COX4-like, a Nuclear-Encoded Mitochondrial Gene Duplicate, Is Essential for Male Fertility in Drosophila melanogaster

Abstract

Recent studies on nuclear-encoded mitochondrial genes (N-mt genes) in Drosophila melanogaster have shown a unique pattern of expression for newly duplicated N-mt genes, with many duplicates having a testis-biased expression and playing an essential role in spermatogenesis. In this study, we investigated a newly duplicated N-mt gene-i.e., Cytochrome c oxidase 4-like (COX4L)-in order to understand its function and, consequently, the reason behind its retention in the D. melanogaster genome. The COX4L gene is a duplicate of the Cytochrome c oxidase 4 (COX4) gene of OXPHOS complex IV. While the parental COX4 gene has been found in all eukaryotes, including single-cell eukaryotes such as yeast, we show that COX4L is only present in the Brachycera suborder of Diptera; thus, both genes are present in all Drosophila species, but have significantly different patterns of expression: COX4 is highly expressed in all tissues, while COX4L has a testis-specific expression. To understand the function of this new gene, we first knocked down its expression in the D. melanogaster germline using two different RNAi lines driven by the bam-Gal4 driver; second, we created a knockout strain for this gene using CRISPR-Cas9 technology. Our results showed that knockdown and knockout lines of COX4L produce partial sterility and complete sterility in males, respectively, where a lack of sperm individualization was observed in both cases. Male infertility was prevented by driving COX4L-HA in the germline, but not when driving COX4-HA. In addition, ectopic expression of COX4L in the soma caused embryonic lethality, while overexpression in the germline led to a reduction in male fertility. COX4L-KO mitochondria show reduced membrane potential, providing a plausible explanation for the male sterility observed in these flies. This prominent loss-of-function phenotype, along with its testis-biased expression and its presence in the Drosophila sperm proteome, suggests that COX4L is a paralogous, specialized gene that is assembled in OXPHOS complex IV of male germline cells and/or sperm mitochondria.

Keywords: COX4L; CRISPR knockout; Drosophila melanogaster; gene duplication; nuclear-encoded mitochondrial gene; spermatogenesis.

Figure 1

Maximum likelihood tree of COX4 and COX4L, using amino acid sequences. Multiple alignments of protein sequences were performed using ClustalW [57] implemented in Geneious (Version 2020.1) [58]. The maximum likelihood phylogenetic tree was constructed using the BlOSUM62 substitution model with 100 bootstrap branch support in PhyML [52], applied in Geneious. Only bootstrap support values ≥ 50 are shown. We used FigTree (Version 1.4.4) (http://tree.bio.ed.ac.uk/software/figtree, accessed on 30 April 2021) to visualize all phylogenies.

Figure 2

(A) Fertility study of COX4L in males. The knockdown of COX4L with bam-Gal4 was semi-fertile, while the COX4-KO males were completely sterile. The COX4L-KO fertility phenotype was completely rescued by bam-Gal4-COX4L-ORF and nos-Gal4-COX4L-ORF (p-Value = 0.73 (nos-Gal4) and p-Value = 0.63 (bam-Gal4), respectively), but not with bam-Gal4-COX4-ORF. (B) Fertility study of COX4L in females. The overexpression of COX4L in the soma did not show any viability effect in females. (C) The overexpression of COX4L with Act5C-Gal4 in the soma showed complete lethality at the embryo stage. (D) The overexpression of COX4L with bam-Gal4 in the germline does not affect female fertility; it does, however, cause significant fertility reduction in males. (*) Means significantly different from the line of control (w¹¹¹⁸) in a t-test (p < 0.05).

Figure 3

Dissected testes of COX4L-KO: (A) Dissected testes of COX4L-KO with an empty seminal vesicle are shown. (B) Sperm bundle with individualization defect. (C) w¹¹¹⁸ control testis with normal spermatogenesis stages and a full seminal vesicle. Additionally, mature sperms are shown moving around the raptured seminal vesicle. (D) Wild-type mobile individualized sperm.

Figure 4

One-day-old male testes of (A,B) w¹¹¹⁸ and (C,D) COX4L-KO stained with MitoTracker™ Deep Red FM and DAPI. Sperm bundles stained with MitoTracker in (B) COX4L-KO show less fluorescence compared to (D) w¹¹¹⁸, due to reduced mitochondrial membrane potential and/or mitochondrial morphology changes (Supplementary Figure S2).