Microarray analysis of BeWo and JEG3 trophoblast cell lines: identification of differentially expressed transcripts.

Trophoblast cell lines are important research tools used as a surrogate for primary trophoblast cells in the study of placental function. Because the cellular origins of transformed trophoblasts are likely to be diverse, it would be of value to understand the unique and shared phenotypes of the cells on a global scale. We have compared two widely used cell lines, BeWo and JEG3, by microarray analysis in order to identify differentially expressed genes. Results indicated that approximately 2700 genes were differentially expressed between the cell lines, with principal differences observed in the biological processes of response to stress, cell adhesion, signal transduction, and protein and nucleobase metabolisms. These data suggest that BeWo and JEG3 cell lines, and perhaps other trophoblast cell lines, are sufficiently dissimilar from each other such that they will be differentially suited for specific experimental paradigms.

Suppression of extravillous trophoblast invasion of uterine spiral arteries by estrogen during early baboon pregnancy.

The present study determined whether estrogen plays a role in regulating invasion and remodeling of the uterine spiral arteries by extravillous trophoblasts during early baboon pregnancy. The level of trophoblast invasion of spiral arteries was assessed on day 60 of gestation (term is 184 days) in baboons untreated or treated on days 25-59 with estradiol or aromatizable androstenedione. The administration of estradiol or androstenedione increased (P<0.01) maternal serum estradiol levels approximately 3-fold above normal. The mean+/-SE percentage of spiral arteries/arterioles invaded by extravillous cytotrophoblasts in estradiol-treated baboons for vessels with diameters of 26-50 microm (0.0+/-0.0), 51-100 microm (1.2+/-0.7) and >100 microm (13.2+/-5.5) was 100%, 90%, and 75% lower (P<0.001), respectively, than in untreated baboons (2.4+/-1.2%; 11.0+/-5.5%, and 54.5+/-8.5%, respectively). Similar results were obtained with androstenedione treatment. However, the distribution of uterine spiral arteries grouped by diameter or number of arteries per basal plate area, i.e. microvessel density, were similar in untreated and estrogen-treated baboons. We suggest, therefore, that the low levels of estrogen exhibited during early primate pregnancy are required to permit normal progression of trophoblast vascular invasion and that the surge in estrogen which occurs during the second-third of normal pregnancy has a physiological role in suppressing further arterial trophoblast invasion. Consequently, we propose that the estrogen-dependent restraint of spiral artery invasion/remodeling ensures optimal blood flow dynamics across the uteroplacental vascular bed to promote normal fetal growth and development.

Influence of maternal diabetes on placental fibroblast growth factor-2 expression, proliferation, and apoptosis.

OBJECTIVE: Type I diabetes mellitus during pregnancy is associated with dysregulation of the oxygen and glucose metabolic pathways, both of which affect placental villous growth and function. Alteration of placental development in women with diabetes may contribute to the increased risk of preeclampsia, macrosomia, or fetal growth restriction. METHODS: To evaluate placental growth in the setting of maternal diabetes, immunohistochemical techniques were used to examine fibroblast growth factor-2 (FGF-2) expression, cell proliferation (Ki67), and apoptosis (Apo-Tag) in placentas from diabetic and nondiabetic patients. RESULTS: Immunostaining for FGF-2 in placentas from diabetic women demonstrated an increase in intensity within the villous stroma and syncytiotrophoblast (P<.05). Associated with these changes in FGF-2 expression, placentas from diabetic women showed no change in villous mitotic activity but did show decreased stromal compartment apoptosis. When expressed as a ratio of Ki67-positive:Apo-Tag-positive nuclei as an index of relative cell turnover, the stromal compartment showed a significant trend towards decreased nuclei turnover (P<.05), suggesting relative tissue growth in diabetic patients. CONCLUSION: Increased FGF-2 expression and decreased stromal cell compartment turnover in the diabetic placenta might be a compensatory mechanism in response to the altered physiologic milieu of maternal diabetes on placental function.

Developmental regulation of morphological differentiation of placental villous trophoblast in the baboon.

The present study determined whether morphological differentiation of placental villous cytotrophoblasts into syncytiotrophoblast during primate pregnancy was developmentally regulated and whether oestrogen has a role in this process. Placental volumetric composition of the cytotrophoblast and syncytiotrophoblast was determined by the test-point counting method on days 45-54, 60, 100, and 170 of gestation (term=184 days) in untreated baboons, on day 60 after placental oestrogen production was prematurely elevated by administration of aromatizable androstenedione or oestradiol, and on day 170 after oestrogen production was suppressed by administration of aromatase inhibitor CGS 20267. Cytotrophoblast and syncytiotrophoblast volumes and oestrogen levels increased (P< 0.01) with advancing gestation. Due to the rise in syncytiotrophoblast volume (12-fold) exceeded that of the cytotrophoblast (threefold), the mean (sem) ratio of syncytiotrophoblast and cytotrophoblast volumes increased (P< 0.001) from 3.4 (0.5) ml on day 60 to 12.1 (2.8) ml on day 170. Androstenedione administration elevated serum oestradiol levels threefold (P< 0.01) and increased the ratio of syncytiotrophoblast: cytotrophoblast volumes on day 60 by 50 per cent (P< 0.03) to that normally observed on day 100. However, the ratio of trophoblast volumes was unaltered in oestradiol-treated and CGS 20267-treated baboons. It is concluded that there is a developmental increase in morphological differentiation of the placental villous trophoblast during primate pregnancy and that androstenedione potentially via its conversion to oestrogen has a role in this process.

Screening for antiproliferative actions of mifepristone. Differential endometrial responses of primates versus rats.

This laboratory has previously shown the capability of the antiprogestin, mifepristone, to noncompetitively inhibit estrogen-induced endometrial proliferation in nonhuman primates. In the following study, use of the rat uterine weight bioassay was compared against a primate (Macaca fascicularis) uterine bioassay to identify the noncompetitive/antiproliferative effects of mifepristone. These uterine bioassays were contrasted for reasons of identifying a comparative laboratory rodent model that could substitute for the need to use primate models in the screening of potential antiprogestins, thereby saving time, cost, and primate resources. Results of the primate experiment showed that mifepristone decreased endometrial proliferation in a dose-dependent manner; importantly, this decrease occurred in the presence of sustained physiologic serum 17 beta-estradiol (E2) levels. However, in the rat model, results showed that mifepristone altered uterine wet weight and blotted weight values only in those animals receiving pharmacological doses of E2 (p < 0.05). Based on the results summarized herein, use of this rat uterine weight bioassay as a substitute for primate models is not recommended for screening and identification of "interesting" antiprogestins. Apparently, the endometrial noncompetitive antiestrogenic/antiproliferative effects of mifepristone, observed repeatedly in these laboratory primates, do not operate in the rat uterine tissue.

Changes in concentrations of hormones, metabolites, and amino acids in plasma of adult horses relative to overnight feed deprivation followed by a pellet-hay meal fed at noon.

Experiment 1 was conducted to characterize the concentrations of prolactin, growth hormone (GH), cortisol, insulin, glucagon, glucose, nonesterified fatty acids (NEFA), urea N, and 10 indispensable amino acids in the plasma of mares (n = 8) and stallions (n = 8) during the last 4 h of a 19-h period of feed deprivation and for 8 h after a noon meal. Experiment 2 was similar to Exp. 1 except that only stallions (n = 8) were used, and they were either fed (n = 4) or not fed (n = 4) at noon in a 2 x 2 Latin square design conducted over two sampling days 7 d apart. In Exp. 1, increases (P < .01) after feeding were observed for plasma concentrations of prolactin, cortisol, insulin, glucagon, glucose, urea N, and all amino acids except methionine; NEFA concentrations decreased (P < .01) after feeding. Episodic increases in GH concentrations were observed for most horses but were not associated with either feeding or gender (P > .1). Plasma urea N concentrations were higher (P < .025) overall in stallions than in mares, and the rise in prolactin concentrations after feeding was greater (P < .01) in stallions than in mares. In Exp. 2, meal-associated increases (P < .01) were observed for plasma concentrations of prolactin, insulin, glucagon, and glucose; NEFA concentrations decreased (P < .01). Except for cortisol, no hormone or metabolite varied with time across days when the stallions were not fed (P > .1), indicating that there was no inherent diurnal or feeding schedule-associated fluctuations in their concentrations. Cortisol concentrations varied (P < .02) over time but did not differ (P > .1) between fed and nonfed stallions. Again, GH concentrations were episodic but did not differ (P > .1) between fed and nonfed stallions. The lack of feeding effects on GH secretion in horses is similar to the response in pigs but differs from that in ruminants, in which GH concentrations generally decline after feeding.

Growth hormone in mares and stallions: pulsatile secretion, response to growth hormone-releasing hormone, and effects of exercise, sexual stimulation, and pharmacological agents.

Short-term patterns of growth hormone (GH) secretion and factors affecting it were studied in mares and stallions. In Exp. 1, hourly blood samples were collected from three mares and three stallions in summer and winter. Although GH concentrations varied in a pulsatile manner in all horses, there was no effect of sex or season (P greater than .1) on plasma GH concentrations and no indication of a diurnal pattern of GH secretion. In Exp. 2, 10-min blood samples were drawn for 8 h from 12 mares; after 6 h, porcine GH-releasing hormone (GHRH) was administered i.v. at 0, 45, 90, or 180 micrograms/mare (three mares per dose). Pulsatile secretion of GH occurred in all mares and averaged 2.4 +/- .3 peaks/6 h; amplitudes were variable and ranged from 2.6 to 74.4 ng/mL. Eight of nine mares responded within 20 min to GHRH injection, but there was no difference (P greater than .1) among the three doses tested. In Exp. 3, plasma GH concentrations in stallions increased (P less than .05) 8- to 10-fold after 5 min of acute physical exercise or exposure to an estrual mare. Restraint via a twitch (5 min) and epinephrine administration (3 mg i.v.) also increased (P less than .05) plasma GH concentrations by approximately fourfold. In Exp. 4 and 5, administration of either .4, 2, or 10 mg of thyrotropin-releasing hormone (TRH) or 100 or 500 mg of sulpiride (a dopamine receptor antagonist) increased (P less than .01) plasma prolactin concentrations but had no effect (P greater than .1) on GH concentrations during the same period of time.(ABSTRACT TRUNCATED AT 250 WORDS)