Previously, it was found that a novel class of neutral fucosylated glycosphingolipids (GSLs) is required for male fertility. These lipids contain very long-chain (C26 -C32) polyunsaturated (4 -6 double bonds) fatty acid residues (VLC-PUFAs). To assess the role of these complex GSLs in spermatogenesis, we have now investigated with which of the testicular cell types these lipids are associated. During postnatal development, complex glycosylated and simple VLC-PUFA sphingolipids were first detectable at day 15, when the most advanced germ cells are pachytene spermatocytes. Their synthesis is most likely driven by ceramide synthase-3. This enzyme is encoded by the Cers3/ Lass3 gene (longevity assurance genes), and out of six members of this gene family, only Cers3 mRNA expression was limited to germ cells, where it was up-regulated more than 700-fold during postnatal testicular maturation. Increasing levels of neutral complex VLC-PUFA GSLs also correlated with the progression of spermatogenesis in a series of male sterile mutants with arrests at different stages of spermatogenesis. Remarkably, fucosylation of the complex VLC-PUFA GSLs was not essential for spermatogenesis, as fucosylation-deficient mice produced nonfucosylated versions of the complex testicular VLC-PUFA GSLs, had complete spermatogenesis, and were fertile. Nevertheless, sterile Galgt1 ؊/؊ mice, with a defective meiotic cytokinesis and a subsequent block in spermiogenesis, lacked complex but contained simple VLC-PUFA GSLs, as well as VLC-PUFA ceramides and sphingomyelins, indicating that the latter lipids are not sufficient for completion of spermatogenesis. Thus, our data imply that both glycans and the particular acyl chains of germinal sphingolipids are relevant for proper completion of meiosis.The testis is composed of two functional compartments as follows: (i) the seminiferous tubules, containing developing germ cells and supporting Sertoli cells, and (ii) the steroidogenic Leydig cells in the interstitium (Fig. 1A) (1, 2). In mature testis, the seminiferous tubules are separated by a blood-testis barrier (BTB) 5 into a basal and an adluminal compartment. Tight junctions contribute to the establishment of the BTB, which is made up of adjacent Sertoli cells and physically segregates post-meiotic germ cells from nutrients and biomolecules in the systemic circulation (3, 4).The development of the male germ cells, taking place within the seminiferous tubules, is a complex and highly regulated process (2). During spermatogenesis, testicular stem cells (undifferentiated spermatogonia) give rise to a lineage of cells that multiply by mitosis (proliferative spermatogonia). These cells differentiate to go through the meiotic division (spermatocytes) and become haploid germ cells (spermatids), which transform into spermatozoa. It is during the meiotic prophase that leptotene spermatocytes transit the BTB. The post-meiotic development (spermiogenesis) involves a dramatic change of nuclear shape, chromatin condensation, the loss of most cell organelles, a...