Chronic Dietary Exposure to a Low-Dose Mixture of Genistein and Vinclozolin Modifies the Reproductive Axis, Testis Transcriptome, and Fertility

Background: The reproductive consequences and mechanisms of action of chronic exposure to low-dose endocrine disruptors are poorly understood.[br/] Objective: We assessed the effects of a continuous, low-dose exposure to a phytoestrogen (genistein) and/or an antiandrogenic food contaminant (vinclozolin) on the male reproductive tract and fertility.[br/] Methods: Male rats were exposed by gavage to genistein and vinclozolin from conception to adulthood, alone or in combination, at low doses (1 mg/kg/day) or higher doses (10 and 30 mg/kg/day). We studied a number of standard reproductive toxicology end points and also assessed testicular mRNA expression profiles using long-oligonucleotide microarrays.[br/] Results: The low-dose mixture and high-dose vinclozolin produced the most significant alterations in adults: decreased sperm counts, reduced sperm motion parameters, decreased litter sizes, and increased post implantation loss. Testicular mRNA expression profiles for these exposure conditions were strongly correlated. Functional clustering indicated that many of the genes induced belong to the “neuroactive ligand-receptor interactions” family encompassing several hormonally related actors (e.g., follicle-stimulating hormone and its receptor). All exposure conditions decreased the levels of mRNAs involved in ribosome function, indicating probable decreased protein production.[br/] Conclusions: Our study shows that chronic exposure to a mixture of a dose of a phytoestrogen equivalent to that in the human diet and a low dose—albeit not environmental—of a common anti-androgenic food contaminant may seriously affect the male reproductive tract and fertility

Assisted Reproductive Technology affects developmental kinetics, <it>H19 </it>Imprinting Control Region methylation and <it>H19 </it>gene expression in individual mouse embryos

<p>Abstract</p> <p>Background</p> <p>In the last few years, an increase in imprinting anomalies has been reported in children born from Assisted Reproductive Technology (ART). Various clinical and experimental studies also suggest alterations of embryo development after ART. Therefore, there is a need for studying early epigenetic anomalies which could result from ART manipulations, especially on single embryos. In this study, we evaluated the impact of superovulation, <it>in vitro </it>fertilization (IVF) and embryo culture conditions on proper genomic imprinting and blastocyst development in single mouse embryos.</p> <p>In this study, different experimental groups were established to obtain embryos from superovulated and non-superovulated females, either from <it>in vivo </it>or <it>in vitro </it>fertilized oocytes, themselves grown <it>in vitro </it>or not. The embryos were cultured either in M16 medium or in G1.2/G2.2 sequential medium. The methylation status of <it>H19 </it>Imprinting Control Region (ICR) and <it>H19 </it>promoter was assessed, as well as the gene expression level of <it>H19</it>, in individual blastocysts. In parallel, we have evaluated embryo cleavage kinetics and recorded morphological data.</p> <p>Results</p> <p>We show that:</p> <p>1. The culture medium influences early embryo development with faster cleavage kinetics for culture in G1.2/G2.2 medium compared to M16 medium.</p> <p>2. Epigenetic alterations of the <it>H19 </it>ICR and <it>H19 </it>PP are influenced by the fertilization method since methylation anomalies were observed only in the <it>in vitro </it>fertilized subgroup, however to different degrees according to the culture medium.</p> <p>3. Superovulation clearly disrupted <it>H19 </it>gene expression in individual blastocysts. Moreover, when embryos were cultured <it>in vitro </it>after either <it>in vivo </it>or <it>in vitro </it>fertilization, the percentage of blastocysts which expressed <it>H19 </it>was higher in G1.2/G2.2 medium compared to M16.</p> <p>Conclusion</p> <p>Compared to previous reports utilizing pools of embryos, our study enables us to emphasize a high individual variability of blastocysts in the <it>H19 </it>ICR and <it>H19 </it>promoter methylation and <it>H19 </it>gene expression, with a striking effect of each manipulation associated to ART practices. Our results suggest that <it>H19 </it>could be used as a sensor of the epigenetic disturbance of the utilized techniques.</p

Assisted Reproductive Technology affects developmental kinetics, Imprinting Control Region methylation and gene expression in individual mouse embryos-1

<p><b>Copyright information:</b></p><p>Taken from "Assisted Reproductive Technology affects developmental kinetics, Imprinting Control Region methylation and gene expression in individual mouse embryos"</p><p>http://www.biomedcentral.com/1471-213X/7/116</p><p>BMC Developmental Biology 2007;7():116-116.</p><p>Published online 18 Oct 2007</p><p>PMCID:PMC2169233.</p><p></p>ically observed (2 PN) and defined the zygote stage. Zygotes obtained after fertilization (Figure group D) or after fertilization (Figure group C) were then cultured in M16 or G1.2/G2.2 medium. The results are expressed as percentage of total zygote (PN) number at day 0 for each experimental group and each culture condition. At each culture day, the number of atretic embryos was determined based upon the observation of necrosis signs

Assisted Reproductive Technology affects developmental kinetics, Imprinting Control Region methylation and gene expression in individual mouse embryos-2

<p><b>Copyright information:</b></p><p>Taken from "Assisted Reproductive Technology affects developmental kinetics, Imprinting Control Region methylation and gene expression in individual mouse embryos"</p><p>http://www.biomedcentral.com/1471-213X/7/116</p><p>BMC Developmental Biology 2007;7():116-116.</p><p>Published online 18 Oct 2007</p><p>PMCID:PMC2169233.</p><p></p>rect sequencing (Figure ) and cloning/sequencing (Figure ). When Single Nucleotide Polymorphism (C/T) was observed by direct sequencing, the proportion of C in the clone sequences was approximately 50%. When only C or T were detected by direct sequencing, all sequences of analyzed clones presented a methylated or an unmethylated status respectively. Reading the direct sequences, for each blastocyst, the presence of C/T, C or T at one CpG position is represented by the black (methylated) and white (unmethylated) lollipops (Figure )

Assisted Reproductive Technology affects developmental kinetics, Imprinting Control Region methylation and gene expression in individual mouse embryos-4

<p><b>Copyright information:</b></p><p>Taken from "Assisted Reproductive Technology affects developmental kinetics, Imprinting Control Region methylation and gene expression in individual mouse embryos"</p><p>http://www.biomedcentral.com/1471-213X/7/116</p><p>BMC Developmental Biology 2007;7():116-116.</p><p>Published online 18 Oct 2007</p><p>PMCID:PMC2169233.</p><p></p> CTCF binding sites are shaded in grey. Only examples are shown for clarity

Assisted Reproductive Technology affects developmental kinetics, Imprinting Control Region methylation and gene expression in individual mouse embryos-6

<p><b>Copyright information:</b></p><p>Taken from "Assisted Reproductive Technology affects developmental kinetics, Imprinting Control Region methylation and gene expression in individual mouse embryos"</p><p>http://www.biomedcentral.com/1471-213X/7/116</p><p>BMC Developmental Biology 2007;7():116-116.</p><p>Published online 18 Oct 2007</p><p>PMCID:PMC2169233.</p><p></p

Assisted Reproductive Technology affects developmental kinetics, Imprinting Control Region methylation and gene expression in individual mouse embryos-0

<p><b>Copyright information:</b></p><p>Taken from "Assisted Reproductive Technology affects developmental kinetics, Imprinting Control Region methylation and gene expression in individual mouse embryos"</p><p>http://www.biomedcentral.com/1471-213X/7/116</p><p>BMC Developmental Biology 2007;7():116-116.</p><p>Published online 18 Oct 2007</p><p>PMCID:PMC2169233.</p><p></p>y and degree of expansion). The results were expressed as a percentage of the total blastocyst number. In the same experimental group (C or D), significant differences of maturity degree were observed according to the culture medium (χtest, P < 0.05

Assisted Reproductive Technology affects developmental kinetics, Imprinting Control Region methylation and gene expression in individual mouse embryos-7

<p><b>Copyright information:</b></p><p>Taken from "Assisted Reproductive Technology affects developmental kinetics, Imprinting Control Region methylation and gene expression in individual mouse embryos"</p><p>http://www.biomedcentral.com/1471-213X/7/116</p><p>BMC Developmental Biology 2007;7():116-116.</p><p>Published online 18 Oct 2007</p><p>PMCID:PMC2169233.</p><p></p>dard sample. The results are presented as proportion of blastocysts with or without detectable transcripts in expressing blastocysts (Figure ). The relative RNA level is expressed according to the equation: RNA quantity = 2 with ΔΔCt = (Ct()-Ct()- Ct()-Ct()). For each group, individual blastocyst expression is represented. The bars indicate the median expression value. The size of symbols is proportional to blastocyst number with the same expression (Figure )

Assisted Reproductive Technology affects developmental kinetics, Imprinting Control Region methylation and gene expression in individual mouse embryos-8

<p><b>Copyright information:</b></p><p>Taken from "Assisted Reproductive Technology affects developmental kinetics, Imprinting Control Region methylation and gene expression in individual mouse embryos"</p><p>http://www.biomedcentral.com/1471-213X/7/116</p><p>BMC Developmental Biology 2007;7():116-116.</p><p>Published online 18 Oct 2007</p><p>PMCID:PMC2169233.</p><p></p>y and degree of expansion). The results were expressed as a percentage of the total blastocyst number. In the same experimental group (C or D), significant differences of maturity degree were observed according to the culture medium (χtest, P < 0.05