Gestational age‐dependent gene expression profiling of ATP‐binding cassette transporters in the healthy human placenta

The ATP‐binding cassette (ABC) transporters control placental transfer of several nutrients, steroids, immunological factors, chemicals, and drugs at the maternal‐fetal interface. We and others have demonstrated a gestational age‐dependent expression pattern of two ABC transporters, P‐glycoprotein and breast cancer resistance protein throughout pregnancy. However, no reports have comprehensively elucidated the expression pattern of all 50 ABC proteins, comparing first trimester and term human placentae. We hypothesized that placental ABC transporters are expressed in a gestational‐age dependent manner in normal human pregnancy. Using the TaqMan^® Human ABC Transporter Array, we assessed the mRNA expression of all 50 ABC transporters in first (first trimester, n = 8) and third trimester (term, n = 12) human placentae and validated the resulting expression of selected ABC transporters using qPCR, Western blot and immunohistochemistry. A distinct gene expression profile of 30 ABC transporters was observed comparing first trimester vs. term placentae. Using individual qPCR in selected genes, we validated the increased expression of ABCA1 (P < 0.01), ABCA6 (P < 0.001), ABCA9 (P < 0.001) and ABCC3 (P < 0.001), as well as the decreased expression of ABCB11 (P < 0.001) and ABCG4 (P < 0.01) with advancing gestation. One important lipid transporter, ABCA6, was selected to correlate protein abundance and characterize tissue localization. ABCA6 exhibited increased protein expression towards term and was predominantly localized to syncytiotrophoblast cells. In conclusion, expression patterns of placental ABC transporters change as a function of gestational age. These changes are likely fundamental to a healthy pregnancy given the critical role that these transporters play in the regulation of steroidogenesis, immunological responses, and placental barrier function and integrity.     

Curcumin and cyclopalladated complexes: a recipe for bifunctional biomaterials

The first examples of binuclear and mononuclear ortho-palladated complexes based on a functionalized 2-phenylquinoline ligand have been synthesized and fully characterized. Conjugating cyclopalladated fragments to curcumin family biologically active beta-diketones gives in one single molecule two different functionalities. The structural variations based on the curcuminoid structure have been tested for their in vitro cytotoxic activity. The activity of complexes comprised of a cyclopalladated fragment conjugated to functionalized bioactive ligands, represents the potential of organometallic systems in generating new bifunctional biomaterials.     

Effect of substrate stiffness on early mouse embryo development

It is becoming increasingly clear that cells are remarkably sensitive to the biophysical cues of their microenvironment and that these cues play a significant role in influencing their behaviors. In this study, we investigated whether the early pre-implantation embryo is sensitive to mechanical cues, i.e. the elasticity of the culture environment. To test this, we have developed a new embryo culture system where the mechanical properties of the embryonic environment can be precisely defined. The contemporary standard environment for embryo culture is the polystyrene petri dish (PD), which has a stiffness (1 GPa) that is six orders of magnitude greater than the uterine epithelium (1 kPa). To approximate more closely the mechanical aspects of the in vivo uterine environment we used polydimethyl-siloxane (PDMS) or fabricated 3D type I collagen gels (1 kPa stiffness, Col-1k group). Mouse embryo development on alternate substrates was compared to that seen on the petri dish; percent development, hatching frequency, and cell number were observed. Our results indicated that embryos are sensitive to the mechanical environment on which they are cultured. Embryos cultured on Col-1k showed a significantly greater frequency of development to 2-cell (68±15% vs. 59±18%), blastocyst (64±9.1% vs. 50±18%) and hatching blastocyst stages (54±25% vs. 21±16%) and an increase in the number of trophectodermal cell (TE,65±13 vs. 49±12 cells) compared to control embryos cultured in PD (mean±S.D.; p<.01). Embryos cultured on Col-1k and PD were transferred to recipient females and observed on embryonic day 12.5. Both groups had the same number of fetuses, however the placentas of the Col-1k fetuses were larger than controls, suggesting a continued effect of the preimplantation environment. In summary, characteristics of the preimplantation microenvironment affect pre- and post-implantation growth.     

Glucocorticoids modulate multidrug resistance transporters in the first trimester human placenta

The placental multidrug transporters, P‐glycoprotein (P‐gp, encoded by ABCB1) and breast cancer resistance protein (BCRP, ABCG2) protect the foetus from exposure to maternally derived glucocorticoids, toxins and xenobiotics. During pregnancy, maternal glucocorticoid levels can be elevated by stress or exogenous administration. We hypothesized that glucocorticoids modulate the expression of ABCB1/P‐gp and ABCG2/BCRP in the first trimester human placenta. Our objective was to examine whether dexamethasone (DEX) or cortisol modulate first trimester placental expression of multidrug transporters and determine whether cytotrophoblasts or the syncytiotrophoblast are/is responsible for mediating these effects. Three models were examined: (i) an ex‐vivo model of placental villous explants (7‐10 weeks), (ii) a model of isolated first trimester syncytiotrophoblast and cytotrophoblast cells and (iii) the BeWo immortalized trophoblast cell line model. These cells/tissues were treated with DEX or cortisol for 24 hour to 72 hour. In first trimester placental explants, DEX (48 hour) increased ABCB1 (P < .001) and ABCG2 (P < .05) mRNA levels, whereas cortisol (48 hour) only increased ABCB1 mRNA levels (P < .01). Dexamethasone (P < .05) and cortisol (P < .01) increased BCRP but did not affect P‐gp protein levels. Breast cancer resistance protein expression was primarily confined to syncytiotrophoblasts. BeWo cells, when syncytialized with forskolin, increased expression of BCRP protein, and this was further augmented by DEX (P < .05). Our data suggest that the protective barrier provided by BCRP increases as cytotrophoblasts fuse to form the syncytiotrophoblast. Increase in glucocorticoid levels during the first trimester may reduce embryo/foetal exposure to clinically relevant BCRP substrates, because of an increase in placental BCRP.     

Effect of zinc administration on thyrotropin releasing hormone-stimulated prolactinemia in healthy men

Previous in vitro studies have demonstrated zinc (Zn⁺⁺) inhibition of basal and of potassium (K⁺) or thyrotropin-releasing hormone (TRH)-stimulated prolactin (PRL) secretion, in a selective, reversible, and dose-dependent manner. Thus, Zn⁺⁺ may regulate physiologically pituitary PRL secretion. Furthermore, studies with patients with uremia, cirrhosis or prolactinoma, have shown the coexistence of hypozincemia and hyperprolactinemia and zinc supplementation did not correct hyperprolactinemia in these patients. In normal individuals Zn⁺⁺ administration produced controversial results on PRL secretion. Here, we investigated whether zinc administration affects TRH-stimulated PRL in healthy men. We found that Zn⁺⁺ administration does not change the TRH-stimulated PRL. Therefore, in normal conditions, Zn⁺⁺ does not inhibit TRH-stimulated prolactinemia. In addition, we found that acute increases of blood PRL and TRH do not alter blood Zn⁺⁺ levels.     

Male pseudohermaphroditism due to nonsalt-losing 3 beta-hydroxysteroid dehydrogenase deficiency: gender role change and absence of gynecomastia at puberty

Adrenal and gonadal functions were evaluated on two adult cousins with male pseudohermaphroditism due to congenital 3 beta-hydroxysteroid dehydrogenase deficiency (3 beta-HSD) without clinical salt-losing. Both patients had been reared as females since birth. Case 1 presented at age 17 with perineal hypospadias virilization without gynecomastia and a female to male gender role change at puberty. Case 2 had previously undergone bilateral orchidectomy in childhood and presented "primary amenorrhea", absence of virilization and a female gender role at the age of 24. In the basal state, as well as after ACTH and hCG stimulation, 3 beta-hydroxy-5-ene-steroid levels were disproportionately elevated, resulting in abnormal 3 beta-hydroxy-5-ene: 3-oxi-4-ene steroids ratios. Normal basal serum cortisol with inadequate cortisol response to ACTH was observed in both patients. Elevated basal plasma renin activity (PRA) and normal basal serum aldosterone (ALDO) were present in both subjects. After ACTH stimulation serum ALDO rose adequately in Case 1 but subnormally in Case 2. Salt restriction resulted in an increase in serum ALDO and no salt loss in Case 1 whereas in Case 2 the substantial rise in PRA and serum ALDO were unable to prevent slight urinary sodium loss. Case 1 had normal basal serum testosterone with subnormal response to hCG stimulation. Incubation of testicular tissue in vitro with [3H]DHEA resulted in large Androstenediol production but diminished testosterone conversion confirming the 3 beta-HSD deficiency in the testes. We conclude that (1) absence of gynecomastia and a female to male gender role change may be observed in the male pubertal presentation of nonsalt-losing 3 beta-HSD deficiency and (2) the different functional behavior of zona glomerulosa in our patients suggests the presence of variable degrees of 3 beta-HSD deficiency in the zona glomerulosa of the nonsalt-losing form.     

Low frequency of CYP2B deletions in Brazilian patients with congenital adrenal hyperplasia due to 21-hydroxylas deficiency

The frequency of large mutations was determined in 131 Brazilian patients with different clinical forms of 21-hydroxylase deficiency, belonging to 116 families. DNA samples were examined by Southern blotting hybridization with genomic CYP21 and C4cDNA probes after Taql and Bg/II restriction. Large gene conversions were found in 6.6% and CYP21B deletions in 4.4% of the alleles. The breakpoint in these hybrid genes occurred after exon 3 in 92% of the alleles. All rearrangements involving CYP21B gene occurred in the heterozygous form, except in a patient with simple virilizing form who presented homozygous CYP21B deletion. Our data showed that in these Brazilian patients, CYP21B deletions were less frequent than in most of the large series previously reported.     

Zinc supplementation does not inhibit basal and metoclopramide-stimulated prolactinemia secretion in healthy men.

Dopamine (DA) and zinc (Zn++) share common mechanisms in their inhibition of prolactin (PRL) secretion. Both substances are present in the same brain areas, where Zn++ is released together with DA, suggesting a modulatory effect of Zn++ on dopaminergic receptors. The aim of the present study was to evaluate the effect of Zn++ supplementation on basal and PRL secretion stimulated by metoclopramide (MCP), a dopaminergic antagonist. Seven healthy men were evaluated in controlled study, where MCP (5 mg) was given intravenously, before and after 3 months of oral Zn++ (25 mg) administration. Our results indicate that chronic Zn++ administration does not change basal or MCP-stimulated plasma PRL secretion suggesting that, in humans, Zn++ does not interfere on PRL secretion mediated through dopaminergic receptors.