Effects of moderate global maternal nutrient reduction on fetal baboon renal mitochondrial gene expression at 0.9 gestation.

Early life malnutrition results in structural alterations in the kidney, predisposing offspring to later life renal dysfunction. Kidneys of adults who were growth restricted at birth have substantial variations in nephron endowment. Animal models have indicated renal structural and functional consequences in offspring exposed to suboptimal intrauterine nutrition. Mitochondrial bioenergetics play a key role in renal energy metabolism, growth, and function. We hypothesized that moderate maternal nutrient reduction (MNR) would adversely impact fetal renal mitochondrial expression in a well-established nonhuman primate model that produces intrauterine growth reduction at term. Female baboons were fed normal chow diet or 70% of control diet (MNR). Fetal kidneys were harvested at cesarean section at 0.9 gestation (165 days gestation). Human Mitochondrial Energy Metabolism and Human Mitochondria Pathway PCR Arrays were used to analyze mitochondrially relevant mRNA expression. In situ protein content was detected by immunohistochemistry. Despite the smaller overall size, the fetal kidney weight-to-body weight ratio was not affected. We demonstrated fetal sex-specific differential mRNA expression encoding mitochondrial metabolite transport and dynamics proteins. MNR-related differential gene expression was more evident in female fetuses, with 16 transcripts significantly altered, including 14 downregulated and 2 upregulated transcripts. MNR impacted 10 transcripts in male fetuses, with 7 downregulated and 3 upregulated transcripts. The alteration in mRNA levels was accompanied by a decrease in mitochondrial protein cytochrome c oxidase subunit VIc. In conclusion, transcripts encoding fetal renal mitochondrial energy metabolism proteins are nutrition sensitive in a sex-dependent manner. We speculate that these differences lead to decreased mitochondrial fitness that contributes to renal dysfunction in later life.

Vulnerability of the fetal primate brain to moderate reduction in maternal global nutrient availability.

Moderate maternal nutrient restriction during pregnancy occurs in both developing and developed countries. In addition to poverty, maternal dieting, teenage pregnancy, and uterine vascular problems in older mothers are causes of decreased fetal nutrition. We evaluated the impact of global 30% maternal nutrient reduction (MNR) on early fetal baboon brain maturation. MNR induced major cerebral developmental disturbances without fetal growth restriction or marked maternal weight reduction. Mechanisms evaluated included neurotrophic factor suppression, cell proliferation and cell death imbalance, impaired glial maturation and neuronal process formation, down-regulation of gene ontological pathways and related gene products, and up-regulated transcription of cerebral catabolism. Contrary to the known benefits from this degree of dietary reduction on life span, MNR in pregnancy compromises structural fetal cerebral development, potentially having an impact on brain function throughout life.

Expression of the placental transcriptome in maternal nutrient reduction in baboons is dependent on fetal sex.

Maternal undernutrition increases the risk of perinatal complications and predisposes offspring to obesity, diabetes, and cardiovascular disease later in life. Emerging evidence suggests that changes in placental function play a role in linking altered maternal nutrition in pregnancy to the subsequent development of adult disease. The susceptibility for disease in response to an adverse intrauterine environment differs distinctly between boys and girls, with girls typically having better outcomes. Here, we tested the hypothesis that regulation of the placental transcriptome by maternal nutrient reduction (NR) is dependent on fetal sex. We used a nonhuman primate model of NR in which maternal global food intake was reduced by 30% in baboons starting at gestational day (GD) 30. At GD 165 (term = GD 183), placental genome expression profiling of 6 control (n = 3 females, 3 males) and 6 nutrient restricted (n = 3 females, 3 males) fetuses was carried out followed by bioinformatic analysis. Surprisingly, there was no coordinated placental molecular response to decreased nutrient availability when analyzing the data without accounting for fetal sex. In contrast, female placentas exhibited a highly coordinated response that included upregulation of genes in networks, pathways, and functional groups related to programmed cell death and downregulation of genes in networks, pathways, and functional groups associated with cell proliferation. These changes were not apparent in the male placentas. Our data support the concept that female placentas initiate complex adaptive responses to an adverse intrauterine environment, which may contribute to increased survival and better pregnancy outcomes in girls.

Effect of 30% nutrient restriction in the first half of gestation on maternal and fetal baboon serum amino acid concentrations.

Mechanisms linking maternal nutrient restriction (MNR) to intra-uterine growth restriction (IUGR) and programming of adult disease remain to be established. The impact of controlled MNR on maternal and fetal amino acid metabolism has not been studied in non-human primates. We hypothesised that MNR in pregnant baboons decreases fetal amino acid availability by mid-gestation. We determined maternal and fetal circulating amino acid concentrations at 90 d gestation (90dG, term 184dG) in control baboons fed ad libitum (C, n 8) or 70% of C (MNR, n 6). Before pregnancy, C and MNR body weights and circulating amino acids were similar. At 90dG, MNR mothers had lower body weight than C mothers (P< 0·05). Fetal and placental weights were similar between the groups. MNR reduced maternal blood urea N (BUN), fetal BUN and fetal BUN:creatinine. Except for histidine and lysine in the C and MNR groups and glutamine in the MNR group, circulating concentrations of all amino acids were lower at 90dG compared with pre-pregnancy. Maternal circulating amino acids at 90dG were similar in the MNR and C groups. In contrast, MNR fetal ß-alanine, glycine and taurine all increased. In conclusion, maternal circulating amino acids were maintained at normal levels and fetal amino acid availability was not impaired in response to 30% global MNR in pregnant baboons. However, MNR weight gain was reduced, suggesting adaptation in maternal-fetal resource allocation in an attempt to maintain normal fetal growth. We speculate that these adaptive mechanisms may fail later in gestation when fetal nutrient demands increase rapidly, resulting in IUGR.

Effects of maternal nutrient restriction, intrauterine growth restriction, and glucocorticoid exposure on phosphoenolpyruvate carboxykinase-1 expression in fetal baboon hepatocytes in vitro.

BACKGROUND: The objective of this study was to develop a cell culture system for fetal baboon hepatocytes and to test the hypotheses that (i) expression of the gluconeogenic enzyme phosphoenolpyruvate carboxykinase-1 (PEPCK-1) is upregulated in hepatocytes isolated from fetuses of nutrient-restricted mothers (MNR) compared with ad libitum-fed controls (CTR), and (ii) glucocorticoids stimulate PEPCK-1 expression. METHODS: Hepatocytes from 0.9G CTR and MNR fetuses were isolated and cultured. PEPCK-1 protein and mRNA levels in hepatocytes were determined by Western blot and quantitative PCR, respectively. RESULTS: Fetuses of MNR mothers were intrauterine growth restricted (IUGR). Feasibility of culturing 0.9G fetal baboon hepatocytes was demonstrated. PEPCK-1 protein levels were increased in hepatocytes isolated from IUGR fetuses, and PEPCK-1 mRNA expression was stimulated by glucocorticoids in fetal hepatocytes. CONCLUSIONS: Cultured fetal baboon hepatocytes that retain their in vivo phenotype provide powerful in vitro tools to investigate mechanisms that regulate normal and programmed hepatic function.

Baboon infant produced by embryo transfer.

An embryo was recovered surgically from a naturally ovulating, naturally inseminated Papio cynocephalus female on day 5 of gestation and transferred surgically to a naturally synchronized, nonmated Papio cynocephalus female on 20 March 1975. A male baboon weighing 875 grams was delivered by cesarean section on 5 September 1975, 174 days after estimated ovulation time.

Microstructural changes of the baboon cerebral cortex during gestational development reflected in magnetic resonance imaging diffusion anisotropy.

Cerebral cortical development involves complex changes in cellular architecture and connectivity that occur at regionally varying rates. Using diffusion tensor magnetic resonance imaging (DTI) to analyze cortical microstructure, previous studies have shown that cortical maturation is associated with a progressive decline in water diffusion anisotropy. We applied high-resolution DTI to fixed postmortem fetal baboon brains and characterized regional changes in diffusion anisotropy using surface-based visualization methods. Anisotropy values vary within the thickness of the cortical sheet, being higher in superficial layers. At a regional level, anisotropy at embryonic day 90 (E90; 0.5 term; gestation lasts 185 d in this species) is low in allocortical and periallocortical regions near the frontotemporal junction and is uniformly high throughout isocortex. At E125 (0.66 term), regions having relatively low anisotropy (greater maturity) include cortex in and near the Sylvian fissure and the precentral gyrus. By E146 (0.8 term), cortical anisotropy values are uniformly low and show less regional variation. Expansion of cortical surface area does not occur uniformly in all regions. Measured using surface-based methods, cortical expansion over E125-E146 was larger in parietal, medial occipital, and lateral frontal regions than in inferior temporal, lateral occipital, and orbitofrontal regions. However, the overall correlation between the degree of cortical expansion and cortical anisotropy is modest. These results extend our understanding of cortical development revealed by histologic methods. The approach presented here can be applied in vivo to the study of normal brain development and its disruption in human infants and experimental animal models.

Developmental expression of Toll-like receptors-2 and -4 in preterm baboon lung.

Preterm babies are susceptible to respiratory infection due to immature lung and immune system. Immune cells express Toll-like receptors (TLRs), which may be important in local host defense of preterm infants. We studied the expression of TLR2 and TLR4 in lung tissues of fetal baboons delivered at 125, 140, and 175 days of gestation (dGA; term=185+/-2 days) and preterm baboons that became naturally infected with bacterial/fungal pathogens. The TLR-mRNA and protein were quantified by Northern and Western blotting, respectively. The expression of both TLRs was significantly low at 125 and 140dGA. At 175dGA, the levels reached equivalent to those in adult baboons. However, in naturally infected baboons, the TLR4-mRNA was reduced (p<0.05); TLR2-mRNA expression remained unaltered. The protein expression of both TLRs was found increased in naturally infected baboons. Our results suggest that the lung TLR expression is developmentally regulated and altered during respiratory infection in preterm babies.

Developmental expression of cell cycle regulators in the baboon fetal adrenal gland.

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)-3beta-hydroxysteroid dehydrogenase (3beta-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 3beta-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.

The baboon as a research model for the study of endometrial biology, uterine receptivity and embryo implantation.

The process of embryo implantation includes attachment of the embryo to the endometrium and penetration through the epithelial layer, decidualization of the basement membrane, invasion of the uterine stroma and access to blood supply. This implantation process is very different in humans when compared to pigs, cattle or rodents. The process of invasion in humans where the embryo gets embedded in decidual tissue and in spiral arteries is more aggressive, but otherwise similar to the process of implantation and invasion in non-human primates such as rhesus monkeys and baboons. For ethical reasons, it is unacceptable to study directly the process of embryo implantation in women, and to this day, this remains one of the 'black boxes' of reproductive science. Indeed for many clinicians practicing reproductive medicine, in fertility centers, the most difficult question and of concern asked by patients is: 'Why do my healthy appearing embryos not implant: is there a problem with my endometrium or uterus?' The olive baboon (Papio anubis anubis) is an excellent animal model for reproductive research. In contrast with smaller non-human primates like rhesus monkeys or cynomolgus monkeys, it is possible in baboons to use transcervical uterine probes (curettes, catheters and hysteroscopic equipment) to perform endometrial biopsy, embryo flushing or transfer and hysteroscopy in a non-invasive way. This can be done easily in multiparous baboons during menstruation, but may be more difficult at the end of the follicular phase (maximal perineal swelling impedes vaginal/cervical access) or during the luteal phase (narrow cervix), in nulliparous baboons and in animals with abnormal internal genitals. In this paper we present an overview regarding the potential of the baboon model to study in vivo uterine receptivity and embryo implantation using invasive and non-invasive approaches.

Serum cortisol binding capacity and cortisol concentration in the pregnant baboon and its fetus during gestation.

The cortisol binding capacity of serum from 11 pregnant baboons (38 samples) and from 7 baboon fetuses delivered prematurely or at term was measured after removal of endogenous steroids. Values for maternal serum collected between 60 and 120 days after mating (59.0 +/- 6.4 mug/100 ml, mean +/- SD) were greater than those for serum collected at term (42.3 +/- 4.9 mug/100 ml). The cortisol-binding capacity of fetal serum collected between 100 and 132 days' gestation was similar to that of the corresponding maternal sample, but at term was only 50% of the maternal value. The rate of clearance of cortisol from both fetal and maternal serum may therefore increase progressively during the last trimester of pregnancy. This effect is likely to be more marked in the fetus. The cortisol binding capacity of 15 serum samples from 9 non-pregnant baboons was 33.4 +/- 5.5 mug/100 ml. Mestranol2 (administered 200 mug/day im for 15 days) significantly increased the serum cortisol binding capacity. The concentration of cortisol in maternal serum from 7 pregnant baboons (10 samples) was 44.0 +/- 8.4 mug/100 ml and was independent of the state of gestation. In fetal serum the cortisol concentration was 4 mug/100 ml before 168 days' gestation and reached 49 mug/100 ml after normal delivery at term. These findings suggest that the mechanisms for production of cortisol by the fetus mature as gestation progresses. The physiological significance of the marked difference between the cortisol concentration and the cortisol binding capacity of fetal serum awaits elucidation.

Biphasic developmental expression of adrenocorticotropin receptor messenger ribonucleic acid levels in the baboon fetal adrenal gland.

We have previously shown an estrogen-dependent developmental regulation of placental oxidation of cortisol to cortisone that results in enhanced fetal pituitary ACTH production and the induction of steroidogenic enzymes in and de novo cortisol production by the fetal adrenal in the second half of baboon pregnancy. However, it is not known whether the receptor for ACTH is simultaneously generated at this time in development to provide a mechanism for mediating the tropic action of ACTH on steroidogenesis in the primate fetal adrenal gland. Therefore, in the present study we determined the levels of ACTH receptor messenger RNA (mRNA) and correlated ACTH receptor expression with appearance of the mRNA for delta5-3beta-hydroxysteroid dehydrogenase/isomerase (3betaHSD), the enzyme protein that signals functional maturation of the definitive cortical zone in the baboon fetal adrenal. A baboon ACTH receptor complementary DNA was cloned and hybridized with polyadenylated RNA isolated from baboon (Papio anubis) fetal adrenals obtained in early (days 58-64; RNA from seven baboon fetuses pooled to yield three samples), mid-(days 99-103; RNA from five baboons pooled to yield four samples), and late (days 165-168; RNA of four individual baboon fetuses) gestation (term = 184 days). Expression of the primary 3.4-kilobase ACTH receptor mRNA transcript, determined by Northern blot and expressed as a ratio of beta-actin mRNA, was minimal early in gestation (mean +/- SE, 0.11 +/- 0.05 arbitrary densitometric units). However, fetal adrenal ACTH receptor mRNA levels increased (P < 0.001, by ANOVA) approximately 13-fold to 1.41 +/- 0.16 at midgestation, then declined by 70% (P < 0.001) to 0.41 +/- 0.10 in late gestation. To determine whether the decrease in ACTH receptor expression by the fetal adrenal in the second half of pregnancy reflected programmed cell death, the integrity of genomic DNA was assessed by 32P-labeled DNA gel electrophoresis and in situ DNA end labeling. Because DNA oligonucleosomes and apoptotic DNA strand breaks characteristic of apoptosis were absent in the adrenal glands of fetal baboons, the decline in ACTH receptor mRNA levels in the fetal adrenal did not seem to reflect programmed cell death. Expression of the single 2.0-kilobase mRNA transcript for 3betaHSD, an enzyme localized specifically in the definitive zone of the fetal adrenal, was minimal in early (0.01 +/- 0.00 arbitrary units) and mid- (0.10 +/- 0.01) gestation. However, 3betaHSD mRNA levels were markedly increased late in gestation to a value (1.38 =/- 0.34) approximately 13-fold greater (P < 0.001) than that in midgestation. These findings indicate that there was a biphasic monomodal developmental expression of the ACTH receptor in the baboon fetal adrenal, which contrasted with the progressive increase in adrenal weight, 3betaHSD expression, and de novo cortisol production previously determined. Because the fetal adrenal is comprised mainly of the fetal cortical zone throughout gestation, the decrease in ACTH receptor expression between mid- and late gestation seems to occur primarily in the latter zone and may signal a selective decline in tropic responsivity of and delta5-C19-steroid, e.g. dehydroepiandrosterone, biosynthesis within the baboon fetal adrenal gland.

Developmental regulation of protein kinase-A and -C activities in the baboon fetal adrenal.

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)

Activation of the baboon fetal pituitary-adrenocortical axis at midgestation by estrogen: enhancement of fetal pituitary proopiomelanocortin messenger ribonucleic acid expression.

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 (P < 0.05) 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 (P < 0.05) 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 (P < 0.05) in baboon fetuses at term (0.57 +/- 0.05) than at midgestation (0.28 +/- 0.03) and increased (P < 0.05) 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)

Activation of the baboon fetal pituitary-adrenocortical axis at midgestation by estrogen: adrenal delta 5-3 beta-hydroxysteroid dehydrogenase and 17 alpha-hydroxylase-17,20-lyase activity.

We have recently demonstrated that treatment of pregnant baboons with androstenedione (delta 4 A) at midgestation to increase estrogen production induced a pattern of placental cortisol (F) metabolism which was similar to that at term and resulted in de novo F production by the fetus, presumably by activation of the fetal hypothalamic-pituitary-adrenocortical axis. The present study was designed to examine the subcellular events in the fetal adrenal that were apparently stimulated by estrogen-induced alterations in transplacental corticosteroid metabolism. Therefore, we determined the effects of estrogen treatment at midgestation and removal of estrogen action near term on the specific activity of the rate-limiting enzymes delta 5-3 beta-hydroxysteroid dehydrogenase (3 beta HSD) and 17-hydroxylase-17,20-lyase (17 alpha-OHase). Fetal adrenals were obtained on day 100 (n = 11) or day 165 (n = 11) of gestation (term = day 184) from untreated animals, on day 100 from animals receiving delta 4 A daily between days 70-100 (n = 9) to increase placental estrogen production, and on day 165 from baboons treated daily between days 130-164 with antiestrogen ethamoxytriphetol (MER-25; n = 7). The activity of 17 alpha-OHase was determined by incubating adrenal microsomes (105,000 x g) with [3H] progesterone, NAD+, and NADH in phosphate buffer. The radiolabeled products 17-hydroxyprogesterone, delta 4 A, and testosterone were purified, and enzyme activity expressed as picograms of product per min/mg tissue. The activity of 3 beta HSD was determined by incubating adrenal microsomes with [3H]pregnenolone and NAD+ in phosphate buffer. The radiolabeled progesterone product was purified, and enzyme activity was expressed as nanograms per min/mg tissue. Treatment with delta 4 A increased estrogen concentration at midgestation 3-fold to levels comparable to those measured near term. Although fetal adrenal weight was greater at term than at midgestation (p less than 0.05), weight was not increased by delta 4 A treatment. The specific activity (mean +/- SE) of fetal adrenal 17 alpha-OHase at midgestation (181 +/- 29) was increased (P less than 0.05) 3-fold by treatment with delta 4 A to levels (591 +/- 105) comparable to those in adrenal microsomes prepared from untreated animals near term (816 +/- 130). Enzyme activity in adrenals of MER-25-treated baboons was 40%, but not significantly lower than that in term controls.(ABSTRACT TRUNCATED AT 400 WORDS)

Developmental regulation of corticosteroid-binding globulin biosynthesis in the baboon fetus.

The present study determined the roles of estrogen and cortisol in maternal and fetal corticosteroid-binding globulin (CBG) levels and fetal hepatic messenger RNA (mRNA) expression in the baboon. Samples of fetal liver, kidney, and brain were obtained from untreated control animals at early (day 60; n = 4), mid (day 100; n = 8), and late (day 165; n = 5) gestation (term = day 184). Maternal and umbilical blood samples were collected on day 100 from baboons in which betamethasone was administered sc to the mother (n = 6) on days 60-99 of gestation and on day 165 from animals (n = 4) in which the fetus was administered betamethasone on days 150-164 of gestation. Maternal serum cortisol concentrations were similar at mid (43 +/- 2 micrograms/dl) and late (42 +/- 3 micrograms/dl) gestation and decreased (P < 0.05) at midgestation (1 +/- 1 micrograms/dl) and term (31 +/- 4 micrograms/dl) after betamethasone treatment. Umbilical serum cortisol levels were also reduced (P < 0.05) at both mid (1 +/- 1 micrograms/dl) and late (14 +/- 5 micrograms/dl) gestation by betamethasone treatment. Fetal serum CBG levels in untreated animals were lower (P < 0.05) on day 165 (444 +/- 29 pmol/ml) than on day 100 (844 +/- 35 pmol/ml) and increased (P < 0.05) at midgestation (1098 +/- 64 pmol/ml), but not at term (551 +/- 24 pmol/ml), after betamethasone treatment. In contrast, maternal serum CBG levels (range, 528-770 pmol/ml) were not altered by gestational age or betamethasone. The human CBG complementary DNA hybridized to a single mRNA species of 1.8 kilobases in baboon fetal liver; however, CBG was not expressed in fetal kidney and was detectable in fetal brain and pancreas only by reverse transcription-PCR. In untreated baboon fetuses, the mRNA levels of hepatic CBG, expressed as a ratio of 18S RNA, progressively decreased (P < 0.05) in early (1.83 +/- 0.17), mid (0.97 +/- 0.12), and late (0.51 +/- 0.04) gestation. These results demonstrate that fetal hepatic CBG mRNA expression and serum CBG concentrations were elevated early in baboon gestation and exhibited a progressive decline during the course of advancing pregnancy. We suggest that the increased levels of fetal CBG in the early stages of gestation reflect stimulation of hepatic CBG synthesis by maternal cortisol, which we previously demonstrated to occur in the fetus as a result of preferential 11 beta-hydroxysteroid dehydrogenase-catalyzed glucocorticoid reduction across the placenta. The decline in fetal CBG may reflect the developmental increase in catabolism of cortisol to bioinactive cortisone in target tissues of the fetus such as the liver.

Regulation of oocyte microvilli development in the baboon fetal ovary by estrogen.

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.

Regulation of immunoreactive inhibin patterns in baboon pregnancy: maternal, placental, and fetal considerations.

To better understand the sources and regulation of circulating inhibin during primate pregnancy, immunoreactive inhibin was measured in sera obtained from the maternal saphenous vein, uterine vein, and the fetus at varying times of baboon pregnancy. In both intact and fetectomized (fetus removed on day 100 of gestation; term = 184 days) animals, maternal serum inhibin concentrations were relatively constant between day 80 (first sampling day) and day 110 of gestation, after which they then steadily increased until days 155-165 (end of sampling). The increase in inhibin concentrations was significantly less in the fetectomized animals than in the intact baboons. Restoration of estrogen levels in the fetectomized animals did not significantly alter the circulating inhibin concentrations. Similarly, administration of the estrogen antagonist MER-25 to intact animals in the last trimester had no effect on maternal serum inhibin concentrations. Inhibin concentrations in uterine venous blood collected on day 100 of gestation were not significantly different from those in the maternal saphenous vein. However, the inhibin concentrations of uterine venous blood collected late in gestation (days 155-165) in either intact or fetectomized animals were significantly higher than the corresponding maternal venous concentrations, suggesting that the uteroplacental tissue becomes a source of circulating inhibin during the third trimester of pregnancy. Consistent with this suggestion was the detection of inhibin alpha-subunit mRNA in the placentae of intact or fetectomized animals obtained late in pregnancy, but its absence at midgestation. Immunoreactive inhibin concentrations were about 16 times higher (6500 +/- 831 mu Leq/mL) in fetal blood than in maternal blood (411 +/- 23 mu Leq/mL) at midgestation. The fetal blood concentrations significantly decreased to about 2800 mu Leq/mL by days 160-165 of gestation, but were still greater than those in the mother (approximately 1000 mu Leq/mL). The umbilical arterial and venous concentrations were the same as the fetal blood concentration of inhibin. The role of the baboon fetal adrenal in inhibin production was studied. Fetal adrenals collected from days 59, 135, and 167 of gestation contained the mRNA for the inhibin alpha-subunit in relatively high abundance. The in utero administration of ACTH for 30 min to five fetuses at midgestation (days 100-110) apparently did not alter the fetal concentration of immunoreactive inhibin. In summary, maternal serum inhibin levels increase during the last trimester of baboon pregnancy. This is suggested to be due to an increasing contribution of placental inhibin secretion, which is regulated not by placental estrogen production but, perhaps, by placental growth.(ABSTRACT TRUNCATED AT 400 WORDS)

Development of the baboon fetal adrenal gland: regulation of the ontogenesis of the definitive and transitional zones by adrenocorticotropin.

Throughout gestation, the primate fetal adrenal gland is comprised of the fetal zone, which expresses the P-450 17alpha-hydroxylase-C17,20 lyase (P-450c17) enzyme that catalyzes the synthesis of C19 steroids used for placental estrogen production. The development of the transitional zone comprised of cortical cells that express the P-450c17 and the 3beta-hydroxysteroid dehydrogenase-isomerase (3betaHSD) enzymes for cortisol production, and the definitive zone, which expresses 3betaHSD, but not P-450c17, for mineralocorticoid synthesis, does not occur until relatively late in gestation. Although ACTH is considered essential to fetal adrenal growth and function, the role that ACTH has in the development of the transitional and definitive zones, is less clear. To answer this question, the width of these zones was determined by immunocytochemical expression of P-450c17 and/or 3betaHSD in fetal adrenal glands obtained on day 100 (mid) of gestation (term = day 184) from baboons in which ACTH was administered to the fetus on days 95-99 of gestation or on day 165 (late) of gestation from baboons in which fetal ACTH was suppressed by treatment of the mother and fetus with betamethasone on days 150-164 of gestation. At midgestation, the fetal adrenal was comprised almost exclusively of fetal zone cells and a small definitive zone (38 +/- 2 microm in width), but was essentially devoid of a transitional zone (7 +/- 2 microm). Treatment with ACTH enhanced (P < 0.05) the width of the transitional zone (67 +/- 4 microm), but not the size of the definitive zone (10 +/- 4 microm). In late gestation, the width of the definitive zone, although 2-fold greater than that on day 100, was smaller (P < 0.05) than that of the transitional zone (120 +/- 15 microm), which greatly exceeded that at midgestation. Treatment with betamethasone in late gestation eliminated the transitional zone, but had no effect on the size of the definitive zone (120 +/- 8 microm). These findings indicate that the development of the baboon fetal adrenal transitional zone late in gestation is dependent on fetal pituitary ACTH. In contrast, the ontogenesis of the definitive zone at midgestation and its growth in late gestation occur in the relative absence of ACTH.

Effect of maternal betamethasone administration at midgestation on baboon fetal adrenal gland development and adrenocorticotropin receptor messenger ribonucleic acid expression.

Although fetal pituitary ACTH is important to fetal adrenal growth and steroidogenesis in the second half of primate pregnancy, its role in adrenal development and function has not been established in vivo in the first half of gestation. In the present study, therefore, baboons were treated at midgestation with betamethasone to determine the effect of fetal pituitary ACTH on fetal adrenal growth, development, and ACTH receptor and P-450 enzyme messenger ribonucleic acid (mRNA) levels. The administration of betamethasone to baboon mothers on days 60-99 of gestation (term = 184 days) decreased fetal pituitary POMC mRNA levels by 54% (P < 0.01) and fetal serum ACTH levels to undetectable values (P < 0.05). The decline in ACTH was associated with decreases in fetal adrenal weight (P < 0.001), cortical cell size (P < 0.05), appearance of apoptosis and cellular disorganization, and a loss of immunocytochemically demonstrable definitive zone-specific delta5-3beta-hydroxysteroid dehydrogenase expression. The concomitant administration of ACTH and betamethasone restored these aspects of adrenal integrity to normal. Moreover, there was approximately a 95% decrease (P < 0.01) in fetal adrenal expression of ACTH receptor, P-450 cholesterol side-chain cleavage, and P-450 17alpha-hydroxylase 17/20-lyase mRNA levels after betamethasone administration. We conclude that fetal pituitary ACTH is necessary for the growth and development of fetal and definitive cortical zones and the marked coordinated increase in ACTH receptor and maintenance of P-450 cholesterol side-chain cleavage/P-450 17alpha-hydroxylase 17/20-lyase expression in the baboon fetal adrenal gland during the first half of gestation.