Generation of Embryonic Stem Cell Lines from Immature Rabbit Ovarian Follicles

In mammalian ovaries, many immature follicles remain after the dominant follicles undergo ovulation. Here we report the successful production of rabbit embryonic stem cells (ESCs) from oocytes produced by in vitro culture of immature follicles and subsequent in vitro maturation treatment. In total, we obtained 53 blastocysts from oocytes that received intracytoplasmic sperm injection followed by in vitro culture. Although only weak expression of POU5f1 was observed in the inner cell masses of in-vitro-cultured follicle-derived embryos, repeated careful cloning enabled establishment of 3 stable ESC lines. These ESC lines displayed the morphological characteristics of primed pluripotent stem cells. The ESC lines also expressed the pluripotent markers Nanog, POU5f1, and Sox2. Further, these ESCs could be differentiated into each of the 3 different germ layers both in vitro and in vivo. These results demonstrate that immature follicles from rabbits can be used to generate ESCs. Moreover, the use of rabbit oocytes as a cell source provides an experimental system that closely matches human reproductive and stem cell physiology.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/140197/1/scd.2012.0300.pd

Head-head interactions of resting myosin crossbridges in intact frog skeletal muscles, revealed by synchrotron x-ray fiber diffraction.

The intensities of the myosin-based layer lines in the x-ray diffraction patterns from live resting frog skeletal muscles with full thick-thin filament overlap from which partial lattice sampling effects had been removed were analyzed to elucidate the configurations of myosin crossbridges around the thick filament backbone to nanometer resolution. The repeat of myosin binding protein C (C-protein) molecules on the thick filaments was determined to be 45.33 nm, slightly longer than that of myosin crossbridges. With the inclusion of structural information for C-proteins and a pre-powerstroke head shape, modeling in terms of a mixed population of regular and perturbed regions of myosin crown repeats along the filament revealed that the myosin filament had azimuthal perturbations of crossbridges in addition to axial perturbations in the perturbed region, producing pseudo-six-fold rotational symmetry in the structure projected down the filament axis. Myosin crossbridges had a different organization about the filament axis in each of the regular and perturbed regions. In the regular region that lacks C-proteins, there were inter-molecular interactions between the myosin heads in axially adjacent crown levels. In the perturbed region that contains C-proteins, in addition to inter-molecular interactions between the myosin heads in the closest adjacent crown levels, there were also intra-molecular interactions between the paired heads on the same crown level. Common features of the interactions in both regions were interactions between a portion of the 50-kDa-domain and part of the converter domain of the myosin heads, similar to those found in the phosphorylation-regulated invertebrate myosin. These interactions are primarily electrostatic and the converter domain is responsible for the head-head interactions. Thus multiple head-head interactions of myosin crossbridges also characterize the switched-off state and have an important role in the regulation or other functions of myosin in thin filament-regulated muscles as well as in the thick filament-regulated muscles

Axial intensity profiles of the C-protein-associated meridional reflections.

<p>The x-ray diffraction pattern was obtained at the Advanced Photon Source. (A) Those of the C1/M1 region. (B–E) Those of the clusters of the C2 to C5 meridional reflections. The experimental intensity profiles are denoted by red dotted curves and the fitted Gaussian functions are shown by black curves. (F) Axial spacings of each C-protein and each myosin reflection when divided by their reflection index. They are shown by blue and red full circles, respectively. Bars on the circles are the standard deviation of the mean from four data sets. The average period of C-protein is 45.33±0.58 nm and that of myosin is 42.96±0.11 nm. In (F), the spacing (44.5 nm) of the reflection which was enhanced by labeling antibody to C-protein <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052421#pone.0052421-Rome1" target="_blank">[40]</a> is shown by the blue open circle. In (A), there are triplet of reflections in 0.0205 nm⁻¹< <i>Z</i><0.0245 nm⁻¹ with a main peak at 1/44.5 nm⁻¹, and the axial positions of the reflections assigned as C1 and M1 are depicted by vertical lines. TN1 denotes the sampled first order troponin-based reflection.</p