Analysis of the Protein Composition of the Mouse Sperm Perinuclear Theca and Characterization of its Major Protein Constituent1

Abstract

The perinuclear theca (PT) is a cytoskeletal structure that covers the nucleus of mammalian spermatozoa and is believed to have a membrane-binding role. The objectives of this study were to analyze the protein composition of the mouse PT, to identify and sequence its major protein component, and to characterize this protein's transcriptional and translational origins during spermatogenesis. The PT was extracted from demembranated and acrosome-depleted mouse sperm heads by alkaline treatment. The protein profile of the PT extract was composed of several polypeptides, of which a 15-kDa subacrosomal protein predominated and was found to be immunocross-reactive with a previously cloned 15-kDa PT protein of the rat (PERF 15) that belongs to a family of lipid-binding proteins. A primer pair designed from rat PERF 15 cDNA was then used to screen a mouse testicular cDNA library by polymerase chain reaction (PCR). The deduced amino acid sequence obtained from the PCR product was almost identical to the testicular-specific rat PERF 15. Developmental Northern blots and in situ hybridization studies performed with riboprobes encoding the mouse PERF 15 cDNA revealed that mRNA levels were highest in round and early elongating mouse spermatids. Immunohistochemistry indicated that PERF 15 began to be expressed in the cytoplasm of mid-pachytene spermatocytes and appeared to reach maximum expression in the distal cytoplasm of late elongating mouse spermatids, long after transcriptional arrest. During the development of round and early elongated spermatids, the immunolabel became progressively concentrated over the anterior half of the spermatid nucleus, suggesting a subacrosomal deposition of PERF 15 during this phase of mouse spermiogenesis.

Mouse sperm acquire a new structure on the apical hook during epididymal maturation

“…A literature search revealed that while the ultrastructure of the rat sperm head was well documented, based primarily on the structure of step 19 spermatid, 1,4,24 the structure of a mature mouse sperm head had not been characterized in detail. 5,25,26 We thus used EM on both whole-mount cauda epididymidal sperm and thin sections of cauda epididymidis to determine the ultrastructure of mouse sperm head, paying special attention to the apical hook ( Figure 2). Images of the whole-mount sperm showed that the triangular rod of the anterior perforatorium was capped by a previously unnoticed circular structure of 160 nm in length by 40 nm in diameter (Figure 2a inset).…”

“…To the best of our knowledge, the HR has not been described before, though electron-dense regions were noted at the apexes of some published EM images of mouse perforatorial cross-sections. 5 The HR is located at the tip of the three corners of the mouse apical hook, and is quite sturdy, not disrupted by repeated sonication, acrosome reaction or harsh chemical conditions. During fertilization, the sperm have to drill through layers of cumulus cells and zona pellucida in order to reach and penetrate the egg.…”

“…The perinuclear theca/perforatoria of rat and mouse sperm have been isolated and characterized. 5,6,27,31 Both of them are composed of a major 15/16-kDa protein, designated PERF15 and several minor proteins. The cloning of rat and mouse Perf15 cDNAs provided the protein sequences that identified PERF15 as a member of the fatty acid-binding protein (FABP) family.…”

“…The cloning of rat and mouse Perf15 cDNAs provided the protein sequences that identified PERF15 as a member of the fatty acid-binding protein (FABP) family. 5,31 Mice lacking PERF15/FABP9 exhibit only minor spermatogenic defects. 32 They have a significant but modest increase in sperm with head abnormalities, including misshaped apical parts and the absence of ventral spur, but the total sperm count and fertility of the mice remained normal.…”

“…The perforatorium in rats and mice is in the shape of a curved triangular rod at the apex of the sperm head, and splits into three interconnected prongs, one dorsal and two ventral, as it passes over the nucleus. 5,6 It is thought to play a mechanical role in egg penetration during fertilization. 1 The postacrosomal sheath covers the posterior portion of the nucleus and thickens to form the ventral spur along the ventral side of the nucleus.…”