NADPH oxidase-generated reactive oxygen species in mature follicles are essential for Drosophila ovulation

Significance Reactive oxygen species (ROS) cause oxidative stress and damage in many pathological conditions, but they can also function as signaling molecules in physiological processes. It is difficult, however, to decipher where ROS come from and which ROS are involved in these processes. In this article, we demonstrate that a NADPH oxidase (NOX) and an extracellular superoxide dismutase (SOD3) function in follicle cells of Drosophila egg chambers to produce hydrogen peroxide, which regulates follicle rupture and ovulation, a process essential for reproduction. NOX and SOD3 are expressed in human follicles and could potentially play similar roles in humans. Our work thus provides potential targets for treating ROS-related infertility or developing novel contraceptive approaches.


SI Results
Previous work used three muscle-Gal4 drivers to demonstrate that NOX functions in ovarian muscles to control muscle contraction and ovulation (1). The three muscle-Gal4 drivers used in the report (c179, c855a, Nrv1-Gal4) were either not at all or not explicitly mapped to the ovarian muscles. Both c179 and c855a are not expressed in ovarian muscles (Fig. S1A-D). Instead, c179 is expressed in seminar receptacle, spermathecae, and neurons innervating oviduct ( Fig. S1A-B). c855a is highly expressed in stage-14 follicle cells (Fig. S1C-D). Nrv1-Gal4 is widely expressed in many tissues in the female reproductive system, including uterus, spermathecae, seminal receptacle, oviduct, follicle cells and ovarian muscles (Fig. S1E-H). These expression patterns made us wonder whether NOX indeed functions in ovarian muscles to control ovulation. To address this question, we used two muscle-specific Gal4 drivers, act57B-Gal4 and Mef2-Gal4 (2), to knock down Nox and measure the egg-laying potential of these females. act57B-Gal4 is specifically detected in peritoneal sheath, oviduct and uterus muscle (Fig. S1I-J), while Mef2-Gal4 is expressed in all muscle cells throughout animal (Fig. S1K-L) (2,3). Females with Nox knockdown in Mef2-Gal4 cells died gradually after fly enclose, likely due to NOX's role in body muscle for normal physiology. In contract, females with Nox knockdown in act57B-Gal4 cells were viable and laid almost normal number of eggs (Table S1). However, females with Nox knockdown in 44E10 or 47A04 cells laid much fewer eggs than control (Table S2). In another experiment, we specifically knocked down Nox in adult muscle cells by shifting newly eclosed flies to the restrictive temperature (29C). These females laid normal number of eggs as control females (Table S1). All these data suggest that ovarian muscle NOX unlikely plays a major role in regulating ovulation as previously reported.

Egg laying and ovulation time
Analysis of egg laying and ovulation time was performed as described (8,10). In short, five-to six-day-old virgin females were fed with wet yeast one day before egg laying, and five females and 10 Oregon-R males were kept in a bottle to lay eggs on molasses-agar plates for two days at 29°C. After egg laying, ovaries were dissected, and mature follicles in these ovaries were quantified. The number of eggs laid on the plates was counted and used to calculate the average time for laying an egg (egg-laying time). The egg-laying time was partitioned into the ovulation time, oviduct time, and the uterus time. The partition ratio was determined based on the percentage of females having eggs in the oviduct or uterus at six hour after mating.

Ex vivo follicle rupture, in situ zymography, and quantitative RT-PCR
The ex vivo follicle rupture assay and in situ zymography were performed as described (8,10). The detailed protocols can be found (11). In brief, virgin females with 47A04-Gal4 or 44E10-Gal4 driving UAS-RFP expression were used to isolate intact mature follicles in Grace's insect medium (Caisson), which were separated into groups of ~30 egg chambers each and cultured in culture media (Grace's medium, 10% fetal bovine serum, and 1X penicillin/streptomycin) supplemented with 20 μM OA (Sigma) or 5 μM ionomycin (Cayman Chemical). For DPI (Cayman Chemical), VAS2870 (Sigma) or BHA (Sigma) treatment, isolated mature follicles were pretreated with indicated chemicals for 30 minutes before adding OA. For in situ zymography, 25 μg/mL of DQ-gelatin conjugated with fluorescein (Invitrogen) was supplemented into the culture media. All cultures were performed at 29°C, the same condition as flies were maintained. Ruptured follicles are defined as those oocytes with more than 80% area lacking follicle-cell covering. Mature follicles with posterior fluorescein signal were considered positive for MMP activity. One data point represents the percent of follicles rupture or with MMP activity per experimental group (~30 egg chambers). Data were represented as mean percentage ± standard error (SE); and Student's T-test was used for statistical analysis.
For quantitative RT-PCR, total RNAs were extracted using Direct-zol™ RNA MicroPrep Kit (Zymo Research) from 60 stage-14 egg chambers isolated from ~10 flies. cDNA synthesis, real-time PCR amplification were previously described (12,10). At least three biological replicates were performed, and data presented as mean ± SE; and Student's T-test was used for statistical analysis. RT-PCR was used to detect Nox expression in follicle cells and oocytes. Stage-14 egg chambers were isolated and treated with or without 20 M OA for three hours. Follicle cells and oocytes from ruptured egg chambers, as well as unruptured egg chambers, were collected for total RNA isolation and RT-PCR analysis. All the primers used for Rps17, Nox, Hnt, Oamb, and Mmp2 are listed in Table S3.

ROS detection
Ovaries were dissected and treated with or without 20 M OA for 30 minutes in culture media before ROS detection. Dihydroethidium (DHE; Invitrogen/Molecular Probes) labeling of fixed tissue was performed as described with minor modifications (13). In brief, the tissues were incubated with 30 M DHE for three minutes, washed with Grace's medium for three times (five minutes each), fixed for six minutes in 4% formaldehyde in 1xPBS, rinsed once in 1xPBS right after fixation, and mounted immediately in 1xPBS. Images were acquired using the exact same settings for control and experimental groups.
L-012 (Wako Chemicals) was used for quantifying O2 − production. Thirty mature follicles isolated as for OA culture were placed in each well of a white 96-well plate with 250 μl culture media plus 200 μM L-012. Plates were placed in a Synergy H1 plate reader (BioTek Instruments, Inc.) and shaked for five seconds before L-012 luminescence reading for 45 minutes with 30-second interval. After 5-minute reading, OA at 20 μM final concentration or ionomycin at 5 μM final concentration were added into each well, shaked for five seconds. Mature follicles were pretreated with BAPTA-AM (Cayman Chemical), DPI, or BHA for 30 minutes before O2 − detection in some experiments. For SOD treatment, different concentrations of SOD extract from bovine erythrocytes (Sigma) were supplemented in the culture media before O2 − detection. For O2 − detection in ovaries, three pairs of ovaries were incubated in each well. Three to four wells/genotype/conditions were used in each experiment, and each experiment was repeated at least twice. The data from one representative experiment were presented as mean ± SE.

Immunostaining and microscopy
Immunostaining was performed following a standard procedure, including ovary dissection, fixation in 4% EM-grade paraformaldehyde for 15 minutes, blocking in PBTG (PBS+ 0.2% Triton X-100+ 0.5% BSA+ 2% normal goat serum), and primary and secondary antibody staining diluted in PBTG. For Vkg::GFP analysis, stage-14 egg chambers were first isolated from ovaries in cold Grace's medium before fixation. were used as primary antibodies. Alexa-488, -568 goat anti-mouse and goat anti-rabbit (1:1000, Invitrogen) were used as secondary antibodies. For non-permeable HA antibody staining, 0.2% Triton X-100 was not added in all the steps before washing off HA primary antibody (1:1000; BioLegend), which was incubated with the ovaries for 2 hours at room temperature. After washing off HA antibody, ovaries were blocked again with PBTG for 1 hour, incubated with DE-Cadherin antibody (1:100; DSHB) overnight. HA and DE-Cadherin antibodies were then recognized by Alexa-568 goat anti-mouse and Alexa-488 goat anti-rat secondary antibodies. Images were acquired using a Leica TCS SP8 confocal microscope for immunostaining or Leica MZ10F fluorescent stereoscope with a sCOMS camera (PCO.Edge) for ex vivo culture assays, and assembled using Photoshop software (Adobe, Inc.) and ImageJ.