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

Deady LD, Sun J - PLoS Genet. (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Neurobiology, University of Connecticut, Storrs, Connecticut, United States of America.

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.

No MeSH data available.


Related in: MedlinePlus

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.
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pgen.1005604.g005: 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.

Mentions: OA-Oamb interaction can induce transient increase of intracellular Ca2+ concentration ([Ca2+]i) [23]. To determine whether OA evokes Ca2+ signaling in mature follicle cells to induce follicle rupture, we first monitored the [Ca2+]i using a genetically encoded calcium sensor (see methods). Fluorescent intensity of the calcium sensor expressed in mature follicle cells rose significantly around six minutes after OA administration in our ex vivo culture system (S7 Fig and S2 Movie). To determine whether Ca2+ is required for OA-induced follicle rupture, we pretreated mature follicles with BAPTA-AM, an intracellular Ca2+ chelator, before OA stimulation. Two hundred μM BAPTA-AM treatment significantly perturbed the OA-induced follicle rupture (Fig 5A–5C). To determine whether Ca2+ is sufficient to induce follicle rupture, we stimulated mature follicles with ionomycin, a potent ionophore for increasing [Ca2+]i. Ionomycin is potent to induce follicle rupture even at 5 μM concentration (Fig 5D–5F), lower than the dose typically used in the field [48]. Taken together these data suggest that the increase of [Ca2+]i is both necessary and sufficient to induce follicle rupture.


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

Deady LD, Sun J - PLoS Genet. (2015)

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.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4608792&req=5

pgen.1005604.g005: 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.
Mentions: OA-Oamb interaction can induce transient increase of intracellular Ca2+ concentration ([Ca2+]i) [23]. To determine whether OA evokes Ca2+ signaling in mature follicle cells to induce follicle rupture, we first monitored the [Ca2+]i using a genetically encoded calcium sensor (see methods). Fluorescent intensity of the calcium sensor expressed in mature follicle cells rose significantly around six minutes after OA administration in our ex vivo culture system (S7 Fig and S2 Movie). To determine whether Ca2+ is required for OA-induced follicle rupture, we pretreated mature follicles with BAPTA-AM, an intracellular Ca2+ chelator, before OA stimulation. Two hundred μM BAPTA-AM treatment significantly perturbed the OA-induced follicle rupture (Fig 5A–5C). To determine whether Ca2+ is sufficient to induce follicle rupture, we stimulated mature follicles with ionomycin, a potent ionophore for increasing [Ca2+]i. Ionomycin is potent to induce follicle rupture even at 5 μM concentration (Fig 5D–5F), lower than the dose typically used in the field [48]. Taken together these data suggest that the increase of [Ca2+]i is both necessary and sufficient to induce follicle rupture.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Neurobiology, University of Connecticut, Storrs, Connecticut, United States of America.

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.

No MeSH data available.


Related in: MedlinePlus