Characterization of germ cell nuclei in freeze-fracture replicas using seminiferous tubules isolated by transillumination

The present investigation was under taken, combining the identification of specific germ cell types present in a particular stage of the seminiferous wave with freeze-fracture techniques to follow the changing pattern of their nuclear pore distribution during spermatogenesis. Male albino adult rats were used. Segments of seminiferous tubules were separated (stages VII-VIII; IX-XI; XII-XIV) by a technique described by Parvinen and Vanha-Perttula where the stages of the cycle were recognized and isolated by transillumination under a dissecting microscope. The tubular segments were immediately fixed in glutaraldehyde, immersed in glycerol, freeze-fractured in a Balzers BAF 301 apparatus at-110°C and examined under a Siemens Elmiskop I EM. The only spermatogonia located in the studied stages (type A) are ovalcells in contact with the basal lamina. Pore distribution is non-random with a relatively in conspicuous aggregation. Preleptotene spermatocytes (stages VII-VIII) are easily identified by being numerous, their location over the basal lamina and the irrelatively small size. They exhibit a striking clustering of pores, in groups of 15-30 separated by pore-free areas (fig.1). Leptotenes (fig.2, 3) (stages IX-XI) and zygotenes (fig.4) (XII-XIII) change the pattern to a more even distributional though clearly maintaining some degree of clustering.