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Ex vivo Follicle Rupture and in situ Zymography in Drosophila   

Edited by
Yanjie Li
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Original research article

A brief version of this protocol appeared in:
eLIFE
Dec 2017

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, Ovulation, Follicle rupture, Octopamine, in situ zymography, Follicle cells

Background

The study of ovulation in Drosophila has largely been limited due to technical challenges in direct visualization and quantification of ovulation events. In the last several decades, multiple indirect methods have been developed with limitations. The first method for the study of ovulation in Drosophila is to push the female’s abdomen and record if an egg was ejected from the ovipositor (Aigaki et al., 1991; Lee et al., 2003). This method is a rough estimate of whether there’s an ovulated egg inside the uterus. The second common assay is to measure the egg-laying capacity of female flies after mating. The egg-laying process is complex, involving development and maturation of a follicle, ovulation, transportation of the ovulated egg through the oviduct, selection of an appropriate egg-laying substrate, and oviposition (Spradling, 1993; Bloch Qazi et al., 2003). If any of these processes are impaired, it will result in defective egg laying. Another indicator that ovulation is impaired is an egg-retention phenotype (Monastirioti et al., 1996; Monastirioti, 2003; Cole et al., 2005). If a female has an excess of mature follicles within her ovaries, it indicates an anovulatory phenotype. However, this can be caused directly by an ovulation defect or indirectly by a defect downstream of ovulation in the egg-laying process. On the other hand, a lack of egg-retention phenotype does not necessarily mean a lack of ovulation defect. A fourth type of assay used to study ovulation is to examine if an egg is present in the reproductive tract (Heifetz et al., 2000; Lee et al., 2009; Lim et al., 2014). Variations in this assay range from quantifying ovulation rate by the percentage of females with an egg in their lower oviduct/common oviduct/uterus post mating (Lee et al., 2009; Lim et al., 2014), to examining the distribution of eggs in each of these separate regions over time (Heifetz et al., 2000). However, each of these assays could be influenced by the speed of oogenesis, ovulation, and oviposition. To account for all the possible drawbacks of each individual assay, we recently combined the egg-retention assay, the egg-laying assay, and the egg location in the female reproductive tract to estimate the average time for ovulating an egg (ovulation time; Sun and Spradling, 2013; Deady et al., 2015 and 2017; Deady and Sun, 2015; Knapp and Sun, 2017). However, this method is tedious and also relies on the indirect measurements of ovulation.

We recently characterized ovulation at a cellular level and discovered that Drosophila ovulation involves a follicle rupture process. During ovulation, posterior follicle cells activate matrix metalloproteinase 2 (Mmp2), which degrades posterior follicle cells allowing for the encased oocyte to rupture into the oviduct (Deady et al., 2015). We also found that this process is initiated by direct octopamine (OA) and octopamine receptor in mushroom body (Oamb) signaling in follicle cells, and the entire process can be recapitulated in our ex vivo culture system (Deady and Sun, 2015). We named this assay ex vivo follicle rupture, in which mature follicles are isolated from the ovary and stimulated with OA to induce follicle rupture. Percent of follicles ruptured can be reported at the end of a short three-hour incubation, which is a direct quantification of follicle rupture. This assay allows for a relatively simple, high-throughput examination of follicle rupture in Drosophila, and is ideal for genetic and pharmacological screens. However, this experiment is done ex vivo, and results should be verified in vivo using some of the assays described above.

Copyright Knapp et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
How to cite:  Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Knapp, E. M., Deady, L. D. and Sun, J. (2018). Ex vivo Follicle Rupture and in situ Zymography in Drosophila. Bio-protocol 8(10): e2846. DOI: 10.21769/BioProtoc.2846.
  2. Deady, L. D., Li, W. and Sun, J. (2017). The zinc-finger transcription factor Hindsight regulates ovulation competency of Drosophila follicles. Elife 6: e29887.
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