Structural Chemistry of Outer Dense Fibers of Rat Sperm

Rat sperm outer dense fibers (ODF) have been studied by several electron microscopic tech niques to determine fine structural features of the ODF cortex and medulla. Cross sections of Triton X-100 demembrananed flagella, fixed in the presence of ruthenium red, reveal a well defined ODF cortex which appears to be composed of a single lamina of 6â€”7nm diameter globular parti des, whereas the medulla appears electron dense with no obvious substructure. Surface replicas and positively or negatively stained whole mount preparations reveal a crossbanding pattern with a 40 nm major period repeat that extends obliquely over the outer dense fiber surface. These periodic ities appear to be confined no the outer dense fiber cortex. Sequential solubilization of spermatozoan organelles in sodium dodecyl sulfate or sodium lauroyl sacrosine results in the isolation of a flagellar subfraction containing only the ODF and attached connecting piece. Further solubilization of the complex in the ionic detergents results in an ODF subfraction which contains 4 polypeptide components. The 3 low molecular weight polypeptide bands have a high cysteine content (10â€”12%)while the high molecular weight com ponent (87,000 daltons) contains â€œ? 3% cysneine. Brief nrypsin or chymotrypsin digestion of dc. membranated flagella gives an ODF subfracnion which by SDS polyacrylamide gel electrophoresis is shown to possess only the 3 low molecular weight polypeptide bands. These ODFs appear no possess an intact medulla but are lacking a cortex, thereby suggesting that the low molecular weight components are located in the medulla. 320 OLSON AND SAMMONS movement in mammalian spermatozoa results from sliding interactions of axonemal doublet microtubules (Lindemann and Gibbons, 1975; Afzelius et al., 1975; Pedersen and Rebbe, 1975) have led to recent suggestions that the ODFs function as passive elastic structures (for review see Fawcett, 1975). In view of the probable role of the ODFs in affecting the bending pattern of the mam malian sperm flagellum, this study was under taken to provide additional data on their structure and composition. Several electron microscopic techniques were employed to resolve new features of ODF substructure and to allow structural information on rat sperm ODF to be correlated with morphological data from other species. In addition, the principal structural polypeptides of the ODF were isolated on a preparative scale and their amino acid compositions are reported. Finally, selec tive proteolytic digestion of flagellar compo nents and correlated electron microscopic analysis were employed to localize specific polypeptides to the ODF medulla. MATERIALS AND METHODS Isolation of Spermatozoa Adult Sprague-Dawley rats were anesthetized with Nembutal. The cauda epididymides were removed, placed in a watch glass and covered with PBS (phos phate buffered saline composed of 0.145 M NaC1 and 0.01 M NaH2PO4/Na2HPO4, pH 7.0). The epididy mides were minced with a razor blade; the spermato zoa released into the PBS were collected with a Pasteur pipette. Spermatozoa were pelleted by centrif ugation at 500 g for 10 mm and used in the following protocols. Isolation ofSperm Tails Sperm flagella were isolated according to a modifi cation of the procedure reported by Calvin (1976). Spermatozoa from each animal were suspended in 7â€”8ml ice cold PBS and sonicated with ten, 10 sec bursts at 100 W with a Braun sonicator to detach the heads and tails. One aliquot of the sperm suspension was mixed with 6 parts of 75% sucrose (w/v), 10 mM sodium phosphate (pH 7.0). Aliquots (3 ml) of the sperm mixture were layered over sucrose step gradi ents containing equal volume steps of 65%, 70% and 75% sucrose (w/v), each containing 20 mM sodium phosphate (pH 7.0). The gradients were centrifuged for 1 h at 100,000 X g in a SW 41 rotor (12 ml gra dients) or a SW 25.1 rotor (32 ml gradients) and the tails that layered at the 65â€”70% interface were removed with a Pasteur pipette, diluted with PBS and pelleted at 50,000 X g for 15 mm. Triton X-100 Extraction Pellets of intact spermatozoa or isolated flagella were extracted in 2 changes of 15 mm each with at least 20 volumes of 1% Triton X-100, 2 mM dithio threitol (DTT), 100 mM NaCI and 25 mM Tris HC1 (pH 9.0). After extraction, the spermatozoa were washed twice in 100 mM NaCl, 25 mM Tris HC1 (pH 7.5) by centrifugation. The final pellets were immediately prepared for electron microscopy or subjected to enzymatic digestion or used for SDS polyacrylamide gel electrophoresis. Enzymatic Digestion ofSperm Tails Pellets of Triton-DTT extracted sperm tails were suspended at room temperature in 20 volumes of PBS containing 1 mg/mI trypsin or chymotrypsin (Sigma Chemical Co.). At 15 mm intervals for periods up to 1 h, duplicate aliquots of the suspension were removed and the nondigested material was pelleted by centrif ugation at 10,000 X g for 10 mm. The pellets were washed twice in PBS by resuspension and centrif ugation and the final pellets were used for SDS poly acrylamide gel electrophoresis and electron micro scopy. isolation ofthe Connecting Piece-Outer Dense Fiber Complex Two procedures were employed to isolate connect ing piece-outer dense fiber complexes. In the first, intact spermatozoa suspended in PBS at a concentra tion of 4â€”6 x 10' cells/mI were mixed with an equal volume of 2% sodium dodecyl sulfate (SDS), 4 mM DTT and 50 mM Tris HC1 (pH 9.0). In some experi ments 2% sodium lauroyl sarcosine (SLS) was substi tuted for the sodium dodecyl sulfate. The suspension was monitored microscopically until the sperm heads detached from the tails and the tails separated into individual fibers joined together at the connecting piece (usually 10â€”15 mm). Aliquots (1 ml) of the sperm extract were layered on top of 40 ml, 30â€”60% linear sucrose gradients containing 0.1% SDS. The tubes were centrifuged at room temperature for 15 mm at 1500 rpm in a Sorval GLC-1 tabletop centri fuge equipped with a swinging bucket head. Two bands were visible after centrifugation, the upper containing the flagellar subfraction and the lower the sperm heads. The bands were removed from the tubes with an ISCO density gradient fractionator. Fractions in which tail components accounted for at least 95% of the particles were pelleted by centrifugation at 50,000 X g for 15 mm and the pellets used for bio chemical and ultrastructural analysis. In the second procedure, pellets of isolated sperm tails were cx tracted in 20 volumes of 1% SDS, 2 mM DTT, 25 mM Tris HC1 (pH 9.0) or 1% SLS, 2 mM DTT, 25 mM Tris HO (pH 9.0). After various extraction times, aliquots of the suspension were removed and the nonsolubilized components were pelleted by centrif ugation at 10,000 X g for 10 mm and the pellets used for electron microscopy and SDS polyacrylamide gel electrophoresis. SPERM DENSE FIBERS 321