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Macrophage ubiquitin-specific protease 2 contributes to motility, hyperactivation, capacitation, and in vitro fertilization activity of mouse sperm

Abstract

Macrophages are innate immune cells that contribute to classical immune functions and tissue homeostasis. Ubiquitin-specific protease 2 (USP2) controls cytokine production in macrophages, but its organ-specific roles are still unknown. In this study, we generated myeloid-selective Usp2 knockout (msUsp2KO) mice and specifically explored the roles of testicular macrophage-derived USP2 in reproduction. The msUsp2KO mice exhibited normal macrophage characteristics in various tissues. In the testis, macrophage Usp2 deficiency negligibly affected testicular macrophage subpopulations, spermatogenesis, and testicular organogenesis. However, frozen–thawed sperm derived from msUsp2KO mice exhibited reduced motility, capacitation, and hyperactivation. In addition, macrophage Usp2 ablation led to a decrease in the sperm population exhibiting high intracellular pH, calcium influx, and mitochondrial membrane potential. Interrupted pronuclei formation in eggs was observed when using frozen–thawed sperm from msUsp2KO mice for in vitro fertilization. Administration of granulocyte macrophage-colony stimulating factor (GM-CSF), whose expression was decreased in testicular macrophages derived from msUsp2KO mice, restored mitochondrial membrane potential and total sperm motility. Our observations demonstrate a distinct role of the deubiquitinating enzyme in organ-specific macrophages that directly affect sperm function.

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Availability of data and material

The datasets in this study are available from the corresponding author upon reasonable request.

Code availability

All software used in this study was obtained legally, and custom code is available from the corresponding author on reasonable request.

Abbreviations

ALDH1A2:

Aldehyde dehydrogenase 1a2

ALH:

Amplitude of lateral head displacement

Ar :

Androgen receptor

BCF:

Beat-cross frequency

BSA:

Bovine serum albumin

CASA:

Computer-assisted sperm motility analysis

CSF1R:

Colony-stimulating factor 1 receptor

CSF2Rα:

Colony-stimulating factor 2 receptor α chain

CTC:

Chlortetracycline

DDX:

DEAD-box helicase

FACS:

Fluorescence-activated cell sorting

GM-CSF:

Granulocyte macrophage-colony stimulating factor

HSD3B:

3-β-Hydroxysteroid dehydrogenase

Hvcn1 :

Voltage-gated hydrogen channel1

IL:

Interleukin

iNOS:

Inducible nitric oxide synthase

IVF:

In vitro fertilization

KD:

Knockdown

KO:

Knockout

Lhcgr :

Luteinizing hormone receptor

LyzM-Cre :

B6.129P2-Lyzstm1(cre)Ifo

M-CSF:

Macrophage-colony stimulating factor

MCT:

Monocarboxylate transporter

MHCII:

Major histocompatibility complex class II

MMP:

Mitochondrial membrane potential

OXPHOS:

Oxidative phosphorylation

PI:

Propidium iodide

Q-FISH:

Quantitative-fluorescent in situ hybridization

qRT-PCR:

Quantitative reverse transcription-polymerase chain reaction

R26GRR:

C57BL/6 N-Gt(ROSA)26Sortm1(CAGEGFP,tdsRed)Utr/Rbrc

RDH10:

Retinol dehydrogenase 10

Slc9c1 :

Solute carrier family 9 member C1

Sox:

Sex-determining region Y-box transcription factor

Stra8 :

Stimulated by retinoic acid 8

TMB:

3,3′,5,5′-Tetramethylbenzidine

TNF:

Tumor necrosis factor

TREM:

Tetramethylrhodamine methyl ester

USP:

Ubiquitin-specific protease

VCL:

Curvilinear velocity

VSL:

Straight-line velocity

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Acknowledgements

The authors would like to thank Dr. Keiji Ueno, Mr. Takeshi Ishino, Ms. Misato Amagasa, Ms. Aya Iida, and Ms. Marina Tanaka from Rakuno Gakuen University, and Dr. Yuko Okamatsu from Hokkaido University. The authors also acknowledge the editing services provided by Uni-edit.

Funding

This work was supported by the Japan Society for the Promotion of Science KAKENHI (15K06805 and 18K06035) and the Rakuno Gakuen University Research Fund (No. 2018-02, 2019-03, and 2020-04).

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Contributions

MH and SK performed the experiments, analyzed the data, and wrote and revised the manuscript. CK, YY, TW, and ET performed the experiments. JO revised the manuscript. MN designed and performed the experiments and revised the manuscript. HK designed and performed the experiments, analyzed data, and wrote and revised the manuscript. All authors read and approved the final version of the manuscript.

Corresponding author

Correspondence to Hiroshi Kitamura.

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The authors have no conflicts of interest/competing interests to declare.

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All animal experiments were approved by the Ethical Review Committee for Animal Experimentation of Rakuno Gakuen University (Approval Numbers VH15A30, VH16A25, VH17A1). We made all efforts to reduce the number of animals used, and to alleviate their suffering.

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Hashimoto, M., Kimura, S., Kanno, C. et al. Macrophage ubiquitin-specific protease 2 contributes to motility, hyperactivation, capacitation, and in vitro fertilization activity of mouse sperm. Cell. Mol. Life Sci. 78, 2929–2948 (2021). https://doi.org/10.1007/s00018-020-03683-9

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  • DOI: https://doi.org/10.1007/s00018-020-03683-9

Keywords

  • USP
  • Granulocyte macrophage-colony stimulating factor
  • Myeloid cells
  • Capacitation
  • Male sterility