Derivation and characterization of novel nonhuman primate embryonic stem cell lines from in vitro-fertilized baboon preimplantation embryos.

The development of nonhuman primate (NHP) embryonic stem cell (ESC) models holds great promise for cell-mediated treatment of debilitating diseases and to address numerous unanswered questions regarding the therapeutic efficacy of ESCs while supplanting ethical considerations involved with human studies. Here we report successful establishment and characterization of 3 novel baboon (Papio cynocephalus) ESC lines from the inner cell mass of intracytoplasmic sperm injection-derived blastocysts. Embryos were cultured in an improved baboon embryo in vitro culture protocol. The inner cell mass of blastocyst was laser-dissected and plated on mouse embryonic fibroblast feeder cell monolayer in the NHP ESC culture medium. Three cell lines with characteristic ESC morphology have been cultured through an extended period (>14 months), with 2 male cell lines (UT-1 and -2) and 1 female cell line (UT-3) displaying normal baboon karyotypes. Reverse transcription-polymerase chain reaction analysis confirmed that all 3 lines express primate ESC pluripotency markers, including OCT-4, NANOG, SOX-2, TERT, TDGF, LEFTYA, and REX-1. All 3 lines demonstrated positive immunocytochemical staining for OCT-4, stage-specific embryonic antigen-3, stage-specific embryonic antigen-4, TRA-1-60, and TRA-1-81. Baboon ESCs injected into NOD/SCID mice formed teratomas with all 3 germ layers. In addition, embryoid body-like spherical structures were derived and initial outgrowth was observed when embedded into extracellular matrix Matrigel. The ESC lines established in this NHP model have the potential to extend our knowledge in the fields of developmental biology, regenerative medicine, and future applications, including preclinical safety assessment of in vivo stem cell therapy.

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

Expression of the molecules that modulate the synthesis and action of estrogen in, or reflect function of, Sertoli cells was determined in the fetal testis of baboons in which estrogen levels were suppressed in the second half of gestation to determine whether this may account for the previously reported alteration in fetal testis germ cell development. P-450 aromatase, estrogen receptor (ER) ß, and α-inhibin protein assessed by immunocytochemistry was abundantly expressed in Sertoli cells of the fetal baboon testis, but unaltered in baboons in which estrogen levels were suppressed by letrozole administration. Moreover, P-450 aromatase and ERα and ß mRNA levels, assessed by real-time RT-PCR, were similar in germ/Sertoli cells and interstitial cells isolated from the fetal testis of untreated and letrozole-treated baboons. These results indicate that expression of the proteins that modulate the formation and action of estrogen in, and function of, Sertoli cells is not responsible for the changes in germ cell development in the fetal testis of estrogen-deprived baboons.

Developmental regulation of the expression of the transferrin receptor and Ki67 in oocytes of the baboon fetal ovary by estrogen.

We previously showed that estrogen regulates baboon fetal ovarian follicle development and oocyte integrity. Because iron incorporated into cells by the transferrin receptor is essential for cell/nuclear function, we determined whether fetal oocyte expression of transferrin receptor and the nuclear protein Ki67 were developmentally regulated by estrogen and associated with DNA integrity/fragmentation. Transferrin-receptor expression was minimal at midgestation and abundant in late gestation and localized to the cytoplasm and surface of oocytes of primordial follicles. Expression of transferrin receptor, however, was negligible in oocytes in fetuses in which serum estradiol-17beta levels were suppressed (>95%) by daily maternal treatment between mid- and late gestation with the aromatase inhibitor letrozole and partially restored by treatment with letrozole and estradiol benzoate. Ki67 was localized to pregranulosa and germ cells at midgestation and throughout the oocyte nucleus in late gestation in estrogen-replete fetuses. In contrast, in estrogen-suppressed fetuses, Ki67 was localized to a limited number of foci around the oocyte nucleus. Apoptosis detected in pregranulosa and germ cells at midgestation was not observed in late gestation in estrogen-replete/-suppressed fetuses. We conclude that estrogen regulates fetal oocyte transferrin-receptor expression and that inhibition of receptor development is associated with alterations in Ki67 expression by the oocyte but not apoptosis. Collectively, these results and our previous studies further define the essential role of estrogen in regulating development of follicles comprised of healthy oocytes by the baboon fetal ovary.

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.

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.

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.

Developmental regulation of baboon fetal ovarian maturation by estrogen.

Ovarian function in adult human and nonhuman primates is dependent on events that take place during fetal development, including the envelopment of oocytes by granulosa (i.e., folliculogenesis). However, our understanding of fetal ovarian folliculogenesis is incomplete. During baboon pregnancy, placental production and secretion of estradiol into the fetus increases with advancing gestation, and the fetal ovary expresses estrogen receptors alpha and beta in mesenchymal-epithelial cells (i.e., pregranulosa) as early as midgestation. Therefore, the current study determined whether estrogen regulates fetal ovarian follicular development. Pregnant baboons were untreated or treated with the aromatase inhibitor CGS 20267, or with CGS 20267 plus estradiol benzoate administered s.c. to the mother on Days 100-164 (term = Day 184). On Day 165, baboon fetuses were delivered by cesarean section and the number of total follicles and interfollicular nests consisting of oocytes and mesenchymal-epithelial cells in areas (0.33 mm(2)) of the outer and inner cortices of each fetal ovary were quantified using image analysis. Maternal and umbilical serum estradiol levels were decreased by >95% with CGS 20267. Treatment with CGS 20267 and estrogen restored maternal estradiol to normal and fetal estradiol to 30% of normal. Although fetal ovarian weight was unaltered, the mean number of follicles +/- SEM/0.33 mm(2) in the inner (59.0 +/- 1.7) and outer (95.3 +/- 2.4) cortical regions of fetal ovaries in untreated animals was 35%-50% lower (P < 0.01) in estrogen-depleted baboons (25.9 +/- 1.4, inner cortex; 62.5 +/- 2.7, outer cortex) and was restored to normal by treatment with CGS 20267 and estrogen. In contrast, the number of interfollicular nests was 2-fold greater (P < 0.01) in fetal ovaries of estrogen-suppressed animals, a change that was prevented by treatment with estrogen. In summary, fetal ovarian follicular development was significantly altered in baboons in which estrogen was depleted during the second half of gestation and restored to normal by estradiol. We propose that estrogen plays an integral role in regulating, and perhaps programming, primate fetal ovarian development.

VIP innervation: sharp contrast in fetal sheep and baboon adrenal glands suggests differences in developmental regulation.

Immunocytochemical technique and light microscopy were used to ascertain the relationship between vasoactive intestinal polypeptide (VIP) and tyrosine hydroxylase in fetal sheep and fetal baboon adrenal cortices and medullae at 85% of gestation. VIP immunostaining was extremely robust in fetal sheep adrenal cortical neurofibers and cells while weak in fibers and nonexistent in cells of fetal baboon. Also, tyrosine hydroxylase-immunopositive cells, present throughout the adrenal cortices of both fetal sheep and baboons, were heavily innervated by VIP-immunoreactive neurofibers in fetal sheep, but not in fetal baboons. Adrenal cortical VIP-immunopositive fibers occurred in greater (P<0.05) frequency in fetal sheep than in fetal baboons (14.82+/-3.10 vs. 0.84+/-0.26 fibers/field), were larger in diameter (2.93+/-0.34 vs. 0.93+/-0.07 microm) and ran for longer distances in the plane of section (127.85+/-5.16 vs. 74.53+/-4.93 microm). VIP immunogenicity in cells (ganglion and chromaffin) and fibers was robust in fetal adrenal medulla of sheep while nonexistent in baboons. VIP fibers in fetal sheep medulla were smaller in diameter compared to fetal sheep cortex (1.22+/-0.13 vs. 2.93+/-0.34 microm, P<0.05), but not compared to extrinsic nerve fibers (1.30+/-0.09 microm). We hypothesize that in fetal sheep of this age, medullary neurofibers derive primarily from extrinsic sources while cortical fibers arise from cortical ganglion cells. We conclude that at 85% of gestation the potential for VIP neural control of paracrine (e.g., glucocorticoid/catecholamine) interactions in both adrenal cortex and medulla is much greater in fetal sheep compared to fetal baboons.

Functional capacity of fetal zone cells of the baboon fetal adrenal gland: a major source of alpha-inhibin.

We have shown that ACTH receptor mRNA expression and steroidogenesis were increased in the transitional zone and decreased in the fetal zone of the baboon fetal adrenal in the second half of gestation. Thus, we proposed that there is a divergence in ACTH receptor-mediated zone-specific steroidogenesis within the fetal adrenal during mid to late gestation. We have also demonstrated that fetal serum alpha-inhibin levels decline with advancing development. It is possible, therefore, that the alpha subunit of inhibin provides a good marker of fetal zone cellular function and that the changes in circulating fetal alpha-inhibin with advancing pregnancy reflect ontogenetic changes in fetal adrenal cortical zone-specific cell function. However, it remains to be determined whether the fetal adrenal is a major source of circulating alpha-inhibin in the fetus and whether alpha-inhibin is expressed in the fetal, definitive, and/or transitional zones. Therefore, the current study compared fetal serum alpha-inhibin levels with immunocytochemical localization of alpha-inhibin in baboon fetal adrenals obtained on Days 60 (early), 100 (mid), and 165 or 182 (late) of gestation (term averages Day 184) from animals untreated or treated with betamethasone, which we previously demonstrated suppressed fetal pituitary ACTH and adrenal weight. Fetal serum alpha-inhibin levels (mean +/- SE) were greater (p < 0.05) at mid (5863 +/- 730 microliter eq/ml) than at late (3246 +/- 379) gestation and were reduced (p < 0. 05) by betamethasone. The inhibin alpha subunit was expressed in abundant quantities in the fetal adrenal cortex, but not in medulla, throughout gestation. At mid and late gestation, alpha-inhibin was expressed throughout the fetal adrenal cortex but most intensely in the innermost area of fetal zone cells. By late gestation, the fetal adrenal exhibited a gradient of alpha-inhibin expression. Thus, the outermost definitive zone cells were devoid of alpha-inhibin, the transitional zone exhibited a relatively low alpha-inhibin content, and fetal zone cells continued to exhibit extensive expression of alpha-inhibin. Betamethasone diminished the intensity of alpha-inhibin expression throughout the fetal adrenal cortex. These results indicate that the fetal adrenal fetal zone is a significant source of circulating alpha-inhibin in the baboon fetus and that alpha-inhibin provides a good marker to study the developmental regulation of fetal zone-specific adrenocortical function.

Differential regulation of baboon SP-A1 and SP-A2 genes: structural and functional analysis of 5'-flanking DNA.

Surfactant protein (SP) A gene transcription is developmentally regulated and stimulated by hormones and factors that increase intracellular cAMP. The baboon (b) genome contains two highly similar SP-A genes, bSP-A1 and bSP-A2. With the use of a ribonuclease protection assay with gene-specific probes, the two bSP-A genes were found to be differentially regulated during baboon fetal lung development in that expression of the bSP-A2 gene appeared to be induced to a high level at a later time in gestation than that of the bSP-A1 gene. Both the bSP-A1 and bSP-A2 genes were found to be highly responsive to the inductive effects of cAMP in baboon fetal lung explants in culture. By DNase I footprinting and electrophoretic mobility shift assays with bacterially expressed thyroid transcription factor-1 (TTF-1) and type II cell nuclear extracts, three TTF-1 binding elements were identified within the 255-bp region flanking the 5'-end of each bSP-A gene; however, these differed in position and spacing for the two bSP-A genes. To functionally define the genomic regions that are required for cAMP regulation of bSP-A gene expression in type II cells, fusion genes composed of various amounts of 5'-flanking DNA from the bSP-A1 and bSP-A2 genes linked to the human growth hormone structural gene as a reporter were transfected into type II cells in primary culture. We found that 255 bp of 5'-flanking DNA, which contain three TTF-1 binding elements, from bSP-A1 and bSP-A2 genes were sufficient to mediate high basal and cAMP-inducible expression in type II cells. We also observed that there were no obvious differences in the magnitude of the responses of these fusion genes to cAMP treatment.

Detection of thioredoxin in human serum and biological samples using a sensitive sandwich ELISA with digoxigenin-labeled antibody.

Thioredoxin is a low molecular weight, redox active protein important in cellular proliferation, signal transduction and antioxidant function. Thioredoxin is secreted by normal as well as neoplastic cells and is potentially involved in paracrine cell communication as suggested by its co-cytokine activity. Thus, the thioredoxin level in biological fluids, cells and tissue homogenates could be an important indicator of physiological or pathophysiological conditions. Hence, an accurate and sensitive measurement is of paramount importance in studies involving thioredoxin. We present here an ultrasensitive enzyme linked immuno-absorbent assay (ELISA) for human thioredoxin using digoxigenin-labelled goat polyclonal anti-human thioredoxin. The assay could detect a minimum level of 15 pg/ml thioredoxin in human serum, cell culture media, and in cell and tissue samples. The assay was optimized for concentration of both antibodies, blocking agent, plates, incubation time and reaction volumes. Excellent linearity and reproducibility were obtained. The assay was applied to different baboon tissues and human serum samples. The intrassay coefficient of variation (CV) was between 6.0 to 14 and the interassay CV was from 1.6 to 11.1. Excellent parallelism of standards with serum samples, tissue homogenates or cell lysates was obtained. More than 90% recovery of human thioredoxin was observed in 10% human serum. The assay is easy to use, rapid, reproducible, but above all it is a quantitative, specific and sensitive way to measure thioredoxin in a variety of biological specimens.

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.

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)

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)

Characterization of electroencephalographic state in fetal baboons.

Long-term recordings of the electroencephalograms (EEGs) from the non-human fetal primate have been visually scored for state. Data were obtained from three fetuses using recently developed techniques for chronic instrumentation of the pregnant baboon. Seven days or more after surgery, nine chart records of 4-5 h in duration were analyzed. These records were made during an interval in gestation from 143 to 153 days (term = 175 to 185 days). Criteria for differentiation of EEG state were based on the presence (state 1) or absence (state 2) of trace alternant, which is the predominant characteristic of EEG activity during quiet sleep in human preterm and term infants. Two patterns of EEG activity were readily distinguished at both standard (30 mm/s) and compressed (30 mm/min) EEG chart speeds. On a minute-by-minute basis, there was an overall concurrence of 82.9% for EEG state when scored from compressed and standard EEG records. From the compressed records, state 1 was present on average 38.3% of the time, with a mean epoch duration of 15.1 min, while epochs of state 2 averaged 25.9 min. We conclude that at least two EEG states are present at this gestation in the fetal baboon. These two states can be reliably defined visually by scoring methods directly comparable to those used for EEG recognition of sleep states in the human newborn infant. To validate the conclusion that these fetal state assignments based on patterns of EEG activity correspond to sleep states, it will be necessary to determine if the oscillations of other physiological parameters are coherent with these patterns.

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)

Mixed lymphocyte culture reactivity of fetal baboons: application for in utero bone marrow transplantation.

Bone marrow transplantation offers a potential cure for patients suffering from genetic diseases such as inborn errors of metabolism. The optimal time to transplant many of these affected individuals would be early in gestation. To date, little information is available on the cellular immune reactivity of fetal primate lymphocytes. Therefore, we tested peripheral blood lymphocytes obtained in utero from baboon fetuses (Papio sp.) for their ability to respond in mixed lymphocyte culture (MLC) against their mothers, against a pool of unrelated animals, and in the case of fetuses given unrelated bone marrow transplants in utero, against their specific bone marrow donors. The majority of fetuses as young as 80 gestational days (182-day normal gestation period) were capable of responding strongly to maternal and unrelated lymphocytes in MLC. Of six fetuses that were transplanted, three did not engraft as indicated by undetectable levels of the donor-specific type B allele of glucose phosphate isomerase in fetal blood samples 1 month post-transplant. The three fetuses that did engraft all lost their grafts before birth. These data demonstrate that fetal lymphocytes obtained in utero can be tested for MLC reactivity and suggest that MLC testing can be used to select appropriate donor-recipient combinations for in utero bone marrow transplantation.

Alpha-myosin heavy chain cDNA structure and gene expression in adult, fetal, and premature baboon myocardium.

To examine cardiac myosin gene structure and expression in a non-human primate model for human heart development and disease, we have constructed a cDNA library from baboon atrium and used baboon beta-myosin heavy chain (beta-MHC)* cDNA probes to isolate atrial MHC clones. The nucleotide sequence of one such clone, lambda BMHC alpha 3, contains sequences that encode part of the light meromyosin region (LMM) and the 3' untranslated region of the baboon alpha-MHC. To study cardiac MHC gene transcription, we constructed probes from the baboon alpha-MHC cDNA for S1 nuclease analyses of RNA from atria and ventricles. To examine translational regulation of cardiac MHC gene expression, we used monoclonal antibodies (MAb) against specific alpha- and beta-MHC epitopes for Western blot analyses. In atria and ventricles from adult baboons, we detected predominantly alpha- and beta-MHC gene transcripts, respectively. In ventricles from fetal baboons at two stages of development (140 and 160 days gestation), we also detected predominantly beta-MHC gene transcripts and isoforms. To investigate changes induced by parturition, we obtained ventricles from baboons that were prematurely delivered at 140 days gestation and supported for 10 days in an extrauterine environment. In contrast to adult and fetal patterns, we observed an increase in alpha-MHC transcripts and isoforms in ventricles of premature baboons. Because alpha-MHC gene expression is increased in premature baboons (total age of 150 days) compared to their older 160 day fetal counterparts, the induction of ventricular alpha-MHC synthesis must have resulted from factor(s) associated with parturition or prolonged mechanical ventilation rather than at predetermined stages of gestational development.

Real-time ultrasonic evaluation of the baboon pregnancy: biometric measurements.

Ultrasonic and direct biometric measurements of nine fetal baboons were made during 23 evaluations. Both the femur length and the biparietal diameter were correlated to gestational age. These initial results have facilitated intrauterine surgery. The data presented will help in determining adequate fetal growth and in dating the baboon pregnancy.