Effects of Heparin-Alpha-Glucosaminide N-Acetyltransferase (Hgsnat) Gene Inactivation in the Testis and Epididymis of Adult Mice

CARVELLI L; PSHEZHETSKY AV; HERMO L; MORALES CR

San Juan

Congreso; SSR (Society for the Study of Reproduction) 2015 Annual Meeting; 2015

The Society for the Study of Reproduction

Introduction: Spermatozoa mature as they transit through the epididymal duct, and acquire motility and the ability to fertilize the egg. The epithelial cells of the epididymis are essential to the maturation process by the endocytosis of substances from the lumen and synthesis and secretion of proteins into the epididymal lumen. Heparan sulfate is a component of the extracellular matrix, basement membranes and the apical cell surface, and it is abundant in the testis and epididymis. After endocytosis, heparan sulfate is degraded in a stepwise fashion in lysosomes by the action of three glycosidases, three sulfatases, and heparin-alpha-glucosaminide N-acetyltransferase (HGSNAT). Although HGSNAT is not a hydrolase, it catalyzes the transmembrane acetylation of the terminal glucosamine residues of heparan sulfate prior to its hydrolysis by α-N-acetylglucosaminidase. A deficiency in HGSNAT results in a variant type of Sanfilippo syndrome (MPS IIIC), a human genetic disorder, which is characterized clinically by morphological and functional disorders of the brain. In the mouse, inactivation of the Hgsnat gene leads to a mild form of MPS IIIC, and animals at late ages show an apparent reduced size of liters. Objectives: To determine the morphological and biochemical effects of Hgsnat inactivation on epithelial cells of the testis and epididymis by routine LM and immunocytochemical analyses and by EM observervations. Materials and Methods: The testes and epididymides of both wild type and Hgsnat -/- adult mice at different ages, i.e., 7 and 11 (n=3 for each age) and 14 months (n=1 for each) were fixed by cardiac perfusion with 2.5% glutaraldehyde buffered in sodium cacodylate and processed for EM analysis. Prior to perfusion, the testis and epididymis of one side were removed and immersed in Bouin?s fixative for LM immunocytochemical studies with different antibodies. Results: In Hgsnat deficient mice, some seminiferous tubules revealed highly vacuolated areas, while other tubules demonstrated a major depletion of the germ cell population. Furthermore, approximately 20% of the tubules decreased in size as compared to wild type mice. These abnormalities increased with age of the mice. In the wild type epididymis, principal cells contained distinct dense spherical lysosomes, which contrasted the numerous empty looking vacuoles of different shapes and sizes that appeared both supranuclearly and infranuclearly in knockout mice. LM immunocytochemical data revealed a specific expression of cathepsin D and prosaposin in these vacuoles suggesting that they were lysosomal in nature. Clear cells became highly vacuolated and lacked their characteristic numerous dense lysosomes. These cells appeared to lose contact with the lumen and be greatly increased in size as they spanned a wider area of the epithelium. Moreover, halo cells were prominent as they were enlarged in size. Numerous spermatozoa were present in the lumen of the epididymal duct, but in addition, small spherical cells and debris were noted. These abnormalities were especially prominent in the caput to caudal regions with the initial segment being less affected.Discussion: Our results show that Hgsnat inactivation affects spermatogenesis and the normal appearance of epididymal epithelial cells, with a phenotype similar to well characterized lysosomal storage disorders. This provides the first evidence that glycan catabolism is important for normal reproductive functions.