[show abstract][hide abstract] ABSTRACT: Genetically modified animals have numerous applications, ranging from basic research to livestock production and agriculture. Recent progress in animal cloning by nuclear transfer has made possible the production of transgenic animals using previously genetically modified cell lineages. However, to produce such lineages, an additional time for in vitro culturing and great manipulation is needed. Herein, we aimed to characterize different aspects of genetically modified cells compared to control cells, and we also analyzed the development rate of embryos produced by nuclear transfer by using them as nuclei donors after short or long periods of in vitro culturing (early versus late passages). We hypothesized that the genetic material inserted in the genome of these cells, associated with the prolonged time in culture, ultimately alters cell growth physiology and cell viability, which leads to impaired nuclei reprogramming potential and consequent reduction in the production of cloned blastocysts. Fetal fibroblasts expressing the enhanced Green Fluorescent Protein gene (eGFP) cultured for different periods in vitro were analyzed with respect to chromosomal numeric abnormalities, nuclear DNA fragmentation, the ratio of BAX and BCL2 gene transcripts, and the intensity of mitochondrial membrane potential, and they were then used as nuclei donors for somatic cell nuclear transfer (SCNT). Early passages were defined as fewer than 11 passages, and late passages were 18th passage (18(th)p) to 21(st)p. No differences were observed in the percentage of cells with chromosomal abnormalities or in the mitochondrial membrane potential analysis. eGFP cells in late passages and control cells in early passages were not different regarding DNA fragmentation; however, control cells in late passages presented higher fragmentation (P < 0.05). The Bax and Bcl2 gene expression ratio in control and transgenic cells presented different patterns regarding cell conditions during culture. For SCNT experiments, no difference was observed between groups reconstructed with early or late-passage cells when fusion (63.1% and 49%), cleavage (67.7% and 69.9%), eight-cell embryo (36.4% and 44.4%) and blastocyst (21.6% and 20.8%) rates were compared. In conclusion, culture behavior was different between control and eGFP cells. However, when different in vitro culturing periods were compared, long-term cultured transgenic fetal fibroblasts remained competent for blastocyst production when used as nuclei donors in the nuclear transfer technique, a feature needed for the genetic manipulation of cell culture experiments aiming for transgenic animal production.
[show abstract][hide abstract] ABSTRACT: To investigate mechanisms of fetal-maternal cell interactions in the bovine placenta, we developed a model of transgenic enhanced Green Fluorescent Protein (t-eGFP) expressing bovine embryos produced by nuclear transfer (NT) to assess the distribution of fetal-derived products in the bovine placenta. In addition, we searched for male specific DNA in the blood of females carrying in vitro produced male embryos. Our hypothesis is that the bovine placenta is more permeable to fetal-derived products than described elsewhere. METHODOLOGYPRINCIPAL FINDINGS: Samples of placentomes, chorion, endometrium, maternal peripheral blood leukocytes and blood plasma were collected during early gestation and processed for nested-PCR for eGFP and testis-specific Y-encoded protein (TSPY), western blotting and immunohistochemistry for eGFP detection, as well as transmission electron microscopy to verify the level of interaction between maternal and fetal cells. TSPY and eGFP DNA were present in the blood of cows carrying male pregnancies at day 60 of pregnancy. Protein and mRNA of eGFP were observed in the trophoblast and uterine tissues. In the placentomes, the protein expression was weak in the syncytial regions, but intense in neighboring cells on both sides of the fetal-maternal interface. Ultrastructurally, our samples from t-eGFP expressing NT pregnancies showed to be normal, such as the presence of interdigitating structures between fetal and maternal cells. In addition, channels-like structures were present in the trophoblast cells. CONCLUSIONSSIGNIFICANCE: Data suggested that there is a delivery of fetal contents to the maternal system on both systemic and local levels that involved nuclear acids and proteins. It not clear the mechanisms involved in the transfer of fetal-derived molecules to the maternal system. This delivery may occur through nonclassical protein secretion; throughout transtrophoblastic-like channels and/or by apoptotic processes previously described. In conclusion, the bovine synepitheliochorial placenta displays an intimate fetal-maternal interaction, similar to other placental types for instance human and mouse.
PLoS ONE 01/2013; 8(5):e64399. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Nitric oxide (NO) is a chemical messenger involved in the control of oocyte maturation. NO stimulates guanylate cyclase (GC) to produce cGMP, which in turn activates cGMP-dependent protein kinase (PKG) and some phosphodiesterases (PDE) that may interfere with cAMP levels, a nucleotide also involved in meiosis resumption. The aim of this study was to to determine the role played by NO on the cGMP/cAMP pathway during meiosis resumption in bovine oocytes. The effects of increasing NO generated by SNAP (S-nitroso-N-acetylpenicillamine ,10-7M to 10-3M ) and of other drugs that may affect the NO/cGMP pathway (Proptoporfirin IX and 8-Br-cGMP) on meiosis resumption were investigated in bovine COCs matured for 9h in a semi-defined medium (TCM199 + 3mg/ml BSA). COCs matured with 10-7 M SNAP associated or not with 100μM ODQ (oxadiazole-one quinoxaline), a GC inhibitor, also had their cGMP and cAMP levels measured during the first hours of maturation (1, 3 and 6h). Quantitative PCR was performed by real-time PCR to determine the effects of NO on expression of genes encoding for enzymes of the NO/GC/cGMP and cAMP pathways during the first 9 h of oocyte maturation. Increasing NO levels using 10-7M SNAP resulted in lower rate of germinal vesicle breakdown (36%GVBD – P<0.05) at 9h IVM, while control group and the treatments with 10-9M and 10-8M SNAP showed about 70% GVBD (P> 0.05). A temporary increase in cGMP levels was also observed with the same treatment (4.51 pmol/COC) at 1 h IVM, which was superior to the control group (2.97 pmol/COC – P<0.05) and was reversed by inhibiting GC activity with 100μM ODQ. Neither cAMP levels nor gene expression were affected by NO. These results suggest that NO acts via GC/cGMP and that even a temporary increase in cGMP levels leads to a delay in meiosis resumption, even when cAMP levels have declined. NO does not act on oocyte maturation by affecting cAMP levels or the expression of genes related to the NO/GC/cGMP and cAMP pathways. Also, to our knowledge this is the first report to detect PKG1, PKG2, PDE5A, ADCY3, ADCY6 and ADCY9 transcripts in bovine oocytes.
[show abstract][hide abstract] ABSTRACT: Nitric oxide is a chemical messenger generated by the activity of the enzyme NO synthase (NOS) and has been implicated in the control of oocyte maturation. Nitric oxide stimulates guanylate cyclase (GC) to produce cyclic guanosine monophosphate (cGMP), which in turn activates cGMP-dependent protein kinase (PKG) and some phosphodiesterases (PDE) that may interfere with cyclic adenosine monophosphate (cAMP) levels, a nucleotide also involved in meiosis resumption. In a previous study, we found that increasing NO levels in the presence of a NO donor (S-nitroso-N-acetylpenicillamine, SNAP) resulted in a delayed resumption of meiosis and a lower rate of germinal vesicle breakdown after 9h of in vitro maturation. A temporary increase in cGMP levels was also observed with the same treatment, which was reversed by inhibiting GC activity with oxadiazolo-quinoxaline-one (ODQ; unpublished data). These results suggest that NO acted via GC/cGMP and that even a temporary increase in the cGMP level led to a delay of meiosis resumption. The aim of the present study was to determine the role played by NO on the expression of genes encoding for enzymes of the NO/GC/cGMP and cAMP pathways during the first 9h of oocyte maturation. Cumulus-oocyte complexes were in vitro matured for 9h in a semi-defined medium (TCM-199+3mgmL(-1) of BSA) with 10 to 7M SNAP associated or not associated with 100µM ODQ, a GC inhibitor. A group of oocytes incubated in the absence of inhibitors was considered the control. Total RNA was extracted from pools of 20 denuded oocytes with TRIzol (Life Technologies, Grand Island, NY, USA) and reverse transcribed into complementary DNA using a high-capacity reverse transcription kit (Applied Biosystems, Foster City, CA, USA). Quantitative PCR was performed by real-time PCR using SYBR Green (Applied Biosystems). The genes that had their expression measured pertained to one of the following groups: 1) genes encoding for enzymes that synthesise NO (NOS2 and NOS3); 2) genes involved in the control of cGMP levels (GUCY1B3 and PDE5A) or the enzymes activated by it (PKG1 and PKG2); or 3) genes involved in the control of cAMP levels (ADCY3, ADCY6, ADCY9, PDE3A, and PDE8A) or one of the enzymes activated by it (PKA1). GAPDH and PPIA were selected as housekeeping genes using qbasePLUS version 2.3 (Biogazelle, Zwijnaarde, Belgium). Data from 5 replicates were analysed using LinRegPCR version 11.1 and SAS version 9.2 (SAS Institute Inc., Cary, NC, USA). All genes were found to be expressed in the three experimental groups; however, a significant difference in gene expression levels was not found among groups. Results suggest that NO does not act on oocyte maturation by affecting the expression of the investigated genes in oocytes. To our knowledge, this is the first report to demonstrate the expression of the ADCY3, ADCY6, and ADCY9 genes in bovine oocytes. Further research is in progress to study the effect of the SNAP treatment on the expression of these genes in cumulus cells.
Reproduction Fertility and Development 12/2012; 25(1):277-8. · 2.58 Impact Factor
[show abstract][hide abstract] ABSTRACT: c1 All correspondence to: F.V. Meirelles. Departamento de Ciências Básicas, Universidade de São Paulo, FZEA, Pirassununga, SP, Brazil., Av. Duque de Caxias Norte 225, Pirassununga, São Paulo, Brasil CEP 13635–900. Tel: +55 19 3565 4112. e-mail: firstname.lastname@example.org
[show abstract][hide abstract] ABSTRACT: The embryonic developmental block occurs at the 8-cell stage in cattle and is characterized by a lengthening of the cell cycle and an increased number of embryos that stop development. The maternal-embryonic transition arises at the same stage resulting in the transcription of many genes. Gene expression studies during this stage may contribute to the understanding of the physiological mechanisms involved in the maternal-embryonic transition. Herein we identified genes differentially expressed between embryos with high or low developmental competence to reach the blastocyst stage using differential display PCR. Embryos were analysed according to developmental kinetics: fast cleavage embryos showing 8 cells at 48 h post insemination (hpi) with high potential of development (F8), and embryos with slow cleavage presenting 4 cells at 48 hpi (S4) and 8 cells at 90 hpi (S8), both with reduced rates of development to blastocyst. The fluorescence DDPCR method was applied and allowed the recovery of 176 differentially expressed bands with similar proportion between high and low development potential groups (52% to F8 and 48% in S4 and S8 groups). A total of 27 isolated fragments were cloned and sequenced, confirming the expected primer sequences and allowing the identification of 27 gene transcripts. PI3KCA and ITM2B were chosen for relative quantification of mRNA using real-time PCR and showed a kinetic and a time-related pattern of expression respectively. The observed results suggest the existence of two different embryonic genome activation mechanisms: fast-developing embryos activate genes related to embryonic development, and slow-developing embryos activate genes related to cellular survival and/or death.
[show abstract][hide abstract] ABSTRACT: This study aimed at assessing the effect of the addition of brain-derived neurotrophic factor (10 ng/ml BDNF) and/or cysteamine (100 μm CYS) during pre-maturation and BDNF, CYS or leptin (10 ng/ml LEP) during maturation culture in vitro on embryo development and oocyte gene expression in cattle. Oocytes were obtained by the aspiration of 2- to 8-mm follicles from slaughtered cows. In Experiment 1, oocytes were pre-matured for 24 h with 10 μm butyrolactone I in the presence or not of BDNF and/or CYS followed by in vitro maturation (IVM), fertilization (IVF) and culture (IVC). In Experiment 2, oocytes were submitted to IVM with BDNF, CYS or LEP or no supplements followed by IVF and IVC. In Experiment 3, oocytes were pre-matured with BDNF and CYS followed by IVM or only in vitro matured with BDNF. Samples for quantitative PCR (qPCR) were collected after pre-maturation (BGV) and after IVM of pre-matured oocytes (BMII) or immediately after follicle aspiration (immature control = GV) and IVM (matured control = MII). Embryo production was not affected by the inclusion of the different factors either during pre-maturation or maturation culture (∼ 43% blastocysts, p>0.05). Transcripts analysis showed that most genes (NLRP5, ZAR1, GPX1, KEAP1, SPHK2, HSP70 and PSMP1) were downregulated (p<0.05) after IVM irrespective of being previously pre-matured. The relative abundance of BAX, BCL2, IGFBP3 and ARFRP1 transcripts was unaffected by pre-maturation or maturation (p>0.05). In conclusion, supplementation of in vitro pre-maturation (BDNF and/or CYS) or maturation media (BDNF, CYS or LEP) did not improve embryo development. Gene expression was not affected by pre-maturation treatment, but some genes were downregulated after maturation, probably related to selective and differential degradation.
Reproduction in Domestic Animals 02/2011; 46(1):e31-8. · 1.39 Impact Factor
[show abstract][hide abstract] ABSTRACT: Ooplasm transfer has been used successfully to treat infertility in women with ooplasmic insufficiency and has culminated in the birth of healthy babies. To investigate whether mitochondrial dysfunction is a factor in ooplasmic insufficiency, bovine oocytes were exposed to ethidium bromide, an inhibitor of mitochondrial DNA replication and transcription, during in-vitro maturation (IVM). Exposure of immature oocytes to ethidium bromide for 24h during IVM hampered meiotic resumption and the migration of cortical granules. However, a briefer treatment with ethidium bromide during the last 4h of IVM led to partial arrest of preimplantation development without affecting oocyte maturation. Ooplasm transfer was then performed to rescue the oocytes with impaired development. In spite of this developmental hindrance, transfer of normal ooplasm into ethidium bromide-treated oocytes resulted in a complete rescue of embryonic development and the birth of heteroplasmic calves. Although this study unable to determine whether developmental rescue occurred exclusively through introduction of unaffected mitochondria into ethidium bromide-damaged oocytes, e.g. ethidium bromide may also affect other ooplasm components, these results clearly demonstrate that ooplasm transfer can completely rescue developmentally compromised oocytes, supporting the potential use of ooplasm transfer in therapeutic applications.
[show abstract][hide abstract] ABSTRACT: Epithelial cells from mammary gland tissue that are cultured in vitro are able to maintain specific functions of this gland, such as cellular differentiation and milk protein synthesis. These characteristics make these cells a useful model to study mammary gland physiology, development and differentiation; they can also be used for production of exogenous proteins of pharmaceutical interest. Bovine mammary epithelial cells were cultured in vitro after isolation from mammary gland tissue of animals at different stages of development. The cells were plated on Petri dishes and isolated from fibroblasts using saline/EDTA treatment, followed by trypsinization. Cells isolated on plastic were capable of differentiating into alveolus-like structures; however, only cells derived from non-pregnant and non-lactating animals expressed β-casein. Real-time qPCR and epifluorescence microscopy analyses revealed that alveolus-like structures were competent at expressing Emerald green fluorescent protein (EmGFP) driven by the β-casein promoter, independent of β-casein expression.
[show abstract][hide abstract] ABSTRACT: Cell cycle synchronization by serum starvation (SS) induces apoptosis in somatic cells. This side effect of SS is hypothesized to negatively affect the outcome of somatic cell nuclear transfer (SCNT). We determined whether apoptotic fibroblasts affect SCNT yields. Serum-starved, adult, bovine fibroblasts were stained with annexin V-FITC/propidium iodide to allow apoptosis detection by flow cytometry. Positive and negative cells sorted by fluorescence activated cell sorting (FACS) and an unsorted control group were used as nuclear donors for SCNT. Reconstructed embryos were cultured in vitro and transferred to synchronized recipients. Apoptosis had no effect on fusion and cleavage rates; however, it resulted in reductions in blastocyst production and quality measured by apoptotic index. However, reconstructed embryos with apoptotic cells resulted in pregnancy rates similar to that of the control on day 30, and generated one live female calf. In conclusion, we showed that apoptotic cells present in serum-starved cultures negatively affect embryo production after SCNT without compromising full-term development. Further studies will evaluate the ability of the oocyte to reprogram cells in specific phases of apoptosis.
Cloning and Stem Cells 09/2009; 11(4):565-73. · 2.66 Impact Factor