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β carbonic anhydrase is required for female fertility in Drosophila melanogaster.

Syrjänen L, Valanne S, Kuuslahti M, Tuomela T, Sriram A, Sanz A, Jacobs HT, Rämet M, Parkkila S - Front. Zool. (2015)

Bottom Line: CAs are present as six structurally divergent enzyme families: α, β, γ, δ, ζ and η. β-CAs have a wide distribution across different species including invertebrates.Whole ovaries of knockdown females looked normal but upon cadherin staining, there was an apparent functional defect in migration of border cells, which are considered essential for normal fertilization.These results indicate that although Drosophila β-CA is dispensable for survival of adult flies, it is essential for female fertility.

View Article: PubMed Central - PubMed

Affiliation: BioMediTech, University of Tampere, Tampere, Finland ; School of Medicine, University of Tampere and Tampere University Hospital, Tampere, Finland ; Department of Otorhinolaryngology, Central Finland Central Hospital, Jyväskylä, Finland.

ABSTRACT

Background: Carbonic anhydrases (CAs, EC 4.2.1.1) are ubiquitous enzymes that catalyze the reversible hydration reaction of carbon dioxide. CAs are present as six structurally divergent enzyme families: α, β, γ, δ, ζ and η. β-CAs have a wide distribution across different species including invertebrates. Previously, we showed that Drosophila melanogaster β-CA is a highly active mitochondrial enzyme. In this study, we investigated the function of Drosophila β-CA by silencing the expression of the β-CA gene using UAS/GAL4-based RNA interference (RNAi) in Drosophila in vivo.

Results: Crossing β-CA RNAi lines over ubiquitous Actin driver flies did not produce any viable progeny, indicating that β-CA expression is required for fly development. RNAi silencing of β-CA ubiquitously in adult flies did not affect their survival rate or function of mitochondrial electron transport chain. Importantly, β-CA RNAi led to impaired reproduction. All β-CA knockdown females were sterile, and produced few or no eggs. Whole ovaries of knockdown females looked normal but upon cadherin staining, there was an apparent functional defect in migration of border cells, which are considered essential for normal fertilization.

Conclusions: These results indicate that although Drosophila β-CA is dispensable for survival of adult flies, it is essential for female fertility.

No MeSH data available.


Related in: MedlinePlus

β-CA RNAi does not alter mitochondrial function in fruit flies. a Mitochondrial oxygen consumption (nmol O2/s mg protein) from female and male Drosophila flies using P + Pr + ADP (CI + III + IV), G3P (CIII + IV) and Ascorbate + TMPD (CIV) as substrates. Number of experiments (n) per group is indicated, data are shown as the mean ± SEM. b) Mitochondrial density measured as citrate synthase activity (UNITS mg/ml of mitochondrial protein) shown as the mean ± SEM from 8 replicate experiments. ADP - Adenosine diphosphate; G3P - Glyceraldehyde 3-phosphate; P – pyruvate; Pr – proline; TMPD - N,N,N',N'-Tetramethyl-p-Phenylenediamine dichloride
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Fig3: β-CA RNAi does not alter mitochondrial function in fruit flies. a Mitochondrial oxygen consumption (nmol O2/s mg protein) from female and male Drosophila flies using P + Pr + ADP (CI + III + IV), G3P (CIII + IV) and Ascorbate + TMPD (CIV) as substrates. Number of experiments (n) per group is indicated, data are shown as the mean ± SEM. b) Mitochondrial density measured as citrate synthase activity (UNITS mg/ml of mitochondrial protein) shown as the mean ± SEM from 8 replicate experiments. ADP - Adenosine diphosphate; G3P - Glyceraldehyde 3-phosphate; P – pyruvate; Pr – proline; TMPD - N,N,N',N'-Tetramethyl-p-Phenylenediamine dichloride

Mentions: Since we have shown earlier that β-CA is a mitochondrial protein [5], we decided to assess whether silencing of β-CA gene expression affects mitochondrial oxygen consumption. Upon knockdown of β-CA we observed a female sterile phenotype (Additional file 1: Table S1); it has been recently shown that mutations in mitochondrial proteins cause general sterility in flies by dysfunction of the electron transport chain (ETC.) [9]. Moreover, complementation of the respiratory phenotype can rescue this and other associated phenotypes [9]. Using high resolution respirometry [10] we measured if mitochondrial respiration in whole fly preparations was affected upon knockdown of the β-CA gene. We did not observe any significant differences using complex I- (pyruvate + proline), complex III- (sn-glycerol-3-phosphate) or complex IV-linked (ascorbate + TMPD) substrates in the presence of ADP (Fig. 3a). Additionally, mitochondrial density, measured by the citrate synthase assay, was not altered by β-CA RNAi (Fig. 3b). In conclusion, ubiquitous silencing of the β-CA gene in adult flies did not affect the mitochondrial oxygen consumption in whole flies.Fig. 3


β carbonic anhydrase is required for female fertility in Drosophila melanogaster.

Syrjänen L, Valanne S, Kuuslahti M, Tuomela T, Sriram A, Sanz A, Jacobs HT, Rämet M, Parkkila S - Front. Zool. (2015)

β-CA RNAi does not alter mitochondrial function in fruit flies. a Mitochondrial oxygen consumption (nmol O2/s mg protein) from female and male Drosophila flies using P + Pr + ADP (CI + III + IV), G3P (CIII + IV) and Ascorbate + TMPD (CIV) as substrates. Number of experiments (n) per group is indicated, data are shown as the mean ± SEM. b) Mitochondrial density measured as citrate synthase activity (UNITS mg/ml of mitochondrial protein) shown as the mean ± SEM from 8 replicate experiments. ADP - Adenosine diphosphate; G3P - Glyceraldehyde 3-phosphate; P – pyruvate; Pr – proline; TMPD - N,N,N',N'-Tetramethyl-p-Phenylenediamine dichloride
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4546311&req=5

Fig3: β-CA RNAi does not alter mitochondrial function in fruit flies. a Mitochondrial oxygen consumption (nmol O2/s mg protein) from female and male Drosophila flies using P + Pr + ADP (CI + III + IV), G3P (CIII + IV) and Ascorbate + TMPD (CIV) as substrates. Number of experiments (n) per group is indicated, data are shown as the mean ± SEM. b) Mitochondrial density measured as citrate synthase activity (UNITS mg/ml of mitochondrial protein) shown as the mean ± SEM from 8 replicate experiments. ADP - Adenosine diphosphate; G3P - Glyceraldehyde 3-phosphate; P – pyruvate; Pr – proline; TMPD - N,N,N',N'-Tetramethyl-p-Phenylenediamine dichloride
Mentions: Since we have shown earlier that β-CA is a mitochondrial protein [5], we decided to assess whether silencing of β-CA gene expression affects mitochondrial oxygen consumption. Upon knockdown of β-CA we observed a female sterile phenotype (Additional file 1: Table S1); it has been recently shown that mutations in mitochondrial proteins cause general sterility in flies by dysfunction of the electron transport chain (ETC.) [9]. Moreover, complementation of the respiratory phenotype can rescue this and other associated phenotypes [9]. Using high resolution respirometry [10] we measured if mitochondrial respiration in whole fly preparations was affected upon knockdown of the β-CA gene. We did not observe any significant differences using complex I- (pyruvate + proline), complex III- (sn-glycerol-3-phosphate) or complex IV-linked (ascorbate + TMPD) substrates in the presence of ADP (Fig. 3a). Additionally, mitochondrial density, measured by the citrate synthase assay, was not altered by β-CA RNAi (Fig. 3b). In conclusion, ubiquitous silencing of the β-CA gene in adult flies did not affect the mitochondrial oxygen consumption in whole flies.Fig. 3

Bottom Line: CAs are present as six structurally divergent enzyme families: α, β, γ, δ, ζ and η. β-CAs have a wide distribution across different species including invertebrates.Whole ovaries of knockdown females looked normal but upon cadherin staining, there was an apparent functional defect in migration of border cells, which are considered essential for normal fertilization.These results indicate that although Drosophila β-CA is dispensable for survival of adult flies, it is essential for female fertility.

View Article: PubMed Central - PubMed

Affiliation: BioMediTech, University of Tampere, Tampere, Finland ; School of Medicine, University of Tampere and Tampere University Hospital, Tampere, Finland ; Department of Otorhinolaryngology, Central Finland Central Hospital, Jyväskylä, Finland.

ABSTRACT

Background: Carbonic anhydrases (CAs, EC 4.2.1.1) are ubiquitous enzymes that catalyze the reversible hydration reaction of carbon dioxide. CAs are present as six structurally divergent enzyme families: α, β, γ, δ, ζ and η. β-CAs have a wide distribution across different species including invertebrates. Previously, we showed that Drosophila melanogaster β-CA is a highly active mitochondrial enzyme. In this study, we investigated the function of Drosophila β-CA by silencing the expression of the β-CA gene using UAS/GAL4-based RNA interference (RNAi) in Drosophila in vivo.

Results: Crossing β-CA RNAi lines over ubiquitous Actin driver flies did not produce any viable progeny, indicating that β-CA expression is required for fly development. RNAi silencing of β-CA ubiquitously in adult flies did not affect their survival rate or function of mitochondrial electron transport chain. Importantly, β-CA RNAi led to impaired reproduction. All β-CA knockdown females were sterile, and produced few or no eggs. Whole ovaries of knockdown females looked normal but upon cadherin staining, there was an apparent functional defect in migration of border cells, which are considered essential for normal fertilization.

Conclusions: These results indicate that although Drosophila β-CA is dispensable for survival of adult flies, it is essential for female fertility.

No MeSH data available.


Related in: MedlinePlus