Paternal inheritance of bisphenol a toxic effects: from sperm alterations to impaired embryo development = Transmisión de la toxicidad del bisfenol A por vía paterna: alteraciones espermáticas y fallos en el desarrollo embrionario
Área de conocimiento
Nowadays, plastic pollution has become one of the major concerns of the society. As a result of that, bisphenol A, a chemical plasticizer used for the manufacture of polycarbonate plastics and epoxy resins, has caught the attention of great many scientific studies. Besides the capacity of this toxicant to interfere with the endocrine system, it has also been reported to exert both genotoxic and epigenotoxic effects. Bearing in mind that the spermatogenesis is a coordinated process that relies on several steroid hormones and that entails deep changes in the chromatin, such as DNA breaks and epigenetic remodelling, it might well be affected by bisphenol A exposure. Hence, there is a great deal of evidence to show that bisphenol A has detrimental effects on male reproductive health. Most of these works are focused on the alterations caused in sperm quality, sexual function and the development of gonads; however, the consequences of paternal bisphenol A exposure for future generations remain still unknown. In this regard, spermatozoa have long been considered as mere vectors to deliver the paternal haploid genome on the oocyte. Only recently have they been stated to harbour more information than just the nuclear DNA and, what is more, this information has been claimed to play a remarkable role in offspring health. Any alteration triggered by bisphenol A during male germline formation may affect the information contained in the spermatozoa, thus being transmitted to the progeny. For this reason, the First Chapter of this Ph.D. Thesis tackles the impact of paternal bisphenol A exposure on embryo development, using zebrafish (Danio rerio) as model species. Adult males were exposed to high doses of bisphenol A during the mitotic phase of spermatogenesis: two weeks of exposure and one week under standard conditions. After treatment, males were mated with non-treated females to obtain the F1 and also the F2 embryos. The evaluation of the embryo development was carried out by routine histology and by using the transgenic line Tg(fli1:EGFP;mlc2a: mCherry). Moreover, some spermatic information, such as global DNA methylation and sperm-borne transcripts, was analysed by UPLC-MS and qPCR, respectively. The results showed that, when treating males with 2000 µg/L BPA, the percentage of malformations, mainly consisting of cardiac edema, increased in both F1 and F2 larvae, a generation who was never in contact with the toxicant. Even though no differences in global DNA methylation were found either in testicles or sperm cells, bisphenol A exposure triggered a decrease in the sperm transcripts encoding insulin receptors in F0 and F1 males. Since the expression of the β subunit of insulin receptor was repressed in F1 and F2 embryos, we concluded that the alteration of spermatic transcripts induced by bisphenol A is transgenerationally inherited. The results derived from this chapter have been published in Environmental Pollution (IF: 4.839). The Second Chapter of this Ph.D. Thesis is focused on finding out the most vulnerable period of male germline formation to bisphenol A exposure. For this purpose, zebrafish were treated with this toxicant during different windows of exposure. The first one entailed the embryonic life, covering the process of primordial germ cell migration to the genital ridge (the first 24 hours of development in this species). The second one affected the same period that chapter I: the mitotic phase of spermatogenesis. And the third one also occurred during adult life, but it included also the meiotic phase and the spermiogenesis (three weeks of exposure). After embryonic exposure, the migration of primordial germ cells as well as their epigenetic status were analysed by whole mount immunostaining. Once embryos reached the adulthood, we assessed the development of the gonads and the breeding capacity of males. Concerning adult exposure, sperm DNA fragmentation and sperm epigenetic profile were evaluated by comet assay and cell immunostaining, respectively. In the F1 progeny, we assessed the percentage of mortality, the DNA repairing capacity and the epigenetic landscape (by whole mount immunostaining) as well as the apoptotic activity (by flow cytometry). The results showed that embryonic exposure to 2000 and 4000 µg/L bisphenol A during the first 24 hours of development alters the expression of two genes involved in primordial germ cell migration (sdf1a and cxcr4b), thus hindering the colonisation of the genital ridges in exposed embryos. However, the histological and morphometrical studies revealed that the impairment of primordial germ cell migration has no long-term effects in the development of the testicles. Regardless of the lower levels of histone acetylation displayed in the spermatozoa of males exposed to 4000 µg/L bisphenol A during embryonic life, they show a proper breeding capacity and generate a normal progeny. Regarding adult male exposure, the treatment with 2000 µg/L bisphenol A during mitotic phase and with 100 and 2000 µg/L bisphenol A during the whole spermatogenesis led to high levels of sperm DNA damage, which have fateful consequences for embryonic development. On the contrary, when males were exposed to 100 µg/L bisphenol A only during mitotic phase, the levels of DNA damage were not that high, so F1 embryos were able to activate DNA repair, showing basal levels of apoptosis. As far as the epigenetic profile of spermatozoa and F1 embryos is concerned, although no changes were observed in the pattern of sperm DNA methylation, males exposed to bisphenol A during the mitotic phase of spermatogenesis showed higher levels of H3K27ac, whereas males treated with the highest dose of the toxicant during the whole spermatogenesis had higher levels of H3K9ac. It is noteworthy that these alterations in the sperm epigenetic marks triggered by male bisphenol A exposure are inherited by F1 embryos, which displayed also an increase in H3K27ac and H3K9ac. In this chapter, it has been proved that the effects of bisphenol A on male germline depend on the window of exposure and that they are much more detrimental when meiosis and spermiogenesis periods are affected. The results obtained from embryonic exposure have been published in Biomolecules (IF: 4.694), whereas those concerning adult male exposure have been submitted to Scientific Reports. The Third Chapter of this Ph.D. Thesis aims to determine the mechanisms by which direct and paternal exposure to bisphenol A impairs heart development. For this purpose, three different experiments were performed: in vitro exposure was performed in rat embryonic cardiomyoblasts, whereas in vivo exposure to bisphenol A was carried out during early embryonic development and adult male life (early spermatogenesis). An immunostaining was performed to assess the levels of histone acetylation, whereas the acetylation of specific gene promoters was assessed by ChIP-qPCR. The expression of a set of genes, which involved heart transcription factors, estrogen receptors and a histone acetyltransferase was analysed by qPCR. The results showed that the treatment with bisphenol A in all these models alter some histone acetylation marks and the expression of the studied genes as well. Nevertheless, there were some differences between embryonic and paternal exposure. The levels of H3K9ac in the promoters of hand2 and esr2b in embryos exposed to bisphenol A were similar to those of control embryos, so in this case, estrogenic effects of bisphenol A, which affect the expression of specific genes, may be more likely related to heart defects than epigenetic changes. On the other hand, we did find out an increase in the enrichment of H3K9ac in the promoters of kat6a, hand2 and esr2b in embryos obtained from males exposed to 2000 µg/L bisphenol A, so the increase in histone acetylation promoted by paternal exposure underlies the overexpression of these three genes. Taking all this data into account, we decided to treat exposed embryos and embryos obtained from exposed males with epigallocatechin gallate. This compound is the major catechin of green tea and it has already been proved to act as an anti-estrogenic substance and as a histone acetyltransferase inhibitor. Our findings showed that the treatment with epigallocatechin gallate successfully counteracted the overexpression of estrogen receptors and histone acetylation, decreasing the levels of hand2 and, thus reducing the cardiac malformations induced by embryonic and paternal bisphenol A exposure. Therefore, in this chapter we have demonstrated not only the mechanisms by which changes in sperm promoted by paternal exposure to bisphenol A have long-term effects on cardiac health of the progeny, but also that epigallocatechin gallate is able to counteract these alterations, allowing a proper heart formation. The results derived from the embryonic exposure to bisphenol A and the neutralisation of direct effects with EGCG have been published in Environmental Pollution (IF: 5.714).
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