Determining requirements for meiotic pairing in Drosophila melanogaster Spermatogenesis

UNCG Author/Contributor (non-UNCG co-authors, if there are any, appear on document)
Christopher Hylton (Creator)
The University of North Carolina at Greensboro (UNCG )
Web Site:
John Tomkiel Dean

Abstract: Diploid germline cells must undergo two consecutive meiotic divisions before differentiating as haploid sex cells. During meiosis I, homologs pair and remain conjoined until segregation at anaphase. Drosophila melanogaster spermatocytes are unique in that the canonical events of meiosis I including synaptonemal complex (SC) formation, double-strand DNA breaks, and chiasmata are absent. Sex chromosomes pair at intergenic spacer sequences within the rDNA. Autosomes pair at numerous euchromatic homologies, but not at heterochromatin, suggesting that pairing may be limited to specific sequences. However, previous work generated from genetic segregation assays or observations of late prophase I/prometaphase I chromosome associations fail to differentiate pairing from maintenance of pairing (conjunction). To begin, the capability of X euchromatin to pair and conjoin with the Y chromosome was examined using an rDNA-deficient X and a series of Dp(1;Y) chromosomes. Genetic assays determined that duplicated X euchromatin can substitute for endogenous rDNA pairing sites; however, segregation was not proportional to homology length. Using fluorescent in situ hybridization (FISH) to early prophase I spermatocytes, pairing was shown to occur with high fidelity at all homologies tested. By comparing genetic and cytological data, we determined that centromere proximal pairings were best at segregation. Segregation was dependent on the conjunction protein Stromalin in Meiosis while the autosomal-specific Teflon was dispensable. Next, the ability of the X euchromatic homology to pair with and segregate from the heterolog chromosome 3 was examined using Dp(1;3) chromosomes containing X euchromatin duplications ranging in size from 21 to 177 Kb. In contrast to duplications of X euchromatin on the Y, duplications of X material on chromosome 3 are not as effective in directing segregation. In early prophase I, however, homologies on the X and chromosome 3 pair. Pairing between homologs is normally released at S2b of prophase I. Using a control probe to only select cells where chromosome 2 has already unpaired, the X and Dp(1;3) was unpaired in a significantly higher number of cells than was the X and Dp(1;Y). This result suggests different mechanisms exist to manage pairings between homologs and pairings between heterologs. The FISH pairing assay was used to score meiotic I nondisjunction (NDJ) and compared to genetic NDJ. Some NDJ frequencies were significantly different between the two methods. Data suggests genetic NDJ calculations are not always a true measure of the meiotic defect. The FISH pairing assay was also used to investigate an uncharacterized male meiotic mutant since the assay provides a rapid identification of the defective meiotic stage. FISH identified a unique defect that caused sister chromatids to segregate to opposite poles during meiosis I. This identification would not have been possible by only monitoring the outcome of meiosis through genetic crosses. The molecular techniques and approaches described within are suggested to be useful in defining the mechanisms regulating the establishment of conjunction and segregation between paired sequences.

Additional Information

Language: English
Date: 2020
Drosophila, Male, Meiosis, Pairing, Spermatogenesis

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