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. 2023 Mar;132(1):1-18.
doi: 10.1007/s00412-023-00784-9. Epub 2023 Jan 17.

A large-scale RNAi screen reveals that mitochondrial function is important for meiotic chromosome organization in oocytes

Karen Jule Nieken  1 , Kathryn O'Brien  1 , Alexander McDonnell  1 , Liudmila Zhaunova  1 , Hiroyuki Ohkura  2
Affiliations

A large-scale RNAi screen reveals that mitochondrial function is important for meiotic chromosome organization in oocytes

Karen Jule Nieken et al. Chromosoma. 2023 Mar.

Abstract

In prophase of the first meiotic division, chromatin forms a compact spherical cluster called the karyosome within the enlarged oocyte nucleus in Drosophila melanogaster. Similar clustering of chromatin has been widely observed in oocytes in many species including humans. It was previously shown that the proper karyosome formation is required for faithful chromosome segregation, but knowledge about its formation and maintenance is limited. To identify genes involved in karyosome formation, we carried out a large-scale cytological screen using Drosophila melanogaster oocytes. This screen comprised 3916 genes expressed in ovaries, of which 106 genes triggered reproducible karyosome defects upon knockdown. The karyosome defects in 24 out of these 106 genes resulted from activation of the meiotic recombination checkpoint, suggesting possible roles in DNA repair or piRNA processing. The other genes identified in this screen include genes with functions linked to chromatin, nuclear envelope, and actin. We also found that silencing of genes with mitochondrial functions, including electron transport chain components, induced a distinct karyosome defect typically with de-clustered chromosomes located close to the nuclear envelope. Furthermore, mitochondrial dysfunction not only impairs karyosome formation and maintenance, but also delays synaptonemal complex disassembly in cells not destined to become the oocyte. These karyosome defects do not appear to be mediated by apoptosis. This large-scale unbiased study uncovered a set of genes required for karyosome formation and revealed a new link between mitochondrial dysfunction and chromatin organization in oocytes.

Keywords: Chromatin; Drosophila; Meiosis; Mitochondria; Oocytes.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
A large-scale screen identified 106 genes required for the integrity of the karyosome. A A stage-5 egg chamber from a control ovary stained for Lamin and DNA. The arrowhead indicates the karyosome, a compact spherical cluster of meiotic chromosomes, in the oocyte nucleus. Other nuclei belong to nurse cells and follicle cells. Bar = 10 μm. B The number of genes selected for the screen. C The karyosome screen workflow. D Summary results of the screen. Among 3916 genes screened, 106 genes showed reproducible karyosome abnormalities when they are silenced by RNAi, while 569 genes showed severe oogenesis defects which prevented examination of the karyosome. E The frequencies of genes with severe oogenesis defects and karyosome abnormalities when silenced, according to expression levels in ovaries. Bins 2, 3, 4, 5, 6, and 7 of the ovary expression level represent genes with 4–10, 11–25, 26–50, 51–100, 101–1000, > 1000 kb−1 million−1 from RNAseq (Mortazavi et al. ; Celniker et al. 2009). ***Significant differences (p < 0.001) in the frequency of genes with severe oogenesis defects in comparison to bin 2. F The frequencies of genes with karyosome abnormalities, excluding genes with severe oogenesis defects that prevented examination of karyosomes, according to different expression levels in ovaries. *Significant differences (p < 0.05) in the frequencies of genes with severe oogenesis defects in comparison to bin 2. ns no significant differences (p > 0.05). G The frequencies of genes resulting in different ovary sizes when silenced, in relation to female fertility. ***Significant differences (p < 0.001) in the frequencies of genes resulting in no or tiny ovaries in comparison to genes showing female fertility. H The frequencies of genes with karyosome abnormalities in relation to female fertility. The two graphs show the frequencies including or excluding genes resulting in no/tiny ovaries, which prevented examination of karyosomes. ***Significant differences (p < 0.001) in the frequencies of genes with abnormal karyosomes in comparison to genes showing female fertility. I The frequencies of genes with karyosome abnormalities according to their ovary size. No/tiny ovaries prevented examination of karyosomes. ***Significant differences (p < 0.001) in the frequencies of genes with abnormal karyosomes in comparison to genes with normal ovaries
Fig. 2
Fig. 2
The 106 genes required for the karyosome are highly interconnected and include genes regulating chromatin, nuclear envelope, and actin. A The physical and/or functional interaction network among the 106 hits. Each node represents a gene identified in this screen and are coloured according to an associated key word indicated in the box. Each line represents a physical and/or functional interaction between two genes in the STRING database. B The numbers of interactions among random 106 genes. One thousand random sets of 106 genes were selected from 3356 genes examined in the screen, and the numbers of the physical and/or functional interactions were plotted. 198 interactions found among the 106 genes identified in the screen is much higher than expected from a random set of 106 genes
Fig. 3
Fig. 3
A list of the 106 genes important for the karyosome identified in the screen. The names of the 106 genes identified in the screen and their orthologue are shown with short summaries. They were grouped under common key words. Twenty-four genes are separately listed, as their karyosome defects caused by gene silencing are dependent on the meiotic checkpoint
Fig. 4
Fig. 4
The karyosome defects of 24 genes are mediated by the meiotic recombination checkpoint. A A diagram of the meiotic recombination checkpoint that induces the karyosome defects in response to persistent DNA double strand breaks (DSBs). Persistent DSBs can be caused by failure of DNA repair or retrotransposition due to failure of piRNA-mediated silencing. B A summary result of rescue experiments by mnk/chk2. C The karyosome morphology in an oocyte in which a gene implicated in DNA repair was silenced by RNAi in the presence (+ mnk) or absence of a heterozygous mnk/chk2 mutation. Bar = 2 μm. D The graph represents the frequencies of the karyosome morphologies in oocytes in which each gene involved in DNA double strand break (DSB) repair was silenced by RNAi in the presence (+ mnk) or absence of a heterozygous mnk/chk2 mutation. E The karyosome morphology in an oocyte in which a gene implicated in piRNA processing was silenced by RNAi. Bar = 2 μm. F The graph represents the frequencies of the karyosome morphologies in an oocyte in which each gene involved in piRNA processing was silenced by RNAi. G Immunostaining of stage-4 oocyte using an antibody against γH2Av which marks DSBs. Bar = 2 μm. H The frequencies of stage 3–6 oocytes with γH2Av foci. ***Significant differences (p < 0.001) in the frequencies of oocytes with γH2Av foci in comparison to the control RNAi
Fig. 5
Fig. 5
Mitochondrial dysfunction leads to distinct karyosome defects. A The numbers of genes showing predominantly attached, predominantly distorted, and only distorted karyosome morphologies upon silencing. B Immunostained nuclei of stage-6 oocytes expressing control and ND-B22 shRNAs using a Lamin antibody and the DNA probe DAPI. RNAi of ND-B22 gene encoding a mitochondria protein predominantly shows an “attached” karyosome morphology, in which meiotic chromosomes, often three masses, are located in proximity to the nuclear envelope. Bar = 2 μm. C Genes showing predominantly attached karyosome morphologies upon silencing. Twelve out of 14 genes in this category encode proteins with roles in mitochondria. D Rescue of the karyosome defects of ND-B22 or l(2)37Bb RNAi by an RNAi-resistant wild-type ND-B22 or l(2)37Bb transgene, respectively. ***Significant differences (p < 0.001) in the frequencies of oocytes with abnormal karyosomes in comparison to the control RNAi. ns; no significant differences (p > 0.05)
Fig. 6
Fig. 6
Silencing of ND-B22 encoding a mitochondrial protein has multiple phenotypic consequences in female meiosis. A Fluorescence in situ hybridisation of the karyosome using peri-centromeric satellites of chromosomes 2 and 3 (cen2 and cen3) in oocytes with control RNAi and ND-B22 RNAi. B The distances between cen2 signals or between cen3 signals. They are categorized into three groups based on the distances. C The distance between cen2 and cen3 signals. The error bars represent the standard errors of the means. ***p < 0.001. **p < 0.01. D The karyosome morphologies in different stages of oocytes expressing shRNA against ND-B22 using an early or late driver. Ovaries were immunostained using a Lamin antibody and DAPI. Bar = 2 μm. E Frequencies of the karyosome morphologies in different stages of oocytes expressing shRNA against ND-B22 using an “early” (nos-Gal4) or “late” (mat-α-tubulin) driver. F The frequency of meiotic cells containing foci of γH2Av, a DSB marker, in various stages of ND-B22 RNAi ovaries. G A progenitor cell undergoes four mitotic divisions to generate 16 interconnected cells. The synaptonemal complex is formed in four cells and completes disassembling in two of the cells first and then in the third cells. The remaining cell (the oocyte) gradually disassembles the synaptonemal complex in later stages. H Morphologies of the synaptonemal complex (C(3)G) in four meiotic nuclei in various stages of oogenesis. The nucleus with the most well formed synaptonemal complex is assigned as nucleus 1, and the nucleus with second, third, and fourth most well formed are assigned as nucleus 2, 3, and 4, respectively
Fig. 7
Fig. 7
Karyosome defects caused by mitochondria dysfunction is not mediated by apoptosis. A Frequencies of genes with different mitochondrial functions that show karyosome defects upon silencing. ** and *Significant differences (p < 0.01 and 0.05, respectively) in the frequency of genes with abnormal karyosomes, in comparison to the other genes with mitochondrial functions. B Immunostaining of apoptotic stage-8 egg chambers in wild-type oocytes after 24 h of starvation, along with healthy stage-8 egg chambers without starvation. All chromatin in ND-B22 RNAi and wild type starved for 24 h shows abnormal morphology associated with apoptosis in this figure, except in the follicle cells. Some examples are indicated by the arrowheads. Bar = 10 μm. C Frequencies of apoptosis and karyosome morphologies in stage-8 egg chambers in wild-type oocytes after different lengths of starvation, along with in ND-B22 RNAi
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