Cytochemical localization of cations in the testis of the syrian hamster, utilizing potassium‐pyroantimonate

Abstract

Pyroantimonate has long been used as a cytochemical technique for the localization of cations. Although the technique was originally alleged to be specific for Na+, other cations, including Mg++, Ba++, Ca++, Zn++ and polycationic amines, form pyroantimonate precipitates. In seminiferous tubules treated with antimonate-osmium fixation, the nuclei of the Sertoli cells and germ cells contained precipitates. In regions of condensed chromatin the precipitates were dense while, in dispersed chromatin, precipitates were sparse or absent. It is postulated that the dense precipitates observed in the condensed chromatin may represent an accumulation of calcium that may facilitate chromatin condensation. The Sertoli cell nucleolar apparatus contained light precipitates in the pars amorpha, moderate precipitates in the nucleolonema, and heavy precipitates in the perinucleolar heterochromatin bodies. The nuclei of the maturation-phase spermatids lacked any Precipitates. The perinuclear ring of the acrosome-phase spermatids contained pyroantimonate deposits. In the maturation-phase spermatids, staining occurred in the postacrosomal dense lamina, in the subacrosomal space between the perforatorium and the inner acrosomal membrane, and in the central mass of the residual bodies. Since the postacrosomal dense lamina is the portion of the spermatozoon that initially fuses with the ovum, it is postulated that its cationic properties in some way facilitate this fusion.Pyroantimonate is a unique anion which can be dissolved as the potassium salt but forms insoluble precipitates with other cations. The low molecular weight of pyroantimonate and its electron-opacity render it useful for ultrastructural localization of cations. Komnick ('62) was the first to use potassium pyroantimonate as a cytochemical technique for localization of cations. Although the method was originally alleged to be specific for the localization of sodium, it was later shown that other cations form pyroantimonate salts. Several lines of evidence indicate that pyroantimonate forms insoluble precipitates with B a + + , Ca++, Mg++, Na+, Zn++, glycogen, histamine, spermine, and histones; potassium-antimonate may form in sites of lowered pH or when the pH of the cytochemical medium is not properly controlled (Bulger, '69; Legato and Langer, '69; Lane and Martin, '69; Tisher et al., '69; Torack and LaValle, '70; Clark and Ackerman, '71; Klein et al., '72; Spicer and Swanson, '72).

Ultrastructural localization of calcium ions in ram spermatozoa before and after cold shock as demonstrated by a pyroantimonate technique

“…In contrast to hamster (Gravis, 1979) and to ram spermatozoa, both human (Chandler & Battersby, 1976) and guinea-pig (Friend, 1977) spermatozoa appeared to have no aggregation of precipitate in the postacrosomal region which might indicate cytochemical differences between the species. Also, ram spermatozoa had no precipitate beneath the acrosome, unlike human (Chandler & Battersby, 1976) and hamster (Gravis, 1979) sperm cells, which might reflect differences in penetration by pyro¬ antimonate. This seems unlikely since it has been suggested that, when pyroantimonate is used with osmium fixation, its penetration rate is "adequate to preserve 'normal' cation distribution" (Simson & Spicer, 1975 (Chandler & Battersby, 1976).…”

“…The sig¬ nificance of calcium in this site is not apparent but it is this region of the plasma membrane that initially fuses with the vitelline membrane (Yanagimachi 8c Noda, 1970;Bedford, 1972). In contrast to hamster (Gravis, 1979) and to ram spermatozoa, both human (Chandler & Battersby, 1976) and guinea-pig (Friend, 1977) spermatozoa appeared to have no aggregation of precipitate in the postacrosomal region which might indicate cytochemical differences between the species. Also, ram spermatozoa had no precipitate beneath the acrosome, unlike human (Chandler & Battersby, 1976) and hamster (Gravis, 1979) sperm cells, which might reflect differences in penetration by pyro¬ antimonate.…”

“…In their review of the technique, Wick & Hepler (1982) noted that when X-ray microanalysis was also used after pyroantimonate, calcium was either the only cellular cation associated with precipitates (13 references cited) or the major cationic component (6 references cited). The pyroantimonate technique has been widely used for studying calcium distribution in many tissues (see Wick & Hepler, 1982) and, in particular, human spermatozoa (Chandler & Battersby, 1976), guinea-pig spermatozoa (Friend, 1977), hamster testis (Gravis, 1979) and hamster ovarian tissue (Weakley, 1979). The precipitation of pyroantimonate with calcium has been con¬ firmed by (1) X-ray microanalysis (e.g.…”

Electron microscopy and X-ray microanalyses of uterine epithelium from lead-injected mice in an experimental delay of implantation

“…Various fixatives containing 2% potassium pyroantimonate (Komnick 1962;Gravis 1979) and various modes of fixation were tried. Mice were divided into groups (three or four mice in each group) for comparing the techniques.…”

“…Therefore we have examined the structural changes that appear in the uterine epithelium 1 day after injecting lead to mice in experimental delay of implantation. Since changes can be caused by a local deposition of lead in the uterine epithelium (Ljung et al 1984) or can be secondary to an effect in, for instance, the ovary (Wide 1983;Franks et al 1989), we also checked whether lead could be detected in the uterine epithelium 1 day after the injection, using X-ray microanalysis of the cation precipitates which appear after a potassium pyroantimonate fixation for electron microscopy (Komnick 1962;Gravis 1979). …”