Synthesis and Processing of Rat Sperm-Associated α-L-Fucosidase1

Abstract

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.

Fucosidases of Sperm and Milt in Darters (Percidae: Etheostomatini)

“…Sperm associated -L-fucosidases have been reported in several other organisms and are considered to be strong candidates for important roles during fertilization [11,12,16,[18][19][20][21][22]. This report describes a sperm associated -Lfucosidase in darters, suggesting that fish too may require -L-fucosidase for fertilization.…”

“…Substantial evidence in the literature supports roles for carbohydrates during fertilization [1][2][3][4][5][6][7][8][9][10]. Research over the past two decades has led to the identification of sperm associated -L-fucosidases in humans [11][12][13][14][15] rats [16][17][18], Drosophila [19][20][21], ascidians [22], Unio elongatulus [23], chimpanzees [24], bulls [25] and Syrian hamsters (Venditti, Swann, and Bean, in revision), but not yet in fishes. Additionally, L-fucose has been shown to be important during fertilization [26][27][28][29][30].…”

Identification, Localization and Involvement of Glycosidases in Sperm-Zona Interaction Using Frozen-Thawed Ejaculated Pig Spermatozoa.

“…However, in spermatozoa, glycosidases are thought to form stable enzyme-substrate complexes by binding with high specificity and affinity to the oligosaccharide residues of the glycoproteins of the ZP without acting as a catalyzer (for review see [2]). Many investigators have found various sperm glycosidases such as α-L-fucosidase in cattle [3,4] and rats [5], α-D-mannosidase in rats [6], mice [7] and humans [8], β-D-galactosidase in rabbits [9] and rats [10][11][12] and N-acetyl-β-D-glucosaminidase (GlcNAc'ase) in sheep [13], rats [10], cattle [14], buffaloes and goats [15], mice [16] and pigs [17]. These glycosidases have been implicated in spermzona interaction.…”

Organization and modifications of sperm acrosomal molecules during spermatogenesis and epididymal maturation

“…On the other hand, acrin3 (MC101) of mouse spermatozoa increases its antigenicity but the molecular size is not changed . Maturation-associated modifications are also reported in several intra-acrosomal proteins, including ␣-L-fucosidase (Hancock et al, 1993) and ␤-galactosidase (Skudlarek et al, 1993;Chayko et al, 2000). Our recent immunoelectron microscopy indicates that guinea pig acrin1 changes its location from the whole M1 domain to the spherical bodies in the M1 during sperm passage through the epididymal duct ( Fig.…”