The probiotic SLAB51 as agent to counteract BPA toxicity on zebrafish gut microbiota -liver-brain axis.

A plethora of studies have so far described the toxic effects of bisphenol A (BPA) on organism health, highlighting the urgent need to find new strategies not only to reduce the presence of this toxicant but also to counteract its adverse effects. In this context, probiotics emerged as a potential tool since they promote organism welfare. Using a multidisciplinary approach, this study explores the effects of SLAB51 dietary administration to counteract BPA toxicity using zebrafish as a model. Adult males and females were maintained under standard conditions (control group; C), exposed for 28 days via the water to an environmental relevant dose of BPA (10 µg/L; BPA), dietary treated with SLAB51 (109 CFU/g of body weight; P) and co-treated with BPA plus SLAB51 (BPA + P). In the gut, exposure to BPA resulted in altered architecture in both males and females, with females also experiencing an increase of pathogenic bacterial species. Co-administration of BPA + P led to the restoration of normal gut architecture, favored beneficial bacteria colonization, and decreased the abundance of pathogenic species. In the liver, male BPA exposure led to steatosis and glycogen depletion, which was partially mitigated by SLAB51 co-administration. In contrast, in females exposed to BPA, the lack of steatosis along with the greater glycogen depletion, suggested an increase in energy demand as supported by the metabolomic phenotype. The analysis of liver metabolites in BPA + P males revealed increased levels of anserine and reduced levels of glutamine, which could lie behind the counteraction of the brain histopathological damage caused by BPA. In BPA + P females, a reduction of retinoic acid was found in the liver, suggesting an increase in retinoids responsible for BPA detoxification. Overall, these results demonstrate that SLAB51 exerts its beneficial effects on the gut microbiota-brain-liver axis through distinct molecular pathways, effectively mitigating the pleiotropic toxicity of BPA.

Loss of the importin Kpna2 causes infertility in male mice by disrupting the translocation of testis-specific transcription factors.

Karyopherins mediate the movement between the nucleus and cytoplasm of specific proteins in diverse cellular processes. Through a loss-of-function approach, we here examine the role of Karyopherin Subunit Alpha 2 (Kpna2) in spermatogenesis. Knockout male mice exhibited reduced body size and sperm motility, increased sperm abnormalities, and led to the dysregulation of testis gene expression and ultimately to infertility. Impaired mRNA expression mainly affected clusters of genes expressed in spermatids and spermatocytes. Downregulated genes included a set of genes that participate in cell adhesion and extracellular matrix (ECM) organization. We detected both the enrichment of some transcription factors that bind to regions around transcription start sites of downregulated genes and the impaired transport of specific factors to the nucleus of spermatid cells. We propose that Kpna2 is essential in the seminiferous tubules for promoting the translocation of testis-specific transcription factors that control the expression of genes related to ECM organization.

An ancient testis-specific IQ motif-containing H gene regulates specific transcript isoform expression during spermatogenesis.

Spermatogenic cells express more alternatively spliced RNAs than most whole tissues; however, the regulation of these events remains unclear. Here, we have characterized the function of a testis-specific IQ motif-containing H gene (Iqch) using a mutant mouse model. We found that Iqch is essential for the specific expression of RNA isoforms during spermatogenesis. Using immunohistochemistry of the testis, we noted that Iqch was expressed mainly in the nucleus of spermatocyte and spermatid, where IQCH appeared juxtaposed with SRRM2 and ERSP1 in the nuclear speckles, suggesting that interactions among these proteins regulate alternative splicing (AS). Using RNA-seq, we found that mutant Iqch produces alterations in gene expression, including the clear downregulation of testis-specific lncRNAs and protein-coding genes at the spermatid stage, and AS modifications - principally increased intron retention - resulting in complete male infertility. Interestingly, we identified previously unreported spliced transcripts in the wild-type testis, while mutant Iqch modified the expression and use of hundreds of RNA isoforms, favouring the expression of the canonical form. This suggests that Iqch is part of a splicing control mechanism, which is essential in germ cell biology.

Bull spermatozoa selected by thermotaxis exhibit high DNA integrity, specific head morphometry, and improve ICSI outcome.

BACKGROUND: Sperm migration by thermotaxis is a guidance mechanism that operates along the oviduct and it has proved to be a valid method for selecting spermatozoa with low DNA fragmentation (SDF) in mice, humans, and stallions. This study aimed to analyse if bull spermatozoa could be selected by thermotaxis and to assess their quality in terms of SDF as well as determine the presence of a specific sperm subpopulation based on sperm morphometry and assess their fertilizing capacity by ICSI. METHODS: We used frozen-thawed sperm from 6 bulls and sperm selection by thermotaxis was performed with TALP medium supplemented with 25 mmol/L of HEPES and 5 mmol/L of caffeine. In these conditions, sperm selection was achieved, obtaining a net thermotaxis of 3.6%. Subsequently, we analysed the SDF of the migrated and not-migrated spermatozoa using the neutral COMET assay, and we evaluated the size of the sperm head using Hemacolor® staining with Motic Images Plus 3 software. Additionally, migrated and not-migrated spermatozoa by thermotaxis were used to fertilize bovine in vitro matured (IVM) oocytes by ICSI, a very inefficient procedure in cattle that is only successful when the oocyte is artificially activated. RESULTS: The results showed lower SDF (χ², P < 0.001, 13.3% reduction, n = 8) and lower head size parameters (length and width, P < 0.01; and perimeter and area, P < 0.001; n = 4) in those spermatozoa migrated in comparison to those not-migrated. The distribution of sperm subpopulations structure varied between groups, highlighting cluster 2, characterized by spermatozoa with small head size, and high ellipticity and elongated heads, as the most abundant in the thermotaxis migrated group. When performed ICSI (without oocyte artificial activation) with the thermotactic sperm, the blastocyst rate was 32.2% ± 9.3% in the group microinjected with the thermotactic spermatozoa vs. 8.3% ± 7.8% in the group of not-migrated sperm (χ², P < 0.05). CONCLUSION: Our results showed that bull sperm selection by thermotaxis has a much higher DNA integrity, small and elongated head size parameters, and different sperm subpopulation structure than the not-selected spermatozoa. Additionally, we evidenced that thermotactic spermatozoa improve ICSI success rates.

Preeclampsia Correlates with an Increase in Cannabinoid Receptor 1 Levels Leading to Macromolecular Alterations in Chorionic Villi of Term Placenta.

Preeclampsia is a human pregnancy-specific disease characterized by abnormal placentation that usually presents with maternal hypertension and proteinuria. The main hallmark of preeclampsia, impaired trophoblast migration, and the subsequent disruption of uterine arteries remodeling lead to several molecular alterations in the placental compartments with those occurring in the chorionic villi being of the utmost importance. Given the essential role of the endocannabinoid system during preimplantation and trophoblast migration, we have combined the histological and hyperspectral imaging analyses to shed light on the involvement of two cannabinoid receptors in the macromolecular alterations related to preeclampsia. The results obtained by immunohistochemistry showed a significant increase in the protein levels of cannabinoid receptor 1 (CB1) in the preeclamptic chorionic villi. However, no changes were reported regarding transient receptor potential vanilloid 1 (TRPV-1) levels either in the bulk placental samples or chorionic villi when comparing control and preeclamptic patients. Histological analysis and Fourier-transform infrared spectroscopy (FTIRI) showed an increase in collagen deposition together with higher levels of lipid peroxidation and phosphorylated compounds in the pathological villi. Since CB1 enhancement has been described as promoting fibrosis and oxidative stress in several tissues, we proposed that the higher receptor abundance in preeclampsia could be triggering similar molecular effects in preeclamptic term placentas.

Sperm Metabolomics through Nuclear Magnetic Resonance Spectroscopy.

This report reviews current knowledge of sperm metabolomics analysis using proton nuclear magnetic resonance spectroscopy (1 H-NMR) with particular emphasis on human and farm animals. First, we present the benefits of NMR over other techniques to identify sperm metabolites and then describe the specific methodology required for NMR sperm analysis, stressing the importance of analyzing metabolites extracted from both the hydrophilic and lipophilic phases. This is followed by a description of advances produced to date in the use of NMR to diagnose infertility in humans and to identify metabolic differences among the sperm of mammalian herbivore, carnivore, and omnivore species. This last application of NMR mainly seeks to explore the possible use of lipids to fuel sperm physiology, contrary to previous theories that glycolysis and oxidative phosphorylation (OXPHOS) are the only sources of sperm energy. This review describes the use of NMR to identify sperm and seminal plasma metabolites as possible indicators of semen quality, and to examine the metabolites needed to maintain sperm motility, induce their capacitation, and consequently, to predict animal fertility.

Effects of bisphenol A exposure during cardiac cell differentiation.

Heart development requires a precise temporal regulation of gene expression in cardiomyoblasts. Therefore, the transcriptional changes in differentiating cells can lead to congenital heart diseases. Although the genetic mutations underlie most of these alterations, exposure to environmental contaminants, such as bisphenol A (BPA), has been recently considered as a risk factor as well. In this study we investigated the genotoxic and epigenotoxic effects of BPA throughout cardiomyocyte differentiation. H9c2 cells (rat myoblasts) were exposed to 10 and 30 µM BPA before and during the last two days of cardiac-driven differentiation. Then, we have analysed the phenotypic and molecular modifications (at transcriptional, genetic and epigenetic level). The results showed that treated myoblasts developed a skeletal muscle cell-like phenotype. The transcriptional changes induced by BPA in genes codifying proteins involved in heart differentiation and function depend on the window of exposure to BPA. The exposure before differentiation repressed the expression of heart transcription factors (Hand2 and Gata4), whereas exposure during differentiation reduced the expression of cardiac-specific genes (Tnnt2, Myom2, Sln, and Atp2a1). Additionally, significant effects were observed regarding DNA damage and histone acetylation levels after the two periods of BPA exposure: in cells exposed to the toxicant the percentage of DNA repair foci (formed by the co-localization of γH2AX and 53BP1) increased in a dose-dependent manner, whereas the treatment with the toxicant triggered a decrease in the epigenetic marks H3K9ac and H3K27ac. Our in vitro results reveal that BPA seriously interferes with the process of cardiomyocyte differentiation, which could be related to the reported in vivo effects of this toxicant on cardiogenesis.

Paternal Inheritance of Bisphenol A Cardiotoxic Effects: The Implications of Sperm Epigenome.

Parental exposure to bisphenol A (BPA) has been linked to a greater incidence of congenital diseases. We have demonstrated that BPA induces in zebrafish males an increase in the acetylation of sperm histones that is transmitted to the blastomeres of the unexposed progeny. This work is aimed to determine whether histone hyperacetylation promoted by paternal exposure to BPA is the molecular mechanism underlying the cardiogenesis impairment in the descendants. Zebrafish males were exposed to 100 and 2000 µg/L BPA during early spermatogenesis and mated with non-exposed females. We analyzed in the progeny the expression of genes involved in cardiogenesis and the epigenetic profile. Once the histone hyperacetylation was confirmed, treatment with epigallocatechin gallate (EGCG), an inhibitor of histone acetyltransferases, was assayed on F1 embryos. Embryos from males exposed to 2000 µg/L BPA overexpressed the transcription factor hand2 and the receptor esr2b, showing their own promoters-as well as that of kat6a-an enrichment in H3K9ac. In embryos treated with EGCG, both gene expression and histone acetylation (global and specific) returned to basal levels, and the phenotype was recovered. As shown by the results, the histone hyperacetylated landscape promoted by BPA in the sperm alters the chromatin structure of the progeny, leading to the overexpression of the histone acetyltransferase and genes involved in cardiogenesis.

The effects of endocrine disruptors on the male germline: an intergenerational health risk.

Environmental pollution is becoming one of the major concerns of society. Among the emerging contaminants, endocrine-disrupting chemicals (EDCs), a large group of toxicants, have been the subject of many scientific studies. Besides the capacity of these compounds to interfere with the endocrine system, they have also been reported to exert both genotoxic and epigenotoxic effects. Given that spermatogenesis is a coordinated process that requires the involvement of several steroid hormones and that entails deep changes in the chromatin, such as DNA compaction and epigenetic remodelling, it could be affected by male exposure to EDCs. A great deal of evidence highlights that these compounds have detrimental effects on male reproductive health, including alterations to sperm motility, sexual function, and gonad development. This review focuses on the consequences of paternal exposure to such chemicals for future generations, which still remain poorly known. Historically, spermatozoa have long been considered as mere vectors delivering the paternal haploid genome to the oocyte. Only recently have they been understood to harbour genetic and epigenetic information that plays a remarkable role during offspring early development and long-term health. This review examines the different modes of action by which the spermatozoa represent a key target for EDCs, and analyses the consequences of environmentally induced changes in sperm genetic and epigenetic information for subsequent generations.

Distribution of DNA damage in the human sperm nucleus: implications of the architecture of the sperm head.

The sperm nucleus is prone to sustain DNA damage before and after ejaculation. Distribution of the damage is not homogeneous, and the factors determining differential sensitivity among nuclear regions have not yet been characterized. Human sperm chromatin contains three structural domains, two of which are considered the most susceptible to DNA damage: the histone bound domain, harboring developmental related genes, and the domain associated with nuclear matrix proteins. Using a quantitative polymerase chain reaction (qPCR) approach, we analyzed the number of lesions in genes homeobox A3 (HOXA3), homeobox B5 (HOXB 5), sex-determining region Y (SRY)-box 2 (SOX2), ß-GLOBIN, rDNA 18S, and rDNA 28S in human sperm after ultraviolet irradiation (400 µW cm-2, 10 min), H2O2treatment (250 mmol l-1, 20 min), and cryopreservation, which showed differential susceptibility to genetic damage. Differential vulnerability is dependent on the genotoxic agent and independent of the sperm nuclear proteins to which the chromatin is bound and of accessibility to the transcription machinery. Immunodetection of 8-hydroxy-2'-deoxyguanosine (8-OHdG) showed that the highest level of oxidation was observed after H2O2treatment. The distribution of oxidative lesions also differed depending on the genotoxic agent. 8-OHdG did not colocalize either with histone 3 (H3) or with type IIα + ß topoisomerase (TOPO IIα + ß) after H2O2treatment but matched perfectly with peroxiredoxin 6 (PRDX6), which is involved in H2O2metabolism. Our study reveals that the characteristics of the sperm head domains are responsible for access of the genotoxicants and cause differential degree of damage to nuclear areas, whereas chromatin packaging has a very limited relevance. The histone-enriched genes analyzed cannot be used as biomarkers of oxidative DNA damage.

Genetic and epigenetic alterations induced by bisphenol A exposure during different periods of spermatogenesis: from spermatozoa to the progeny.

Exposure to bisphenol A (BPA) has been related to male reproductive disorders. Since this endocrine disruptor also displays genotoxic and epigenotoxic effects, it likely alters the spermatogenesis, a process in which both hormones and chromatin remodeling play crucial roles. The hypothesis of this work is that BPA impairs early embryo development by modifying the spermatic genetic and epigenetic information. Zebrafish males were exposed to 100 and 2000 µg/L BPA during early spermatogenesis and during the whole process. Genotoxic and epigenotoxic effects on spermatozoa (comet assay and immunocytochemistry) as well as progeny development (mortality, DNA repairing activity, apoptosis and epigenetic profile) were evaluated. Exposure to 100 µg/L BPA during mitosis slightly increased sperm chromatin fragmentation, enhancing DNA repairing activity in embryos. The rest of treatments promoted high levels of sperm DNA damage, triggering apoptosis in early embryo and severely impairing survival. Regarding epigenetics, histone acetylation (H3K9Ac and H3K27Ac) was similarly enhanced in spermatozoa and embryos from males exposed to all the treatments. Therefore, BPA male exposure jeopardizes embryonic survival and development due to the transmission of a paternal damaged genome and of a hyper-acetylated histone profile, both alterations depending on the dose of the toxicant and the temporal window of exposure.

Embryonic Exposure to Bisphenol A Impairs Primordial Germ Cell Migration without Jeopardizing Male Breeding Capacity.

A large amount of chemicals are released to the environment each year. Among them, bisphenol A (BPA) is of utmost concern since it interferes with the reproductive system of wild organisms due to its capacity to bind to hormone receptors. Additionally, BPA epigenotoxic activity is known to affect basic processes during embryonic life. However, its effects on primordial germ cells (PGCs) proliferation and migration, both mechanisms being crucial for gametogenesis, remain unknown. To investigate the effects of BPA on PGCs migration and eventual testicle development, zebrafish embryos were exposed to 100, 2000 and 4000 µg/L BPA during the first 24 h of development. Vasa immunostaining of PGCs revealed that exposure to 2000 and 4000 µg/L BPA impaired their migration to the genital ridge. Two pivotal genes of PGCs migration (cxcr4b and sdf1a) were highly dysregulated in embryos exposed to these doses, whereas DNA methylation and epigenetic marks in PGCs and their surrounding somatic cells were not altered. Once embryos reached adulthood, the morphometric study of their gonads revealed that, despite the reduced number of PGCs which colonized the genital ridges, normal testicles were developed. Although H3K9ac decreased in the sperm from treated fishes, it did not affect the progeny development.

Cardiogenesis impairment promoted by bisphenol A exposure is successfully counteracted by epigallocatechin gallate.

Exposure to the emerging contaminant bisphenol A (BPA) is ubiquitous and associated with cardiovascular disorders. BPA effect as endocrine disruptor is widely known but other mechanisms underlying heart disease, such as epigenetic modifications, remain still unclear. A compound of green tea, epigallocatechin gallate (EGCG), may act both as anti-estrogen and as inhibitor of some epigenetic enzymes. The aims of this study were to analyze the molecular processes related to BPA impairment of heart development and to prove the potential ability of EGCG to neutralize the toxic effects caused by BPA on cardiac health. Zebrafish embryos were exposed to 2000 and 4000 µg/L BPA and treated with 50 and 100 µM EGCG. Heart malformations were assessed at histological level and by confocal imaging. Expression of genes involved in cardiac development, estrogen receptors and epigenetic enzymes was analyzed by qPCR whereas epigenetic modifications were evaluated by whole mount immunostaining. BPA embryonic exposure led to changes in cardiac phenotype, induced an overexpression of hand2, a crucial factor for cardiomyocyte differentiation, increased the expression of estrogen receptor (esr2b), promoted an overexpression of a histone acetyltransferase (kat6a) and also caused an increase in histone acetylation, both mechanisms being able to act in sinergy. EGCG treatment neutralized all the molecular alterations caused by BPA, allowing the embryos to go on with a proper heart development. Both molecular mechanisms of BPA action (estrogenic and epigenetic) likely lying behind cardiogenesis impairment were successfully counteracted by EGCG treatment.

Tolerance to paternal genotoxic damage promotes survival during embryo development in zebrafish (Danio rerio).

Spermatozoa carry DNA damage that must be repaired by the oocyte machinery upon fertilization. Different strategies could be adopted by different vertebrates to face the paternal genotoxic damage. Mammals have strong sperm selection mechanisms and activate a zygotic DNA damage response (DDR) (including cell cycle arrest, DNA repair and alternative apoptosis) in order to guarantee the genomic conformity of the reduced progeny. However, external fertilizers, with different reproductive strategies, seem to proceed distinctively. Previous results from our group showed a downregulation of apoptotic activity in trout embryos with a defective DNA repairing ability, suggesting that mechanisms of tolerance to damaged DNA could be activated in fish to maintain cell survival and to progress with development. In this work, zebrafish embryos were obtained from control or UV-irradiated sperm (carrying more than 10% of fragmented DNA but still preserving fertilization ability). DNA repair (γH2AX and 53BP1 foci), apoptotic activity, expression of genes related to DDR and malformation rates were analyzed throughout development. Results showed in the progeny from damaged sperm, an enhanced repairing activity at the mid-blastula transition stage that returned to its basal level at later stages, rendering at hatching a very high rate of multimalformed larvae. The study of transcriptional and post-translational activity of tp53 (ZDF-GENE-990415-270) revealed the activation of an intense DDR in those progenies. However, the downstream pro-apoptotic factor noxa (ZDF-GENE-070119-3) showed a significant downregulation, whereas the anti-apoptotic gene bcl2 (ZDF-GENE-051015-1) was upregulated, triggering a repressive apoptotic scenario in spite of a clear genomic instability. This repression can be explained by the observed upregulation of p53 isoform Δ113p53, which is known to inhibit bcl2 transcription. Our results showed that tp53 is involved in DNA damage tolerance (DDT) pathways, allowing the embryo survival regardless of the paternal DNA damage. DDT could be an evolutionary mechanism in fish: tolerance to unrepaired sperm DNA could introduce new mutations, some of them potentially advantageous to face a changing environment.

Transgenerational inheritance of heart disorders caused by paternal bisphenol A exposure.

Bisphenol A (BPA) is an endocrine disruptor used in manufacturing of plastic devices, resulting in an ubiquitous presence in the environment linked to human infertility, obesity or cardiovascular diseases. Both transcriptome and epigenome modifications lie behind these disorders that might be inherited transgenerationally when affecting germline. To assess potential effects of paternal exposure on offspring development, adult zebrafish males were exposed to BPA during spermatogenesis and mated with non-treated females. Results showed an increase in the rate of heart failures of progeny up to the F2, as well as downregulation of 5 genes involved in cardiac development in F1 embryos. Moreover, BPA causes a decrease in F0 and F1 sperm remnant mRNAs related to early development. Results reveal a paternal inheritance of changes in the insulin signaling pathway due to downregulation of insulin receptor ß mRNAs, suggesting a link between BPA male exposure and disruption of cardiogenesis in forthcoming generations.