Biology of Reproduction 43:525-542 (1990)
Key Words: testis spermatogenesis cycle stages morphometry
The stages of the cycle in the rat seminiferous epithelium are illustrated for testes fixed by vascular perfusion and embedded in plastic resins. Improved cellular resolution in plastic sections permitted a clearer demarcation of the stages than in paraffin. Quantitative data are presented to support the recognition of stages, particularly those in transition. Stages IV, V, VII, XI and XII had the highest frequencies of transitional characteristics. Stage IV was redefined to be more consistent with the occurrence of a high percentage of mitotic figures. The presence of mitosis was also used in the discrimination of Stages V and VI, as no difference was found in the percent nuclear area covered by the acrosomic system at this transition. The acrosomic system covered less of the round spermatid nucleus in the methacrylate than in epoxy or paraffin. Although the resolution of cellular detail was greatly improve with the use of plastics, the thinner sections contained fewer classical identifying features; thus additional characteristics were required for staging, such as the diameter of the nucleus at the acrosomal-nuclear junction (steps 10-13 spermatids). A binary decision key is provided to improve consistency among laboratories in the identification of the stages in plastic-embedded testes.
The first description of 14 stages in the cycle of the rat seminiferous epithelium presented an excellent overview of the cellular associations and the general evolution of cells in time (Leblond and Clermont, 1952a). This morphologic classification of the seminiferous cycle has served as the foundation for many studies of the biochemistry and physiology of spermatogenesis (Parvinen et al., 1986) and provided the standard to which testicular histopathology is compared. However, recognition of the stages in the cycle can be difficult for some individuals depending upon their training and optical acuity, as well as the type of histologic fixatives and stains that are used (Clermont and Leblond, 1955). Because of these difficulties, additional clarification of the stages was introduced to facilitate their routine use in paraffin sections (Leblond and Clermont, 1952b; Clermont and Perey, 1957). In particular, precise acrosomic changes associated with spermatid development were shown to be the most accurate characteristics by which stages could be divided (Leblond and Clermont, 1952b; Clermont and Perey, 1957). Because spermatogenesis is a continuous process, the seminiferous tubules were often found in transition between two stages, which required even further definition (Perey et al., 1961). Thus, consistent and precise recognition of the stages in paraffin histologic sections requires a knowledge not only of the general characteristics (Leblond and Clermont, 1952a), but also perception of the subtle changes of the epithelium through transition.
Although these early descriptions of the stages have been adequate for most purposes, they were based upon observations of tissues fixed and processed using classical histologic methods. More advanced techniques of histology and ultrastructure have been developed to improve the preservation of cells and to increase the resolution of cellular details (Russell, 1983; Chapin et al., 1984). Improvements in resolution through the use of electron microscopy have contributed much to our understanding of the evolution of spermatids through the seminiferous cycle (Clermont and Rambourg, 1978; Clermont et al., 1980; Lalli and Clermont, 1981; Tang et al., 1982; Morales and Clermont, 1982). However, there is a gap between the ability to recognize stages in thick paraffin and ultrathin plastic sections, and attempts to clarify the light microscopic characteristics in plastic remain limited and insufficiently illustrated (Clermont and Rambourg, 1978; Russell and Frank, 1978; Kerr et al., 1984).
Recognition of germ cells in plastic histologic sections can be more difficult than in paraffin (Dym and Cavicchia, 1978), probably due to the lack of clear illustrations of the stages and more precise definition of the transitions between stages. This difficulty led to the combination of stages in one study because of the "lack of reliable criteria to differentiate stages I-IV in the present material ..." (Wing and Christensen, 1982). Therefore, this study presents a comprehensive evaluation of the stages in the cycle of the seminiferous epithelium as observed in testes fixed by vascular perfusion and embedded in plastic resins. Special attention is given to the recognition of stages that are in transition and a binary decision key is provided to improve consistency among laboratories in the identification of the stages.