摘要：

EFFECT OF DIVALENT IONS IN ACROSOME REACTION INDUCED BY GLYCOSAMINEGLYCANS IN PORCINE SPERMATOZOA N. M. DELGADO, A. CARRANCO, H. MERCHANT, and R. REYES Magnesium, calcium, and zinc at the concentration of 10 pM are capable of inducing a "true" acrosome reaction in the pig spermatozoa judged b y the criteria of the fusion of the acrosome and the plasmatic membrane at the anterior region or the sperm nucleus. The optimal percent of acrosome reaction reached b y any of the ions tested as a whole was 50%.When glycosamine- glycan sulfate (GAGs) plus 10 pM of Mg*, Ca*. or Zn* was added, they reach to 70-80% of acrosome reaction. At the electrom microscope, thin sections taken from pig spermatozoa +treated with ions, GAGs, or ion GAGs under optimal experimental conditions revealed the same pattern of acrosomal reaction. Results suggest the important role that divalent cations play in general in the induction of the acrosome reaction and question the so-called essential role of calcium ions. Key Words: Divalent ions; Glycosamineglycan; Folicular fluid; Phospholipase; Acrosome membrane. INTRODUCTION A new current in the field of biology of reproduction implies the participation of glycosamine- glycan sulfate (GAGs) as one of the principal effectors of acrosomal reaction [ l o , 11, 151 and in other events of the process of fertilization [3, 7-9, 131. Through this methodology we have been able to show that at least in the case of the pig spermatozoa there was no need of Ca" ions for the achievement of the acrosomal reaction. However, there is no doubt of the importance that divalent cations play in some important steps of the process of fertiliza- tion as zinc [8, 141. Therefore, it seems necessary to evaluate the participation of divalent cations in a mechanism performed without the participation of divalent ions and proteolytic activity such as the acrosomal reaction in the pig spermatozoa induced by GAGS extracted from pig follicular fluid. Received May 31, 1984. From the Secci6n Enzimologia, Unidad de Investigacih Biomddica del Centro Mddico Nacional, Instituto Mexicano del Seguro Social, Mdxico, D.F. 03020, Mexico (N.M.D., A.C., R.R.), and the Departamento de Biologia del Desarrollo, Instituto de Investigaciones BiomBdicas, Universidad Nacional Aut6noma de MBxico, MBxico, D.F. Mexico (H.M.). Address reprint requests to: N. M. Delgado, S e c c i h d e Enzimologia, Unidad de Investigacih Biomddica del Centro MBdico Nacional, Instituto Mexicano del Seguro Social, Apartado postal 73-231, Mdxico, D.F. 03020, Mexico. ARCHIVES O F ANDROLOGY 14:193-198(1985) Copyright 0 1985 by Hemisphere Publishing Corporation 193 194 N. M Delgado e t al. MATERIAL AND METHODS Porcine ovaries and follicular fluid and the extraction of GAGs were obtained as described previously [ 151 with a slight modification that consisted in giving exhaustive washing to the GAGs t o get rid of ions. Ejaculated pig spermatozoa were obtained from Duroc adult animals. All were sexually experienced and previously shown t o be fertile. The sperm cell was washed twice with a salt medium (296 mOs/kg) containing 0.1 15 M sodium chloride, 0.020 M Tris (hydroxymethylamino) methane (chloride salt), 0.0125 monopotassium phosphate, 0.0025 M dipotassium phosphate, adjusted t o pH 7.4, and resuspended t o a final concentration of 1o8 cells/ml. To separate aliquots containing lo8 cells/ml, 0 (blank) and 0.5 mg of GAGs free of ions extracted from porcine follicular fluid were added; in addition, separate aliquots of pig spermatozoa were treated with 10 pM of Ca*, Mg*, Zn", or a mixture of ion-GAGS. The suspensions were incubated by mixing them at 37'C. Aliquots were extracted at preestablished times [ 1 5 1 . The evaluation of the percent of spermatozoa with acrosome reaction and electron microscopy were performed as previously described [ 151 . The highest inorganic quality Merck reagent and standard solution of divalent ions for atomic absorption spectroscopy were used. RESULTS AND DISCUSSION The acrosome reaction of mammalian spermatozoa is characterized by a sequence of events

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