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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 adrenergic signaling is required for ovulation and follicle cell trimming in vivo.(A-C) Egg laying (A), mature follicles in ovary (B), and the average ovulation and uterus time (C) is shown for control females or those expressing OambRNAi in mature follicle cells driven by R44E10-Gal4. Student’s T-test was used (A-B; *** P<0.001; **P<0.01; * P<0.05). (D-F) Follicle cell trimming is significantly reduced when follicular Oamb is knocked down by R44E10-Gal4 driving OambRNAi1 expression (44E10>OambRNAi1). Representative images show trimmed follicles in control (D) but not Oamb-knockdown (E) ovaries. Trimmed follicles are outlined with dashed yellow lines, and the posterior leading edge of the follicle-cell layer is marked by a straight red line. Quantification of trimmed follicles (F). (G-L) Follicle cell trimming is also significantly reduced in TβH (G-I) or Tdc2 (J-L) mutant females. See Tables 1 and 2 for the number of females analyzed and statistics.
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pgen.1005604.g003: Follicular adrenergic signaling is required for ovulation and follicle cell trimming in vivo.(A-C) Egg laying (A), mature follicles in ovary (B), and the average ovulation and uterus time (C) is shown for control females or those expressing OambRNAi in mature follicle cells driven by R44E10-Gal4. Student’s T-test was used (A-B; *** P<0.001; **P<0.01; * P<0.05). (D-F) Follicle cell trimming is significantly reduced when follicular Oamb is knocked down by R44E10-Gal4 driving OambRNAi1 expression (44E10>OambRNAi1). Representative images show trimmed follicles in control (D) but not Oamb-knockdown (E) ovaries. Trimmed follicles are outlined with dashed yellow lines, and the posterior leading edge of the follicle-cell layer is marked by a straight red line. Quantification of trimmed follicles (F). (G-L) Follicle cell trimming is also significantly reduced in TβH (G-I) or Tdc2 (J-L) mutant females. See Tables 1 and 2 for the number of females analyzed and statistics.

Mentions: To determine whether follicular adrenergic signaling is required for ovulation in vivo, we first analyzed the fecundity of females lacking follicular Oamb. Follicular Oamb-knockdown females with either R47A04-Gal4 or R44E10-Gal4 drivers laid significantly fewer eggs than control flies (Fig 3A and Table 1). The egg-laying defect is not caused by oogenesis problems, as mature follicles are abundant in these ovaries. In fact, Oamb-knockdown flies generally had more mature follicles in their ovaries (Fig 3B), indicating an ovulation defect. Indeed, Oamb-knockdown flies had a much longer ovulation time compared to control flies but did not show defects in transporting ovulated eggs into the uterus or ejecting them out of the uterus (Fig 3C and Table 1). These data strongly suggest that follicular Oamb is required for ovulation in vivo.


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

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

Follicular adrenergic signaling is required for ovulation and follicle cell trimming in vivo.(A-C) Egg laying (A), mature follicles in ovary (B), and the average ovulation and uterus time (C) is shown for control females or those expressing OambRNAi in mature follicle cells driven by R44E10-Gal4. Student’s T-test was used (A-B; *** P<0.001; **P<0.01; * P<0.05). (D-F) Follicle cell trimming is significantly reduced when follicular Oamb is knocked down by R44E10-Gal4 driving OambRNAi1 expression (44E10>OambRNAi1). Representative images show trimmed follicles in control (D) but not Oamb-knockdown (E) ovaries. Trimmed follicles are outlined with dashed yellow lines, and the posterior leading edge of the follicle-cell layer is marked by a straight red line. Quantification of trimmed follicles (F). (G-L) Follicle cell trimming is also significantly reduced in TβH (G-I) or Tdc2 (J-L) mutant females. See Tables 1 and 2 for the number of females analyzed and statistics.
© Copyright Policy
Related In: Results  -  Collection

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

pgen.1005604.g003: Follicular adrenergic signaling is required for ovulation and follicle cell trimming in vivo.(A-C) Egg laying (A), mature follicles in ovary (B), and the average ovulation and uterus time (C) is shown for control females or those expressing OambRNAi in mature follicle cells driven by R44E10-Gal4. Student’s T-test was used (A-B; *** P<0.001; **P<0.01; * P<0.05). (D-F) Follicle cell trimming is significantly reduced when follicular Oamb is knocked down by R44E10-Gal4 driving OambRNAi1 expression (44E10>OambRNAi1). Representative images show trimmed follicles in control (D) but not Oamb-knockdown (E) ovaries. Trimmed follicles are outlined with dashed yellow lines, and the posterior leading edge of the follicle-cell layer is marked by a straight red line. Quantification of trimmed follicles (F). (G-L) Follicle cell trimming is also significantly reduced in TβH (G-I) or Tdc2 (J-L) mutant females. See Tables 1 and 2 for the number of females analyzed and statistics.
Mentions: To determine whether follicular adrenergic signaling is required for ovulation in vivo, we first analyzed the fecundity of females lacking follicular Oamb. Follicular Oamb-knockdown females with either R47A04-Gal4 or R44E10-Gal4 drivers laid significantly fewer eggs than control flies (Fig 3A and Table 1). The egg-laying defect is not caused by oogenesis problems, as mature follicles are abundant in these ovaries. In fact, Oamb-knockdown flies generally had more mature follicles in their ovaries (Fig 3B), indicating an ovulation defect. Indeed, Oamb-knockdown flies had a much longer ovulation time compared to control flies but did not show defects in transporting ovulated eggs into the uterus or ejecting them out of the uterus (Fig 3C and Table 1). These data strongly suggest that follicular Oamb is required for ovulation in vivo.

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