Expression and localization of alpha-fetoprotein mRNA and protein in human early villous trophoblasts.

Alpha-fetoprotein (AFP) is a major plasma protein produced during human fetal life. It is a good marker for several possible disorders affecting gestation. We previously reported that afp gene expression, which takes place mainly in yolk sac and fetal liver, also occurs in normal human placenta, specifically in early pregnancy. The aim of the present study was to determine the precise location of AFP synthesis sites within the placental villi. In situ hybridization and immunohistochemical experiments were performed on sections obtained from placentas of first-trimester and full-term pregnancies. We found that the pattern of afp gene expression was restricted to specific villous trophoblastic areas in early placentas. Both afp transcripts and AFP protein were mainly located in discontinuous regions, at junctions between two villi and at budding sites. In contrast, no AFP expression was detected in the cytotrophoblastic extravillous proliferative zone or in other placental cell types. According to the earlier studies, no AFP synthesis was detected in placental villous tissue from full-term pregnancies, using in situ hybridization and immunohistochemistry.

Human c-Myc isoforms differentially regulate cell growth and apoptosis in Drosophila melanogaster.

The human c-myc proto-oncogene, implicated in the control of many cellular processes including cell growth and apoptosis, encodes three isoforms which differ in their N-terminal region. The functions of these isoforms have never been addressed in vivo. Here, we used Drosophila melanogaster to examine their functions in a fully integrated system. First, we established that the human c-Myc protein can rescue lethal mutations of the Drosophila myc ortholog, dmyc, demonstrating the biological relevance of this model. Then, we characterized a new lethal dmyc insertion allele, which permits expression of human c-Myc in place of dMyc and used it to compare physiological activities of these isoforms in whole-organism rescue, transcription, cell growth, and apoptosis. These isoforms differ both quantitatively and qualitatively. Most remarkably, while the small c-MycS form truncated for much of its N-terminal trans-activation domain efficiently rescued viability and cell growth, it did not induce detectable programmed cell death. Our data indicate that the main functional difference between c-Myc isoforms resides in their apoptotic properties and that the N-terminal region, containing the conserved MbI motif, is decisive in governing the choice between growth and death.

Alpha-fetoprotein gene expression in early and full-term human trophoblast.

Alpha-fetoprotein (AFP) is a major serum glycoprotein synthesized during fetal life mainly by the yolk sac and the fetal liver. At term, it reaches high concentrations in the maternal intervillous blood, which is in direct contact with the placental trophoblastic microvillous membrane, and this suggests the placental origin of the AFP at the fetal-maternal interface. We used several experimental approaches to investigate the expression of AFP gene and fetal protein production in early gestation and term placentas. RT-PCR and immunological studies clearly identified AFP messenger RNA and AFP protein in the placental villi from first trimester of pregnancy. The AFP gene was also expressed in highly purified cytotrophoblasts from early placentas, and enzymo-immunoassay showed that AFP protein was synthesized and secreted by early cytotrophoblasts. AFP was also detected in the cytoplasm of these cells by immuno-cytochemistry. However, none of these methods detected any expression of the AFP gene in full-term placental villi or in cultured trophoblasts. These findings demonstrate that both AFP mRNA and protein are present in trophoblastic cells early in pregnancy. The absence of AFP gene expression in term placental villi also suggests, that the AFP at the fetal-maternal interface is attributable to a notable transplacental passage of AFP from fetal blood in late pregnancy.

Phytoestrogens as modulators of steroid action in target cells.

Although numerous reports exist on the potential beneficial role of nutritional phytoestrogens in human health, their molecular mechanism in target cells is still not completely understood. Phytoestrogens promote estrogen and antiestrogen effects by interacting with numerous molecules, carrier proteins, enzymes and membrane and nuclear receptors, directly or indirectly involved in the transfer of estrogen signals. The hypothesis that the ER beta subtype plays a key role in antiproliferative effect of phytoestrogens, especially in breast cancer, is examined here. This review focus on the effects of phytoestrogens in developmental processes such as those linked to reproductive function, tumorigenesis and angiogenesis.

Molecular basis for the inhibition of Drosophila eye development by Antennapedia.

Hox genes encoding homeodomain transcriptional regulators are known to specify the body plan of multicellular organisms and are able to induce body plan transformations when misexpressed. These findings led to the hypothesis that duplication events and misexpression of Hox genes during evolution have been necessary for generating the observed morphological diversity found in metazoans. It is known that overexpressing Antennapedia (Antp) in the head induces antenna-to-leg as well as head-to-thorax transformation and eye reduction. At present, little is known about the exact molecular mechanism causing these phenotypes. The aim of this study is to understand the basis of inhibition of eye development. We demonstrate that Antp represses the activity of the eye regulatory cascade. By ectopic expression, we show that Antp antagonizes the activity of the eye selector gene eyeless. Using both in vitro and in vivo experiments, we demonstrate that this inhibitory mechanism involves direct protein-protein interactions between the DNA-binding domains of EY and ANTP, resulting in mutual inhibition.

Corticosteroid-binding globulin status at the fetomaternal interface during human term pregnancy.

The status of the corticosteroid-binding globulin (CBG) at the fetomaternal interface, especially in the maternal intervillous blood space (I), was investigated and compared to that of CBG in the maternal (M) and fetal (umbilical arteries [A] and vein [V]) peripheral circulations at term. Immunoquantitation of plasma CBG showed that the CBG concentration in I was 30% less than that in M (P < 0.001) and threefold higher than that in umbilical cord blood (P < 0.001). The microheterogeneity of CBG studied by immunoaffinoelectrophoresis in the presence of concanavalin A and Western blotting indicated that the CBG in I was mainly of maternal origin and different from fetal CBG. A CBG mRNA, but no classic 50- to 59-kDa CBG, was found in isolated term trophoblastic cells. The steroid environment of the CBG in I differed greatly from that in the peripheral maternal and fetal circulations, because the progesterone:cortisol molar ratio in I was 75-fold higher than that in M and 7- to 10-fold higher than that in the fetal circulation. Binding studies revealed that the affinity constants of CBG for cortisol in I, A, and V were significantly lower than that in M plasma (P < 0.02) in their respective hormonal contexts. The binding parameters for I-CBG stripped of endogenous steroids and lipids were close to those for M-CBG but different from those of fetal CBG (P < 0.001). These data reflect the physiological relevance of the CBG-steroid interaction, especially with very CBG-loaded progesterone at the fetomaternal interface during late pregnancy.

Drosophila Pax-6/eyeless is essential for normal adult brain structure and function.

A role for the Pax-6 homologue eyeless in adult Drosophila brain development and function is described. eyeless expression is detected in neurons, but not glial cells, of the mushroom bodies, the medullar cortex, the lateral horn, and the pars intercerebralis. Furthermore, severe defects in adult brain structures essential for vision, olfaction, and for the coordination of locomotion are provoked by two newly isolated mutations of Pax-6/eyeless that result in truncated proteins. Consistent with the morphological lesions, we observe defective walking behavior for these eyeless mutants. The implications of these data for understanding postembryonic brain development and function in Drosophila are discussed.

Evidence for progesterone receptors in the human fetoplacental vascular tree.

The presence of progesterone receptors (PR) throughout the human term fetoplacental vascular tree was investigated. By reverse transcription-polymerase chain reaction (RT-PCR), we showed expression of PR mRNAs in stem villi vessels, chorionic arteries and veins, and umbilical arteries and veins. Binding studies and Scatchard analysis revealed a single class of high-affinity binding sites for (3)H-R5020 (promegestone) in cytosolic extracts of all placental vessels, with K(d) values in the range of 2.5-4 nM. High levels of PR were detected in placental vessels compared to other vascular tissues. Thus, maximum binding capacities of stem villi vessels, chorionic arteries and veins, and umbilical arteries and veins were 247 +/- 25, 377 +/- 58, 295 +/- 40, 371 +/- 118, and 672 +/- 144 fmol/mg protein, respectively. Endothelial cell elimination in chorionic arteries did not significantly modify the number of PR. RT-PCR and binding studies also assessed PR expression in cultured placental vascular smooth muscle cells isolated from stem villi vessels. All these data suggested that most of the PR of fetoplacental vessels were from the media. In conclusion, we report here the first evidence of the presence of PR in the muscular layer of human term fetoplacental vessels. This finding, together with the high progesterone concentrations in cord blood, suggests that the interactions between the PR and its ligand may play a role in the physiology and physiopathology of human fetoplacental vascularization.

Does high polyunsaturated free fatty acid level at the feto-maternal interface alter steroid hormone message during pregnancy?

Polyunsaturated fatty acids (PUFA) are important in pregnancy, fetal development and parturition. We measured free fatty acids (FFA), albumin and alpha-fetoprotein (AFP) in the maternal and fetal circulations of women undergoing elective Caesarean section at term. We also studied the impact of PUFAs on estrogen (ER) and progesterone receptors (PR) binding properties in vitro in the myometria of pregnant women and ex vivo in human myometrial cells in culture. FFA in intervillous blood (I) (feto-maternal interface) and maternal peripheral blood (M) were similar, while those in the umbilical vein (V) and arteries (A) were 2-4 fold lower (P<0.001). PUFA levels were low in M and 3 fold higher in I, A and V (P< 0.001); consequently C20:4 and C22:6 were most abundant in intervillous space. Albumin was uniformly distributed throughout the maternal-fetal unit, but there was a transplacental gradient in AFP. The AFP in the intervillous space had a special conformation (less immuno-reactive, more anionic), suggesting loading with PUFA. Physiological concentrations of C20:4 stimulated estradiol binding, but inhibited progestin binding. C20:4 inhibited progesterone binding by decreasing the number of binding sites, with no change in apparent affinity, in vitro in myometrial tissue and ex vivo in myometrial cells. Thus PUFA may modulate the steroid hormone message, so that the high C20:4 concentration at the maternal-fetal interface at term may help amplify the estrogen signal and inhibit the progesterone signal.

Estrogen receptors (ERalpha/ERbeta) in normal and pathological growth of the human myometrium: pregnancy and leiomyoma.

The distributions of the mRNAs for estrogen receptors (ERalpha and ERbeta) and their binding properties in myometria of pregnant and nonpregnant women and in leiomyoma were studied. RT-PCR analysis indicated that the term pregnancy myometria had little ERalpha mRNA, whereas the amounts of ERbeta mRNAs in pregnant or nonpregnant myometria appeared to be similar. Both ERalpha and ERbeta mRNA were greater in certain leiomyoma than in normal nonpregnant myometria. The binding kinetics revealed that two specific binding sites (with high or low affinity) for 17beta-estradiol were present in the nonpregnant myometrium. Only the low-affinity binding sites were detectable in late-pregnancy myometria and in leiomyoma, and their capacities were increased two- to threefold (P < 0.001) in leiomyoma. The pregnancy- and leiomyoma-related changes in myometrial ER status, especially the low concentration of ERalpha mRNA and the lack of high-affinity ER in pregnant women, plus the increased ERalpha and ERbeta mRNAs and the increased low-affinity ER in some leiomyoma, suggest that the redistribution of ER subtypes is associated with the pathological and/or normal growth of the myometrium.

Point mutations within and outside the homeodomain identify sequences required for proboscipedia homeotic function in Drosophila.

The Drosophila homeotic gene proboscipedia (pb) encodes a homeodomain protein homologous to vertebrate HoxA2/B2 required for adult mouthparts formation. A transgenic Hsp70-pb (HSPB) element that rescues pb mutations also induces the dominant transformation of antennae to maxillary palps. To identify sequences essential to PB protein function, we screened for EMS-induced HSPB mutations leading to phenotypic reversion of the HSPB transformation. Ten revertants harbor identified point mutations in HSPB coding sequences. The point mutations that remove all detectable phenotypes in vivo reside either within the homeodomain or, more unexpectedly, in evolutionarily nonconserved regions outside the homeodomain. Two independent homeodomain mutations that change the highly conserved Arginine-5 in the N-terminal hinge show effects on adult eye development, suggesting a previously unsuspected role for Arg5 in functional specificity. Three additional revertant mutations outside the homeodomain reduce but do not abolish PB+ activity, identifying protein elements that contribute quantitatively to pb function. This in vivo analysis shows that apart from the conserved motifs of PB, other elements throughout the protein make important contributions to homeotic function.

Ras1-mediated modulation of Drosophila homeotic function in cell and segment identity.

Mutations of the Drosophila homeotic proboscipedia gene (pb, the Hox-A2/B2 homologue) provoke dose-sensitive defects. These were used to search for dose-sensitive dominant modifiers of pb function. Two identified interacting genes were the proto-oncogene Ras1 and its functional antagonist Gap1, prominent intermediaries in known signal transduction pathways. Ras1+ is a positive modifier of pb activity both in normal and ectopic cell contexts, while the Ras1-antagonist Gap1 has an opposite effect. A general role for Ras1 in homeotic function is likely, since Ras1+ activity also modulates functions of the homeotic loci Sex combs reduced and Ultrabithorax. Our data suggest that the modulation occurs by a mechanism independent of transcriptional control of the homeotic loci themselves, or of the Ras1/Gap1 genes. Taken together our data support a role for Ras1-mediated cell signaling in the homeotic control of segmental differentiation.

A homeodomain point mutation of the Drosophila proboscipedia protein provokes eye loss independently of homeotic function.

The Drosophila homeotic gene proboscipedia (pb: HoxA2/B2 homolog) is required for adult mouthparts development. Ectopic PB protein expression from a transgenic Hsp70-pb minigene (HSPB) results in transformation of adult antennae to maxillary palps. In contrast, most tissues appear refractory to PB-induced effects. To study the basis of homeotic tissue specificity we are isolating and studying mutations that modify dominant HSPB-induced phenotypes. One HSPB point mutation (Arg5 of the homeodomain to His) removes homeotic activity in the mouthparts and antennae, but provokes a dose-sensitive eye loss. We show that eye loss can be induced by PB proteins that no longer effectively bind to DNA. The dose-sensitive eye loss thus appears to be mediated by specific, context-dependent protein-protein interactions.

High polyunsaturated fatty acid, thromboxane A2, and alpha-fetoprotein concentrations at the human feto-maternal interface.

Polyunsaturated fatty acids (PUFA) like arachidonic (C20:4) and docosahexaenoic (C22:6) acids are essential for harmonious fetal development. This study evaluates, at near term, the distributions of free fatty acids (FFA) and their fetal carrier protein, alpha-fetoprotein (AFP) in the maternal (M) and fetal circulation (umbilical arteries (A) and vein (V)), focusing on the feto-material interface where maternal intervillous blood (I) contacts the fetal trophoblast. FFA concentrations in intervillous and maternal blood were similar, while those in umbilical arteries and vein were 2- to 4-fold lower (P < 0.001). There were more saturated FFA in umbilical vein (41%) and arteries (44%) blood than in maternal (30%) and intervillous (32%) blood (P < 0.001). Monounsaturated FFA predominated (P < 0.001) in maternal (43%) blood, but not in intervillous (35%), umbilical vein (33%) and arteries (31%) blood. Di-triunsaturated FFA were similar in intervillous and maternal (25%) blood and lower in umbilical vein and arteries (16%) (P < 0.001). PUFA were low in maternal (2.5%) blood and higher in intervillous and umbilical vein and arteries (9%, P < 0.001); consequently, C20:4 (40 microM) and C22:6 (16 microM) were the most abundant in the intervillous space. The AFP concentrations and AFP lectin-reactive isoforms were similar in intervillous and umbilical vein and arteries blood, but immuno-electrophoresis revealed a particular AFP conformation (less immuno-reactive, more anionic) in the intervillous space, suggesting that AFP is heavily loaded with PUFA at the feto-maternal interface. Prostacyclin derived from C20:4 was similar in all compartments but the thromboxane A2 concentration was 10-fold higher in intervillous blood than in maternal and umbilical vein and arteries blood. Thus the feto-maternal interface has a specific pattern of cell signalling molecules that might critically influence parturition.

Interactions between phytoestrogens and human sex steroid binding protein.

The interactions of human Sex steroid binding protein (SBP) and the lignans [Nordihydrogaiaretic acid (NDGA) enterolactone (Ent), enterodiol (End)] and isoflavonoid phytoestrogens [Equol (Eq), diazein Dad), genistein (Gen)] were studied. The phytoestrogens had different dose-dependent inhibitory effects on steroid binding by SBP. Their relative efficiencies were: Ent> or = NDGA = Eq > Gen for displacing E2 and Eq > Ent > NDGA > Gen for displacing T. End and Dad were much less active. Scatchard analysis suggested that NDGA had similar non- competitive effects on T and E2 binding by reducing the number of binding sites without changing the association constants. But Eq seemed to inhibit E2 binding non-competitively and T binding competitively. NDGA binding to SBP reduced the immunorecognition of SBP by monospecific anti-SBP antibodies, suggesting that NDGA changed SBP immunoreactivity. Unlike NDGA, Eq binding to SBP caused no immunological changes in SBP, indicating qualitative differences in the effects of the lignan and isoflavonoid. Our results indicate that phytoestrogens may modulate the SBP activity and so influence the role of this protein in the delivery of hormonal information to sex steroid-dependent cells.

Postprandial free fatty acids stimulate activity of human corticosteroid binding globulin.

The effect of postprandial variation of free fatty acids (FFA) on serum corticosteroid binding globulin (CBG) properties and cortisol (hydrocortisone) concentrations were explored in 11 women (20-30 yr) during 8 h after an oral load of tallow (26% C16:0, 18% C18:0, and 43% C18:1), oleic-sunflower (oleic-SF; 73% C18:1), sunflower (SF; 67% C18:2), and mixed oil (MO; 39% C18:1 and 48% C18:2). Serum FFA increased little after SF and MO but more than doubled in the late postprandial period (6 and 8 h) after oleic-SF (due to monounsaturated FFA) or tallow (due to saturated and monounsaturated FFA). CBG concentrations remained unchanged, but in relation with the postprandial elevation of serum FFA, CBG binding activity was increased after tallow or oleic-SF as a result of a combined two- to threefold increase in affinity constant and a 50% reduction in binding sites. Immunological and in vitro binding studies showed the changes in CBG behavior to be conformational and to be mediated mainly by monounsaturated FFA, especially C18:1. The modifications of CBG properties were associated with sustained high concentrations of cortisol (suppression of midday decrease) 6 and 8 h after tallow or oleic-SF. Thus dietary FFA may have an impact on bioavailability of glucocorticoids.

Levels of homeotic protein function can determine developmental identity: evidence from low-level expression of the Drosophila homeotic gene proboscipedia under Hsp70 control.

The autonomous selector capacity of the homeotic proboscipedia (pb) gnee of the Drosophila Antennapedia Complex was tested. We introduced into the germline a P element containing a transcriptional fusion of a mini-gene for pb (normally required for formation of the labial and maxillary palps of the mouthparts) and the Hsp70 promoter. Uninduced expression of this Hsp70:pb element (HSPB) directs a novel, fully penetrant dominant transformation of antennae toward maxillary palps. Gene dosage experiments varying the number of HSPB elements indicate that the extent of the dominant transformation depends on the level of PB protein. At the same time, expression from the transgene also rescues recessive pb mutations. Finally, HSPB function may override the dominant antennal transformations caused by Antennapedia (Antp) mutations in a dose-sensitive manner, directing a switch of the antennal disc-derived appendage from ectopic leg to ectopic maxillary palp. This switch correlated with strikingly reduced ANTP protein accumulation when PB concentrations exceeded a genetically defined threshold level. These observations support a crucial role for quantitative aspects of pb function in determining segmental identity, including cross-regulatory events involved in this determination.

In vivo effect of free fatty acids on the specific binding of glucocorticosteroids to corticosteroid binding globulin and liver receptors in immature rats.

Stimulating lipase activity with heparin (200 IU/kg b.w.) increased the plasma free fatty acid (FFA) concentration of immature rats (15 days). The effect of this elevated FFA concentration on glucocorticoid binding to corticosteroid binding globulin (CBG), and liver cytosol glucocorticoid receptor (GR), was analyzed. The plasma FFA concentration increased 2-fold, 10 minutes (P < 0.001), 20 minutes (P < 0.01), and 60 minutes (P < 0.01) post-heparin. The corticosterone (B) and progesterone concentrations were unchanged 60 minutes post-injection. The binding activity of immature rat CBG for B dropped 50% (P < 0.001) 60 minutes post-heparin injection, decreased B binding and increased plasma FFA were correlated (r = -0.8). The decreased B binding resulted from a 2-fold decrease in the apparent number of CBG binding sites; the affinity constant (Ka) remained unchanged. The liver cytosol endogenous FFA content of immature rats was also increased 2-fold, 60 minutes after heparin-induced lipolysis. The increased cytosol FFA, with no significant change in glucocorticoid, was accompanied by a significant decrease in dexamethasone binding to liver cytosol glucocorticoid receptor. The decrease resulted from a significantly lower apparent Ka for dexamethasone and fewer receptor binding sites (n). There was a good inverse correlation between Ka (r = -0.93) and n (r = -0.90) and the increased liver cytosol FFA content. Thus the higher plasma FFA induced in vivo by lipase activation or a standard FFA mixture probably causes conformational changes in CBG and GR, reducing glucocorticoid binding to immature rat CBG and liver GR.