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. 2018 May 20;8(10):e2846.
doi: 10.21769/BioProtoc.2846.

Ex vivo Follicle Rupture and in situ Zymography in Drosophila

Elizabeth M Knapp  1 , Lylah D Deady  1 , Jianjun Sun  1   2
Affiliations

Ex vivo Follicle Rupture and in situ Zymography in Drosophila

Elizabeth M Knapp et al. Bio Protoc. .

Abstract

Ovulation, the process of releasing a mature oocyte from the ovary, is crucial for animal reproduction. In order for the process of ovulation to occur, a follicle must be fully matured and signaled to rupture from the ovary. During follicle rupture in both mammals and Drosophila, somatic follicle cells are enzymatically degraded to allow the oocyte to be liberated from the follicle. Here, we describe a detailed protocol of our newly developed ex vivo follicle rupture assay in Drosophila, which represents a first assay allowing direct quantification of follicles' capacity to respond to ovulation stimuli and rupture. This assay can be modified to stimulate rupture with other reagents (for example, ionomycin) or to query enzymatic activity (in situ zymography). In addition, this assay allows genetic or pharmacological screens to identify genes or small molecules regulating follicle rupture in Drosophila.

Keywords: Drosophila; Follicle cells; Follicle rupture; Octopamine; Ovulation; in situ zymography.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1.
Figure 1.. PYREX 9-well spot plate setup
Video 1.
Video 1.. Dissection example
Video 2.
Video 2.. Analysis of in situ zymography assay.
Open images acquired at the end of each experiment (both bright-field and fluorescent images) in ImageJ. Count the total number of follicles using the multi-point tool in the bright-field image. In this example, there are a total of 29 follicles. Then, count the total number of follicles with posterior green fluorescence in the green-fluorescent image. Before taking the image, most of the follicles identified with enzymatic activity were segregated toward the top of the image. In this example, there are a total of 12 follicles with posterior green fluorescence; therefore, 41.4% of follicles had enzymatic activity.
Video 3.
Video 3.. Analysis of ex vivo follicle rupture assay.
Open images acquired at the end of each experiment (both bright-field and fluorescent images) in ImageJ. Count the total number of follicles using the multi-point tool in the bright-field image. In this example, there are a total of 27 follicles. Then, count the total number of ruptured follicles in the fluorescent image. In this example, there are a total of 20 ruptured follicles; therefore, 74% of follicles ruptured in this example.
Figure 2.
Figure 2.. Examples of follicle cell layer coverage of oocyte.
Scale bar is estimated 100 μm.
See this image and copyright information in PMC

References

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