Mechanism of Action of an Environmentally Relevant Organochlorine Mixture in Repressing Steroid Hormone Biosynthesis in Leydig Cells.

Within Leydig cells, steroidogenesis is induced by the pituitary luteinizing hormone (LH). The binding of LH to its receptor increases cAMP production, which then activates the expression of genes involved in testosterone biosynthesis. One of these genes codes for the steroidogenic acute regulatory (STAR) protein. STAR is part of a complex that shuttles cholesterol, the precursor of all steroid hormones, through the mitochondrial membrane where steroidogenesis is initiated. Organochlorine chemicals (OCs) are environmental persistent organic pollutants that are found at high concentrations in Arctic areas. OCs are known to affect male reproductive health by decreasing semen quality in different species, including humans. We previously showed that an environmentally relevant mixture of OCs found in Northern Quebec disrupts steroidogenesis by decreasing STAR protein levels without affecting the transcription of the gene. We hypothesized that OCs might affect STAR protein stability. To test this, MA-10 Leydig cell lines were incubated for 6 h with vehicle or the OCs mixture in the presence or absence of 8Br-cAMP with or without MG132, an inhibitor of protein degradation. We found that MG132 prevented the OC-mediated decrease in STAR protein levels following 8Br-cAMP stimulation. However, progesterone production was still decreased by the OC mixture, even in the presence of MG132. This suggested that proteins involved in steroid hormone production in addition to STAR are also affected by the OC mixture. To identify these proteins, a whole cell approach was used and total proteins from MA-10 Leydig cells exposed to the OC mixture with or without stimulation with 8Br-cAMP were analyzed by 2D SDS-PAGE and LC-MS/MS. Bioinformatics analyses revealed that several proteins involved in numerous biological processes are affected by the OC mixture, including proteins involved in mitochondrial transport, lipid metabolism, and steroidogenesis.

Adipose Tissue Transcriptome Is Related to Pollutant Exposure in Polar Bear Mother-Cub Pairs from Svalbard, Norway.

Being at the food chain apex, polar bears (Ursus maritimus) are highly contaminated with persistent organic pollutants (POPs). Females transfer POPs to their offspring through gestation and lactation; therefore, young cubs present higher POPs concentrations than their mothers. Recent studies suggest that POPs affect the lipid metabolism in female polar bears; however, the mechanisms and impact on their offspring remain unknown. Here, we hypothesized that exposure to POPs differentially alters genome-wide gene transcription in the adipose tissue from mother polar bears and their cubs, highlighting molecular differences in response between adults and young. Adipose tissue biopsies were collected from 13 adult female polar bears and their twin cubs in Svalbard, Norway, in April 2011, 2012, and 2013. Total RNA extracted from biopsies was subjected to next-generation RNA sequencing. Plasma concentrations of summed polychlorinated biphenyls, organochlorine pesticides, and polybrominated diphenyl ethers in mothers ranged from 897 to 13620 ng/g wet weight and were associated with altered adipose tissue gene expression in both mothers and cubs. In mothers, 2502 and 2586 genes in total were positively and negatively, respectively, correlated to POP exposure, whereas in cubs, 2585 positively and 1690 negatively genes. Between mothers and cubs, 743 positively and negatively genes overlapped between mothers and cubs suggesting partially shared molecular responses to ΣPOPs. ΣPOP-associated genes were involved in numerous metabolic pathways in mothers and cubs, indicating that POP exposure alters the energy metabolism, which, in turn, may be linked to metabolic dysfunction.

Inferring and modeling inheritance of differentially methylated changes across multiple generations.

High-throughput methylation sequencing enables genome-wide detection of differentially methylated sites (DMS) or regions (DMR). Increasing evidence suggests that treatment-induced DMS can be transmitted across generations, but the analysis of induced methylation changes across multiple generations is complicated by the lack of sound statistical methods to evaluate significance levels. Due to software design, DMS detection was usually made on each generation separately, thus disregarding stochastic effects expected when a large number of DMS is detected in each generation. Here, we present a novel method based on Monte Carlo sampling, methylInheritance, to evaluate that the number of conserved DMS between several generations is associated to an effect inherited from a treatment and not randomness. Moreover, we developed an inheritance simulation package, methInheritSim, to demonstrate the performance of the methylInheritance method and to evaluate the power of different experimental designs. Finally, we applied methylInheritance to a DNA methylation dataset obtained from early-life persistent organic pollutants (POPs) exposed Sprague-Dawley female rats and their descendants through a paternal transmission. The results show that metylInheritance can efficiently identify treatment-induced inherited methylation changes. Specifically, we identified two intergenerationally conserved DMS at transcription start site (TSS); one of those persisted transgenerationally. Three transgenerationally conserved DMR were found at intra or integenic regions.

Cell-lineage specificity of primary cilia during postnatal epididymal development.

STUDY QUESTION: Where are primary cilia (PC) organelles located during postnatal epididymal development? SUMMARY ANSWER: Our findings unveil the existence of PC sensory organelles in different epididymal cell types according to postnatal development stage. WHAT IS KNOWN ALREADY: Primary cilia are sensory organelles that orchestrate major signaling pathways during organ development and homeostasis. Epididymal PC have been detected in the horses, donkey and mules but their cell-lineage specificity has never been investigated in this organ. STUDY DESIGN, SIZE, DURATION: A longitudinal study was performed by examining tissue from n = 3 to n = 10 transgenic mice at different times of postnatal development. Tissues were fixed by intracardiac perfusion and the epididymides collected. PARTICIPANTS/MATERIALS, SETTING, METHODS: Transmission electron microscopy and confocal microscopy/3D reconstruction were used on a double transgenic mouse model expressing endogenous fluorescence in PC and centrioles (Arl13b-mCherry/Centrin2-GFP). Several PC parameters (i.e. length, orientation relative to the lumen) were quantified by using an image-processing pipeline. Epididymal tissues and serum-free cultures of DC2 immortalized epididymal principal murine cell lines were used to identify primary ciliary signaling components. MAIN RESULTS AND THE ROLE OF CHANCE: We report here a constitutive localization of PC in peritubular myoid cells and a dynamic profiling in epithelial cells throughout postnatal epididymal development. While PC are present at the apical pole of the undifferentiated epithelial cells from birth to puberty, they are absent from the apical pole of the epithelium in adults, where they appear exclusively associated with cytokeratin 5-positive basal cells. We determined that PC from epididymal cells are associated with polycystin 1 (PC1), polycystin 2 (PC2), and Gli-3 Hedgehog signaling transcription factor. No inter-individual variability was observed within each age group. LIMITATIONS, REASONS FOR CAUTION: As our present study is descriptive and performed exclusively in the mouse, future functional studies will be required to unravel the contribution of these organelles in the control of reproductive functions. WIDER IMPLICATIONS OF THE FINDINGS: Acknowledging the important roles played by PC sensory organelles in organ homeostasis and development in humans, our work opens new avenues of research concerning the cellular control of epididymal functions, which are essential to male fertility. STUDY FUNDING/COMPETING INTEREST(S): Study funded by an NSERC operating grant to CB (RGPIN-2015-109194). No competing interest to declare.

Histomorphologic Analysis of the Late-term Rat Fetus and Placenta.

Histological examination of the rat placenta and fetus is uncommon. Toxicological studies mainly rely on gross examination of the fetus and on fetal and placental weights. These are often insufficient to assess the fetal and placental toxicity of xenobiotics. The small size of the fetus makes its dissection labor-intensive. Thus, our objective was to develop a simple and accurate technique to evaluate the rat fetus and placenta. Sprague-Dawley rat fetuses at gestational day 19.5 ( n = 18) and their placentas ( n = 32) were fixed in formalin. Placentas were cut transversally in the center. Fetuses were cut following a freehand whole-body serial sectioning diagram adapted from Wilson's method. Sections were stained with hematoxylin-eosin-phloxine-saffron, and histomorphometry was used to measure the area of the fetal placental region (27.2 ± 1.7 mm2), including the labyrinth (22.2 ± 1.0 mm2) and the basal zone (4.8 ± 0.8 mm2). Our whole-fetus serial sectioning technique resulted in 12 precise cutting planes that fit on 3 histological slides, enabling the examination of most organs without labor-intensive dissection. Quantitative analysis of placental areas improves the understanding of the pathogenesis of treatment-related changes. This technique provides a standardized method for future research in pertinent fields such as developmental biology and toxicology.

Genome-wide analysis of sperm DNA methylation from monozygotic twin bulls.

Monozygotic (MZ) twins are of great interest to elucidate the contributions of pre- and postnatal environmental factors on epigenetics in the expression of complex traits and diseases. Progeny testing recently revealed that MZ twin bulls do not necessarily lead to identical genetic merit estimates (i.e. breeding values). Therefore, to explain differences in offspring productivity of MZ twin bulls despite their identical genetic backgrounds, we hypothesised that paternal sperm epigenomes vary between MZ twin bulls. In the present study, semen characteristics and global sperm DNA methylome were profiled for four pairs of MZ twin bulls. Some MZ twin pairs had divergent semen quality (sperm morphology, motility and viability). Comparative genome-wide DNA methylome surveys were performed using methyl-sensitive enrichment and microarray identification. Between 2% and 10% of all probes (400000) were differentially methylated between MZ twin pairs. In addition, there were 580 loci differentially methylated across all pairs of MZ twins. Furthermore, enrichment analysis indicated a significant enrichment for fertility associated quantitative trait loci (P=0.033). In conclusion, differences in the sperm epigenome may contribute to incongruous diverging performances of daughters sired by bulls that are MZ twins.

Novel technical strategies to optimize cryopreservation of goat semen using cholesterol-loaded cyclodextrin.

Artificial insemination is well-established in dairy cattle, with sires housed in commercial studs for processing. In some species, however, sires located on-farm are used for artificial insemination by shipping their semen to an off-site laboratory for processing within 24 h of collection. To expedite semen transport from the farm to laboratory, protocols must be uncomplicated. For goat semen, an obstacle is the seminal plasma, which must be removed because it contains proteins that impede sperm quality. Our objective is to develop a simple strategy to transiently store goat semen for 24 h prior to freezing. Cholesterol-loaded cyclodextrin (CLC) has been demonstrated to improve sperm tolerance to cryopreservation. Therefore, we hypothesized that CLC improves goat sperm resistance to seminal plasma damage, over 24 h prior to cryopreservation. We first evaluated the ability of CLC to protect goat sperm against seminal plasma damage by treating fresh semen with or without seminal plasma prior to cryopreservation. Second, fresh goat semen with seminal plasma was extended in skim milk-based extender ± CLC and held for 24 h at 5 °C prior to freezing. Our results indicate that CLC treatment improves goat sperm resistance to seminal plasma-mediated injury and protects sperm quality over 24 h prior to freezing (P < 0.05). Although the in vivo fertility of semen must first be assessed, it is possible that protocols for goat semen cryopreservation can be simplified by including CLC and eliminating seminal plasma removal. Processing and distribution of goat semen for AI would thereby be facilitated.

Cholesterol-Loaded Cyclodextrin Increases the Cholesterol Content of Goat Sperm to Improve Cold and Osmotic Resistance and Maintain Sperm Function after Cryopreservation.

The success of semen cryopreservation depends on sperm membrane integrity and function after thawing. Cholesterol-loaded cyclodextrin (CLC) is used for in vitro incorporation of cholesterol to protect cells against cold temperatures. We hypothesized that CLC treatment also enhances sperm cholesterol content to increase tolerance to osmotic shock and cryoresistance, thereby improving fertility. We confirmed the fact that treatment of goat semen with 3 mg/ml CLC increases sperm cholesterol content using both the Liebermann-Burchard approach and filipin III labeling of membrane cholesterol. Sperm were then treated with or without CLC and cryopreserved. After thawing, sperm cholesterol dramatically fell, even in the presence of CLC, which explains the mechanism of cryocapacitation. CLC treatment, however, maintained a normal prefreeze cholesterol level in sperm after cryopreservation. Furthermore, fresh sperm treated with CLC and subjected to either cold shock or incubated in hypo-, iso-, and hyperosmotic media, designed to mimic stresses associated with freezing/thawing, displayed increased temperature and osmotic tolerance. CLC treatment also improved sperm viability, motility, and acrosome integrity after thawing. Furthermore, CLC treatment did not affect the sperm's ability to undergo in vitro capacitation according to chlortetracycline fluorescence and protein tyrosine phosphorylation. A pilot field trial demonstrated that artificial insemination with sperm that underwent increased cholesterol levels following CLC treatment yielded higher fertility ( ITALIC! P< 0.1) and proliferation ( ITALIC! P< 0.05) rates in vivo than untreated semen from the same ejaculate samples. These observations suggest that CLC treatment could be used to improve cryoprotection during the freezing and thawing of goat sperm.

In boar sperm capacitation L-lactate and succinate, but not pyruvate and citrate, contribute to the mitochondrial membrane potential increase as monitored via safranine O fluorescence.

Having ascertained using JC-1 as a probe that, in distinction with the controls, during capacitation boar sperm maintains high mitochondrial membrane potential (ΔΨ), to gain some insight into the role of mitochondria in capacitation, we monitored ΔΨ generation due to externally added metabolites either in hypotonically-treated spermatozoa (HTS) or in intact cells by using safranine O as a probe. During capacitation, the addition to HTS of L-lactate and succinate but not those of pyruvate, citrate and ascorbate + TMPD resulted in increase of ΔΨ generation. Accordingly, the addition of L-lactate and succinate, but not that of citrate, to intact sperm resulted in ΔΨ generation increased in capacitation.

In vitro exposure of Leydig cells to an environmentally relevant mixture of organochlorines represses early steps of steroidogenesis.

Leydig cell steroidogenesis is mainly regulated by LH via increased cAMP production leading to STAR protein activation. STAR is essential for cholesterol shuttling inside mitochondria where steroidogenesis is initiated. Accumulating evidence suggest that persistent organochlorine compounds disrupt testicular function, but the mechanism of action remains poorly characterized. Here we report that in vitro exposure of MA-10 and MLTC-1 Leydig cells to an environmentally relevant mixture of 15 organochlorines impairs steroidogenesis. While having no effect on cell viability and basal steroid production, the organochlorine mixture caused a 50% decrease in cAMP-induced progesterone production. The mixture also reduced cAMP-induced 30 kDa STAR protein by 50% while having no effect on basal STAR protein. Basal or cAMP-induced Star mRNA levels and promoter activity were unaffected by the mixture, indicating that the organochlorine mixture acted at the translational/posttranslational level. Further supporting this is the fact that in COS-7 cells overexpressing STAR, the organochlorine mixture caused a decrease in the 30 kDa form of STAR and an accumulation of the 37 kDa forms. In addition to STAR, we found that the organochlorine mixture also decreases the levels of CYP11A1 and ADXR, two proteins essential for the conversion of cholesterol into pregnenolone. In conclusion, our data show that organochlorine exposure disrupts Leydig cell function by targeting different components of the steroidogenic pathway.

The Association between Long-Term DDT or DDE Exposures and an Altered Sperm Epigenome-a Cross-Sectional Study of Greenlandic Inuit and South African VhaVenda Men.

BACKGROUND: The organochlorine dichlorodiphenyltrichloroethane (DDT) is banned worldwide owing to its negative health effects. It is exceptionally used as an insecticide for malaria control. Exposure occurs in regions where DDT is applied, as well as in the Arctic, where its endocrine disrupting metabolite, p,p'-dichlorodiphenyldichloroethylene (p,p'-DDE) accumulates in marine mammals and fish. DDT and p,p'-DDE exposures are linked to birth defects, infertility, cancer, and neurodevelopmental delays. Of particular concern is the potential of DDT use to impact the health of generations to come via the heritable sperm epigenome. OBJECTIVES: The objective of this study was to assess the sperm epigenome in relation to p,p'-DDE serum levels between geographically diverse populations. METHODS: In the Limpopo Province of South Africa, we recruited 247 VhaVenda South African men and selected 50 paired blood serum and semen samples, and 47 Greenlandic Inuit blood and semen paired samples were selected from a total of 193 samples from the biobank of the INUENDO cohort, an EU Fifth Framework Programme Research and Development project. Sample selection was based on obtaining a range of p,p'-DDE serum levels (mean=870.734±134.030 ng/mL). We assessed the sperm epigenome in relation to serum p,p'-DDE levels using MethylC-Capture-sequencing (MCC-seq) and chromatin immunoprecipitation followed by sequencing (ChIP-seq). We identified genomic regions with altered DNA methylation (DNAme) and differential enrichment of histone H3 lysine 4 trimethylation (H3K4me3) in sperm. RESULTS: Differences in DNAme and H3K4me3 enrichment were identified at transposable elements and regulatory regions involved in fertility, disease, development, and neurofunction. A subset of regions with sperm DNAme and H3K4me3 that differed between exposure groups was predicted to persist in the preimplantation embryo and to be associated with embryonic gene expression. DISCUSSION: These findings suggest that DDT and p,p'-DDE exposure impacts the sperm epigenome in a dose-response-like manner and may negatively impact the health of future generations through epigenetic mechanisms. Confounding factors, such as other environmental exposures, genetic diversity, and selection bias, cannot be ruled out. https://doi.org/10.1289/EHP12013.

A Novel ENU-Induced Mfn2 Mutation Causes Motor Deficits in Mice without Causing Peripheral Neuropathy.

Mitochondrial fission and fusion are required for maintaining functional mitochondria. The mitofusins (MFN1 and MFN2) are known for their roles in mediating mitochondrial fusion. Recently, MFN2 has been implicated in other important cellular functions, such as mitophagy, mitochondrial motility, and coordinating endoplasmic reticulum-mitochondria communication. In humans, over 100 MFN2 mutations are associated with a form of inherited peripheral neuropathy, Charcot-Marie-Tooth disease type 2A (CMT2A). Here we describe an ENU-induced mutant mouse line with a recessive neuromuscular phenotype. Behavioral screening showed progressive weight loss and rapid deterioration of motor function beginning at 8 weeks. Mapping and sequencing revealed a missense mutation in exon 18 of Mfn2 (T1928C; Leu643Pro), within the transmembrane domain. Compared to wild-type and heterozygous littermates, Mfn2L643P/L643P mice exhibited diminished rotarod performance and decreases in activity in the open field test, muscular endurance, mean mitochondrial diameter, sensory tests, mitochondrial DNA content, and MFN2 protein levels. However, tests of peripheral nerve physiology and histology were largely normal. Mutant leg bones had reduced cortical bone thickness and bone area fraction. Together, our data indicate that Mfn2L643P causes a recessive motor phenotype with mild bone and mitochondrial defects in mice. Lack of apparent nerve pathology notwithstanding, this is the first reported mouse model with a mutation in the transmembrane domain of the protein, which may be valuable for researchers studying MFN2 biology.

Exposure to environmental contaminants and folic acid supplementation intergenerationally impact fetal skeleton development through the paternal lineage in a rat model.

Persistent organic pollutants (POPs) are ubiquitous in the environment, which is of concern since they are broadly toxic for wildlife and human health. It is generally accepted that maternal prenatal folic acid supplementation (FA) may beneficially impact offspring development, but it has been recently shown that the father's exposures also influence the health of his offspring. Bone is an endocrine organ essential for whole-body homeostasis and is susceptible to toxicants. Herein, we tested the hypotheses that prenatal paternal exposure to POPs induces developmental bone disorders in fetuses across multiple generations and that FA supplementation attenuates these disorders. We used a four-generation rat model, in which F0 founder females were divided into four treatment groups. F0 females were gavaged with corn oil or an environmentally-relevant POPs mixture and fed either a control diet (2 mg FA/kg), or FA supplemented diet (6 mg FA/kg) before mating and until parturition (four treatments in total). After the birth of the F1 litters, all F0 females and subsequent generations received the FA control diet. Staining with alcian blue and alizarin red S of male and female fetal skeletons was performed at Gestational Day 19.5. Paternal direct and ancestral exposure to POPs delayed bone ossification and decreased the length of long limb bones in fetuses. Maternal FA supplementation did not counteract the POPs-associated delayed fetal ossification and reduced long bone length. In conclusion, prenatal paternal POPs exposure causes developmental bone abnormalities over multiple generations, which were not corrected by maternal FA supplementation.

ACRBP (Sp32) is involved in priming sperm for the acrosome reaction and the binding of sperm to the zona pellucida in a porcine model.

In boar sperm, we have previously shown that capacitation is associated with the appearance of the p32 tyrosine phosphoprotein complex. The principal tyrosine phosphoprotein involved in this complex is the acrosin-binding protein (ACRBP), which regulates the autoconversion of proacrosin to intermediate forms of acrosin in both boar and mouse sperm. However, the complete biological role of ACRBP has not yet been elucidated. In this study, we tested the hypothesis that tyrosine phophorylation and the presence of the ACRBP in the sperm head are largely necessary to induce capacitation, the acrosome reaction (AR) and sperm-zona pellucida (ZP) binding, all of which are necessary steps for fertilization. In vitro fertilization (IVF) was performed using matured porcine oocytes and pre-capacitated boar sperm cultured with anti-phosphotyrosine antibodies or antibodies against ACRBP. Anti-ACRBP antibodies reduced capacitation and spontaneous AR (P<0.05). Sperm-ZP binding declined in the presence of anti-phosphotyrosine or anti-ACRBP antibodies. The localisation of anti-ACRBP antibodies on the sperm head, reduced the ability of the sperm to undergo the AR in response to solubilized ZP or by inhibiting the sarco/endoplasmic reticulum Ca2+-ATPase. These results support our hypothesis that tyrosine phosphorylated proteins and ACRBP are present upon the sperm surface in order to participate in sperm-ZP binding, and that ACRBP upon the surface of the sperm head facilitates capacitation and the AR in the porcine.

Early-Life Exposure to Environmental Contaminants Perturbs the Sperm Epigenome and Induces Negative Pregnancy Outcomes for Three Generations via the Paternal Lineage.

Due to the grasshopper effect, the Arctic food chain in Canada is contaminated with persistent organic pollutants (POPs) of industrial origin, including polychlorinated biphenyls and organochlorine pesticides. Exposure to POPs may be a contributor to the greater incidence of poor fetal growth, placental abnormalities, stillbirths, congenital defects and shortened lifespan in the Inuit population compared to non-Aboriginal Canadians. Although maternal exposure to POPs is well established to harm pregnancy outcomes, paternal transmission of the effects of POPs is a possibility that has not been well investigated. We used a rat model to test the hypothesis that exposure to POPs during gestation and suckling leads to developmental defects that are transmitted to subsequent generations via the male lineage. Indeed, developmental exposure to an environmentally relevant Arctic POPs mixture impaired sperm quality and pregnancy outcomes across two subsequent, unexposed generations and altered sperm DNA methylation, some of which are also observed for two additional generations. Genes corresponding to the altered sperm methylome correspond to health problems encountered in the Inuit population. These findings demonstrate that the paternal methylome is sensitive to the environment and that some perturbations persist for at least two subsequent generations. In conclusion, although many factors influence health, paternal exposure to contaminants plays a heretofore-underappreciated role with sperm DNA methylation contributing to the molecular underpinnings involved.

Localization of Hsp60 and Grp78 in the human testis, epididymis and mature spermatozoa.

Molecular chaperones of the heat shock proteins (HSP) family are important in numerous cellular processes. In this study, the expression of Hsp60 and Grp78 proteins was investigated in the male reproductive tract. The cellular distribution of Hsp60 and Grp78 proteins was analysed in the human testis and epididymis by immunohistochemical approaches. DNA microarray technology was used to analyse HSP60 and GRP78 gene expression along human epididymis. The cellular localization of these chaperone proteins in ejaculated spermatozoa was investigated by indirect immunofluorescence and by Western blot following sperm sub-cellular fractionation. In the human testis, Hsp60 was detected in spermatogonia, whereas a strong Grp78 staining was observed in spermatocytes and round spermatids. Grp78 protein was also observed in the epididymal epithelium, whereas no Hsp60 staining was observed in this organ by immunohistochemistry. The presence of both Hsp60 and Grp78 RNA in human epididymis was confirmed by microarrays. In ejaculated spermatozoa, Hsp60 was localized in the mid-piece, whereas Grp78 was detected in the neck region. These results indicate that in addition to being expressed in human testis spermatogenic cells, both Hsp60 and Grp78 proteins persist in ejaculated spermatozoa. These findings are in agreement with the involvement of Hsp60 and Grp78 during spermatogenesis and in sperm functions such as fertilization.

Beyond fertilisation: How the paternal environment influences future generations.

In light of the relatively ignored role of paternal influences on offspring development and increasing societal concerns regarding possible health consequences of chemical exposures, our team has addressed the overall hypothesis that environmentally-relevant levels of contaminants have long-lasting effects that are transmitted through the paternal lineage. This review focuses on our research examining the impact of developmental exposure to toxicants and nutrients on the phenotype and epigenome of the male and of his subsequent generations. This report is intended to encourage animal andrologists as well as the domestic animal production industry to increase their consideration of the sire's environment in the context of agricultural productivity.

Maternal folic acid supplementation does not counteract the deleterious impact of prenatal exposure to environmental pollutants on lipid homeostasis in male rat descendants.

Prenatal exposure to persistent organic pollutants (POPs) has been associated with the development of metabolic syndrome-related diseases in offspring. According to epidemiological studies, father's transmission of environmental effects in addition to mother's can influence offspring health. Moreover, maternal prenatal dietary folic acid (FA) may beneficially impact offspring health. The objective is to investigate whether prenatal FA supplementation can overcome the deleterious effects of prenatal exposure to POPs on lipid homeostasis and inflammation in three generations of male rat descendants through the paternal lineage. Female Sprague-Dawley rats (F0) were exposed to a POPs mixture (or corn oil) +/- FA supplementation for 9 weeks before and during gestation. F1 and F2 males were mated with untreated females. Plasma and hepatic lipids were measured in F1, F2, and F3 males after 12-h fast. Gene expression of inflammatory cytokines was determined by qPCR in epididymal adipose tissue. In F1 males, prenatal POPs exposure increased plasma lipids at 14 weeks old and hepatic lipids at 28 weeks old and prenatal FA supplementation decreased plasma total cholesterol at 14 weeks old. Prenatal POPs exposure decreased plasma triglycerides at 14 weeks old in F2 males. No change was observed in inflammatory markers. Our results show an impact of the paternal lineage on lipid homeostasis in rats up to the F2 male generation. FA supplementation of the F0 diet, regardless of POPs exposure, lowered plasma cholesterol in F1 males but failed to attenuate the deleterious effects of prenatal POPs exposure on plasma and hepatic lipids in F1 males.

Modulation of bovine sperm signalling pathways: correlation between intracellular parameters and sperm capacitation and acrosome exocytosis.

In the present study, the viability, intracellular pH (pHi), cAMP ([cAMP]i), calcium concentration and protein phosphotyrosine content were evaluated in relation to the acrosomal and capacitation status of freshly ejaculated bull spermatozoa. These parameters were evaluated before and after incubation with the capacitation inducer heparin, the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX), the phosphotyrosyl-protein phosphatase inhibitors phenylarsine oxide (PAO) and sodium orthovanadate, and hydrogen peroxide. The results obtained were integrated to address the physiological interactions between the different signalling events affecting sperm capacitation and acrosome reaction. As expected, heparin promoted the expression of the 'B' pattern of chlortetracycline binding, increased pHi, [cAMP]i and the phosphotyrosine content of sperm proteins. The effects of heparin were enhanced by IBMX. Both PAO and sodium orthovanadate stimulated protein phosphotyrosine content and acrosomal exocytosis, although only PAO affected pH, Ca2+ and cAMP levels. Intracellular pH was increased while both Ca2+ and [cAMP]i were decreased. Physiological concentrations of H2O2 increased [cAMP]i and promoted acrosomal exocytosis. A significant positive correlation was found between sperm capacitation, protein phosphotyrosine content and stored Ca2+ concentration, whereas the acrosome reaction was correlated with pHi and Ca2+ concentration. This study presents the first global analysis of the major elements individually described during sperm capacitation and acrosome reaction signalling pathways, supported by statistical correlations.