THELOCALIZATIONOFGLYCOGENIN THESPERMATOZOAOFVARIOUS INVERTEBRATEANDVERTEBRATESPECIES WINSTON A. ANDERSON and PAULPERSONNE FromtheLaboratoiredeBiologieCellulaire4,FacultedesSciences,Orsay, France ;theDepartment ofAnatomyandLaboratoryofHumanReproduction, HarvardMedicalSchool, Boston, Massachusetts02115 ;andtheLaboratoiredeBiologieAnimale, CollegeScientifiqueUniversitaire, Amiens,France ABSTRACT Withtheperiodicacid-thiosemicarbazide-silverproteinateprocedureforthedetectionof polysaccharidesinthinsections,glycogenislocalizedinthecavities ofcentriolesandbasal bodies,withintheaxoneme(andsurroundingit),inmitochondria, andinthe"packing" cytoplasmofthemiddlepieceofspermatozoaofseveralinvertebrate andvertebratespe- cies .Thecytochemicallocalizationofglycogenisverified byextractionwitha-amylase solution .Thesefindingsestablishtheexistenceofstored glycogeninsperm .Thepolysac- charidepresumablyservesasanendogenoussourceofenergyintheabsence ofextracellular metabolites,undereitheraerobicoranaerobicconditions . Otherhypothesesonthephysio- logicalsignificanceofintracellularglycogen storesinspermarediscussed .Spermthat storeglycogencontainsomeenzymesofglycogenmetabolism . Inthepresenceofglucose-l- phosphate,ATP,andMg++ions,anamylophosphorylasecatalyzesthe invivosynthesisof glycogen.Thenewlyformedproductresemblesy-particles,andisdigestible witha-amylase . INTRODUCTION Spermatozoaarehighlyspecializedformotility andtheotheractivitiesessentialtofertilizationof ova .Toreduceitsvolumeandprotectthegenome fromdamageduringexposuretovariousenviron- mentsencounteredintransittotheovum, the spermnucleusbecomeshighlycondensedand metabolicallyinert .Tofavoritsmotility,thede- velopingspermatozoandivestsitselfofmostofits cytoplasm.Thereremainsseeminglyvery little spacefortheintracellularstorageofsubstrates whichcouldprovidetheenergyessentialfortheir motility .Insomespecies,thespermatozoa are releasedintoaspecializedinternalenvironment richinexogenousnutrients .Forexample,mam- malianspermatozoanormally metabolizecar- bohydratesaddedtotheseminalplasmabythe accessoryglands(Mann,1964) .However,under aerobicconditionsandintheabsence ofgly- colyzablematerial,thesespermutilizeendogenous lipidsastheirmainsourceofenergy .Thus,mam- malianspermhavetheenzymesystemscapable ofutilizingeitherenvironmentalcarbohydrateor endogenouslipidsdependingupontheavailabil- ityofexogenoussubstrateandoxygen . Ontheotherhand,somespeciesdischargetheir spermatozoaintoanaqueousenvironmentthat isdevoidofmetabolizable compounds.These spermmust,therefore,dependexclusively upon endogenous substrates. According toAfzelius andMohri(1966),themotilityofsea urchin spermatozoa,forexample,dependsuponenergy obtainedbytheaerobicoxidationofphospho- 2 9 on February 16, 2018 jcb.rupress.org Downloaded from
Transcript
THE LOCALIZATION OF GLYCOGEN IN
THE SPERMATOZOA OF VARIOUS
INVERTEBRATE AND VERTEBRATE SPECIES
WINSTON A. ANDERSON and PAUL PERSONNE
From the Laboratoire de Biologie Cellulaire 4, Faculte des Sciences, Orsay, France; the Departmentof Anatomy and Laboratory of Human Reproduction, Harvard Medical School, Boston,Massachusetts 02115 ; and the Laboratoire de Biologie Animale, College Scientifique Universitaire,Amiens, France
ABSTRACT
With the periodic acid-thiosemicarbazide-silver proteinate procedure for the detection ofpolysaccharides in thin sections, glycogen is localized in the cavities of centrioles and basalbodies, within the axoneme (and surrounding it), in mitochondria, and in the "packing"cytoplasm of the middle piece of spermatozoa of several invertebrate and vertebrate spe-cies. The cytochemical localization of glycogen is verified by extraction with a-amylasesolution. These findings establish the existence of stored glycogen in sperm . The polysac-charide presumably serves as an endogenous source of energy in the absence of extracellularmetabolites, under either aerobic or anaerobic conditions . Other hypotheses on the physio-logical significance of intracellular glycogen stores in sperm are discussed . Sperm thatstore glycogen contain some enzymes of glycogen metabolism . In the presence of glucose-l-phosphate, ATP, and Mg++ ions, an amylophosphorylase catalyzes the in vivo synthesis ofglycogen. The newly formed product resembles y-particles, and is digestible with a-amylase .
INTRODUCTIONSpermatozoa are highly specialized for motilityand the other activities essential to fertilization ofova. To reduce its volume and protect the genomefrom damage during exposure to various environ-ments encountered in transit to the ovum, thesperm nucleus becomes highly condensed andmetabolically inert . To favor its motility, the de-veloping spermatozoan divests itself of most of itscytoplasm. There remains seemingly very littlespace for the intracellular storage of substrateswhich could provide the energy essential for theirmotility. In some species, the spermatozoa arereleased into a specialized internal environmentrich in exogenous nutrients . For example, mam-malian spermatozoa normally metabolize car-bohydrates added to the seminal plasma by the
accessory glands (Mann, 1964) . However, underaerobic conditions and in the absence of gly-colyzable material, these sperm utilize endogenouslipids as their main source of energy. Thus, mam-malian sperm have the enzyme systems capableof utilizing either environmental carbohydrate orendogenous lipids depending upon the availabil-ity of exogenous substrate and oxygen .
On the other hand, some species discharge theirspermatozoa into an aqueous environment thatis devoid of metabolizable compounds. Thesesperm must, therefore, depend exclusively uponendogenous substrates. According to Afzeliusand Mohri (1966), the motility of sea urchinspermatozoa, for example, depends upon energyobtained by the aerobic oxidation of phospho-
lipids in their middle piece . Other work, however,indicates that some invertebrate sperm may useendogenous carbohydrate such as glycogen asan energy source (Spikes, 1949 ; Personne andAndre, 1964 ; Lanza and Quattrini, 1964 ; Ander-son, 1968) . Although the fine structure of inver-tebrate sperm has been extensively investigated,the presence of glycogen has been reported in onlya few-Testacella and Helix (Personne and Andre,1964; Andre, 1965), Lumbricus (Anderson et al.,1967), Paracentrotus (Anderson, 1968). Based onmorphological findings, glycogen-like granuleshave been demonstrated in sperm of the Platy-helminthes and Chilopoda (Silveira and Porter,1964; von Bonsdorff and Telkka, 1965 ; Horst-mann, 1968) .
The recent development of satisfactory methodsfor the demonstration of polysaccharides at theelectron microscope level (Hanker et al ., 1964 ;Seligman et al ., 1965 ; Thiery, 1967) now makes itpossible to localize polysaccharides in cells andtissues in which this substance formerly escapeddetection. In the present study, the periodicacid- thiosemicarbazide -silver proteinate (PA-TSC-SP) technique of Thiery has been appliedto sperm of a wide variety of invertebrate andvertebrate species in order to determine how com-mon glycogen is and where it is localized. Glyco-gen is demonstrated in the cavity of centrioles andbasal bodies, in the matrix within the axoneme(and around it), and in the mitochondria and thesurrounding cytoplasm. The cytochemical iden-tification of glycogen has been verified in someinstances by enzyme extraction procedures . The
30 THE JOURNAL OF CELL BIOLOGY • VOLUME 44, 1970
methods of Takeuchi and Kuriaki (1955) and ofGuha-Wegmann (1959) have been utilized todemonstrate in the sperm of certain species thepresence of an active phosphorylase capable ofcatalyzing the synthesis of glycogen (Personneand Anderson, 1969 a) . The observations are dis-cussed in relation to the conditions of spermstorage and the environment in which fertilizationtakes place in the several species studied .
MATERIALS AND METHODS
Animal MaterialThe various specimens whose spermatozoa were
investigated were collected in France or in the UnitedStates or purchased from dealers in those countries .They include the following :
ANNE LID A : Lumbricus terrestris; Hirudo medici-nalis.MOLLUSCA : (a) Marine Lamellebranchia : My-
All micrographs, except Fig . 4 B, were taken from sections that were treated with theperiodic acid-thiosemicarbazide-silver proteinate procedure (PA-TSC-SP) for the demon-stration of glycogen according to Thiery (1967) .
FIGURE 1 Transverse sections through flagella of sperm of Hirudo. After the PA-TSC-SP treatment, intensely stained glycogen granules (arrows) occupy the matrix between theouter doublets of the axoneme and the flagellar membrane. X 40,000.
FIGURE 2 Following oxidation in 5%o periodic acid solution for 10 min at 33°C, rinsingin distilled water for 30 min, and treatment of thin sections for 60 min with 0.5% ce-amylasesolution, the glycogen granules totally disappear from sperm tails . Following the PA-TSC-SP treatment and staining in 2% aqueous uranyl acetate solution for 5 min, thematrix surrounding the axonemes (A) remains clear (arrows) . X 70,000 .
FIGURE 3 Glycogen granules (G) remain intact around the axonemes (A) of sperm tailsin control sections that were incubated in heat-inactivated a-amylase solutions and treatedin the same manner as those of Fig . 2 . X 60,000 .
Electron MicroscopySmall blocks of tissue (1-3 mm3), were fixed by
immersion, or isolated sperm were suspended infixative according to the following procedures .Samples of testes, seminal vesicles, and sperm re-ceptacles of terrestrial and freshwater invertebrateswere fixed for 30-60 min at room temperature in a2% glutaraldehyde solution . The fixative contained5.6 ml biological grade glutaraldehyde (36%),50 ml 0.1 M sodium cacodylate buffer at pH 7 .2,44.4 ml of distilled water, and 1 .5 g of sucrose . Afterfixation, the samples were rinsed in a solution con-taining 5.0 g of sucrose in 100 ml 0 .1 M sodium caco-dylate solution. Tissues from vertebrates and fromsome marine invertebrates were fixed for 30-45 minat 4 ° or at 27 °C in 2% glutaraldehyde in 0 .1 Msodium cacodylate made up in filtered seawater .The fixative contained 1 .5 g of sucrose, and was ad-justed to pH 7.0. In other instances, good preserva-tion of tissues from vertebrates and invertebrateswas obtained after fixation in the formaldehyde-glutaraldehyde plus trinitrocresol mixture of Itoand Karnovsky (1968) . Subsequent to aldehydefixation, all specimens were postfixed in 2% osmiumtetroxide solution . After postfixation, samples weredehydrated in acetone-water solutions and embeddedin Epon or in an Epon-Araldite mixture (Voelz andDworkin, 1962 ; Anderson and Ellis, 1965) .
3 2
Cytochemical Procedures for the Demonstration
of GlycogenTHE PERIODIC ACID-THIOSEMICARBAZIDE-
SILVER PROTEINATE PROCEDURE (PA-TSC-sP) : The details of this technique and the lengthsof the respective reactions for the optimal demonstra-tion of glycogen and of other polysaccharides in thinsections have been fully worked out by Thiery (1967) .In this study, except for some minor adjustments,Thiery's procedure has been precisely followed .Thin sections of aldehyde-osmium tetroxide-fixedtissues were collected and transported to the respec-tive incubation media within the lumen of poly-ethylene rings according to Marinozzi (1961) .Sections were floated for 30-45 min on the surfaceof a solution of I % periodic acid in distilled water .Thin sections were then rinsed thoroughly (approxi-mately 30 min) in several changes of distilled water,and refloated for 30-45 min on 1 % thiosemicarba-zide in 10% acetic acid . Subsequently, the sectionswere washed in three changes (approximately 10 mineach) of 10% acetic acid solution, then for 5 min in5 and 1 % acetic acid solutions and finally in distilledwater. The sections were then refloated for 30 minon 1 0/0 silver proteinate in distilled water . Incubationin silver proteinate solution was performed in thedark. The sections were thoroughly rinsed in severalchanges of distilled water and collected on coppergrids .THE PERIODIC ACID-THIOSEMICARBAZIDE
AND THE PERIODIC ACID-THIOCARBOHYDRA-ZIDE PLUS OSMIUM TETROXIDE PROCEDURES(PATO, PATCO) OF SELIGMAN ET AL ., (1965)AND THE PAS-PHOSPHOTUNGSTIC ACID PROCE-DURE OF THIERY (1967) : These were also triedin the detection of glycogen in thin sections . Resultswith these techniques were identical with thoseobtained with the PA-TSC-SP procedure. However,since the PA-TSC-SP procedure yielded the most
FIGURE 4 Transverse sections through the sperm tails of Littorina are shown in thismicrograph. The glycogen granules (0) are located around the axoneme (A) . The inset(B) shows a specimen from sections that were stained for approximately 60 min in asaturated uranyl acetate solution in 40% ethanol . Glycogen granules are not stained, buta pepsin-digestible halo (arrow) surrounds the axoneme . X 55,000 .
FIGURE 5 A longitudinal section through a portion of the sperm tail of Littorina is shownhere. Several layers of 0-glycogen particles (G) are tightly packed between the axoneme(A) and the flagellar membrane. X 110,000.
FIGURE 6 A transverse section through the flagellum of a spermatozoon of Littorina isshown here . 18 f3-particles (as numbered) generally constitute the first row of glycogenparticles (G) surrounding the axoneme (A) . X 150,000 .
consistent and accurate results with a minimum ofbackground precipitation, it was mainly used in thedemonstration of glycogen in thin sections of sperma-tozoa.
Enzymatic Extraction of Glycogenfrom Thin Sections
Cytochemical techniques for the enzymatic ex-traction of glycogen from Epon-embedded sectionswere performed according to Monneron and Bern-hard (1966) . Experimental and control sectionswere similarly carried in polyethylene rings accord-ing to Marinozzi (1961) . After periodate oxidationand rinsing according to Monneron and Bernhard(1966), thin sections were incubated in 1, 0 .5, 0 .1,and 0.05% a- or ,l3-amylase solutions in 0.1 M
phosphate buffer at pH 7 .0. The sections were in-
cubated at 37 °C for 30 min, 1, 2, and 3 hr . Thinsections were also incubated in saliva for 3-4 hr at37 °C .
Control sections were incubated in heat-inacti-vated a- or /3-amylase solutions and saliva, for thesame times and under the same conditions . Enzyme-and saliva-treated, as well as control sections werethen stained according to procedures describedabove for the detection of glycogen or were doublestained in uranyl acetate and lead citrate solutions .
In some instances, tissues which were fixed for 30-60 min in 2% formalin in 0 .1 M phosphate bufferat pH 7.0 and rinsed for 8-16 hr in buffer were in-cubated en bloc for 1-3 hr in 0 .5-1 % a-amylase solu-tion or in saliva . Control samples were incubated inphosphate buffer only . After incubation, the tissueswere rinsed in buffer, postfixed in 2% Os04 solu-
FIGURES 7 and 8 Longitudinal sections through the terminal portion of the mitochondrial sheath (MP)and the tail segment (TP) of sperm of Pomacea (Fig. 7) and of Planorbis (Fig. 8) are shown in these micro-graphs. Tightly packed l3-particles comprise the thick mantle around the axoneme . In sperm of Planorbis,j3-particles extend into the axoneme within the mitochondrial sheath (arrow, Fig . 8), but in sperm ofPomacea glycogen is not observed in the axoneme (arrow, Fig . 7) within the middle piece . X 70,000;X 60,000 .
tion, and prepared for electron microscopy by rou-tine procedures.
Chemical Isolation of Glycogenfrom Spermatozoa
Spermatozoa that were isolated from the her-maphroditic duct of Lymnaea and Otala were used inthis study . After boiling the spermatozoa in 30%KOH solution, an ethanol precipitable fractionwas obtained. This material was purified by re-peated ethanol precipitation of the water-solubleextract. Drops of the extract suspended in 55%ethanol were spread on Formvar-coated grids.Excess fluid was removed from the surface of thegrid and was replaced with a drop of 10/o phospho-tungstic acid (PTA) in 55% ethanol or in distilledwater at pH 6.9 . After 1-5 min, the PTA solutionwas removed and the grids were allowed to dry atroom temperature .
Demonstration of AmylophosphorylaseActivity Associated withGlycogen SynthesisThe Takeuchi and Kuriaki (1955) and the Guha
and Wegmann (1959 ; 1961) procedures were used todemonstrate the synthesis of glycogen by an amylo-phosphorylase. Isolated spermatozoa were incubatedin the following medium : 25 mg of glucose-l-phos-phate, 5 mg of ATP, 10 mg of NaF, 4 mg of MgSO4,5 ml 0.2 as acetate buffer at pH 6 .0, and 7.5 ml ofdistilled water . Sodium fluoride was added in orderto inhibit phosphorylase phosphatase activity, thusblocking the conversion of active phosphorylase ato inactive phosphorylase b . ATP and Mg++ ionsactivate phosphorylase kinase which catalyzes theconversion of inactive phosphorylase b to activephosphorylase a . Unfixed spermatozoa were alsoincubated in medium containing AMP (20 mg),but lacking ATP and MgSO4. Control specimenswere incubated in medium from which glucose-l-phosphate was omitted . Experimental and con-trol specimens were incubated at 37 °C from 30 minto 3 hr. After fixation in glutaraldehyde and osmiumtetroxide solutions, samples were prepared for elec-tron microscopy by the above-mentioned procedures .Pre-existing and newly formed glycogen were demon-strated in thin sections by the PA-TSC-SP procedureof Thiery (1967). Some control and experimentalsections were incubated for 1 hr in medium contain-ing either 0 .5% a- or 13-amylase in phosphate bufferat pH 7 .0 .
A detailed study on the involvement of an amylo-phosphorylase in glycogen synthesis in sperm ofpulmonate gastropods will be presented in a sepa-rate report (Personne and Anderson, 1969) .
FIGURES 9 and 10 Longitudinal and transverse sec-tions through the tail piece of sperm of Lepas are shownhere . A single row of glycogen granules (arrows) existson one side of the axoneme (A), while a stack of granulesis located at the opposite side . X 120,000.
YYINSTON A. ANDERSON AND PAUL PERSONNE Glycogen in Spermatozoa
Spermatozoa of the annelids Lumbricus andHirudo conform to the plan of organization ofother flagellate spermatozoa in that they aresubdivided into acrosome, nucleus, middle piece,and tail segments (Anderson et al ., 1967) . Theshort cylindrical middle piece lies immediatelybehind the helical nucleus of Hirudo and the uni-formly cylindrical nucleus of Lumbricus . The basalbody of the axoneme is located at the base of themiddle piece . Glycogen granules are consistentlyfound in the sperm tails of Hirudo (Fig. 1) and ofLumbricus (Anderson et al ., 1967) . The irregularlyshaped granules are approximately 200-300 Ain diameter and are identical to 0-glycogengranules (nomenclature according to Drochmans,1962) . In thin sections prepared by conventionalprocedures for electron microscopy, the densityof the periaxonemal granules is only moderatelyintensified with lead stains . These granules aremuch more opaque after treatment with the PA-TSC-SP procedure. The granules are distributedperipheral to the doublets and immediatelybeneath the flagellar membrane . In Lumbricus,the 0-particles are distributed along the entirelength of the flagellum, whereas in Hirudo theyterminate at the junction of the axoneme with anhomogeneous rodlike tip of the tail . This rodlikespecialization is moderately contrasted aftertreatment with the PA-TSC-SP procedure .
For assessing further the specificity of the stain-ing procedure in the detection of glycogen, thinsections were exposed to glycogen-extraction solu-tions. Following treatment in 0 .5% a-amylasesolution for 60 min at 37°C, the periaxonemalglycogen granules are totally extracted . Whensections are treated first with a-amylase solution,followed by the PA-TSC-SP procedure, the re-gion surrounding the outer doublets now appearsclear (Fig. 2). When thin sections are treatedwith proteinases (according to Monneron andBernhard, 1966; Anderson and Andre, 1968),the axonemal components are extracted whileglycogen particles remain intact . In controlsections that were incubated in heat-inactivateda-amylase solution or in water, the glycogenparticles remained unaltered around the axoneme(Fig. 3) .Spermatozoa of the molluscs Littorina and
Purpura do not conform morphologically in all
36 TAE JOURNAL OF CELL BIOLOGY • VOLUME 44, 1970
respects to the basic plan of flagellate sperma-tozoa. The axoneme originates at the base of theacrosome and runs the entire length of the cell .Anteriorly, it is surrounded by the nucleus for40-60 µ, and by the mitochondrial derivative(Walker and MacGregor, 1968). Behind themiddle piece, the flagellum continues for about30-40 u to form the tail . Axonemes of Littorinaand Purpura have the 9 + 2 arrangement of micro-tubules . Walker and MacGregor (1968) have alsodescribed multiple coarse fibers around the axo-neme, but in our opinion, what they interpretedas periaxonemal fibers are, in fact, deposits ofglycogen . The particles are closely apposed tothe doublets, and are present throughout thelength of the tail (Figs. 4 and 6) . Glycogen gran-ules are clearly absent from that portion of theaxoneme that is enveloped by the mitochondrialsheath or by the nucleus . The granules are notstained appreciably even after staining in uranylacetate solution for extensive periods (30-60 min) ;however, moderately opaque, pepsin-extractableproteinaceous material is seen around the axo-neme in such preparations (Fig . 4, inset). Uponclose examination of sections treated by the PA-TSC-SP procedure the staining of the glycogenparticles is punctate, suggesting the presence ofsubunits (Fig. 6) . It is not certain, however, thatthis is other than a meaningless pattern of pre-cipitation of the stain . The glycogen granules donot contact the doublets and are not presentwithin the axoneme of these spermatozoa . Theextensive deposits of glycogen do not appear toimpede movement of the tail piece, for living sper-matozoa of Littorina are actively motile through-out their entire length.
Glycogen granules are present within the tailpiece of spermatozoa of all freshwater pulmonategastropods studied so far, Lymnaea, Helisoma, andPlanorbis (Fig . 8) . Similarly, in typical flagellatespermatozoa of prosobranch gastropods, glycogengranules are also located around the axoneme ofthe tail piece (Fig . 7) . In spermatozoa of thesefreshwater gastropods then, the glycogen gran-ules form a thick mantle around the peripheraldoublets. Glycogen particles are also observedwithin the axoneme of the tail piece of thesespermatozoa .
Unlike other crustacean spermatozoa, those ofcirriped Crustacea are flagellate (Brown, 1966)and motile . Significant amounts of glycogenare present within the spermatozoa of Lepas .
FIGURE 11 Transverse sections through flagella of atypical sperm of Viviparus are shown in this micro-graph. Beta glycogen granules are located between the central pair and peripheral doublets of the ax-oneme (arrows) . X 60,000.
WINSTON A. ANDERSON AND PAUL PER5QN;jA Glycogen in Spermatozoa
Glycogen particles are disposed in a single layeron one side of the axoneme, while on the oppositeside they are densely packed in a prominent ridge(Figs. 9 and 10) .
Intra-Axonemal Distribution of Glycogen
Atypical, vermiform spermatozoa of proso-branch gastropods possess multiple flagella .Glycogen granules are randomly distributed be-tween the peripheral doublets and the centralpair of microtubules in each flagellum (Fig . 11) .
Material with the staining properties of glyco-gen is consistently present within the axoneme ofspermatozoa in some pulmonate gastropods(Figs . 12-14). The granules conform to the usualdescription of $-particles and are intensely stained
after the PA-TSC-SP treatment (Personne andAnderson, in preparation ;' Anderson and Per-sonne, 1969) . They are not randomly distributedin the axoneme, for they consistently appearbetween the central tubules and the peripheraldoublets (Figs . 13-14). In some species, thegranules seem to be arrayed in parallel heliceswithin the axoneme . They disappear after treat-ment with a-amylase solution .
Spermatozoa of the lamellibranch molluscsPecten, Mytilus, Ostrea, Spisula, and of the gastro-pods Gibbula, Patella, and Diodora are divided intohead, a short middle piece, and a simple flagel-lum. According to Franzen (1956), these sperma-tozoa are classified as "primitive ." The middle
piece is composed of relatively unmodified mito-chondria surrounding the basal body and theproximal regions of the axoneme .
Spermatozoa of most Lamellibranchii and ofthe Gastropoda named above are similar in theirgeneral features to those of Mytilus and of Crassos-trea which have been described, respectively, byLongo and Dornfeld (1967) and by Galtsoff andPhilpott (1960) ; but they vary in shape of head andnumber of mitochondria . In sperm of Mytilus,an axial rod projects into a cylindrical space withinthe nucleus.
' P. Personne and W. A. Anderson. 1969. Existencede glycogcne intraflagellaire daps le spermatozoidedes Gasteropodes Pulmones.
3 8 THE JOURNAL OF CELL, BIOLOGY . VOLUME 44, 1970
After double staining of thin sections withuranyl acetate and lead citrate, no unusual struc-tures were observed . The mitochondria and cen-trioles were surrounded by material of low den-sity or by finely granular or homogeneouslydense material . After processing sections with thePA-TSC-SP procedure, a-amylase digestibleglycogen particles are consistently observed withinthe middle piece . The glycogen granules aresimilar in appearance to those already describedin sperm of other invertebrate species. The par-ticles are arranged in compact clusters around themitochondria and centrioles, and within thecavity of the centrioles (Figs . 15-18). In sperma-tozoa of Pecten, Mytilus and Spisula, glycogen par-ticles are often in intimate contact with the outermembrane of the mitochondria (Figs . 17 and 18),in close apposition to the wall of the proximal anddistal centrioles (Figs. 15 and 16), and withinthe central canal (of Mytilus sperm) and in periph-eral indentations of the nucleus . Glycogen par-ticles are not found in the flagellum of maturespermatozoa of these lamellibranch molluscs .Glycogen is absent from the middle piece of
spermatids and of immature spermatozoa ob-tained from the testis . Mature spermatozoa in-cubated in seawater for 6 hr contain varyingamounts of glycogen . Glycogen disappears fromsperm of some species under these conditions ;in others, it is undiminished .
Intramitochondrial Localization of Glycogen
Spermatozoa of pulmonate gastropods possessan enormous middle piece that is largely com-posed of a highly modified mitochondrial sheathcontaining a paracrystalline component . Usingclassical techniques for the demonstration ofpolysaccharides, Personne and Andre (1964)and Andre (1965) demonstrated the presence ofglycogen within a distinct compartment of themitochondrial sheath . Other cytochemical testsindicate that the paracrystalline region is proteina-ceous (Personne, 1965) and may contain therespiratory enzymes . In the present study, glyco-gen reserves are observed within the mitochon-drial derivative of spermatozoa of all pulmonategastropods studied so far (Figs . 12, 13, and 19) .The number of glycogen-containing compartmentsvaries with the species, but, in all cases, they runa helical course from the neck to the tail piece(Fig. 20). Although the glycogen stores are en-cased by the mitochondrial derivative, the corn-
FIGURE 12 This micrograph illustrates a longitudinal section through the middle piece (M) of sperm ofPlanorbis. Opaque granules (arrow) with staining properties of glycogen are linearly arranged within theaxoneme (A) of this spermatozoon . X 90,000.
partment in which they reside is membrane-bounded and is thus set off from the other compo-nents of the mitochondrion .Although the helical compartment is already
identifiable within the mitochondrial derivative ofspermatids and of immature spermatozoa, glyco-gen is usually absent at these early stages (Fig . 21) .In Lymnaea and Planorbis, glycogen polymerizationseems to commence in the tail piece and subse-quently appears in the helical compartments ofthe mitochondrial derivative. It must be empha-sized that in sperm of pulmonate gastropods glyco-gen accumulation commences after the comple-tion of spermatozoan maturation . Therefore,intracellular glycogen reserves are most prominentin mature sperm in the hermaphroditic duct .With the hot KOH procedure, ethanol-pre-
cipitable glycogen is extracted from spermatozoaof pulmonate gastropods . When samples of glyco-gen so precipitated are spread on Formvar-coatedgrids and treated with 1 % PTA in 55 % ethanol,,Q-glycogen granules are revealed .
Phosphorylase activity has been demonstrated inspermatozoa of pulmonate gastropods at the lightand electron microscope levels (Personne, 1966) .Under normal in vivo conditions, the enzyme pre-sumably functions in the catabolism of glycogen .Under certain defined conditions in vitro, phos-phorylase can also catalyze the synthesis of glyco-gen (Cori and Cori, 1945 ; Mordoh et al ., 1965 ;Wanson and Drochmans, 1968) . Demonstrationof phosphorylase activity associated with the invivo synthesis of glycogen has been reported byTakeuchi and Kuriaki (1955) and by Guha andWegmann (1959, 1961). Thus, when sperm areincubated in medium containing glucose-l-phos-phate ATP, Mg++ ions, and NaF, the concentra-tion of glycogen in sperm cells increases (Personne
FIGURE 18 Transverse sections through the middle(M) and tail segments of sperm of Planorbis are demon-strated . Intensely stained granules (arrow) are locatedin the matrix between the doublets of the axoneme (A)and the central pair of microtubules . X 90,000 .FIGURE 14 A transverse section through the tail pieceof a spermatozoon of Lymnaea is illustrated here . Gly-cogen granules are arranged as a thick mantle aroundthe axoneme . Opaque granules (arrow) are shown atthe level of the secondary fibers within the axoneme .X 120,000 .
FIGURE 15 A longitudinal section through the basal region of a spermatozoon of Mytilus is demon-strated in this micrograph . Glycogen granules (arrows) occupy the concavity of the proximal centriole(C) and of the basal body (B) . Glycogen granules are also present, in the matrix between the nucleus (N)and mitochondria (M) and around the centrioles . X 130,000 .
FIGURE 16 This micrograph illustrates a transverse section through the centriole (C) of a spermatozoonPecten . The glycogen granules are tightly packed in the lumen of the centriole (arrow) and are closelyapposed to its outer wall. X 130,000 .
and Anderson, 1969) . The newly formed productis finely granular (approximately 40 A in diame-ter) and closely resembles the gamma particlesdescribed by Drochmans (1962) . The fine-grainedproduct of glycogen synthesis lies adjacent to pre-existing glycogen deposits . When AMP is substi-tuted for ATP, the phosphorylase activity is weak .The newly formed particles believed to be glyco-gen are extracted after treatment with a-amylaseand partially extracted by $-amylase . Controlspecimens incubated in substrate-free mediumshow no increase in glycogen concentration . Afuller report on the localization of amylophos-phorylase activity in spermatozoa will be publishedseparately (Personne and Anderson, 1969) .
Glycogen in Atypical Spermatozoa ofProsobranch GastropodsProsobranch snails produce long threadlike,
ofuniflagellate sperm and enormous numbersatypical, vermiform spermatozoa. Atypical sper-matozoa lack distinct acrosomal and nuclear com-partments and a well defined middle piece . Inaddition, these vermiform spermatozoa aremultiflagellate and capable of vigorous movementsthroughout their entire length . Though metaboli-cally active and motile, they are incapable ofeffecting fertilization . Detailed studies on the de-velopment and structural polymorphism of thesespermatozoa have been made by Hanson et al .(1952), Kaye (1958), Gall (1961), and Yasuzumi(1960) .Atypical spermatozoa of Pomacea and Viviparus
(Paludina) are quite similar in ultrastructure . Theyare composed of an anterior cylindrical portionand a posterior tuft of flagella . The anteriorcylinder is composed of several rectangular orpyramidal structures that surround a central canal .
WINSTON A . ANDERSON AND PAUL PERSONNE Glycogen in Spermatozoa
FrGumes 17 and 18 Intensely stained glycogen granules (arrows) are closely apposed to the peripheralleaflet of the mitochondrial envelope of these spermatozoa of Pecten Fig. 17) and Mytilus (Fig. 18) . Thegranules are tightly packed between adjacent mitochondria (M) and around the centriole (C) . X 80,000.
FIGURE 19 Transverse sections through the middle and tail piece of sperm of Lymnaea are illustratedhere . In the middle piece, glycogen granules are ensheathed within special compartments (arrows) of themitochondrial derivative (M) . X 40,000 .
Mitochondria, dense rods, and several axonemesoccupy this canal . Glycogen particles are dis-tributed among the structures forming the anteriorcylinder, within the axonemes, and in the centralcanal itself (Figs . 22-23) . Glycogen granules ap-pear within and around the axonemes of flagellaat the base of these vermiform spermatozoa(Fig . 24) .
Distribution of Glycogen in Spermatozoaof Chordates
Mature spermatozoa of the Ascidiacea, Cionaand Molgula, all contain rosettes of a-glycogengranules . These glycogen particles are located be-neath the plasmalemma surrounding the nucleusand mitochondria . Glycogen is absent from theflagella of these spermatozoa .
Beta particles appear in the "packing" cyto-plasm around the mitochondria in spermatozoa ofthe toad fish, Opsanus tau. Spermatozoa of theguppy, Lebistes reticulatus contain both a- and
/3-glycogen granules. In this case too, the glycogenis located in the cytoplasm adjacent to the mito-chondria .
Sperm of the amphibians Rana and Bufo retainsome peripheral cytoplasm, and /3-particles arerandomly distributed in this residual cytoplasm .
The following table (Table I) presents a summa-tion of the sperm of various taxa that do and donot contain glycogen. The classification (Types I,II, III) under Mollusca is not based on interna-tional rules, but is a convenience used by us toplace sperm with similar mitochondrial deriva-tives into three distinct groups . Thus, Type I in-cludes "primitive" spermatozoa with relatively un-changed mitochondria ; Type II, sperm possessingan elongate, membranous mitochondrial deriva-tive; and Type III, sperm possessing a paracrys-talline mitochondrial derivative .
DISCUSSION
Previous electron microscope observations on
WINSTON A. ANDERSON AND PAUL PERSONNE Glycogen in Spermatozoa
FIGURE 20 Tangential sections through the middle piece of mature spermatozoa isolated from the her-maphroditic duct of Lymnaea . The glycogen granules occupy compartments (arrows) that course low-pitched helices within the paracrystalline mitochondrial sheath . X 30,000 .
FIGURE 21 Transverse sections through the middle piece of immature sperm in the ovotestis of Lymnaea .After treatment with the PA-TSC-SP procedure, the compartments encased by the mitochondrial sheathappear clear (arrows) and lack glycogen deposits . X 30,000 .
spermatozoa of some of the species examined inthis report have consistently failed to demonstratethe presence of glycogen . Failure to detect glyco-gen could have resulted from (1) the inability ofroutine techniques to demonstrate the very lowconcentrations of polysaccharide present, (2) amasked state of the polysaccharide, or (3) the ex-traction of glycogen following inadequate fixation .In addition, it must be emphasized that the factorof maturity is important in the accumulation ofglycogen, since in specimens examined by us gly-cogen stores appeared only in spermatozoa thathave completed their differentiation . Double fixa-tion by aldehydes and osmium tetroxide mayfacilitate glycogen preservation by fixing associ-ated proteins, but double fixation alone does notaugment the contrast of the polysaccharides . It ap-pears then, that negative results for glycogen withroutine methods are not indicative of its absence .
44 THE JOURNAL OF CELL BIOLOGY • VOLUME 44, 1970
Cytochemical tests that augment the contrast ofglycogen at the electron microscope level are nowwell developed (Hanker et al ., 1964; Seligman etal ., 1965 ; Thiery, 1967) . It must be emphasizedthat the PA-TSC-SP procedure is a direct modifi-cation of the PA-TSC-Os0 4 procedure originallydescribed by Hanker et al. (1964) and by Seligmanet al. (1965). In thie procedure, the aldehyde-thio-semicarbazones formed as a result of the perio-date oxidation of 1 , 2 glycol groups in polysaccha-rides and reaction with thiosemicarbazide arerevealed by their affinity for silver proteinate . AsThiery clearly indicated, when the thiosemicarba-zide reaction is short (30-45 min), the PA-TSC-SPprocedure is specific for components containinghigh concentrations of polysaccharide and for rela-tively pure polysaccharide complexes as glycogen .Based on its specificity and reproducibility, the per-iodic acid-thiosemicarbazide-silver proteinate pro-
FIGURE 22 A longitudinal section through the anterior, cylindrical region of an atypical sperm ofPomacea . Glycogen granules are present within and around the axoneme (A), and in the matrix betweenthe structures composing the walls of the cylinder (arrows) . X 50,000 .
cedure of Thiery (1967) proves most reliable forthe detection of small amounts of glycogen .
Significance of Glycogen in Spermatozoa
Depending upon the species, the survival timeof spermatozoa within gamete storage organsvaries from weeks to months . Spermatozoa areconsequently specialized to survive under condi-tions of high sperm density and of low oxygentension . The relative absence of oxygen and ofexogenous glycolyzable material suggests thatspermatozoan survival in these forms dependsneither upon aerobic oxidative processes nor uponthe utilization of extracellular glycolyzable sub-stances, but probably upon the anaerobic utiliza-tion of endogenous substrates . Glycogen located inthe middle piece and flagellum of spermatozoa ofsome species may well be an important endogenousenergy source .
Spermatozoa of some species are released intoan aqueous environment that is devoid of metabo-lizable substances and varies extensively in itsdegree of oxygenation . These sperm reportedlydepend entirely upon aerobic oxidative metabolic
processes (Rothschild, 1951 ; Rothschild andCleland, 1951 ; Afzelius and Mohri, 1966); yetsperm of some species survive for several hours inthe absence of oxygen (Barron, 1932) . Moreover,biochemical analyses establish the existence of sig-nificant amounts of glycogen within spermatozoaof some of these species. Sperm of Ciona and Phal-lusia (Ascidiacea), respectively, yield 2 .8 and 1 .6%
glycogen on a dry weight basis (Restivo andReverberi, 1957). Mature sperm of Saxostrea con-tain as much as 1 % glycogen . These findings indi-cate that sperm of these species have stored glyco-gen. The presence of intracellular glycogen wouldseem to indicate a capacity for survival where noexogenous substrates are available . Under condi-tions in which aerobic oxidative processes are in-hibited, sperm of the oyster metabolize exogenousmaterials (Humphrey, 1950) . In the presence offructose diphosphate, glycolytic processes accountor 0 .3 µl of lactic acid/10 8 sperm/hour. It seemspossible, therefore, that, like most cells, sperm ofsome marine species can break down carbohy-drates either aerobically or anaerobically .
Spermatozoa that effect internal fertilization
WINSTON A . ANDERSON AND PAUL PERSONNE Glycogen in Spermatozoa 45
FIGURE 93 A transverse section through the anterior cylindrical region of an atypical spermatozoon ofViviparus . Glycogen granules are present between the components of the walls of the cylinder, and in thecentral canal surrounding the axonemes (A) and mitochondria (M) . X 40,000 .
FIGURE 94 A longitudinal section through the basal region of an atypical sperm of Pomacea . /Q-particlesoccupy the central canal of the anterior cylinder and in the flagella that extend from it (arrows) . X40,000.
are usually released in seminal plasma that is richin hexoses and other metabolizable substances .Mammalian spermatozoa possess the full comple-ment of enzymes for catalyzing glycolytic activitiesunder aerobic or anaerobic conditions, as well asfor oxidative processes under appropriate cpndi-tions (Mann, 1964) . Since the prefertilizationmilieu is rich in metabolizable material, mam-malian sperm do not rely on endogenous substratesfor energy production except under unusual condi-tions . It is not surprising, therefore, that sperm ofthese species lack stored glycogen .
Spermatozoa of gastropod molluscs that effectinternal fertilization possess relatively large storesof intracellular glycogen which is located withinthe mitochondrial derivative, as well as withinand/or surrounding the axoneme . Thus, the glyco-gen stores are located in close proximity to theenergy-producing and energy-utilizing compo-nents. The glycogen presumably serves as a sourceof energy for metabolism and motility . The exist-ence of an active phosphorylase in the glycogen
46
THE JOURNAL OF CELL BIOLOGY • VOLUME 44, 1970
compartment (Personne, 1966 ; Personne andAnderson, 1969) and of glucose-6-phosphatase ac-tivity in glycogen-containing mitochondria (An-derson et al ., 1968 ; Anderson, 1968) stronglysuggests the presence of pathways for glycogenmetabolism in these cells .
The Distribution and Localization of Glycogen
in Spermatozoa
The appearance and form of glycogen in sper-matozoa vary with the species . However, it seemsapparent that #-particles represent the most com-mon form observed in both vertebrates and in-vertebrates. Among the species studied, a-particlesare present only in spermatozoa of some pulmo-nate gastropods, in the Ascidiacea, Ciona andMolgula, and in the poeciliid fish, Lebistes reticulatus .
The arrangement and localization of glycogenin sperm is somewhat related to the degree ofmodification of their middle piece . In the so called"primitive" spermatozoa possessing typical mito-
chondria at the base of the nucleus (Franzen,1956), glycogen particles are tightly packed withinall available space in the middle piece. Althoughglycogen is mainly located in the "packing" matrixbetween the mitochondria, it is even present insome instances within the centriole and basal body,and in indentations of the nucleus in sperm of somespecies. The presence of glycogen in centrioles andbasal bodies may have no special metabolic signifi-
48 THE JOURNAL OF CELL BIOLOGY • VOLUME 44, 1970
Reference
cance but may simply reflect storage in all availa-ble space. In the "primitive" spermatozoa, theglycogen is closely apposed to the externalenvelope of the mitochondria and to the base ofthe kinetic apparatus . The "primitive" sperm inquestion that store glycogen have some of the en-zymes needed to break it down, such as glucose-6-phosphatase (Anderson, 1968) .
Glycogen is usually absent from the middle
ChordataAscidiacea
Arbacia lixula
Ciona intestinalisMolgula manhattensis
Negative
Perinuclear, perimito-chondrial
Pisces Gasterosteus aculeatus
NegativeXiphophorus helleriPlatypocelia maculataLebistes reticulatesPerimitochondrialOpsanus tau
piece of more complex flagellate sperm . Instead,it is located adjacent to the axonemal componentsin the distal segments of the sperm tail in the re-gion between the peripheral doublets and theflagellar membrane . This particular arrangementof glycogen is characteristic of spermatozoa of suchwidely separated species as terrestrial and fresh-water annelids (Lumbricus, Hirudo), marine gastro-pods (Purpura), typical sperm of freshwater proso-branchs (Viviparus; Pomacea), and in sperm of thecirriped crustacean, Lepas. The close topographi-cal relationship of glycogen to the axoneme sug-gests a functional relationship between the two .
Glycogen granules are specifically located inmembrane-bounded compartments in typicalmitochondria (Anderson, 1968) and in the com-plex paracrystalline mitochondrial derivative ofsperm of pulmonate gastropods (Lanza andQuattrini, 1964 ; Personne and Andre, 1964 ;Andre, 1965) . The presence of glycolytic enzymes(glyceraldehyde - 3 - phosphate dehydrogenase,LDH) in the immediate proximity of the glycogengranules was shown in a previous paper (Andersonand Personne, 1969) . The presence of phosphoryl-ase activity in the glycogen compartment alsoindicates the existence of glycogen synthetic anddegradation enzymes within this compartment(Personne and Anderson, 1969) . Hypotheses onthe interrelations of glycogen and of othermetabolic pathways have already been discussed(Anderson et al ., 1968) .
The intra-axonemal localization of glycogen inspermatozoa of pulmonate and prosobranch gas-tropods is highly unusual . In these sperm, granuleswith the staining properties of glycogen and whichare extractable with ex-amylase are located at thelevel of the secondary fibers between the centraland peripheral doublets . The functional signifi-cance of this arrangement is unknown .
REFERENCES
AFZELIUS, B. A ., and H. MOHRI. 1966 . Mitochondriarespiring without exogenous substrate. A study ofaged sea urchin spermatozoa . Exp . Cell Res. 42:10 .
ANDERSON, W. A. 1968 . Cytochemistry of sea urchingametes. I . Intramitochondrial localization ofglycogen, glucose-6-phosphatase and adenosinetriphosphatase activity in spermatozoa of Para-centrotus lividus. J. Ultrastruct . Res . 24:398 .
ANDERSON, W. A., and R. A . ELLIS . 1965 . Ultrastruc-ture of Trypansoma lewisi : flagellum, microtubulesand the kinetoplast. J. Protozool. 12:483 .
ANDERSON, W. A., and J. ANDRE. 1968 . The extrac-
Spermatozoa of some species (e .g . insects, birds,and mammals) do not contain stores of glycogengranules per se, even though, on the basis of theirability to flagellate actively, they are consideredmature. In some specimens (e .g . insects, Baccettiet al ., 1969 a, b ; Cantacuzene, personal communi-cation) the coarse fibers of the axoneme containpolysaccharide material that bears some similarityto glycogen. It may be unlikely, however, thatpolysaccharides in structural components of theaxoneme are metabolized in energy produc-tion .
Glycogen storage, therefore, seems to be a rela-tively widespread phenomenon among sperma-tozoa of several invertebrate and vertebrate species(Anderson and Personne, 1969) . Evidence thatintracellular glycogen reserves may represent animportant energy source will be presented in asubsequent report (Anderson and Personne, inpreparation) .
The authors express appreciation to Dr . Don W .Fawcett, Department of Anatomy and Departmentof Reproduction and Human Reproductive Biology,Harvard Medical School, Boston, Massachusetts,and Dr. Jean Andre, Laboratoire de Biologic Cellu-laire 4, Faculte des Sciences, 91-Orsay, France, fortheir constructive criticism of this manuscript .
This study was supported in part by grant ERA-174 from the Centre National de la Recherche Scien-tifique and in part by Contract No . NIH 69-2107from the Institute of Child Health and Human De-velopment, National Institutes of Health, UnitedStates Public Health Service . It was also supportedby a Postdoctoral Fellowship (Grant No . PF-342)awarded to Dr . Anderson from the American Can-cer Society, Inc ., New York .
Received for publication 7 April 1969, and in revised form18 August 1969.
tion of some cell components with pronase andpepsin from thin sections of tissue embedded in anepon-araldite mixture . J. Microsc. 7 :343 .
ANDERSON, W. A., and P . PERSONNE . 1969. Recentcytochemical studies on some invertebrate andvertebrate spermatozoa . Proceedings of the FirstInternational Symposium of Comparative Sperm-atology, Rome and Siena. B. Baccetti, editor .Accademia dei Lincei, Rome .
ANDERSON, W. A., A. WEISSMAN, and R. A. ELLIS.1967. Cytodifferentiation during spermiogenesisin Lumbricus terrestris. J. Cell Biol . 32 :11 .
WINSTON A. ANDERSON AND PAUL PERSONNE Glycogen in Spermatozoa
ANDERSON, W. A ., P . PERSONNE, and J . ANDRE .1968. Chemical compartmentalization in Helixspermatozoa . J. Microsc. 7 :367 .
ANDRE, J . 1965 . Quelques donnees recentes sur lastructure et la physiologic des mitochondries ;glycogene, particules elementaires, acides nucle-iques . Arch . Biol . 76 :277.
BACETTI, B., E. BIGLIARDI, R . DALLAS, and F. Ro-SATI. 1969 a . The outer coarse fibers in the axialfilament complex of insect spermatozoa . J. Mi-crosc. 8 :32 a.
BACCETTI, B., K. DALLAI, and F. ROSATI . 1969 b .The spermatozoon of Arthropoda. III . The low-est holometabolic insects . J. Microsc . 8 :233 .
BARRON, E . S. G. 1932. The effect of anaerobiosison the eggs and sperm sea urchin, starfish andNereis and fertilization under anaerobic condi-tions . Biol . Bull. 62 :46 .
VON BONSDORFF, C.-H., and A. TELKKA . 1965. Thespermatozoon flagella of Diphyllobothrium latum(fish tapeworm) . Z. Zellforsch. 66:643.
BROWN, G. G. 1966. Ultrastructural studies of crus-tacean spermatozoa and fertilization. DoctoralDissertation, University of Miami.
CORI, C. F., and G. T. CORI. 1945. The activity andcrystallization of phosphorylase b. J. Biol. Chem .158 :341 .
DROCHMANS, P. 1962. Morphologic du glycogene .Etude au microscope electronique de colorationsnegatives du glycogene particulaire . J. Ultra-struct. Res. 6 :141 .
FRANZEN, A . 1956. On spermiogenesis, morphologyof the spermatozoon, and biology of fertilizationamong invertebrates. Zool. Bidrag., Uppsala . 31 :355 .
GALL, J . G. 1961 . Centriole replication . A study ofspermatogenesis in the snail Viviparus. J. Biophys.Biochem . Cytol . 10 :163.
GALTSOFF, P. S., and D . E. PHILPOTT. 1960 . Ultra-structure of the spermatozoon of the oyster, Cras-sostrea virginica . J . Ultrastruct . Res . 3 :241 .
GUHA, S., and R. WEGMANN. 1959. Etudes sur l'ac-tivite phosphorylasique . I. Une nouvelle methodede mise en evidence de la phosphorylase. Ann .Histochem . 4 :103 .
GUHA, S., and R. WEGMANN. 1961 . Phosphorylase in
HANSON, J., J . T. RANDALL, and S . T. BAYLEY .1952. The microstructure of the spermatozoa ofthe snail Viviparus . Exp . Cell Res. 3 :65.
HORSTMANN, E. 1968. Die spermatozoen von Geophi-lus linearis Koch (Chilopoda) . Z. Zellforsch . 89 :410 .
HUMPHREY, G . F. 1950. The metabolism of oysterspermatozoa . Aust. J. Exp. Biol. Med. Sci. 28 :1 .
ITO, S ., and M. J . KARNOVSKY. 1968. Formaldehyde-
KAYE, J . S. 1958. Changes in the fine structure ofmitochondria during spermatogenesis . J. Morphol .102 :347.
LANZA, B., and D . QUATTRINI. 1964. Observazionisul contenuto in glicogeno degli spermi e degliepiteli della gonade e delle vie sussuali ermafrodiedi Vaginalus borellianus (Colosi) (Mollusca, Gas-tropoda, Solifera) . Boll . Soc . Ital . Biol . sper . 40 :1154.
LONGO, F. J., and E . J . DORNFELD. 1967 . The finestructure of spermatid differentiation in the mus-sel, Mytilus edulis . J. Ultrastruct . Res. 20:462 .
MANN, T. 1964. In Biochemistry of Semen and theMale Reproductive Tract. Methuen and Co .,Inc., London .
MARINOZZI, V. 1961 . Silver impregnation of ultra-thin sections for electron microscopy . J. Biophys .Biochem . Cytol. 9 :121 .
MONNERON, A., and W. BERNHARD . 1966 . Actionde certaines enzymes sur des tissue inclus en epon .J. Microsc . 5 :697 .
MORDOH, J ., L. F. LELOIR, and C. R. KRISMAN .1965 . In vitro synthesis of particulate glycogen .Proc. Nat . Acad. Sci . 53 :86 .
PERSONNE, P. 1965. Etude cytochemique du derivemitochondrial du spermatozoide de la Testacelle :recherche des proteines et des lipides . J. Microsc.4:627.
PERSONNE, P. 1966 . Role des mitochondries dans lemetabolisme du glycogene . J. Microsc. 5:70A .
PERSONNE, P., and W. A. ANDERSON. 1969 . Localisa-tion mitochondrial d'enzymes liees au metabolismedu glycogene daps le spermatozoide de l'escargot .J. Cell Biol . 44 :20.
PERSONNE, P ., and J . ANDRE. 1964 . Existence deglycogene mitochondrial dans le spermatozoidede la Testacelle . J. Microsc . 3 :643.
REsTivo, F., and G . REVERSERL 1957. Ricerce suglispermi di Ciona a di Patella . Localizzazione dellacitochromo-ossidasi, della succino-deidrogenasi edel glicogeno . Acta Embryol. Morphol. Exp . 1 :164 .
H. DMOCHOwsICI, and L . I . KATZOFF. 1965 . Histo-chemical demonstration of some oxidized macro-molecules with thiosemicarbohydrazide (T.C .H .)or thiosemicarbazide (T .S.C .) and osmium tetrox-ide . J. Histochem . Cytochem . 13 :629 .
SILVEIRA, M., and K. PORTER . 1964 . The spermato-zoids of flatworms and their microtubular systems .Protoplasma . 49 :240 .
chick-embryo liver . J. Histochem . Cytochem . 9 :454 . ROTHSCHILD, L., and K. W. CLELAND. 1952. TheHANKER, J. S ., L .UENO, and A. M.
P. SEAMAN,SELIGMAN .
H. P. WEISS, H . physiology of sea urchin spermatozoa . The natureand localization of the endogenous substrate . J.Exp . Biol. 29 :66 .
SELIGMAN, A . M., J . S . HANKER, H . WASSERKRUG,
1964. Osmiophilicreagent : new cytochemical principle for light andelectron microscopy . Science. 140:1039 .
SPIKES, J. D. 1949 . Metabolism of sea urchin sperm .
Amer. Natural. 83 :285 .TAKEUCHI, T ., and H . KURIAKI. 1955. Histochem-
ical detection of phosphorylase in animal tissues .
J. Histochem. Cytochem . 3 :153 .THIERY, J . P. 1967 . Mise en evidence des polysac-
charides sur coupes fines en microscopic elec-tronique . J. Microsc . 6 :987 .
VOELZ, H., and M . DWORKIN . 1962 . Fine structureof Myxococcus xanthus during morphogenesis . J.Bacteriol. 84 :943 .
WALKER, M., and H. C. MACGREGOR . 1968. Sper-matogenesis and the structure of the mature spermin Nucella lapillus (L) . J. Cell Sci. 3 :95 .
WANSON, J . C., and P. DROCHMANS . 1968. Detectionof phosphorylase with the electron microscope.Control of Glycogen Metabolism . Fed. Europ.Biochem . Soc . Symp. 4th. Oslo . 16 9 .
YASUZuM!, G . 1960 . Spermatogenesis in animals asrevealed by electron microscopy . VII . Spermatiddifferentiation in the crab, Eriocher japonicus . J .Biochem . Cytol. 7 :73 .
WINSTON A. ANDERSON AND PAUL PERSONNE Glycogen in Spermatozoa
