Early-life exposure to the endocrine disruptor 17-α-ethinylestradiol induces delayed effects in adult brain, liver and ovotestis proteomes of a self-fertilizing fish.

Early-life represents a critically sensitive window to endocrine disrupting chemicals, potentially leading to long-term repercussions on the phenotype later in life. The mechanisms underlying this phenomenon, referred to as the Developmental Origins of Health and Disease (DOHaD), are still poorly understood. To gain molecular understanding of these effects, we exposed mangrove rivulus (Kryptolebias marmoratus) for 28 days post hatching (dph) to 4 and 120 ng/L 17-α-ethinylestradiol, a model xenoestrogen. After 28 days, fish were raised for 140 days in clean water and we performed quantitative label-free proteomics on brain, liver and ovotestis of 168 dph adults. A total of 820, 888 and 420 proteins were robustly identified in the brain, liver and ovotestis, respectively. Effects of 17-α-ethinylestradiol were tissue and dose-dependent: a total of 31, 51 and 18 proteins were differentially abundant at 4 ng/L in the brain, liver and ovotestis, respectively, compared to 20, 25 and 39 proteins at 120 ng/L. Our results suggest that estrogen-responsive pathways, such as lipid metabolism, inflammation, and the innate immune system were affected months after the exposure. In addition, the potential perturbation of S-adenosylmethionine metabolism encourages future studies to investigate the role of DNA methylation in mediating the long-term effects of early-life exposures. SIGNIFICANCE: The Developmental Origins of Health and Disease (DOHaD) states that early life stages of humans and animals are sensitive to environmental stressors and can develop health issues later in life, even if the stress has ceased. Molecular mechanisms supporting DOHaD are still unclear. The mangrove rivulus is a new fish model species naturally reproducing by self-fertilization, making it possible to use isogenic lineages in which all individuals are highly homozygous. This species therefore permits to strongly reduce the confounding factor of genetic variability in order to investigate the effects of environmental stress on the phenotype. After characterizing the molecular phenotype of brain, liver and ovotestis, we obtained true proteomic reaction norms of these three organs in adults after early life stages have been exposed to the common endocrine disruptor 17-α-ethinylestradiol (EE2). Our study demonstrates long-term effects of early-life endocrine disruption at the proteomic level in diverse estrogen-responsive pathways 5 months after the exposure. The lowest tested and environmentally relevant concentration of 4 ng/L had the highest impact on the proteome in brain and liver, highlighting the potency of endocrine disruptors at low concentrations.

Lower selfing rates in metallicolous populations than in non-metallicolous populations of the pseudometallophyte Noccaea caerulescens (Brassicaceae) in Southern France.

BACKGROUND AND AIMS: The pseudometallophyte Noccaea caerulescens is an excellent model to study evolutionary processes, as it grows both on normal and on heavy-metal-rich, toxic soils. The evolution and demography of populations are critically impacted by mating system and, yet, information about the N. caerulescens mating system is limited. METHODS: Mean selfing rates were assessed using microsatellite loci and a robust estimation method (RMES) in five metallicolous and five non-metallicolous populations of N. caerulescens in Southern France, and this measure was replicated for two successive reproductive seasons. As a part of the study, the patterns of gene flow among populations were analysed. The mating system was then characterized at a fine spatial scale in three populations using the MLTR method on progeny arrays. KEY RESULTS: The results confirm that N. caerulescens has a mixed mating system, with selfing rates ranging from 0·2 to 0·5. Selfing rates did not vary much among populations within ecotypes, but were lower in the metallicolous than in the non-metallicolous ecotype, in both seasons. Effective population size was also lower in non-metallicolous populations. Biparental inbreeding was null to moderate. Differentiation among populations was generally high, but neither ecotype nor isolation by distance explained it. CONCLUSIONS: The consequences of higher selfing rates on adaptation are expected to be weak to moderate in non-metallicolous populations and they are expected to suffer less from inbreeding depression, compared to metallicolous populations.

Molecular, Biochemical and Ultrastructural Changes Induced by Pb Toxicity in Seedlings of Theobroma cacao L.

Pb is a metal which is highly toxic to plants and animals, including humans. High concentrations of Pb have been observed in beans of T. cacao, as well as in its products. In this work, we evaluated the molecular, biochemical, and ultrastructural alterations in mature leaves and primary roots of seedlings of two progenies of T. cacao, obtained from seed germination in different concentrations of Pb (0, 0.05, 0.1, 0.2, 0.4, 0.8 g L(-1)), in the form of Pb(NO3)2. The progenies resulted from self-fertilization of Catongo and a cross of CCN-10 x SCA-6. The Pb, supplied via seminal, caused alterations in the ultrastructures of the mesophyll cells and in the amount of starch grains in the chloroplasts. The dosage of substances reactive to thiobarbituric acid showed that Pb induced lipid peroxidation. The activity of guaiacol peroxidases and the expression of genes associated to synthetase of phytochelatin, SODcyt and PER increased in response to Pb. In addition, there was alteration in the expression of stress-related proteins. The progeny of CCN-10 x SCA-6 was more tolerant to Pb stress when compared to Catongo, since: (i) it accumulated more Pb in the roots, preventing its translocation to the shoot; (ii) it presented higher activity of peroxidases in the roots, which are enzymes involved in the elimination of excess of reactive oxygen species; and (iii) increased expression of the gene in the phytochelatin biosynthesis route. The results of the proteomic analysis were of paramount importance to differentiate the defense mechanisms used by both progenies of T. cacao.

A stable JAZ protein from peach mediates the transition from outcrossing to self-pollination.

BACKGROUND: Variations in floral display represent one of the core features associated with the transition from allogamy to autogamy in angiosperms. The promotion of autogamy under stress conditions suggests the potential involvement of a signaling pathway with a dual role in both flower development and stress response. The jasmonic acid (JA) pathway is a plausible candidate to play such a role because of its involvement in many plant responses to environmental and developmental cues. In the present study, we used peach (Prunus persica L.) varieties with showy and non-showy flowers to investigate the role of JA (and JA signaling suppressors) in floral display. RESULTS: Our results show that PpJAZ1, a component of the JA signaling pathway in peach, regulates petal expansion during anthesis and promotes self-pollination. PpJAZ1 transcript levels were higher in petals of the non-showy flowers than those of showy flowers at anthesis. Moreover, the ectopic expression of PpJAZ1 in tobacco (Nicotiana tabacum L.) converted the showy, chasmogamous tobacco flowers into non-showy, cleistogamous flowers. Stability of PpJAZ1 was confirmed in vivo using PpJAZ1-GFP chimeric protein. PpJAZ1 inhibited JA-dependent processes in roots and leaves of transgenic plants, including induction of JA-response genes to mechanical wounding. However, the inhibitory effect of PpJAZ1 on JA-dependent fertility functions was weaker, indicating that PpJAZ1 regulates the spatial localization of JA signaling in different plant organs. Indeed, JA-related genes showed differential expression patterns in leaves and flowers of transgenic plants. CONCLUSIONS: Our results reveal that under stress conditions ­ for example, herbivore attacks ­ stable JAZ proteins such as PpJAZ1 may alter JA signaling in different plant organs, resulting in autogamy as a reproductive assurance mechanism. This represents an additional mechanism by which plant hormone signaling can modulate a vital developmental process in response to stress.

[Role of ethylene in the control of gametophyte-sporophyte interactions in the course of the progamic phase of fertilization].

We investigated dynamics of the content of 1-aminocyclopropane-l-carboxylic acid (ACC) and ethylene production in male gametophyte development and germination in fertile (self-compatible and self-incompatible) and sterile clones of petunia. Fertile male gametophyte development was accompanied by two peaks of ethylene production by anther tissues. The first peak occurred during the microspore development simultaneously with the degeneration of both the tapetal tissues and the middle layers of the anther wall. The second peak coincided with dehydration and maturation of pollen grains. In the anther tissues of the sterile line of petunia, tenfold higher ethylene production was observed at the meiosis stage compared with that in fertile male gametophytes. This fact correlated with the degeneration of both microsporocytes and tapetal tissues. Exogenously applied ethylene (1-100 ppm) induced a degradation of the gametophytic generation at the meiosis stage. According to the obtained data, ethylene synthesis in germinating male gametophyte is provided by a 100-fold ACC accumulation in mature pollen grains. The male gametophyte germination, both in vitro, on the culture medium, and in vivo, on the stigma surface, was accompanied by an increase in ethylene production. Depending on the type of pollination, germination of pollen on the stigma surface and the pollen tube growth in the tissues of style were accompanied by various levels ofACC and ethylene release. The male gametophyte germination after self-compatible pollination was accompanied by higher content of ACC as compared with the self-incompatible clone, whereas, after the self-incompatible pollination, we observed a higher level of ethylene production compared with compatible pollination. For both types of pollination, ACC and ethylene were predominantly produced in the stigma tissues. Inhibitor of ethylene action, 2,5-norbornadiene (NBN), blocked both the development and germination of the male gametophyte. These results suggest that ethylene is an important factor in male gametophyte development, germination, and growth at the progamic phase of fertilization.

Bisphenol A modulates expression of sex differentiation genes in the self-fertilizing fish, Kryptolebias marmoratus.

Endocrine disrupting chemicals (EDCs) have been a major concern in the normal reproduction and development of aquatic organisms. In the teleost, steroid hormones are synthesized via the steroidogenesis pathway, and play a key physiological role in the regulation of gonadal sex differentiation. The protogynous hermaphroditic fish, Kryptolebias marmoratus is the only vertebrate capable of reproducing through internal self-fertilization. To uncover the effect of bisphenol A (BPA) on sex differentiation genes on transcription, we investigated the expression patterns of several sex differentiation-related genes such as dax1, dmrt1, mis, sf1, figlα, StAR and wt1 after BPA exposure with controls (E2 and TMX). In response to 17ß-estradiol (E2) exposure, a testis-specific gene, dmrt1 mRNA was down-regulated in the gonad of the secondary male but the expression of the female-specific gene, dax1 mRNA was significantly elevated in the brain and gonad. A high level of StAR mRNA was detected in the brain and gonad of both hermaphrodite and secondary males, suggesting that the elevated expression of dax1 and StAR genes would be involved in E2 exposure. As expected, upon BPA exposure, the dmrt1 and MIS mRNA level decreased in both hermaphrodite and secondary males, while the female-specific gene, figlα mRNA level increased in the gonad of both genders. BPA showed an opposite mode of action on the expression of dax1 (induction, P>0.05) and sf1 mRNA (inhibition, P>0.05) in the brain and gonad against both genders. The sensitivity of dax1 to BPA on expression was relatively high in the secondary male. The wt1 mRNA was up-regulated in most tissues except in the liver of BPA-exposed secondary males. Regarding the time course study, the figlα mRNA level increased at 6 h after BPA exposure. In addition, BPA elevated the expression of StAR, dax1, and wt1 mRNA but repressed sf1 mRNA. In this paper, we demonstrated that BPA may modulate the expression of sex differentiation and steroidogenesis pathway genes, and this finding would provide a better understanding on the modulation of transcription upon BPA exposure in steroidogenesis and sex differentiation in the hermaphroditic fish, K. marmoratus.