Vol. 40 BASIC AMINO-ACIDS OF PROTEINS 481(18) Dakin, H. D. (1920). J. biol. Chem. 44, 499.(19) Osborne, T. B., Van Slyke, D. D., Leavenworth,

C. S. & Vinograd, M. (1915). J. biol. Chem. 22,259.

(20) Stokes, J. L., Gunness, M., Dwyer, I. M. & Caswell,M. C. (1945). J. biol. Chem. 160, 35.

(21) Dunn, M. S., Camien, M. N., Shankman, S. & Rock-land, L. B. (1945). J. biol. Chem. 159, 653.

(22) Ross, A. F. (1942). J. biol. Chem. 143, 685.

(23) Ross, A. F. (1941). J. biol. Chem. 138, 741.(24) Dunn, M. S., Shankman, S. & Camien, M. N. (1945).

J. biol. Chem. 161, 669.(25) Hier, S. W., Graham, C. E., Freides, R. & Klein, D.

(1945). J. biol. Chem. 161, 705.(26) Doermann, A. H. (1945). J. biol. Chem. 160, 95.(27) Block, R. J. & Bolling, D. (1945). Arch. Biochem. 6,

419.(28) Sharp, J. G. (1939). Biochem. J. 33, 679.

Studies on the Metabolism of Semen3. FRUCTOSE AS A NORMAL CONSTITUENT OF SEMINAL PLASMA.SITE OF FORMATION AND FUNCTION OF FRUCTOSE IN SEMEN

BY T. MANN, Moiteno Institute, Univernity of Cambridge

(Received 2 April 1946)

Previous investigations (Mann, 1945a, b, c) haveshown that the glycolytic pathways and enzymesinvolved in the carbohydrate metabolism of sper-matozoa resemble those of other animal tissues andyeast. These studies have established the participa-tion of hexokinase, zymohexase and of certainphosphopherases and dehydrogenases in spermglycolysis and they disclosed that adenosinetri-phosphate (ATP) forms a link between the activityof spermatozoa and glycolysis. In all these experi-ments washed spermatozoa were used. It remainedto be. seen what is the glycolytic mechanism whichoperates in whole semen, i.e. in semen which has notbeen divided artificially into its two natural com-ponents, spermatozoa and seminal plasma.

Since the early researches by Iwanow (1931) andRedenz (1933) it has been known that in oneimportant aspect at least the washed spermatozoashow a strikingly different behaviour from wholesemen. Whereas washed spermatozoa can surviveanaerobically only in presence of glucose or someother glycolyzable carbohydrate, in whole sementhe spermatozoa continue living without additionalsugar, at the expense of a reducing and yeast-fermentable carbohydrate already present in theseminal plasma. In the extensive literature dealingwith the subject of seminal sugar, this substancehas hitherto been described either as glucose orsimply as the reducing sugar of semen (Killian,1933; Bernstein, 1933; Goldblatt, 1935; Shergin,1937; McKenzie, Miller & Bauguess, 1938; Davis &Cole, 1939; Huggins, Scott, & Heinen, 1942; Moore& Mayer, 1941; MacLeod & Hotchkiss, 1942;Salisbury & Van Demark, 1945), and the onlyreference to the probable occurrence of fructose insemen is found in a paper by Yamada (1933) whosurveyed human tissues and body fluids for the

presence of fructose by means of a colour test with'Cryogenine' (the antipyretic drug 'Cryog6nineLumiere').

In view of the inadequate evidence as to theidentity of the reducing carbohydrate present insemen, an investigation of its chemical nature wasundertaken. The sugar has been putified and shownto be identical with d(-)-fructose (preliminarycommunication, Mann, 1946). In this study it willbe demonstrated that fructose accounts for practi-cally the whole of the yeast-fermentable carbo-hydrate present in the seminal plasma of severalspecies, including bull, ram, rabbit, boar and man.Evidence will also be given that fructose originatesin the accessory glands of reproduction and thatspermatozoa come in contact with it during theirpassage through the male generative tract. Finally,it will be shown that fructose in whole semen is thenatural substrate which provides life energy to thespermatozoa by its glycolytic breakdown to lacticacid.

EXPERIMENTAL

MethodsThe experiments were carried out with ejaculated semen.The semen of animals was collected by Dr A. Walton at theAnimal Research Station, Cambridge, by the method ofWalton (1945). Several samples of bull semen were kindlysupplied by Veterinary Officers in charge of Cattle In-semination Centres in England. The semen was used wholeor it was separated by centrifugation into seminal plasmaand spermatozoa. The spermatozoa were washed free of theseminal plasma by means of a Ca-free Ringer's solutioncomposed as follows: 100 nil. 0.9% NaCl+4 ml. 1.15%KCI +1 ml. 2.11% KH2PO4 +1 ml. 3.82% MgSO4. 7H20+2 ml. 1.3% NaHCO.. In some experiments this solutionwas further supplemented by 0.5% fructose ('Ringer.fructose') or by 20 ml. 0.25 x-phosphate buffer, pH 7.4

T. MANN('Ringer-phosphate'), or by both fructose and phosphate('Ringer-fructose-phosphate').

Total reducing sugar was determined by the method ofHagedorn & Jensen (1923); with this method the reducingvalue of fructose is only about 2% lower than that of anequivalent amount of glucose (Baranowski, 1935). Thevalue obtained by the reduction of ferricyanide in theHagedorn-Jensen method will be referred to as the 'totalreducing sugar'; it represents the sum of yeast-fermentablesugar and some reducing but non-fermentable substances.

Yeast-fermentable sugar. In order to distinguish betweenthe yeast-fermentable and non-fermentable components ofthe seminal plasma the following technique was adopted.Fresh baker's yeast (2 g.) was suspended in 20 ml. tap water,left 1 hr. at roomtemperature and then centrifuged. Theyeast was resuspended in 20 ml. of 0-05M-phosphate buffer(pH 6) or in 20 ml. water. Portions of 2 ml. of the washedyeast suspension were pipetted into two conical centrifugetubes, A and B. A had no further additions, B received1 ml. seminal plasma. Both tubes were incubated for 2 hr.at 300. The incubation was terminated by centrifugation,and the centrifuged yeast washed with 2 ml. water. Thesupernatant solutions and wash-fluids were then trans-ferred quantitatively into two 10 ml. volumetric flasks, and1 ml. fresh seminal plasma was added to A. After dilutingthe content of each flask with water to the 10 ml. mark,suitable samples (1-5 ml.) were removed and examined forreducing sugar content. The difference in the reducingvalue between A and B represents the amount of 'yeast-fermentable sugar'.The time required for 2 ml. yeast suspension to remove

the whole yeast-fermentable sugar from 1 ml. seminalplasma was rather long (up to 2 hr.); much longer than thetime needed for yeast to remove the sugar from 1 ml. bloodplasma. This is only partly due to the fact that the seminalplasma contains several times more sugar than the bloodplasma. The main reason is that the yeast-fermentablesugar present in the seminal plasma is not glucose butfructose. Baker's yeast available locally was found tobe much less effective towards fructose than towardsglucose.

Fructose. The identification of fructose as the sugarpresent in the seminal plasma was carried out by thepurification of the sugar and by the preparation of themethylphenyl-fructosazone which has been shown byNeuberg (Neuberg, 1902, 1904; Neuberg & Strauss, 1902;Langstein & Neuberg, 1907) to be one of the few chemicalderivatives by means of which fructose can be identifiedand distinguished from glucose and from other sugars.According to Neuberg, pure methylphenyl-fructosazonehas m.p. 1530.For rapid quantitative determinations of fructose in

small samples of semen (0 05-0-5 ml.) two colorimetricmethods were adapted, those of Roe (1934) and of Stewart,Scarborough & Davidson (1938). Both gave satisfactoryresults provided that the proportions of the solutions usedfor deproteinizing, 10% (w/v) ZnSO4.7H2O and 0-5N-NaOH, were carefully adjusted so as to yield mixtures ofpH 74-7-5. The routine procedure was similar to that usedfor blood (Somogyi, 1930): The semen was diluted with waterto 8 ml., 1 ml. ZnSO4 and 1 ml. NaOH added, the mixtureheated for 1 min. in boiling water and filtered; 2 ml. of theclear filtrate were used for fructose estimation. Roe'smethod could be used also for trichloroacetic acid filtrates.

Glucose. When fructose was established as the principalyeast-fermentable sugar of semen, it became necessary todecide whether fructose occurs in the semen alone or withother yeast-fermentable sugars, particularly glucose, thusrequiring a method for the accurate determination of smallquantities of glucose in presence of excess of fructose. Thiswas accomplished enzymically by glucose oxidase, anenzyme extracted from moulds, which rapidly oxidisesglucose but scarcely attacks fructose. Two sources of theenzyme were used, one a purified preparation of 'Notatin'kindly supplied to Prof. D. Keilin by Prof. Raistrick andDr Birkinshaw (Keilin & Hartree, 1945); the other a crudebut very potent preparation (QO2 =1000) obtained fromAspergillus niger in the following manner. The fungus wasgrown as described by Mann (1944). After 2 days of growthat 300, 500 ml. filtered culture medium, pH about 4-5-5,were concentrated in vacuo to 50 ml., dialyzed overnight,and the enzyme precipitated with 5 vol. of cold acetone.The acetone-dried preparation was stable at 00. It wasdissolved before use in 0-2M-phosphate buffer, pH 6, so as togive a 0.5% solution. For the estimation of glucose, twoincubation flasks were used. Both flasks received 2 ml.seminal plasma + 1 ml. 0-2M-phosphate buffer, pH 6, and inaddition 0-1 ml. glucose oxidase and 1 drop of a catalasepreparation were added to one flask. The flasks were thenshaken in air at 200 for 2 hr. The samples were then de-proteinized and the reducing sugar was determined. Thedifference in the reducing value between the oxidase-treated and untreated sample gives directly the glucosecontent in 2 ml. seminal plasma.

Other methods. The methods employed for the estimationof lactic acid and of acid-soluble phosphate were the sameas previously described (Mann, 1945b, c). In a few experi-ments the 'total carbohydrate' was estimated by theorcinol method of Tillmans & Philippi (1929) and glycogenby the method of Good, Kramer & Somogyi (1933). How-ever, a full interpretation of the data obtained by means ofthe last two methods will be possible only when the seminal'glycogen' has been purified and its chemical natureproperly investigated.

RESULTSPURIFICATION AND IDENTIFICATION OF

FRUCTOSE IN SEMEN

For the purification of fructose bull seminal plasmawas chosen because it has an even higher fructosecontent than the seminal plasma of other mammals.Altogether 120 ml. bull seminal plasma were used,containing 780 mg. fructose. This amount repre-sented a mixture of seven samples, each originatingfrom three to four bulls. The quantitative analysisQf the seven samples gave the following results:

Reducing sugar 785 635 498 447 1090 531 1098(mg./100 ml.)

Fructose 779 592 480 376 1040 435 1062(colorim.)(mg./100 ml.)

The seminal plasma was made just acid to methylred with 011N-HCI and precipitated with 5 vol. ofethanol. The clear filtrate was concentrated by

482 I946

FRUCTOSE IN SEMEN

distillation in vacuo at 400 and the thick syrup

extracted with several successive portions of boilingethanol till the residue gave only a very weakSeliwanoff reaction. The combined ethanolic ex-

tracts, 250 ml. in all, were concentrated in vacuo,

and the extraction of the syrup was repeated withboiling ethanol. This extract, on concentration,yielded a syrup which when dissolved in water andfiltered through a thin layer of kieselguhr gave

15 ml. of a perfectly clear and almost colourlesssolution containing 1030 mg. dry matter. Of thismaterial 630 mg., or 65 %, was fructose as assessedby reduction of ferricyanide and by colorimetricdetermination. This concentrated solution of fruc-tose obtained from serqinal plasma was used for thefollowing tests.

Optical activity. A portion was diluted with waterto a concentration of 0 848% reducing sugar, andthe solution, examined in the polarimeter in a

2 dm. tube, gave a=-156, [a]M = _ 92.2°. Purefructose in aqueous solution had [a] °=2 -92.3°.

Yeast-fermentation test. A 1 ml. sample (42 mg.seminal fructose) suitably diluted with phosphatebuffer, pH 6, was divided into two portions ofwhich one was incubated for 2 hr. with yeast, theother serving as control. In both samples thereducing sugar and fructose were determined. Theyeast-incubated sample no longer contained any

fructose or reducing sugar.

Absence of glucose. 1 ml. suitably diluted was

examined for the presence of glucose by means ofglucose oxidase but there was no change in eitherreducing value or fructose content as the result ofthis treatment.

Phenylosazone. 1 ml. was used for the prepara-

tion of phenylosazone. Two recrystallizations fromethanol gave 19 mg. of osazone; m.p. 2050.

Methylphenyl-fructosazone. The remaining solutionof seminal fructose was concentrated in-vacuo over

P205 to 3-3 ml. Insoluble material was centrifugedoff, and 3 ml. of the clear solution (450 mg. reducingsugar) was treated in a small tube with 1 g. a-

methylphenylhydrazine and then ethanol addeddrop by drop till a clear solution was obtained.After addition of 1 ml. 50% acetic acid the tube wasimmersed in boiling water for 5 min. and then cooledin ice. Within 30 min. there appeared a mass ofyellow needle-shaped crystals. In addition, a brownoily material was deposited in the bottom of thetube. After 2 hr. the crystalline material was

separated from the oil, and washed on a sinteredglass filter with ice-cold water. It was recrystallizedfrom 15% (v/v) ethanol giving 200 mg. of the re-

crystallized osazone; m.p. 1510. Methylphenyl-osazone prepared from pure fructose had exactlythe same m.p. (1510). An attbmpt to obtain in thismanner methylphenylosazone from glucose was

negative.Biochem. 1946, 40

A further batch of methylphenylosazone wasobtained from the oily residue. This was washedseveral times with cold water (by decanting), driedover H2SO4 and then dissolved in absolute ethanol.The solution (3 ml.) was filtered into a test-tube andimmersed in a freezing mixture of acetone and solidcarbon dioxide. The crystallization began alnostat once and after 14 hr. at 00 the crystals wereseparated. A little more crystalline osazone wasobtained from the filtrate by gradual addition ofwater. However, the product obtained in thismanner had m.p. 148°, even after one recrystalliza-tion. The total yield of methylphenyl-fructosazonefrom the seminal sugar was 56 %. The yield obtainedby Neuberg & Strauss (1902) from ascites fluid was66 %; they reported even higher values in some oftheir other preparations.

Search for other sugars in seminal plamaPhosphofructose. Bull seminal plasma contains

more fructose than the plasma of other animals. Itis also distinguished by a remarkably high thoughvariable content of organic acid-hydrolyzable phos-phorus compounds (130-200 mg. P/100 ml.). Thepossibility was envisaged that some of this organicphosphorus may occur in the form of phospho-fructose. However, the presence of more than anegligible quantity of fructose in the phosphory-lated form may be discounted in view of the follow-ing facts. When the seminal plasma is treated withyeast (2 hr., 30°) only 2-25 mg./100 ml. fructoseremain out of several hundred mg./100 ml. fructoseoriginally present, yet the disappearance of fructoseis not accompanied by any appreciable diminutionin the above-mentioned organic P-fraction. More-over, if the seminal plasma is precipitated with Baacetate in presence of ethanol, the precipitate con-tains very little fructose, practically all of whichremains in the supernatant solution. Under theseconditions monophospho- and diphospho-fructosewould both be precipitated as Ba-salts.The possibility was also considered that apart

from fructose the seminal plasma may containsome ketotrioses, either phosphorylated or phos-phate-free, which give the same colour reactions asfructose and its derivatives. However, this appearedunlikely in view of the very low content of bi-sulphite-binding substances. Trichloroacetic acidextracts from bull and ram seminal plasma treatedwith NaHSO3 according to Clift & Cook (1932) usedup less than 0-2 ml. O1OlN-12/ml. semen.

Glucose. Yet another question to be consideredwas that the seminal plasma might contain someglucose in addition to the large amount of fructose.Table 1 shows that this can be ruled out. When theseminal plasma is shaken aerobically with glucoseoxidase and the reducing value of such a sample iscompared with that of fresh seminal plasma, th-e

32

483VoI. 40

T. MANNdifference found is very small indeed. Glucoseadded to seminal plasma disappears after treatmentwith glucose oxidase.

Table 1. The effect of glucose oxidase on total reducingsugar and fructose content of seminal plama

(Total reducing sugar estimated by reduction of ferri-cyanide; fructose determined colorimetrically. Resultsexpressed in terms of mg. fructose/100 ml. seminal plasma.)

Before incubation After incubationwith glucose with glucose

oxidase oxidase

Reducing ReducingBull: sugar Fructose sugar Fructose

Sem. plasma 559 513 546 505Sem. plasma 1359 539 546 535+0-8% glucose

Ram:Sem. plasmaSem. plasma

Rabbit:Sem. plasmaBlood serumBlood serum+1% glucose

Man:Sem. plasma

Boar:Sem. plasma

Concentratiojplama

The quantitativeout in samples ofranging from 0*05 noview ofthe identificaof methylphenylosadescribed in the prregard the valuesmethods as representable, the determinicarried out simultaestimLation of fructoisame standard soluifor both riethods.

Comparative figuyeast-fermentable stplasma of severalstrikingly high levelrabbit and man eglucose in blood. Ir9 mg./100 ml. fruct(of the fructose concmust be remembereejaculate of boar (2Xlarger than that of 1quantity of fructoseis about the same:

Table 2. Total reducing sugar, yeast-fermentablesugar and fructose content of seminal plama

(Results expressed as mg. fructose/100 ml. seminal plasma.)Total

reducingsugar

Bull 7851090

Ram 458315472525

Rabbit 438618

Man 260262362

Boar 31

Fructose7791040340268285466318515100120204

9

Yeast-fermentable

sugar779105535026939446630052110012820810

Non-fermentable

residue.16351084678591389716013415421

682 565 682 565231 191 220 190 There is on the whole a close agreement between

the values for fructose and for yeast-fermentable618 515 610 515 sugar. This furnishes an additional proof that142 15 glucose, if at all present, occurs in the seminal

1142 - 16 - plasma only as a slight admixture with fructose.However, occasionally a discrepancy has been

265 100 257 100 observed between the content of yeast-fermentablesugar and that of fructose, indicating the presenceof a small amount of yet another yeast-fermentable

31 9 30 9 component. On the other hand, there is in alm,ostIn offructose in seminal every instance a distinct difference between theof various animals total reducing sugar and the yeast-fermentablesugar; this points to the existence in the seminalassay of fructose was carried plasma of some reducing but non-fermentable sub-semen from various animals, stances. The chemical nature of these substancesil. in bull to 1 ml. in boar. In remains to be investigated.ktion of fructose by the isolation Another characteristic feature of the seminal6zone and by other means as sugar is the variability of the fructose content in thereceding parts, it was safe to semen of the same species. Considerable variationsobtainable by colorimetric were observed in the semen from the same animal

ting fructose. Whenever practic- collected on different occasions, in spite of the factLation of reducing sugar was that all estimLations of fructose were carried out as6neously with the colorimetric soon after ejaculation as possible. These findingsse; in all such experiments the agree with statements by earlier investigators whotion of pure fructose was used recorded similar variations in reducing sugar in

semen (Goldblatt, 1935; Shergin, 1937; McKenzieires for total reducing sugar, et al. 1938; Davis & Cole, 1939; Ross, Miller &agar and fructose in the seminal Kurzrok, 1941; Moore & Mayer, 1941). The varia-species in Table 2 reveal the tions in the content of seminal fructose are difficultof fructose, which in bull, ram, to explain. If they were solely due to fructolysislxceeds several times that of taking place in semen already before ejaculation,i boar, on the other hand, only i.e. inside the male body, one would expect thatDse was found, or about 1/100 samples of semen comparatively poor in fructose3entration in bull. However, it would have at the same time a rather high content3d that the volume of a single of lactic acid. This, however, is not so (Table 3). It50-500 ml.) is almost 100 times seems, therefore, that the level of fructose in thebull so that in both animals the semen is not controlled by that mechanism whichcontained in a single ejaculate so strictly regulates the level ofglucose, for instance,20-50 mg. in blood and some other body fluids.

484 I946

FRUCTOSE IN SEMEN

Table 3. Fructose and lactic acid contentof whole semen (ram)

(Semen deproteinized within 10 sec. after ejaculation.)Ram Fructose Lactic acidno. (mg./100 ml.) (mg./loo ml.)1 188 43

274 252 280 43

428 563 238 424 300 375 176 226 340 55

500 62

Fructose in accessory organs of reproduction

As shown above, freshiy ejaculated semen oframor bull contains several hundred mg./100 ml.fructose. If this high content of fructose is con-

nected with the presence of spermatozoa as such,one would expect in the testes and in the epididymisan even higher concentration of fructose. Yet boththese organs were found to contain only 1-4 mg./100 ml. fructose. Moreover, the epididymal semen,

i.e. the thick suspension of spermatozoa which can

be obtained directly from the ram or bovineepididymis, gives a barely perceptible Seliwanoffreaction. This shows that the site of fructose forma-tion in the male body is situated along the genera-

tive passage between the epididymis and theurethral outlet.Huggins & Johnson (1933), to whom we owe an

extensive study of the secretions produced by theprostate gland and the seminal vesicles in man, havenoted that the high concentration of reducing sugar

in the semen is due principally to the seminalvesicles. Similar observations were made on the bull(Bernstein, 1937), boar (McKenzie et al. 1938) andram (Moore & Mayer, 1941). In view of theseobservations it seemed desirable to determine thefructose content in the various accessory glands ofreproduction. Table 4 shows that in the bull andram the seminal vesicles contain a high concentra-tion of fructose. On the other hand, in rabbit,

Table 4. Distribution offructose inorgans of reproduction

Fructose (mg./100 ml.)

TestesEpididymisProstate glandSeminal vesiclesFluid from seminalvesiclesAmpullaeUterus masculinusCowper's gland

Bull335

480840

Ram1

38

160570

Rat11

1010

Rabbit

1

230

Cat3435

12--O-0

Trace

which does not possess seminal vesicles, the prostate(GL. vesicularis) is rich in fructose. The form inwhichfructose occurs in the accessory glands is underinvestigation.

Function of fructo8e in semen

The main function of fructose is to supply the lifeenergy to the spermatozoa in the form of an easilyglycolyzable material. The incubation of a freshlyejaculated whole semen is followed by a progressivefall in fructose, and lactic acid accumulates as theresult of fructolysis (Table 5). The rate of fructolysisis greater in N2 than in air, and the ratio betweenlactic acid produced and fructose used up isappreciably higher in N2 than in air (Table 5, Exp.1). Aerobically, fructolysis is not the only source ofenergy for the spermatozoa which, even if deprivedof fructose, can survive in O owing to utilization ofother substances mainly of undetermined nature.Anaerobically, however, the spermatozoa verylargely depend on fructose and the cessation offructolysis invariably terminates their activity. Inorder to deplete ram semen of most of its fructose itis usually enough to incubate the semen in N2 for afew hours at 30-37'. If the anaerobic fructolysis isallowed to proceed almost to depletion of fructoseand if at this point fresh 'Ringer-fructose-phosphate'is added, this enables the spermatozoa to retain theiranaerobic activity and to continue to produce lacticacid with unabated vigour (Table 5, Exp. 2).Reinforcement of the fructose content by extrafructose added at the start of the incubation periodhas a similar effect (Table 5, Exp. 3). It must bestressed, however, that the survival under anaerobicconditions in semen can only be achieved if the pHof semen is not allowed to fall too low; for thisreason sugar was added to semen in thp form of'Ringer-fructose-phosphate' solution which has ahigh buffering capacity.

Since there is such a close connexion between thedisappearance of fructose and formation of lacticacid one might expect perhaps that the addition ofexcess carbohydrate to whole semen would not onlyprolong the duration of fructolysis but would at thesame time enhance the rate of lactic acid formation.However, that is not so, at least not in semen whichalready has a high concentration of fructose.- Itcan be seen from Exp. 3, Table 5, that during thefirst hour of anaerobic incubation, the yield of lacticacid formed is not markedly affected by thepresence of additional sugar. Only later, when thesemen has depleted its own sugar reserve, thebeneficial effect of extraneous sugar becomes moreapparent. In Table 6 fructose disappearance inwhole semen is compared with lactic acid formationboth in absence and in presence of various addi-tional sugars. It can be seen that in presence of

32-2

VoI. 40 485

486 T. MANN I946

Table 5. Fructolyi8 in whole semen

Exp. 1. Ram semen incubated at 300; anaerobically in Thunberg tubes filled with N2; aerobically by shaking in Barcroftmanometers filled with air. -.^-1-ucosls

Exp. 2. Ram semen; anaerobic incubation at 37°.

Additions to semen

Fresh semen

Same semen after 120 min. 1 vol. Ringer-phosphateincubation

1 vol. Ringer-fructose-phosphate

Fructose(mg./100 ml.)

4903121604133621594

Lactic acid(mg./100 ml.)

112290396500220292336

Fructose(mg./100 ml.)

Incubation , AA

(min.) Content Decrease0 465

120 35 4300 3590 9 260 535

90 287 248

Yructose 1088accounted for as

lactic acid(%)

1008686706542

Lactic acid(mg./100 ml.)

Content Increase118394 276394407 17394593 199

Exp. 3. 1 ml. samples of semen diluted with 1 ml. Ringer-phosphate (R. phosph.), Ringer-fructose-phosphate (R. fruct.phosph.) and Ringer-phosphate containing 0-5% glucose instead of fructose (R. gluc. phosph.), respectively. Otherwiseconditions as in Exp. 2. T

Additions to semen

R. phosph.

*R. fruct. phosph.

R. gluc. phosph.

-r rucmob(mg./100 ml.)

Incubation , -A

(min.) Content Decrease0 393 -

(mg./100 ml.)

Content Increase127 -

60 57 336 284120 42 351 291180 9 384 28960 582 311 161

120 450 443 240180 389 504 29760 318 75 164120 265 128 236180 187 206 315

157164162161240297164236315

Table 6. Effect of additional carbohydrate on the rate of glycoly8i8and on the fructo8e content of whole semen

(1% carbohydrate added to whole semen and mixture incubated at 300.)

Carbohydrate addedto whole semen

NoneNoneGlucoseFructoseMannoseGlycogenGalactosePyruvateArabinoseMaltoseLactoseSucroseRaffinose

Incubation(hr.)0

333333.333333

Fructose(mg./100 ml.)

33141274

27743592831128131

Lactic acid(mg./lOO ml.)

201397390432394405369560351401393390371

Decrease nfructose(%)0

8818

17878292916261

Ratio:Lactic acid formed

Fructose broken down

0-673-30

3-600-720-611-180-500-990-96

Anaerobically

Aerobicaly

Incubation(min.)

0150*300450150300450

FRUCTOSE IN SEMEN

added glucose or mannose the rate of fructose dis-appearance from semen is considerably diminished,but the rate of lactic acid formation remainsunimpaired. In other words, the addition of glucoseor mannose to whole semen has a suppressing in-fluence on the lactic acid formation from fructose,but this decrease is made good by lactic acidproduced from glucose or mannose respectively.The 'preserving effect' on the seminal fructose isevident only with glycolyzable sugars such asglucose and mannose. Glycogen and other non-glycolyzable sugars have no effect.The rate of fructolysis in semen is highly de-

pendent on temperature (Table 7). At 5-100, which

Table 7. Effect of temperature on the rate offructoly8is in whole semen

(Ram semen stored in narrow test-tubes at constanttemperature.)

Incubation I

Temp. Hr.

50 48100 48240 4300 3370 2

C-

Fructose(mg./

100 ml.)4143351131258350

Incubation

Exp. 1

Lacticacid(mg./

100 ml.)204242278364421423

Exp. 2

Decrease Fructose loss

in accounted forfructose by lactic acid(%) (%)0

18 4877 2570 5580 6987 60

k _ Fructose

remp. Hr. (mg./100 ml.)0 265

50 1 259100 1 254150 1 222240 1 40280 1 11300 1 10

Decrease infructose(%)

0

48

17659596

is the usual temperature for storing semen, an

appreciable proportion of seminal fructose stillremains after 2 days' incubation. At 30370, on theother hand, the disappearance of fructose is only a

matter of a few hours.Apart from fructose, the semen contains some

reducing substances which are'not fermented byyeast as well as a certain amount of carbohydratematerial which resembles glycogen. So far, however,no evidence could be produced that any of thesecompounds can be utilized by the spermatozoa as an

additional source of glycolysis (Table 8).The seminal 'glycogen', i.e. the alkali-resistant

polysaccharide which behaves on ethanol-precipita-tion and hydrolysis like the glycogen of otheranimal tissues, seldom exceeds 0-1 % in ram' semen;

of seven samples of ram semen, in five the glycogencontent ranged from 0-019 to 0-078 %, the remain-ing two had 0-118 and 0-190% respectively. Even

on prolonged incubation at 370, under conditionswhere fructose is completely exhausted, the level ofglycogen in semen underwent little change. Thechemical properties and function of seminal glyco-gen remain to be investigated.With regard to the enzymic mechanism in semen

which regulates the fructose metabolism, this issimilar in many ways to the mechanism of glucosemetabolism previously described in studies onwashed spermatozoa (Mann, 1945c). There it wasshown that glucose is first phosphorylated byadenosinetriphosphate and that the monophos-phohexose thus formed is further metabolizedthrough diphosphofructose, phosphotriose, phos-phoglyceric acid and pyruvic acid to lactic acid.Table 9 shows that glucose, fructose and mannoseare metabolized by washed spermatozoa at a ratewhich is of an equally high order for all threesugars; galactose and glycogen are glycolyzed onlyvery slowly. Also, the hexokinase reaction can bedemonstrated for glucose, fructose and mannosealike when these sugars are incubated with washedspermatozoa in presence of fluoride and adenosinetriphosphate (ATP). Table 10 shows that almosthalf of the readily hydrolyzable phosphorus of ATPis esterified within i hr. at 300, and to the sameextent with glucose, fructose and mannose. Amongthe various other sugars examined, only galactoseseems to undergo esterification with ATP. How-ever, a small degree of esterification with ATP canbe observed even without additional sugar (Exp. 2,Table 10). This is probably due to small amounts ofcarbohydrate present in the spermatozoa despitethe washing.The hexokinase reaction also takes place in whole

semen (Table 11), the chief reactants being fructosecontained in the seminal plasma and preformedATPin the spermatozoa. As a result of this reactionphosphohexose accumulates and can be precipi-tated as Ba-salt at pH 8 in the presence of 80%ethanol. Emphasis must be laid upon the peculiardependence which was formerly shown to existbetween the level of ATP and the glycolysis inwashed spermatozoa. This dependence is an equallyoutstanding feature in whole semen; here, too, themaintenance of the ATP level is intimately linked,at any rate anaerobically, with the unhamperedprogress of glycolysis (Table 12).

DISCUSSION

Generally, the evidence for the occurrence of freefructose in the animal body has been rather scanty.The presence of fructose in the embryo is one of thefew instances where the nature of this sugar wasadequately proved. A laevorotatory constituent infoetal fluids was first noticed by Claude Bemard(1855) but its identity with fructose was recognized

VoI. 40 487.

_ . .

Table 8. Change8 in various'carbohydrate fractions brought about by anaerobic glyColysi8 in whole semen

Exp. 1. 10 ml. ram semen divided into 2 equal parts, A and B. A centrifuged immediately; B centrifuged after 3 hr.incubation at 370 in N2. Centrifuged seminal plasma subdivided into 2 equal parts, one examined without furthertreatment, and the other after 2 hr. treatment with washed yeast; Results expressed in mg./100 ml. seminal plasma.

Total carbohydrate in non-deproteinized seminal plasma

Total carbohydrate inprotein-free filtrates

Total reducing sugar inprotein-free filtrates

Fructose

ASeminal plasma from fresh

Yeast- Non-fermentable fermentable

508 370

491

457

447

BSeminal plasma from semen allowed to

semen glycolyze for 3 hr., 37°, in N2

Yeast- Non-Together fermentable fermentable Together

878 51 368 419

l7O 661

78 535

19 466

49 167 216

19

21

97

11

116

32

Exp. 2. 20 ml. ram semen divided into 2 equal parts, A and B. A used fresh; B after 3 hr. incubation at 370, in N2.Glycogen estimated in 5 ml. samples of whole semen. Free fructose and 'Embden ester' estimated in filtrates aftertreatment of whole semen with trichloroacetic acid. The 'Embden ester' was separated as follows. Ba acetate added atpH 8, precipitate discarded, supernatant solution treated with 5 vol. ethanol, left overnight, and centrifuged. Theprecipitate dissolved in dilute HCI, Ba removed with Na2SO4, the Ba-free extracts analyzed. Results in mg./100 ml.whole semen. B

Free fructoseGlycogen:Reducing sugar

'Embden ester':Reducing sugarFructosePoP30Ptot.

AFresh semen

330

118

213017

Semen allowed toglycolyze for3 hr. at 370

23

101

3660212

(PO=phosphorus determined as phosphate which reacts directly with ammonium molybdate; P7 and P30=phosphoruswhich appears as orthophosphate after 7 and 30 min. hydrolysis with N-HCI; Pt0t. =phosphorus as total phosphate afterincineration of trichloroacetic acid extract.)

only some time later; fructose was definitely shownto be a normal constituent of both the allantoicand amniotic fluid and in urine of newly-bomanimals (Majewski, 1858; Gruber & Grunbaum,1904; Paton, Watson & Kerr, 1907; Langstein &Neuberg, 1907); it was also stated to be present infoetal blood (Orr, 1924). Fructose has also beendemonstrated in certain pathological dysfunctionslike fructosuria and in transudates (Rosin & Laband,1902; Neuberg & Strauss, 1902; Moraczewski, 1907;Adler, 1911; Barrenscheen, 1922). In most, if not inall of these instances, the concentration of fructosewas sufficiently high to make practicable a properidentification of the sugar by such means aspurification, optical activity measurements and thepreparation of specific derivatives. Occasionally,however, claims were made of the alleged presenceof fructose in animal tissues and body fluids wherethe chemical criteria applied were less satisfactory.Not infrequently statements were based merely on

positive reactions with certain colour-producingsubstances such as resorcinol (Seliwanoff, 1887),diphenylamine (Ihl, 1885) and cryogenine (Yamada,1933). Unfortunately, these colour reactions, unlesssupplemented by more direct and specific methods,seldom disclose the true chemical nature of thesugar studied, since they give positive results notonly with fructose but also with a variety of othersubstances such as the Neuberg ester (6-phospho-fructose), Harden-Young ester (2:6-diphospho-fructose), sucrose, methylglyoxal, dihydroxyace-tone, and numerous ketoses and ketose derivatives.Among the body fluids, semen is distinguished by

a very high content of reducing sugar which thepresent study proves to be d(-)-fructose. Thepurified seminal sugar shows the same reducingpower and the same specific optical activity as purefructose, and it yields the crystalline methylphenyl-fructosazone typical of fructose. It also gives thephenylosazone and it is yeast-fermnentable but,

488 T. MANN I946

FRUCTOSE IN SEMENTable 9. Glycoty8i8 in wa8hed spermatozoa

(Spermatozoa washed free from seminal plasma and madeup to the original vol. of whole semen with Ringer-phosphate.)Exp. 1. Bull spermatozoa, incubated 30 min. at 300 with

0-02M-sugar. T.nn+4n ol ;,l

No substrateAnaerobically:

GlucoseFructoseMannose

Aerobically:GlucoseFructose

iLac,cic acia(mg./100 ml.)

1-5

30542-530-5

24-524-5

Exp. 2. Ram spermatozoa, incubated anaerobically for2 hr. at 370 with 0 1 M-sugar. T AA S1

No substrateGlucoseFructoseMannoseGalactoseGlycogen

Lactic acid(mg./100 ml.)

7-0447-0417-0405 0103-032-0

unlike glucose, it is not acted upon by glucoseoxidase. In addition, it reacts in a characteristicmanner with the Seliwanoff reagent and with otherketose-reagents.At the site of their origin, in the testes and in the

epididymis, the spermatozoa, still immotile, havehardly any fructose at their disposal. However,when they traverse the male generative passagesfrom the testes onwards they receive the comple-mentary secretions of several accessory organs ofreproduction-seminal ducts, seminal vesicles,prostate gland, Cowper's gland and other urethralglands. These secretions, which together constitutethe seminal plasma, form the natural environmentand nutrient medium for the reproductive cells.Fructose is one of the numerous constitutents of theseminal plasma. Its concentration varies greatlybut in some species it may amount' to as much as1 g./100 ml. plasma. The seminal vesicles are themain 'contributors of fructose and the final con-centration of fructose in the whole semen asejaculated largely depends on the contributionmade by these organs. In some animals, which donot possess seminal vesicles, e.g. rabbit, part of theprostate provides the source of fructose. In the semenitself fructose occurs in a free, non-esterified form.This, however, does not exclude the possibility thatfructose may be the end-product of enzymicreactions involving more complex compounds.

Once in contact with spermatozoa, fructosediffuses readily into the cells and enters the charac-teristic chain of reactions initiated by the hexo-kinase-catalyzed reaction with adenosinetriphos-phate, leading finally to lactic acid. However, even

under purely anaerobic conditions the productionof lactic acid does not account for the total fructosemetabolized. In this respect semen behaves likeother animal tissues, except that whereas themajority of tissues have glucose or glycogen at theirdisposal, semen uses fructose which is the naturallyavailable sugar. It should be pointed out, however,that although normally spermatozoa utilize fructosethey are well able to glycolyze other sugars as well.If glucose or mannose, for instance, is added to thesemen, this offers the spermatozoa a choice ofseveral glycolyzable substrates and they actuallymake use of all of them so that whereas in untreatedsemen the entire lactic acid would have been theoutcome of fructolysis, in presence of additionalsugar only a certain proportion of lactic acid isderived from the seminal fructose and the rest is theresult of glycolysis of the extraneous sugar. This

Table 10. Pho8phorylation of carbohydrate byadeno8inetriphoaphate in washed sperm

Exp. 1. 109 ram spermatozoa + 0-422 mg. pyro-P of ATP +0-05M-sugar +0 04N-NaF; anaerobic incubation 30 min.at 300, in 95% N2 +5% C02.

No sugarGlucoseFructoseMannose

Incuba-tion P0(min.) (mg.)

0 0-19830 0-25130 0-25130 0-261

P7(mg.)0-6200-4280-4180 475

P30(mg.)0-6260-52105000-525

Pyro-Pesterifiedwith sugar

(mg.) (%)0 00-182 430-202 480-145 35

Exp. 2. 109 ram spermatozoa + 0-283 mg. labile P ofATP+0-03M-sugar+0005N-NaF; incubation in test-tubes open to air, 30 min. at 300. Pvrn P

No sugarNo sugarGlucoseFructose

Incu-bation(min.)

0303030

Po(mg.)0-2220-2720-2430-222

P7(mg.)0-5050 4950-3810-376

.ryru-.resterifiedwith sugar

r

(mg.) (%)0 00-011 40X124 440-129 45

Exp. 3. 109 ram spermatozoa +0-251 mg. pyro-P ofATP +0 03M-sugar + 0'04N-NaF; incubation in test-tubes opentn air Rn miin A.. RAO

No sugarNo sugarGlucoseFructoseMannoseGalactoseArabinoseSucroseMaltoseRaffinoseLactose

Incu-bation(min.)

0

30303030303030303030

PO(mg.)0-2440-2820-2320-2250-2510-2520-2940-2930-2940-2940-318

P7(mg.)0-4950-4470-3830-3690-3880-3980-4660-4730-4780-4480-478

Pyro-Pesterifiedwith sugar

(mg.) (%)0 00-048 190-112 440-156 620-107 420-097 390-029 110-022 90-017 60-047 190-017 6

VoIl. 40 489

-co air auVUu. av U ov .

Table 11. Pho8phorylationa in whole semen

Exp. 1. Ram semen diluted with equal volume of 'Ringer-fructose-phosphate' and incubated at 300, then centrifuged andthe sperm extracted with trichloroacetic acid. Results are expressed in mg. P/100 ml. non-diluted semen; NaF, 0-02N;iodoacetate (IAA), 0-001 N. pvn-p

Additions Incubationto semen min.

0 20NaF 20IAA 20

Po

8-08-0

11-2

P7

15-711-613-2

P30

16-513-615-9

icyr-v-.Lesterified

Pa1k. with sugar8-0 08-0 4-1

12-8 2-5

Exp. 2. Incubated 30 min. at 370: (A) 5 ml. ram semen without any additions, (B) 5 ml. ram semen containing 0-02 x-NaF.Incubation terminated by centrifugation. Centrifuged sperm extracted with trichloroacetic acid. Extracts precipitatedwith Ba acetate at pH 8 in presence of 10% ethanol, precipitates discarded. Supernatant solutions treated with 5 vol.of ethanol, the precipitate analyzed for P-fractions and fructose (from phosphofructose). Results in mg./100 ml. semen.

PO P7 P30 Ptot. Pt0t.-P30 FructoseA 0 0 0-10 3-05 2-95 0-2B 0 0-50 0-70 6-15 5-45 3-4

(Palk =alkali labile P phosphorus which appears as orthophosphate after exposure to 0-1 -NaOH for 20 min. at 200;POI P7, P30 and Pt t.=symbols explained in footnote to Table 8.)

Table 12. Dependence of the ATP-level on thefructoly8i8 in whole 8emen

Exp. 1. 150 min. anaerobic incubation at 370.

Inhibitoradded to ram

semen

0-015N-NaF0-001 N-iodo-acetate

Anaerobicincubation Fructose

at 370 (mg./(min.) 100 ml.)

432150 38150 168150 248

Lacticacid(mg./

100 ml.)158346158128

ATP-amino-N(mg./

100 ml.)1-440-740-280-35

Exp. 2. 10 ml. ram semen allowed to glycolyze 2 hr. at370, i.e. till the fructose content was reduced to 2 mg.

Then divided into two equal parts of which one (A) was

diluted with 1 vol. Ringer-phosphate, and the other (B)with 1 vol. Ringer-phosphate containing 27-8 mg. fruc-tose. Deproteinized with trichloroacetic acid after 2 hr.incubation at 37°. Results are expressed in mg./100 ml.semen.

Fructose

Beforeincubation

A 2

B 29-8

Afterincubatiou

0

16-7

Lactic acid ATP-amino-NAfter After

incubation incubation10-2 0-3122-6 0-92

explains why it has been possible in the past toemploy glucose successfully as the nutrient com-

ponent of artificial media for storing mammaliansemen. The ability of spermatozoa to make equaluse of fructose, glucose and mannose has itsfoundation in the fact that all three sugars enter thecycle of glycolysis by way of the same hexokinase-reaction with ATP, followed by the formation ofmonophosphohexose. From this point onwards thebreakdown leads through diphosphofructose, phos-photriose, phosphoglyceric acid, pyruvic acid tolactic acid, and in this respect the process in whole,semen is very much like that in washed spermatozoa

to which sugar was added artificially (Mann,1945a, b, c).The progress of fructolysis in semen depends on

several factors, such as the actual concentration offructose in the seminal plasma, the density ofspermatozoa, pH and temperature. In the field ofartificial insemination it has been a practice of longstanding to store the semen of farm animals at a

comparatively low temperature of 5-10', since atthis temperature the spermatozoa were known toremain alive much longer than at body temperature.It is now probable that this is due to the slower rateof fructose utilization at lower temperature.At present it is difficult to form an opinion as to

why the animal body should choose fructose insteadof glucose or glycogen as the natural sugar of semen.One may recall at this point another body fluid,milk, where the occurrence of lactose creates a

similar problem (Kay, 1945). There may be, ofcourse, several causes for the presence of fructose insemen but at least one already established fact mustbe taken into consideration. This relates to therather specific behaviour of spermatozoa towardsfructose as compared with that of most otheranimal tissues, few of which, according to Dickens& Greville (1932, 1933) who made a thorough com-

parative study of glucose and fructose metabolismin numerous animal organs, are capable of meta-bolizing fructose anaerobically to lactic acid. Thisshould enable the spermatozoa to draw freely on thefructose. reservoir as provided by the organs ofreproduction without any serious competition fromother animal tissues.

SUMMARY1. Contrary to the views- generally held, it was

found that the reducing carbohydrate of the seminalplasma is not glucose but d(-)-fructose.

P30-P7

0-82-02-7

Palk-Po0-00-01-6

I946490 T. MANN

Vol. 40 FRUCTOSE IN SEMEN 4912. Fructose has been purified from seminal

plasma and identified by its reducing value, opticalactivity, preparation ofmethylphenyl-fructosazone,yeast-fermentation test and by other means.

3. In the seminal plasma of several species,including bull, ram, rabbit, boar and man, fructoseaccounts for practically the whole of the yeast-fermentable reducing sugar. Little glucoge, if any,is present in the seminal plasma; this was shown byapplying a method which makes use of mouldglucose oxidase which oxidizes glucose qiuanti-tatively but leaves fructose untouched.

4. The level of fructose in seminal plasma variesfrom one species to another and even within thesame species there are individual differences. Thehighest values were observed in bull where .theconcentration sometimes exceeds 1 g. fructose/100 ml. seminal plasma.

5. Sperm obtained directly from the epididymiscontains hardly any fructose. During the passagethrough the male generative tract the semen acquiresfructose from the accessory glands of reproduction,of which the seminal vesicles are the chief contri-butors of fructose. In some animals which have no

seminal vesicles proper, fructose is generated in theprostate.

6. The main function of fructose in semen is tosupply the spermatozoa with readily glycolyzablematerial. On storage, the content of seminal fruc-tose falls progressively and lactic acid accumulates.

7. Normally spermatozoa utilize fructose, as thisis the chief sugar available in seminal plasma, buttheir enzymic equipment enables them to meta-bolize equally efficiently glucose and mannose. Theglycolysis of these three sugars in sperm is initiatedby a hexokinase interaction with adenosinetri-phosphate. The monophosphohexose thus formed isfurther metabolized through diphosphohexose,phosphotriose, phosphoglyceric acid and pyruvicacid to lactic acid.

This work has been carried out on behalf of the Agri-cultural Research Council. I wish to thank Prof. D. Keilin,F.R.S., and Dr A. Walton for their valuable help and kindinterest in this work. For the collection of the material Iam greatly indebted to Dr A. Walton and to the followingveterinary officers in charge of Cattle Insemination Centresin England: Mr R. Clarke, Mr E. D. Conn, Mr L. E. A.Rowson, Mr G. Smith and Mr D. L. Stewart.

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