A comparative morphological study of human germ cells in vitro or in situ within seminiferous tubules

For many infertile couples, intracytoplasmic germ cell/spermatozoon injecti on into unfertilized eggs may be their only hope for producing their own bi ological children. Thus far, success with injection of pre-spermatozoan ger m cells such as round spermatids has not been as great as that of spermatoz oon injection. This could be due in part to the difficulty of identifying y ounger (less mature) male germ cells in testicular biopsy dispersions. To i mprove the identification of various types of live, dispersed, human testic ular cells in vitro, a comparative study of the morphological characteristi cs of human spermatogenic germ cells in vitro or in situ within seminiferou s tubules was conducted. Live human testicular tissue was obtained from an organ-donating, brain-dead person with a high density of various germ cells . A cell suspension was obtained by enzymatic digestion, and cells were cul tured for 3 days in an excessive volume (100-fold medium:cells; v:v) of HEP ES-TC 199 medium at 5 degrees C and observed live with Nomarski optics (int erference-contrast microscopy). For comparative purposes, testes from ten m en obtained at autopsy were fixed, embedded in epoxy resin, sectioned at 20 mu m, and observed unstained by Nomarski optics. This approach allowed com parison of morphological characteristics of individual germ cells seen in v itro or in situ in the human testis. In both live and fixed preparations fr om control men with varied daily sperm production rates, Sertoli cells have oval to pear-shaped nuclei with indented nuclear envelopes and large nucle oli, which makes their appearance distinctly different from germ cells. The size, shape, and chromatin pattern of nuclei, and the presence of meiotic metaphase figures, acrosomic vesicles/structures, tails, and/or mitochondri a in the middle piece of germ cells are characteristically seen in live cel ls in vitro and in those cells observed in the fixed seminiferous tubules. Hence, this comparative approach allows verification of the identity of ind ividual germ cells seen in vitro and provides a checklist of distinguishing characteristics of live human germ cells, to be used by scientists and tec hnical staff in infertility clinics when selecting specific germ cells from a testicular aspirate or enzymatically digested biopsy.