Search

As letters form the vocabulary of a language, biochemical 'symbols' (the building blocks of oligo- and polymers) make writing molecular messages possible. Compared to nucleotides and amino acids, sugars have chemical properties that facilitate to reach an unsurpassed level of oligomer diversity. These glycans are a part of the ubiquitous cellular glycoconjugates. Cyto- and histochemically, the glycans' structural complexity is mapped by glycophenotyping of cells and tissues using receptors ('readers', thus called lectins), hereby revealing its dynamic spatiotemporal regulation: these data support the concept of a sugar code. When proceeding from work with plant (haem)agglutinins as such tools to the discovery of endogenous (tissue) lectins, it became clear that a broad panel of biological meanings can indeed be derived from the sugar-based vocabulary (the natural glycome incl. post-synthetic modifications) by glycan-lectin recognition in situ. As consequence, the immunocyto- and histochemical analysis of lectin expression is building a solid basis for the steps toward tracking down functional correlations, for example in processes leading to cell adhesion, apoptosis, autophagy or growth regulation as well as targeted delivery of glycoproteins. Introduction of labeled tissue lectins to glycan profiling assists this endeavor by detecting counterreceptor(s) in situ. Combining these tools and their applications strategically will help to take the trip toward the following long-range aim: to compile a dictionary for the glycan vocabulary that translates each message (oligosaccharide) into its bioresponse(s), that is to crack the sugar code.

An experimental observation on selecting binding partners underlies the introduction of the term 'lectin'. Agglutination of erythrocytes depending on their blood-group status revealed the presence of activities in plant extracts that act in an epitope-specific manner like antibodies. As it turned out, their binding partners on the cell surface are carbohydrates of glycoconjugates. By definition, lectins are glycan-specific (mono- or oligosaccharides presented by glycoconjugates or polysaccharides) receptors, distinguished from antibodies, from enzymes using carbohydrates as substrates and from transporters of free saccharides. They are ubiquitous in Nature and structurally widely diversified. More than a dozen types of folding pattern have evolved for proteins that bind glycans. Used as tool, this capacity facilitates versatile mapping of glycan presence so that plant/fungal and also animal/human lectins have found a broad spectrum of biomedical applications. The functional pairing with physiological counterreceptors is involved in a wide range of cellular activities from cell adhesion, glycoconjugate trafficking to growth regulation and lets lectins act as sensors/effectors in host defense.

The availability of regulatory sequences directing tissue-specific expression of transgenes in genetically modified mice and large animals is a prerequisite for the development of adequate models for human diseases. The rat insulin 2 gene (Ins2) promoter, widely used to achieve transgene expression in pancreatic beta-cells of mice, also directs expression to extrapancreatic tissues and performs poorly in isolated pancreatic islets of human, mouse, and pig. To evaluate whether the full 5' untranslated region (UTR) of the porcine insulin gene (INS) confers robust and specific expression in beta-cells we generated an expression cassette containing 1500bp of the porcine INS 5' UTR and the 3' UTR of the bovine growth hormone gene (GH). The cassette was designed to allow easy exchange of the sequences to be expressed and easy removal of the vector backbone from the expression cassette. To evaluate the properties of the cassette, we initially inserted a cDNA encoding human betacellulin, a growth factor known to affect structural and functional parameters of beta-cells. After confirming the functionality and specificity of the construct in vitro, transgenic mouse lines were generated by pronuclear DNA microinjection. Using RT-PCR, immunohistochemistry and immunofluorescence, we show that transgenic mice expressed human betacellulin exclusively in beta-cells. Confirming the proposed insulinotropic effect of betacellulin, transgenic mice showed improved glucose tolerance. We conclude that the newly designed expression cassette containing 1500bp of the porcine insulin promoter 5' UTR confers robust and specific transgene expression to beta-cells in vitro and in transgenic mice.

Lentiviral vectors are a powerful tool for the genetic modification of livestock species. We previously generated transgenic founder cattle with lentiviral integrants carrying enhanced green fluorescent protein (EGFP) under the control of the phosphoglycerate kinase (PGK) promoter. In this study, we investigated the transmission of LV-PGK-EGFP integrants through the female and male germ line in cattle. A transgenic founder heifer (#562, Kiki) was subjected to superovulation treatment and inseminated with semen from a non-transgenic bull. Embryos were recovered and transferred to synchronized recipient heifers, resulting in the birth of a healthy male transgenic calf expressing EGFP as detected by in vivo imaging. Semen from a transgenic founder bull (#561, Jojo) was used for in vitro fertilization (IVF) of in vitro matured (IVM) oocytes from non-transgenic cows. The rates of cleavage and development to blastocyst in vitro corresponded to 52.0 +/- 4.1 and 24.5 +/- 4.4%, respectively. Expression of EGFP was observed at blastocyst stage (day 7 after IVF) and was seen in 93.0% (281/302) of the embryos. 24 EGFP-expressing embryos were transferred to 9 synchronized recipients. Analysis of 2 embryos, flushed from the uterus on day 15, two fetuses recovered on day 45, and a healthy male transgenic calf revealed consistent high-level expression of EGFP in all tissues investigated. Our study shows for the first time transmission of lentiviral integrants through the germ line of female and male transgenic founder cattle. The pattern of inheritance was consistent with Mendelian rules. Importantly, high fidelity expression of EGFP in embryos, fetuses, and offspring of founder #561 provides interesting tools for developmental studies in cattle, including interactions of gametes, embryos and fetuses with their maternal environment.

Currently, our knowledge of early mammalian embryogenesis, stem cell differentiation and development is largely based on studies performed in mouse models. However, in important aspects, e.g. the timing of epigenetic reprogramming and embryonic genome activation, livestock species probably reflect far more closely the situation in men and other non-rodent mammals. A major challenge is the fact that in mammals, the development of individual zygotes is highly variable and vulnerable, and the outcome is uncertain. Valid indicators of the highly heterogeneous development and health status, and the actual developmental potential of individual oocytes, zygotes or embryos would be crucially important to tap the full power of holistic transcriptome and proteome analyses. Fluorescent reporter proteins opened new vistas for embryology and stem cell research: they can be used as reporters for the activity of gene promoters or tagged to functional proteins to study their intracellular localization in living cells, tissues and organisms. Fluorescent reporter genes may be used to microscopically observe key processes of early development. Thus, novel information related to developmental potential can be obtained from living embryos before processing them, e.g. for "-omic" studies. This review summarizes the main current reporter gene techniques and gene transfer approaches, which might be suitable for the investigation of early embryogenesis in livestock mammals. The potential of promoter reporter genes is exemplified by a bovine model system for quantitative monitoring of transcriptional reactivation of the so-called pluripotency gene POU5F1 in cloned bovine embryos.

The development of somatic cell nuclear transfer (SCNT) embryos critically depends on appropriate reprogramming and expression of pluripotency genes, such as Pou5f1/POU5F1 (previously known as Oct4/OCT4). To study POU5F1 transcription activation in living bovine SCNT embryos without interference by maternal POU5F1 mRNA, we generated chromosomally normal fetal fibroblast donor cells stably carrying a mouse Pou5f1 promoter-driven enhanced green fluorescent protein (EGFP) reporter gene at a single integration site without detectable EGFP expression. Morphologic and quantitative analyses of whole-mount SCNT embryos by confocal microscopy revealed robust initial activation of the Pou5f1 reporter gene during the fourth cell cycle. In Day 6 SCNT embryos EGFP expression levels were markedly higher than in Day 4 embryos but varied substantially between individual embryos, even at comparable cell numbers. Embryos with low EGFP levels had far more morphologically abnormal cell nuclei than those with high EGFP levels. Our data strongly suggest that bovine SCNT embryos consistently start activation of the POU5F1 promoter during the fourth cell cycle, whereas later in development the expression level substantially differs between individual embryos, which may be associated with developmental potential. In fibroblasts from phenotypically normal SCNT fetuses recovered on Day 34, the Pou5f1 reporter promoter was silent but was activated by a second round of SCNT. The restoration of pluripotency can be directly observed in living cells or SCNT embryos from such Pou5f1-EGFP transgenic fetuses, providing an attractive model for systematic investigation of epigenetic reprogramming in large mammals.

Leptin has been shown to exert positive effects during the maturation of bovine oocytes, influencing blastocyst development, apoptosis, and the transcript levels of developmentally important genes. The present study was conducted to characterize further the mechanisms of leptin action on oocytes and the role of cumulus cells (CCs) in this context. In the first series of experiments, cumulus-oocyte complexes (COCs) were matured in serum-free medium that contained 0, 1 or 10 ng/ml leptin or in medium that was supplemented with 10% (v/v) estrus cow serum (ECS). Leptin concentrations of 1 and 10 ng/ml stimulated the meiotic progression of oocytes. Moreover, TUNEL staining demonstrated that these leptin doses reduced the proportion of apoptotic CCs. In the second series of experiments, COCs or denuded oocytes (DOs) were matured in the presence of 0 or 10 ng/ml leptin. The percentages of COCs and DOs with extruded polar bodies were increased by leptin. In contrast, positive effects of leptin on fertilization rates and blastocyst development were only observed after treatment of COCs but not of DOs. Leptin treatment of COCs consistently enhanced blastocyst development even after parthenogenetic activation of oocytes or after the removal of CCs before fertilization. The proportion of polyspermic oocytes was not affected by leptin treatment or oocyte denudation. In the third series of experiments, COCs were matured in the presence of 0, 1 or 10 ng/ml leptin. The transcript levels of specific genes were determined by reverse transcriptase-quantitative PCR (RT-qPCR) analysis of cumulus cells and single oocytes. Leptin treatment increased the levels of FAS, FASLG, and STAT3 transcripts in oocytes, but did not affect the LEPR, BAX, and BIRC4 mRNA concentrations. In cumulus cells, leptin treatment increased the mRNA levels for LEPR, STAT3, BAX, BIRC4, and FAS, but did not alter FASLG mRNA abundance. In conclusion, leptin differentially regulates gene expression in oocytes and cumulus cells. Moreover, leptin enhances both oocyte maturation and developmental capacity via cumulus cell-independent and -dependent mechanisms.

Like various other diurnal birds of prey, the world's largest eagle, the Harpy (Harpia harpyja), presents an atypical bird karyotype with 2n=58 chromosomes. There is little knowledge about the dramatic changes in the genomic reorganization of these species compared to other birds. Since recently, the chicken provides a "default map" for various birds including the first genomic DNA sequence of a bird species. Obviously, the gross division of the chicken genome into relatively gene-poor macrochromosomes and predominantly gene-rich microchromosomes has been conserved for more than 150 million years in most bird species. Here, we present classical features of the Harpy eagle karyotype but also chromosomal homologies between H. harpyja and the chicken by chromosome painting and comparison to the chicken genome map. We used two different sets of painting probes: (1) chicken chromosomes were divided into three size categories: (a) macrochromosomes 1-5 and Z, (b) medium-sized chromosomes 6-10, and (c) 19 microchromosomes; (2) combinatorially labeled chicken chromosome paints 1-6 and Z. Both probe sets were visualized on H. harpyja chromosomes by multicolor fluorescence in situ hybridization (FISH). Our data show how the organization into micro- and macrochromosomes has been lost in the Harpy eagle, seemingly without any preference or constraints.

We propose the use of single nucleotide polymorphisms (SNPs) instead of polymorphic microsatellite markers for individual identification and parentage control in cattle. To this end, we present an initial set of 37 SNP markers together with a gender-specific SNP for identity control and parentage testing in the Holstein, Fleckvieh and Braunvieh breeds. To obtain suitable SNPs, a total of 91.13 kb of random genomic DNA was screened yielding 531 SNPs. These, and 43 previously identified SNPs, were subjected to the following selection criteria: (1) the frequency of the minor allele must be larger than 0.1 in at least two of the three examined breeds, and (2) markers should not be linked closely. Allele frequencies were estimated by analysing sequencing traces of pooled DNA or by genotyping individual DNA samples. The selected SNP loci were physically mapped by radiation hybrid mapping or by fluorescence in situ hybridization, and tested against the neutral mutation hypothesis. The presented marker set theoretically allows probabilities of identity less than 10(-13) for individual verification and exclusion powers exceeding 99.99% for parentage testing.

Chicken chromosome paints for macrochromosomes 1-10, Z, and the nine largest microchromosomes (Griffin et al. 1999) were used to analyze chromosome homologies between chicken (Gallus gallus domesticus: Galliformes), domestic pigeon (Columba livia: Columbiformes), chaffinch (Fringilla coelebs: Passeriformes), and redwing (Turdus iliacus: Passeriformes). High conservation of syntenies was revealed. In general, both macro- and microchromosomes in these birds showed very low levels of interchromosomal rearrangements. Only two cases of rearrangements were found. Chicken chromosome 1 corresponds to chromosome 1 in pigeon, but to chromosomes 3 and 4 in chaffinch and chromosomes 2 and 5 in redwing. Chicken chromosome 4 was shown to be homologous to two pairs of chromosomes in the karyotypes of pigeon and both passerine species. Comparative analysis of chromosome painting data and the results of FISH with (TTAGGG)n probe did not reveal any correlation between the distribution of interstitial telomere sites (ITSs) and chromosome rearrangements in pigeon, chaffinch and redwing. In chaffinch, ITSs were found to co-localize with a tandem repeat GS (Liangouzov et al. 2002), monomers of which contain an internal TTAGGG motif.

Sulfate is one of the most important macronutrients in cells and the major sulfur source in many organisms as well as one of the most abundant anions in the serum. As sulfate is a hydrophilic anion, movement across the lipid bilayer is mediated by transporters that regulate efflux and influx. Here, we report the molecular cloning, mapping, and functional analysis of the bovine solute carrier/sulfate transporter SLC26a2 gene, the first member of this family to be cloned in cattle. A recombinant phage library was screened, and single phages harbouring the SLC26a2 gene was isolated and sequenced. A fragment of 6295 base pairs (bp) of the bovine SLC26a2 gene harbouring exon 2 and exon 3 was used for further analysis. Similar to the human, ovine, mouse, and rat SLC26a2 gene, the bovine ortholog consists of two coding exons. The open reading frame harbours 2202 nucleotides (nt), coding for a protein of 734 amino acids with a calculated molecular weight of 81.5 kilodaltons (kDa) and a statistical isoelectric point (p I ) of 8.77. The bovine SLC26a2 gene was mapped to chromosome 7q23–q24 (BTA 7q23–q24) by fluorescence in situ hybridisation (FISH) analysis. Two point mutations were identified comparing the DNAs of 300 Holstein Frisian cattle, one of them resulting in an isoleucine to serine amino acid exchange at position 520. The Ile520Ser exchange influences the sulfate uptake as measured in primary fibroblasts isolated from testis and in immortalized fibroblastoid bovine cell lines.

Chromosome painting probes specific for macrochromosomes 1, 2, 3, 4, 5, and Z were applied to both mitotic and lampbrush chromosomes of the chicken (Gallus gallus domesticus). Five autosomal macrobivalents and sex chromosome Z in the lampbrush phase were identified and their correspondence to the target chromosomes in the metaphase of mitosis was shown. Nascent transcripts on lateral loops of the target lampbrush chromosome were intensively labelled when the hybridization was performed without RNase A treatment according to the DNA/(DNA + RNA) hybridization protocol.

Development of early embryonic stages before activation of the embryonic genome depends on sufficiently stored products of the maternal genome, adequate recruitment and degradation of mRNAs, as well as activation, deactivation, and relocation of proteins. By application of an isobaric tagging for relative and absolute quantification (iTRAQ)-based approach, the proteomes of bovine embryos at the zygote and 2-cell and 4-cell stage with MII oocytes as a reference were quantitatively analyzed. Of 1072 quantified proteins, 87 differed significantly in abundance between the four stages. The proteomes of 2-cell and 4-cell embryos differed most from the reference MII oocyte, and a considerable fraction of proteins continuously increased in abundance during the stages analyzed, despite a strongly attenuated rate of translation reported for this period. Bioinformatic analysis revealed particularly interesting proteins involved in the p53 pathway, lipid metabolism, and mitosis. Verification of iTRAQ results by targeted SRM (selected reaction monitoring) analysis revealed excellent agreement for all five proteins analyzed. By principal component analysis, SRM quantifications comprising a panel of only five proteins were shown to discriminate between all four developmental stages analyzed here. For future experiments, an expanded SRM protein panel will provide the potential to detect developmental disturbances with high sensitivity and enable first insights into the underlying molecular pathways.

Arrangements of chromosome territories in nuclei of chicken fibroblasts and neurons were analysed employing multicolour chromosome painting, laser confocal scanning microscopy and three-dimensional (3D) reconstruction. The chicken karyotype consists of 9 pairs of macrochromosomes and 30 pairs of microchromosomes. Although the latter represent only 23% of the chicken genome they contain almost 50% of its genes. We show that territories of microchromosomes in fibroblasts and neurons were clustered within the centre of the nucleus, while territories of the macrochromosomes were preferentially located towards the nuclear periphery. In contrast to these highly consistent radial arrangements, the relative arrangements of macrochromosome territories with respect to each other (side-by-side arrangements) were variable. A stringent radial arrangement of macro- and microchromosomes was found in mitotic cells. Replication labelling studies revealed a pattern of DNA replication similar to mammalian cell nuclei: gene dense, early replicating chromatin mostly represented by microchromosomes, was located within the nuclear interior, surrounded by a rim of late replicating chromatin. These results support the evolutionary conservation of several features of higher-order chromatin organization between mammals and birds despite the differences in their karyotypes.

Uromodulin-associated kidney disease (UAKD) is a dominant heritable renal disease in humans which is caused by mutations in the uromodulin (UMOD) gene and characterized by heterogeneous clinical appearance. To get insights into possible causes of this heterogeneity of UAKD, we describe the new mutant mouse line UmodC93F, leading to disruption of a putative disulfide bond which is also absent in a known human UMOD mutation, and compare the phenotype of this new mouse line with the recently published mouse line UmodA227T. In both mutant mouse lines, which were both bred on the C3H background, the Umod mutations cause a gain-of-toxic function due to a maturation defect of the mutant uromodulin leading to a dysfunction of thick ascending limb of Henle's loop (TALH) cells of the kidney. Umod mutant mice exhibit increased plasma urea and cystatin levels, impaired urinary concentration ability, reduced fractional excretion of uric acid, and nephropathological alterations including uromodulin retention in TALH cells, interstitial fibrosis and inflammatory cell infiltrations, tubular atrophy, and occasional glomerulo- und tubulocystic changes, a phenotype highly similar to UAKD in humans. The maturation defect of mutant uromodulin leads to the accumulation of immature uromodulin in the endoplasmic reticulum (ER) and to ER hyperplasia. Further, this study was able to demonstrate for the first time in vivo that the severity of the uromodulin maturation defect as well as onset and speed of progression of renal dysfunction and morphological alterations are strongly dependent on the particular Umod mutation itself and the zygosity status.

The spatial organization of chromatin in the nucleus contributes to genome function and is altered during the differentiation of normal and tumorigenic cells. Although nuclear actin-related proteins (Arps) have roles in the local alteration of chromatin structure, it is unclear whether they are involved in the spatial positioning of chromatin. In the interphase nucleus of vertebrate cells, gene-dense and gene-poor chromosome territories (CTs) are located in the center and periphery, respectively. Here we analyzed the chicken DT40 cells in which Arp6 had been knocked out conditionally, and showed that the radial distribution of CTs was impaired in these knockout cells. Arp6 is an essential component of the SRCAP chromatin remodeling complex, which deposits the histone variant H2A.Z into chromatin. The redistribution of CTs was also observed in H2A.Z-deficient cells in gene-rich microchromosomes, but to lesser extent in gene-poor macrochromosomes. These results indicate that Arp6 and H2A.Z contribute to the radial distribution of CTs through different mechanisms. Microarray analysis suggested that the localization of chromatin to the nuclear periphery per se is insufficient for the repression of most genes.

BACKGROUND: The causes, modes, biological role and prospective significance of cell death in preimplantation development in humans and other mammals are still poorly understood. Early bovine embryos represent a very attractive experimental model for the investigation of this fundamental and important issue. METHODS AND FINDINGS: To obtain reference data on the temporal and spatial occurrence of cell death in early bovine embryogenesis, three-dimensionally preserved embryos of different ages and stages of development up to hatched blastocysts were examined in toto by confocal laser scanning microscopy. In parallel, transcript abundance profiles for selected apoptosis-related genes were analyzed by real-time reverse transcriptase-polymerase chain reaction. Our study documents that in vitro as well as in vivo, the first four cleavage cycles are prone to a high failure rate including different types of permanent cell cycle arrest and subsequent non-apoptotic blastomere death. In vitro produced and in vivo derived blastocysts showed a significant incidence of cell death in the inner cell mass (ICM), but only in part with morphological features of apoptosis. Importantly, transcripts for CASP3, CASP9, CASP8 and FAS/FASLG were not detectable or found at very low abundances. CONCLUSIONS: In vitro and in vivo, errors and failures of the first and the next three cleavage divisions frequently cause immediate embryo death or lead to aberrant subsequent development, and are the main source of developmental heterogeneity. A substantial occurrence of cell death in the ICM even in fast developing blastocysts strongly suggests a regular developmentally controlled elimination of cells, while the nature and mechanisms of ICM cell death are unclear. Morphological findings as well as transcript levels measured for important apoptosis-related genes are in conflict with the view that classical caspase-mediated apoptosis is the major cause of cell death in early bovine development.

Transgenic mouse lines expressing Cre recombinase in a cell-specific and tissue-specific manner are essential tools for studying gene function and for developing suitable models for human diseases. Here, we used an expression cassette containing the full 5' untranslated region of the porcine insulin gene to generate a mouse line expressing Cre recombinase specifically in pancreatic β-cells by pronuclear DNA microinjection. We obtained a founder animal that transmitted the construct to its descendants in a Mendelian fashion and whose descendants showed a clear activation of β-galactosidase expression in pancreatic β-cells after crossing into the ROSA26 lacZ reporter mouse line. Cre expression in other organs was negative except for the kidney, intestine, and the cerebral pons where β-galactosidase activity was detected in a small percentage of the cells. This new mouse line is a valuable tool for recombination of floxed alleles in pancreatic β-cells in vivo.

Oocyte maturation is a complex process and a critical issue in assisted reproduction techniques (ART) in humans and other mammals. We used a sensitive 2-D DIGE saturation labeling approach including an internal pooled standard for quantitative proteome profiling of immature versus in vitro matured bovine oocytes in six independent samples. The study comprised 48 2D gel images representing 24 DIGE experiments. From 250 ng sample analyzed per gel, quantitative analysis revealed an average of 2244 spots in pH 4-7 images and 1291 spots in pH 6-9 images. Thirty-eight spots with different intensities were detected in total. Spots of a preparative gel from 2200 oocytes were identified by nano-LC-MS/MS analysis. The ten spots which could be unambiguously identified include the Ca2+-binding protein translationally controlled tumor protein, enzymes of the Krebs and pentose phosphate cycles, clusterin, 14-3-3 epsilon, elongation factor-1 gamma, and redox enzymes such as polymorphic forms of GST Mu 5 and peroxiredoxin-3. The cellular distribution of two proteins was determined by confocal laser scanning microscopy. The interesting protein candidates identified by this study may help to improve the in vitro maturation process in order to increase the rate of successful in vitro fertilization and other ART in cattle and other mammals.

A histochemical study using fluorescein isothiocyanate (FITC)-labelled lectins to identify glycoconjugates present in the efferent ductules and the three segments of the ductus epididymis (initial, middle and terminal segment) of dogs was carried out. The lectins used were: mannose-binding lectins (Con A, LCA and PSA), galactose-binding lectins (PNA, RCA), N-acetylgalactosamine-binding lectins (DBA, SBA, SJA and GSL I), N-acetylglucosamine-binding lectins (WGA and WGAs), fucose-binding lectins (UEA) and lectins which bind to complex carbohydrate configurations (PHA E and PHA L). The lectin-binding pattern in the canine epididymis presents similarities and differences to those observed in other mammalian species. The ductuli efferentes distinctly stained with most of the lectins used, whereas in the ductus epididymis a segment specific staining pattern was observed. Whereas principal cells of the ductus epididymis stained clearly with several FITC-labelled lectins (WGA, UEA and PHA-L), basal cells showed only a significant binding of Con A.

Karyotypes of most bird species are characterized by around 2n = 80 chromosomes, comprising 7-10 pairs of large- and medium-sized macrochromosomes including sex chromosomes and numerous morphologically indistinguishable microchromosomes. The Falconinae of the Falconiformes has a different karyotype from the typical avian karyotype in low chromosome numbers, little size difference between macrochromosomes and a smaller number of microchromosomes. To characterize chromosome structures of Falconinae and to delineate the chromosome rearrangements that occurred in this subfamily, we conducted comparative chromosome painting with chicken chromosomes 1-9 and Z probes and microchromosome-specific probes, and chromosome mapping of the 18S-28S rRNA genes and telomeric (TTAGGG)( n ) sequences for common kestrel (Falco tinnunculus) (2n = 52), peregrine falcon (Falco peregrinus) (2n = 50) and merlin (Falco columbarius) (2n = 40). F. tinnunculus had the highest number of chromosomes and was considered to retain the ancestral karyotype of Falconinae; one and six centric fusions might have occurred in macrochromosomes of F. peregrinus and F. columbarius, respectively. Tandem fusions of microchromosomes to macrochromosomes and between microchromosomes were also frequently observed, and chromosomal locations of the rRNA genes ranged from two to seven pairs of chromosomes. These karyotypic features of Falconinae were relatively different from those of Accipitridae, indicating that the drastic chromosome rearrangements occurred independently in the lineages of Accipitridae and Falconinae.

The development of somatic cell nuclear transfer (SCNT) embryos critically depends on appropriate reprogramming and expression of pluripotency genes, such as Pou5f1/POU5F1 (previously known as Oct4/OCT4). To study POU5F1 transcription activation in living bovine SCNT embryos without interference by maternal POU5F1 mRNA, we generated chromosomally normal fetal fibroblast donor cells stably carrying a mouse Pou5f1 promoter-driven enhanced green fluorescent protein (EGFP) reporter gene at a single integration site without detectable EGFP expression. Morphologic and quantitative analyses of whole-mount SCNT embryos by confocal microscopy revealed robust initial activation of the Pou5f1 reporter gene during the fourth cell cycle. In Day 6 SCNT embryos EGFP expression levels were markedly higher than in Day 4 embryos but varied substantially between individual embryos, even at comparable cell numbers. Embryos with low EGFP levels had far more morphologically abnormal cell nuclei than those with high EGFP levels. Our data strongly suggest that bovine SCNT embryos consistently start activation of the POU5F1 promoter during the fourth cell cycle, whereas later in development the expression level substantially differs between individual embryos, which may be associated with developmental potential. In fibroblasts from phenotypically normal SCNT fetuses recovered on Day 34, the Pou5f1 reporter promoter was silent but was activated by a second round of SCNT. The restoration of pluripotency can be directly observed in living cells or SCNT embryos from such Pou5f1-EGFP transgenic fetuses, providing an attractive model for systematic investigation of epigenetic reprogramming in large mammals.

Cattle can serve as a model organism to resolve central questions in mammalian reproduction that cannot be clarified in the mouse model due to notable species-specific peculiarities of early mouse embryogenesis. As part of a project on structural, molecular, and functional deficiencies of bovine oocytes, we started to systematically investigate fertilization and the onset of embryo development in vitro by 3-dimensional multicolor fluorescence microscopy. We are using 3D visualisation as key approach to clarify the multiple parallel and sequential processes and events of fertilization as well as to identify and classify errors and failures. Moreover, we aim to gain insights into the mechanisms of aberrations by linking processes at the cellular and the molecular level. We studied class I and II oocytes collected from slaughterhouse ovaries and matured for 23 h in vitro. Oocytes were fixed at different times from 4 to 12 h postinsemination with formaldehyde in a microtubule-stabilising buffer containing taxol in such a way that the 3-dimensional cell architecture was maintained, and were stained for DNA, microtubules, and f-actin microfilaments. In addition, serine 10-phosphorylated histone H3 was used as a marker for chromosome condensation and the spindle midbody. For 3-dimensional imaging of the oocytes in toto, confocal serial sections were captured at 1-µm intervals using a 40× objective (NA = 1.3). For imaging details, we used a high spatial sampling density (pixel size: 50 × 50 nm, z-step size: 200 nm) close to the Nyquist criterion and image restoration by maximum likelihood estimation (MLE) deconvolution. A series of more than 500 three-dimensional snapshots of fertilized oocytes at different points in time gives a first detailed view on the spatial and temporal course of the sperm entry, the formation of the paternal pronucleus and the sperm aster, completion of oocyte meiosis and the formation of the maternal pronucleus, as well as dynamic changes of the cytoskeleton. Moreover, we can document a spectrum of abnormalities including spontaneous parthenogenetic oocyte activation, polyspermy, and aberrations of meiosis I and II. The latter include irregular spindle formation and chromosome segregation, the occurrence of chromatin bridges and abnormal spindle positioning and rotation (e.g. leading to nonextrusion of a first or a second polar body or the extrusion of two second polar bodies). Our microscopic investigation in the bovine system contributes to unraveling the origins of irregular cleavage, aneuploidy, and mosaicism in mammals. Three-dimensional high-speed microscopy of oocytes and zygotes in affordable timeframes could be of great value in improving the differential diagnosis of oocyte and sperm dysfunction, as well as in identifying and dissecting problems, limitations, and potential risks of reproductive technologies (ART).This work is supported by the Deutsche Forschungsgemeinschaft (DFG FOR 1041).

Development of early embryonic stages before activation of the embryonic genome depends on sufficiently stored products of the maternal genome and adequate activation, deactivation, and relocation of proteins. To establish protein function, several posttranslational events (e.g. proteolytic activation, phosphorylation, or secretion) are frequently essential and thereby prevent prediction of protein abundance from transcript abundance. Consequently, proteomic studies are indispensable to characterise the molecular processes governing early embryonic development and to establish corresponding regulatory networks. Here, we present a quantitative proteome analysis of bovine zygotes and embryos at the 2-cell and 4-cell stage. Cumulus-oocyte complexes (COC) were prepared from bovine ovaries obtained from a local abattoir and selected for a compact layer of cumulus cells. In vitro maturation, fertilization, and embryo production were performed according to standard procedures. For quantitative isobaric tags for relative and absolute quantitation (iTRAQ)-liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, protein from batches of 50 MII oocytes (serving as a reference), zygotes, 2-cell and 4-cell stage embryos, respectively, was extracted. Quantitative proteome analysis of iTRAQ-labelled tryptic peptides was performed on an Orbitrap XL instrument (Thermo Fisher, Waltham, MA, USA) coupled to an Eksigent nano-liquid chromatography system (AB Sciex, Framingham, MA, USA). The tandem MS data were analysed by MASCOT and filtered for a false discovery rate (FDR) of

Leptin has been shown to exert positive effects during the maturation of bovine oocytes, influencing blastocyst development, apoptosis, and the transcript levels of developmentally important genes. The present study was conducted to characterize further the mechanisms of leptin action on oocytes and the role of cumulus cells (CCs) in this context. In the first series of experiments, cumulus-oocyte complexes (COCs) were matured in serum-free medium that contained 0, 1 or 10 ng/ml leptin or in medium that was supplemented with 10% (v/v) estrus cow serum (ECS). Leptin concentrations of 1 and 10 ng/ml stimulated the meiotic progression of oocytes. Moreover, TUNEL staining demonstrated that these leptin doses reduced the proportion of apoptotic CCs. In the second series of experiments, COCs or denuded oocytes (DOs) were matured in the presence of 0 or 10 ng/ml leptin. The percentages of COCs and DOs with extruded polar bodies were increased by leptin. In contrast, positive effects of leptin on fertilization rates and blastocyst development were only observed after treatment of COCs but not of DOs. Leptin treatment of COCs consistently enhanced blastocyst development even after parthenogenetic activation of oocytes or after the removal of CCs before fertilization. The proportion of polyspermic oocytes was not affected by leptin treatment or oocyte denudation. In the third series of experiments, COCs were matured in the presence of 0, 1 or 10 ng/ml leptin. The transcript levels of specific genes were determined by reverse transcriptase-quantitative PCR (RT-qPCR) analysis of cumulus cells and single oocytes. Leptin treatment increased the levels of FAS, FASLG, and STAT3 transcripts in oocytes, but did not affect the LEPR, BAX, and BIRC4 mRNA concentrations. In cumulus cells, leptin treatment increased the mRNA levels for LEPR, STAT3, BAX, BIRC4, and FAS, but did not alter FASLG mRNA abundance. In conclusion, leptin differentially regulates gene expression in oocytes and cumulus cells. Moreover, leptin enhances both oocyte maturation and developmental capacity via cumulus cell-independent and -dependent mechanisms.

Bloom's syndrome (BS) is an autosomal disorder characterized by predisposition to a wide variety of cancers. The gene product whose mutation leads to BS is the RecQ family helicase BLM, which forms a complex with DNA topoisomerase IIIalpha (Top3alpha). However, the physiological relevance of the interaction between BLM and Top3alpha within the cell remains unclear. We show here that Top3alpha depletion causes accumulation of cells in G2 phase, enlargement of nuclei, and chromosome gaps and breaks that occur at the same position in sister chromatids. The transition from metaphase to anaphase is also inhibited. All of these phenomena except cell lethality are suppressed by BLM gene disruption. Taken together with the biochemical properties of BLM and Top3alpha, these data indicate that BLM and Top3alpha execute the dissolution of sister chromatids.

Growth factors are involved in development and function of the mammary gland. The aim of this study was the localization of fibroblast growth factor 1 (FGF-1) and its mRNA in the bovine mammary gland during different developmental and functional stages. Mammary tissue was obtained from German Brown Swiss cows (n = 23) during defined stages of mammogenesis (before and during pregnancy), lactogenesis, peak lactation and involution. The distribution of FGF-1 mRNA was studied using non-radioactive in situ hybridization, the corresponding FGF-protein was analysed using immunohistochemistry [avidin-biotin peroxidase complex (ABC)-method]. A moderate to distinct staining for FGF-mRNA was found in the epithelium of ducts and developing alveoli during mammogenesis. Post-partum at the same cellular locations, a considerable amount of FGF-1 mRNA, was seen that decreased during lactation. Also during early involution clear staining for FGF-mRNA could still be observed. Immunoreactive FGF-1 was found in considerable concentration in the epithelium of the mammary gland in heifers. The staining intensity generally decreased somewhat during mammogenesis and lactation, but could be always clearly demonstrated in the secretory epithelial cells of alveoli and glandular ducts. Also during the first day after the end of milking, the epithelium displayed a moderate to distinct epithelial immunostaining. Notably, After 4 weeks of involution, in many alveoli a shedding of the FGF-1 positive luminal cell layer was found. In our localization studies, no strict correlation between FGF-1 mRNA and its corresponding protein was found. The various reasons for this finding are discussed.

Several fibroblast growth factors (FGFs) are implicated in proliferation and differentiation of both somatic and germ cells during testicular development, as well as in spermatogenesis of adult testis. The expression of FGF2 was studied in the adult bovine testis using quantitative RT-PCR, RNA in situ hybridization, and immunohistochemistry. Quantitative RT-PCR revealed consistent levels of FGF2 mRNA in parenchymal samples of the bovine testis. In situ hybridization localized FGF2 transcripts only in a constant fraction of Leydig and Sertoli cells as well as in modified Sertoli cells of the terminal segments. Immunohistochemistry revealed (a) no FGF2 protein in Sertoli cells (b) moderate cytoplasmic staining in Leydig cells and spermatogonia and (c) strong nuclear and faint cytoplasmic staining in myofibroblasts, in epithelial cells of straight tubules and rete testis and in blood vessels. These observations indicate a pleiotropic effect of FGF2 on the control of spermatogenesis in a paracrine and/or autocrine manner.

Separation of X- and Y-bearing sperm cells, together with artificial insemination using sex-specific semen, makes it possible to pre-determine the sex of calves. This has the potential to considerably improve cattle breeding, genetic resource management and particularly the efficiency of dairy and meat production. However, the broad use of sexed semen will depend on availability, price, fertilizability and in particular the actual sorting purity of sperm doses. To validate the accuracy of sperm sexing in Bos taurus, we have developed a simple, fast and reliable dual colour fluorescence in situ hybridization (FISH) test, where Y-bearing spermatozoa are identified by a DNA fragment hybridizing to a large pericentromeric repetitive DNA block on the bovine Y chromosome (locus DYZI, Yp13-q12). To avoid an underestimation of Y signals, we used a second DNA probe identifying a large subcentromeric block of complex repetitive DNA on the bovine autosome 6 (locus D6Z1, 6q12-15) as a positive control. Bovine sperm were fixed with methanol:acetic acid and denatured by simply immersing in 3 M NaOH, yielding consistent hybridization results and good preservation of sperm morphology. The FISH protocol was evaluated on unsorted sperm as well as on sperm samples sexed using the Beltsville technology, which separates X- and Y-bearing spermatozoa by staining with Hoechst 33342 and flow sorting according to their DNA content (Johnson et al. 1987).

The chicken Ig-like receptors (CHIR) have been described as two Ig domain molecules with long cytoplasmic tails containing inhibitory motifs. In this study, we demonstrate that CHIR form a large family, with multiple members showing great sequence variability among members as well as a great diversity in domain organization and properties of the transmembrane and cytoplasmic segments. We characterize various novel receptor types with motifs indicative of inhibitory, activating, or both functions. In addition to the inhibitory receptors with two ITIM, receptors with a single immunoreceptor tyrosine-based switch motif or receptors lacking a cytoplasmic domain were isolated. Activating receptors with a short cytoplasmic domain and a transmembrane arginine assembled with the newly identified chicken FcεRIγ chain. Three bifunctional receptor types were characterized composed of one or two C2-type Ig-like domains, a transmembrane region with a positively charged residue and combinations of cytoplasmic motifs such as ITIM, immunoreceptor tyrosine-based switch motif, and YXXM. RT-PCR revealed distinct expression patterns of individual CHIR. All receptor types shared a conserved genomic architecture, and in single Ig domain receptors a pseudoexon replaced the second Ig exon. Southern blot analyses with probes specific for the Ig1 domain were indicative of a large multigene family. Of 103 sequences from the Ig1 domain of a single animal, 41 unique sequences were obtained that displayed extensive variability within restricted Ig regions. Fluorescence in situ hybridization localized the CHIR gene cluster to microchromosome 31 and identified this region as orthologous to the human leukocyte receptor complex.

A bovine BAC clone containing a processed laminin receptor pseudogene (LAMR1P) has been isolated and characterized. A 2,901-bp sequence was produced from the clone, of which 1,187 bp represented seven identifiable exon-like domains, but no intervening sequences. The pseudogene sequence reveals several transversions and transitions, as well as insertions and deletions. A premature stop codon motif is present at nucleotide position 115 located in the exon-2-like domain. Physical mapping of the gene was performed by FISH and RH panel mapping and assigned LAMR1P to BTA4q24→q26 with the closest linkage to BM6458 (19 cR, LOD score of 11.6). The functional laminin receptor putatively plays an important role in the transmission of bovine spongiform encephalopathy (BSE). In this process, the receptor supposedly acts as the binding site for prion proteins to enter mammalian cells. Considering the existence of several human laminin receptor pseudogenes forming a complex family, any knowledge of even pseudogene sequences might be helpful to isolate the functional bovine laminin receptor gene.

Chicken genome mapping is important for a range of scientific disciplines. The ability to distinguish chromosomes of the chicken and other birds is thus a priority. Here we describe the molecular cytogenetic characterization of each chicken chromosome using chromosome painting and mapping of individual clones by FISH. Where possible, we have assigned the chromosomes to known linkage groups. We propose, on the basis of size, that the NOR chromosome is approximately the size of chromosome 22; however, we suggest that its original assignment of 16 should be retained. We also suggest a definitive chromosome classification system and propose that the probes developed here will find wide utility in the fields of developmental biology, DT40 studies, agriculture, vertebrate genome organization, and comparative mapping of avian species.

Hyaluronidases are enzymes responsible for the breakdown of hyaluronan (HA). The human genome contains six hyaluronidase-like genes sharing about 40% amino acid identity with one another. The genes are organized into two groups: HYAL1, HYAL2, and HYAL3 are clustered on HSA3p21.3 and are equivalent to the genes for tumor suppressor for small cell lung carcinoma LUCA1, LUCA2, and LUCA3, respectively (Wei et al., 1996). This cluster has been reported to be part of the Tumor Suppressor Genes clone (TSGs) spanning F370 kb containing the gene encoding putative tumor suppressor protein FUS2 (Lerman and Minna, 2000). The other group containing HYAL4, SPAM1/PH-20 and the expressed pseudogene HYALP1 was assigned to HSA7q31.3. Knowledge about the genes’ functions resulted from the identification of mutations in the HYAL1 gene causing mucopolysaccharidosis type IX in man. In this report we document the mapping of the porcine HYAL3 gene by FISH and hybrid panel analyses and reveal the location on SSC13q21. Materials and methods

The recognition of carbohydrate sequences by complementary receptors has been shown to be a critical factor when the plasma membrane of the sperm head binds to the zona pellucida (ZP), an extracellular coat surrounding the oocyte. ZP3, one of the three glycoproteins in the ZP, is the egg protein recognized by sperm. A human sperm surface protein, HUMSPRMTK (Mr = 95,000) homolog has been identified on the basis of its specific affinity for ZP3 (Burks et al., 1995). It is expressed only in testicular germ cells, which has been assigned to chromosome HSA2q. Detailed mapping and the availability of the primary sequence would further our understanding of the molecular basis of infertility and for development of a spermtargeted contraceptive. The latter based on the fact that synthetic blocking of the intracellular domain inhibits phosphotyrosine activity (Leyton and Saling, 1989), thus preventing fertilization. Here we report the localization of the porcine sperm protein zona receptor tyrosine kinase gene to chromosome 3q11→q12. Materials and methods

The acid hydrolase GUSB (s-D-glucuronoside glucuronosohydrolase, E.C. 3.2.1.31) plays an essential role in catabolism of glucuronic acid containing glycosaminoglycans (GAGs). It cleaves s-glucuronosyl residues at the non-reducing end of oligosaccharides from GAGs. GUSB degrades sulphated glycosaminoglycans such as heparan, dermatan and chondroitin and is involved in the catabolic pathway of hyaluronic acid. GUSB deficiency causes the lysosomal storage disorder Mucopolysaccharidosis type VII. Clinical and biochemical findings of MPSVII in human were first described by Sly et al. (1973). Fyfe et al. (1999) reported a G→A transition at position 1074 of an MPSVII affected cat GUSB cDNA. GUSB is already assigned to human chromosome 7q21.11. Here we report the localization of the porcine GUSB gene to chromosome SSC3p16→p14 by FISH and confirm the position by screening of hybrid panels.

In the nucleus of animal and plant cells individual chromosomes maintain a compartmentalized structure. Chromosome territories (CTs), as these structures were named by Theodor Boveri, are essential components of the higher-order chromatin architecture. Recent studies in mammals and non-mammalian vertebrates indicate that the radial position of a given CT (or segments thereof) is correlated with its size, its gene-density and its replication timing. As a representative case, chicken cell nuclei show highly consistent radial chromatin arrangements: gene-rich, early replicating microchromosomes are clustered within the nuclear interior, while gene-poor, later replicating macrochromosomes are preferentially located at the nuclear periphery. In humans, chromosomes 18 and 19 (HSA18 and 19) territories that are of similar size show a distinctly different position in the cell nuclei of lymphocytes and lymphoblastoid cells: the gene-rich and early replicating HSA19 CTs are typically found close to the nuclear center, while the gene-poor and later replicating HSA18 CTs are preferentially located at the nuclear periphery. Recent comparative maps between human and chicken chromosomes revealed that the chicken macrochromosomes 2 and Z contain the genes homologous to HSA18, while the genes on HSA19 are located onto the chicken microchromosomes. These data lend tentative support to the hypothesis that differences in the radial nuclear positions of gene-rich, early replicating and gene-poor, later replicating chromatin have been evolutionarily conserved during a period of more than 300 million years irrespective of the evolution of highly divergent karyotypes between humans and chicken.

Three-dimensional (3-D) super-resolution fluorescence microscopy has allowed major progress in studies of the functional nuclear organization (Markaki et al. 2010 Cold Spring Harb. Symp. Quant. Biol. 75, 475–492; Markaki et al. 2012 Bioessays 34, 412–426). We have exploited these new possibilities to explore nuclear organization at different stages of bovine pre-implantation development (4-cell, 8-cell, 16-cell, morula, and blastocyst stage). In particular, we studied the topography of RNA polymerase II and the distribution of transcriptionally competent and noncompetent chromatin using antibodies against H3K4me3 and H3K27me3, respectively. For comparison, we have started analyses of mouse pre-implantation embryos and embryonic stem cells as well. Our results support the chromosome territory-interchromatin compartment (CT-IC) model (Cremer and Cremer 2010 Cold Spring Harb. Perspect. Biol. 2, a003889; Cremer et al. 2012 In: Epigenetic Regulation and Epigenomics 451–483). In all cell types, the nuclear space is occupied by chromosome territories (CTs; Koehler et al. 2009 Exp. Cell Res. 315, 2053–2063), the interchromatin compartment (IC), and one or several nucleoli. The CTs are built up from interconnected, megabase-sized chromatin domains (CDs). These ~1-Mbp CDs may consist of a series of ~100-kbp CDs (Cremer et al. 2000 Crit. Rev. Eukaryot. Gene Expr. 10, 179–212), which globally form a compact chromatin core surrounded by a layer of decondensed chromatin, called the perichromatin region. Current evidence supports the hypothesis that the perichromatin region represents the nuclear compartment, where transcription, co-transcriptional splicing, DNA-replication, and DNA-repair take place (Rouquette et al. 2010 Int. Rev. Cell Mol. Biol. 282, 1–90). The IC provides a contiguous, crowded compartment, which starts with channels at nuclear pores and pervades the chromatin compartment both between and within CTs. Small-scale chromatin loops of the perichromatin region can protrude into the interior of IC channels allowing direct contacts between CDs in cis and trans. At other sites the IC expands to wider, chromatin-free lacunas with splicing speckles and nuclear bodies. This model is in line with a fractal higher-order chromatin arrangement at all levels from CTs, chromosome arms and bands to ~1 Mbp CDs organized as fractal globules (Mirny 2011 Chromosome Res. 19, 37–51). This work is supported by the DFG (ZA 425/1-3, CR 59/29-2).

A high proportion of bovine oocytes fertilized in vitro fail to develop beyond the first 4 cleavage cycles. The first mitotic division of the zygote and proper segregation of chromosomes and cytoplasmic components seems to be a particularly delicate task. Notably, zygotes cleaving with a delay of only a few hours seem to have a very low chance of developing to the blastocyst stage. But what exactly goes wrong, how often, and why? To answer such questions we have to visualize in greater detail basic structures and processes such as the sperm aster, DNA replication, migration and apposition of the 2 pronuclei, synchronous chromosome condensation and breakdown of the nuclear envelopes, assembly of the first mitotic spindle and chromosome congression, anaphase, and cytokinesis. Oocytes fertilized in vitro were fixed at different time points around the first cleavage and stained for DNA, Ser10-phosphorylated histone H3, microtubules, and microfilaments. Zygotes were imaged in toto by recording confocal serial sections at 1-µm intervals using a 40× objective (NA = 1.3). Details were recorded with high spatial sampling densities (pixel size 50 × 50 nm, z-step size of 200 nm) close to the Nyquist criterion and restored by maximum likelihood estimation deconvolution using the real point spread function. We present a series of 3-D confocal images captured at different stages of the first cleavage. The images reveal new insights into the formation, structure, and function of the first mitotic spindle and the occurrence of spindle aberrations, irregular chromosome segregation, and abnormal cytokinesis. The microscopic findings guide us to candidate proteins for localization analyses and functional studies based on 3-D fluorescence live-cell imaging of zygotes and early embryos. This work is supported by the Deutsche Forschungsgemeinschaft (DFG FOR 1041).

As part of a study on structural, molecular, and functional deficiencies of both in vitro and in vivo matured oocytes, the outcome of in vitro fertilization and embryo culture was evaluated by noninvasive time-lapse monitoring. Oocytes for IVM were recovered from abattoir-derived ovaries (G1). In vivo matured oocytes were obtained by ovum pick-up from six FSH (Pluset®, Laboratorios Calier, Spain) superstimulated Simmental heifers (Reichenbach et al. 2010 Reprod Domest Anim. 45, 41) shortly before ovulation (G2). Gonadotropin-releasing hormone (GnRH; Receptal®, MSD, Germany) was applied 18 h before ovum pickup (OPU). After IVM (G1) for 23 h or directly after OPU (G2), all oocytes were fertilized and cultured in vitro for 7 days. To continuously monitor multiple individual embryos in parallel, a well-of-wells culture dish system and a transmitted-light microscope equipped with a digital camera placed in the incubator (Primo Vision, Cryo Innovation Inc., Budapest, Hungary) was used. Each embryo was photographed every 5 min over the whole culture period to precisely determine the time and morphological pattern of the first cleavage and to observe the development up to the hatching blastocyst stage. In a first set of experiments, 128 (G1) and 64 (G2) embryos were cultured and monitored under identical conditions. In a statistical analysis (ANOVA, SAS, SAS Institute Inc., Cary, NC, USA) only embryos with a morphologically regular first and second cleavage (G1: n = 86; G2; n = 42; Total n = 128) were included. Fixed effects in the model were time of first cleavage (class 1: ≤26 hours post-insemination (hpi), class 2: >26 to 28 hpi, class 3: >28 to 30 hpi, class 4: >30 to 32 hpi, class 5: >32 hpi) and in vitro versus ex vivo maturation on blastocyst outcome. The total blastocyst rate was lower after in vitro maturation (40/86) than after in vivo maturation (29/42), while the time of first cleavage was not different. The time until the onset of the first cytokinesis ranged in both oocyte groups from 25–37 hpi. Notably, zygotes that cleaved between 26 and 28 hpi (class 2) showed the highest blastocyst rate (35/47), while the rate decreased in a statistically significant way (P

Recent findings on oogenesis, folliculogenesis, and ovarian aging in cows make the bovine system an attractive model for elucidating ovarian function and dysfunction as well as reproductive aging in women. The aim of the present study was to investigate the influence of donor age on the developmental competence of in vitro v. in vivo matured bovine cumulus–oocyte complexes (COC) obtained by ultrasound-guided repeated ovum pickup (OPU). Two groups (G1 and G2) of German Simmental heifers (14 months old at the beginning of the experiment, n = 5 and n = 7), first-lactation young cows (2–4 y old, n = 5 and n = 3), and old cows (10–15 y old, n = 5 and n = 3) were subjected to twice-weekly OPU without hormonal prestimulation 32 (G1) and 6 times (G2). Afterward, animals in G1 were punctured at 5-week intervals 9 times after FSH superstimulation to obtain in vivo matured COC at the metaphase II stage. Data were analysed using a mixed model (SAS). In the twice-weekly OPU for G1 and G2 combined, significantly (P

An alternative approach to classic techniques for the generation of transgenic livestock is the use of viral vectors. Using lentiviral vectors (LV) we previously generated transgenic founder cattle with integrants carrying phosphoglycerate kinase (PGK) promoter-enhanced green fluorescent protein (eGFP) expression cassettes (Hofmann et al. 2004 Biol. Reprod. 71, 405-409). The aim of this work was to investigate the transmission of LV-PGK-eGFP integrants through the female and male germ line of transgenic founder cattle in resulting embryos, fetuses, and offspring. The female founder animal was superovulated and artificially inseminated with a nontransgenic bull. Six of the 16 embryos obtained were transferred to synchronized recipient heifers, resulting in 2 pregnancies and birth of 1 healthy male transgenic calf, expressing eGFP as detected by in vivo imaging and real-time PCR. Cryopreserved semen of the founder bull and matured COC of nontransgenic cows were used for in vitro embryo production as previously described by Hiendleder et al. (2004 Biol. Reprod. 71, 217-223). The rates of cleavage and development to blastocysts in vitro corresponded to 52.3 ± 3.8% and 23.5 ± 4.6%, respectively. In vivo expression of eGFP was observed at blastocyst stage (Day 7 after IVF) and was seen in 93.8% (198/211) of all blastocysts. Twenty-four eGFP-positive embryos were transferred to 9 synchronized recipients. Analysis of 2 embryos flushed on Day 15, 2 fetuses recovered on Day 45, and a healthy male transgenic calf revealed consistent high-level expression of eGFP in all tissues investigated. These observations show for the first time transmission of lentiviral integrants through the germ line of female and male transgenic founder cattle. Although eGFP transgenic cattle have been produced before by nuclear transfer from transfected cells, lentiviral transgenesis has the advantage that only one copy of the provirus is integrated at a particular chromosomal integration site. High-fidelity expression of eGFP in embryos, fetuses, and offspring of founders provides an interesting tool for developmental studies in cattle, including interactions of gametes, embryos, and fetuses with their maternal environment.

The series of events associated with oocyte growth and maturation determines its ability to undergo successful fertilization, cleavage, and embryonic development. Among the molecules involved in these events, leptin has been identified as a modulator of oocyte function. The latest studies have focused on long-term effects of leptin during maturation of bovine oocytes on apoptosis and gene expression in in vitro-produced blastocysts. Briefly, blastocysts originating from leptin-treated oocytes exhibited decreased transcript levels of BCL2 associated X-protein (BAX), but increased mRNA concentrations for leptin receptor (LEPR), signal transducer and activator of transcription 3 (STAT3), and baculoviral inhibitor of apoptosis protein repeat-containing 4 (BIRC4, also known as XIAP) (Boelhauve et al. 2005 Biol. Reprod. 73, 737-744). In the present study, we analyzed single oocytes and their surrounding cumulus cells matured in the presence of 0, 1, and 10 ng/mL leptin. Transcript levels of LEPR, STAT3, BAX, BIRC4, FASLG (encoding Fas ligand), and FAS (encoding Fas receptor) were determined by reverse transcriptase-quantitative PCR (RT-qPCR) analysis of matured oocytes and cumulus cells. Four IVM replicates with four individual samples were collected 22 h after the start of IVM. Total RNA was isolated using a modified TriZol protocol, and reverse transcribed using the enzyme Superscript II-RT and random hexamer primers. Quantitative PCR was conducted with SYBR-Green as a double-stranded DNA-specific fluorescent dye in an ABI 7000 SDS apparatus. Treatment of COCs with 1 or 10 ng/mL leptin increased transcript levels of LEPR (2-fold; P < 0.001), STAT3 (2-fold; P < 0.001), BAX (2-fold; P < 0.001) and FAS (2-fold; P < 0.001) in cumulus cells. Interestingly, the transcript level of the well-known inhibitor of apoptosis, BIRC4, was increased about 4-fold (P < 0.001). In oocytes, leptin treatment increased the mRNA abundance of STAT3 (10 ng/mL; P < 0.05), FAS (1 ng/mL; P < 0.05 and 10 ng/mL; P < 0.001), and FASLG (10 ng/mL; P < 0.05). In conclusion, physiological doses of leptin during maturation of COCs have effects on the expression of developmentally important and apoptosis-associated genes in the oocyte and surrounding cumulus cells. The higher level of BIRC4 mRNA in leptin-treated cumulus cells was associated with a reduced proportion of cumulus cell apoptosis, which might explain the positive and long-lasting effects of leptin on the developmental competence of bovine oocytes.

Leptin is a pleiotrophic peptide that has been implicated in the events associated with oocyte maturation and acquisition of developmental competence. Previous studies indicated that leptin supplementation during oocyte maturation has long-term effects, increasing blastocyst development and reducing the proportion of TUNEL-positive cells per blastocyst. Moreover, blastocysts derived from leptin-treated oocytes showed increased BIRC4 and reduced BAX expression (Boelhauve et al. 2005 Biol. Reprod. 73, 737-744). The objective of the current study was to determine the mechanism of leptin action during bovine oocyte maturation. In the first series of experiments cumulus-oocyte complexes (COCs) were matured in serum-free medium containing 0, 1, or 10 ng/mL leptin or 10% estrous cow serum (ECS) as positive control. Leptin reduced the proportion of cumulus cells undergoing cell death through apoptosis as determined by TUNEL staining (6.8 � 0.4, 1.8 � 0.4, 1.5 � 0.4, and 6.3 � 0.5% for 0, 1, or 10 ng/mL leptin or 10% ECS, respectively; P < 0.0001), but had no effect on the frequency of apoptotic oocytes. Nuclear maturation was also enhanced by leptin. The proportion of oocytes with extruded polar bodies (64.0 � 2.9, 75.7 � 2.9, 73.8 � 2.9, and 63.3 � 2.9% for 0, 1, or 10 ng/mL leptin or 10% ECS; P < 0.05) and the proportion of DAPI-stained metaphase II oocytes (72.9 � 2.9, 90.5 � 2.9, 85.8 � 2.9, and 82.0 � 2.9% for 0, 1, or 10 ng/mL leptin or 10% ECS; P < 0.05) were increased by 1 and 10 ng/mL leptin. There was no effect of ECS in any of the parameters examined. A second series of experiments tested whether the maturation-promoting activity of leptin was mediated by cumulus cells. Denuded oocytes (DO) and COCs were matured in serum-free medium containing 0 or 10 ng/mL leptin. The percentage of oocytes with extruded polar bodies (COCs: 59.1 � 3.7% vs. 75.7 � 3.7%, and DO: 45.5 � 3.7% vs. 54.8 � 3.7% for 0 or 10 ng/mL leptin; P < 0.01) was increased by leptin regardless of cumulus cells. Even though leptin did not affect cleavage rate, it increased blastocyst development. The proportion of COCs that developed to the blastocyst stage increased from 22.3 � 4.6% in the control group to 35.2 � 4.1% in the leptin-treated group. On the other hand, DO matured without cumulus cells did not acquire developmental competency, and this was not reversed by leptin supplementation (1.0 � 5.0% vs. 1.0 � 4.7% for 0 or 10 ng/mL leptin; P < 0.05; leptin � oocyte interaction P < 0.05). In conclusion, leptin enhanced oocyte maturation by acting directly in the oocyte and indirectly through cumulus cells. This suggests that leptin modulates the release of cumulus-derived factors secreted in the oocyte through gap junction coupling and in the extracellular environment.

The enzyme s-N-acetylhexosaminidase B (EC: 3.2.1.52) is a tetramer of two beta-a and two beta-b chains which are derived from the cleavage of a precursor chain, which is a product of the s-N-acetylhexosaminidase B gene (HEXB) (Mahuran, 1999). It catalyses hydrolysis of terminal non-reducing N-acetyl-D-hexosamine residues. Mutations in HEXB gene cause Sandhoff disease in humans (Srivastava and Beuter, 1973). The human HEXB gene stretches over 37 kb and consists of 14 exons (Proia, 1988). Human HEXB has been assigned to chromosome 5q13 (Boedecker et al., 1975). Here we report the localization of the porcine HEXB gene to chromosome 2 confirming homology between the q arms of chromosomes HSA5 and SSC2.

Methylmalonyl-CoA mutase (MCM) is a nuclear encoded mitochondrial matrix enzyme which catalyses the isomerisation between methylmalonyl-CoA and succinyl-CoA. Inborn genetic defects in the MUT gene encoding human MCM give rise to a serious disorder of propionic acid and methylmalonic acid metabolism termed mut methylmalonic aciduria (Crane et al., 1994). The clinical manifestation of the disease varies from minor symptoms to severe neuro-muscular disorders (Ledley et al., 1997). The molecular cloning of the porcine MUT gene and its chromosomal localization will enable further investigations of the gene and its significance in neuromuscular disorders in pig and piglet survival. The human and mouse orthologous genes were assigned to HSA6p21→p12 and MMU17C–D, respectively (Ledley et al., 1988; Sertic et al., 1990; Threadgill et al., 1990). Materials and methods