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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

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 Mmp2RNAi 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 Mmp2RNAi or Timp driven by R47A04-Gal4 prevents follicle rupture in response to OA or NE. All experiments were performed in four replicates except Mmp2RNAi, 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.
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pgen.1005604.g004: 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 Mmp2RNAi 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 Mmp2RNAi or Timp driven by R47A04-Gal4 prevents follicle rupture in response to OA or NE. All experiments were performed in four replicates except Mmp2RNAi, 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.

Mentions: The crucial role of Mmp2 in trimming of posterior follicle cells [37] prompted us to investigate the relationship between follicular adrenergic signaling and Mmp2 activity. It is unlikely that adrenergic signaling regulates Mmp2 expression, as Mmp2 was readily detected in the posterior follicle cells of TβH mutants (S6A and S6B Fig). To test whether OA regulates Mmp2 activity, we examined gelatinase enzymatic activity in the OA-induced ex vivo ovulation assay using in situ zymography [37,38]. About 20% of mature follicles cultured in a control medium had gelatinase activity at their posterior end (Figs 4A, 4C, S6C and S6G). In contrast, more than 70% of mature follicles stimulated with OA had gelatinase activity (Figs 4B, 4C, S6D and S6G). The entire eggshells of ruptured oocytes were coated with Mmp-activated gelatin-fluorescein (Figs 4B and S6D), as we observed in vivo [37]. In addition, OA-induced gelatinase activity was blocked in mature follicles with Oamb knockdown or misexpression of Timp, an endogenous inhibitor of Mmp2 [47], in follicle cells (S6E–S6G Fig). These data indicate that OA-Oamb signaling is sufficient to induce Mmp2 activation.


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

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

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 Mmp2RNAi 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 Mmp2RNAi or Timp driven by R47A04-Gal4 prevents follicle rupture in response to OA or NE. All experiments were performed in four replicates except Mmp2RNAi, 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.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4608792&req=5

pgen.1005604.g004: 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 Mmp2RNAi 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 Mmp2RNAi or Timp driven by R47A04-Gal4 prevents follicle rupture in response to OA or NE. All experiments were performed in four replicates except Mmp2RNAi, 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.
Mentions: The crucial role of Mmp2 in trimming of posterior follicle cells [37] prompted us to investigate the relationship between follicular adrenergic signaling and Mmp2 activity. It is unlikely that adrenergic signaling regulates Mmp2 expression, as Mmp2 was readily detected in the posterior follicle cells of TβH mutants (S6A and S6B Fig). To test whether OA regulates Mmp2 activity, we examined gelatinase enzymatic activity in the OA-induced ex vivo ovulation assay using in situ zymography [37,38]. About 20% of mature follicles cultured in a control medium had gelatinase activity at their posterior end (Figs 4A, 4C, S6C and S6G). In contrast, more than 70% of mature follicles stimulated with OA had gelatinase activity (Figs 4B, 4C, S6D and S6G). The entire eggshells of ruptured oocytes were coated with Mmp-activated gelatin-fluorescein (Figs 4B and S6D), as we observed in vivo [37]. In addition, OA-induced gelatinase activity was blocked in mature follicles with Oamb knockdown or misexpression of Timp, an endogenous inhibitor of Mmp2 [47], in follicle cells (S6E–S6G Fig). These data indicate that OA-Oamb signaling is sufficient to induce Mmp2 activation.

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