Comparison of DNA damage in granulosa cells of women undergoing controlled ovarian stimulation in in vitro fertilization protocols with the recombinant human follicle-stimulating hormones Corneumon®, Gonal-F®, Pergoveris® and Puregon®: a randomized trial.

PURPOSE: To compare the DNA damage in granulosa cells (GCs) of women undergoing ovarian-stimulated cycles with four widely used recombinant human follicle-stimulating hormones (rhFSH) in in vitro fertilization (IVF) protocols (Corneumon®, Gonal-F®, Pergoveris® and Puregon®). METHODS: A randomized trial was carried out at a Mexican hospital. GCs were isolated from 18 women with infertility undergoing assisted reproductive techniques (ART). Four controlled ovarian stimulation (COS) protocols including Corneumon®, Gonal-F®, Pergoveris® or Puregon® were used. GCs DNA damage was assessed by the Comet assay. Two parameters were measured: comet tail length (CTL), and Olive tail moment (OTM, the percentage of DNA in the tail multiplied by the distance between the center of the tail and head). RESULTS: Use of the different hrFSH in COS caused variable and statistically significant levels of DNA damage in GCs of infertile women. CTL was similar in the Corneumon® and Pergoveris® groups (mean values of 48.73 and 55.18, respectively) and Corneumon® CTL was significantly lower compared to the Gonal-F® and Puregon® groups (mean values of 61.98 and 91.17, respectively). Mean OTM values were significantly lower in Corneumon® and Pergoveris® groups, compared to Gonal-F® and Puregon® groups (25.59, 27.35, 34.76, and 47.27, respectively). CONCLUSION: Use of Corneumon® and Pergoveris® in COS caused statistically significantly lower levels of DNA damage in GCs of infertile women undergoing ART, which could potentially correlate with better reproductive outcomes.

Chronic stress decreases fertility parameters in female rats.

Multiple effects of stress on health have been reported; however, reproductive alterations in oocytes and cumulus cells have not been fully described. In females, chronic stress has been shown to produce alterations in the estrous cycle, to decrease oocyte in vivo maturation, and to increase the percentage of abnormal oocytes. The aim of this study was to evaluate whether the oocytes from chronically stressed female rats could recover and mature in vitro by providing them with all the necessary culture conditions, as well as to evaluate the functionality of the GAP junctions, and the viability and DNA integrity of the cumulus cells, which are crucial for the complete maturation and development of the oocyte. For this, rats were stressed daily by cold water immersion (15 °C) during 15 min for 30 consecutive days. Corticosterone serum levels in rats increased as an indicator of stress. Chronic stress decreased the percentage of in vitro matured oocytes because the cumulus cells presented irreparable damage to their DNA that led to their death, being unable to establish bidirectional communication with the oocyte for its meiotic resumption through the GAP junctions, which were also damaged. These findings could partially explain an association between stress and infertility.

miRNAs: A potentially valuable tool in pesticide toxicology assessment-current experimental and epidemiological data review.

miRNAs are responsible for the regulation of many cellular processes such as development, cell differentiation, proliferation, apoptosis, and tumor growth. Several studies showed that they can also serve as specific, stable, and sensitive markers of chemical exposure. In this review, current experimental and epidemiological data evidencing deregulation in miRNA expression in response to fungicides, insecticides or herbicides were analyzed. As shown by Venn's diagrams, miR-363 and miR-9 deregulation is associated with fungicide exposure in vitro and in vivo, while let-7, miR-155, miR-181 and miR-21 were found to be commonly deregulated by at least three different insecticides. Furthermore, let-7, miR-30, miR-126, miR-181 and miR-320 were commonly deregulated by 3 different herbicides. Notably, these 5 miRNAs were also found to be deregulated by one or more insecticides, suggesting their participation in the cellular response to pesticides, regardless of their chemical structure. All these miRNAs have been proposed as potential biomarkers for fungicide, insecticide, or herbicide exposure. These results allow us to improve our understanding of the molecular mechanisms of toxicity upon pesticide exposure, although further studies are needed to confirm these miRNAs as definitive (not potential) biomarkers of pesticide exposure.

DNA damage in cumulus cells generated after the vitrification of in vitro matured porcine oocytes and its impact on fertilization and embryo development.

BACKGROUND: The evaluation of the DNA damage generated in cumulus cells after mature cumulus-oocyte complexes vitrification can be considered as an indicator of oocyte quality since these cells play important roles in oocyte developmental competence. Therefore, the aim of this study was to determine if matured cumulus-oocyte complexes exposure to cryoprotectants (CPAs) or vitrification affects oocytes and cumulus cells viability, but also if DNA damage is generated in cumulus cells, affecting fertilization and embryo development. RESULTS: The DNA damage in cumulus cells was measured using the alkaline comet assay and expressed as Comet Tail Length (CTL) and Olive Tail Moment (OTM). Results demonstrate that oocyte exposure to CPAs or vitrification reduced oocyte (75.5 ± 3.69%, Toxicity; 66.7 ± 4.57%, Vitrification) and cumulus cells viability (32.7 ± 5.85%, Toxicity; 7.7 ± 2.21%, Vitrification) compared to control (95.5 ± 4.04%, oocytes; 89 ± 4.24%, cumulus cells). Also, significantly higher DNA damage expressed as OTM was generated in the cumulus cells after exposure to CPAs and vitrification (39 ± 17.41, 33.6 ± 16.69, respectively) compared to control (7.4 ± 4.22). In addition, fertilization and embryo development rates also decreased after exposure to CPAs (35.3 ± 16.65%, 22.6 ± 3.05%, respectively) and vitrification (32.3 ± 9.29%, 20 ± 1%, respectively). It was also found that fertilization and embryo development rates in granulose-intact oocytes were significantly higher compared to denuded oocytes in the control groups. However, a decline in embryo development to the blastocyst stage was observed after CPAs exposure (1.66 ± 0.57%) or vitrification (2 ± 1%) compared to control (22.3 ± 2.51%). This could be attributed to the reduction in both cell types viability, and the generation of DNA damage in the cumulus cells. CONCLUSION: This study demonstrates that oocyte exposure to CPAs or vitrification reduced viability in oocytes and cumulus cells, and generated DNA damage in the cumulus cells, affecting fertilization and embryo development rates. These findings will allow to understand some of the mechanisms of oocyte damage after vitrification that compromise their developmental capacity, as well as the search for new vitrification strategies to increase fertilization and embryo development rates by preserving the integrity of the cumulus cells.

Effects of methylparaben on in vitro maturation of porcine oocytes.

Parabens (PBs) are compounds widely used in industry for food and personal care products as antimicrobials and preservatives. Their indiscriminate use has resulted in their detection in different ecosystems so that humans and other organisms are highly exposed. Methylparaben (MePB), compared with other PBs, is mostly detected in food, personal care and baby care products. PBs could be linked to the generation of hormonal disorders and fertility impairment since their recent classification as endocrine disruptors. The knowledge of the effects that MePB can exert is of great importance as, in terms of reproduction, information is limited. Therefore, the objective of the present study was to evaluate the effect of MePB on porcine oocyte viability and in vitro maturation (IVM), as well as to determine the lethal concentration at 50% (LC50 ) and the maturation inhibition concentration at 50% (MIC50 ). Oocytes were exposed to different MePB concentrations 0 (control), 50, 100, 500, 750 and 1000 µm during 44 h of IVM. Cytoplasmic alterations and reduced cumulus cell expansion were observed in oocytes exposed to MePB; however, viability was not affected. In addition, oocyte maturation decreased in a concentration-dependent manner after exposure to MePB. The estimated LC50 was 2028.38 µm, whereas MIC50 was 780.31 µm. To our knowledge, this is the first study that demonstrates that MePB altered porcine oocyte morphology, and caused a reduction in cumulus cell expansion, both of which resulted in decreased oocyte maturation. Therefore, MePB exposure may be one of the factors involved in fertility impairment in mammals, including that of humans.

GPR30 expression and function in breast cancer cells are induced through a cis­acting element targeted by ETS factors.

The capacity that G protein­coupled receptor 30 (GPR30) has demonstrated for triggering estrogen­dependent signaling pathways has attracted the interest of breast cancer researchers; however, the reported expression profiles and functions of GPR30 in breast cancer are inconsistent. The main purpose of the present investigation was to identify transcriptional mechanisms underlying the expression of GPR30 that allow a better understanding of its role in breast cancer progression. In the cell lines used as different polarity models in the present study, it was determined immunologically that GPR30 is expressed in normal mammary gland cells and that this expression decreased considerably during breast cancer development, where cell identity is lost. However, it was also determined that, in spite of low GPR30 expression levels in breast cancer cells with little differentiation, this membrane estrogen receptor (ER) is able to increase cell viability and suppress migration in cells that have acquired metastatic capacity. In addition, through transient expression assays in breast cancer cells, it was revealed that a transcriptional mechanism dependent on protein kinase A and susceptible to retinoic acid in ER­positive cells induces GPR30 expression through a cis­regulatory element for E26 transformation­specific transcription factors, located between ­631 and ­625 bp from the GPR30 translation start codon. Overall, these results suggested that in vitro transcriptional regulation of GPR30 expression in breast cancer cells may serve a relevant role in the conservation of an epithelial phenotype, and also may be important to avoid the transition to metastasis.

Low bone mineral density and renal malformation in Mexican patients with Turner syndrome are associated with single nucleotide variants in vitamin D-metabolism genes.

Turner syndrome (TS) is a common genetic disorder. TS-phenotype includes short stature, gonadal dysgenesis, cardiac and kidney malformations, low bone mineral density (low-BMD) and thyroiditis. TS-phenotype varies from patient to patient and the cause is not clear, the genomic background may be an important contributor for this variability. Our aim was to identify the association of specific single nucleotide variants in the PTPN22, VDR, KL, and CYP27B1 genes and vitamin D-metabolism, heart malformation, renal malformation, thyroiditis, and low-BMD in 61 Mexican TS-patients. DNA samples were genotyped for SNVs: rs7975232 (VDR), rs9536282 (KL), rs4646536 (CYP27B1), and rs1599971 (PTPN22) using the KASP assay. Chi-square test under a recessive model and multifactorial dimensionality reduction method were used for analysis. We found a significant association between renal malformation and the rs9536282 (KL) variant and between rs4646536 (CYP27B1) and low-BMD, these variants may have modest effects on these characteristics but contribute to the variability of the TS phenotype. In addition, we identified gene-gene interactions between variants in genes KL, CYP27B1 and VDR related to vitamin D-metabolism and low-BMD in TS-patients. Our results support the idea that the genetic background of TS-patients contributes to the clinical variability seen in them.

Perfluorooctanoic acid disrupts gap junction intercellular communication and induces reactive oxygen species formation and apoptosis in mouse ovaries.

Perfluorooctanoic acid (PFOA) is a member of the perfluoroalkyl acid family of compounds. Due to the presence of strong carbon-fluorine bonds, it is practically nonbiodegradable and highly persistent in the environment. PFOA has been detected in the follicular fluid of women, and positively associated with reduced fecundability and infertility. However, there are no reports concerning the experimental evaluation of PFOA on oocyte toxicity in mammals. The aim of the present study was to determine if PFOA is able to induce oxidative stress in fetal ovaries and cause apoptosis in oocytes in vitro. In addition, since inhibition of the gap junction intercellular communication (GJIC) by PFOA has been demonstrated in liver cells in vivo and in vitro, the effect of PFOA on the GJIC between the oocyte and its supportive cumulus cells was studied. Results show that PFOA induced oocyte apoptosis and necrosis in vitro (medium lethal concentration, LC50 = 112.8 µM), as evaluated with Annexin-V-Alexa 508 in combination with BOBO-1 staining. Reactive oxygen species (ROS) levels, as assessed by DCFH-DA, increased significantly in fetal ovaries exposed to » LC50 (28.2 µM, a noncytotoxic and relevant occupational exposure concentration) and LC50 PFOA ex vivo. This perfluorinated compound also caused the blockage of GJIC in cumulus cells-oocyte complexes (COCs) obtained from female mice exposed in vivo, as evaluated by calcein transfer from cumulus cells to the oocyte. The ability of PFOA of disrupting the GJIC in COCs, generating ROS in the fetal ovary and causing apoptosis and necrosis in mammal's oocytes, might account for the reported association between increasing maternal plasma concentrations of PFOA with reduced fertility in women.

Endocrine disruptor effect of perfluorooctane sulfonic acid (PFOS) and perfluorooctanoic acid (PFOA) on porcine ovarian cell steroidogenesis.

Previous studies with perfluorooctanesulfonic acid (PFOS) and perfluorooctanoic acid (PFOA) indicate that they act as endocrine disruptors, in addition to inducing alterations and damaging reproductive health; however, the biological mechanisms by which these disorders are produced are not yet understood. The aim of this study was to analyze the effect of PFOS and PFOA on in vitro steroidogenic secretion in porcine theca and granulosa cells, with or without gonadotropic stimulation. Granulosa and theca cells were isolated and cultured. Cell nature was performed by immunocytochemistry. PFOS and PFOA effect on steroid secretion was analyzed by chemiluminescence. In the present study, alterations in steroidogenic secretion were found when administering PFOS (0.12, 1.2, 12, 120 or 240µM) or PFOA (0.012, 0.12, 1.2, 12 or 24µM) to theca and granulosa cells. When theca and granulosa cells were stimulated with 500ng/mL LH or 500ng/mL FHS, respectively and immediately followed with 1.2µM of PFOS or PFOA, the perfluorinated compounds inhibited the secretion of steroid hormones in both stimulated cell types. The results indicate that PFOS and PFOA act on steroidogenic ovarian cells as endocrine disruptors, which could affect the dependent functions of sexual steroids.

Oxidative stress as a damage mechanism in porcine cumulus-oocyte complexes exposed to malathion during in vitro maturation.

Malathion is one of the most commonly used insecticides. Recent findings have demonstrated that it induces oxidative stress in somatic cells, but there are not enough studies that have demonstrated this effect in germ cells. Malathion impairs porcine oocyte viability and maturation, but studies have not shown how oxidative stress damages maturation and which biochemical mechanisms are affected in this process in cumulus-oocyte complexes (COCs). The aims of the present study were to determine the amount of oxidative stress produced by malathion in porcine COCs matured in vitro, to define how biochemical mechanisms affect this process, and determine whether trolox can attenuate oxidative damage. Sublethal concentrations 0, 750, and 1000 µM were used to evaluate antioxidant enzyme expressions, reactive oxygen species (ROS production), protein oxidation, and lipid peroxidation, among other oxidation products. COCs viability and oocyte maturation decreased in a concentration-dependent manner. Malathion increased Cu, Zn superoxide dismutase (SOD1), glutathione-S-transferase (GST), and glucose 6 phosphate dehydrogenase (G6PD) protein level and decreased glutathione peroxidase (GSH-Px) and catalase (CAT) protein level. Species reactives of oxygen (ROS), protein oxidation and Thiobarbituric acid reactive substances (TBARS) levels increased in COCs exposed to the insecticide, but when COCs were pre-treated with the trolox (50 µM) 30 min before and during malathion exposure, these parameters decreased down to control levels. This study showed that malathion has a detrimental effect on COCs during in vitro maturation, inducing oxidative stress, while trolox attenuated malathion toxicity by decreasing oxidative damage.

Sebox plays an important role during the early mouse oogenesis in vitro.

Oogenesis is a highly complex process that requires the exquisite temporal and spatial regulation of gene expression at multiple levels. Skin-embryo-brain-oocyte homeobox (Sebox) gene encodes a transcription factor that is highly expressed in germinal vesicle stage oocytes and that plays an essential role in early embryogenesis at the 2-cell stage in the mouse. As Sebox is also expressed in mouse fetal ovaries, the aim of the present study was to study its role during the early oogenesis in vitro. Expression of Sebox was low in 15.5 to 17.5 days post coitum (dpc) ovaries, showed a peak at 18.5 dpc and then its expression decreased dramatically in newborn ovaries. Sebox expression was efficiently knocked down (>80%) in fetal mouse ovary explants in culture using RNAi technology. When fetal ovary explants were transfected with Sebox-specific RNAi, the number of oocytes at germinal vesicle stage and showing a diameter of 40-70 µm was decreased significantly to 75% after 7 days of culture relative to the negative control, and to 22.4% after 10 days of culture, thus indicating that Sebox plays an important role in the early oogenesis in mice.

Role of thyroid hormones and mir-208 in myocardial remodeling in 5/6 nephrectomized rats.

BACKGROUND AND AIMS: Thyroid hormones exert important effects on heart remodeling through mir-208. The process may have a role in myocardial changes in chronic kidney disease where thyroid abnormalities are common. In this study the effect of T4 supplementation on left ventricle (LV) remodeling in 5/6 nephrectomized rats (5/6Nx) was analyzed. METHODS: 5/6Nx rats and 5/6Nx under T4 supplementation (5/6Nx + T4) were compared with control (C) and thyroidectomized (Tx) rats. After 8 weeks of follow-up, LV was analyzed for α-MHC, ß-MHC, TGF-ß, and mir-208 expression, hydroxyproline content, and myocardial fibrosis. Serum collagenase activity was also analyzed. RESULTS: Heart weight increased in 5/6Nx rats compared to C, which was prevented with T4 supplementation (C, 1.5 ± 0.04; 5/6Nx, 1.8 ± 0.09; 5/6Nx + T4, 1.6 ± 0.07 g, p <0.05). The same pattern was seen for LV wall thickness, hydroxyproline content, LV fibrosis, and mRNA TGF-ß expression (C, 0.47 ± 0.17; 5/6Nx, 10.55 ± 3.4; 5/6Nx + T4, 3.01 ± 0.52, p <0.01). Tx rats had reduction in heart weight, increased LV wall thickness, and fibrosis. Collagenase activity did not change in any group. mRNA expression of α-, ß-MHC, and TGF-ß increased in 5/6Nx in comparison to C and 5/6Nx + T4. Expression of mir-208 decreased in 5/6Nx groups, and levels were restored with T4 supplementation (4.21 ± 0.28, 3.39 ± 0.29, and 4.26 ± 0.37 RU, respectively, p <0.01). CONCLUSIONS: Decreased plasma level of thyroid hormones or sensitivity at tissue level observed in chronic kidney disease induced by 5/6Nx has an important effect in heart remodeling processes, some of it related or mediated by mir-208 and TGF-ß expression in the heart.

Effect of porcine follicular fluid proteins and peptides on oocyte maturation and their subsequent effect on in vitro fertilization.

The follicular fluid (FF) is a microenvironment that contains molecules involved in oocyte maturation, ovulation, and fertilization. Characterizing the proteins and peptides present in the FF could be useful for determining which proteins and peptides to use as a supplement for culture media. Biologically active peptides produced during the maturation or degradation of functional proteins are called cryptides. The aim of this study was to identify the proteins and cryptides in porcine FF that could stimulate porcine oocyte in vitro maturation (IVM) and in vitro fertilization (IVF) when added to culture maturation medium. Five FF protein fractions (F1-F5) were obtained by ionic exchange chromatography, resolved by SDS-PAGE, and identified by tandem mass spectrometry. These fractions had effects on IVM and/or IVF. The F1 fraction, which was composed of immunoglobulin fragments, cytokeratin, transferrin, and plasminogen precursor increased IVM and IVF. The F2, F3, and F4 fractions reduced the percentage of oocytes in first metaphase. Additionally, the F3 fraction, which was composed of immunoglobulins and transthyretin, interfered with germinal vesicle breakdown. The F5 fraction, which was mainly composed of serum albumin and keratin, favored germinal vesicle breakdown and promoted IVM. Most of the 31 proteins which were associated with the immune response and inflammatory processes could be related to oocyte maturation and fertilization. Some of the identified proteins were present in more than one fraction; this could be explained by a change in their isoelectric points, because of the loss of part of the amino acid sequence or a change in the glycosylation status of the protein. Improved oocyte IVM and IVF will increase embryo production, which in turn will contribute to the efficiency of assisted reproduction in various mammalian species.

Effect of malnutrition on the expression of cytokines involved in Th1 cell differentiation.

Malnutrition is a common cause of secondary immune deficiency and has been linked to an increased susceptibility to infection in humans. Malnutrition specifically affects T-cell-mediated immune responses. The aim of this study was to assess in lymphocytes from malnourished children the expression levels of IL-12, IL-18 and IL-21, molecules that induce the differentiation of T cells related to the immunological cellular response (Th1 response) and the production of cytokines related to the immunological cellular response (Th1 cytokines). We found that the expression levels of IL-12, IL-18 and IL-21 were significantly diminished in malnourished children compared to well-nourished children and were coincident with lower plasmatic levels of IL-2 and IFN-γ (Th1 cytokines). In this study, we show for the first time that the gene expression and intracellular production of cytokines responsible for Th1 cell differentiation (IL-12, IL-18 and IL-21) are diminished in malnourished children. As expected, this finding was related to lower plasmatic levels of IL-2 and IFN-γ. The decreased expression of Th1 cytokines observed in this study may contribute to the deterioration of the immunological Type 1 (cellular) response. We hypothesize that the decreased production of IL-12, IL-18 and IL-21 in malnourished children contributes to their inability to eradicate infections.

Deregulation of the Sod1 and Nd1 genes in mouse fetal oocytes exposed to mono-(2-ethylhexyl) phthalate (MEHP).

Mono-(2-ethylhexyl) phthalate (MEHP) is the active metabolite of the diester (DEHP), a well-known reproductive toxicant. Since, different molecular mechanisms underlying this toxicity are not well understood, the effects of MEHP on cell viability and gene expression were assessed in murine fetal oocytes cultured in vitro. Oocyte survival decreased significantly after a 24h exposure to MEHP and hence, the effects of MEHP on changes in gene expression were analyzed using cDNA libraries and differential screenings. In these assays, the genes that suffered the most severe deregulation corresponded to those encoding Cu-Zn superoxide dismutase (Sod1) and a mitochondrial respiratory chain protein (Nd1). Indeed, functional assays on somatic cells transfected with a Sod1 luciferase reporter construct demonstrated its specific MEHP dose-dependent up-regulation, confirming that the expression of this gene is deregulated in response to MEHP.

Differential effects of herbicides atrazine and fenoxaprop-ethyl, and insecticides diazinon and malathion, on viability and maturation of porcine oocytes in vitro.

Exposure to pesticides may be a major cause of reproductive dysfunction in humans and animals. Atrazine and fenoxaprop-ethyl, widely used herbicides, and malathion and diazinon, organophosphate insecticides, are considered only slightly toxic to vertebrates; however, there is evidence of greater effects on reproductive function. The aim of this study was to evaluate the effect of these pesticides on oocyte viability and in vitro maturation. Gametes were matured in increasing concentrations of the pesticides and then stained with MTT to evaluate viability and bisbenzimide to assess the maturation stage, in the same oocyte. Atrazine had no effect on viability but maturation was significantly reduced, while fenoxaprop-ethyl affected both parameters. The insecticides affected viability and maturation but to a different degree. The four pesticides showed a more pronounced effect on maturation than on viability, due to a blockage at germinal vesicle stage.

Identification and characterization of novel mammalian spermatogenic genes conserved from fly to human.

Spermatogenesis is a complex and highly regulated developmental process by which round spermatogonial stem cells undergo mitotic proliferation and meiosis, followed by extraordinary differentiation into highly specialized elongated mature sperm. Extensive differences in terms of sperm production such as testicular structure and organization, hormonal regulation are reported between humans and insects, yet it is not known to what extent components of the process could be conserved and furthermore to what extent the underlying genetic regulators could be shared from insects to mammals. We hence take a genomic approach to identify genes which are expressed in the testes of both fly and mouse through in silico analysis and are phylogenetically conserved across metazoans. Fifty eight testis-enriched, phylogenetically conserved from fly to mouse genes were identified. Among them, 12 genes are novel. Detailed characterization of their murine and human homologs indicate most of them are testis-restricted or enriched and developmentally regulated, thus suggesting that they are important regulators of sperm development in mammals and potential human fertility factors. Our results reveal the existence of spermatogenic homologs with similar testicular expression across a large evolutionary distance, further functional study will be needed to explore the functional conservation among those spermatogenic orthologs.

Effects of the insecticides malathion and diazinon on the early oogenesis in mice in vitro.

Malathion and diazinon are two of the most commonly used organophosphorous (OP) agrochemicals. Several studies show that these pesticides exert several effects on mammalian spermatogenesis. Nevertheless, there are no studies concerning their effects on oogenesis. The objective of this study was to evaluate the effects of these insecticides on the viability of in vitro cultured mouse oocytes during the early oogenesis and to get a further understanding of the molecular mechanisms by which OP insecticides act and affect germinal cells. Oocytes were cultured from fetal ovaries for 10 days, when most oocytes had reached the diplotene stage (germinal vesicle stage). Cultures were exposed to different concentrations of malathion or diazinon for 24 h, and the effect on oocyte viability was assessed. Gene expression in oocytes exposed to the insecticides was analyzed by generating cDNA libraries and performing differential screenings. Results show a significant decrease in oocytes survival after 24-h exposure to 250 microM malathion or 900 nM diazinon, and the effect of these insecticides on the regulation of genes encoding proteins involved in transcription (BP75), translation (ribosomal protein S5), and mitochondrial function (cytochrome oxidase subunits I and III), providing evidence for OP insecticides as toxicants for mammals oocytes during the early oogenesis.

Gene expression analysis on the early development of pig embryos exposed to malathion.

Malathion is a widely used pesticide and there is evidence that it could alter mammal's germ and somatic cells, as well as cell lines. There are not enough studies showing how the nonacute malathion doses affect gene expression. This study analyzes gene expression alterations in pig morular embryos exposed in vitro, for 96 h, to several malathion concentrations after in vitro fertilization. cDNA libraries of isolated morular embryos were created and differential screenings performed to identify target genes. Seven clones were certainly identified. Genes related to mitochondrial metabolism as cytochrome c subunits I and III, nuclear genes such as major histocompatibility complex I (MHC I), and a hypothetical protein related with a splicing factor were the target of malathion's deregulation effect. The widespread use of malathion as a pesticide should be regarded with reproductive implications and more detailed analysis would yield more about molecular mechanisms of malathion injury on embryo cells.

Differential gene expression in lymphocytes from malnourished children.

Malnutrition, which is widespread in developing countries, may be particularly devastating during childhood, when tissue development is occurring and nutrient requirements are great. Since protein-energy malnutrition potentially involves many cellular alterations, we have evaluated gene expression changes in lymphocytes from malnourished children using differential hybridization cloning. A cDNA library was generated from well-nourished children and differential screenings were performed with cDNAs obtained from well-nourished and malnourished children who presented with bacterial gastrointestinal infections. Differential expression was detected for genes involved in cell development and differentiation, and for genes involved in lymphocyte and mitochondrial functions. The genes detected in the present study suggest mechanisms for the changes in cell growth and immune function that are associated with protein-energy malnutrition. Two down-regulated genes in malnourished children may represent mechanisms of protection against immunosuppression. This finding clearly merits further investigation.