Marjorie D. Skudlarek

Biosynthesis, Processing, and Subcellular Localization of Rat Spermβ-d-Galactosidase

Abstract During spermatogenesis, spermatids synthesize constituent proteins present in mature spermatozoa; however, little information exists on the molecular processes involved. In previous studies, this laboratory reported the characterization of rat sperm β-d-galactosidase. In this paper, we report the localization of this enzyme along with its biosynthesis and processing. An antibody against rat luminal fluid β-d-galactosidase was used to immunolocalize the enzyme in the testis and in epididymal spermatozoa. We found that β-d-galactosidase is localized within the acrosomal cap of spermatids and in the acrosome and cytoplasmic droplet of epididymal spermatozoa. A combination of germ cell radiolabeling, immunoprecipitation, SDS-PAGE, and autoradiography revealed that spermatids produce two forms of β-d-galactosidase, 90 and 88 kDa. During pulse-chase analysis, a 56-kDa form appeared. Treatment of β-d-galactosidase immunoprecipitates from testicular spermatozoa with N-glycanase or Endo H revealed that both the 90- and 88-kDa forms become a 70-kDa polypeptide on SDS-PAGE. Since Endo H or N-glycanase treatment provided similar results, the presence of extensive N-linked high mannose/hybrid-type glycans on these proteins is indicated. Treatment of the 56-kDa form of β-d-galactosidase with Endo H or N-glycanase resulted in the appearance of 52- and 50-kDa forms, respectively. This result suggests that the 56-kDa form contains N-linked high mannose/hybrid as well as complex oligosaccharides. During epididymal maturation, the 90-kDa form of β-d-galactosidase persists in caput epididymal spermatozoa and is gradually converted to a major 74-kDa form in cauda spermatozoa. In addition to the 90- to 74-kDa forms, cauda spermatozoa show a 56- to 52-kDa form on Western immunoblots. Since only the high-molecular weight forms of β-d-galactosidase are present on immunoblots of isolated sperm heads, we suggest that they are acrosomal in origin and that the 56-kDa form, which is processed to 52 kDa in cauda spermatozoa, is associated with the cytoplasmic droplet.

Identification and androgen regulation of egasyn in the mouse epididymis

The expression and androgen regulation of egasyn, the endoplasmic reticulum-targeting protein of beta-D-glucuronidase, was examined in the mouse-epididymis. The proximal (caput) and distal (corpus & cauda) epididymal tissue extracts were prepared by homogenization and sonication in buffered Triton X-100 solution, and high speed centrifugation. The supernatant when resolved by 2D-PAGE under non-denaturing conditions and stained for esterase activity showed that the distal (but not proximal) epididymis of the normal mouse contain several specific forms of esterases. These forms include a series of four variants (pI 5.2-5.75) with high mobility (HM) and esterase activity, and three faintly staining variants (beginning at pI 6.0) with low mobility (LM). Several lines of evidence indicate that the specific esterases seen in the corpus/cauda epididymidis are egasyn-esterases. Firstly, these molecular forms were not seen in the distal epididymal extracts from the egasyn-deficient mouse. Secondly, the HM forms can be immunoprecipitated with anti-egasyn antibody, suggesting the presence of free egasyn. Finally, the LM forms disappeared after heat treatment (56 degrees C for 8 min), a condition known to dissociate egasyn:beta-D-glucuronidase complex. This result indicates that a small amount of egasyn is complexed with beta-D-glucuronidase. Immunoblotting (Western blot) studies (using anti-egasyn antibody) following resolution of egasyn released from the egasyn:beta-D-glucuronidase complex revealed a single band of an apparent molecular weight 64 kDa in the distal (but not proximal) epididymis, indicating that the mouse epididymal egasyn is identical or very similar to the liver egasyn. Castration of mice lead to the appearance of free and complexed egasyn forms in the proximal epididymis. Testosterone supplementation to the castrated mice resulted in the disappearance of the induced egasyn forms from the caput epididymidis. Taken together, these results indicate that the expression of egasyn in the epididymis is region-specific and is differentially regulated by androgens.