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The air-liquid interface culture of the mechanically isolated seminiferous tubules embedded in agarose or alginate improves in vitro spermatogenesis at the expense of attenuating their integrity

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Abstract

Optimization of tissue culture systems able to complete male germ cell maturation to post-meiotic stages is considered as an important matter in reproductive biology. Considering that hypoxia is one of the factors limiting the efficiency of organ culture, the aim of this study was to use isolated seminiferous tubules (STs), having more surface and less thickness, in an organotypic culture system in order to improve oxygen diffusion and reduce hypoxia. The mechanically separated STs embedded in agarose or alginate and 1–3-mm3 testicular tissue fragments of 3 adult mice were separately placed on the flat surface of agarose gel that was half-soaked in the medium. Survival and differentiation of germ cells using PLZF and SCP3 markers, identity of Sertoli cell using GATA4, cell proliferation with the Ki67 marker, and ST integrity using a ST scoring were evaluated up to 36 d at different culture times, each corresponding to the duration of one spermatogenic cycle. We observed a significantly reduced ST integrity in STs embedded in agarose or alginate on day 9 (versus tissue fragments p ≤ 0.05). There was no difference in the number of PLZF-positive cells between groups, but the number of SCP3 (in all-time points) and GATA4-positive cells was significantly higher in the culture of embedded STs. Although embedding STs can be useful for the progress of in vitro spermatogenesis, it makes them sensitive to degeneration. Further improvements are required to modify the air-liquid interface method to maintain ST integrity.

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Acknowledgments

The authors thank all the staff members of Institut de Recherche Expérimentale et Clinique who were involved in this project.

Funding

This study was supported by the Université Catholique de Louvain and funds from the Salus Sanguinis foundation and Saint-Luc foundation.

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Authors and Affiliations

  1. Gynecology-Andrology Research Unit, Institut de Recherche Expérimentale et Clinique, Medical School, Université Catholique de Louvain, 1200, Brussels, Belgium

    Keykavos Gholami, Maxime Vermeulen, Federico Del Vento, Francesca de Michele, Maria Grazia Giudice & Christine Wyns

  2. Gametogenesis Research Center, Kashan University of Medical Sciences, Kashan, Iran

    Keykavos Gholami

  3. Department of Gynecology-Andrology, Cliniques Universitaires Saint-Luc, Avenue Hippocrate 10, 1200, Brussels, Belgium

    Christine Wyns

Authors
  1. Keykavos Gholami
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  2. Maxime Vermeulen
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  3. Federico Del Vento
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  4. Francesca de Michele
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  5. Maria Grazia Giudice
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  6. Christine Wyns
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Corresponding author

Correspondence to Christine Wyns.

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The authors declare that they have no conflicts of interest.

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Editor: Tetsuji Okamoto

Electronic supplementary material

FIG S1

Histological staining at different magnifications showing seminiferous tubules integrity of STs embedded in alginate or agarose and within tissue fragments after 27 days of culture. The central and marginal areas are magnified in the right and left insets respectively showing intraepithelial vacuoles (orange arrow) of varying sizes in STs in marginal areas and a relatively good integrity in central areas. STs with poor score (score 3) are located more in marginal areas and peripheral degeneration was observed in all groups. (PNG 9782 kb)

High Resolution Image (TIF 3614 kb)

FIG S2

A) the pattern of PLZF expression in central (right) and marginal areas (left) is shown. The more number of PLZF-positive cells was observed in marginal areas. A) Quantification of tubules positive for PLZF staining in well-preserved tubules located in central areas. Scale bars: 500 μm (Alginate and tissue in the middle), 1 mm (Agarose in the middle), 50 μm (all groups, left and right). (PNG 5678 kb)

High Resolution Image (TIF 899 kb)

FIG S3

A) Expression of GATA4 is more limited to marginal and submarginal areas in tissue culture but it was observed in all areas in STs embedded in alginate or agarose. B) Number of GATA4 - positive cells in centrally-located tubules at different culture times. Scale bars: 500 μm (Alginate and tissue in the middle), 1 mm (Agarose in the middle), 50 μm (all groups, left and right), ⁎: p ≤ 0.0181, ⁎ ⁎: p ≤ 0.0050. (PNG 5009 kb)

High Resolution Image (TIF 790 kb)

FIG S4

A) SCP3 expression is more seen in sub marginal/marginal areas of tissue fragments but distribution of SCP3 is almost observed in all parts in the mechanically-separated STs embedded in alginate or agarose. The expression of SCP3 partly disappeared in the central areas of tissue fragment. B) Quantification of SCP3 expression by counting the number of SCP3-positive cells per well-preserved ST only located in central areas in all groups. Scale bars: 500 μm (all groups in the middle), 50 μm (all groups, left and right), ⁎: p ≤ 0.0325, ⁎ ⁎: p ≤ 0.0019. (PNG 5359 kb)

High Resolution Image (TIF 837 kb)

FIG S5

A) Cell proliferation was observed in the different groups. The expression pattern of Ki67 in groups is shown in marginal / sub marginal (on the left) and central areas (on the right). Quantification of Ki67 expression by counting the number of Ki67-positive cells per well-preserved ST only located in central areas in all groups. Scale bars: 500 μm (all groups in the middle), 50 μm (all groups, left and right). (PNG 5032 kb)

High Resolution Image (TIF 810 kb)

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Gholami, K., Vermeulen, M., Del Vento, F. et al. The air-liquid interface culture of the mechanically isolated seminiferous tubules embedded in agarose or alginate improves in vitro spermatogenesis at the expense of attenuating their integrity. In Vitro Cell.Dev.Biol.-Animal 56, 261–270 (2020). https://doi.org/10.1007/s11626-020-00437-6

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  • DOI: https://doi.org/10.1007/s11626-020-00437-6

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