SonoAVC: a novel method of automatic volume calculation.

OBJECTIVES: To assess the ability of the new software SonoAVC to measure follicular volume and to compare these volume calculations with those made by conventional methods. METHODS: Three-dimensional ultrasound imaging was used to acquire volumetric data from the ovaries of 51 women undergoing controlled ovarian stimulation as part of in-vitro fertilization treatment. All assessments were performed on the day of oocyte retrieval and the true volume of each follicle was ascertained by manual measurement of the follicular aspirate. SonoAVC was used to automatically measure the volume of follicles and to provide three perpendicular diameters (xyz diameters), which were used to estimate volume using the sphere formula. The sphere formula was also used to estimate the volume from manual measurements of follicle diameter derived from conventional two-dimensional (2D) displays. Virtual Organ Computer-aided AnaLysis (VOCAL) was also used to measure volume, and the validity of each technique was compared using limits of agreement. RESULTS: Two hundred and twenty-four follicles with a mean follicular volume of 3.7 (range, 0.4-16.2) cm(3) were studied. SonoAVC provided highly accurate automatic follicular volume measurements in all cases. Volume estimations made from the automatic maximal follicular diameter measurements (xyz diameters) were less valid. VOCAL proved highly valid but was less accurate than SonoAVC. Volumes estimated from manually derived follicular diameter measurements were the least accurate. CONCLUSIONS: SonoAVC provides highly valid, automatic measurements of follicular volume. These measurements are more accurate than volumes estimated from 2D manual measurements, automated measurements of follicular diameter and those calculated using VOCAL.

Generation and analysis of mice with a targeted disruption of the arylamine N-acetyltransferase type 2 gene.

Arylamine N-acetyltransferases (NATs) are polymorphic xenobiotic metabolising enzymes, linked to cancer susceptibility in a variety of tissues. In humans and in mice there are multiple NAT isoforms. To identify whether the different isoforms represent inbuilt redundancy or whether they have unique roles, we have generated mice with a null allele of Nat2 by gene targeting. This mouse line conclusively demonstrates that the different isoforms have distinct functions with no compensatory expression in the Nat2 null animals of the other isoforms. In addition, we have used the transgenic line to show the pattern of Nat2 expression during development. Although Nat2 is not essential for embryonic development, it has a widespread tissue distribution from at least embryonic day 9.5. This mouse line now paves the way for the teratological role of Nat2 to be tested.

Disruption of the talin gene arrests mouse development at the gastrulation stage.

Studies on cultured cells show that the cytoskeletal protein talin plays a key role in cell spreading and the assembly of cell-extracellular matrix junctions. To examine the role of talin in vivo, we have generated mice with a targeted disruption of the talin gene. Heterozygotes are normal, but no surviving homozygous mutant animals were obtained, proving that talin is required for embryogenesis. Mutant embryos develop normally to the blastocyst stage and implant, but there is a gross disorganization of the embryos at gastrulation (6.5-7.5 days post coitum), and they die around 8.5-9.5 days post coitum. The embryonic ectoderm is reduced in size, with fewer cells, and is incompletely organised compared with wild-type embryos. The mutant embryos show disorganised extraembryonic tissues, and the ectoplacental and excocoelomic cavities are not formed. This seems to be because embryonic mesoderm accumulates as a mass on the posterior side of the embryos and fails to migrate to extraembryonic regions, although mesodermal cells are evident in the embryo proper. Spreading of trophoblast cells derived from cultured mutant blastocysts on fibronectin and laminin is also considerably reduced. Therefore, the fundamental deficit in these embryos seems to be a failure of cell migration at gastrulation.

Integrin-mediated cell adhesion: the cytoskeletal connection.

Members of the integrin family of cell adhesion molecules play a pivotal role in the interaction between animal cells and the extracellular matrix. This article reviews the evidence (i) that the integrin beta-subunit cytoplasmic domain is important in the localization of integrins to focal adhesions, and for integrin-mediated cell adhesion/spreading; and (ii) that the integrin beta-subunit can be linked to F-actin via the actin-binding proteins talin, alpha-actinin and filamin. Talin has two or more actin-binding sites, and three binding sites for the cytoskeletal protein vinculin. Because vinculin can also bind F-actin, it may cross-link talin and actin, thereby stabilizing the interaction. In addition, vinculin contains a binding site for VASP (vasodilator-stimulated phospho-protein), a protein which may serve to recruit a profilin/G-actin complex to talin, which has actin-nucleating activity. Evidence that talin, vinculin and alpha-actinin are important in the assembly of focal adhesions, obtained using antisense technology and protein microinjection, is reviewed. To analyse the role of talin in focal adhesions, we have disrupted both copies of the talin gene in mouse embryonic stem (ES) cells. Undifferentiated talin (-/-) ES cell mutants are unable to assemble focal adhesions when plated on fibronectin, whereas vinculin (-/-) ES cells are able to do so. Finally, the role of small GTP-binding proteins in the assembly of focal adhesions is discussed, along with our recent studies using streptolysin-O-permeabilized Swiss 3T3 cells which suggest that the GTP-binding protein ADP-ribosylation factor-1 (ARF-1) is important in targeting the protein paxillin to focal adhesions.

Disruption of the talin gene compromises focal adhesion assembly in undifferentiated but not differentiated embryonic stem cells.

We have used gene disruption to isolate two talin (-/-) ES cell mutants that contain no intact talin. The undifferentiated cells (a) were unable to spread on gelatin or laminin and grew as rounded colonies, although they were able to spread on fibronectin (b) showed reduced adhesion to laminin, but not fibronectin (c) expressed much reduced levels of beta1 integrin, although levels of alpha5 and alphaV were wild-type (d) were less polarized with increased membrane protrusions compared with a vinculin (-/-) ES cell mutant (e) were unable to assemble vinculin or paxillin-containing focal adhesions or actin stress fibers on fibronectin, whereas vinculin (-/-) ES cells were able to assemble talin-containing focal adhesions. Both talin (-/-) ES cell mutants formed embryoid bodies, but differentiation was restricted to two morphologically distinct cell types. Interestingly, these differentiated talin (-/-) ES cells were able to spread and form focal adhesion-like structures containing vinculin and paxillin on fibronectin. Moreover, the levels of the beta1 integrin subunit were comparable to those in wild-type ES cells. We conclude that talin is essential for beta1 integrin expression and focal adhesion assembly in undifferentiated ES cells, but that a subset of differentiated cells are talin independent for both characteristics.

Talin contains three actin-binding sites each of which is adjacent to a vinculin-binding site.

We have determined the sequence of chicken talin (2,541 amino acids, M(r) 271,881) which is very similar (89% identity) to that of the mouse protein. Alignments with the Caenorhabditis elegans and Dictyostelium discoideum talin sequences show that the N- and C-terminal regions of the protein are conserved whereas the central part of the molecule is more divergent. By expressing overlapping talin polypeptides as fusion proteins, we have identified at least three regions of the protein which can bind F-actin: residues 102-497, 951-1,327 and 2,269-2,541. The N-terminal binding site contains a region with homology to the ERM family of actin-binding proteins, and the C-terminal site is homologous to the yeast actin-binding protein Sla2p. Each of the actin-binding sites is close to, but distinct from a binding site for vinculin, a protein which also binds actin. The Pro1176 to Thr substitution found in talin from Wistar-Furth rats does not destroy the capacity of this region of the protein to bind actin or vinculin. Microinjection studies showed that a fusion protein containing the N-terminal actin-binding site localised weakly to stress fibres, whereas one containing the C-terminal site initially localised predominantly to focal adhesions. The former was readily solubilised, and the latter was resistant to Triton extraction. The N-terminal talin polypeptide eventually disrupted actin stress fibres whereas the C-terminal polypeptide was without effect. However, a larger C-terminal fusion protein also containing a vinculin-binding site did disrupt stress fibres and focal adhesions. The results suggest that, although both the N- and C-terminal regions of talin bind actin, the properties of these two regions of the protein are distinct.