The Use of Streptomyces Hyaluronidase in …Histochemistry of Acid M ucosaccharides Kazuyori Yamada':< and Satoru Shimizu料 Summary Streptomyces hyaluronidase was used as a means

Connective Tissue - 61ー

Vo1.7， No. 2: 61~71

The Use of Streptomyces Hyaluronidase in Connective

Tissue Specimens for Electron Microscopic

Histochemistry of Acid M ucosaccharides

Kazuyori Yamada':< and Satoru Shimizu料

Summary

Streptomyces hyaluronidase was used as a means of selectively digesting connective

tissue specimens for the electron microscopic indentification of hyaluronic acid. These

specimens included human umbilical cord and rabbit aorta. Testicular hyaluronidase was

also employed as a means of similar digestion in the same specimens for comparison.

The effects of digestions wIth Streptomyces and testicular hyaluronidases upo日 th巴dialyzed

iron reaction of acid mucosaccharid巴scan consistently be interpreted in terms of not only

the substrate specificities of the enzymes but the biochemically known nature of acid

mucosaccharides present in each connective tissue. Streptomyces hyaluronidase is thought

to depolymerize selectively and eliminate hyァaluronicacid in connective tissue specimens

for electron microscopy as in those for light microscopy and to be useful for the electron

microscopic identification of hyaluronic acid in mucosaccharide histochemistry.

In trod uction

A novel hyaluronidase with an absolute substrate specificity has been puri五吋 from

cultur百 ofStreptomyces hyalurolyticus nov. sp.14l， 15>， and has proved to be useful for the

histochemical identi五cationof hyaluronic acid in the system of light microscopy24)， 26> ，27> .

Biochemical assay studies have demonstrated that digestion with Streptomyces hyaluronidase

rusul ts in the selecti刊 degradationand elimination of hyaluronic acid in histologic sections

of di妊巴rentacid mucosaccharide-containing tissues for light microscopy27). Furthermore，

the Streptomyces enzym巴 was found to fail to affect practically no other mucosac-

charides than hyaluronic acid such as neutral and su!fated acid mucosaccharides in the

system of light microscopy30). The use of Streptomyces hyaluronidase in sp己cimens for

electron microscopic histochemistry of connective tissue mucosaccharides should， likewise，

be promising for gaining insight into the precise localization of hyaluronic acid at the

ultrastructural levels. In electron microscopic histochemistry of mucosaccharides， dig巴stion

experimen ts wi th mucosaccharidases such as testicular hyaluronidases6)， 7)，9)，13)，16>， siali-

* Lahor且toryof Histology， D邑partmentof Anatomy， Nagoya University School of Medicine， 65， Tsuruma-cho， Showa-ku， Nagoya， Japan.

** Lahoratory of U1trastructure Research， Aichi Cancer Center Res号arch Institute， 1159-81， Kanokoden， Tashiro-cho， Chikusa-ku， Nagoya， Japan. Received， May 2.， 1975

- 62一 Connective Tissue

dases2l，3l，6l and chondroitinases5l，28l can be performed upon at 1east three forms of tissue

specimens; tiny b1ocks， thick sections and u1trathin sections25l，29l. In 1ight microscopy of

acid mucosaccharides， however， the usefu1ness of Streptomyces hya1uronidase for the

identification of hya1uronic acid has been confirmed in thick sections of connective tis-

sues27l， 30l . Hence， i t seems 1ike1y tha t digestion exp巴riments with the Streptomyces

enzyme must be done pertinent1y in thick sections of mucosaccharide-containing connec-

tive tissues which are to be processed for e1ectron microscopy.

This communication demonstrates the use of Streptomyces hya1uronidase as a means

of se1ective digestion of hya1uronic acid in thick sections of connective tissues for e1ectron

microscopy of acid mucosaccharides， particu1arly as it gives reasonab1e resu1ts， when the

Rinehart-Abu1-Haj solution18l of dia1yzed iron is emp10y巴das a staining reagent of choice.

In order to be sure that the Streptomyces enzyme acts exclusive1y upon hya1uronic acid，

digestion with testicu1ar hya1uronidase was performed in paralle1 in the same connective

tissue specimens as those emp10yed for digestion with the Streptomyces enzyme. The

resu1 ts obtained in the pres巴ntstudy are taken to indicate that Streptomyces hya1uronidase

is usefu1 for the histochemica1 identification of hya1uronic acid in connective tissue speci-

mens for e1ectron microscopy as in thos巴 forlight microscopy.

Materials and Methods

Surgica1 specimens of human umbilica1 cords and autopsey specimens of rabbit aortas

were used. Immediate1y after being removed from donors， thes邑 tissueswere p1aced in

either of the two 五xatives; (a) chilled (40C) cacody1ate bu妊ered (pH 7.2) 2.5%

glutara1dehyde-2.0% paraforma1dehyde9l and (b) chilled (40C) cacody1ate bu狂ered(pH

7.2) 2.5% glutara1dehyde9l. In the fixatives， the tissues were sliced by a razor b1ade into

small strips with a thickness of approximat巴1y1-2 mm and fixed at 40C for 1-2 hr. After

五xation，the materia1s in the form of small strips were rinsed in 7.5% sucrose and then

sectioned at 40-60μwith a tissu巴 sectionerof Smith-Farquhar type or in a cryostat. The

thick sections thus obtained were rinsed in 7.5% sucrose and kept intact or subjected to.

the procedures of either the enzyme digestion or contro1 experiments described be1ow.

Subsequent1y， th巴 tissuesections were reacted 12-24 hr for the Rinehart-Abu1-Haj solu-

tion18l of dia1yzed iron at room temperature. The dia1yzed iron stock solution was

prepared according to the Hardin-Spicer modi五cation4lof the origina1 method18l. Before

use， 1 vo1um巴 ofglacia1 acetic acid and 4 vo1umes of the dia1yzed iron stock solution4l

were mixed， yie1ding a staining solution of pH 1.8-2.0. After staining with the dia1yzed

iron reagent， the tissue sections were rinsed in cacody1ate buffer (pH 7.2) and reflxed

1-1.5 hr at room temperature in cacody1ate buffered (pH 7.2) 2.0% osmium tetroxide.

The tissue specimens were then rinsed in distilled water， dehydrated with graded ethanol

series of ascending concentrations， embedded in Epon 8121!l and sectioned with a LKB or

Porter-B1um ultramicrotome. The u1trathin sections doub1y stained with urany1 acetate21l'

and 1ead19l were examined in a Hitachi HU ll-C or HS-4 e1ectron microscope.

1. Digestion experiments with hya1uronidases

(a) Digestion with Streptomyces hya1uronidase:

K. Yamada and S. Simizu : The Use of Streptomyces Hyaluronidas~ in Connective - 63一

1t was found appropriate to use 40μthick sections of glutaraldehyde帽paraformaldehyde

'.fixed tissues to digest hyaluronic acid and eliminate it in tissues consistently. Satisfactory

results were obtained by digestion procedures similar in incubation conditions to those

employed for tissue sections for light microscopy27J ，3OJ. Tissue sections were incubated at

39-410 C for 4-6 hr in 0.1 M phospha t巴 buffer(pH 5.0) solution which contains Strepto圃

myces hyaluronidas (Amano Pharmaceutical Company， Nishiharu， Aichi-ken， .Japan) at

an activity concentration of 100-150 TRU (Turbidity Reducing Unit)/ml.

(b) Digestion with t巴sticularhyaluronidase:

As in the case with digestion with Streptomyces hyaluronidase， 40μthick s巴ctionsof

glutaraldehyde-paraformaldehyde五xed tissues were used for digestion e五periments with

testicular hyaluronidase. Tissue sections were incubated at 370C for 4-6 hr in 0.1 M

phosphate buffer (pH 5.5) containing testicular hyaluronidase (Sigma Chemical Company，

St. Louis， U. S. A.) at an activity concentration of 1.0-1.5 mg/ml.

II. Control位 perimentsfor enzyme digestions

Two types of control experiments were conducted for the enzym日 digestionexperi-

ments.

(a) Controls with bu妊ersolutions without enzymes:

Some control tissue sections were incubated at 39-410C or 370C for 4-6hr in O.lM

phosphate buffers (pH 5.0 or 5.5) without enzymes.

(b) Controls with bu首位 solutionswith inactivated enzymes:

Other con trol tissue sections wer巴 immerseda t 39-410 C or 370 C for 4-6 hr in 0.1 M

phosphate buffers (pH 5.0 or 5.5) with heat-inactivated Streptomyces or testicular enzymes.

The results of these control experiments were compared with those obtained with

intact tissue sections which underwent none of th巴 experimental procedures.

Results

The two types of the present connective tissues， human umbilical cord and rabbit

aorta consist of a number of different histologic structures; umbi1ical cord (stroma， arterial

and venous stroma， lakes etc.) and aorta (interfibrillar spaces in intima， media and

adventitia etc.). 1n the present study， electron microscopic observations were focussed

upon such histologic structures as constitute the major proportions of each connective

tissue; stroma and arterial stroma in human umbilical cord and interelastic spaces of the

media in rabbit aorta.

1. Human umbilical cord

In the intact controls of human・umbilical cord tissues， the stroma reacts positively

for dialyzed iron and dialyzed iroロ reactivestructur百 aredemonstrated not only in the

spaces b巴tweenfibrils but in close association with fibrils (Figs. 1 and 2). As high power

views of the stroma disclose， thes巴 dialyzediron reactive structures are found to consist

of五ne granules (Fig. 3). In the spaces between五brils，dialyzed iron reactive五ne

granules are aligned in五lamentous figur巴sof di妊erentthickness巴s(50-200A) and th回日

五guresform ramifying meshworks which are here and there continuous with 的 ril-

associated structures consisting of similarly reactive fine granules (Fig. 3). Under the

- 64ー Connective Tissue

histochemical conditions employed in the present study， the dialyzed iron reaction of

undifferentiated mesenchymal cells appears to de capricious， and no systematic observations

were made on the cytochemical features of such cells. In the arterial stroma of the

umbilical cord tissues， dialyzed iron reactive五ne granules exhibit dual (fibril-associated

and unassociated) distribution patterns which are essentially identical with those recorded

for the stroma of the umbilical cord tissues.

Digestion with Streptomyces hyaluronidase results in the disappearanc巴 of ramifying

meshworks of五lamentous五guresconsisting cif dialyzed iron reactive fine granules in the

spaces between fibrils throughout the stroma tissues (Figs. 4 and 5). In the stroma of

the umbilical cord tissues undergone digestion with Streptomyces hyaluronidase，五bril-

associated dialyzed iron r巴active五negranules are relatively smaller in amount and weaker

in stainability， as compared with those observed in the stroma of intact control tissues

(Figs. 4 and 5). Digestion with Streptomyces hyaluronidase yields a nearly identical

effect upon the dialyzed iron reactive fine granules exhibiting dual distribution patterns

in the arterial stroma of the umbilical cord tissues.

In the human umbilical cord tissues digested with testicular hyaluronidase， ramifying

meshworks of filamen tous figures consisting of dialyzed iron reactive fine granules are

never observed in the spaces between fibrils throughout the stroma tissues (Figs. 6 and

7)， as in the case with the tissues digested with Streptomyces hyaluronidase. Digestion

with testicular hyaluronidase gives， likewise， ris巴 toa suppressive effect upon fibril-

associated dialyzed iron reactive fine granules which is significantly more marked than the

effect induced by digestion with Streptomyces hyaluronidase (Figs. 6 and 7). Thus，

dialyzed iron reactive五negranules associated with fibrils are far smaller in amount and

apparently less intense in stainability， in comparison with those in the tissues digested

with Streptomyces hyaluronidas巴. Similarly， digestion with testicular hyaluronidase results

in a markedly suppressive e妊ectupon dialyzed iron reactive fine granules exhibiting dual

distribution patterns in the arterial stroma of the umbilical cord tissues.

II. Rabbit aorta

In the intact controls of rabbit aorta tissues， dialyzed iron reactiv

K. Yamada and S. Simizu : The Use of Streptomyces Hyaluronidase in Connective - 65 -

dialyzed iron reactive五negranules in the interelastic spaces of the media are strikingly

diminished in amount and stainability and are substantially smaller in amount and more

feeble in stainability than those in the tissues undergone digestion with Streptomyces

hyaluronidase (Fig. 10).

III. Con trol experimen ts.

(a) Controls with bu妊ersolutions without enzymes

1n the two types (umbilical cord and aorta) of control tissues treated with bu妊er

solutions without enzymes， dialyzed iron reactive structures are detected which are largely

similar in composition， stainability and distribution patterns to those observed in the two

types of intact control tissues. There are， however， exceptions such as dialyzed iron

reactive fine granules in some parts of the spaces between fibrils in the stroma of human

umbilical cord tissues incubated in bu百ersolutions (pH 6.6) without enzymes. Dialyzed

iron reactive fine granules in such sites are smaller in amount and weaker in stainability，

as compared with those in intact control tissues.

。)Controls with buffer solutions with heat-inactivated enzymes:

1n the two types (umbilical cord and aorta) of control tissues subjected to treatment

with buffer solutions with heat-inactivated enzymes， dialyzed iron reactive structures are

essen tially comparabl巴 inmorphological characteristics to those observed in the two types

of intact control tissues.

Discu日sion

1n a series of reagents for the electron microscopic histochemical demonstration of

acid mucosaccharides such as colloidal ironω，23)， colloidal thoriumll ，l7J and ruthenium redI2)，

the Rinehart-Abul-Haj solutionl8) of dialyzed iron appears to be one of the best in terms

of the staining selectivity for acid mucosaccharides， the property of penetrating into

tissues， the size of granulations of products of reaction with acid mucosaccharides and

the usability in combination with mucosaccharidase digestion procedures4) ，23) ，29) ，31>; staining

with the dialyzed iron solution has been shown to demonstrate selectively acid mucosacc司

harides rich in carboxyl groups and sulfate esters4) ，23)， and the from of iron which binds

specifically to the substrates has been reported to be th巴 complexion FeOH十+10) Thus，

all the dialyzed iron reactive structures observed in the two types (umbilical cord and

aorta) of the present connective tissues are thought to contain acid mucosaccharides rich

in carboxyl groups and sulfate esters.

1n biochemical system， Streptomyces hyaluronidase is known to exhibit an absolute

substrate specificity; hyaluronic acid is the only acid mucosaccharide which can be deg-

raded by this enzyme and various types of isomeric chondroitin sulfates， chondroitin，

heparin， keratosulfate and chitin were shown to be una妊ectedby this enzyme 14)，15) In

the system routinely employed in light microscopic histochemistry of mucosaccharides，

such absolute substrate specificity of Streptomyces hyaluronidase has been certi五edas a

resul t of previous histochemica124)， 25)，26) ，27) ，30) and biochemicaj24)， 27) studies on the na ture

of the enzyme.

1t has biochemically been well recognized that testicular hyaluronidase splits endo-s-

- 66一 Connective Tissue

N-acetyl-D-glucosaminidic residues specifical1y22). Under the conditions of light micro-

scopic histochemistry， likewise， such substrate specificity of the testicular enzyme has been

confirmed and the enzyme has been known to depolymerize and eliminate， in tissues， al1

the acid mucosaccharides containing endo-s-N-acetyl-D-glucosaminidic residues such as

hyaluronic acid， chondroi tin and chondroi tin sulfa tes A and C7)， 20)，30)，33)，34).

In the present study， the fol1owing structural componets have been found to be

extinguished by digestion with Streptomyces hyaluronidase; (a) dialyzed iron reactive

fine granules forming ramifying meshworks of filamentous figures in the spaces between

fibrils and certain moieties of fibril-associated structures in the stroma and arterial stroma

of human unbilical cord tissues and (b) some proportion of fibril-associated and unas-

sociated dialyzed iron reactive fine granules in the interelastic spaces of the media of

rabbit aorta tissues. In view of the staining selectivity of the dialyzed iron r巴agent4) ， 23) 3!l

and the histochemical1y confirmed substrate specificity of Streptomyces hyaluronidase24) ，25)，

26)，27)，30)， al1 of these dialyzed iron reactive fine granules are conceived to consist of hy-

aluronic acid.

In the stroma and arterial stroma of human umbilical cord tissues and in the

interelastic spaces of the media of rabbit aorta tissues， dig巴stionwith testicular hyalu-

ronidase results in further disappearance of structural components consisting of dialyzed

iron reactive fine granules， in addtion to the components extinguished by digestion with

Streptomyces hyaluronidase. In other words， graded effects upon dialyzed iron reactive

fine granules forming various structures have been induced by digestions with Streptomyces

versus (vs.) testicular hyaluronidases in the human umbilical cord and rabbit aorta tissues.

In light of the histochemical1y known substrate specificity of testicular hyaluronidase白川，)

30) ，33) ，34)， those dialyzed iron reactive fine granules additional1y巴xtinguishedby digestion

with the testicular enzyme can be interpreted to consist of chondroitin， chondroitin sulfates

A andj or C in the umbilical cord and aorta tissues.

According to the previous data of biochemical analyses of various mucosaccharides in

animal tissues， the major acid mucosaccharides involved in human umbilical cord tissues

are hyaluronic acid and chondroitin sulfate C20)，32)， while as those possibly involved in

mammalian aorta tissues chondroitin sulfates A and C， hyaluronic acid， dermatan sulfate

and heparan sulfate being ennumerated20) ，32) ，33) ，34). If these data on the biochemical com-

positions of acid mucosaccharides in the two types of tissues examined in the present study

are taken into consideration， th巴 presenteffects of digestions with Streptomyces and testi・

cular hyaluronidases upon the dialyzed iron r巴actionof acid mucosacchrid己scan consistently

be interpreted in terms of the substrate spεCl五citiesof the two hyaluronidases， which have

been confirmed histochemical1y in connective tissue sections fOI" ligh t microscopy.

All the data obtained in the present study are wel1 comprehended by the conc巴pt

that digestion of thick tissue sections with Streptomyc巴shyaluronidase provides a promising

means for the electron microscopic histochemical identification of hyaluronic acid. In

electron microscopic histochemistry of mucosaccharides， however， digestion exp巴riments

wi th mucosaccharidas巴scan be p巴rformedupon at least three forms of tissue specimens;

tiny blocks， thick sections and ultrathin sections25)， 20). 1n the present study， moreover，

K. Yamada and S. Simizu : The Use of Streptomyces Hyaluronidase in Connective - 67ー

a limited number of mammalian connective tissues have been employed as materials for

testing the utility of Streptomyces hyaluronidase in electron microscopic histochemistry of

mucosaccharides Accordingly， further extensive studies on different forms (tiny blocks， thick

古巴ctions，ultrathin sections etc.) of various mucosaccharide-containing connective tissues

in a number of animal species ar巴 needed，in order to determine critically the usefulness

of Streptomyces hyaluronidase for the electron microscopic histochemical identification of

hyaluronic acid and to find out the most appropriate histochemical conditions for proce-

dures of digestion with the Streptomyces enzyme. Thes巳 statementsof discussion ar巴 to

b巴 stressed，particularly because in the present study the dialyzed iron reaction of acid

mucosaccharides in some structural components examin吋 isfound， though exceptionally，

to be affected by prior treatment with bu在住 solutionswithout enzymes. The true nature

of such enigmatic phenomenon remains to be巴lucidatedprecisely， even though either pos.

sible liberation of acid mucosaccharides into the buffer solutions27l，30) or perplexing buffer

interference of dialyzed iron reaction in staining acid mucosaccharides20) might be responsible

for it.

References

1) Berlin， J. D.: The localization of acid mucopDlysaccharides in the GDlgi complex of intestinal

goblet cells. 1. Cell Biol.， 32: 760-766， 1967.

2) Gasic， G. and Berwick， L. : Hale stain for sialic acid-cDut乱iningmucins; adaptation to electron

microscopy. 1. Cell Biol.， 19: 223-228， 1963.

3) Groniowski， J. W.， Biczyskowa， W. and Walski， M.: Electron microscopic studies on the

surfac色 coatof the nephron. 1. Cell Biol.， 40: 585-60l， 1969.

4) Hardin， 1. H. and Spicer， S. S.: Ultrastructural localization of dialyzed iron reactive muco・

substance in rabbit het巴rophils，basophils and eosinophils. 1. Cell Biol.， 48: 368-386， 1971.

5) Hoshino， M. and Yamada， K.: E茸ectsof digestion with chondroitinases upon mucosaccharide

stainings of rabbit carti1age as revealed by electron microscopy. Histochemie， 32: 221-229，

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'6) Jones， D. B.: Mucosubstances of the glomerulus. Lab. Invest.， 21: 119-125， 1969.

7) Knese， K. -H. : Cytologische Beobachtung an Sk巴letzellenuber di巴 Bi1dungder Kohlenhydrat

Protein Komplexe. Z. mikr・anat.Forsch.， 81: 233-294， 1969.

8) Leppi， T.1. and Stoward， P.1.: On the use of testicular hyaluronidas巴 forid巴ntifying acid

mucins in tissue s巴ctions. 1. Histochem.， cytochem.， 13: 406-407， 1965.

9) Leppi， T.1. and Kinnison， P. A.: The connective tissue ground substance in the mous己 uterine

cervix: An electron microscopic histochemical study. Anat. Rec.， 170: 97-118， 1971

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distribution of protein-polysaccharide in cartilage matrix. J. CelI Biol.， 32: 365-377， 1969.

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Oflicial Note of Amano Pharmaceutical Company Ltd.， 1， 1971.

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& Wilkins Co.， Baltimore， 1967， p. 251-303.

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gl ucosaminidas巴 ofbeef Iiver. J. biol. Chem.， 239: 59-63， 1964.

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Description of Plate Figures

Figs. 1 and 2. Parts of the stroma of human umbilical cord tissues. Dialyzed iron reactive五ne

granules are ass:Jciated with曲 rilsand are aligned in五lamentousfigures forming

ramifying meshworks (arrows) in the spaces bεtween 的 rils. Dialyzed iron

stained. X 31，000.

Fig. 3. A high pJwer view of the stroma of human umbilical cord tissu巴s. Ramifying

meshworks of五lamentousfigures (arrows) cons!sting of dialyzed iron reactive fine

granules in the spaces between出 rilsand五bril・associatedaccumulations of simi-

larly reactive五negranules are obvious. Dialyzed iron stained. X55，000.

Figs. 4 and 5. Parts of the stroma of human umbilical cord tissues. In the spaces between五brils

no dialyzed iron reactive structures can be found at all. Fibrils are associated

with dialyzed iron reactive fine granules which are smaller in amount and less

intense in stainability than thos巴 inFigs. 1 and 2. Streptomyces hyaluronidase

digested and dialyz巴diron stained. X 31，000.

Figs. 6 and 7. Parts of tbe stroma of human umbilical cord tissues. In the spaces between出rils

no dialyzed iron reactive structures can be discerned at all. Fibrils are associated

with dialyzed iron reactive五negranules which are far smaller in amount and less

intense in stainability than those in Figs. 4 and 5. Testicular hyaluronidase

digested and dialyzed iron stained. X 31，000.

Fig. 8. Part of the media in rabbit aorta tissues. In the interelastic spaces dialyzed iron

reactive自negranules are mostly associated with collagenous的 rilsand occasionally

distributed in the spaces between五brillarand cellular elements. E (elastic五ber)，

C (cellular element). Dialyzed iron stained. X 14，000.

Fig. 9. Part of the media in rabbit aorta tissues. In th邑 interelasticspaces dialyzed iron

reactive fine granules are smaller in amount and less intens巴 instainability than

those in Fig. 8. E (elastic五ber)，C (cellular element). Streptomyces hyaluro-

nidase digested. X 14，000.

Fig. 10. Part of the media in rabbit aorta tissues. In the interelastic spaces dialyzed iron

reactive fine granules are apparently smaller in amount and less intens巴 instain-

ability than those in Fig. 9. E (elastic曲er)，C (cellular element). Testicular

hyaluronidase digested and dialyzed iron stained. X 14，000.

- 70ー Connective Tissue

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K. Yamada and S. Simizu : The Use of StreptoIllyces Hyaluronidase in Connective ー 71ー