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, in recent years, encouraged 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 whether 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 the 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.

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 ß-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.

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.

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 the ovary. 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 ovary is 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 to establish 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.

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.

Transforming growth factor beta1 regulates follistatin mRNA expression during in vitro bovine granulosa cell differentiation.

In order to test the hypothesis that transforming growth factor beta (TGF-beta) acts by FS regulation on bovine granulosa cells in in vitro differentiation, we analyzed the effect of TGF-beta1 on follistatin mRNA expression in three differentiation states of bovine granulosa cells. We showed a positive regulation of FS mRNA after TGF-beta1 (1 ng/ml) treatment of freshly isolated granulosa cells from small-medium antral follicles (2-8 mm). This effect was abolished by the addition of exogenous follistatin (100 ng/ml), suggesting that this effect could be mediated by activin. Although these cells showed a similar effect on FS mRNA expression after treatment with activin-A, a soluble form of activin receptor type IIA was unable to inactivate the TGF-beta effect. When we tested the TGF-beta effect on FS mRNA in different granulosa cell states, TGF-beta1 regulation was associated with progesterone production only in freshly isolated cells. The amount of total activin-A produced by first passage cells (dedifferentiated cells), was ten times smaller than the one measured in a conditioned medium from freshly isolated cells (mature cells). The TGF-beta1-dependent FS mRNA expression persisted in first passage cells without changes with FS addition. On the other hand, the BGC-1 granulosa cell line (immature cells) produced large amounts of activin-A regulated by TGF-beta1 and an invariable steady state of FS mRNAs. In summary, our results showed that FS mRNA expression is regulated by TGF-beta1 independently of activin effects in differentiated granulosa cells.

Exposure of bovine oocytes to the endogenous metabolite 2-methoxyestradiol during in vitro maturation inhibits early embryonic development.

Catecholestrogens are endogenous metabolites that have been shown to modulate granulosa, theca, and luteal cell function in some species. The present study was aimed at determining the possible role of these steroids on oocyte maturation. Cumulus-enclosed bovine oocytes were matured for 24 h, fertilized, and then cultured for 8 days. Whereas estradiol was without effect, addition of catecholestrogens (2-hydroxyestradiol, 4-hydroxyestradiol, and 2-methoxyestradiol [2-MOE2]) to the maturation medium did not affect the cleavage rate but was associated with a decrease in blastocyst production on Day 8. Although 2-MOE2 was also able to inhibit blastocyst formation when added during embryo culture, the effects were less pronounced than those seen when the steroid was added only during maturation. In agreement with the known ability of 2-MOE2 to bind tubulin at the colchicine site, marked alterations were observed in the spindle assembly of oocytes exposed to 2-MOE2 during maturation, which lead to gross chromosomal aberrations after fertilization and consequent developmental arrest at the morula stage. Moreover, that the blastocyst rate was not affected when meiosis was blocked with roscovitine during 2-MOE2 exposure is consistent with the idea that altered nuclear maturation is the cause of the low developmental competence. Because 2-MOE2 could be increased in follicular fluid in response to aryl hydrocarbon-receptor ligands, such as some environmental contaminants, our results show that abnormally high intraovarian levels of catecholestrogens could have a deleterious effect on oocyte maturation and early embryonic development arising from the alterations in the meiotic spindle.

Paradoxical effects of tumour necrosis factor-alpha on rat granulosa cell DNA synthesis.

Tumour necrosis factor-alpha (TNF-alpha) has been proposed as an intraovarian modulator of granulosa cell function. The effect of TNF-alpha on DNA synthesis in cultured rat granulosa cells was examined. Tumour necrosis factor-alpha stimulated thymidine incorporation when added in the presence of transforming growth factor-beta (TGF-beta). In contrast, the co-mitogenic effect of follicle-stimulating hormone (FSH) and TGF-beta was inhibited in a dose-dependent manner by TNF-alpha. Inhibition of FSH-dependent DNA synthesis by TNF-alpha was also found when cultures were co-stimulated with activin A. The inhibitory action of TNF-alpha on FSH-treated cultures was not associated with changes in cell viability. The inhibitory effects of TNF-alpha could not be solely explained by a decrease in cAMP levels, since TNF-alpha was also able to inhibit the stimulation by dibutyryl-cAMP and TGF-beta on granulosa cell DNA synthesis. These results suggest that TNF-alpha regulation of granulosa cell growth is elicited either independently or downstream from gonadotrophin-induced cAMP production. The actions of TNF-alpha could be only partially mimicked by a cell-permeable analogue of ceramide, thus indicating that actions of this cytokine can not be fully ascribed to an activation of sphingomyelinase. Data presented here indicate that, in addition to its previously demonstrated inhibitory effects on gonadotrophin-induced cell differentiation, TNF-alpha may also exert a marked inhibition on hormonally stimulated immature granulosa cell proliferation. In contrast to this inhibitory action, this cytokine could amplify the mitogenic action of putative intraovarian growth regulators such as TGF-beta. These observations add further support to the notion that TNF-alpha plays a physiological role as a paracrine modulator of follicle development and may be also relevant to the alteration of ovarian function during physiopathological processes.

Autocrine role of transforming growth factor beta1 on rat granulosa cell proliferation.

We evaluated the effects of transforming growth factor beta1 (TGFbeta1), alone or in combination with FSH and estradiol, on DNA synthesis in primary cultures of immature rat granulosa cells. 3H-Thymidine incorporation was significantly stimulated by TGFbeta1 (5.6-fold). This effect was enhanced by FSH (20 ng/ml, 27.7-fold) or estradiol (100 ng/ml, 13.4-fold) or by a combination of both hormones (59.2-fold). Measurement of TGFbeta bioactivity showed the presence of significant amounts of TGFbeta in conditioned medium from granulosa cell cultures, and most of the activity was present in the latent form. FSH alone or in combination with estradiol produced a marked suppression of the production of latent and active TGFbeta. Activated conditioned medium from control cultures of granulosa cell elicited a 1.4-fold increase in thymidine incorporation. This effect was markedly amplified by FSH (3-fold) and estradiol (4.3-fold) and by a combination of both (8.7-fold). The peptide containing the cell-binding domain of fibronectin (RGDSPC) partially inhibited thymidine incorporation stimulated by TGFbeta1. Fibronectin did not synergize with FSH, and the interaction between TGFbeta1 and FSH was even observed in the presence of this protein. The conclusions reached were as follows: 1) TGFbeta1 is an autocrine stimulator of rat granulosa cell DNA synthesis, 2) FSH and estradiol produce a suppression of latent and active TGFbeta production but markedly amplify TGFbeta action, presumably at a postreceptor level, and 3) the stimulatory effects of TGFbeta1 may be only partly mediated by the increased fibronectin secretion.

Funcion de distintas formas de fibronectina en el desarrollo de foliculos bovinos in vitro / Role of different forms of fibronectin in in vitro bovine follicular development

En el presente estudio, se analizó la posibilidad de que las distintas formas de fibronectina (FN), producidas como resultado de la maduración alternativa (alternative splicing) del mensajero, ejerzan funciones diferenciales en el desarrollo folicular. En particular se determinó la presencia de la región ED-I, ausente en la FN plasmática, tanto a nivel de mensajero como de proteína, durante este proceso. El análisis de los niveles de FN en fluidos foliculares correspondientes a distintas etapas de desarrollo mostró marcadas variaciones en la concentración de FN ED-I+ y los de permanecieron relativamente constantes. En folículos corespondientes a la fase de selección se observó una correlación inversa entre los niveles de FN ED-I+ y los de estradio (p<0.001). El tratamiento con estradiol no tuvo efecto sobre el splicing alternativo de FN en cultivos de células de la granulosa bovinas, mientras que el AMPc tuvo un efecto inhibitorio sobre la incorporación de ED-I. Por otra parte, el factor de crecimiento transformante tipo beta (TGF-beta) estimuló tanto la produción de FN total como la inclusión de la región ED-I. Este efecto fue verificado tanto a nivel de la proteína (Western blots) como del ARN mensajero (Northern blots). Un péptido correspondiente a la región ED-I tuvo un efecto estimulatorio sobre el crecimento de una línea de células de la granulosa bovinas (BGC-1) mientras que el péptido correspondiente e las regiones flanqueantes no tuvo efecto. Los datos presentados en este estudio plantean una nueva forma de regulación mediante la cual cambios cualitativos en la estructura primaria de la FN podrían mediar algunas de las acciones de gonadotrofinas y factores intraováricos durante el desarrollo folicular. (AU)

Funcion de distintas formas de fibronectina en el desarrollo de foliculos bovinos in vitro / Role of different forms of fibronectin in in vitro bovine follicular development

En el presente estudio, se analizó la posibilidad de que las distintas formas de fibronectina (FN), producidas como resultado de la maduración alternativa (alternative splicing) del mensajero, ejerzan funciones diferenciales en el desarrollo folicular. En particular se determinó la presencia de la región ED-I, ausente en la FN plasmática, tanto a nivel de mensajero como de proteína, durante este proceso. El análisis de los niveles de FN en fluidos foliculares correspondientes a distintas etapas de desarrollo mostró marcadas variaciones en la concentración de FN ED-I+ y los de permanecieron relativamente constantes. En folículos corespondientes a la fase de selección se observó una correlación inversa entre los niveles de FN ED-I+ y los de estradio (p<0.001). El tratamiento con estradiol no tuvo efecto sobre el splicing alternativo de FN en cultivos de células de la granulosa bovinas, mientras que el AMPc tuvo un efecto inhibitorio sobre la incorporación de ED-I. Por otra parte, el factor de crecimiento transformante tipo beta (TGF-beta) estimuló tanto la produción de FN total como la inclusión de la región ED-I. Este efecto fue verificado tanto a nivel de la proteína (Western blots) como del ARN mensajero (Northern blots). Un péptido correspondiente a la región ED-I tuvo un efecto estimulatorio sobre el crecimento de una línea de células de la granulosa bovinas (BGC-1) mientras que el péptido correspondiente e las regiones flanqueantes no tuvo efecto. Los datos presentados en este estudio plantean una nueva forma de regulación mediante la cual cambios cualitativos en la estructura primaria de la FN podrían mediar algunas de las acciones de gonadotrofinas y factores intraováricos durante el desarrollo folicular.