COMBINED USE OF PROACROSIN IMMUNOCYTOCHEMISTRY AND AUTOSOMAL DNA IN-SITU HYBRIDIZATION FOR EVALUATION OF HUMAN EJACULATED GERM-CELLS

The recently reported human pregnancies and births after fertilising o ocytes with round spermatids recovered from the ejaculate of men with non-obstructive azoospermia have underscored the need for a more accur ate evaluation of the nuclear and cytoplasmic maturation status of eja culated germ cells. In this study we describe our first experience wit h a method combining the immunocytochemical visualisation of proacrosi n with autosomal DNA fluorescence in situ hybridisation (FISH) to asse ss ejaculated germ cells from patients with a spermiogenesis defect. T he proacrosin immunoreactivity, analysed with the use of the monoclona l antibody 4D4, has been detected in cells of round spermatid size pre senting a haploid FISH figure as well as in larger cells whose ploidy corresponds to primary and secondary spermatocytes. These observations are in agreement with previously published results obtained, with the use of the same antibody, by immunocytochemical analysis of histologi cal sections of testicular tissue. All the cells of round spermatid si ze possessing proacrosin immunoreactivity were found to be haploid by FISH. On the other hand, some of the haploid cells of round spermatid size did not possess proacrosin immunoreactivity. The structural patte rn of proacrosin immunoreactivity was highly variable both in spermati ds and in younger spermatogenic cells. These data show that cell size is the main criterion to be used for the identification of ejaculated round spermatids, whereas the presence of the developing acrosome repr esents only an auxiliary criterion. The scoring of acrosomal developme nt in ejaculated spermatids may be useful as part of pre-treatment dia gnosis before the inclusion of infertile couples in a spermatid concep tion programme.