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

<p>(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 <i>Mmp2</i>-knockdown (G) and <i>Timp</i>-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 <i>Oamb</i> mutant follicles. Representative images show <i>Oamb</i>^<i>+/-</i> (J) and <i>Oamb</i>^<i>-/-</i> (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.</p

Disulfide reduction of R2-VWA-Ig did not affect receptor-PA binding.

<p>PA₈₃, R2-VWA, and R2-VWA-Ig were mixed, as indicated in 20 mM Tris-HCl (pH 8.5), 150 mM NaCl, 1 mM MgCl₂, in the presence or absence of 10 mM TCEP. The samples were subjected to native gel electrophoresis followed by Coomassie blue staining.</p

Reducing agents inhibited PA-mediated LF_N translocation across the plasma membrane.

<p>PA prepore was incubated with CHO-ANTXR2 cells at 4°C followed by the addition of ³⁵S-LF_N to the cells. The cells were treated with 10 mM DTT or TCEP, as indicated. Translocation was triggered by addition of a pH 5.0 buffer to the cells. The cells were either directly lysed (pronase−) or treated with pronase (pronase+) followed by lysis. ³⁵S-LF_N was detected by autoradiography.</p

ANTXR2 ectodomain is composed of R2-VWA and R2-Ig.

<p><b>A</b>. Schematic of ANTXR2. ANTXR2 is composed of R2-VWA (VWA, 38–218) and R2-Ig (Ig, 219–318), a single-pass transmembrane domain (TM, 319–343) and a cytoplasmic domain (Cyto, 344–489). <b>B</b>. The crystal structure of R2-VWA domain (1SHU) as displayed in Swiss-PDB-Viewer. Residues Cys175, the Cys39–Cys218 disulfide bond, and Mn²⁺ ion of MIDAS are shown and labeled. <b>C</b>. Sequence alignment of the ectodomains of ANTXR1 (R1) and ANTXR2 (R2). The seven conserved Cys residues are highlighted and numbered in R2. Identical residues are labeled with asterisk (*).</p

A Follicle Rupture Assay Reveals an Essential Role for Follicular Adrenergic Signaling in <i>Drosophila</i> Ovulation

<div><p>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 <i>Drosophila</i> and mammalian ovulation at both the cellular and molecular levels. During ovulation in <i>Drosophila</i>, 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 <i>ex vivo</i>, 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 <i>ex vivo</i> and ovulation <i>in vivo</i>. 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 <i>ex vivo</i> follicle rupture assay and demonstrates the role for follicular adrenergic signaling in Mmp2 activation and ovulation in <i>Drosophila</i>, which is likely conserved in other species.</p></div

The effect of follicular adrenergic signaling for follicle trimming.

<p>* P<0.05</p><p>**P<0.01</p><p>***P<0.001</p><p>The effect of follicular adrenergic signaling for follicle trimming.</p

A novel <i>ex vivo</i> follicle rupture assay in <i>Drosophila</i>.

<p>(A) A schematic diagram representing the female reproductive system and <i>ex vivo</i> experiments. Mature follicle cells are marked by fluorescent proteins (red), and octopaminergic neurons are shown in green [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005604#pgen.1005604.ref003" target="_blank">3</a>]. (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 <i>R47A04-Gal4</i> driving <i>UAS-RFP</i> (<i>47A04>RFP</i>) 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.</p

Disulfide reduction of R2-VWA-Ig inhibited release of K⁺.

<p><b>A</b>. PA prepore, R2-VWA, and R2-VWA-Ig were incubated as indicated (receptor/PA ratio = 2/1) in 20 mM Tris-HCl (pH 8.5), 150 mM NaCl, 1 mM MgCl₂, in the presence or absence of 10 mM TCEP. Release of K⁺ ions from KCl-charged liposomes was monitored with a K⁺-selective probe in the solution. <b>B</b>. PA prepore (10 µg/ml) was incubated at 4°C with CHO-ANTXR2 cells, with various concentrations of TCEP as indicated. The cells were washed with 20 mM Tris-HCl (pH 8.0) and 150 mM NaCl to remove unbound PA, and acidification was initiated by addition of a pH 5.0 buffer into the cell cultures at 4°C for various times. The supernatants were collected and the content of K⁺ ion was measured by the K⁺-selective probe and read with a pH meter as mV. DNI, a PA dominant negative mutant that is defective in pore formation, served as negative control.</p

Disulfide reduction did not affect formation of SDS-resistant PA₆₃ oligomers at low pH.

<p><b>A</b>. PA prepore, R2-VWA, and R2-VWA-Ig were mixed as indicated (receptor/PA ratio is 2/1) in 20 mM Tris-HCl (pH 8.5), 150 mM NaCl, 1 mM MgCl₂, in the presence or absence of 10 mM TCEP. Acidification was initiated by addition of 1/10 volume 1 M sodium acetate (pH 5.0). The samples were subjected to SDS-PAGE and stained with Coomassie blue. <b>B</b>. PA prepore was incubated at 4°C with CHO-ANTXR2 cells in the presence or absence of reducing agents (10 mM DTT or TCEP as indicated). The cells were washed to remove unbound PA, and acidification was initiated by addition of a pH 5.0 buffer into the cell cultures. After 10 min at 4°C the cells were harvested, and lysates were applied to SDS-PAGE, followed by western blotting with anti-PA antibody.</p

Purified ANTXR2 ectodomain, R2-VWA-Ig, is a soluble monomeric protein containing three disulfide bonds.

<p><b>A</b>. Gel filtration of the purified R2-VWA-Ig. R2-VWA-Ig was expressed and purified as a fusion protein with an N-terminal MBP tag and a C-terminal His₆-tag. After removal of the MBP- and His₆- tags, R2-VWA-Ig ran as a monomer (∼30 kDal) in a Superdex-75 column in 20 mM TrisHCl (pH 8.0) and 150 mM NaCl. <b>B</b>. SDS-PAGE of the purified R2-VWA-Ig. The purified R2-VWA-Ig was treated with or without 10 mM DTT in SDS sample buffer, and run on SDS-PAGE. <b>C</b>. left panel, cysteine standard curve; at OD₄₁₂ was plotted after L-cysteine at various concentrations was incubated with 500 µM DTNB in 6 M guanidium HCl and 0.1 M TrisHCl, pH 8.0; right panel, 10 µM of R2-VWA-Ig (WT) or R2-VWA-Ig (C175A) was incubated with 500 µM DTNB in 6 M guanidium HCl and 0.1 M TrisHCl, pH 8.0. OD₄₁₂ was recorded and –SH/molecule was calculated based on the cysteine standard curve and protein concentration.</p