Effect of sialylation and complexity of FSH oligosaccharides on inhibin production by granulosa cells

Granulosa cell inhibin A and B production is regulated by FSH and gonadal factors. This gonadotrophin is released as a mixture of glycoforms, which induce different biological responses in vivo and in vitro. Our aim was to determine the effect of recombinant human FSH glycosylation variants on inhibin A and B production by rat granulosa cells. Preparative isoelectrofocusing was used to isolate more acidic/sialylated (pH5.00) rhFSH charge analogues. Concanavalin-A was used to isolate unbound and firmly bound rhFSH glycoforms on the basis of their oligosaccharide complexity. Granulosa cells, obtained from oestrogen-primed immature rats, were cultured with either native rhFSH or its glycosylation variants. Inhibin A and B were determined using specific ELISAs. Results were expressed as mean±SEM. Under basal conditions inhibin A was the predominant dimer produced (inhibin A: 673±55; inhibin B: 80±4 pg/mL). More acidic/sialylated charge analogues stimulated inhibin B production when compared to inhibin A at all doses studied; in contrast, less acidic/sialylated charge analogues stimulated inhibin A production and elicited no effect on inhibin B. Glycoforms bearing complex oligosaccharides showed a potent stimulatory effect on inhibin B when compared to inhibin A production (i.e. dose 1ng/mL: 4.9±0.5vs0.9±0.1 fold stimulation, P<0.001). Glycoforms bearing hybrid type oligosaccharides favoured inhibin A production (i.e. dose 4ng/mL 2.9±0.1vs1.6±0.1 fold stimulation, P<0.05). These results show that the sialylation degree as well as the complexity of oligosaccharides present in the rhFSH molecule may be considered additional factors that differentially regulate dimeric inhibin production by rat granulosa cells.Fil: Loreti, Rosana Nazareth. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Gobierno de la Ciudad de Buenos Aires. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Fundación de Endocrinología Infantil. Centro de Investigaciones Endocrinológicas "Dr. César Bergada"; ArgentinaFil: Ambao, Veronica Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Gobierno de la Ciudad de Buenos Aires. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Fundación de Endocrinología Infantil. Centro de Investigaciones Endocrinológicas "Dr. César Bergada"; Argentina. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; ArgentinaFil: Andreone, Luz. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Gobierno de la Ciudad de Buenos Aires. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Fundación de Endocrinología Infantil. Centro de Investigaciones Endocrinológicas "Dr. César Bergada"; Argentina. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; ArgentinaFil: Trigo, Romina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Gobierno de la Ciudad de Buenos Aires. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Fundación de Endocrinología Infantil. Centro de Investigaciones Endocrinológicas "Dr. César Bergada"; Argentina. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; ArgentinaFil: Bussmann, Ursula Agnes. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Campo, Stella Maris. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Gobierno de la Ciudad de Buenos Aires. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Fundación de Endocrinología Infantil. Centro de Investigaciones Endocrinológicas "Dr. César Bergada"; Argentina. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; Argentin

Establishment and characterization of a novel tissue-specific dna construct and culture system with potential for avian bioreactor generation

Transgenic chickens are of great interest for the production of recombinant proteins in their eggs. However, the use of constitutive strong promoters or the tissue-specific ovalbumin promoter for the generation of the transgenic chickens have different drawbacks that have to be overcome in order to make chicken bioreactor an efficient production system. This prompted us to investigate the use of an alternative tissue-specific promoter, the vitellogenin promoter, which could overcome the difficulties currently found in the generation of chicken bioreactors. In the present work we establish and characterize a DNA construct consisting of a fragment of the 5´-flanking region of the chicken vitellogenin II gene cloned in a reporter vector. This construct is capable of showing the ability of the promoter to drive expression of a reporting gene in a tissue-specific manner and in a way that closely resembles physiologic regulation of vitellogenin, making it an ideal candidate to be used in the future for generation of avian bioreactors. Besides, we validate an in vitro culture system to test the performance of the DNA construct under study that could be used as a practical tool before generating any transgenic chicken. These results are important since they provide the proof of concept for the use of the vitellogenin promoter for future genetic modification of chickens bioreactors with improved characteristics in terms of quality of the recombinant protein produced.Fil: Pérez Sáez, Juan Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Bussmann, Leonardo Edmundo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Bussmann, Ursula Agnes. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentin

An aryl hydrocarbon receptor agonist amplifies the mitogenic actions of estradiol in granulosa cells: evidence of involvement of the cognate receptors

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that, besides mediating toxic responses, may have a central role in ovarian physiology. Studying the actions of AHR ligands on granulosa cells function, we have found that beta-naphthoflavone amplifies the comitogenic actions of FSH and 17beta-estradiol in a dose-dependent manner. This amplification was even greater in cells that overexpress the AHR and was reversed by cotreatment with the AHR antagonist alpha-naphthoflavone, suggesting that this effect is mediated by the AHR. The estrogen receptor is likewise implicated in this phenomenon, because a pure antiestrogen abolished the described synergism. However, the more traditional inhibitory AHR-estrogen receptor interaction was observed on the estrogen response element-driven transcriptional activity. On the other hand, alpha-naphthoflavone inhibited dose-dependently the mitogenic actions of FSH and 17beta-estradiol. Beta-naphthoflavone induced the expression of Cyp1a1 and Cyp1b1 transcripts, two well-characterized AHR-inducible genes that code for hydroxylases that metabolize estradiol to catecholestrogens. Nevertheless, the positive effect of beta-naphthoflavone on proliferation was not caused by increased metabolism of estradiol to catecholestrogens, because these compounds inhibited the hormonally stimulated DNA synthesis. This latter inhibition exerted by catecholestrogens suggests that these hydroxylases would play a regulatory point in granulosa cell proliferation. Our study indicates that AHR ligands modulate the proliferation of rat granulosa cells, and demonstrates for the first time that an agonist of this receptor is able to amplify the comitogenic action of classical hormones through a mechanism that might implicate a positive cross-talk between the AHR and the estrogen receptor pathways.Fil: Bussmann, Ursula Agnes. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Bussmann, Leonardo Edmundo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Barañao, Jose Lino Salvador. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentin

Regulation of aryl hydrocarbon receptor expression in rat granulosa cells

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that mediates most of the toxic and endocrine-disruptive actions of aromatic compounds in theovary. Paradoxically, this receptor has been shown to play important roles in normal female reproductive function as well. Although knowledge of AHR expression regulation in the ovaryis of crucial significance to understand the receptor biology and its function in reproductive physiology, there are only limited data in this area. The purpose of the present study was toestablish the possible regulation that AHR might undergo in ovarian cells. Here we show that the hormones FSH and estradiol are able to reduce AHR protein and transcript levels in granulosa cells in a way that parallels the changes observed in ovarian tissue across the rat estrous cycle. These findings suggest that estradiol and FSH would be cycle-associated endogenous modulators of AHR expression. In addition, we show that in granulosa cells the receptor is rapidly downregulated via proteasomal degradation following treatment with AHR ligands. However, prolonged treatment with an agonist caused an increase in Ahr mRNA levels. These actions would constitute a regulatory mechanism that both attenuates AHR signal rapidly and replenishes the cellular receptor pool in the long term. In conclusion, our results indicate that AHR expression is regulated by classical hormones and by its own ligands in granulosa cells.Fil: Bussmann, Ursula Agnes. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Barañao, Jose Lino Salvador. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentin

Interaction between the aryl hydrocarbon receptor and transforming growth factor-beta signaling pathways: evidence of an asymmetrical relationship in rat granulosa cells

The aryl hydrocarbon receptor (AHR) mediates toxic responses to environmental contaminants and plays pivotal physiological roles in various biological processes as well, particularly in ovarian function. It is well documented that expression and function of the AHR is negatively regulated by transforming growth factor-beta (TGF-beta) in many cell types. In addition, several studies indicate that AHR activity inhibits TGF-beta expression and function in some systems. However, the interplay between these two signals is highly dependent upon the cell type being studied, precluding a generalization about the outcome of such interaction. Therefore, the goal of the present study was to determine the effect of TGF-beta on AHR expression and activation in granulosa cells, an ovarian cell type where the growth factor is mitogenic and AHR activation has been associated with promotion of proliferation as well. In addition, we conducted experiments aimed at evaluating the effect of AHR ligands on TGF-beta action in our system. Results presented herein demonstrate that AHR expression is not regulated by TGF-beta in rat granulosa cells, neither at the mRNA level nor at the protein level. Moreover, we find that the growth factor does not alter the transcriptional function of the AHR. Conversely, we show that activation of AHR by an agonist deregulates TGF-beta function in granulosa cells, inhibiting its transcriptional activity and its mitogenic action. The described one-sided interplay between TGF-beta and AHR signaling pathway may help provide a mechanistic explanation to some of the physiological outcomes of AHR or TGF-beta activation in granulosa cells.Fil: Bussmann, Ursula Agnes. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Barañao, Jose Lino Salvador. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentin

Improvement of chicken primordial germ cell maintenance in vitro by blockade of the Aryl Hydrocarbon Receptor endogenous activity

Primordial germ cells (PGCs) are the undifferentiated progenitors of gametes. Germline competent PGCs can be developed as a cell-based system for genetic modification in chickens, which provides a valuable tool for transgenic technology with both research and industrial applications. This implies manipulation of PGCs, which encouraged in recent years a lot of research focused on the study of PGCs and the way of improving their culture. The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that besides mediating toxic responses to environmental contaminants plays pivotal physiological roles in various biological processes. Since a novel compound that acts as an antagonist of this receptor has been reported to promote expansion of hematopoietic stem cells, we conducted the present study with the aim of determining if addition of an established AHR antagonist to the standard culture medium used nowadays for in vitro chicken PGCs culture improves ex vivo expansion. We have found that addition of α-naphthoflavone in culture medium promotes the amplification of undifferentiated cells and that this effect is exerted by blockade of AHR action. Our results constitute the first report of the successful use of a readily available AHR antagonist to improve avian PGCs expansion and they further extend the knowledge of the effects of AHR modulation in undifferentiated cells.Fil: Pérez Sáez, Juan Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Bussmann, Leonardo Edmundo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Barañao, Jose Lino Salvador. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Bussmann, Ursula Agnes. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentin

Aryl hydrocarbon receptor activation leads to impairment of estrogen-driven chicken vitellogenin promoter activity in LMH cells

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that mediates most of the toxic effects of environmental contaminants. Among the multiple pleiotropic responses elicited by AHR agonists, the antiestrogenic and endocrine-disrupting action of the receptor activation is one of the most studied. It has been demonstrated that some AHR agonists disrupt estradiol-induced vitellogenin synthesis in the fish liver via a mechanism that involves crosstalk between the AHR and the estrogen receptor (ER). Chicken hepatocytes have become a model for the study of AHR action in birds and the induction of the signal and its effect in these cells are well established. However, the impact of AHR activation on estradiol-regulated responses in the chicken liver remains to be demonstrated. The aim of the present study was, therefore, to determine the effect of AHR action on ER-driven transcription in a convenient model of chicken liver cells. For this purpose, we designed a reporter construct bearing the 5′ regulatory region of the chicken vitellogenin II gene and used it to transfect chicken hepatoma LMH cells. We found that β-naphthoflavone represses ER-driven vitellogenin promoter activity and that this action is mediated by the AHR. This inhibitory crosstalk between both pathways appears to be unidirectional, since estradiol did not alter the transcript levels of an AHR target gene. Besides, and highly relevant, we show that LMH cell line transfected with a reporter construct bearing the chicken vitellogenin promoter sequence is a useful and convenient model for the study of AHR–ER interaction in chicken liver-derived cells.Fil: Bussmann, Ursula Agnes. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Pérez Sáez, Juan Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Bussmann, Leonardo Edmundo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Barañao, Jose Lino Salvador. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentin

Involvement of thyroid hormones in the alterations of T-cell immunity and tumor progression induced by chronic stress

Stress alters the neuroendocrine system, immunity, and cancer. Although the classic stress hormones are glucocorticoids and catecholamines, thyroid hormones have also been related to stress. We recently reported that chronic restraint stress impairs T-cell mediated immunity and enhances tumor growth in mice.Fil: Frick, Luciana Romina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Rapanelli, Maximiliano. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Bussmann, Ursula Agnes. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Klecha, Alicia Juana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Barreiro Arcos, María Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Genaro, Ana Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Cremaschi, Graciela Alicia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; Argentin