Brain and other tissues from a patient with extensive neonatal ascites and clinical symptoms suggestive of a severe neurovisceral storage disorder were examined following autopsy for the accumulation of oligosaccharides. This carbohydrate analysis revealed the presence of large amounts (3--21) mumol/g fresh weight) of sialic acid in brain, liver, and kidney tissue as the major abnormality. Exhaustive characterization of the accumulating material by gel filtration, gas-liquid chromatography, thin-layer chromatography, and GLC-mass spectrometry positively identified the saccharide as free N-acetylneuraminic acid. Based on the accumulation of only free N-acetylneuraminic acid in the tissue of this patient, and normal activities of lysosomal enzymes involved in the catabolism of cellular glycoproteins, this storage disorder appears to result from a previously unreported defect in glycoconjugate metabolism.

Caprine beta-mannosidosis, an autosomal recessive disorder of glycoprotein catabolism, as yet undetected in man, was expressed in a 96/150 day gestation goat fetus. Deficiency of plasma, kidney, brain, liver and skin fibroblast acidic beta-mannosidase activity was associated with the accumulation of tissue oligosaccharides. Characteristic lucent cytoplasmic lysosomal storage vacuoles were present in the brain, thyroid, kidney and other tissues. Axonal spheroids were present in the central nervous system. The biochemical and morphological prenatal expressions of beta-mannosidosis which were documented by this investigation may facilitate the identification of the disease in man.

alpha-L-Fucosidase (alpha-fucosidase) is the most active of a number of glycosidases measured in rat epididymal sperm through use of artificial 4-methylumbelliferyl substrates. In addition, enzyme activity can be detected in purified populations of testicular germ cells; in comparison to round spermatids, caput epididymal sperm show at least a 1.8-fold increase in alpha-fucosidase activity per cell. Metabolic labeling of cultured testicular germ cells, followed by immunoprecipitation and SDS-PAGE, reveals synthesis of alpha-fucosidase as a polypeptide of 54 kDa in pachytene spermatocytes and round spermatids but no synthesis in condensing spermatids. This polypeptide is N-glycosylated but does not undergo further processing as determined by pulse/chase labeling and treatment with endoglycosidase H. In contrast, the alpha-fucosidase synthesized by cultured clone 9 cells (a rat liver-derived cell line) undergoes processing from a 54-kDa precursor to a slightly larger intermediate centered at 56 kDa on SDS-PAGE (via carbohydrate modification) and finally to a 52-kDa mature polypeptide. Immunoblotting confirms the presence of the 54-kDa form of alpha-fucosidase in testicular germ cells and shows the existence of a 52-kDa mature polypeptide in epididymal sperm. In addition, immunoreactive polypeptide is more prominent in caput epididymal sperm preparations; this is consistent with the increase in enzyme activity. In the absence of alpha-fucosidase synthesis beyond the round spermatid stage of development, it appears that alpha-fucosidase may be acquired from the luminal fluid by sperm during transit through the excurrent duct system. This hypothesis is supported by evidence that metabolically labeled cultured epididymal epithelial cells synthesize and secrete into culture medium an immunoprecipitable 58-kDa alpha-fucosidase polypeptide. Analysis of sperm isolated from the first part of the caput epididymis indicates that both acquisition and processing of sperm-associated alpha-fucosidase takes place prior to or concomitant with arrival of sperm in the epididymis.

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A number of hsp70-like proteins are associated with developing male germ cells. One of these molecules, P70, is not sensitive to heat stress and is germ cell-specific, and its expression is developmentally regulated. We have characterized the association of the rat P70(rP70) with differentiating germ cells in the testis and with posttesticular sperm. An antibody originally raised against human sperm proacrosin (designated C3; Sigel et al., 1987: J Reprod Immunol 11:307-319) was found to immunostain rP70 by immunoblot analysis and was used in subsequent studies of the rP70 molecule. The C3 antibody reacted with P70 isoforms in rat, human, mouse, guinea pig, boar, and rooster testicular homogenates. In the developing rat testis, abundant rP70 protein levels were first detected on postnatal day 22, with upregulation to adult levels occurring after postnatal day 28. Purified populations of adult rat pachytene spermatocytes, round spermatids, and elongating spermatids, isolated by unit gravity velocity sedimentation, all expressed rP70. Posttesticular sperm exhibited a loss of the rP70 molecule; caput epididymal sperm were weakly immunoreactive for rP70, but no immunoreactivity was observed in either cauda epididymal sperm or epididymal fluid. In contrast to human ejaculated sperm, rat ejaculated sperm did not express rP70. The loss of P70 from rat posttesticular sperm may reflect species-specific differences in P70 functions, which are thought to include a role in the structural modifications that occur during germ cell differentiation.

Cultured skin fibroblasts established from goats affected with beta-mannosidosis, an inherited neurovisceral storage disorder, showed an absence of lysosomal beta-mannosidase activity and the corresponding accumulation of a trisaccharide (TS) with the structure Man beta (1----4)GlcNAc beta (1----4)GlcNAc (0.4 mumol/g) and lesser amounts (0.15 mumol/g) of a Man beta (1----4)GlcNAc disaccharide (DS). By using purified storage TS isolated from fibroblasts metabolically labelled with [3H]GlcN, no conversion of TS into DS could be demonstrated in homogenates of affected cells at either lysosomal pH (4.4) or cytosolic pH (6.1), or in the culture medium (pH 7.0) of affected cells. Both TS and DS were secreted into the culture medium by affected fibroblasts. When affected fibroblasts were treated with tunicamycin before labelling with [3H]GlcN, the accumulation of both labelled TS and DS was completely inhibited. Treatment of both affected and normal goat fibroblasts with swainsonine resulted in the inhibition of lysosomal alpha-mannosidase activity and in the accumulation of the same labelled oligosaccharides in both. The major storage pentasaccharide from both normal and affected swainsonine-treated fibroblasts was sensitive to digestion with alpha-mannosidase and endo-beta-N-acetylhexosaminidase D, suggesting a branched mannose structure and a chitobiose core. In the absence of evidence for the existence of unusual N-linked glycoprotein-associated chitotriose oligosaccharide structures in affected goat fibroblasts, it must be concluded that degradative pathways for N-linked oligosaccharides are similar in both normal and affected goat fibroblasts, and that these pathways differ from catabolic pathways in human fibroblasts.

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