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

For many infertile couples, intracytoplasmic germ cell/spermatozoon injection into unfertilized eggs may be their only hope for producing their own biological children. Thus far, success with injection of pre-spermatozoan germ cells such as round spermatids has not been as great as that of spermatozoon injection. This could be due in part to the difficulty of identifying younger (less mature) male germ cells in testicular biopsy dispersions. To improve the identification of various types of live, dispersed, human testicular cells in vitro, a comparative study of the morphological characteristics of human spermatogenic germ cells in vitro or in situ within seminiferous 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 cultured for 3 days in an excessive volume (100-fold medium:cells; v:v) of HEPES-TC 199 medium at 5 degrees C and observed live with Nomarski optics (interference-contrast microscopy). For comparative purposes, testes from ten men obtained at autopsy were fixed, embedded in epoxy resin, sectioned at 20 mu m, and observed unstained by Nomarski optics. This approach allowed comparison of morphological characteristics of individual germ cells seen in vitro or in situ in the human testis. In both live and fixed preparations from control men with varied daily sperm production rates, Sertoli cells have oval to pear-shaped nuclei with indented nuclear envelopes and large nucleoli, 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 mitochondria in the middle piece of germ cells are characteristically seen in live cells in vitro and in those cells observed in the fixed seminiferous tubules. Hence, this comparative approach allows verification of the identity of individual germ cells seen in vitro and provides a checklist of distinguishing characteristics of live human germ cells, to be used by scientists and technical staff in infertility clinics when selecting specific germ cells from a testicular aspirate or enzymatically digested biopsy.