The overall goal of this method is to automate the analysis of a Caenorhabditis elegans germline to study its nuclei, protein, and cytoskeletal distribution. This method can help answer the key questions in the C.Elegans field about the number of nuclei and sperm, nuclear distribution, and cytoskeletal structure of the germline. The main advantages of this technique are it reduces the analysis time, increases the sample size, and eliminate the human error associated with the manual analysis.
To image the samples, place slides with stained germlines into the slide holder above the 63X objective of a confocal microscope, and locate a germline. Focus on the top of the germline, and mark it with the confocal microscope software. When the total germline thickness has been established, define the thickness of each slice up to 0.5 micrometers, and mark the complete germline.
Then, start the acquisition, scanning each slice eight times and averaging the values to improve the image quality. When all of the images have been obtained, import the images to an appropriate image analysis software program, and use the surface function tool to select the mitotic region. An image of the distal end of the germline will appear.
Cancel the automatic surface creation wizard, and manually draw a region of interest around the mitotic region at the first and last slices of the image from the three-dimensional stack. Click Create Surface, followed by Mask Channel, to mask all of the channels except the DAPI channel. Using the spot function tool, define the size parameters of the mitotic region with an XY diameter of two micrometers and with the Z diameter undefined.
Click subtract background to subtract the background. To define the minimum threshold, increase the minimum 3D rendering threshold on a DAPI-stained wild-type germline until the first spot appears outside of the germline, and generate a 3D model for each region. After saving the images, click table to view the number of nuclei, as generated automatically by the software, and export the images as TIFF files.
For the transition zone, create surface as explained before. Then, click Mask Channel to mask all the channels except the DAPI channel. Using spot function, define an XY diameter of two micrometers and Z diameter of 1.5 micrometers, and click subtract background to subtract the background.
To define the minimum threshold, increase the minimum 3D rendering threshold on a DAPI-stained wild-type germline until the first spot appears outside of the germline, and generate a 3D model for each region. After saving the images, click table to view the number of nuclei, as generated automatically by the software, and export the images as TIFF files. For scoring the sperm number, select the spermatheca as the region of interest, as demonstrated for the nuclei regions, and use the spot function tool to define the XY diameter between 0.75 and one micrometers with an undefined Z diameter to detect each sperm.
Select the Background Subtract box, and use the background correction values calculated by the software to subtract the background. Then, adjust the 3D rendering threshold to detect all of the DAPI-stained sperm in the spermatheca. For chromosome counting in the oocytes, select the oocytes and define the XY diameter as less than 0.75 micrometers, and define the threshold before developing the 3D models.
Then, save the images for export as TIFF files. For cytoskeletal germline reconstruction, identify the mitotic region of interest in the germline and create a surface mask as previously demonstrated. Click Mask Channels to mask all of the channels except the phalloidin channel, and use the surface function tool to define a surface detail of 0.25 micrometers.
After subtracting the background, adjust the 3D rendering threshold to save the developed 3D image for export as a TIFF file. The nuclei distribution is dependent on the stage of differentiation. For example, at the mitotic region, the inner actin appears as a solid mass upon which the nuclei are organized.
In a wild-type germline, the mitotic region contains approximately 250 nuclei and is tightly packed with nuclei compared to the rest of the germline. As the nuclei move away from the distal end, however, they appear closer to the circumference of the germline, with the change in distribution starting at the transition zone and finishing as the nuclei enter the third stage of prophase in meiosis. At the transition zone, the germline inner actin assumes a more cylindrical structure, becoming a hollow cylinder as it reaches the pachytene.
The second layer of actin covers the inner layer, giving shape to the germline. This cylinder within a cylinder actin structure disappears toward the oocyte region, at which point the actin appears as thick fibers covering the oocytes. In the spermatheca, the actin also forms into thick bundles of fiber, although these fibers appear to be packed more closely together than in the oocyte region.
Three-dimensional rendering of the proximal end of the germline, as demonstrated, reveals an average of 151 sperm in each spermatheca. Once mastered, this analysis can be completed in 10 minutes per germline. While attempting this procedure, it is important to remember to define the analysis parameters according to the magnification of the objective used to visualize the germline in your experiment.
After watching this video, you should have a good understanding of how to perform an automatic analysis of a germline and to study cytoskeletal structure and to score the number of the nuclei within the germline.
