Biosynthesis of rat sperm outer dense fibers during spermiogenesis. In vivo incorporation of [3H]leucine into the fibrillar complex

The temporal incorporation profile of [3H]leucine into the outer dense fiber polypeptides was determined after the intratesticular injection of the radioisotope. Groups of four rats were killed on alternate days after injection, and the outer dense fibers were isolated from the caput epididymal sperm. The radioactivity incorporated into the whole sperm and into the isolated fibers showed a sharp peak at 10 days after injection. Therefore, considering the known kinetics of spermatogenesis in the rat, the maximal incorporation of radioactivity into the fibers occurred during the second half of spermiogenesis. The radioactivity incorporated into the six major polypeptides of the fibers separated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate accounted for 95 percent of the total radioactivity associated with the isolated fibrillar complex. Furthermore, analysis of the time-course incorporation of [3H]leucine into the polypeptides of the fibers indicated that the maximal incorporation into each of the six major components took place within the same period of time. Using two different procedures, the specific activity of each major polypeptide was determined at the time of maximal incorporation. It was found that the specific activity of the most abundant components (molecular weights of 30,400 plus 26,000) was approximately twice that of the other polypeptides.     

Antibodies to rat sperm tail polypeptides recognize Sertoli cell secretory proteins

We have previously reported that (a) polyclonal antisera raised against rat Sertoli cell secretory protein S70 and S45-S35 heterodimeric protein recognize outer dense fiber polypeptides from rat sperm tail, and (b) protein S70 is antigenically related to polypeptides S45 and S35, the disulfide-linked components of the heterodimeric protein. We now report that polyclonal antisera generated against three different outer dense fiber polypeptides recognize (a) the putative antigen of the sperm tail and (b) Sertoli cell secretory protein S70 and its antigenically-related polypeptides. Immunogold electron microscopy shows that outer dense fibers of epididymal sperm crossreact with anti-S70 serum as well as with an antiserum raised against the polypeptide D complex of extracted outer dense fibers. Electron microscopy demonstrates that outer dense fibers consist of filamentous, coil-coiled units aligned side-by-side with each other. Results of this study strengthen the antigenic homology between Sertoli cell secretory proteins and outer dense fiber polypeptides of the sperm tail.     

Polypeptide composition of rat sperm outer dense fibers. A simple procedure to isolate the fibrillar complex

Treatment of caput or cauda epididymal rat sperm with a low concentration (0.05%) of the cationic detergent cetyltrimethylammonium bromide and 30 mM 2-mercaptoethanol solubilized most of the sperm structures except for the sperm head and the outer dense fiber-connecting piece complex. The latter were purified, and about 10% of these complexes are formed by nine fibers attached to the connecting piece. Of these fibers, two are shorter than the other seven and presumably correspond to fibers 3 and 8 (Fawcett, D.W. (1975) Dev. Biol. 44, 394-436). Electron microscopy confirmed the purity of the isolated outer dense fibers and revealed their characteristic irregular cross-sectional shape. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed six major polypeptides (Mr = 87,000, 30,400, 26,000, 18,400, 13,000, and 11,500) with a high content of serine, aspartic and glutamic acids, proline, cysteine, leucine, and tyrosine. Furthermore, several lines of evidence indicate a close structural relationship between the components of 30,400 and 26,000 Da. The six major components of the fibers are phosphorylated at serine residues. These results indicate that the major components of rat sperm outer dense fibers are a unique family of phosphoproteins.     

Structural Proteins of Rabies Virus

Purified rabies virions, unlabeled or labeled with radioactive amino acids or d-glucosamine, were dissociated into their polypeptides by treatment with sodium dodecyl sulfate in a reducing environment and fractionated by electroiphoresis in sodium dodecyl sulfate-containing polyacrylamide gel. The molecular weights of individual polypeptides were estimated by comparison of their rate of migration with that of protein markers of known molecular weight. Purified viral nucleocapsid and a mixture of envelope components, isolated from virions disrupted by sodium deoxycholate, were analyzed by the same procedure. The number of molecules per virion of each polypeptide was estimated from the proportions of the separated components, the known molecular weight of the viral ribonucleic acid, and the chemical composition of the nucleocapsid. The protein moiety of the nucleocapsid particle was estimated to consist of 1,713 molecules of a major polypeptide (molecular weight, 62,000 daltons) and 76 molecules of a minor polypeptide (molecular weight, 55,000 daltons). In addition to 1,783 molecules of a glycoprotein component (molecular weight, 80,000 daltons), the viral envelope contains 789 and 1,661 molecules, respectively, of two other polypeptides (molecular weight, 40,000 and 25,000 daltons).     

Comparative analysis of proteins from the fibrous sheath and outer dense fibers of rat spermatozoa

The protein composition of the fibrous sheath (FS) and the outer dense fibers (ODF), two cytoskeletal components of the tail of spermatozoa, was compared by using polyacrylamide gel electrophoresis and immunochemistry applied to Western blots and to spermatozoa. Isolated FS and ODF, the purity of which were verified by electron microscopy (EM), were denatured and either run on sodium dodecyl sulfate-polyacrylamide gels or used to raise antibodies. The gels revealed at least 18 and 14 polypeptide bands for the FS and ODF, respectively. The major bands of the FS had molecular masses of 75, 27.5, and 14.4 kDa, whereas the major bands of the ODF-connecting piece had molecular masses of 32-26, 20, 14.4, 84, and 80 kDa. Several prominent FS and ODF bands were found to comigrate on gels, and the 14.4 kDa polypeptides had similar electrophoretic properties. Anti-FS serum reacted with the majority of Western blot-transferred FS polypeptides, but also cross-reacted strongly with a major 14.4 kDa ODF polypeptide and with less affinity to other major ODF polypeptides. Anti-ODF serum reacted with the majority of ODF polypeptides, but also cross-reacted strongly with a major 14.4 kDa FS polypeptide, and with less affinity to several other FS polypeptides including the 75 kDa band. Antibodies affinity-purified from the 14.4 kDa FS polypeptide only cross-reacted with the 14.4 kDa ODF polypeptide, whereas antibodies purified from the 14.4 kDa ODF polypeptide cross-reacted with 14.4, 27.5, 57, and 63 kDa FS polypeptides. The immunocross-reactions observed on Western blots were confirmed by immunocytochemical methods applied to spermatozoa. This study demonstrates that the FS and ODF, both composed of many polypeptides, several having similar molecular weights, are related cytoskeletal structures as they have epitopes in common, and both contain 14.4 kDa polypeptides with common antigenic and electrophoretic properties.     

Purification of an 80,000-dalton protein that is a component of the isolated microvillus cytoskeleton, and its localization in nonmuscle cells

The microvillus cytoskeleton, isolated from chicken intestinal epithelial cell brush borders, is known to contain five major protein components, the 110,000-dalton polypeptide, villin (95,000 daltons), fimbrin (68,000 daltons), actin (43,000 daltons), and calmodulin (17,000 daltons). In this paper we describe our first step in studying the minor components of the isolated core. We have so far identified and purified an 80,000-dalton polypeptide that was present in the isolated structure in approximately 0.7% the molar abundance of actin. Antibodies to the 80,000-dalton component did not react with other microvillus core proteins, and, when used in indirect immunofluorescence microscopy, they stained the microvilli of intestinal epithelial cells fixed in situ. The 80,000-dalton component therefore appears to be a newly-identified, authentic component of intestinal microvilli in vivo and of isolated microvillus cores. Immunological studies demonstrate that the 80,000-dalton component is widely distributed in nonmuscle cells. Indirect immunofluorescence microscopy reveals that it is particularly enriched in surface structures, such as blebs, microvilli, and retraction fibers of cultured cells.     

Antigenic homology between rat sperm tail polypeptides and Sertoli cell secretory proteins

A high performance liquid chromatographic procedure has been used for the purification of rat Sertoli cell secretory protein S70 and S45-S35 heterodimeric protein to determine their role during spermatogenesis. These two proteins display binding affinity for each other and appear antigenically related. We have observed that: 1. S70 and S45-S35 heterodimeric protein coelute during purification, 2. polyclonal antiserum raised against protein S70 recognizes common antigenic determinants in polypeptides S45 and S35, the disulfide-linked components of the heterodimeric protein, and 3. a monoclonal antibody that recognizes polypeptide S35 but does not crossreact with either protein S70 or polypeptide S45, immunoprecipitates the S70/S45-S35 heterodimeric protein complex. In immunofluorescent experiments, antisera raised against protein S70 and polypeptide components of S45-S35 heterodimeric protein immunoreact with two major sperm intracellular structures: the acrosome and periaxonemal outer dense fibers of sperm tail. Immunoreactivity was not detected on the sperm plasma membrane surface of unfixed, living sperm. Outer dense fibers extracted from sperm tails by a combined treatment with cetylthrimethylammonium bromide and 2-mercaptoethanol, yielded a characteristic polypeptide pattern. In immunoblotting experiments sperm tail polypeptides were recognized by polyclonal antisera raised against Sertoli cell secretory proteins. We conclude that Sertoli cell secretory proteins S70 and S45-S35 heterodimeric protein are antigenically related to each other and to keratin-like polypeptides from sperm tail.     

Polypeptides of a Light-Harvesting Complex of the Diatom Phaeodactylum tricornutum Are Synthesized in the Cytoplasm of the Cell as Precursors

A light-harvesting fucoxanthin-chlorophyll a/c-protein complex has been isolated from the diatom Phaeodactylum tricornutum by detergent extraction of thylakoid membranes coupled with sucrose density gradient centrifugation. The isolated complex was devoid of photochemical activity and displayed spectral characteristics consistent with light harvesting function. It has three major polypeptides of apparent molecular weights 18,000, 19,000, and 19,500 as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Using protein synthesis inhibitors, these polypeptides were shown to be synthesized on 80S cytoplasmic ribosomes. Antibodies raised to a mixture of the 19,000 and 19,500 dalton components of the complex were used to demonstrate structural similarity among the three polypeptide components. Immunoprecipitation from primary translation products synthesized in a reticulocyte lysate system primed with P. tricornutum poly(A) RNA, indicates that the polypeptide components are synthesized as precursors 3,000 to 5,000 daltons larger than the mature polypeptides.     

Protein constituents of the mouse spermatozoon: I. An electrophoretic characterization

Total protein constituents of the mouse spermatozoon have been fractionated and characterized by polyacrylamide gel electrophoresis. Three spermatozoan fractions were obtained following homogenization with 1% sodium dodecylsulfate (SDS) and sucrose gradient centrifugation: SDS-soluble proteins, SDS-insoluble tail components, and SDS-insoluble head components. Purities of these fractions were assessed at greater than 95% using Nomarksi differential interference microscopy. Subsequently, the SDS-insoluble sperm heads were further fractionated into five protein subclasses by ultracentrifugation and ion-exchange chromatography. SDS-Polyacrylamide gel electrophoresis indicates that each of these spermatozoan fractions contains distinct protein species. Furthermore, the electrophoretic profiles are highly reproducible and show no evidence of cross-contamination or proteolysis. The SDS-soluble fraction, which includes proteins from the plasma membrane, acrosome, axoneme, matrix and cristae of the mitochondria, contains one major 39,000-molecular weight band and numerous minor bands with molecular weights ranging from ～30,000 to greater than 100,000. In contrast, electrophoresis of the SDS-insoluble tail proteins reveals the presence of at least nine prominent bands with apparent molecular weights between 21,000 and 89,000. Ultrastructural analysis suggests that this fraction contains proteins from the outer dense fibers, fibrous sheath, outer mitochondrial membranes, and structural elements of the neck region of the sperm tail. Two subfractions of the SDS-insoluble sperm heads each contain one of the two mouse protamines. In addition, the acidic and moderately basic head fractions each contain a limited number of distinct protein bands with molecular weights ranging from 14,000 to 76,000. These proteins are apparently derived from either the spermatozoan nucleus or the associated perinuclear material, since all other sperm head structures are solubilized during SDS treatment. One- and two-dimensional electrophoresis on acetic acid-urea polyacrylamide gels indicates that the moderately basic fraction may contain minor components that resemble certain histones and/or spermatidal basic nuclear proteins.     

Isolation, structure and protein composition of the perforatorium of rat spermatozoa

Heads of spermatozoa were sonically separated from tails and treated in 1 N NaOH until the perforatoria were partially detached from the nucleus. Their complete detachment was then assured by repeatedly passing the suspension through a 22-gauge needle. The perforatoria were then separated from nuclei on sucrose gradients and the purity of the fraction was verified by electron microscopy. The isolated perforatoria were denatured and used to raise antibodies or run on polycrylamide gels. Such gels revealed many polypeptide bands, six of which were most prominent (Mr approximately 13,000, 13,400, 16,000, 33,000, 35,000, and 43,000). Of these, the 16,000 Mr polypeptide was major. Anti-perforatorium serum reacted with the perforatoria of fixed spermatozoa, with a substance found between the plasmalemma and the outer acrosomal membrane of the acrosomal head cap and with the inner component of the ventral spur, but not with the postacrosomal dense lamina. This observation indicated that the perforatorium and dense lamina, although structurally continuous to form the perinuclear theca, are biochemically distinct. On Western blots, the anti-perforatorium serum reacted with the prominent polypeptides of the perforatorium and cross-reacted with some less prominent polypeptides of the fibrous sheath (FS) and outer dense fibers (ODF), most notably with a 16,000 Mr polypeptide found in both. Likewise anti-FS or anti-ODF serum cross-reacted with some major and minor polypeptides of the perforatorium, again most notably with a major 16,000 Mr polypeptide. The immunocross-reactions observed on Western blots were confirmed by immunocytochemical methods applied to spermatozoa. These results demonstrate that the perforatorium is composed of several polypeptides, is immunologically distinct from the postacrosomal dense lamina, may be immunologically similar to a substance found between the plasmalemma and outer acrosomal membrane and to a substance found on the inner aspect of the ventral spur, has antigenic determinants in common with the FS and ODF, and may share a 16,000 Mr polypeptide with these two cytoskeletal structures of the flagellum.     

黄毛鼠精子尾部主段超微结构

黄毛鼠精子尾主段具有纤维鞘。纤维鞘具背腹两条纵柱和环肋。纤维鞘之内分布着从中段延伸下来的外周致密纤维。在主段的近中段端,有９条外周致密纤维。随后,外周致密纤维逐渐变细,且逐一终止,在９条外周致密纤维中,最早终止的是Ｆ８,随后的终止顺序是Ｆ３、Ｆ４、Ｆ７、Ｆ２、Ｆ９、Ｆ５、Ｆ６、Ｆ１。在主段的近末段端,没有外周致密纤维。根据外周致密纤维的数量,可以将主段分为１０个区域。从近中段端至近末段端,这１０个     

Production of antibody to individual polypeptides derived from purified hepatitis B surface antigen.

Purified preparations of hepatitis B surface antigen (HBsAg) were solubilized with sodium dodecyl sulfate and urea under reducing conditions and subsequently fractionated by preparative sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis (PAGE). Pools of the individual fractions eluted from the preparative PAGE were concentrated and purified further by analytical PAGE. Five purified polypeptides were isolated from HBsAg, types adw and ayw, with molecular weights of 19,000, 24,000, 27,000, 35,000, and 40,000. Each preparations was emulsified in Freund complete adjuvant and injected into guinea pigs. Antibody to each HBsAg type was measured by radioimmunoassay. The 19,000 molecular weight polypeptide derived from ayw particles and the 27,000 molecular weight subunit obtained from both types failed to elicit an antibody response. The other three polypeptides derived from the ayw particles elicited group-specific antibody responses. Similar group-specific reactivities were observed in the testing of anti-adw 35,000 and anti-adw 40,000 molecular weight polypeptide sera. However, guinea pigs immunized with the 19,000 and the 24,000 molecular weight polypeptides of the adw type produced antibody that reacted preferentially with adw particles. This indicates that either these subunits carry predominately d determinants or that, because of the low levels of material used for inoculation, no immune response or an undetectable one was elicited to the a or w components.     

Identification of flagellar and associated polypeptides of Helicobacter (formerly Campylobacter) pylori

Flagella of Helicobacter pylori were isolated from intact organisms by shearing and differential centrifugation. Treatment of the flagella with the detergent Triton X-100 removed the flagellar sheath, which was confirmed by electron microscopy, and the remaining naked flagella were shown by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) to consist primarily of a single 54 kilodalton (kDa) polypeptide. This was confirmed by immunogold labelling and electron microscopy of detergent treated whole organisms, using a mouse antiserum specific for the 54 kDa polypeptide. Polypeptides solubilised from crude flagellar preparations by detergent treatment were found to have molecular weights of 26, 30, 58, 62, 66 and 80 kDa. These polypeptides are possible components of the flagellar sheath and they may represent outer membrane proteins, based on the assumption that the flagellar sheath is related in composition to the outer membrane of the organism. Analysis and definition of these components of the surface structures of the organism are important in understanding the interaction between the organism and its host in pathogenesis.     

A structural model of human erythrocyte protein 4.1

Limited proteolysis and specific chemical cleavage methods have enabled a detailed structural characterization of human erythrocyte protein 4.1. This protein is composed of two chemically very similar polypeptide chains (a and b) with apparent molecular masses of 80,000 and 78,000 daltons. Cleavage of protein 4.1 at cysteine residues by 2-nitro-5-thiocyanobenzoic acid produces a series of doublets which differ by approximately 2,000 daltons and have identical peptide maps. Alignment of these peptides by mapping analysis has localized 4 cysteine residues within a 17,000-dalton segment on both a and b polypeptides. Mild chymotryptic treatment at 0 degrees C cleaves protein 4.1 primarily in three central locations and generates two families of unrelated peptides. Analysis of these fragments in two-dimensional gels and by peptide mapping reveals an unusual polarity in protein 4.1 structure in that each polypeptide chain contains two segments, one relatively acidic the other basic, that are segregated at opposite ends of the molecule. The basic region is digested into a cysteine-rich 30,000-dalton domain which resists further breakdown while the acidic region is readily degraded into smaller fragments. The peptides derived from the acidic region all appear as doublets suggesting that protein 4.1 a and b polypeptides differ close to the terminus of the acidic end. Similar phosphorylation sites occur on both polypeptides within a segment some 24,000-34,000 daltons from the acidic terminus.     

Proteins of bovine leukemia virus. I. Characterization and reactions with natural antibodies.

The bovine leukemia virus (BLV) was purified from a chronically infected fetal lamb kidney cell line. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of this virus revealed the presence of eight distinguishable viral components with molecular weights ranging from 80,000 to 11,000. The major component is a non-glycosylated protein having a molecular weight of 24,000 (p24). At least three heavier polypeptides were found, one of them representing a glycoprotein (gp 60). In addition, four minor polypeptides with respective molecular weights of 19,000, 16,000, 13,000, and 11,000 were identified. In a complement fixation assay using naturally occurring antibodies of a leukemic cow, four polypeptides, which included gp 60, p35, p24, and p16, were found to be reactive.     

76 and 14 kDa polypeptides, two major components released from amphibian urinary bladder epithelium. Purification and characterization

Exocytotic processes play a major role in the hormonal control of water permeability in the amphibian urinary bladder. Different treatments such as antidiuretic hormone (ADH) stimulation, incubation with phorbol ester or mild detergent and mechanical stretch of the bladder, consistently induce a liberation of two major polypeptides of 76 and 14 kDa molecular mass into the luminal medium. Each of these polypeptides represents 3 to 5% of the total protein of epithelial cell homogenates and 20 to 50% of the released material. Proportions of released 76 kDa polypeptide in urinary bladders of toads (Bufo marinus) and frogs (Rana esculenta) were similar but, in the frog extracts, two bands ("doublet") were resolved at the level of 76 kDa. In high performance liquid chromatography (HPLC), using gel filtration and ion exchange chromatography, the frog 76 kDa protein was resolved into two polypeptides of 80,000 to 100,000 and 60,000 to 80,000 daltons while the 14 kDa protein included two polypeptides, each with a molecular mass of approximately 14,000 daltons. Isoelectric focusing of the material released during a mechanical stretch of the tissue ("stretch extract") or of isolated purified proteins from the frog urinary bladder showed that the 14 kDa polypeptides were resolved in two major groups of polypeptides, one in the range of pH 7.4 to 7.8, the other at pH 5.6. The lower band of the 76 kDa doublet also comprised some diffuse bands (5.0 less than pI less than 5.2) while the other polypeptide of the doublet presented a sharp band at pH 6.2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Multiple glycolytic enzymes are tightly bound to the fibrous sheath of mouse spermatozoa

The fibrous sheath is a cytoskeletal structure located in the principal piece of mammalian sperm flagella. Previous studies showed that glyceraldehyde 3-phosphate dehydrogenase, spermatogenic (GAPDHS), a germ cell-specific glycolytic isozyme that is required for sperm motility, is tightly bound to the fibrous sheath. To determine if other glycolytic enzymes are also bound to this cytoskeletal structure, we isolated highly purified fibrous sheath preparations from mouse epididymal sperm using a sequential extraction procedure. The isolated fibrous sheaths retain typical ultrastructural features and exhibit little contamination by axonemal or outer dense fiber proteins in Western blot analyses. Proteomic analysis using peptide-mass fingerprinting and MS/MS peptide fragment ion matching identified GAPDHS and two additional glycolytic enzyme subunits, the A isoform of aldolase 1 (ALDOA) and lactate dehydrogenase A (LDHA), in isolated fibrous sheaths. The presence of glycolytic enzymes in the fibrous sheath was also examined by Western blotting. In addition to GAPDHS, ALDOA, and LDHA, this method determined that pyruvate kinase is also tightly bound to the fibrous sheath. These data support a role for the fibrous sheath as a scaffold for anchoring multiple glycolytic enzymes along the length of the flagellum to provide a localized source of ATP that is essential for sperm motility.     

Immunochemical characterization of a human high molecular weight — melanoma associated antigen identified with monoclonal antibodies

Sodium dodecyl sulfate polyacrylamide gel analysis of a high molecular weight (HMW) human melanoma associated antigen (MAA) defined by murine monoclonal antibodies revealed a number of distinct polypeptides ranging from 80,000 up to 280,000 daltons, in addition to an extremely heterogeneous group of components distributed over a wide range in apparent molecular weight (300,000-700,000 daltons). The 280,000 dalton and the larger heterogeneous molecular weight material are glycosylated since they are labeled with 3H-sugars. The HMW-MAA is readily solubilized in the absence of detergents and the entire series of polypeptides fractionates together in the void volume of a Sephadex G200 column. Peptide maps of the various polypeptides of the HMW-MAA, generated by Staphylococcus aureus V-8 protease, are essentially the same except that some of the proteolytic fragments derived from the lower molecular weight polypeptides (80,000 daltons) are present in greater amounts than are similar fragments derived from the larger molecular weight polypeptides; the latter finding suggests that the complexity in molecular weight of the MAA may reflect combinations of several base subunits. Proteolytic cleavage of the HMW-MAA generates a number of peptides ranging in molecular weight from 77,000 daltons to less than 12,000 daltons, which still react with monoclonal antibodies and can distinguish monoclonal antibodies specific for different antigenic determinants of this MAA.     

Flagellar adenosine triphosphatases from annelid spermatozoa: electrophoretic identification of dyneins

Axonemes of sperm flagella were prepared from the annelid, Tylorrhynchus heterochaetus. Dialysis of the axonemes against 1 mm Tris-HCl buffer (pH 8.3)-0.1 mm EDTA-0.1 mm dithiothreitol (Tris-EDTA solution) caused disintegration of typical 9 + 2 microtubules into each doublet, resulting in extraction of one-third of the protein and almost all ATPase activity. Agarose polyacrylamide gel electrophoresis of the extract showed the presence of three kinds of dyneins actively stained for ATPase (designated as bands I, II, and III) and two non-ATPase proteins (bands IV, V). The polypeptide components of each dynein molecule and intact axoneme were analyzed by subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis to obtain the following results: (1) In the highmolecular-weight region, the intact axonemes yield two major polypeptides with molecular weights of 365,000 and 345,000 (designated as bands A and B, respectively) and three minor polypeptides, 310,000, 290,00, and 270,00 (C1, C2, C3). (2) All three dyneins contain A-band polypeptide as a common polypeptide component. In addition, band I dynein and band II dynein also contain B and C1 polypeptides, and C3 polypeptide, respectively, as high-molecular-weight components. (3) Band III dynein also contains four polypeptides in the lower molecular-weight region, which migrate similarly with those of 21 S dynein from sea urchin sperm flagella or 18 S dynein from Chlamydomonas.     

Adenylate kinases 1 and 2 are part of the accessory structures in the mouse sperm flagellum

Proper sperm function depends on adequate ATP levels. In the mammalian flagellum, ATP is generated in the midpiece by oxidative respiration and in the principal piece by glycolysis. In locations where ATP is rapidly utilized or produced, adenylate kinases (AKs) maintain a constant adenylate energy charge by interconverting stoichiometric amounts of ATP and AMP with two ADP molecules. We previously identified adenylate kinase 1 and 2 (AK1 and AK2) by mass spectrometry as part of a mouse SDS-insoluble flagellar preparation containing the accessory structures (fibrous sheath, outer dense fibers, and mitochondrial sheath). A germ cell-specific cDNA encoding AK1 was characterized and found to contain a truncated 3' UTR and a different 5' UTR compared to the somatic Ak1 mRNA; however, it encoded an identical protein. Ak1 mRNA was upregulated during late spermiogenesis, a time when the flagellum is being assembled. AK1 was first seen in condensing spermatids and was associated with the outer microtubular doublets and outer dense fibers of sperm. This localization would allow the interconversion of ATP and ADP between the fibrous sheath where ATP is produced by glycolysis and the axonemal dynein ATPases where ATP is consumed. Ak2 mRNA was expressed at relatively low levels throughout spermatogenesis, and the protein was localized to the mitochondrial sheath in the sperm midpiece. AK1 and AK2 in the flagellar accessory structures provide a mechanism to buffer the adenylate energy charge for sperm motility.