BIOLOGY OF REPRODUCTION 49, 166-175 (1993) Nuclear Status of Human Sperm Cells by Transmission Electron Microscopy and Image Cytometry: Changes in Nuclear Shape and Chromatin Texture during Spermiogenesis and Epididymal Transit JACQUES AUGER 2 and JEAN-PIERRE DADOUNE 1 Groupe d'Etude de la Formation et de la Maturation du Gam&te male, UFR Biomddicale des Saints-Pdres University Rene Descartes, Paris, France ABSTRACT Computer-assisted transmission electron microscopy (TEM) image cytometry was used to investigate changes in nuclear shape and chromatin texture of human sperm during normal spermiogenesis and epididymal transit. Analysis was performed on a large series of micrographs of longitudinal sections of nuclei from spermatids and epididymal caput and cauda sperm. Thirteen param- eters characterizing nuclear shape and chromatin texture were measured. Quantitative data showed that from early spermiogen- esis to the end of epididymal transit, a decrease in nuclear area and width occurred concomitantly with not only an increase in chromatin condensation but also an increase in heterogeneity of the degree of condensation. The oriented spatial arrangement of chromatin along the major nuclear axis as measured by gradient parameters indicated that in humans, condensation of chro- matin begins in the posterior pole and proceeds apically; this is an important difference between humans and other mammalian species. Most parameters did not differ substantially in sperm from testis and caput epididymis, but did change as the cells moved from the caput to the cauda epididymis, indicating completion of nuclear maturation. Discriminant functions of basic parameters, as followed by canonical transformation and cluster representation, automatically classified the nuclei in a sequence that was found to concur with the biological maturation sequence during normal spermiogenesis and epididymal transit. INTRODUCTION Dramatic biochemical and ultrastructural changes in nu- clei occur as mammalian spermatids develop into mature spermatozoa. Histones of spherically shaped nuclei pos- sessing fine granular chromatin are replaced by more basic proteins, the transition proteins, in elongating and con- densing nuclei; and these basic nucleoproteins are then re- placed by even more basic sperm-specific nucleoproteins, the protamines, in mature spermatids with compact chro- matin [1-3]. Nuclear maturation continues in the epididy- mis through an increase in formation of protamine disul- fide bonds [4]. In the mouse and rat, spermatid nuclei abruptly undergo developmental stages characterized by increased resistance to disruption by various mechanical and chemical agents, and these alterations are correlated with changes in the basic nuclear proteins [5]. It has been sug- gested that the functional status of ejaculated sperm nuclei could result from these complex structural and biochemi- cal alterations. Thus, penetration through the oocyte vest- ments could be facilitated for elongated spermatozoa with dramatically condensed chromatin [6]. Subsequently, the early stages of embryogenesis might depend on nuclear proteins and chromatin organization [7, 8]. Various reports in mam- malian species support the hypothesis of a close relation- ship between nuclear maturity and fertility of ejaculated Accepted March 1, 1993. Received February 19, 1992. 'Correspondence: J-P. Dadoune, Laboratoire d'Histologie, UFR Biomedicale des Saints-P&res, 45 Rue des Saints-P6res, 75270 Paris cedex 06, France. FAX: 42-86-85- 12 2 Present address: CECOS (Centre d'Etude et de Conservation du Sperme Hu- main), H6pital de Bicetre, 94270 Le Kremlin Bictre, France. sperm [9-11]. This relationship could depend on events that occur during spermiogenesis and epididymal maturation. In the present investigation, changes in nuclear shape and chromatin texture of human sperm were analyzed through use of a new quantitative approach combining transmission electron microscopy (TEM) computer-assisted image cytometry, and multivariate statistical analysis. Simi- lar methods have proven useful in other cell types for quantitative assessment of nuclear changes occurring dur- ing cell cycling [12, 13]. Size, shape, and chromatin texture parameters were found to be salient indicators of stage-re- lated nuclear transformations from early spermiogenesis to the end of epididymal transit. Furthermore, a combination of the most discriminant parameters, as followed by cluster representation, automatically classified the nuclei in a se- quence that was found to concur with the biological se- quence of normal differentiation and maturation of human sperm. MATERIALS AND METHODS Collection of Tissue and Cell Samples Normal testicular and epididymal tissue samples were obtained using two methods: 1) testicular biopsies from four healthy patients aged 20-25 who were undergoing opera- tions for hydrocoel, and 2) removal of testis and epididymis from four men aged 25-40 with proven brain death. Epi- didymal spermatozoa were collected from caput and cauda epididymis by cutting and mincing into fragments in Hanks' solution. 166 Downloaded from https://academic.oup.com/biolreprod/article-abstract/49/1/166/2762228 by guest on 26 May 2020