Edmundo Bonilla

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 μM 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.

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.

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.

Deregulation of gene expression in fetal oocytes exposed to doxorubicin.

Doxorubicin is an effective anticancer drug but its use is limited due to its adverse side effects such as infertility and cardiomyopathy. Some possible mechanisms of the action of doxorubicin have been postulated, but the initial gene deregulation response has not been investigated. Fetal life stages are critical periods in mammalian oogenesis. This study analyzes gene expression alterations in mouse fetal oocytes exposed in vitro to this anticancer agent. cDNA libraries were generated from isolated fetal oocytes and differential screenings performed with cDNAs from in vitro doxorubicin-treated and -untreated oocytes. Differentially expressed genes were assessed by real-time RT-PCR to quantify the extent of their transcriptional control in doxorubicin-exposed oocytes. The results show that doxorubicin alters the expression of genes involved in the mitochondrial respiratory chain, intracellular transport and cell differentiation. Finally, the up-regulation of a differentially expressed gene (metaxin) mediated by its promoter was evaluated in a functional assay. When treated with doxorubicin, somatic cells transfected with a genetic construct including the promoter of metaxin and a reporter gene showed increases in expression similar to those observed in fetal oocytes. This demonstrates the direct effect of agent on the regulation of a specific gene.

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.

Changes in cyclin B localisation during pig oocyte in vitro maturation

The localisation of cyclin B throughout in vitro maturation of pig oocytes was determined by indirect immunofluorescence using a monoclonal antibody specific for an epitope of the human cyclin B. Maturation of pig oocytes was induced by addition of Pergonal (2 UI/ml of FSH/LH) and beta-oestradiol to the medium where isolated ovarian follicles were cultured for up to 72 h. Immature gametes with an intact germinal vesicle were observed during the first 30 h of culture. Only 10% were competent to reinitiate meiosis and showed germinal vesicle breakdown (GVBD) after 36 h. However, after 48-72 h, 60% of the oocytes accomplished their maturation and showed metaphase chromosomes. Immature oocytes showed cyclin B immunofluorescent staining in the cytoplasm, whereas mature oocytes showed the immunofluorescent label concentrated in the nucleus. Metaphase chromosomes showed an intense immunofluorescence. The migration of cyclin B to the nucleus and its association with metaphase chromosomes in pig oocytes able to progress through meiosis resembled the subcellular localisation of cyclin B and the distribution of maturation promoting factor (MPF) in mitotic dividing cells.

In vitro fertilizing capacity of frozen-thawed boar semen

We describe a porcine semen cryopreservation technique and assess the in vitro fertilizing capacity of the frozen-thawed spermatozoa. The thawed spermatozoa did not lose the physiological properties of motility, viability, and acrosome reaction or capacity to fertilize in vitro. Immediately after thawing, the spermatozoa showed 51% mean motility, 60% viability, and 5% induced acrosome reaction. After 2.5 h of incubation in TALP medium, the spermatozoa exhibited 61% motility, 63% viability and 40% induced acrosome reaction. The average in vitro fertilization capacity of thawed spermatozoa was 68% compared with that of spermatozoa from fresh semen (85%). The percentage of polyspermy was highly variable, with frozen-thawed samples ranging from 0 to 28% and fresh samples from 0 to 30%. The results obtained with frozen semen from 5 boars of different breeds did not show considerable variation. This suggests that the freezing-thawing technique is reproducible and adequate for in vitro fertilization.

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.

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 Mael in early oogenesis and during germ-cell differentiation from embryonic stem cells in mice in vitro

In a previous study, we have identified a set of conserved spermatogenic genes whose expression is restricted to testis and ovary and that are developmentally regulated. One of these genes, the transcription factor Mael, has been reported to play an essential role in mouse spermatogenesis. Nevertheless, the role of Mael in mouse oogenesis has not been defined. In order to analyse the role of Mael in mouse oogenesis, the expression of this gene was blocked during early oogenesis in mouse in vitro using RNAi technology. In addition, the role of Mael during differentiation of embryonic stem cells (ESC) into germ cells in vitro was analysed. Results show that downregulation of Mael by a specific short interfering RNA disrupted fetal oocyte growth and differentiation in fetal ovary explants in culture and the expression of several germ-cell markers in ESC during their differentiation. These results suggest that there is an important role for Mael in early oogenesis and during germ-cell differentiation from embryonic stem cells in mouse in vitro.