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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

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.
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pgen.1005604.g001: 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.

Mentions: In Drosophila and other insects, the biogenic monoamines tyramine (TA) and octopamine (OA) act as functional counterparts to mammalian epinephrine and norepinephrine and regulate a variety of behaviors, including the fight-or-flight response, motivation, aggression, and reproduction [17,18]. Analogous to the adrenergic innervation in mammalian ovaries, Drosophila octopaminergic neurons innervate ovaries and the female reproductive tract (Fig 1A; [3,19,4]). OA released from these neurons is essential for ovulation, as mutations that disrupt the enzymes required for OA synthesis, tyrosine decarboxylase 2 (Tdc2) and tyramine β-hydroxylase (TβH), completely block ovulation [20–22].


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

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

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.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4608792&req=5

pgen.1005604.g001: 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.
Mentions: In Drosophila and other insects, the biogenic monoamines tyramine (TA) and octopamine (OA) act as functional counterparts to mammalian epinephrine and norepinephrine and regulate a variety of behaviors, including the fight-or-flight response, motivation, aggression, and reproduction [17,18]. Analogous to the adrenergic innervation in mammalian ovaries, Drosophila octopaminergic neurons innervate ovaries and the female reproductive tract (Fig 1A; [3,19,4]). OA released from these neurons is essential for ovulation, as mutations that disrupt the enzymes required for OA synthesis, tyrosine decarboxylase 2 (Tdc2) and tyramine β-hydroxylase (TβH), completely block ovulation [20–22].

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