A Follicle Rupture Assay Reveals an Essential Role for Follicular Adrenergic Signaling in Drosophila Ovulation

Abstract

Ovulation is essential for the propagation of the species and involves a proteolytic degradation of the follicle wall for the release of the fertilizable oocyte. However, the precise mechanisms for regulating these proteolytic events are largely unknown. Work from our lab and others have shown that there are several parallels between Drosophila and mammalian ovulation at both the cellular and molecular levels. During ovulation in Drosophila, posterior follicle cells surrounding a mature oocyte are selectively degraded and the residual follicle cells remain in the ovary to form a corpus luteum after follicle rupture. Like in mammals, this rupturing process also depends on matrix metalloproteinase 2 (Mmp2) activity localized at the posterior end of mature follicles, where oocytes exit. In the present study, we show that Mmp2 activity is regulated by the octopaminergic signaling in mature follicle cells. Exogenous octopamine (OA; equivalent to norepinephrine, NE) is sufficient to induce follicle rupture when isolated mature follicles are cultured ex vivo, in the absence of the oviduct or ovarian muscle sheath. Knocking down the alpha-like adrenergic receptor Oamb (Octoampine receptor in mushroom bodies) in mature follicle cells prevents OA-induced follicle rupture ex vivo and ovulation in vivo. We also show that follicular OA-Oamb signaling induces Mmp2 enzymatic activation but not Mmp2 protein expression, likely via intracellular Ca2+ as the second messenger. Our work develops a novel ex vivo follicle rupture assay and demonstrates the role for follicular adrenergic signaling in Mmp2 activation and ovulation in Drosophila, which is likely conserved in other species.

Fig 1. A novel ex vivo follicle rupture assay in Drosophila.

(A) A schematic diagram representing the female reproductive system and ex vivo experiments. Mature follicle cells are marked by fluorescent proteins (red), and octopaminergic neurons are shown in green [3]. (B-C) Representative images show mature follicles after three-hour culture without (B) or with (C) OA. Follicles are imaged with incident light shown in blue and follicle cells are marked by R47A04-Gal4 driving UAS-RFP (47A04>RFP) expression in red. White arrowheads indicate ruptured follicles here and in subsequent figures. (D) The cumulative percentage of ruptured follicles throughout the 19-hour culture period. Twenty-seven and 50 mature follicles were used in the control (0 μM) and experimental (5 μM) group, respectively. (E) A time-lapse image shows the entire follicle rupturing process after 20 μM of OA stimulation. The dotted yellow line outlines the rupturing follicle and the straight red line marks the posterior leading edge of the follicle-cell layer. Time is in minutes. (F) The kinetics of the rupturing process is similar between follicles. Data were pooled from two independent experiments, and nine out of 28 follicles isolated from ten females were analyzed. (G-I) Percentage of ruptured follicles after three-hour culture with different concentrations of OA (G), TA (H), and NE (I). Errors are standard deviations. The number of follicles analyzed was in the parenthesis above the charts in this figure and all the following figures. All conditions have three replicates except for 0 μM NE, which has four replicates.

Fig 2. Follicular Oamb is required for OA/NE-induced follicle rupture.

(A-D) Representative images show mature follicles (marked by R44E10>GFP in follicle cells in red) after three-hour culture with 20 μM of OA (A-B) or NE (C-D). Mature follicles are from control (A and C) and Oamb mutant (B and D) females. (E) Quantification of Oamb mutant mature follicles in response to OA or NE stimulation. Four replicates were used for each genotype, except in Oamb ^-/- group with NE treatment, which has three replicates. (F-G) Quantification of follicle rupture after three-hour OA or NE treatment (20 μM). Mature follicles were derived from TβH (F) or Tdc2 (G) mutant females and marked by 47A04>RFP. All treatments have three replicates except for TβH ^+/- with NE treatment and Tdc2 ^-/-, which have four replicates. (H-J) Oamb knockdown with R47A04-Gal4 blocks follicle rupture. Representative images show control (H) and Oamb ^RNAi1 (I) mature follicles after three-hour culture with 20 μM of OA. Quantification of follicle rupture (J). The number of replicates for each condition in (J) is 3, 3, 3, 4, and 2. (K-M) Oamb knockdown with R44E10-Gal4 blocks follicle rupture induced by OA or NE. Representative images show control (K) and Oamb ^RNAi2 (L) mature follicles after a three-hour culture with 20 μM of OA. Quantification of follicle rupture (M). The number of replicates for each condition in (M) is 6, 5, 4, and 3. Student’s T-test was used (*** P<0.001; ** P<0.01; * P<0.05).

Fig 3. Follicular adrenergic signaling is required for ovulation and follicle cell trimming in vivo.

(A-C) Egg laying (A), mature follicles in ovary (B), and the average ovulation and uterus time (C) is shown for control females or those expressing Oamb ^RNAi in mature follicle cells driven by R44E10-Gal4. Student’s T-test was used (A-B; *** P<0.001; **P<0.01; * P<0.05). (D-F) Follicle cell trimming is significantly reduced when follicular Oamb is knocked down by R44E10-Gal4 driving Oamb ^RNAi1 expression (44E10>Oamb ^RNAi1). Representative images show trimmed follicles in control (D) but not Oamb-knockdown (E) ovaries. Trimmed follicles are outlined with dashed yellow lines, and the posterior leading edge of the follicle-cell layer is marked by a straight red line. Quantification of trimmed follicles (F). (G-L) Follicle cell trimming is also significantly reduced in TβH (G-I) or Tdc2 (J-L) mutant females. See Tables 1 and 2 for the number of females analyzed and statistics.

Fig 4. Adrenergic signaling activates Mmp2 to regulate ovulation.

(A-C) In situ zymography shows increased Mmp activity in mature follicles after three-hour culture with 20 μM of OA. Mmp activity is indicated by Gelatin-fluorescein (green in A and B). The percentage of follicles with posterior Mmp activity is quantified in (C; *** P < 0.001). Three and four replicates were used for OA- and OA+ groups, respectively. (D-F) Expression of Mmp2 ^RNAi or Timp driven by R44E10-Gal4 prevents follicle rupture in response to OA or NE (*** P <0.001 and ** P < 0.01). The number of replicates used for each condition is 6, 5, 6, 4, 3, and 3. (G-I) Expression of Mmp2 ^RNAi or Timp driven by R47A04-Gal4 prevents follicle rupture in response to OA or NE. All experiments were performed in four replicates except Mmp2 ^RNAi, which have three replicates. (J-L) Ovaries are shown for the Oamb mutant (J), the Oamb mutant with ectopic expression of Mmp2 driven by R44E10-Gal4 (K), and the Oamb heterozygous with ectopic Mmp2 expression (L). Mature eggs were released into the female abdominal cavity.

Fig 5. Intracellular Ca²⁺ is the second messenger downstream of follicular adrenergic signaling.

(A-C) Pretreatment of BAPTA-AM blocks OA-induced follicle rupture. Representative images show mature follicles treated with DMSO (A) or BAPTA-AM (B) followed a three-hour stimulation with 20 μM of OA. Ruptured follicles were quantified in C. Three replicates are used for each condition. (D-F) Ionomycin is sufficient to induced follicle rupture. Representative images show follicles after three-hour culture with ethanol (D) or 5 μM of ionomycin. Ruptured follicles after different doses of ionomycin treatment are quantified in F. All conditions have three replicates except in 5 μM, which has four replicates. (G-H) Representative images of Mmp2-knockdown (G) and Timp-overexpressing (H) follicles treated with 5 μM of ionomycin for three hours. (I) Quantification of ruptured follicles with Mmp2 knockdown or Timp overexpression in mature follicle cells in response to 20 μM of OA or 5 μM of ionomycin stimulation. All conditions have three replicates except for Timp overexpression with ionomycin treatment, which has six replicates. (J-L) Ionomycin, but not OA, is sufficient to induce rupture in Oamb mutant follicles. Representative images show Oamb ^+/- (J) and Oamb ^-/- (K) follicles after three-hour culture with ionomycin. (L) Quantification of ruptured follicles after three-hour culture with 20 μM of OA or 5 μM of ionomycin. The number of replicates for each condition is 4, 4, 3, and 5. (M) A cartoon showing the model of follicular adrenergic signaling in Mmp activity and follicle rupture. Octopaminergic neurons are shown in green.