboons at term (E----F = 13%; F----E = 28%). The dominance of transuterofetoplacental conversion of F----E over the conversion of E----F in term and in androstenedione-treated animals at midgestation was maintained when transfer constants were corrected for fetal metabolic contributions. We conclude that the increase in placental estrogen production induced by androstenedione administration at midgestation alters the pattern of transuterofetoplacental F-E metabolism, supporting the hypothesis that estrogen regulates placental corticosteroid metabolism.

Pregnancy alters the pattern of maternal cortisol (F) metabolism and increases the maternal serum cortisol-binding capacity (CBC) of baboons. To determine whether these changes are associated with alterations in F clearance,the metabolic clearance rate (MCR) and interconversion (p) of F and cortisone (E) were measured by continuous infusion of (3H)F and (14C)E in 9 regularly menstruating and 7 pregnant baboons (Papio papio). In nonpregnant animals, the values (X +/- SE) for MCR-E (488 +/- 48 1/day) were greater (P less than 0.001) than those for MCR-F (214 +/- 22 1/day). The p value for the conversion of E leads to F (62.8 +/- 4.7%) was greater (P less than 0.001) than that for the reaction F leads to E (41.6 +/- 3.7%), indicating that F formation is favored. Consistent with MCR-E greater than MCR-F, the per cent of F bound to proteins other than albumin (75 +/- 2) was greater (P less than 0.001) than the per cent of E bound (52 +/-3). The production rate (MCR x peripheral concentration; mug/min) of F (55.1 +/- 7.9) was greater (P less than 0.001) than that of E (28.5 +/-3.9) with essentially all of the F being secreted directly (secretion rate 51.2 +/- 7.9 mug/min). Essentially all of the E produced was derived from circulating F, vitually none being secreted directly (secretion rate 4.6 +/- 3.9 mug/min). Pregnancy did not alter the MCR-F (190 +/- 23 1/day), MCR-E 525+/- 51 1/day), per cent of F (79 +/- 3), or per cent of E (49 +/-3) bound,or F (57.2 +/- 9.2 mug/min) or E (35.5 +/- 4.9 mug/min) production rates. CBC (mug F/100 ml) was significantly (P less than 0.01) elevated (25.3 +/- 2.3, nonpregnant vs 35.1 +/- u.6, pregnant). In addition, p E leads to F was increased (75.5 +/- 1.8%) as was p F leads to E (54.3 +/- 3.7%; P less than 0.01). We have concluded that the MCR-F during pregnancy is more dependent on alterations in maternal metabolism than on the increased serum CBC characteristic of gestation. We suggest that the latter factor may be important in regulating the physiologic levels of the other steroids which bind to it.

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.

We measured the concentration of [3H]estradiol (E2) and its conversion to [3H]estrone (E1) in endometrium and myometrium at diestrus, estrus, and Days 2-6 of pseudopregnancy (PSP) in the rat. Animals (N = 5-10/group) were anesthetized with pentobarbital and a constant infusion of [3H]E2 initiated via a jugular vein. Following attainment of isotopic equilibrium, a blood sample was obtained from the inferior vena cava, the endometrium and myometrium isolated, and [3H]E2 and [3H]E1 content determined after purification by paper chromatography. The metabolic clearance rate of E2 and the peripheral conversion of E2 to E1 (% Cr E2----E1), remained relatively constant throughout the study period. Regardless of the reproductive status of the rat, endometrial and myometrial [3H]E2 and [3H]E1 concentrations (cpm/g) were greater (P less than 0.001) than respective values (cpm/ml) in serum. At estrus, the concentration of [3H]E2 in the endometrium was lower (P less than 0.05) than that at diestrus. In contrast, endometrial % Cr E2----E1 and weight of the endometrium were both greater (P less than 0.05) at estrus than at diestrus. Similar patterns were measured in the myometrium. During PSP, the concentration of [3H]E2 in the endometrium increased (P less than 0.05) from Day 2 to Day 6 whereas concentrations in the myometrium remained relatively constant. Endometrial weight increased (P less than 0.05) between Days 2-4 of PSP and was associated with a concomitant decrease (P less than 0.05) in the % Cr E2----E1 from 59 to 22%. The % Cr E2----E1 in the myometrium (2-4%) was constant between Days 2-4 of PSP but increased to 10% by Day 6 in association with a decrease (P less than 0.05) in weight of the myometrium. We have demonstrated in vivo that growth and differentiation of the uterine endometrium during early pseudopregnancy in preparation for implantation/deciduogenesis is associated with decreased endometrial % Cr E2----E1. Because ovarian E2 production is markedly decreased during early PSP, we suggest that a decrease in uterine metabolism of E2 may act, in part, to maintain endometrial E2 at concentrations sufficient to permit receptor interaction.

The metabolic clearance rate (MCR), transfer constants (p), production (PR) and secretion (SR) rates of cortisol (F) andrortisone (E) were determined by the continuous infusion of {1,23H}F and {4-14C}E into 5 neonates delivered prior to the parturition by cesarean section (164–179 days; term = 184 days) and into 5 newborns delivered spontaneously per vagina at term (166 – 187 days). In spontaneously delivered animals, MCR-E (X ± SE, 34.3 ± 7.0 1/day/kg was greater (P < 0.001) than MCR-F (14.9 ± 1.5 1/day/kg), pF to E (59.7 ± 8.9%) exceeded (P < 0.001) pE to F (17.8 ± 3.0%) and the percentage of F bound to serum proteins other than albumin (57.5 ± 6.2) was greater (P < 0.001) than that of E (27.0 ± 10.3) Although the serum E level (25.6 ± 3.6 μg/100 ml) was similar to that of F (33.5 ± 8.0 μg/100 ml), the PR-E (6.4 ± 1.3 μ/min/kg) was greater (P < 0.001) than PR-F (3.3 ± 0.5 μ/min/kg). Approximately eighty-five percent of the E and 65% of the F produced orginated by secretion. In animals delivered by cesarean section, the serum F concentration (32.4 ± 6.7 μ/100ml), pE to F (13.4 ± 2.8%) pF to E (80.0 ± 12.2%) PR-E (4.5 ± 0.2 μ/min/kg) and SR-E (3.9 ± 0.3 μ/min/kg) were not different from values for spontaneously delivered animals. Serum E levels (35.9 ± 1.6 μ/100 ml) were higher but MCR-F (6.7 ± 0.6 1/day/kg) and MCR-E (18.2 ± 0.41/day/kg) lower in neonates delivered by cesarean section. Serum Cortisol binding capacity (μg F bound/100 ml) was greater (P < 0.025) in neonates delivered by cesarean section (23.6 ± 2.6) than in spontaneously delivered animals (14.4 ± 2.0). As a result of these changes in F and E dynamics, PR-F (1.4 ± 0.3 μ/min/kg) and SR-F (0.9 ± 0.2 μ/min/kg) in neonates delivered by cesarean section were lower (P< 0.01) than corresponding values in spontaneously delivered newborns. It is concluded that the greater F secretion in animals delivered spontaneously than those delivered by cesarean section probably results from increased fetal adrenal 3β-hydroxysteroid dehydrogenase-isomerase activity, which as previously reported, occurs in late gestation in this species.

We determined whether the reduction in placental progesterone (P4) production observed after administration of the antiestrogen ethamoxytriphetol (MER-25) to pregnant baboons was associated with a decline in activity and/or content of the placental mitochondrial cholesterol side-chain cleavage system (P-450scc). Pregnant baboons (Papio anubis) were untreated (n = 9) or administered MER-25 (25 mg/kg BW, orally; n = 6) daily on days 140-170 of gestation (term = 184 days). Placentas were obtained by cesarean section on day 170 of gestation, and P-450scc activity and cytochrome P-450 content were determined on mitochondria-rich fractions. Administration of MER-25 to pregnant baboons resulted in a 40% reduction (P less than 0.01, by Student's t test) in the mean (+/- SE) peripheral serum P4 concentration (6.3 +/- 0.3 ng/ml) compared to that in untreated (10.4 +/- 0.3 ng/ml) baboons. P-450scc activity, as determined by formation of pregnenolone (P5) and P4 during a 30-min incubation (picomoles per mg protein), was 37% lower (P less than 0.01) in placental mitochondria obtained from MER-25-treated baboons (179.8 +/- 25.0) than in that from untreated (285.4 +/- 13.4) baboons. Mitochondrial cytochrome P-450 content, assessed by spectral analysis, was 28% lower (P less than 0.02) in antiestrogen-treated (46.7 +/- 2.1 pmol/mg protein) than in untreated (64.8 +/- 5.2 pmol/mg protein) baboons. The initial (time zero) free cholesterol content (nanomoles per mg protein) of mitochondrial-rich preparations was not significantly different in antiestrogen-treated (189.3 +/- 13.0) and untreated (225.0 +/- 15.1) animals. Collectively, these results suggest that the decline in placental P4 production observed in baboons in response to MER-25 occurs at least in part as a result of a decrease in cytochrome P-450scc activity. The loss in P-450scc activity appears to be an intramitochondrial event and not a result of depletion of the total mitochondrial cholesterol pool. We propose, therefore, that one mechanism by which estrogen may regulate the production of P4 by the placenta during primate pregnancy is via the maintenance of placental mitochondrial cytochrome P-450, the terminal oxidase of cytochrome P-450scc.

Invasive surgical procedures are often used to study the reproductive and adrenocortical endocrine systems in primates. Anesthetic agents must, therefore, be used that have the least confounding effects on these systems. The present study was designed to characterize various adrenocortical endocrine responses of female baboons (Papio anubis), each treated for 120 minutes with an infusion of ketamine HCl (6 mg/min) in 5% dextrose in water (0.40 ml/min), a combination of ketamine and acetylpromazine (0.6 mg acetylpromazine and 6 mg ketamine HCl/min) in 5% dextrose in water, or inhalation of vaporized halothane (1.0% halothane, N2O 25%, 1 liter/min; O2 75%, 3 liters/min). Blood samples were collected throughout the treatment period, and serum was assayed for prolactin (PRL), dehydroepiandrosterone (DHA), dehydroepiandrosterone sulfate (DHAS), and cortisol (F). No significant elevations in DHA, F, or PRL concentrations were found following infusion of ketamine alone. Only serum DHAS concentrations were significantly altered after long-term exposure to ketamine. Acetylpromazine increased PRL concentrations tenfold to levels significantly greater than those in ketamine- and halothane-treated animals but had no effect on serum DHA, DHAS, or F. Treatment with halothane had no effect on serum PRL, DHA, or DHAS but did suppress F (>40%) concentrations over time. These data indicate that ketamine is best suited for the collection of biological samples when deep analgesia is not required but that halothane is preferable in the latter situation.

Dehydroepiandrosterone (D) and dehydroepiandrosterone sulphate (DS) concentrations were determined by radioimmunoassay in peripheral sera of non-pregnant, pregnant (55 days to term) and newborn baboons and in umbilical sera of animals delivered by Caesarean section close to term. D concentrations (mean ± sd, μg/100 ml, N) in non-pregnant animals (1.61 ± 1.32, 23) were not different (P > 0.05) from those during pregnancy (1.80 ± 1.21, 101). DS concentrations, expressed as unconjugated D, in non-pregnant (13.5 ± 6.0, 23) and pregnant (15.1 ± 7.5, 101) animals were also similar (P > 0.05). However, both D (P < 0.01) and DS (P < 0.005) levels increased with gestational age such that serum D (2.46±1.39, 23, P < 0.05) and DS (18.9 ± 5.7, 23, P < 0.001) levels between 150 and 180 days gestation were greater than in non-pregnant animals. These increases may be important since oestrogen production rises rapidly during late gestation and both D and DS can serve as oestrogen precursors. In both non-pregnant (P < 0.005) and pregnant (P < 0.001) animals D and DS concentrations (ratio 1:8) were correlated. Cord serum D levels (2.4 ± 1.4, 5) were not different from those of maternal serum (P > 0.05), while cord DS levels (40.3 ± 14.8) were greater (P < 0.001) than those of maternal serum. This may reflect rapid equilibration of D but not DS between foetal and maternal circulations. In sera from neonates, D (19.4 ± 14.6, 8) and DS 567 ± 570, 8) concentrations were greater (P < 0.001) than those in maternal serum, indicating marked post-partal changes in clearance or production of both compounds. The high levels of D and DS in cord and newborn sera are compatible with the view that the baboon foetus makes appreciable contributions of oestrogen precursors in pregnancy.

We measured uterine extraction (i.e. metabolism) and transuteroplacental interconversion of cortisol (F) and cortisone (E) to determine whether metabolism across the uterus changes during pregnancy and contributes to the MCR of these corticosteroids. On day 100 (n = 4) or 170 (n = 3) of pregnancy (term = day 184), baboons (Papio anubis; 14-18 kg) were sedated with ketamine, and a constant infusion (0.38 ml/min) of 8-12 microCi [3H]F and 9-15 microCi [14C]E in 80 ml 0.9% NaCl-1% ethanol was initiated (time zero) via a maternal antecubital vein. At 60 min, animals were laparotomized, and at 70, 80, and 90 min, blood samples were obtained from right and left uterine veins and from a maternal saphenous vein. At 95 min, a transverse incision was made in the uterus, the fetus was isolated, and blood samples were obtained from the umbilical vein and artery. The cord was then clamped, and the fetus was delivered. Radio-labeled F and E were extracted from serum and purified by sequential paper chromatography, and metabolic parameters were calculated. Endogenous F and E levels were determined by RIA. In the mother, the percent conversions of E to F at midgestation (mean +/- SE; 72 +/- 4) and late gestation (65 +/- 3) were similar and exceeded (P less than 0.01) respective values for oxidation of F to E (51 +/- 7 and 46 +/- 7, respectively), indicating that maternal corticosteroid metabolism favors F formation and is unchanged during the second half of gestation. In contrast, corticosteroid metabolism across the uterus and placenta (transuteroplacental) was altered during pregnancy. At midgestation, transuteroplacental conversion of E to F (37 +/- 9) exceeded (P less than 0.05) the reverse reaction (18 +/- 3), whereas oxidation of F to E at term (28 +/- 4) was 7-fold greater (P less than 0.05) than reduction of E to F (4 +/- 1). At midgestation, essentially all of the F and E in umbilical vein was derived from maternal F. This contrasts with that observed in near-term baboons in which only 41 +/- 9% of the F and 64 +/- 8% of the E entering the fetal circulation was of maternal origin. As a result of uterine, placental, and fetal metabolism, 30% of the F and 15% of the E in maternal circulation were extracted by the uterus at both mid- and late gestation. We conclude that transuteroplacental corticosteroid metabolism changes from reduction at midgestation to oxidation at term.(ABSTRACT TRUNCATED AT 400 WORDS)

Since interactions between progesterone (P4), Cortisol (F), cortisone (E) and corticosteroid binding globulin (CBG) may influence the metabolic clearance rates (MCR) of these steroids, the effect of altering circulating F concentrations on clearance of the steroids was determined. MCR of P4, F and E were determined by the iv constant infusion method in 6 pregnant and 6 nonpregnant baboons (Papio papio). Serum F concentrations were altered by iv infusion of 5 mg F/90 min or im injection of betamethasone (3 mg bi-daily for 2 days). Mean MCR-P4 (1/d/kg ± SE) was greater (P < 0.01) in pregnant (92.8 ± 8.5) than in nonpregnant (53.9 ± 4.4) animals while mean MCR-F was similar in both groups (10.8 ± 1.2 vs 13.0 ± 1.5, respectively). Mean MCR-E was also similar in pregnant (30.8 ± 4.9) and nonpregnant (34.1 ± 4.5) baboons. Mean serum F concentrations (/gmg/100 ml ± SE) in 4 nonpregnant (42.0 ± 8.6) and 4 pregnant (52.2 ± 10.0) baboons were increased (P < 0.05) 60% by F administration but MCR-P4, -F and -E were unaltered. Betamethasone treatment reduced (P < 0.05) serum F 75% in both groups. In nonpregnant baboons, betamethasone treatment reduced (P < 0.01) MCR-P4 (37.3 ± 3.9), MCR-F (7.4 ± 1.6) and MCR-E (18.5 ± 3.7). Betamethasone treatment of pregnant animals reduced (P < 0.01) MCR-P4 (56.5 ± 7.4), MCR-F (6.3 ± 0.8) and MCR-E (14.6 ± 2.6). Infusion of F into betamethasone-treated animals increased serum F levels and increased MCR-P4, -F and -E. It is concluded that variations in serum F levels affect the clearance of F, E and P4 presumably because of the mutual interactions of these steroids with CBG.

The metabolism of I.V. administered 4-[14C]-cortisol (F) was examined in 3 premature (cesarean section at 160, 167, and 174 days gestation, term = 184 days) and 8 spontaneously delivered term (184 ± 2 days) baboon neonates (Papio papio). Three premature (Group 1) and 4 spontaneously delivered animals (Group 2) were studied 2–18 h after delivery and, the remainder on the 5th (N = 2) or 12th (N = 2) day of life (Group 3). [14C]-Metabolites were isolated by ehromatography and crystallization. More than 10% of 14C was in liver, 3–9% in intestine and brain, ≤ 2% in lung, kidney, heart, spleen, and urine, and < 0.2%/g in blood and muscle 30 min. after injection. Most 14C (77.5 ± 1.6%) in all tissues was unconjugated. Extrahepatic tissues were primarily responsible for oxidation of F to cortisone. Formation of tetrahydrocortisol (THF), tetrahydrocortisone (THE), two hydroxylated metabolites and glucuronoside conjugates was restricted to liver. Alterations in F metabolism (decreased hydroxylated metabolites, increased THF and THE glucuronides) were not evident until 5–12 days after delivery. It is concluded that extrahepatic 11β-hydroxysteroid dehydrogenase is important in achieving a low F:E ratio in fetal blood. The similarity in F catabolic patterns of premature and day 1 term newborns makes it unlikely that changes in metabolism can account for the increased fetal blood F concentrations prior to parturition.

The objective of this study was to determine the influence of fetectomy at midgestation in baboons upon the peripheral serum concentrations of progesterone (P), estrone (E1), and estradiol (E2). Maternal saphenous venous blood was collected at 1- to 4-day intervals between 90 days gestation and the 24-h postpartum period in five baboons in whom the fetus but not the placenta was removed at 100–109 days gestation and in four untreated baboons. Serum P, E1, and E2 concentrations were determined by RIA. Baboons fetectomized at midgestation spontaneously delivered their placentas on day 169 ± 8 (mean ± SE) of gestation. The mean length of gestation for our baboon colony is 184 days. Fetectomy resulted in a marked decline (64–80%; P < 0.001) in serum P concentrations from mean values of 6.9–9.7 ng/ml in the five baboons before fetectomy to 1.7–3.5 ng/ml thereafter. Between 90 days gestation and term the mean (± SE) serum P concentration in untreated baboons was 7.7 ± 0.3 ng/ml, and although P concentrations fl...

Studies on the ability of granulosa to secrete progesterone after hormonal treatment have not addressed the possibility of variations in response associated with the stage of follicular development. Accordingly, granulosa were isolated from follicular fluid obtained from apparently healthy ovaries of patients undergoing tubal bypass. Follicle size was estimated by laparoscopic and ultrasonic measurements, and granulosa cells were prepared by centrifugation and treatment with EGTA and hypertonic sucrose solution to minimize aggregation. Cell viability was determined and 10(6) cells/ml medium 199D were incubated for 24 h without or with ovine LH (1 microgram/ml) or 17 beta-estradiol (5 micrograms/ml). Progesterone secretion by granulosa increased from 6 to 30 ng/24 h/10(6) cells and correlated with follicle sizes of 5--25 mm. Compared to untreated cells obtained from the same follicle, granulosa from follicles ranging from 5--15 mm secreted 2.5 times more progesterone when treated with LH. This effect of LH diminished with further increases in follicle size, with LH having no effect on cells from follicles larger than 20 mm. Compared to untreated cells from the same follicles, 17 beta-estradiol caused a progressive inhibition of progesterone secretion by granulosa that correlated with follicle sizes between 5--18 mm, with almost 60% inhibition occurring in cells from 18-mm follicles. Thereafter, the inhibitory effect of 17 beta-estradiol diminished, and no effect was found in granulosa from follicles larger than 22 mm. These results indicate that LH and 17 beta-estradiol have maximal in vitro effects on progesterone secretion by granulosa from follicles with diameters of 15--18 mm. The change in response to LH and 17 beta-estradiol observed in samples from larger follicles may be due to the fact that these cells already have maximal binding of LH and 17 beta-estradiol to their receptors. Alternatively, it is possible that cells from larger follicles are desensitized to LH and 17 beta-estradiol.

We have previously shown that ACTH and PRL stimulate baboon fetal adrenal dehydroepiandrosterone (DHA) production both in vitro and in vivo and that estrogen diminishes the responsivity of the adrenal to tropic peptides in vitro. In the present study we determined the effects of increasing placental estrogen production by the administration of androstenedione at midgestation on DHA production by the baboon fetus in vivo. Pregnant baboons were untreated (n = 8) or treated (n = 9) with increasing numbers of androstenedione implants inserted in the mother at 8-day intervals between days 70-100 of gestation (term = day 184). On day 100, the fetuses were exteriorized, and a constant infusion of saline (0.1 ml/min) was initiated via a catheter inserted into a femoral vein of the fetus. At 40 min, a bolus injection of either 30 nmol ACTH or 40 nmol ovine PRL was administered to fetuses. ACTH or PRL (0.2 nmol/min.0.1 ml saline) were then infused for an additional 25 min. The concentrations of serum estradiol (E2) in the uterine vein (20.2 +/- 1.5 ng/ml; mean +/- SE) and estrone (E1) in umbilical vein (11.9 +/- 3.1 ng/ml) of androstenedione-treated baboons were 2-fold greater (P less than 0.05) than respective values in untreated baboons. Baseline concentrations of DHA in the femoral vein of the fetus were similar in all treatment groups (overall mean, 120 +/- 20 ng/ml) and greater (P less than 0.05) than values (27 +/- 3) in the mother. In untreated control baboons, basal DHA concentrations in the fetus were increased (P less than 0.05) by 69 +/- 17% and 94 +/- 29% after fetal injection of ACTH (n = 4) or PRL (n = 4), respectively. In contrast, neither PRL (n = 5) nor ACTH (n = 4) had any effect on serum DHA when injected into androstenedione-treated baboons. Regardless of treatment, injection of ACTH or PRL into the fetus had no effect on DHA concentrations in the mother. Collectively, these findings indicate that the ability of the fetal adrenal to increase DHA production in response to an acute infusion of ACTH or PRL was abolished in baboons in which placental estrogen production was increased prematurely at midgestation. Therefore, we suggest that during the second half of gestation in the baboon a regulatory system may exist in utero, in which there is feedback control of the placental product estrogen on the formation of the fetal adrenal precursor DHA.

It has been suggested that pituitary factors other than ACTH modulate adrenal steroidogenesis during maturation of the pituitary-adrenocortical axis. Therefore, we determined whether hormones other than ACTH influence the production of cortisol (F), dehydroepiandrosterone (DHA), and DHA-sulfate (DHAS) in baboon infants studied between 8 and 24 months of age. Animals (three males, two females) were sedated with ketamine and peripheral blood samples taken 20, 10, and 0 min before a 90-min constant iv infusion of 448 pmol/min of either ACTH, ovine PRL, ovine GH, 2.4 nmol/min human CG (hCG), or normal saline. Serum F, DHA, and DHAS concentrations of blood samples obtained during the infusion (70, 80, 90 min) were averaged and compared with average pretreatment values. Each animal received each of the treatment protocols which included a minimum recovery period of 3-6 weeks. The serum concentrations of F, DHA, and DHAS did not vary with age and averaged (mean +/- SE) 24 +/- 2, 1.9 +/- 0.2, and 36 +/- 5 micrograms/dl, respectively. Compared to pretreatment values, ACTH increased (P less than 0.05) mean serum F concentrations by 155 +/- 20%; PRL, GH, hCG, and saline had no effect. In contrast, serum DHA concentrations were stimulated (P less than 0.05) by both ACTH (131 +/- 20%) and PRL (58 +/- 18%); GH, hCG, and saline had no effect. Similar findings were observed for serum DHAS concentrations. These findings indicate that the majority of serum androgens in young baboons is of adrenal origin. Therefore, we conclude that PRL, in addition to ACTH, may also be an adrenocorticotrophic factor in baboon infants. However, in contrast to ACTH, the action of PRL on the adrenal is apparently specific for androgen production.