Transgenic expression of full-length tafazzin restores male fertility and the normal cardiolipin profile of TAZ-deletion mutant flies. (A) Male fertility of the tafazzin mutant flies without (TAZ−/−) or with (TAZ−/−; TG-TAZ) the transgenic expression of tafazzin gene driven by GAL4 relative to the fertility of wild-type flies (WT). Data from 3 independent experiments. (B) Fluorescence HPLC cardiolipin profiles from WT, TAZ−/−, and (TAZ−/−; TG-TAZ) flies. The fluorescence yield (ordinate) is plotted against retention time (abscissa). In normal Drosophila, because of cardiolipin remodeling, peak 2, which contains a mixture of cardiolipin molecules with palmitoleoyl (16:1) and linoleoyl (18:2) residues (see ref. 25), is the dominant component. This peak is markedly reduced in the TAZ−/− mutant and is restored to nearly normal levels in the transgenic flies.

Cross-section electron micrographs through preindividualized (A and B) and mature (C and D) portions of spermatogenic cysts from wild-type (A and C) and TAZ deletion mutant (B and D) testes. Preindividualized spermatogenic cysts are similar in TAZ−/− (B) and wild-type (A) testes. These cysts are identifiable by their light-staining major mitochondrial derivatives (big arrow), in which an electron-dense material accumulates, enlarged minor mitochondrial derivatives (small arrow), and the presence of extensive cytoplasm between the axonemes (arrow head). After individualization, wild-type cysts contain highly ordered arrays of elongated spermatids that have undergone a significant reduction in the amount of cytoplasm that can be seen between the sperm tails (C). TAZ mutant cysts fail to undergo proper individualization and in them spermatid arrangements are grossly disorganized (D). (Scale bar, 1 μm.)

The P element insertion in EY05103 inactivates the gene CG6718 encoding the calcium-independent phospholipase A2, iPLA2VIA. (A) The Drosophila genomic region containing the gene (top bar, adapted from Flybase). The Flybase annotation indicates that CG6718, through alternative splicing, produces 4 mRNA transcripts (RA, RB, RC, and RD) that encode 2 protein products with different N termini (PA and that of PB, PC, or PD). The position of the EY05103 P element insertion is indicated by an open triangle above the genomic region. Two arrowheads below the genomic region and above the mRNAs schematics indicate the positions of the primers used for RT-PCR. (B) Agarose gel electrophoresis of the products generated by RT-PCR from the CG6718-mRNAs in wild type (WT) and the EY05103 (EY) fly strain. Lane EY, homozygous EY05103 insertion strain; lane WT, w¹¹¹⁸ strain; lane M, DNA size marker. RA, RB, RC, and RD correspond to the CG6718 transcripts annotated in the Flybase. The asterisk indicates a new alternatively spliced transcript not yet annotated in the Flybase. Control indicates the amplified transcript fragment of an irrelevant gene (CG34133) as an internal control. The identity of each fragment was confirmed by DNA sequencing.

Inactivation of iPLA₂ suppresses the male-sterile phenotype of tafazzin-deficient flies and partially restores the MLCL/CL ratio. (A) Relative male fertilities of w¹¹¹⁸ (WT), tafazzin mutant (TAZ−/−), EY05103 P element insertion strain (iPLA₂−/−), double mutant (TAZ−/−; iPLA₂−/−) and the precise P element excision strain [TAZ−/−; EY(−)]. That the excision in the TAZ−/−; EY(−) strain was precise and restored the normal iPLA₂ gene was determined by PCR amplification of the relevant chromosomal region and DNA sequencing through the previous insertion site. The male fertility of WT flies is defined as 100%. Data from 3 independent experiments were obtained. (B) Fluorescence HPLC profiles of MLCL and CL isolated from WT, iPLA₂−/−, TAZ−/−, and the double-mutant flies. The fluorescence yield (ordinate) is plotted against retention time (abscissa). The vertical dotted lines separate the regions with peaks representing CL (to the right) from those with peaks corresponding to monolyso-CL species (to the left). The arrows indicate the dominant CL peak containing mature cardiolipin molecules with palmitoleoyl (16:1) and linoleoyl (18:2) residues due to remodeling (25). (C) Quantification of the MLCL/CL ratio in WT, iPLA₂−/−, TAZ−/−, and double-mutant flies. Data are from the profiles obtained from 4 independent experiments. (D) Treatment of BTHS lymphoblasts with an iPLA2 inhibitor (BEL) partially restores their MLCL/CL ratio. The lymphoblast cultures were treated with 2.5 μM bromoenol lactone (BEL) for 48 h and their MLCL and CL contents were determined by fluorescence HPLC. The data are presented as the average obtained from 2 BTHS lymphoblast cell lines and 2 normal control (NC) lymphoblast cell lines.