The Development of Regionalized Lipid Ditfusibility in the Germ Cell Plasma Membrane during Spermatogenesis in the Mouse

The lipids and proteins of sperm cells are highly regionalized in their lateral distribution. Fluorescence recovery after photobleaching studies of sperm membrane component lateral diffusibility have shown that the sperm plasma membrane is also highly regionalized in the extents and rates of diffusion of its surface components. These studies have also shown that regionalized changes in lateral diffusibility occur during the differentiative processes of epididymal maturation and capacitation. Unlike mammalian somatic cells, sperm cells exhibit large nondiffusing lipid fractions. In this paper, we will show that both regionalized lipid diffusibility and nondiffusing lipid fractions develop with the morphogenesis of cell shape during spermatogenesis in the mouse. Pachytene spermatocytes and round spermatids show diffusion rates and the nearly complete recoveries (80-90%) typical of mammalian somatic cells. In contrast, stage 10-11 condensing spermatids, testicular spermatozoa, cauda epididymal spermatozoa, as well as the anucleate structures associated with these later stages of spermatogenesis (residual bodies and the cytoplasmic droplets of condensing spermatids and testicular spermatozoa), exhibit large nondiffusing fractions. Both the diffusion rates and diffusing fractions observed on the anterior and posterior regions of the head of stage 10-11 condensing spermatids are the same as the values obtained for these regions on testicular spermatozoa. Possible mechanisms of lipid immobilization and possible physiological implications of this nondiffusing lipid are discussed. M ATURE spermatozoa are highly differentiated cells, whose surface components, both protein and lipid, are highly regionalized in their lateral distributions (4, 14, 18, 21, 25, 26, 30, 32, 33, 44, 45). These surface regionalizations, which presumably reflect the specialized functions of specific surface regions, evolve with the further differentiation of the spermatozoa during epididymal maturation and capacitation (4, 32, 35, 37). A critical unanswered question is how spermatozoa restrict the free random diffusion of their membrane components to effect lateral surface regionalization. To address this question, several recent studies have used the technique of fluorescence recovery after photobleaching (FRAP) l to measure the lateral diffusion rates of both proteins and lipids within or between distinct surface regions of the plasma membrane of mammalian spermatozoa (31, 55, 58, 59). In the context of the present study, these experiments have provided at least three noteworthy observations. 1. Abbreviations used in this paper: Ct6diI, 1, l'-dihexadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate; D, diffusion coefficient; FRAP, fluorescence recovery after photobleaching; %R, percent recovery. First, lipids are not completely free to diffuse laterally in the plasma membrane of mammalian spermatozoa. That is, a significant fraction of the lipid is immobile and does not recover in a FRAP measurement. This has been observed by Wolf and Voglmayr (55) in ram spermatozoa for the fluorescent lipid analogue 1,1'-dihexadecyl-3,3,Y,Y-tetramethylindocarbocyanine perchlorate (C16diI), by Wolf et al. (58) in mouse spermatozoa for Ct6diI, and by D. E. Koppel (personal communication) in guinea pig spermatozoa for C16diI and nitrobenzoxadiazole-labeled phospholipids. In contrast, the lipids of most homeothermic cells show complete or nearly complete diffusibility (12, 37, 53). Thus, these large nondiffusing fractions appear to be a trademark of mammalian spermatozoa. This in turn raises the questions: What is the cause of these nondiffusing fractions? Is this cause related to the mechanisms of surface regionalization? Do nondiffusing lipids play a role in sperm physiology? Second, Wolf and Voglmayr (55) have shown in ram and Wolf et al. (58) in mouse that the lateral diffusibility of Cl6diI differs over the morphologically distinct regions of the sperm surface. Significant differences between regions are observed for both lipid probe diffusion coefficient (D) © The Rockefeller University Press, 0021-9525/86/11/1745/6 $1.00 The Journal of Cell Biology, Volume 103, November 1986 1745-175