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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
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3) Groniowski, J. W., Biczyskowa, W. and Walski, M.: Electron microscopic studies on the
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4) Hardin, 1. H. and Spicer, S. S.: Ultrastructural localization of dialyzed iron reactive muco・
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'6) Jones, D. B.: Mucosubstances of the glomerulus. Lab. Invest., 21: 119-125, 1969.
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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