Centromeric dots in crane-fly spermatocytes: meiotic maturation and malorientation

During the first meiotic division in crane-fly spermatocytes, the two homologs of a metaphase bivalent each bear two sister kinetochores oriented toward the same pole. We have previously reported treatments that increase the percentage of metaphase bivalents in which one or both homologs have bipolar malorientations: kinetochore microtubules extending from a homolog toward both poles. The maloriented homologs lag at anaphase. Treatments that induce this behavior include: (a) recoverey from exposure to low temperatures or Colcemid or Nocodazole concentrations that prevent spindle formation but allow nuclear membrane breakdown, and (b) exposure to 6 degrees C, a temperature that permits spindle assembly but slows progression through meiosis. Giemsa staining methods reveal two 0.5 micron diameter dots at the centromeric region of each metaphase homolog; these often are more separated in maloriented homologs. This investigation was undertaken to assess whether this separation precedes the establishment of bipolar malorientation, and hence may be a cause of it, or is only a consequence of forces resulting from bipolar malorientation. Analysis showed that, in untreated cells, the average center-to-center distance between sister centromeric dots increases during the course of meiosis I. After the above-mentioned treatments, center-to-center distances similar to those normally seen in untreated half-bivalents at anaphase I were seen in bivalents, both after and before nuclear membrane breakdown. Longer exposure to temperatures that arrested meiosis increased the degree of dot separation. Based on our data, we conclude that normal orientation during the first meiotic division is aided by the close apposition of centromeric dots, and that a time-dependent maturation occurs causing centromeric dots to separate for the second meiotic division and facilitating orientation of sister kinetochores to opposite poles. If centromeric maturation occurs either prior to or during early stages of the first meiotic division, then it may contribute to persisting bipolar malorientation.