Rat spermatogenic cell beta-D-galactosidase: characterization, biosynthesis, and immunolocalization.
In this study we have demonstrated that the rat sperm acrosomal beta-d-galactosidase is expressed in late spermatocytes and spermatids (round, elongated/condensed) during spermatogenesis. The enzyme is an exoglycohydrolase which, along with other exoglycohydrolases and proteases, is thought to aid in penetration of the zona pellucida, the extracellular glycocalyx that surrounds the mammalian egg. The presence of the enzyme in spermatocytes was confirmed by multiple approaches using biochemical, biosynthetic, and immunohistochemical protocols. The germ cells (spermatocytes, round spermatids, and elongated/condensed spermatids), purified from rat testis, were found to contain beta-galactosidase and four other glycohydrolases (beta-d-glucuronidase, alpha-d-mannosidase, alpha-l-fucosidase, and beta-N-acetylglucosaminidase). With the exception of alpha-l-fucosidase, the other enzymes assayed demonstrated a two- to threefold higher activity per cell in spermatocytes than in round spermatids. Immunoblotting approaches of affinity-purified germ cell extracts demonstrated several molecular forms of beta-galactosidase in spermatocytes and round spermatids; one of these forms (62 kDa) was seen only in round spermatids. The biosynthetic approach demonstrated that the enzyme is synthesized in spermatocytes and round spermatids in culture in high-molecular-weight precursor forms (90/88 kDa) which undergo processing to lower molecular weight mature forms in a cell-specific manner. The net result is the formation of predominantly 64- and 62-kDa forms in spermatocytes and round spermatids, respectively. The conversion of precursor forms to mature forms in the diploid and haploid cells in culture is rapid with t(1/2) of 6.5 and 9.0 h, respectively. Immunohistochemical approaches revealed an immunopositive reaction in the Golgi membranes, Golgi-associated vesicles, and lysosome-like structures in the late spermatocytes and early round spermatids. The forming/formed acrosome in round and elongated spermatids was also immunoreactive.
Skudlarek
M D
MD
Department of Obstetrics & Gynecology, Department of Cell Biology, Vanderbilt University School of Medicine, Room D-3243 MCN, Nashville, Tennessee 37232-2633, USA.
Abou-Haila
A
A
Tulsiani
D R
DR
D000118:Acetylglucosaminidase / Q000378:metabolism; D000818:Animals; D005966:Glucuronidase / Q000378:metabolism; D007150:Immunohistochemistry; D008297:Male; D008361:Mannosidases / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D013087:Spermatids / Q000201:enzymology; D013090:Spermatocytes / Q000201:enzymology; D013091:Spermatogenesis / Q000502:physiology*; D013094:Spermatozoa / Q000201:enzymology*; D013737:Testis / Q000166:cytology / Q000201:enzymology; D005644:alpha-L-Fucosidase / Q000378:metabolism; D043323:alpha-Mannosidase; D001616:beta-Galactosidase / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism*
D008361:Mannosidases; D001616:beta-Galactosidase; D043323:alpha-Mannosidase; D005966:Glucuronidase; D005644:alpha-L-Fucosidase; D000118:Acetylglucosaminidase