214 PRE-IMPLANTATION EMBRYO SEX DETERMINATION BY AMPLIFICATION OF AMELOGENIN GENE IN WATER BUFFALOES (BUBALUS BUBALIS)

2016 ◽  
Vol 28 (2) ◽  
pp. 238
Author(s):  
J.-H. Shang ◽  
C.-Y. Yang ◽  
H.-Y. Zheng ◽  
J. Qin ◽  
Y.-P. Gu ◽  
...  
Keyword(s):  
Y Chromosome ◽  
Natural Science ◽  
Water Buffalo ◽  
Tooth Enamel ◽  
Mammalian Species ◽  
Early Embryo ◽  
Specific Pattern ◽  
Specific Sequence ◽  
Embryo Sex

Sex-specific sequence variability of the amelogenin (AMEL) gene has been observed in a variety of mammalian species. AMEL is a multi-copy gene expressed in abundance in the development of mammalian tooth enamel matrix. Its homologous genes are located on the X- or Y-chromosome, respectively. In our study, a pair of primers was designed to allow amplification of a single fragment of 312 bp from buffalo cows. Because the gene is slightly differently encoded on the Y-chromosome, the same primers were expected to produce 2 fragments of 312 and 249 bp from bulls. The primers were successfully applied to single, in vitro-produced Day-6 to Day-8 water buffalo blastocysts by direct lysis of the whole blastocysts, followed by PCR. The amplification on presumed female and male embryos consistently displayed the same sex-specific pattern. Of 100 blastocysts, 43 were determined to be females, 51 males, and 6 unknown (samples missed). We concluded that PCR amplification of the AMEL gene can be used for early embryo sex identification of water buffalo. This work was supported by the National Natural Science Foundation of China under Grant No. 31160456 and the Guangxi Natural Science Foundation of China under Grants No. 2013GXNSFAA019075 and 2014GXNSFAA118116.

219 EXPRESSION PATTERN OF THE SUB-CORTICAL MATERNAL COMPLEX IN OVINE OOCYTES AND PRE-IMPLANTATION EMBRYOS

2013 ◽  
Vol 25 (1) ◽  
pp. 258 ◽  
Author(s):  
D. Bebbere ◽  
L. Bogliolo ◽  
F. Ariu ◽  
O. Murrone ◽  
A. Strina ◽  
...  
Keyword(s):  
Expression Pattern ◽  
Protein Complex ◽  
Mammalian Species ◽  
Expression Patterns ◽  
Embryonic Cell ◽  
Specific Pattern ◽  
Pcr Analysis ◽  
Cell Stage ◽  
Oocyte Growth

The sub-cortical maternal complex (SCMC) is a multi-protein complex located in the sub-cortex of the oocyte. In mouse, it assembles during oocyte growth and is essential for zygotes to progress beyond the first embryonic cell divisions (Li et al., 2008). At least 4 proteins contribute to the complex: oocyte expressed protein (OOEP), maternal antigen that embryo requires (MATER), transducin-like enhancer of split 6 (TLE6), and ES cell associated transcript 1 (ECAT1), all encoded by maternal effect genes. In mouse, the relative transcripts are degraded during meiotic maturation and ovulation, whereas the SCMC proteins persist in the early embryo. Whereas MATER expression has been studied in several species, the existence of the genes encoding the other components has been assessed in few mammalian species and their pattern of expression during pre-implantation development has been analysed only in mouse (Li et al. 2008 Dev. Cell 15, 416–425). In a previous work (Bebbere et al. 2008 Reprod. Fertil. Dev. 20, 908–915), we assessed MATER existence and pattern of expression in the ovine species. The aim of the present work was to assess the existence of OOEP, TLE6, and FILIA in the ovine species and to analyse the expression pattern of the 4 genes in the oocytes and during pre-implantation embryo development. Total RNA was isolated and reverse transcribed from pools of immature (GV) and in vitro matured (IVM) metaphase II (MII) oocytes, from in vitro fertilized and cultured (IVFC) embryos at the 2-, 4-, 8-, and 16-cell stage and from blastocysts. Three pools of 10 oocyte/embryos were analysed for each class. Primers were designed on the basis of the sequences conserved among orthologs and amplify intron-spanning regions. The PCR products were sequenced, and the alignment, performed with BLASTn, confirmed the homology with the orthologous genes present in public databases. Real-time PCR analysis revealed that all 4 transcripts are present at its highest level in the GV oocyte but decrease during embryo pre-implantation development with a gene-specific pattern. Conversely to the pattern of expression observed in mouse, all 4 transcripts persisted until the 8-cell stage embryo, disappearing only at the 16-cell stage. No transcripts were detected at the blastocyst stage. This study confirms the existence of transcripts related to SCMC also in the ovine species, but highlights species-specific patterns of expression in the 2 species, possibly related to the different time of activation of the embryo genome in mouse and in sheep. The observed expression patterns suggest an involvement of the protein complex in oocyte maturation and in the very first phases of life, possibly in the transition from the maternal to embryonic program of development.

Why we should not select the faster embryo: lessons from mice and cattle

2015 ◽  
Vol 27 (5) ◽  
pp. 765 ◽  
Author(s):  
Alfonso Gutierrez-Adan ◽  
Carlee R. White ◽  
Ann Van Soom ◽  
Mellissa R. W. Mann
Keyword(s):  
In Vitro Culture ◽  
Embryo Quality ◽  
Mammalian Species ◽  
Sex Selection ◽  
Developmental Competence ◽  
Bovine Embryo ◽  
Timely Manner ◽  
Developmental Rates ◽  
Embryo Sex

Many studies have shown that in vitro culture can negatively impact preimplantation development. This necessitates some selection criteria for identifying the best-suited embryos for transfer. That said, embryo selection after in vitro culture remains a subjective process in most mammalian species, including cows, mice and humans. General consensus in the field is that embryos that develop in a timely manner have the highest developmental competence and viability after transfer. Herein lies the key question: what is a timely manner? With emerging data in bovine and mouse supporting increased developmental competency in embryos with moderate rates of development, it is time to question whether the fastest developing embryos are the best embryos for transfer in the human clinic. This is especially relevant to epigenetic gene regulation, including genomic imprinting, where faster developing embryos exhibit loss of imprinted methylation, as well as to sex selection bias, where faster developmental rates of male embryos may lead to biased embryo transfer and, in turn, biased sex ratios. In this review, we explore evidence surrounding the question of developmental timing as it relates to bovine embryo quality, mouse embryo quality and genomic imprint maintenance, and embryo sex.

259 A RAPID, NON-PCR-BASED BOVINE EMBRYO BIOPSY SEXING ASSAY

2009 ◽  
Vol 21 (1) ◽  
pp. 227 ◽  
Author(s):  
R. Bleher ◽  
W. Erwin ◽  
A. M. Paprocki ◽  
C. M. Syverson ◽  
R. Koppang ◽  
...  
Keyword(s):  
Y Chromosome ◽  
Blastocyst Stage ◽  
Bright Spot ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Gender Assignment ◽  
Specific Sequence ◽  
Laboratory Equipment

Sex determination of bovine embryos in conjunction with embryo transfer is an established method to maximize efficiency by producing offspring with the preferred gender. Present methodology for sexing embryos is based on PCR, which requires molecular laboratory equipment, trained personnel, and several hours before a result becomes known. We developed a simple, rapid, non-PCR procedure for identification of Y-chromosomes in bovine blastomeres recovered via biopsy. A biopsy (n = 5 to 8 blastomeres) was taken from a blastocyst-stage embryo and transferred onto a plastic slide. After drying and fixation, the cells were denatured and incubated with a peptide nucleic acid probe designed to target unique Y-chromosome specific sequence. The probe was conjugated to a fluorescent dye (CY-3) which enables Y-chromosome detection as a bright spot within blastomere nuclei when using a fluorescent microscope. The absence of signal indicates female embryonic DNA. From placement of the biopsy onto a plastic slide, the methodology required approximately 75 minutes to determine embryo gender. The accuracy of the biopsy sexing procedure was demonstrated by parallel gender determination of the same embryo using an established PCR method designed for the bovine amelogenin locus. Based on 18 in vitro-produced bovine embryos generating a result for both assays, there was a 94.4% match (17/18) of gender assignment. The present technology represents a simple alternative to PCR-based embryo sexing technology. Research is ongoing for future development of live embryo sexing determination.

Dynamic proteome signatures in gametes, embryos and their maternal environment

2011 ◽  
Vol 23 (1) ◽  
pp. 81 ◽  
Author(s):  
Georg J. Arnold ◽  
T. Frohlich
Keyword(s):  
Mass Spectrometry ◽  
De Novo ◽  
Expression Profiles ◽  
Mammalian Species ◽  
Early Embryo ◽  
Label Free ◽  
Maternal Environment ◽  
Stable Isotope Labelling ◽  
Proteomic Data

Comprehensive molecular analysis at the level of proteins represents a technically demanding, but indispensable, task since several post-transcriptional regulation mechanisms disable a valid prediction of quantitative protein expression profiles from transcriptome analysis. In crucial steps of gamete and early embryo development, de novo transcription is silenced, meaning that almost all macromolecular events take place at the level of proteins. In this review, we describe selected examples of dynamic proteome signatures addressing capacitation of spermatozoa, in vitro maturation of oocytes, effect of oestrous cycle on oviduct epithelial cells and embryo-induced alterations to the maternal environment. We also present details of the experimental strategies applied and the experiments performed to verify quantitative proteomic data. Far from being comprehensive, examples were selected to cover several mammalian species as well as the most powerful proteomic techniques currently applied. To enable non-experts in the field of proteomics to appraise published proteomic data, our examples are preceded by a customised description of quantitative proteomic methods, covering 2D difference gel electrophoresis (2D-DIGE), nano-liquid chromatography combined with tandem mass spectrometry, and label-free as well as stable-isotope labelling strategies for mass spectrometry-based quantifications.

In Vitro Methods for the Assessment of L-Cysteine Conjugate Toxicity

1994 ◽  
Vol 22 (2) ◽  
pp. 72-80
Author(s):  
Lorraine D. Buckberry ◽  
Harriet J. Adcock ◽  
Jeremy Adler ◽  
Ian S. Blagbrough ◽  
Peter J. Gaskin ◽  
...  
Keyword(s):  
Mechanism Of Action ◽  
Mammalian Species ◽  
Mercapturic Acid ◽  
Assay Method ◽  
Cellular Toxicity ◽  
In Vitro Methods ◽  
Metabolic Route ◽  
Assay Methods ◽  
Chang Liver

L-Cysteine conjugates are normally metabolised via N-acetylation to produce a mercapturic acid. However, a recently identified metabolic route (C-S lysis) may lead to the generation of an unstable thiol which has been demonstrated to be responsible for toxicity in various mammalian species. Human Chang liver cells were challenged with a number of established L-cysteine conjugates. The cellular toxicity of these compounds was determined using a range of assay procedures, which provided differing information, depending on the assay method used. These observations were then investigated in order to establish which system would provide the most reliable indication of C-S lyase toxicity and whether any information on the mechanism of action could be obtained by these assay methods.

scholarly journals Novel Role for Albumin in Innate Immunity: Serum Albumin Inhibits the Growth of Blastomyces dermatitidis Yeast Form In Vitro

2003 ◽  
Vol 71 (11) ◽  
pp. 6648-6652 ◽  
Author(s):  
Steven Giles ◽  
Charles Czuprynski
Keyword(s):  
Molecular Weight ◽  
Inhibitory Activity ◽  
Mammalian Species ◽  
Blastomyces Dermatitidis ◽  
Low Molecular Weight ◽  
Rat Serum ◽  
Yeast Form ◽  
Domain Iii

ABSTRACT In this study we found that serum inhibitory activity against Blastomyces dermatitidis was principally mediated by albumin. This was confirmed in experiments using albumin from several mammalian species. Analbuminemic rat serum did not inhibit B. dermatitidis growth in vivo; however, the addition of albumin restored inhibitory activity. Inhibitory activity does not require albumin domain III and appears to involve binding of a low-molecular-weight yeast-derived growth factor.

scholarly journals Single cell RNA-seq reveals genes vital to in vitro fertilized embryos and parthenotes in pigs

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Zhi-Qiang Du ◽  
Hao Liang ◽  
Xiao-Man Liu ◽  
Yun-Hua Liu ◽  
Chonglong Wang ◽  
...  
Keyword(s):  
Embryo Development ◽  
Gene Networks ◽  
Regulatory Networks ◽  
Rna Binding ◽  
Expression Patterns ◽  
Early Embryo ◽  
Specific Factor ◽  
Early Embryos ◽  
Genome Activation

AbstractSuccessful early embryo development requires the correct reprogramming and configuration of gene networks by the timely and faithful execution of zygotic genome activation (ZGA). However, the regulatory principle of molecular elements and circuits fundamental to embryo development remains largely obscure. Here, we profiled the transcriptomes of single zygotes and blastomeres, obtained from in vitro fertilized (IVF) or parthenogenetically activated (PA) porcine early embryos (1- to 8-cell), focusing on the gene expression dynamics and regulatory networks associated with maternal-to-zygote transition (MZT) (mainly maternal RNA clearance and ZGA). We found that minor and major ZGAs occur at 1-cell and 4-cell stages for both IVF and PA embryos, respectively. Maternal RNAs gradually decay from 1- to 8-cell embryos. Top abundantly expressed genes (CDV3, PCNA, CDR1, YWHAE, DNMT1, IGF2BP3, ARMC1, BTG4, UHRF2 and gametocyte-specific factor 1-like) in both IVF and PA early embryos identified are of vital roles for embryo development. Differentially expressed genes within IVF groups are different from that within PA groups, indicating bi-parental and maternal-only embryos have specific sets of mRNAs distinctly decayed and activated. Pathways enriched from DEGs showed that RNA associated pathways (RNA binding, processing, transport and degradation) could be important. Moreover, mitochondrial RNAs are found to be actively transcribed, showing dynamic expression patterns, and for DNA/H3K4 methylation and transcription factors as well. Taken together, our findings provide an important resource to investigate further the epigenetic and genome regulation of MZT events in early embryos of pigs.

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