103. ROLE OF CANDIDATE STEM/PROGENITOR CELLS IN A MOUSE MODEL OF ENDOMETRIAL MENSTRUAL BREAKDOWN AND REPAIR

Adult stem/progenitor cells (SPC) identified in human and mouse endometrium may be responsible for its remarkable regenerative capacity (1), however a functional role for SPC in menstruation is yet to be established. This study aimed to identify label retaining cells (LRC) as candidate epithelial SPC involved in the rapid re-epithelisation of the uterine surface in a mouse model; tissue SPCs are quiescent and will retain label (BrdU), while label is diluted out as transit amplifying cells proliferate. Mice were ovariectomised and endometrial breakdown and repair induced, mimicking menstruation in women (2). BrdU (50µg/g) was administered intraperitoneally 8.5 days before endometrial repair. Tissue was collected to assess initial labelling, and following four chase periods prior to and during endometrial repair (n=3-5 animals/group) and immunostained for BrdU. LRC were categorised as minimal (<50% nuclear label), partial (50-75%) and full (100%) and counted in the luminal (LE) and glandular (GE) epithelial compartments for each group. The majority of LE (91.4±1.9%; mean±SEM) and 35±3.8% of GE were initially labelled. During breakdown and repair the percentage of full LE LRC (38±13.1% vs 1.3±1.1%) and partial LE LRC (49.1±4.3% vs 1.8±0.7%) significantly decreased (p≤0.01) whilst minimal LRC significantly increased (12.9±3.5% vs 85.2±4.3%; p≤0.001) indicating dilution of BrdU label as cells proliferated. In contrast full, partial and minimal GE LRC did not significantly change throughout endometrial breakdown and repair. The rapid dilution of BrdU label in the LE suggests that the transit amplifying population are responsible for the rapid proliferation observed, whilst the lack of change of BrdU in the GE suggests that GE may be a source of SPC. Double immunofluorescence and confocal microscopy are currently underway to further characterise the LRC population in this model. This study provides some of the first insights into the contribution of candidate SPC to endometrial repair.

Transcripts for the acetylcholine receptor and acetylcholine esterase show distribution differences in cultured chick muscle cells.

In situ hybridization of chick cultured muscle cells using exonic DNA probes for both AChR alpha-sub-unit and the catalytic subunit of AChE, revealed major differences in the distribution of label both over nuclei and in their surrounding cytoplasm, although some overlap in these distributions exists. For the AChR alpha-subunit there is a highly skewed distribution of labeled nuclei, with 35% of the nuclei being relatively inactive (less than 0.25 times the mean label) and approximately 10% being very heavily labeled (greater than 2.5 times the mean label). In contrast the nuclei labeled with the exonic probe for the AChE transcripts had a more Gaussian distribution, yet with some slight skewness in the direction of a few heavily labeled nuclei. There was also a difference in the cytoplasmic distribution of the label. The AChR alpha-subunit mRNA was mainly within 4 microns of labeled nuclei while the AChE mRNA was more widely distributed throughout the cytoplasm, possibly within a 10 microns rim around labeled nuclei. An intronic probe for the AChE gave the identical distribution of nuclear label to that of the exonic probe (but without any cytoplasmic label). In addition, calibration of the technique indicated that per myotube the AChE transcript is about sixfold more abundant than the AChR alpha-subunit transcript.

In vitro uptake of tritium from uridine-5-H3 into RNA (and DNA) in normal and murine leukemia virus infected mouse embryo tissue

The multiplication and release into the medium of a murine leukemic virus (GC) occurs in mouse embryo tissue cultures. Autoradiographic analysis of such infected and uninfected cultures after 24-h labeling periods with uridine-5-H3 demonstrated extensive nuclear and cytoplasmic activity. Such activity was always more extensive in infected cultures, indicating that they were more metabolically active than uninfected cultures. The cytoplasmic activity was apparently in single-stranded RNA, since it was completely removed with pancreatic RNase. A portion of the nuclear label which was RNase-resistant was in DNA, since RNase followed by DNase, or the reverse, removed all label.Autoradiographic analysis of cultures given pulse labels of uridine-5-H3 from 5 to 60 min revealed only nuclear label, always more extensive in infected cultures. This indicates that the synthesis of "rapidly labeled" RNA is enhanced by viral infection; this occurs even within 2 h after infection. Nuclear activity observed after 5- and 10-min pulses was removed by buffers containing RNase or DNase. Possibly this label was in diffusible low molecular weight species of RNA. After 30-min pulses, activity was exclusively associated with pancreatic RNase-sensitive single-stranded RNA. After 60-min labeling, tritium from uridine-5-H3 was taken up into nuclear DNA in addition to RNA.

BONE FORMATION INDUCED IN MOUSE THIGH BY CULTURED HUMAN CELLS

Cultured FL human amnion cells injected intramuscularly into cortisone-conditioned mice proliferate to form discrete nodules which become surrounded by fibroblasts. Within 12 days, fibroblastic zones differentiate into cartilage which calcifies to form bone. Experiments were conducted to test the hypothesis that FL cells behave as an inductor of bone formation. In the electron microscope, FL cells were readily distinguished from surrounding fibroblasts. Transitional forms between the two cell types were not recognized. Stains for acid mucopolysaccharides emphasized the sharp boundary between metachromatic fibroblastic and cartilaginous zones and nonmetachromatic FL cells. 35S was taken up preferentially by fibroblasts and chondrocytes and then deposited extracellularly in a manner suggesting active secretion of sulfated mucopolysaccharides. FL cells showed negligible 35S utilization and secretion. FL cells, labeled in vitro with thymidine-3H, were injected and followed radioautographically, during bone formation. Nuclear label of injected FL cells did not appear in adjacent fibroblasts in quantities sufficient to indicate origin of the latter from FL cells. The minimal fibroblast nuclear labeling seen may represent reutilization of label from necrotic FL cells. It is suggested that FL cells injected into the mouse thigh induced cartilage and bone formation by host fibroblasts.