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First published online 12 January 2005
doi: 10.1242/dev.01609

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Label-retaining epithelial cells in mouse mammary gland divide asymmetrically and retain their template DNA strands

Laboratory of Mammary Gland Biology and Tumorigenesis, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892, USA

View larger version (11K): [in a new window]   Fig. 1. Diagram showing the experimental procedures. Mice entering puberty and exactly 3 weeks of age on the day (Friday) of surgery were anesthetized, their #4 and #9 mammary fat pads were cleared of endogenous mammary epithelium and mammary fragments were implanted immediately into the cleared pads. The mice were allowed to recover for 10 days then the wound clips were removed (Monday). They were injected with 1.0 µg estradiol and 3HTdR i.p., for 5 days, Monday to Friday. On the following Monday, two mice were removed to provide mammary tissues (#3,4,8,9 fat pads) and small intestine for autoradiographical analysis. Thereafter for the next 3 weeks, the remaining mice received 1.0 mg estradiol each Monday, Wednesday and Friday. Then, when they were 10 weeks old (Friday), an additional two mice were removed to provide tissue samples to determine by autoradiography the number of label-retaining cells (LREC) present. On the following Monday, the mice were placed into three groups receiving different hormone combinations and were inoculated with 5BrdU. i.p. 24 hours apart, on Monday and Tuesday. One mouse from each group (3) was removed Wednesday morning to provide tissues for evaluation of 5BrdU incorporation and 3HTdR retention (LREC arrow). The remaining mice (9) received hormones as indicated until Friday and on the following Monday were euthenized to provide tissues for evaluation of 5BrdU incorporation and 3HTdR retention (End chase arrow).

View larger version (32K): [in a new window]   Fig. 2. Blue bars, total percentage of 5BrdU-labeled cells; pink bars, percentage of 3HTdR label-retaining cells; green bars indicate the percentage of double label (3HTdR/5BrdU)-containing nuclei. After the two day 5BrdU pulse (post-pulse), 7.3±0.9% of the nuclei were 5BrdU-positive including 1.8% of the total number of 3HTdR label-retaining cells (82.8±0.9% of LREC). Following a 5 day chase (post chase), the percentage of 5BrdU-positive nuclei increased to nearly 12%, while the percent of doubly labeled 3HTdR/5BrdU nuclei dropped to 0.6%(14.7±3.0% of LREC) and the singly labeled 3HTdR-retaining cells rose to roughly 1.6% (85.2±3.0% of LREC) of the total nuclei counted. *The percent of LREC in the population after 5BrdU incorporation did not vary significantly, remaining near 2.0%.

View larger version (89K): [in a new window]   Fig. 3. (A-E) Nuclei positive for 3HtdR alone (A,E) or 5BrdU alone (B-D). (F-J) 5BrdU/3HTdR-labeled cell nuclei. Doubly labeled 5BrdU/3HTdR nuclei, singly labeled 3HTdR-positive nuclei and 5BrdU-labeled nuclei were often juxtaposed, suggesting that their labeling resulted from a recent mitotic event (double arrows in K-M). (E,N) Singly labeled 3HtdR labeled nuclei in 5BrdU-labeled mammary tissues. Scale bar: 10 µm.

View larger version (97K): [in a new window]   Fig. 4. LREC (arrows) included cells staining positive for estrogen receptor (A) and progesterone receptor (B). Scale bar: 10 µm.

View larger version (74K): [in a new window]   Fig. 5. The progeny of parity-induced mammary epithelial cells marked by the expression of ß-galactosidase (blue) included LREC that were doubly labeled by 5BrdU and 3HtdR (arrows) in mammary transplant outgrowths in the cleared fat pads of doubly pulsed female hosts. This indicated that during the process of expansive self-renewal some of these cells become asymmetrically dividing LREC. The red brackets outline the region of the image where the plane of section is tangential to the long axis of the duct, thus exposing the suprabasal epithelial layer. Scale bar: 10 µm.