Temporal expression, polar distribution and transition of an epitope domain in the perinuclear theca during mouse spermatogenesis

Abstract

A novel domain of epitopes is expressed by a family of high-Mr proteins at the anterior pole of the germ cell nucleus during spermiogenesis, and later by two low-Mr proteins at the anterior and posterior poles of the nucleus during sperm maturation in the epididymis. Initially, monoclonal antibodies (mAbs) PNT-1 (IgG2b) and PNT-2 (IgG2a) bound to antigens present in a cap-like configuration at the apical pole of the spermatid nucleus at step 5 of spermiogenesis. The distribution of epitopes on the nucleus expanded posteriorly until, in testicular sperm they covered the anterior pole down to the distal limits of the subacrosomal perforatorium. By contrast, sperm from the epididymis and vas deferens bound both mAbs in two distinct regions on the nucleus, one on the dorsal margin of the anterior pole, and the other in a ventral zone at the posterior pole. On SDS-PAGE and isoelectric focusing (IEF) immunoblots, both mAbs bound three major proteins with Mr of approximately 80,000, 77,000 and 75,000 from spermatids and testicular sperm, and proteins of Mr 50,000 and 48,000 in epididymal and vas deferens sperm. Both the high- and low-Mr protein families were recovered in germ cell nuclear/perinuclear matrices. Their mobilities on SDS-PAGE were not altered by exo- or endoglycosidases or by aminoethylation in denaturing conditions. mAb PNT-1 bound to the sperm proteins with a Ka of 3.53 × 10(12) M-1 and mAb PNT-2 with a Ka of 2.08 × 10(12) M-1. From competition binding data, mAbs PNT-1 to -10 appeared to recognize six adjacent or overlapping epitopes on the same proteins. These data suggest the high-Mr proteins, the thecins, present at the anterior pole of haploid germ cells are processed at the onset of sperm maturation to yield two low-Mr proteins that then occupy two distinct domains at the anterior and posterior poles of the nucleus.