Barriers to male transmission of mitochondrial DNA in sperm development - PubMed (original) (raw)

Barriers to male transmission of mitochondrial DNA in sperm development

Steven Z DeLuca et al. Dev Cell. 2012.

Abstract

Across the eukaryotic phylogeny, offspring usually inherit their mitochondrial genome from only one of two parents: in animals, the female. Although mechanisms that eliminate paternally derived mitochondria from the zygote have been sought, the developmental stage at which paternal transmission of mitochondrial DNA is restricted is unknown in most animals. Here, we show that the mitochondria of mature Drosophila sperm lack DNA, and we uncover two processes that eliminate mitochondrial DNA during spermatogenesis. Visualization of mitochondrial DNA nucleoids revealed their abrupt disappearance from developing spermatids in a process requiring the mitochondrial nuclease, Endonuclease G. In Endonuclease G mutants, persisting nucleoids are swept out of spermatids by a cellular remodeling process that trims and shapes spermatid tails. Our results show that mitochondrial DNA is eliminated during spermatogenesis, thereby removing the capacity of sperm to transmit the mitochondrial genome to the next generation.

Copyright © 2012 Elsevier Inc. All rights reserved.

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Figures

Figure 1

Figure 1. Paternal mtDNA is Excluded from Mature Sperm and Fertilized Embryos

(A) Sequence of nucleotides deleted in mt:ND2del1 (yellow highlight), and the BgIII restriction site (red font) that was targeted in the generation of mt:ND2del1. Arrows show the 3' portion of PCR primers that anneal to wild type (mt:ND2), but not mutant (mt:ND2del1) mtDNA. (B) Cross scheme and experimental design. (C) PCR genotyping of parental lines that were crossed to follow wild type paternal mtDNA in (D,E). (D) qPCR measuring paternal mtDNA (mt:ND2) transferred to deletion mutant eggs (mt:ND2del1). The mean paternal mtDNA copy number (red dot) in relation to a standard curve (blue dots) is graphed on a “per embryo” basis. Error bar represents standard deviation in three embyro extracts. (E) qPCR measuring the amount of mtDNA (mt:ND2) in sperm stored by mt:ND2del1 females. Y chromosome (red a triangle) and sperm mtDNA (red circle) copy number in representative sperm storage organ extract in relation to standard curves (blue) are graphed on a “per female” basis. In (D,E), standard curves were generated by adding 100 to 12500 copies of mt:ND2 (blue circles) or Y chromosomal DNA (blue triangles) to control extracts, and graphed on a “per embryo” or “per female” basis. See also Fig. S1.

Figure 2

Figure 2. mtDNA Nucleoids Disappear During Spermatogenesis

(A) Schematic summary of post-meiotic sperm development depicting nuclei (green), mitochondria (red), and investment cones (purple). (B) Onion stage spermatids stained for DNA (PicoGreen: green), and mitochondria (mito-YFP: red). Each nucleus (n) is paired with a large mitochondrial structure, the nebenkern (mt). (C–E) Elongating spermatid bundles stained for DNA (PicoGreen: green) and actin (phalloidin: purple). (C) Elongation R stage bundle, 560 μm long. A cluster of normal spermatid nuclei (n), apically discarded spermatid nuclei (n'), and a somatic cell nucleus belonging to a cyst cell (n”) form large staining foci. mtDNA nucleoids (magnified in inset) form much smaller foci throughout the tail bundle. (D,E) Two late elongation stage bundles, 1729 μm (D) and 1747 μm (E) long. Numbers indicate distance (μm) of image from basal tip of bundle. Scale bars are 10 μm. (F) Nucleoid density (number of nucleoids/μm) along the lengths of 4 representative spermatid bundles (each bundle is a different colored line) of the indicated length (μm). (G) Average number of nucleoids per onion stage spermatid (O), elongating spermatid < 1700 μm (E1), elongating spermatid 1700–1800 μm (E2), and elongating spermatid > 1800 μm (E3). Error bars indicate standard deviation in a least 3 cysts. See also Fig. S2.

Figure 3

Figure 3. mtDNA Nucleoid Elimination During Spermatid Elongation Requires EndoG

(A) Schematic showing the approximate location of the MB07150 minos transposon insertion (red triangle) in relation to the catalytic residues of EndoG (yellow oval). (B) Quantitative RT-PCR measuring EndoG mRNA abundance in adult male flies. The approximate positions of the 3 primer sets used to measure the abundance of different regions of EndoG mRNA are diagramed in (A). Error bars represent standard deviation in 3 extracts. (C–E) Spermatid bundles stained for DNA (green), and actin (purple). (C) Late elongation stage EndoGMB07150 mutant bundle, 1830 μm long. Numbers indicate distance (μm) of image from basal tip of bundle. (D) Cystic bulge of spermatid bundles of the indicated genotype during spermatid individualization. Numbers indicate % of the length of the bundle that the cystic bulge has traveled. Rare spermatid nuclei (n) culled during the individualization process. (E) Elongation stage EndoGI mutant bundle, 1280 μm long. Numbers indicate distance (μm) of image from basal tip of bundle. Scale bars are 10 μm. See also Fig. S3.

Figure 4

Figure 4. Residual mtDNA Nucleoids are Removed from EndoG Mutant Sperm During Spermatid Individualization

(A–A”,B–B”) Two individualizing sperm-tail bundles stained for DNA (green) and actin (purple). EndoGMB07150/+ (A–A”) and EndoGMB07150/Df (B–B”) bundles imaged ahead of the cystic bulge (A”, B”), at the cystic bulge (A', B') and behind the cystic bulge (A, B). Arrowheads indicate examples of mtDNA nucleoids. Scale bars are 10 μm. (C) Number of nucleoids in cystic bulges at different stages of individualization in bundles from control and EndoG mutants. (D) Focal section through a wild type cystic bulge stained for mitochondria (DJ–GFP: red) and actin (purple). (E) Schematic showing early mtDNA elimination in wild type and late mtDNA elimination in EndoG mutant spermatids; DNA (green), mitochondria (red), and investment cones (purple).

Comment in

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