Spermatogenesis in Sciara coprophila. I. Chromosome orientation on the monopolar spindle of meiosis I

Abstract

Meiosis I of spermatogenesis in the fungus fly, Sciara coprophila, has a monopolar spindle which collects the maternal and supernumerary L chromosome sets, while the paternal chromosomes migrate away from the single pole to be excluded in a bud. By inspection, the metacentric paternal chromosome IV moves with its centromere lagging rather than leading the direction of motion. Therefore, we wondered if all paternal homologues move in such a reverse orientation. To determine the orientation of the other homologues which are acrocentrics (chromosomes II, III, X), their centromeres were localized by use of the DAPI C-bonding technique. In addition, we characterized centromeric heterochromatin on polytene chromosomes by C-banding and in situ hybridization of satellite DNA isolated by Ag+-Cs2SO4 (rho CsC1 satellite I=1.698 g/ml; rho CsC1 satellite II=1.705 g/ml). The two satellite fractions were localized to the centromeric heterochromatin of all the chromosomes, and to a varying degree to all chromosome telomeres. By DAPI C-banding we could precisely locate each centromere band on polytene chromosomes, and these results agreed with those of satellite cRNA in situ hybridization. We then applied the DAPI C-banding technique to primary spermatocyte preparations, and determined that all paternal chromosomes migrate at anaphase I with their centromeres lagging rather than leading movement to the cell periphery. Since in polytene chromosomes the X chromosome contains a moderately fluorescent band on its noncentromeric end as well, in order to clarify its DAPI C-banding result in primary spermatocytes, we did in situ hybridization of (3)H nick-translated cloned rDNA, since rDNA is a convenient marker for the centromeric heterochromatin of the X. These data and the DAPI C-banding results indicate that the X as well as all th other paternal homologues display a reverse orientation (centromeres lag) as they migrate away from the single spindle pole to the cell periphery. - One model explaining this unusual paternal chromosome orientation is that there may be unique neocentromeric-like attachments to the non-centromeric free ends of these chromosomes. These attachments could serve to pull the paternal chromosomes to the cellular periphery as anaphase I progresses. In order to test this model, we analyzed anaphase I spermatocytes after a terminal block of heterochromatin had been removed from metacentric paternal chromosome IV by X-irradiation. We observed that when metacentric paternal chromosome IV is broken, it maintains its inverted "V" orientation rather than assuming a rod-like configuration. These data imply that there are no unique, terminal neocentromeric attachments to paternal chromosome IV as it progresses to the cellular periphery.