The Culture and Correlative Electron Microscopy of Pollen Mother Cells in Meiosis: Development of Techniques and Some Observations on Selected Topics

Abstract

Reliable techniques for the living cell culture and correlative light and electron microscopy (EM) of meiotic pollen mother cells (PMCs) of Iris spuria, Allium triquetrum and Tradescantia flumenensis are described in detail. Living PMCs were successfully cultured in a slide chamber on agar/sucrose medium. Cells were covered with an inert oil to prevent their dehydration, and some cells were cultured from metaphase I to tetrad cell formation over a 20 hour period. Other PMCs were fixed with glutaraldehyde and flat embedded using a modification of the agar sandwich technique of Molè-Bajer and Bajer (1968). This technique was developed to permit the preselection of PMCs at known meiotic stages, for subsequent EM examination. Serial thin sections were cut at known planes of section; and 3-D reconstructions of MT distribution, and MT counts from transverse sections were completed. It was also possible to examine sections of an Iris anaphase I PMC which had been previously studied in life. Anaphase I and II chromosome velocities were analysed in the three species. Mean velocities were approximately 0.5 μm/min with some variation from cell to cell and between sister half-spindles. In Allium anaphase I there was also variation in chromosome velocity within the half-spindle; and this variation was found not to be related to chromosome position on the metaphase I plate. Spindle elongation was zero in Allium anaphase I and in Iris anaphase II, but was detectable in Allium anaphase II (40%) and in "Iris anaphase I (l5%). The extent of spindle elongation in Tradescantia could not be determined. The kinetochore region in the first meiotic division consisted of two closely appressed, but structurally (and functionally) distinct, sister kinetochores. At meiosis II, the two sister kinetochores were separate from each other and faced opposite poles. The kinetochore arrangement probably changes from side-by-side (meiosis I) to back-to-back (meiosis II) during chromosome recondensation at prophase II in these cells. Bundles of non-kinetochore microtubules (nkMTs) span the interzone between sister chromosome units at metaphase I and II and anaphase II. Bundles of kinetochore MTs (kMTs) do not increase in divergence at any stage of meiosis studied; there was little interaction between nkMTs and kMTs, and MT-MT cross bridges were rare. These observations are not consistent with models of chromosome movement based on MT sliding or zipping. No relationship was found between nkMT distribution and spindle elongation, and the several different nkMT distributions which have been reported for other cell types may be variations on a structural theme. Spindle endoplasmic reticulum (ER) in meiosis II was found to be derived largely from invaginations and evaginations of the nuclear envelope. Growth of existing spindle ER was proposed to account for the doubling in the amount of ER observed between interphase and prometaphase II. Randomly oriented elements of ER, in early prometaphase II spindles may become passively aligned along the interpolar axis and then actively transported polewards at later stages of prometaphase II and metaphase II. Suggestions for future research are offered.