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mTORC1/C2 regulate spermatogenesis in Eriocheir sinensis via alterations in the actin filament network and cell junctions

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Abstract

Spermatogenesis is a finely regulated process of germ cell proliferation and differentiation that leads to the production of sperm in seminiferous tubules. Although the mammalian target of rapamycin (mTOR) signaling pathway is crucial for spermatogenesis in mammals, its functions and molecular mechanisms in spermatogenesis remain largely unknown in nonmammalian species, particularly in Crustacea. In this study, we first identified es-Raptor (the core component of mTOR complex 1) and es-Rictor (the core component of mTOR complex 2) from the testis of Eriocheir sinensis. Dynamic localization of es-Raptor and es-Rictor implied that these proteins were indispensable for the spermatogenesis of E. sinensis. Furthermore, es-Raptor and es-Rictor knockdown results showed that the mature sperm failed to be released, causing almost empty lumens in the testis. We investigated the reasons for these effects and found that the actin-based cytoskeleton was disrupted in the knockdown groups. In addition, the integrity of the testis barrier (similar to the blood-testis barrier in mammals) was impaired and affected the expression of cell junction proteins. Further study revealed that es-Raptor and es-Rictor may regulate spermatogenesis via both mTORC1- and mTORC2-dependent mechanisms that involve es-rpS6 and es-Akt/es-PKC, respectively. Moreover, to explore the testis barrier in E. sinensis, we established a cadmium chloride (CdCl2)–induced testis barrier damage model as a positive control. Morphological and immunofluorescence results were similar to those of the es-Raptor and es-Rictor knockdown groups. Altogether, es-Raptor and es-Rictor were important for spermatogenesis through maintenance of the actin filament network and cell junctions in E. sinensis.

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Acknowledgements

The authors want to show their appreciation to all members of the Sperm Laboratory in Zhejiang University for their support, encouragement, and assistance.

Funding

This project was supported by the National Natural Science Foundation of China (no. 32072954 and no. 32102786).

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Authors and Affiliations

  1. The Sperm Laboratory, College of Life Sciences, Zhejiang University, Hangzhou, 310058, China

    Zhen-Fang Li, Shuang-Li Hao, Lan-Min Wang, Hong-Yu Qi, Jia-Ming Wang & Wan-Xi Yang

  2. The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, 310003, China

    Fu-Qing Tan

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  1. Zhen-Fang Li
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Correspondence to Shuang-Li Hao or Wan-Xi Yang.

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The animal use in this study was approved by the Animal Experimental Ethical Inspection of the First Affiliated Hospital, College of Medicine, Zhejiang University (Reference Number: 2019–1061).

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441_2022_3680_MOESM1_ESM.tif

Supplementary file1 Fig. S1 Multiple sequence alignment of es-Raptor and other species of Raptor GenBank accession numbers for the analyzed sequences were as follows: HsRaptor, Homo sapiens (AAI36653.1), MmRaptor, Mus musculus (NP_083174.2), XtRaptor, Xenopus tropicalis (NP_001123843.1), DrRaptor, Danio rerio (XP_021327418.1), DmRaptor, Drosophila melanogaster (NP_001284924.1) (TIF 7003 KB)

441_2022_3680_MOESM2_ESM.tif

Supplementary file2 Fig. S2 Multiple sequence alignment of es-Rictor and other species of Rictor GenBank accession numbers for the analyzed sequences were as follows: HsRictor, Homo sapiens (AAI44510.1), MmRictor, Mus musculus (NP_084444.3), DrRictor, Danio rerio (XP_009293661.1), DmRictor, Drosophila melanogaster (NP_001285433.1) (TIF 7610 KB)

441_2022_3680_MOESM3_ESM.tif

Supplementary file3 Fig. S3 Structural domains and phylogenetic trees (a) The es-Raptor protein had the Raptor_N domain (46-199 aa) and seven WD domains (931-1263 aa). The sequence numbers of the selected species were as follows: Drosophila melanogaster (NP_001284924.1), Homo sapiens (AAI36653.1), Mus musculus (NP_083174.2), Xenopus tropicalis (NP_001123843.1), Danio rerio (XP_021327418.1), Sus scrofa (JAA53799.1), Penaeus monodon (XP_037776024.1), Lepeophtheirus salmonis (CAF2941010.1), Manduca sexta (XP_037293727.1), Parasteatoda tepidariorum (XP_042895958.1), Belonocnema treatae (XP_033214113.1), Solenopsis invicta (XP_025995586.1), Culex quinquefasciatus (XP_038108826.1), Bombyx mori (NP_001177772.1). (b) The es-Rictor protein domains contained RICTOR_N domain (108-474 aa)、RICTOR_M domain (562-785 aa)、Ras GEF N_2 domain (786-900 aa)、RICTOR V domain (962-1034 aa). The sequence numbers of the selected species were as follows: Drosophila melanogaster (NP_001285433.1), Homo sapiens (AAI44510.1), Mus musculus (NP_084444.3), Danio rerio (XP_009293661.1), Bos taurus (NP_001137568.1), Sus scrofa (XP_005672486.2), Penaeus monodon (XP_037803156.1), Lepeophtheirus salmonis (CAF2942537.1), Monomorium pharaonis (XP_012523043.2), Chelonus insularis (XP_034935765.1), Araneus ventricosus (GBM31652.1), Apis florea (XP_012347111.1) (TIF 8155 KB)

441_2022_3680_MOESM4_ESM.tif

Supplementary file4 Fig. S4 Specificity of the es-Raptor and es-Rictor antibodies (a) Western blot results of the total protein of E. sinensis testis using the es-Raptor antibody. (b) Western blot results of the total protein of E. sinensis testis using the es-Rictor antibody (TIF 3552 KB)

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Li, ZF., Hao, SL., Wang, LM. et al. mTORC1/C2 regulate spermatogenesis in Eriocheir sinensis via alterations in the actin filament network and cell junctions. Cell Tissue Res 390, 293–313 (2022). https://doi.org/10.1007/s00441-022-03680-3

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