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.

Steroid signaling in mature follicles is important for Drosophila ovulation

“…To determine whether ecdysteroid signaling interferes with this pathway to regulate follicle rupture, we measured the activation of Mmp2 in control and steroid-defective follicles with in situ zymography. Consistent with our previous report (12), approximately 80% of control follicles showed gelatinase activity at their posterior end after OA stimulation for 3 or 6 h; however, only 40% of shd follicles showed gelatinase activity ( Fig. 5 B and D and Fig.…”

“…The mechanisms underlying ovulation in insects, such as Drosophila, were thought to be highly divergent from mammals, in which ovulation is regulated by luteinizing hormone and progesterone, which has not been identified in insects so far. However, our recent work demonstrated the involvement of follicle rupture and matrix metalloproteinase in Drosophila ovulation, reminiscent to mammalian ovulation (1,9,12). In addition, it seems that adrenergic regulation of calcium signaling is also conserved in regulating ovulation in both Drosophila and mammals (12,44,45).…”

“…With R44E10-Gal4 transcription factor driving UAS-shd RNAi expression in mature follicle cells (12), shd expression is severely knocked down in mature follicles but normal in early egg chambers ( Fig. 1D and Fig.…”

“…OA activates the Oamb receptor in mature follicle cells, which sequentially leads to an increase of intracellular calcium, activation of Mmp activity, and follicle rupture (Fig. 5A) (12). To determine whether ecdysteroid signaling interferes with this pathway to regulate follicle rupture, we measured the activation of Mmp2 in control and steroid-defective follicles with in situ zymography.…”

“…Mmp2 activation is stimulated by the monoamine octopamine (OA), which is equivalent to norepinephrine in mammals and is essential for ovulation (10,11). OA binds to its receptor Octopamine receptor in mushroom body (Oamb) in mature follicle cells to induce an intracellular calcium rise, Mmp2 activation, and follicle rupture (12). Drosophila ovulation is also regulated by multiple ovarian extrinsic factors, including secretions from the oviduct (13), female reproductive glands (14), and male accessory glands (15,16).…”