Figure 1.
(A) Schematic diagram of the sprn gene, transcripts, and protein products showing the site of transposon insertion, the region the antibody was raised against, the conserved PH/Ran protein binding homology domain and the primers used for RT-PCR. For the transcripts, the coding exons are orange, the UTRs grey, and the introns depicted as thin lines. (B) Expression of sprn mRNA shown by RT-PCR from adult ovaries, testes, and third instar larval brains. Three distinct bands of approximately 500 bp were detected in testes but not in ovaries or larval brains. Two of the bands are likely the predicted 483 bp and 534 bp products from sprn-RB/RC and sprn-RE/RF, respectively. The third band is not readily accounted for, but could be a new alternative splice product. (C) Western blot of extracts from wild-type OreR or homozygous sprnMB12149 mutant adult testes, ovaries, and third instar larval brains. Sprn in testes but not in ovaries or larval brains, anti-Sprn antibody recognizes a band around 68 kD consistent with predicted size, and Sprn is undetectable in the sprnMB12149 mutant.
Figure 2.
Sprn is expressed in the nebenkern throughout spermatid elongation.
Endogenous Sprn expression is first detectable, though weak, in meiosis I and II, when mitochondria form first aggregates (arrowheads). Sprn expression increases at the spermatid nebenkern and persists throughout spermatid elongation (arrows), but is absent in mature sperm. ATP-synthase labels inner membranes of mitochondria. γ-tubulin labels the centriole as follows: in each spermatocyte, there are two v-shaped centriole pairs (four centrioles); in meiosis I, each daughter cell has one pair of centrioles (two centrioles); in meiosis II, each daughter cell has one centriole and after meiosis, each spermatid has only one centriole. Scale bars, 10 µm.
Figure 3.
Sprn localizes in the nebenkern lumen.
(A) Sprn is absent from sprnMB12149 mutant nebenkerns (arrows). Scale bars, 10 µm. (B) Superresolution images showing Sprn localizes in the matrix of the nebenkern in spermatids. In the onion stage (upper panel), when two big mitochondrial derivatives wrap around one another to form the nebenkern, Sprn does not overlap with the mitochondrial inner membrane marker ATP synthase complex V subunit alpha (arrows). And in late spermatids (lower panel), when the mitochondrial derivative becomes a slender and cylindrical tube, Sprn is in the matrix of mitochondria, as shown localizing in-between the inner membranes marked by ATP synthase complex V subunit alpha in the longitudinal section (arrows). Scale bars, 5 µm.
Figure 4.
Ectopically expressed Sprn localizes to mitochondria.
Sprn-Myc, expressed in Kc167 cells, colocalizes with mitochondrial marker ATP synthase (arrows). Sprn or Myc signal was absent from untransfected cells (arrowheads). Scale bars, 10 µm.
Table 1.
Lethal analysis of ectopic in vivo UAS-Sprn-GFP expression.
Figure 5.
Sprn mutant males have normal fertility and sperm tail length.
(A) The number of progeny produced by single w1118 or sprnMB12149 males in the fertility test. The sprnMB12149 mutant males produced more progeny, but the difference was not significant according to Student's t test (p = 0.0905). Error bars represent standard error of the means (SEM), numbers inside the bars indicate the total number of males tested. (B) sprnMB12149 has normal sperm tail length compared to wild-type (w1118). Error bars represent standard error of the means (SEM), Student's t test was used to determine the statistical significance (p = 0.438). Values inside the bars indicate the total number of sperm that were measured.