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Molecular basis for maternal inheritance of human mitochondrial DNA

Nature Genetics volume 55, pages 1632–1639 (2023)Cite this article

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Abstract

Uniparental inheritance of mitochondrial DNA (mtDNA) is an evolutionary trait found in nearly all eukaryotes. In many species, including humans, the sperm mitochondria are introduced to the oocyte during fertilization1,2. The mechanisms hypothesized to prevent paternal mtDNA transmission include ubiquitination of the sperm mitochondria and mitophagy3,4. However, the causative mechanisms of paternal mtDNA elimination have not been defined5,6. We found that mitochondria in human spermatozoa are devoid of intact mtDNA and lack mitochondrial transcription factor A (TFAM)—the major nucleoid protein required to protect, maintain and transcribe mtDNA. During spermatogenesis, sperm cells express an isoform of TFAM, which retains the mitochondrial presequence, ordinarily removed upon mitochondrial import. Phosphorylation of this presequence prevents mitochondrial import and directs TFAM to the spermatozoon nucleus. TFAM relocalization from the mitochondria of spermatogonia to the spermatozoa nucleus directly correlates with the elimination of mtDNA, thereby explaining maternal inheritance in this species.

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Fig. 1: Mitochondria of human spermatozoa contain no mtDNA.
Fig. 2: Mature sperm cells contain a specific isoform of TFAM.
Fig. 3: Mature sperm cells contain TFAM in the nucleus but not in the mitochondria.
Fig. 4: MTS, but not UTR of TFAM mRNA, has a role in TFAM localization.
Fig. 5: TFAM import to mitochondria is prevented in spermatozoa.
Fig. 6: Sperm TFAM relocalization during spermatogenesis.

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Data availability

Source data are provided with this paper. The mass spectrometry proteomics data have been deposited into the MassIVE (massive.ucsd.edu) and ProteomeXchange (proteomexchange.org) data repository with the accession number MSV000092433 and PXD043765, respectively. Mitochondrial and nuclear DNA sequencing read counts, extracted from the whole-genome sequencing datasets and used for inference of mtDNA copy number, are included in Supplementary Table 1. However, whole-genome sequencing of nuclear and mtDNA datasets is not central to the research findings and conclusions presented in this manuscript. In accordance with international laws and OHSU IRB regulations and the terms of the consent signed by the research participants, protecting the anonymity of research participants is one of the key ethical requirements for our clinical research. Because the genetic identity of gamete donors can be revealed by whole-genome sequencing information, these datasets cannot be publicly uploaded or shared outside of the OHSU network. Our priority is to protect the privacy of our participants while facilitating responsible and controlled access to the research data for legitimate research purposes. Therefore, we are willing to provide this information on a case-by-case basis upon approval by the OHSU IRB and Research Integrity chairs. Approved requestors will be directed to an OHSU compliance officer to initiate a nondisclosure agreement, ensuring the confidentiality of our research participants. Upon successful approval, the requestor will be escorted by a team member at all times and granted access to an OHSU computer in a shared office where they can access and review the whole-genome sequencing datasets. All requests should be initiated with OHSU Research Integrity and will follow their established process, which may take upwards of 3–6 months. To request access, interested parties should contact Kara Drolet, Associate VP, ORIO, at irb@ohsu.edu. Source data are provided with this paper.

Code availability

Scripts for Picard tools (v2.26.9) used on GATK for preprocessing raw sequencing reads to produce BAM were available in the following GitHub repository: http://broadinstitute.github.io/picard. Details of other code/software packages used in the study have been provided in the Methods.

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Acknowledgements

This study was supported by the NIH grant R35GM131832 (to D.T.), grants PID2020-115091RB-I00, MCIN/AEI/10.13039/501100011033, and PI2020/09-4 from CIBERNED, Instituto de Salud Carlos III (ISCIII), Spain (to R.T.), and OHSU Institutional funds (to S.M.). We thank NYULH DART Microscopy Laboratory, A. Liang, C. Petzold and K. Dancel-Manning for consultation and assistance with TEM work; this core is partially funded by NYU Cancer Center Support Grant NIH/NCI P30CA016087. The authors are indebted to C. Van Dyken, D. Battaglia, the Oregon National Primate Research Center staff, OHSU Reproductive Endocrinology and IVF clinic for their expertise and services in obtaining monkey and human gametes for this study. We are grateful to all study participants for sperm and blood donations, and Y. Li and D. Frana from the OHSU Center for Embryonic Cell and Gene Therapy and B. Sereda and other staff members from the OHSU Fertility Consultants and Andrology Division in the Department of Obstetrics and Gynecology for their assistance in procurement and preparation of sperm and tissue donations. We thank Thomas Jefferson University BioImaging facility and M. Covarrubias for help with confocal microscopy experiments. M. Anikin and W. T. McAllister (Rowan University) are acknowledged for their critical reading of the manuscript and fruitful discussion.

Author information

Author notes

  1. These authors contributed equally: William Lee, Angelica Zamudio-Ochoa, Gina Buchel.

Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA, USA

    William Lee, Angelica Zamudio-Ochoa, Gina Buchel & Dmitry Temiakov

  2. Neurobiology Unit, Institut d’Investigacions Biomèdiques de Barcelona (IIBB-CSIC-IDIBAPS) and Centro de Investigación Biomédica en Red de Enfermedades Neurodegenerativas (CIBERNED), Barcelona, Spain

    Petar Podlesniy, Margalida Puigròs, Anna Calderon & Ramon Trullas

  3. Center for Embryonic Cell and Gene Therapy, Oregon Health & Science University, Portland, OR, USA

    Nuria Marti Gutierrez, Aleksei Mikhalchenko, Amy Koski & Shoukhrat Mitalipov

  4. Molecular & Cellular Oncogenesis Program, The Wistar Institute, Philadelphia, PA, USA

    Hsin-Yao Tang

  5. Department of Pathology and Genomic Medicine, Thomas Jefferson University, Philadelphia, PA, USA

    Li Li

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Contributions

N.M.G. and L.L. performed tissue collection. G.B., W.L., and A.Z.O. carried out cloning. G.B. performed Northern blots and mapped mRNA UTRs. P.P., M.P., A.C. and R.T. conducted ddPCR. A.Z.O. undertook protein purification. W.L. managed cell culture, and carried out transduction, IH and CISH procedures. H.Y.T. conducted LC–MS/MS analysis. A.M. performed WGS and data analysis. A.K. provided regulatory oversight. W.L., A.Z.O., G.B., R.T., S.M. and D.T. developed the experimental design. D.T. completed the original draft of the writing. D.T., W.L., A.Z.O., R.T., M.A. and S.M. reviewed and edited the writing.

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Correspondence to Dmitry Temiakov.

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Extended data

Extended Data Fig. 1 Mitochondria of human spermatozoa contain no mtDNA.

a. Schematics of the mtDNA copy number assessment in sperm cells using Whole Genome Sequencing (WGS). b. Box plots of mtDNA copy number (mtDNA/per cell) analysis in bulk spermatozoa and blood samples (n = 3); n represents a biologically independent number of samples. Center line, median; box bounds, 25th and 75th percentiles; whiskers, minimum to maximum within 1.5 interquartile range; data points outside whiskers, outliers. c. Illustration of a mitochondrial genome coverage breadth in bulk blood cells (top) and spermatozoa (bottom) sample (donor 2) enriched for mtDNA using long-range PCR amplifying full-length mtDNA in one reaction. Location of the forward (F) and reverse (R) PCR primers are shown by black arrows. A peak in the read density observed in the region of priming of the PCR primers (black arrows, position ~2,000 nt) in spermatozoa but not in the blood cells likely reflects the presence of degraded mtDNA molecules. d. Schematic of human mtDNA showing the location of sequences targeted by different primer pairs. Pink arrows indicate the mtDNA common deletion region. The name and location of the amplicons are shown in blue. e. Representative one-dimension droplet scatter plot illustrating the accuracy of ddPCR in mtDNA detection. The pJET plasmid containing only the mt64-ND1 target mtDNA fragment was used; amplification was performed using mt64-ND1 and mt79-COX3 primers. Blue dots above the green line indicate amplicon-positive droplets. Green lines indicate amplitude thresholds to distinguish amplicon-positive from amplicon-negative droplets, red lines - amplicon-negative droplets. In the multiplex assay for the haploid genomes sample, the lower positive droplets are for the TBP1 amplicon, and the upper positive droplets are for the TEFM amplicon. f. Representative one-dimension droplet scatter plots were obtained using different primer pairs after ddPCR of DNA in the same spermatozoa sample. The absolute mtDNA copy number is given for each primer pair at the top of the panel.

Extended Data Fig. 2 Mapping of the UTRs of the sperm TFAM isoform.

a. Western blot analysis of the proteins indicated in mitochondria of HEK cells and spermatozoa (SPZ). b. Schematics of the RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE) experiment. PNK – Polynucleotide kinase. P – 5’ phosphate group. OH – 3’ hydroxyl group. An – PolyA mRNA tail. Gppp – 5’ mRNA cap. RT-PCR – Reverse transcription polymerase chain reaction. PCR – Polymerase chain reaction. c. The sequence of the 3’ UTR of the sperm TFAM cDNA. d. Schematic illustration of polyadenylation of the TFAM mRNA in somatic cells. PAS – polyadenylation signal. e. Schematic illustration of polyadenylation of the TFAM mRNA in sperm cells. Putative polyadenylation signal (PAS) and UGUAN elements are indicated.

Source data

Extended Data Fig. 3 Identification of TFAM peptides by LC-MS/MS analysis.

a. LC-MS/MS analysis of HEK cells. The rectangular box represents the protein, and regions with peptide identification using MaxQuant are indicated by yellow boxes. The red trace shows the MS/MS spectra count of the identified peptides relative to the peptide with the highest spectra count. Identified peptide sequences are also highlighted in yellow and the MS/MS spectra counts are indicated by the gray lines under the sequence and summarized below the figure. b. LC-MS/MS analysis of human spermatozoa. Note that the SGAELCSGCGSR peptide was identified in the human sperm TFAM sample using pFind and, therefore, is not shown in this panel. c. LC-MS/MS analysis of Rhesus monkey spermatozoa.

Extended Data Fig. 4 TFAM expression pattern in different human tissues.

a. Exon IT is detected in testis mRNA only. Histograms of RNA-seq data from 10 different tissues have been visualized using the Genome Data Viewer (NCBI, chr10: 58,385,053 –58,389,712). The schematic of the TFAM pre-mRNA is shown below the histograms. b. TFAM peptides matching the mitochondrial pre-sequence are not detected in somatic tissues. The uniquely-mapping protein-calling peptides are shown as blue bars and multi-mapping and non-tryptic peptides are yellow bars. Light blue, 1–4 observations. Dark blue, five or more observations. Proteomic data were obtained from 1099 proteomic experiments. Source: Peptide Atlas (db.systemsbiology.net).

Extended Data Fig. 5 TFAM localization in spermatozoa and somatic cells.

a. Cryo immunogold electron microscopy of spermatozoa head. Red arrows indicate gold particles. A -acrosome. b. Cryo immunogold electron microscopy of spermatozoon midpiece. M- mitochondria. c. Staining of human testicular tissue. Merge image. Red- staining with anti-TFAM antibody, blue- DAPI, green -staining with anti-TOM20 antibody. Red arrows - spermatogonia, yellow arrows – spermatocytes, white arrows – spermatids. The basement membrane is indicated by a dashed line, L- seminiferous tubule lumen. Close-up images of spermatogonia (1), spermatocytes (2) and spermatids (3) correspond to the white squares indicated. d. Confocal microscopy of HeLa cells transduced with TFAM-mScarlet fusion having somatic 5’ and 3’ UTRs. e. Confocal microscopy of HeLa cells transduced with TFAM-mScarlet fusion lacking UTRs.

Extended Data Fig. 6 Expression of TFAM, H2B, TEFM, and TOM20 in somatic cells and mature spermatozoa.

a. Confocal microscopy of spermatozoa transduced with TFAM-mScarlet fusion having sperm 3’ and 5’ UTR regions b. Confocal microscopy of HeLa cells transduced with H2B-mScarlet c. Confocal microscopy of spermatozoa transduced with H2B-mScarlet d. Confocal microscopy of spermatozoa transduced with TEFM-mScarlet e. Confocal microscopy of spermatozoa transduced with TOM20-mScarlet.

Extended Data Fig. 7 Import of the TFAM variants in HeLa cells and spermatozoa.

a. Confocal microscopy of spermatozoa transduced with MTSpol-mScarlet b. Confocal microscopy of HeLa cells transduced with MTSpol-mScarlet c. Confocal microscopy of HeLa cells transduced with MTSPol-Δ42TFAM-mScarlet d. Confocal microscopy of HeLa cells transduced with MTSTFAM-Scarlet.

Extended Data Fig. 8 Expression of the phosphomimicking TFAM variant in HEK293 cells.

a. Confocal microscopy of HEK293 cells transduced with TFAMS31AA/S34AA-mScarlet b. Confocal microscopy of HEK293 cells transduced with TFAMS31DD/S34DD-mScarlet.

Supplementary information

Reporting Summary

Supplementary Tables 1–4

Supplementary Table 1: MtDNA copy number (CN) analysis of the blood and spermatozoa samples by WGS; Supplementary Table 2: Deep sequencing of enriched mtDNA from the blood and spermatozoa samples; Supplementary Table 3: Oligonucleotides used in the study; Supplementary Table 4: Plasmids generated during the study.

Source data

Source Data Fig. 2

Unprocessed western and southern blots for Fig. 2a,b.

Source Data Extended Data Fig. 2

Unprocessed western blots for Extended Data Fig. 2a.

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Lee, W., Zamudio-Ochoa, A., Buchel, G. et al. Molecular basis for maternal inheritance of human mitochondrial DNA. Nat Genet 55, 1632–1639 (2023). https://doi.org/10.1038/s41588-023-01505-9

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