Drosophila melanogaster is a powerful model system widely used to elucidate a variety of biological processes. For instance, the use of Drosophila female and male germ lines has largely contributed to understanding the mechanisms underlying cell division and stem cell biology.
The group of Dr. Maria Grazia Giansanti at the Institute of Molecular Biology and Pathology (IBPM-CNR) in Rome makes use of a whole-mount procedure for dissection and immunofluorescence analysis of Drosophila testes. Following the protocol described in Sechi et al., 2019 adult testes are dissected in PBS and fixed in ice-cold methanol (5 min) and ice-cold acetone (2 min). Here, in particular, we show testes which were permeabilized, blocked in PBS with 0.1% Triton X-100 and 3% BSA and then stained for alpha-tubulin (secondary antibody: FITC) and Cyclin B (secondary antibody: Alexa Fluor-647). After DNA staining with DAPI samples were mounted with ProLong Glass Antifade (Invitrogen).
CrestOptics X-Light V3 spinning disk confocal has been used to acquire images of the Drosophila testes. The microscope set-up used was equipped with Eclipse Ti2 microscope (Nikon), Celesta laser source (Lumencor), Prime 95B sCMOS camera (Photometrics). In order to visualize the whole organ, we compared acquisitions using 20x air objective (0.75 NA, 1 mm WD) and 40x silicone oil objective (1.25 NA, 300 um WD, kindly provided by Nikon). In Figure 1, we report both acquisitions and we emphasize that 40x silicone oil objective clearly allows higher-resolution and definition of the small subcellular structures (see zoomed area) compared to 20x air objective. To better appreciate the 3D cellular organization inside the testes, we also show a whole organ in 3D volume view and movies (Figure 2).
For detailed and high-resolution views, we further used 100x oil objective (1.49 NA, TIRF 120 um WD) and run Z-stack acquisitions. We focused of two regions of interest due to the presence of different stages of spermatogenesis. The inner twisted area contains mature sperm tails whereas spermatogonia and spermatocytes occupy the first third of the testis. Both areas are shown in 2D,3D volume views and the outer region in 3D rendering movie (Figure 3).
As described in Sechi et al., 2019, the whole-mount technique can be applied to the analysis of male meiotic division and spermatogenesis in Drosophila mutants, allowing to detect defects in centriole separation, spindle organization, chromosome segregation and cytokinesis. Moreover, the correct timing of the G2/M transition is crucial for proper meiotic progression and, hence, for spermatocyte maturation. Among meiotic regulators it is worth mentioning Cyclin B, which controls the correct timing of the G2/M that completes meiotic prophase (Baker et al., 2015). Here, we have highlighted Cyclin B distribution along the whole testis (Figures 1, 2) and more specifically at single-cell level (Figure 3). Altogether, these data enable protein visualization at high resolution in a whole-mount testis and allow following quantitative analysis (e.g., localization, fluorescence intensity measurements) according to scientific needs.
Figure 1: Whole testes acquisition performed with X-Light V3 spinning disk confocal. Denoised maximum intensity projections (MIP) of Z-stacks are shown (alpha-tubulin (cyan), Cyclin B (magenta) and DNA (white)). Scale bar, 100 um.
Figure 2: Whole testes acquisition performed with X-Light V3 spinning disk confocal and 40x silicone oil objective. 3D view and movies of a deconvolved image is shown (alpha-tubulin (green), Cyclin B (cyan) and DNA (white)).
Figure 3: Regions of interest acquired with X-Light V3 spinning disk confocal with 100x Oil objective. Scale bar, 50 um.
Inner region: MIP, 3D view, orthogonal view
Outer region: MIP, 3D view, orthogonal view
Outer region: Movie
The application note has been prepared in collaboration with Dr. Anna Frappaolo, Dr. Stefano Sechi , Dr. Angela Karimpour Ghahnavieh and Dr. Maria Grazia Giansanti