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Live-cell imaging and ultrastructural analysis reveal remarkable features of cultured porcine gonocytes

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Abstract

Gonocytes in the neonatal testis have male germline stem cell potential. The objective of the present study was to examine the behavior and ultrastructure of gonocytes in culture. Neonatal porcine testis cells were cultured for 4 weeks and underwent live-cell imaging to explore real-time interactions among cultured cells. This included imaging every 1 h from day 0 to day 3, every 2 h from day 4 to day 7, and every 1 h for 24 h at days 14, 21, and 28. Samples also underwent scanning electron microscopy, transmission electron microscopy, morphometric evaluations, immunofluorescence, and RT-PCR. Live-cell imaging revealed an active amoeboid-like movement of gonocytes, assisted by the formation of extensive cytoplasmic projections, which, using scanning electron microscopy, were categorized into spike-like filopodia, leaf-like lamellipodia, membrane ruffles, and cytoplasmic blebs. In the first week of culture, gonocytes formed loose attachments on top of a somatic cell monolayer and, in week 2, formed grape-like clusters, which, over time, grew in cell number. Starting at week 3 of culture, some of the gonocyte clusters transformed into large multinucleated embryoid body–like colonies (EBLCs) that expressed both gonocyte- and pluripotent-specific markers. The number and diameter of individual gonocytes, the number and density of organelles within gonocytes, as well as the number and diameter of the EBLCs increased over time (P < 0.05). In conclusion, cultured porcine gonocytes displayed extensive migratory behavior facilitated by their various cytoplasmic projections, propagated, and transformed into EBLCs that increased in size and complexity over time.

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Acknowledgments

We thank Brian Andries and Tatjana Ometlic at the Prairie Swine Centre for their assistance in the collection of neonatal porcine testes. We also thank Drs. Muhammad Anzar and Patrick Krone for their insightful discussions.

Funding

This study was supported by grants from the Natural Sciences and Engineering Research Council (NSERC) of Canada to A. Honaramooz. The University of Saskatchewan College of Graduate and Postdoctoral Studies and the University of Saskatchewan Western College of Veterinary Medicine provided scholarships to A.H. Awang-Junaidi and M.A. Fayaz. The Malaysian Ministry of Higher Education also provided financial support to A.H. Awang-Junaidi.

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  1. Awang Hazmi Awang-Junaidi

    Present address: Department of Veterinary Preclinical Sciences, Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor Darul Ehsan, Malaysia

Authors and Affiliations

  1. Department of Veterinary Biomedical Sciences, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, S7N 5B4, SK, Canada

    Awang Hazmi Awang-Junaidi, Mohammad Amin Fayaz, Daniel J. MacPhee & Ali Honaramooz

  2. WCVM Imaging Centre, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, S7N 5B4, SK, Canada

    Eiko Kawamura & LaRhonda Sobchishin

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  1. Awang Hazmi Awang-Junaidi
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Contributions

A.H.A.J. contributed to conceiving and designing of the study, performed the experiments, and wrote the first draft of the manuscript. M.A.F. contributed to the experimental analyses and revising the manuscript. E.K. and L.S. contributed to the live-cell imaging and ultrastructural studies. D.J.M. contributed to designing of the study and revising the manuscript. A.H. supervised the work and contributed to conceiving and designing of the study, as well as writing and revising the manuscript.

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Correspondence to Ali Honaramooz.

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The authors declare that they have no competing interests.

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All experimental procedures involving animals were approved by the University of Saskatchewan’s Institutional Animal Care and Use Committee.

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Portions of this study were presented as an abstract at the 51st Annual Meeting of Society for the Study of Reproduction, 2018.

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Supplementary Video 1

Organization of neonatal porcine testis cells in prolonged culture at 0–24 h (00:04 s), 25–48 h (00:09 s), 17–120 h (00:15 s), D7 (00:20 s), D14 d (00:27 s), D21 (00.33 s) and D28 (00:39 s) (time lapse 15 fps). Images were taken every 1 h for 24 h from day 0 to day 3, every 2 h for 24 h from day 4 to day 7, and finally every 1 h for 24 h at days 14, 24, and 28. (WMV 42141 kb)

Supplementary Video 2

Cytoplasmic projections and amoeboid-like movement of gonocytes at 1-week (a) in the media (00:04 s) and (b) on top of somatic cell monolayer (00:12 s). The images were taken every 2.5 s for 30 s (time lapse 1 fps) and 5 min for gonocytes (time lapse 4 fps) floating in the media and on the somatic monolayer, respectively. (WMV 15346 kb)

Supplementary Video 3

The stages in the formation of embryoid body–like colony (EBLC) formation. The images were taken every 2.5 s for 5 min (time lapse 4 fps). (a) Gonocytes first settled on the somatic cell monolayer either singles or paired (00:04 s). (b) Continuous migration of gonocytes towards each other results in the formation of morula-like (grape-like) colonies (00:11 s). (c) The morula-like colonies increased in size over time (00:18 s). (d) The EBCL formed after ~14 days of culture (00:24 s). Cytoplasmic fusions of adjacent gonocytes seemed to transform the morula-like colonies into an embryoid body–like colony (00:34 s). (e) Continued fusion of migrating gonocytes increased the mass of the EBLC (00:44 s). (g) Note the presence of extensive cytoplasmic projections possessed by EBLC which seemed to assist with its movement, and further attachment of migrating gonocytes (00:56 s). (h) Higher magnification of active movement of cytoplasmic projections of EBLC (01:06). (WMV 122001 kb)

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Awang-Junaidi, A.H., Fayaz, M.A., Kawamura, E. et al. Live-cell imaging and ultrastructural analysis reveal remarkable features of cultured porcine gonocytes. Cell Tissue Res 381, 361–377 (2020). https://doi.org/10.1007/s00441-020-03218-5

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  • DOI: https://doi.org/10.1007/s00441-020-03218-5

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