In eukaryotes, the nucleocytoplasmic export of bulk poly(A)+-mRNAs through the nuclear pore complex is mediated by the ubiquitously expressed NXT1-NXF1 heterodimer. In humans, NXT1 has an X-chromosomal paralog, NXT2, which exhibits testis-enriched expression, suggesting a role in spermatogenesis. Here, we report the in vivo interaction of NXT2 with crucial components of the nuclear export machinery, including NXF1, the testis-specific NXF1 paralogs NXF2 and NXF3, and the nuclear pore complex proteins NUP93 and NUP214. Further, NXT2's NTF2-like domain mediates binding to NXF2 and NXF3. By identifying infertile men with loss-of-function variants in NXT2 and NXF3, we link the impaired NXT2-NXF activity to disturbed germ cell development. The predominant absence of germ cells in men with NXT2 deficiency indicates its critical function already during fetal or first steps of germ cell development. In contrast, loss of NXF3 affects later stages of spermatogenesis resulting in quantitatively and qualitatively impaired sperm production.

The authors have declared no competing interest.

This work was carried out within the frame of the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) funded Clinical Research Unit Male Germ Cells (CRU326, project no. 329621271, grants to F.T. and N.N.).

I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.

Yes

The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

The Ethics Committees/Institutional Review Board of the University of Muenster (Ref. No. Muenster: 2012-555-f-S and 2010-578-f-S) gave ethical approval for this work. The Ethics Committee/Institutional Review Board of Nijmegen University gave ethical approval for this work (NL50495.091.14 version 5.0). The Ethics committee of Newcastle upon Tyne Hospitals NHS Foundation Trust (Newcastle, UK) gave ethical approval for this work (REC ref. 18/NE/0089).

I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.

Yes

I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).

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I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.

Yes

Sequencing data of the MERGE study is available upon request to the corresponding author. Access to this data is limited for each case and specific consent of the respective samples. Sequencing data from the Nijmegen cohort have been deposited in the European Genome-phenome Archive(EGA) under the accession code EGAS00001005417 [https://ega-archive.org/studies/EGAS00001005417] and will be made available upon reasonable request for academic use and within the limitations of the provided informed consent by the corresponding author upon acceptance. Every request will be reviewed by the Newcastle University Male Infertility Genomics Data Access Committee; the researcher will need to sign a data access agreement after approval. All other data supporting the findings of this study are available from the corresponding author upon reasonable request. AlphaFold2 structure accession code for NXT2 is AF-Q9NPJ8-F1 [https://alphafold.ebi.ac.uk/entry/Q9NPJ8]. The mass spectrometry proteomics data have been deposited the ProteomeXchange Consortium via the PRIDE56 partner repository with the dataset identifier PXD052904 [https://www.ebi.ac.uk/pride/] and will be released upon acceptance of the manuscript. Genetic variants identified in NXT2 and NXF3 were submitted to ClinVar (SCV005043065-SCV005043067; [https://www.ncbi.nlm.nih.gov/clinvar/]). Source data are provided with this paper

https://www.ncbi.nlm.nih.gov/clinvar/

https://ega-archive.org/studies/EGAS00001005417

https://alphafold.ebi.ac.uk/entry/Q9NPJ8

https://www.ebi.ac.uk/pride/