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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

Follicular Oamb is required for OA/NE-induced follicle rupture.(A-D) Representative images show mature follicles (marked by R44E10>GFP in follicle cells in red) after three-hour culture with 20 μM of OA (A-B) or NE (C-D). Mature follicles are from control (A and C) and Oamb mutant (B and D) females. (E) Quantification of Oamb mutant mature follicles in response to OA or NE stimulation. Four replicates were used for each genotype, except in Oamb-/- group with NE treatment, which has three replicates. (F-G) Quantification of follicle rupture after three-hour OA or NE treatment (20 μM). Mature follicles were derived from TβH (F) or Tdc2 (G) mutant females and marked by 47A04>RFP. All treatments have three replicates except for TβH+/- with NE treatment and Tdc2-/-, which have four replicates. (H-J) Oamb knockdown with R47A04-Gal4 blocks follicle rupture. Representative images show control (H) and OambRNAi1 (I) mature follicles after three-hour culture with 20 μM of OA. Quantification of follicle rupture (J). The number of replicates for each condition in (J) is 3, 3, 3, 4, and 2. (K-M) Oamb knockdown with R44E10-Gal4 blocks follicle rupture induced by OA or NE. Representative images show control (K) and OambRNAi2 (L) mature follicles after a three-hour culture with 20 μM of OA. Quantification of follicle rupture (M). The number of replicates for each condition in (M) is 6, 5, 4, and 3. Student’s T-test was used (*** P<0.001; ** P<0.01; * P<0.05).
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pgen.1005604.g002: Follicular Oamb is required for OA/NE-induced follicle rupture.(A-D) Representative images show mature follicles (marked by R44E10>GFP in follicle cells in red) after three-hour culture with 20 μM of OA (A-B) or NE (C-D). Mature follicles are from control (A and C) and Oamb mutant (B and D) females. (E) Quantification of Oamb mutant mature follicles in response to OA or NE stimulation. Four replicates were used for each genotype, except in Oamb-/- group with NE treatment, which has three replicates. (F-G) Quantification of follicle rupture after three-hour OA or NE treatment (20 μM). Mature follicles were derived from TβH (F) or Tdc2 (G) mutant females and marked by 47A04>RFP. All treatments have three replicates except for TβH+/- with NE treatment and Tdc2-/-, which have four replicates. (H-J) Oamb knockdown with R47A04-Gal4 blocks follicle rupture. Representative images show control (H) and OambRNAi1 (I) mature follicles after three-hour culture with 20 μM of OA. Quantification of follicle rupture (J). The number of replicates for each condition in (J) is 3, 3, 3, 4, and 2. (K-M) Oamb knockdown with R44E10-Gal4 blocks follicle rupture induced by OA or NE. Representative images show control (K) and OambRNAi2 (L) mature follicles after a three-hour culture with 20 μM of OA. Quantification of follicle rupture (M). The number of replicates for each condition in (M) is 6, 5, 4, and 3. Student’s T-test was used (*** P<0.001; ** P<0.01; * P<0.05).

Mentions: To identify the receptor responsible for OA/NE-induced follicle rupture, we focused on Oamb, which is essential for ovulation [24] and is the most highly expressed OA receptor in mature follicles (S1 Fig). We verified the requirement of Oamb in ovulation with a new mutant allele (OambMI12417), in which a MiMIC vector with a splice acceptor [46] was inserted in the coding intron of Oamb gene to disrupt the correct mRNA splicing (S4 Fig). Females bearing this mutant allele laid significantly fewer eggs and took a much longer time to ovulate an egg (Table 1). We then isolated mature follicles from these females and applied OA stimulation ex vivo. Oamb mutant follicles showed severe defects in OA-induced follicle rupture compared to control follicles (Fig 2A, 2B and 2E). In addition, the Oamb mutation abolished the NE-induced follicle rupture (Fig 2C–2E). The defective response of Oamb mutant follicles to OA/NE stimulation is not likely due to defective OA signaling in the oviduct or other organs, because follicles from TβH or Tdc2 mutant females are fully competent to OA/NE-induced follicle rupture (Fig 2F and 2G). These data indicate that Oamb in mature follicles is likely responsible for OA/NE-induced follicle rupture.


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

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

Follicular Oamb is required for OA/NE-induced follicle rupture.(A-D) Representative images show mature follicles (marked by R44E10>GFP in follicle cells in red) after three-hour culture with 20 μM of OA (A-B) or NE (C-D). Mature follicles are from control (A and C) and Oamb mutant (B and D) females. (E) Quantification of Oamb mutant mature follicles in response to OA or NE stimulation. Four replicates were used for each genotype, except in Oamb-/- group with NE treatment, which has three replicates. (F-G) Quantification of follicle rupture after three-hour OA or NE treatment (20 μM). Mature follicles were derived from TβH (F) or Tdc2 (G) mutant females and marked by 47A04>RFP. All treatments have three replicates except for TβH+/- with NE treatment and Tdc2-/-, which have four replicates. (H-J) Oamb knockdown with R47A04-Gal4 blocks follicle rupture. Representative images show control (H) and OambRNAi1 (I) mature follicles after three-hour culture with 20 μM of OA. Quantification of follicle rupture (J). The number of replicates for each condition in (J) is 3, 3, 3, 4, and 2. (K-M) Oamb knockdown with R44E10-Gal4 blocks follicle rupture induced by OA or NE. Representative images show control (K) and OambRNAi2 (L) mature follicles after a three-hour culture with 20 μM of OA. Quantification of follicle rupture (M). The number of replicates for each condition in (M) is 6, 5, 4, and 3. Student’s T-test was used (*** P<0.001; ** P<0.01; * P<0.05).
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pgen.1005604.g002: Follicular Oamb is required for OA/NE-induced follicle rupture.(A-D) Representative images show mature follicles (marked by R44E10>GFP in follicle cells in red) after three-hour culture with 20 μM of OA (A-B) or NE (C-D). Mature follicles are from control (A and C) and Oamb mutant (B and D) females. (E) Quantification of Oamb mutant mature follicles in response to OA or NE stimulation. Four replicates were used for each genotype, except in Oamb-/- group with NE treatment, which has three replicates. (F-G) Quantification of follicle rupture after three-hour OA or NE treatment (20 μM). Mature follicles were derived from TβH (F) or Tdc2 (G) mutant females and marked by 47A04>RFP. All treatments have three replicates except for TβH+/- with NE treatment and Tdc2-/-, which have four replicates. (H-J) Oamb knockdown with R47A04-Gal4 blocks follicle rupture. Representative images show control (H) and OambRNAi1 (I) mature follicles after three-hour culture with 20 μM of OA. Quantification of follicle rupture (J). The number of replicates for each condition in (J) is 3, 3, 3, 4, and 2. (K-M) Oamb knockdown with R44E10-Gal4 blocks follicle rupture induced by OA or NE. Representative images show control (K) and OambRNAi2 (L) mature follicles after a three-hour culture with 20 μM of OA. Quantification of follicle rupture (M). The number of replicates for each condition in (M) is 6, 5, 4, and 3. Student’s T-test was used (*** P<0.001; ** P<0.01; * P<0.05).
Mentions: To identify the receptor responsible for OA/NE-induced follicle rupture, we focused on Oamb, which is essential for ovulation [24] and is the most highly expressed OA receptor in mature follicles (S1 Fig). We verified the requirement of Oamb in ovulation with a new mutant allele (OambMI12417), in which a MiMIC vector with a splice acceptor [46] was inserted in the coding intron of Oamb gene to disrupt the correct mRNA splicing (S4 Fig). Females bearing this mutant allele laid significantly fewer eggs and took a much longer time to ovulate an egg (Table 1). We then isolated mature follicles from these females and applied OA stimulation ex vivo. Oamb mutant follicles showed severe defects in OA-induced follicle rupture compared to control follicles (Fig 2A, 2B and 2E). In addition, the Oamb mutation abolished the NE-induced follicle rupture (Fig 2C–2E). The defective response of Oamb mutant follicles to OA/NE stimulation is not likely due to defective OA signaling in the oviduct or other organs, because follicles from TβH or Tdc2 mutant females are fully competent to OA/NE-induced follicle rupture (Fig 2F and 2G). These data indicate that Oamb in mature follicles is likely responsible for OA/NE-induced 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