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. 2021 Feb 10;11:3458. doi: 10.1038/s41598-021-82792-2

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Effect of oxygen concentration for in vitro rat spermatogenesis. (A) Photos of cultured testis tissues at week 6 in different O₂ concentrations with or without PC. Scale bar; 1 mm. (B) cVenus expression area ratio in each group at week 6. n = 6–7 (P7). *P < 0.05. (C) Histological pictures of 6 week cultured tissue stained with PAS. Red, yellow, and green arrowheads indicate pachytene spermatocyte, round spermatids at step 1–4, and step 5–6, respectively. Panels in 2nd and bottom row are magnified image. Scale bar; 50 µm (top and 3rd row), 10 µm (2nd and bottom row). (D) Immunohistochemical staining with anti-Venus (green) and PNA (red), along with nuclear stain with Hoechst (blue). Area in white rectangles in top panels was enlarged in bottom panels. Scale bar; 50 µm (top panels), 10 µm (bottom panels). (E) Ratio of seminiferous tubules classified with the most differentiated germ cell type contained was shown by band graph. Round spermatids of step 1–4 appears as having acrosome of dot shape, while step 5–6 having cap shape. n = 5–12 (P7).