Biochemical studies on the male reproductive system of Drosophila melanogaster
Creators: name: Ingman-Baker, Jane
Publisher: name: University of British Columbia
Year: 2010

description: Testes and paragonial glands of Drosophila melanogaster wild type males were labelled in vitro using ³⁵S-methionine, and the proteins synthesized were analysed by 2-dimensional gel electrophoresis (O'Farrell, 1975). Testes and paragonial glands were also labelled in vivo by feeding male or larvae on ³⁵S-labelled yeast and then dissecting the adult males. Approximately 1200 proteins were resolved by autoradiography of the gels. The in vitro method was shown to be more sensitive and to allow faithful synthesis of all proteins produced in vivo. ³H-proline was also used to label testes, and no significant differences from the pattern obtained with ³⁵S-methionme were found. Different laboratory stocks were analyzed to examine the degree of genetic heterozygosity of testicular proteins. The variation between patterns was very low, facilitating subsequent studies in which flies of defined genetic constitution, but with different genetic backgrounds, were compared. Testes and paragonial glands from X/0 and X/Y/Y males were labelled in vitro with ³⁵S-methionine, and the proteins synthesized were compared to those produced by wild-type males of identical autosomal background. No differences attributable to the Y chromosome could be detected in the testes or paragonial gland samples. Non-equilibrium pH gradient 2 dimensional gels (O'Farrell et al., 1977) were also run on testis proteins from X/0, X/Y and X/Y/Y males. These gels will resolve basic as well as acidic proteins and once again no differences attributable to the Y chromosome were seen. Pure sperm was manually dissected from in vivo labelled males and the proteins analyzed. Ninety-two proteins were detected, and all were synthesized in comparable amounts by X/0, X/Y and X/Y/Y males, showing that the Y chromosome does not code for any of these structural sperm proteins. It is postulated that no Y chromosome products were detected because they are organizational factors, or regulatory proteins only present in very small amounts in the adult testes. ³⁵S labelled males were also mated to unlabel led females, and the proteins of the transferred sperm were analyzed by 2DPAGE. The contributions of the testes and paragonial gland to the ejaculate were determined. Testes at various stages of development were also cultured in vitro in ³⁵S-methiomne containing media. A profile of the proteins synthesized during development revealed that the spectrum of proteins synthesized at different stages between third instar larvae and the imago were remarkably similar, despite the morphological changes taking place in the organ. Sperm proteins were localized on the patterns, and the quantitative changes occurring during this period were examined. The basic proteins of the testis were studied in an attempt to biochemically identify a Drosophila protamine. Sperm was isolated by dissection, and the acid soluble proteins were separated on a 15% modified Laemmli SDS gel. No unusually small basic protein was seen upon staining, but a protein was present which comigrated with trout histone H4. This suggests that D. melanogaster males may retain somatic histones in the nucleus during the condensation of the sperm head. Testes were labelled in vitro with ³H-arginine and the basic proteins were analyzed on a 15% modified Laemmli SDS gel. The gel was autoradiographed and a prominent doublet was seen at the front of the gel, suggesting that a small highly basic protein is synthesized in the testis.