Luc Grobet

Molecular definition of an allelic series of mutations disrupting the myostatin function and causing double-muscling in cattle

We have determined the entire myostatin coding sequence for 32 double-muscled cattle sampled from ten European cattle breeds. Seven DNA sequence polymorphisms were identified, of which five would be predicted to disrupt the function of the protein, one is a conservative amino acid substitution, and one a silent DNA sequence variant. Four additional DNA sequence polymorphisms were identified in myostatin intronic sequences. In all but two breeds, all double-muscled animals were either homozygous or compound heterozygotes for one of the five loss-offunction mutations. The absence of obvious loss-of-function mutations in the coding sequence of the two remaining breeds points either towards additional mutations in unexplored segments of the gene, or towards locus heterogeneity of double-muscling.

The mh gene causing double-muscling in cattle maps to bovine chromosome 2.

While the hereditary nature of the “double-muscling” phenotype (a generalized muscular hypertrophy documented in several cattle breeds) is well established, its precise segregation mode has remained controversial. Both monogenic models (autosomal dominant or recessive) and oligogenic models have been proposed. Using a panel of 213 bovine microsatellite markers, and an experimental pedigree obtained by backcrossing “double-muscled (Belgian Blue)xconventional (Friesian)” F1 dams to double-muscled sire, we have mapped a locus on bovine Chromosome (Chr) 2 that accounts for all the phenotypic variance in the backcross generation. This locus, referred to as mh (muscular hypertrophy), has been positioned with respect to a map composed of seven Chr 2-specific microsatellites, at 2 cM from the closest marker. This result confirms the validity in the Belgian Blue population of the monogenic model involving an autosomal mh locus, characterized by a wild-type “+” and a recessive “mh” allele, causing the double-muscling phenotype in the homozygous condition. The linkage relationship between the mh locus and the Chr 2 markers was confirmed in three informative pedigrees collected from the general Belgian Blue Cattle population, reinforcing the notion of genetic homogeneity of the double-muscling trait in this breed. This work paves the way towards marker-assisted selection for or against the double-muscling trait, and towards positional cloning of the corresponding gene.

Microsatellite mapping of the bovine roan locus: a major determinant of White Heifer Disease

In the Belgian Blue Cattle breed, coat color variation is mainly under the influence of a single autosomal locus, the roan locus, characterized by a pair of codominant alleles: r+ (black) and R (white). Heterozygous r+R animals have intermingled black and white hairs, yielding the “blue” phenotype typical of the breed. Major interest for the roan locus stems from its pleiotropic effect on fertility, owing to the critical role of the R allele in the determinism of White Heifer Disease. We describe the linkage mapping of the roan locus to bovine Chromosome (Chr) 5, in the interval between microsatellite markers BP1 and AGLA293, with an associated lodscore of 11.2. Moreover, we map a candidate gene, the Steel locus coding for the mast cell growth factor, to bovine Chr 5.

Lower intracellular concentration of cryoprotectants after vitrification than after slow freezing despite exposure to higher concentration of cryoprotectant solutions

STUDY QUESTION What is the intracellular concentration of cryoprotectant (ICCP) in mouse zygotes during vitrification (VIT) and slow-freezing (SLF) cryopreservation procedures? SUMMARY ANSWER Contrary to common beliefs, it was observed that the ICCP in vitrified zygotes is lower than after SLF, although the solutions used in VIT contain higher concentrations of cryoprotectants (CPs). WHAT IS KNOWN ALREADY To reduce the likelihood of intracellular ice crystal formation, which has detrimental effects on cell organelles and membranes, VIT was introduced as an alternative to SLF to cryopreserve embryos and gametes. Combined with high cooling and warming rates, the use of high concentrations of CPs favours an intracellular environment that supports and maintains the transition from a liquid to a solid glass-like state devoid of crystals. Although the up-to-date publications are reassuring in terms of obstetric and perinatal outcomes after VIT, a fear about exposing gametes and embryos to high amounts of CPs that exceed 3-4-fold those found in SLF was central to a debate initiated by advocates of SLF procedures. STUDY DESIGN, SIZE, DURATION Two experimental set-ups were applied. The objective of a first study was to determine the ICCP at the end of the exposure steps to the CP solutions with our VIT protocol (n = 31). The goal of the second investigation was to compare the ICCP between VIT (n = 30) and SLF (n = 30). All experiments were performed in triplicates using mouse zygotes. The study took place at the GIGA-Research Institute of the University of Liège. PARTICIPANTS/MATERIALS, SETTING, METHODS Cell volume is modified by changes in extracellular osmolarity. Hence, we estimated the final ICCP after the incubation steps in the VIT solutions by exposing the cells to sucrose (SUC) solutions with defined molarities. The ICCP was calculated from the SUC concentration that produced no change in cell volume, i.e. when intra- and extracellular osmolarities were equivalent. Cell volume was monitored by microscopic cinematography. ICCP was compared between SLF and VIT based on the principle that a high ICCP lowers the probability of (re)crystallization during warming but increases the probability of over-swelling of the cell due to fast inflow of water. The survival rates of mouse zygotes after SLF or VIT were compared using either (i) various warming rates or (ii) various concentrations of SUC in the warming dilution medium. MAIN RESULTS AND THE ROLE OF CHANCE The ICCP in mouse zygotes during the VIT procedure prior to plunging them in liquid nitrogen was ∼2.14 M, i.e. one-third of the concentration in the VIT solution. After SLF, the warming rate did not affect the zygote survival rate. In contrast, only 3/30 vitrified zygotes survived when warmed slowly but as many as 30/30 zygotes survived when warming was fast (>20 000°C/min). Vitrified zygotes showed significantly higher survival rates than slow-frozen zygotes when they were placed directly in the culture medium or in solutions containing low concentrations of SUC (P < 0.01). These two experiments demonstrate a lower ICCP after VIT than after SLF. LIMITATIONS, REASONS FOR CAUTION The results should not be directly extrapolated to other stages of development or to other species due to possible differences in membrane permeability to water and CPs. WIDER IMPLICATIONS OF THE FINDINGS The low ICCP we observed after VIT removes the concern about high ICCP after VIT, at least in murine zygotes and helps to explain the observed efficiency and lack of toxicity of VIT. STUDY FUNDING / COMPETING INTEREST(S) The study was funded by the FNRS (National Funds for Scientific Research). The authors declare that they have no competing interests.

Cryopréservation d’ovocytes et d’embryons par congélation ou vitrification dans le cadre de l’assistance médicale à la procréation

La cryopreservation est un procede permettant de garder les fonctions cellulaires ou tissulaires intactes en reduisant la temperature en dessous de celle a laquelle se deroulent normalement les reactions biochimiques.

High-resolution, human-bovine comparative mapping based on a closed YAC contig spanning the bovine mh locus

Abstract. A closed YAC contig spanning the mh locus was assembled by STS content mapping with seven microsatellite markers, eight genes or EST, and nine STS corresponding to YAC ends. The contig comprises 27 YACs, has an average depth of 4.3 YACs, and spans an estimated 1.2 Mb. A linkage map was constructed based on five of the microsatellite markers anchored to the contig and shown to span 7 cM, yielding a ratio of 160 kb/1 cM for the corresponding chromosome region. Comparative mapping data indicate that the constructed contig spans an evolutionary breakpoint connecting two chromosome segments that are syntenic but not adjacent in the human. Consolidation of human gene order by means of whole genome radiation hybrids and its comparison with the bovine order as inferred from the contig confirm conservation of gene order within segments.

Three-dimensional reconstruction of the pharyngeal tonsil innervation pattern in sheep.

The pharyngeal tonsil has recently been identified as a new participant in airborne contamination by the ovine scrapie agent. In the context of scrapie pathogenesis, we conducted a three-dimensional reconstruction of the innervation pattern in the lymphoid compartments of this tonsil. This model confirmed that very few nerve fibres penetrated the lymphoid follicles and suggested that the nerve fibre distribution in the interfollicular and subepithelial areas is more suitable with neuro-invasion through direct contact between these nerve fibres and prion-transporting cells prior to or after prion amplification in the germinal centre of the pharyngeal tonsil lymphoid follicles.