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Maternally contributed Nlrp9b expressed in human and mouse ovarian follicles contributes to early murine preimplantation development

  • Embryo Biology
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Journal of Assisted Reproduction and Genetics Aims and scope Submit manuscript

Abstract

Purpose

The aim of the study is to investigate presence and role of the gene encoding the maternally contributed nucleotide-binding oligomerization domain (NOD)–like receptors with a pyrin domain (PYD)–containing protein 9 (NLRP9) in human and mouse ovaries, respectively, and in preimplantation mouse embryo development by knocking down Nlrp9b.

Methods

Expression levels of NLRP9 mRNA in human follicles were extracted from RNA sequencing data from previous studies. In this study, we performed a qPCR analysis of Nlpr9b mRNA in mouse oocytes and found it present. Intracellular ovarian distribution of NLRP9B protein was accomplished using immunohistochemistry. The distribution of NLRP9B was explored using a reporter gene approach, fusing NLRP9B to green fluorescent protein and microinjection of in vitro–generated mRNA. Nlrp9b mRNA function was knocked down by microinjection of short interference (si) RNA targeting Nlrp9b, into mouse pronuclear zygotes. Knockdown of the Nlrp9b mRNA transcript was confirmed by qPCR.

Result

We found that the human NLRP9 gene and its corresponding protein are highly expressed in human primordial and primary follicles. The NLRP9B protein is localized to the cytoplasm in the blastomeres of a 2-cell embryo in mice. SiRNA-mediated knockdown of Nlrp9b caused rapid elimination of endogenous Nlrp9b mRNA and premature embryo arrest at the 2- to 4-cell stages compared with that of the siRNA-scrambled control group.

Conclusions

These results suggest that mouse Nlrp9b, as a maternal effect gene, could contribute to mouse preimplantation embryo development. It remains to investigate whether NLRP9 have a crucial role in human preimplantation embryo and infertility.

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Acknowledgements

The authors wish to thank past and current members of the Lykke-Hartmann Laboratory (AU) for their scientific discussions.

Funding

This work was supported by grants from the Carlsberg Foundation (CF18-0474 to MA), Aarhus University Research Foundation (to LS and KLH) and the Independent Research Fund Denmark – Medical Sciences (grant number 6120-00027B), the Novo Nordisk Foundation (NNF16OC0022480 and NNF170C0026820), Kong Christian Den Tiendes Fond, Th. Maigaards Eft. Fru Lily Benthine Lunds Fond, Toyota Fonden, Augustinus Fonden and Fonden til Lægevidenskabens Fremme (to KLH).

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Authors and Affiliations

  1. Department of Biomedicine, Aarhus University, Aarhus, Denmark

    Mahboobeh Amoushahi, Line Lawaetz Steffensen, Adelya Galieva, Jens Agger, Anders Heuck, Piotr Siupka, Morten S. Nielsen, Lone Sunde & Karin Lykke-Hartmann

  2. The Fertility Clinic, Horsens Hospital, Horsens, Denmark

    Erik Ernst

  3. The Fertility Clinic, Aarhus University Hospital, 8200, Aarhus, Denmark

    Erik Ernst

  4. Lundbeck Foundation Research Initiative on Brain Barriers and Drug Delivery, Aarhus, Denmark

    Morten S. Nielsen

  5. Department of Clinical Genetics, Aarhus University Hospital, Aarhus, Denmark

    Lone Sunde & Karin Lykke-Hartmann

  6. Department of Clinical Medicine, Aarhus University, Aarhus, Denmark

    Karin Lykke-Hartmann

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  1. Mahboobeh Amoushahi
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Contributions

KLH and LS conceived the study. AG and AH performed the cloning work. EE provided human tissue. MA performed the IHC. JA and AH performed the qPCR analysis. PS, MA, MSN and KLH performed the confocal analysis. LLS, MA and KLH performed the embryo work and siRNA microinjections. All of the authors analyzed and interpreted the results. KLH wrote the manuscript. All of the authors approved the final manuscript.

Corresponding author

Correspondence to Karin Lykke-Hartmann.

Ethics declarations

Written informed consent was obtained from all patients. The study was approved by Danish Scientific Ethical Committee (approval number: KF299017 and J7KF/01/170/99) and the Danish Data Protection Agency. All of the procedures were approved by the Ethics Committee for the Use of Laboratory Animals in Aarhus University (2015−15−0201−00800 to KLH).

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Amoushahi, M., Steffensen, L.L., Galieva, A. et al. Maternally contributed Nlrp9b expressed in human and mouse ovarian follicles contributes to early murine preimplantation development. J Assist Reprod Genet 37, 1355–1365 (2020). https://doi.org/10.1007/s10815-020-01767-w

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