[Show abstract][Hide abstract] ABSTRACT: In vitro oocyte maturation (IVM) protocols can be improved by adding chemical supplements to the culture media. Tretinoin is considered an important retinoid in embryonic development and its association with lipid-core nanocapsules (TTN-LNC) represents an innovative way of improving its solubility, and chemical stability, and reducing its toxicity. The effects of supplementing IVM medium with TTN-LNC was evaluated by analyzing production of reactive oxygen species (ROS), S36-phosphorilated-p66Shc levels and caspase activity in early embryonic development, and expression of apoptosis and pluripotency genes in blastocysts. The lowest concentration tested (0.25μM) of TTN-LNC generated higher blastocyst rate, lower ROS production and S36-p66Shc amount. Additionally, expression of BAX and SHC1 were lower in both non-encapsulated tretinoin (TTN) and TTN-LNC-treated groups. Nanoencapsulation allowed the use of smaller concentrations of tretinoin to supplement IVM medium thus reducing toxic effects related with its use, decreasing ROS levels and apoptose frequency, and improving the blastocyst rates.
Full-text · Article · Oct 2015 · Reproductive Toxicology
[Show abstract][Hide abstract] ABSTRACT: Natural products continue to be an invaluable resource of anticancer drug discovery in recent years. Propolis is known for its biological activities such as antimicrobial and antitumor effects. This study assessed the effects of Brazilian red propolis (BRP) on apoptosis and migration potential in human bladder cancer cells. The effect of BRP ethanolic extract (25, 50, and 100 μg/mL) on 5637 cells was determined by MTT, LIVE/DEAD, and migration (scratch assay) assays. Apoptosis induction was investigated through flow cytometry and gene expression profile was investigated by qRT-PCR. Results showed cytotoxicity on MTT and LIVE/DEAD assays, with IC50 values of 95 μg/mL in 24 h of treatment. Cellular migration of 5637 cells was significantly inhibited through lower doses of BRP ethanolic extract (25 and 50 μg/mL). Flow cytometry analyses showed that BRP induced cytotoxicity through apoptosis-like mechanisms in 5637 cells and qRT-PCR revealed increased levels of Bax/Bcl-2 ratio, p53, AIF, and antioxidant enzymes genes. Data suggest that BRP may be a potential source of drugs to bladder cancer treatment.
Full-text · Article · Nov 2014 · Evidence-based Complementary and Alternative Medicine
[Show abstract][Hide abstract] ABSTRACT: Tretinoin is a retinoid derivative that has an antiproliferative effect on several kinds of tumours. Human lung adenocarcinoma epithelial cell lines (A549) exhibit a profound resistance to the effects of tretinoin. Nanocarriers seem to be a good alternative to overcomecellular resistance to drugs. The aim of this study was to test whether tretinoin-loaded lipid-core nanocapsules exert anantitumor effect on A549 cells. A549 cells were incubated with free tretinoin (TTN), blank nanocapsules (LNC) and tretinoin-loaded lipid-core nanocapsules (TTN-LNC). Data from evaluation of DNA content and Annexin V binding assay by flow cytometry showed that TTN-LNC induced apoptosis and cell cycle arrest at the G1-phase while TTN did not. TTN-LNC showed higher cytotoxic effects than TTN on A549 cells evaluated by MTT and LIVE/DEAD cell viability assay. Gene expression profiling identified up-regulated expression of gene p21 by TTN-LNC, supporting the cell cycle arrest effect. These results showed for the first time that TTN-LNC are able to overcome the resistance of adenocarcinoma cell line A549 to treatment with TTN by inducing apoptosis and cell cycle arrest, providing support for their use in applications in lung cancer therapy.
Full-text · Article · Feb 2014 · European journal of pharmaceutics and biopharmaceutics: official journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik e.V
[Show abstract][Hide abstract] ABSTRACT: Foram coletadas 100 amostras de conteúdo fecal de aves de corte, 100 de produtos de frango (coxa, sobrecoxa, asa, dorso, carne moída e fígado) e 100 de fezes de humanos, e analisadas para pesquisa de Campylobacter. Realizou-se a determinação da espécie e da presença dos genes cdt, responsáveis pela codificação da toxina citoletal distensiva (CDT), através da técnica da PCR. A bactéria foi isolada de 61% das amostras de fezes de frango, 20% de produtos de frango e 3% de fezes de humanos. A maioria dos isolados foi determinada como C. jejuni . Destes, 93,5% apresentaram os genes para a toxina CDT. Apesar de a ocorrência de Campylobacter em fezes de humanos ter sido baixa, a prevalência em frangos de corte e produtos de frango foi elevada, fato que, aliado à presença dos genes cdt na maioria dos isolados, representa risco potencial para os consumidores. Esses resultados são indicativos da necessidade de medidas preventivas no sistema de produção e de boas práticas de fabricação na indústria, de forma a minimizar a contaminação dos produtos e diminuir o risco para os consumidores.
Full-text · Article · Feb 2014 · Arquivo Brasileiro de Medicina Veterinária e Zootecnia
[Show abstract][Hide abstract] ABSTRACT: Here we investigated alterations in the protein profile of Hep-2 treated with red propolis using two-dimensional electrophoresis associated to mass spectrometry and apoptotic rates of cells treated with and without red propolis extracts through TUNEL and Annexin-V assays. A total of 325 spots were manually excised from the two-dimensional gel electrophoresis and 177 proteins were identified using LC-MS-MS. Among all proteins identified that presented differential expression, most were down-regulated in presence of red propolis extract at a concentration of 120 μg/mL (IC50): GRP78, PRDX2, LDHB, VIM and TUBA1A. Only two up-regulated proteins were identified in this study in the non-cytotoxic (6 μg/mL) red propolis treated group: RPLP0 and RAD23B. TUNEL staining assay showed a markedly increase in the mid- to late-stage apoptosis of Hep-2 cells induced by red propolis at concentrations of 60 and 120 μg/mL when compared with non-treated cells. The increase of late apoptosis was confirmed by in situ Annexin-V analysis in which red propolis extract induced late apoptosis in a dose-dependent manner. The differences in tumor cell protein profiles warrant further investigations including isolation of major bioactive compounds of red propolis in different cell lines using proteomics and molecular tests to validate the protein expression here observed.
Full-text · Article · Nov 2013 · Food and chemical toxicology: an international journal published for the British Industrial Biological Research Association
[Show abstract][Hide abstract] ABSTRACT: The aim of this study was to determine the binding patterns of Canavalia ensiformis (ConA), Canavalia boliviana (ConBol) and Canavalia brasiliensis (ConBr) lectins to bovine sperm and their effects on sperm motility, viability, lipid peroxidation, reactive oxygen species production and fertilization ability. ConA bound to whole spermatozoa, with the exception of the equatorial segment, ConBol did not interact with the acrosome region and ConBr exhibited a fragmented binding pattern. The three lectins decreased sperm motility but did not affect cell viability or lipid peroxidation. Nevertheless, ROS production was increased in comparison to controls and a reduction in the cleavage and blastocyst ratio was induced in comparison to controls. In conclusion, this study determined that structurally similar lectins interact differently with bovine sperm and affect sperm motility, viability, lipid peroxidation, ROS production and fertilization ability in various ways.
Full-text · Article · Mar 2013 · Reproductive Toxicology
[Show abstract][Hide abstract] ABSTRACT: In this study the correlation between seminal plasma protein profile and the sperm motility and sperm viability in rabbits submitted to treatment with an expression vector containing EPO gene and with human recombinant EPO was evaluated. In rabbits submitted to treatment with EPO expression vector, two protein bands were associated to sperm motility 48kDa associated to low motility (P<0.05) and 18kDa to high motility (P<0.05) and this protein band was also associated to high sperm viability (P<0.05). In rabbits submitted to treatment with human recombinant EPO, a protein factor, 63kDa, was associated to high sperm motility (P<0.05) while two protein factors, 26 and 40kDa, were associated to high sperm viability (P<0.05). These results suggest that gene doping leads to changes in rabbit seminal plasma protein, altering sperm motility and sperm viability.
Full-text · Article · Feb 2013 · Arquivo Brasileiro de Medicina Veterinária e Zootecnia
[Show abstract][Hide abstract] ABSTRACT: Erythropoietin (EPO) gene therapy can be used for several purposes; however, its effects on reproductive performance are unknown. The aim of this study was to evaluate the toxicological effects of non-viral (EPO) gene transfer on sperm motility, viability, morphology and concentration. Rabbit EPO cDNA was cloned into a pTarget mammalian expression vector. Rabbits were administered with: (1) pTarget/EPO vector, (2) recombinant human EPO (rHuEpo) and (3) saline (control). Both pTarget/EPO and rHuEpo significantly increased (P < 0.05) hematocrit levels 1 week after injection and they remained significantly higher than the control for up to 5 weeks (P < 0.05), showing that both EPO treatments were effective in stimulating the production of red blood cells in rabbits. The EPO gene transfer or rHuEPO administration had no significant effect (P > 0.05) on sperm motility, vigor, viability, concentration or morphology in the testis.
[Show abstract][Hide abstract] ABSTRACT: This study evaluated pre-freezing and post-thawing boar sperm quality in distinct portions of the ejaculates and identified protein bands potentially related to boar sperm tolerance to freezing. The sperm-rich portion of the ejaculate was fractioned in: the first 10 mL (P1); and its remaining volume (P2). Those portions were frozen and the bands present in both portions were evaluated by one-dimensional electrophoresis. Inter-and intra-boar ANOVA evaluated how the presence of bands related to pre-freezing and post-thawing sperm motility and mem-brane integrity and to post-thawing acrosome integrity. Post-thawing sperm motility, mem-brane integrity and acrosome integrity were greater for P1 than for P2 (P b 0.05). The presence of the 19 kDa band was associated with improved pre-freezing sperm motility and membrane integrity, but with reduced post-thawing acrosome integrity (P b 0.05). The pres-ence of the 44 kDa band was associated with reduced post-thawing sperm motility and acro-some integrity (P b 0.05). Post-thawing acrosome integrity was enhanced when the 80 kDa band were present (P b 0.05). Reduced pre-freezing sperm membrane integrity and post-thawing sperm motility were associated to the presence of the 100 kDa band (P b 0.05). The presence of the 18 kDa band in P2 was associated with reduction in both post-thawing sperm motility and membrane integrity (P b 0.05), but such an effect was not observed in P1 (P > 0.05). Thus, sperm from P1 presented greater tolerance to freezing than sperm from P2 and the 18, 19, 44, 65, 80 and 100 kDa bands may be potential markers for boar sperm toler-ance to cryopreservation.
[Show abstract][Hide abstract] ABSTRACT: Single nucleotide polymorphisms (SNPs) in the p53 gene have been studied extensively in humans. The aims of this study were to determine the frequency of the Arg/Pro SNP in p53 in Thoroughbred mares on one stud in Brazil and to correlate p53 genotypes with reproductive performance. SNPs were detected by PCR-restriction fragment length polymorphism in blood samples from 105 horses and confirmed by sequencing. The allele frequency in Thoroughbred mares at codon 72 in exon 4 was 73.3% Arg/Pro, 17.1% Arg/Arg and 9.6% Pro/Pro. The presence of Arg/Pro was significantly associated with abortion (P=0.02), while Pro/Pro mares had a lower probability of abortion (P<0.05). Using a logistic regression model, the dominance effect was significant (P=0.044; odds ratio 7.94) for abortion and additive effects were not significant (P=0.26). p53 may play a role in equine reproduction.
Full-text · Article · Mar 2012 · The Veterinary Journal
[Show abstract][Hide abstract] ABSTRACT: In this study, polymerase chain reaction (PCR) reamplification of the first PCR product (2nd-PCR) and a qPCR assay were used to detect the sex determining region Y (SRY) gene from circulating cell-free fetal DNA (ccffDNA) in blood plasma of pregnant mares to determine fetal sex. The ccffDNA was isolated from plasma of 20 Thoroughbred mares (5-13 y old) in the final 3 mo of pregnancy (fetal sex was verified after foaling). For controls, plasma from two non-pregnant mares and two virgin mares were used, in addition to the non-template control. The 182 bp nucleotide sequence corresponding to the SRY-PCR product was confirmed by DNA sequencing. Based on SRY/PCR, 8 of 11 male and 9 of 9 female fetuses were correctly identified, resulting in a sensitivity of 72.7% (for male fetuses) and an overall accuracy of 85%. Furthermore, using SRY/2nd-PCR and qPCR techniques, sensitivity and accuracy were 90.9 and 95%, respectively. In conclusion, this study is apparently the first report of fetal sex determination in mares using ccffDNA.
[Show abstract][Hide abstract] ABSTRACT: SummaryThe gene expression of Bax, Bcl-2, survivin and p53, following in vitro maturation of equine oocytes, was compared in morphologically distinct oocytes and cumulus cells. Cumulus-oocyte complexes (COC) were harvested and divided into two groups: G1 - morphologically healthy cells; and G2 - less viable cells or cells with some degree of atresia. Total RNA was isolated from both immature and in vitro matured COC and real-time reverse transcription polymerase chain reaction (qRT-PCR) was used to quantify gene expression. Our results showed there was significantly higher expression of survivin (P < 0.05) and lower expression of p53 (P < 0.01) in oocytes compared with cumulus cells in G1. No significant difference in gene expression was observed following in vitro maturation or in COC derived from G1 and G2. However, expression of the Bax gene was significantly higher in cumulus cells from G1 (P < 0.02).
[Show abstract][Hide abstract] ABSTRACT: Transgenic animals have been successfully produced by mass gene transfer techniques such as sperm-mediated gene transfer (SMGT). The aim of this work was to demonstrate transgene transmission by SMGT in chickens using dimethylsulfoxide (DMSO) or N,N-dimethylacetamide (DMAc) as transfectants after seminal plasma removal to prevent DNase activity. Sperm samples were prepared by repetitive washes, and after each wash sperm motility, seminal plasma proteins, exogenous DNA integrity and its uptake by spermatozoa were evaluated. Laying hens were inseminated using spermatozoa transfected with pEGFP-N1 vector in the presence of DMSO or DMAc. Transgene transmission in newborn chicks was evaluated by in vivo enhanced green fluorescent protein (EGFP) expression, RT-PCR and PCR analysis. DNA internalization was limited to sperm samples washed twice. The presence of DMSO or DMAc during transfection had no effect on fertilization or hatching rates. PCR analysis detected the presence of EGFP DNA in 38% of newborn chicks from the DMSO group and 19% from the DMAc group. EGFP mRNA was detected in 21% of newborn chicks from the DMSO group, as against 8.5% from the DMAc group. However, in vivo expression of EGFP was only observed in a single animal from the DMSO group. Our data revealed that the plasmid DNA-DMSO combination coupled with sperm washes can be an efficient method for transfection in chickens.
Full-text · Article · Sep 2011 · Journal of Biosciences
[Show abstract][Hide abstract] ABSTRACT: The objective was to evaluate the effect of three cryopreservation methods on the in vitro maturation (IVM) and membrane integrity (MIn) of immature equine oocytes. An open pulled straw (OPS) method, a novel solid surface vitrification (SSV) process, and the addition of a synthetic ice blocker were evaluated. Compared with the control group (N=269), the OPS (N=159) and the SSV (N=202) cryopreservation methods decreased both IVM (50.9 vs. 13.3 and 9.4%, respectively; P<0.001) and MIn (76.6 vs. 31.1 and 33.7%; P<0.001) of immature equine oocytes. However, inclusion of 0.1% ice blocker in the OPS vitrification process increased the rates of both IVM (30.5%; P<0.01) and MIn (45.8%; P<0.05) of the oocytes (N=59). Including 0.1% ice blocker in the SSV process improved the IVM rate (20.9%; P<0.05), whereas MIn remained compromised in this group (N=67). However, increasing the concentration of the ice blocker (to 1.0%) in the cryopreservation methods did not significantly improve rates of IVM. In conclusion, the addition of a synthetic ice blocker (0.1%) to both cryopreservation processes significantly increased rates of both IVM and MIn of immature equine oocytes cryopreserved by OPS.
[Show abstract][Hide abstract] ABSTRACT: The objectives were to investigate whether: 1) nanotransfectants are more effective than other common transfection methods for SMGT; 2) NanoSMGT is able to transmit exogenous DNA molecules to bovine embryos; and 3) halloysite clay nanotubes (HCNs) can be used as a transfection reagent to improve transgene transmission. Four transfection systems were used: naked DNA (without transfectant), lipofection, nanopolymer, and halloysite clay nanotubes. Plasmid uptake by sperm and its transfer to embryos were quantified by conventional and real-time PCR, as well as EGFP expression by fluorescence microscopy. Furthermore, sperm motility and viability, and embryo development were investigated. Mean number of plasmids taken up was affected (P < 0.05) by transfection procedure, with the nanopolymer being the most effective transfectant (∼ 153 plasmids per spermatozoon). None of the treatments affected sperm motility or viability. The mean number of plasmids transmitted to four-cell stage embryos was higher (P < 0.05) in nanopolymer and HCNs than liposomes and naked DNA groups. The number of embryos carrying the transgene increased from 8-10% using naked DNA or liposomes to 40-45% using nanopolymer or HCN as transfectants (P < 0.05). There were no significant differences among transfection procedures regarding blastocyst formation rate of resulting embryos. However, no EGFP-expressing embryo was identified in any treatment. Therefore, nanotransfectants improved transgene transmission in bovine embryos without deleterious effects on embryo development. To our knowledge, this was the first time that bovine embryos carrying a transgene were produced by NanoSMGT.
[Show abstract][Hide abstract] ABSTRACT: The objective was to introduce exogenous DNA into commercially sex-sorted bovine sperm using nanopolymer for transfection. In the first experiment, the optimal concentration and ratio of linear-to-circular plasmid was determined for NanoSMGT in unsorted sperm. A second experiment was conducted to transfect exogenous DNA into sex-sorted sperm. Exogenous DNA uptake occurred in a dose-dependent manner (P < 0.05). The optimal amount of DNA was 10 μg/10(6) cells. The ratios of linear-to-circular plasmid do not influence the uptake by unsorted sperm cells and none of the tested treatments affected sperm motility and viability. Commercially sex-sorted bovine sperm were able to uptake exogenous DNA using nanopolymer; however, both X- and Y-sorted sperm had decreased DNA uptake in comparison to unsorted sperm (P < 0.05). Neither sperm motility nor viability were affected by nanotransfection. In conclusion, nanopolymer efficiently introduced exogenous DNA into commercially sex-sorted bovine sperm; we inferred that these sperm could be used for production of embryos of the desired sex, a technique named NanoSMGT.
[Show abstract][Hide abstract] ABSTRACT: The objective of this study was to describe the steps of the method to recover equine oocytes by scraping the follicle wall and to determine recovery rates of compact Cumulus-oocytes by scraping. Equine ovaries were obtained at a slaughterhouse. After removal of the albuginea tunica, follicles having 10 to 30 mm in diameter were dissected without the ovarian stroma. The follicles were opened individually in Petri dishes, and the granulose cell layer was scraped with a bone currett. Cumulus-oocytes complexes were identified using a dissection microscope and classified according to the Cumulus oophorus morphology and ooplasm appearance. From a total of 527 dissected ovaries, were obtained 3.5 follicles dissected/ ovary and 2.5 oocytes/ovary. The recovery rate was 71.0% (oocytes/follicles), whereas 70.2% of these were classified as having compact Cumulus-oocytes and homogenous ooplasma. The average of compact Cumulus-oocytes per ovary was 1.75. The scraping of the granulose layer of follicle having 10 to 30 mm in diameter allows the recovery of equine oocytes with compact Cumulus, which are considered adequate for in vitro maturation studies.
KEY WORDS: Equine oocytes, scraping of follicle wall, in vitro maturation. Este trabalho teve como objetivo determinar a eficiência da técnica de curetagem da parede folicular (scraping) para a obtenção de complexos Cumulus oophorus compactos, bem como descrever as etapas do processo. Submeteram-se ovários eqüinos coletados em abatedouro à dissecação da túnica albugínea, para identificação e individualização dos folículos com diâmetro entre 10 e 30 mm. Esses foram abertos individualmente em placa de Petri e, posteriormente, realizou-se raspagem da camada granulosa com auxílio de cureta e exame do líquido folicular em lupa estéreo-microscópica. Classificaram-se os oócitos quanto à integridade do ooplasma e as características das células do Cumulus oophorus. De um total de 527 ovários dissecados, obtiveram-se 3,5 folículos/ovário e uma média de 2,5 oócitos/ovário, compreendendo uma taxa de recuperação de 71,0% (oócitos/folículos), sendo 70,2% classificados como Cumulus oophorus compactos e ooplasma homogêneo. A média de oócitos Cumulus oophorus compactos por ovário foi de 1,7. A utilização da curetagem da camada granulosa de folículos entre 10 e 30 mm de diâmetro comprovou ser satisfatória para a obtenção de um elevado percentual de oócitos eqüinos com Cumulus oophorus compacto,o que é adequados para estudos de maturação in vitro.
PALAVRAS-CHAVE: Curetagem da parede folicular, maturação in vitro, oócitos eqüinos, scraping.
[Show abstract][Hide abstract] ABSTRACT: 1. INTRODUÇÃO O processo de maturação, que deve conferir ao oócito a capacidade de ser fertilizado assim como a de desenvolvimento embrionário, ocorre em duas fases. A primeira de crescimento, com síntese de proteínas e formação de organelas, e a segunda com modificações nucleares e citoplasmáticas (Fissore et al., 2002; Fernades, 2004). As taxas de maturação in vitro (MIV) de oócitos eqüinos são baixas, quando comparadas as de outras espécies, obtêm-se entre 40 a 60% de oócitos atingindo o estágio de metáfase II (Dell Aquila et al., 1997). As razões para a falha dos oócitos eqüinos em completar a meiose durante a MIV são desconhecidas, podendo estar ligadas a alterações na cascata bioquímica envolvida na meiose (Goudet et al., 1998; Fernandes, 2004). Uma grande variedade de meios de cultivo vem sendo utilizada para avaliar o efeito de diferentes componentes do soro, macromoléculas, hormônios, fatores de crescimento e adição de células somáticas na MIV em eqüinos (Carneiro, 2002). Os compostos tióis de baixo peso molecular, como a cisteamina e b-mercaptoetanol, quando presentes na MIV de oócitos bovinos e ovinos, estimulam a síntese de glutationa (GSH), aumentando os índices de desenvolvimento embrionário (Matos et al., 1996; Matos et al., 2003). Resultados similares foram obtidos em suínos (Grupent et al., 1995) e búfalos (Gasparrini et al., 2003). Pois, o GSH presente nas células de mamíferos tem um importante papel de proteção contra o dano oxidativo (Meister & Tate, 1976). O presente estudo teve como objetivo examinar o efeito da suplementação de cisteamina ao meio de maturação in vitro, através da análise da viabilidade de membrana e taxa de maturação de oócitos eqüinos.