Spermatogenesis is a cyclic process during which, within each epithelial area, various generations of germ cells undergo a series of developmental steps according to a fixed time schedule. The cycle of the seminiferous epithelium can be subdivided into stages. In the mouse, 12 such stages have been described that can be distinguished from one another by steps in spermatid development. The best way to recognize the stages in seminiferous tubule cross-sections is to use Bouin's-fixed testes of normal mice and sections stained with the Periodic acid Schiff (PAS) technique and hematoxylin. Unfortunately, this is not always possible. Sometimes PAS staining cannot be used, such as when immunohistochemistry is carried out. Moreover, not all germ cell types may be present in some instances, as in young or mutant mice. We summarize here all stage-identifying criteria that can be used in the ideal situation as well as in hematoxylin-only stained sections and/or when germ cell types are missing.

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BackgroundExposure to high doses of ionizing radiation (IR) can lead to localized radiation injury of the skin and exposed cells suffer dsDNA breaks that may elicit cell death or stochastic changes. Little is known about the DNA damage response after high-dose exposure of the skin. Here, we investigate the cellular and DNA damage response in acutely irradiated minipig skin.Methods and FindingsIR-induced DNA damage, repair and cellular survival were studied in 15 cm2 of minipig skin exposed in vivo to ∼50 Co-60 γ rays. Skin biopsies of control and 4 h up to 96 days post exposure were investigated for radiation-induced foci (RIF) formation using γ-H2AX, 53BP1, and active ATM-p immunofluorescence. High-dose IR induced massive γ-H2AX phosphorylation and high 53BP1 RIF numbers 4 h, 20 h after IR. As time progressed RIF numbers dropped to a low of <1% of keratinocytes at 28–70 days. The latter contained large RIFs that included ATM-p, indicating the accumulation of complex DNA damage. At 96 days most of the cells with RIFs had disappeared. The frequency of active-caspase-3-positive apoptotic cells was 17-fold increased 3 days after IR and remained >3-fold elevated at all subsequent time points. Replicating basal cells (Ki67+) were reduced 3 days post IR followed by increased proliferation and recovery of epidermal cellularity after 28 days.ConclusionsAcute high dose irradiation of minipig epidermis impaired stem cell replication and induced elevated apoptosis from 3 days onward. DNA repair cleared the high numbers of DBSs in skin cells, while RIFs that persisted in <1% cells marked complex and potentially lethal DNA damage up to several weeks after exposure. An elevated frequency of keratinocytes with persistent RIFs may thus serve as indicator of previous acute radiation exposure, which may be useful in the follow up of nuclear or radiological accident scenarios.

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