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Analysis of hyaluronidase diffusion for histochemical purposes
Aeta hlstoehem. 64, :W- 25 (1979)
Department of Transplantology, Institute of Biostructure, Medical Academy at \Varsaw, \Varsaw, Poland
Analysis of hyaluronidase diffusion for histochemical purposes By JANUSZ KOMENDER and
EWA
LESIAK-CYGANOWSKA
With 5 fIgures (Received July 13, 1978)
Summary Observations on hyaluronidase diffusion from natural and artificial objects to precipitated medium are described. The conclusions from this investigation are: (1) hyaluronidase diffusing from biological objects in a precipitated medium IS an active enzyme. (2) hyaluronidase diffusion may be utllized for localization of the enzyme. (3) certain parameters of the reaction hyaluronidase - hyaluronic acid may be measured WIth the use of the notion "dIffusion radius".
The known attempts of histochemical determination of hyaluronidase are based on the different methods. SZEMPLINSKA et al. (1962) used the substrate film technique. KOMENDER et al. (1973) determined hyaluronidase activity in histological section by incubation in the medium containing substrate, decreasing the substrate concentration was information concerning the activity of enzyme. GOULD and BERSTEIN (1975) localised precisely hyaluronidase in sperm by immunohistochemical reaction. In this paper a trial was undertaken to determine the localization of hyaluronidase by analizing the activity of the enzyme diffusing from the biological objects.
Material and methods Testes of mature rats of the Wlstar strain weighing about 200 g were used for the experiments, they were placed in 0.9 % NaCl at + 4 DC, cut into small pieces so as to obtain a suspension with fragments of the seminiferous tubules and single cells. This suspension was fixed In cold acetone and placed in a medium composed of gelatin 4.0 g hyaluronic acid 0.2 g, sodium chlorIde 8.7 gj 100 ml and 0.2 M acetate buffer pH = 4.0 up to 100 mI.
Course of exper,iment 1. The tubules and cell suspension were mixed with the incubation medium and smears about 1 mm thick were prepared. 2. The preparatlOn, were fIxed m 4 % buffered formahn at +4 DC. 3. After washing away the formalin preparations were incubated in 0.2 M acetate buffer pH = 4.0 at 37 DC.
21
Analysis of hyaluronidase diffusion for histochemical purpose 4. After incubation and washing of the preparations in 0.2 M acetate buffer pH were stamed with 0.5 % toluidmo blue at pH = 4.0 and 25°C for 30 mm. 5. Exce~s of dye was washed out with distilled water.
4.0 they
Sephadex preparation
+
I. Sephadex G·IOO beads were placed in hyaluromdase solutIOn 10 mg/ml and stored at 4°C for 24 h. 2. The Sephadex beads imbibed with hyaluromdase were placed m the above described medium. 3. Smears of the medium WIth the Sephadex beads were made on cover slip~. 4. The further procedure coinCIded with the course of the expenment (items 2 to 6). For checkmg the specificity of the reaction control tests were performed with Sephadex beads Imblded with trypsin or collagenase under conditions optImal for these enzymes.
Reagents 1. Hyaluronic acid (Koch & LIght) 2. Collagenase and Testicular Hyaluronidase (Sera and Vaccine Laboratories, 'Varsaw, Poland) 3. Trypsine (Difco) 4. Gelatine (Difco) 5. Sephadex G-IOO (Pharmacia) 6. Toluidme blue (BDH) 7. The remaining reagents: sodium chloride, acetic acid, sodium acetate, formalin, sodium phosphate were product of the Chemical Reagents Laboratories, Gliwice, Poland.
Measurements of diffusion radius The preparations containmg Sephadex beads or seminiferous tubules and single cells were mspected in microscope to observe diffusion of enzyme into the medIUm. If a clear, unstained area around them wa, observed, its diameter (SIze) was measured. The measurements were taken at several places on the same object, from the edge to the distinct border between the clear area and the background. Th,s diameter was named in paper "diffUSIOn radius". For each experimental point 50 measurements of the diffusion radIUS were taken. Mean values were calculated from data obtained and result was expresed in ftm. In order to obtain a better colour contrast during measurement of the preparations, a monochromatIC f,lter [A = 555 nm] was used. The total magnification of mICroscopic p,ctures was X 100.
Results Hyaluronidase diffusion from seminiferous tubules and single cells In material prepared according to the above described method clear areas could be seen, without metachromatic staining, around the seminiferons tubules (fig. I a, b, c). These light areas are sites where hyaluronic acid had been depolymerized by the enzyme diffusing from the cells. As seen in the microphotographs, the diffusion zone is not of equal dimensions, in spite of the uniform experiml:lntal procedure and the use of material from the same organ. Evaluation of free hyaluronidase diffusion from artificial objects For establishing optimal conditions for observation of hyaluronidase diffusion, Sephadex G-IOO beads imbibed hyaluronidase and incubated as described above
2~
J.
KOllIE N V E l t
a nd
EWA LESIAK · CYflAKO W RK A
1
2
Fig. 1. Seminifer ou s tubules (a , b) and group o f separate d cells (e ) from rat t estes , incubated as described in text. Around t h ese objects a cle ar, unstained area is visible. T olui d ine blue. x 100. F ig. 2. Sephadex G-IOO b eads filled with hyal uronidase (a) and with scnbeel in t ext. Toluidme blue . x lOO.
trip~ine
(b), incubated a s de -
were used (fig. 2) . With their aid several factores influencing the extent of diffusion could be ascertained. a) Dependence of diffusion radius on the size of the object
Advantage was taken of the differ ent size of the Sephadex beads (51,0 to 180,0 f-lm in diametre) to check whether the size of the object affect s the hyaluronidase diffusion radius. The results are shown diagram (fig. 3). The r adius length depends on dimensions of the analysed obj ect, it was therefore, decided to use in further studies beads with a diamet er exceeding 150 flm. b) Influence of incubation time on diffusion radius
In order t o establish the optimal incubation time the relation was checked between size o f the clear areas (diffusion radius) and the time of incubation of the pre-
Analysis of hyaluronidase chffusion for hIstochemical purposes
23
10
8
50 DIA,.,nCf~
100 OF SEPHAD£X
200
150 BEAD
JIm
Fig. 3. Relation 'of dIffusion radius to diameter of Sephadex beads. 6
E
-'
~
lJ)
::::>
a '<{
It:
"
<: 0
Vi
::::>
l:::
2i
2
<:
'<{ ~
~
30'
90
120' MINUTES
Fig. 4. Relation of diffusion radius to time of incubation. Curves were obtained in three separated experiments.
parations. The results are shown in fig. 4. It appeared that the diffusion radius length increases linearly with incubation time up to 90 min of incubation. Between 90 and 120 min the radius increase is rather slow, and after 120 min there is practically no more change. It was, therefore, considered that the optimal incubation time is 120 min. c) Dependence of diffusion radius on enzyme concentration
The Sephadex beads imbibed with enzyme of 2, 5, 10, 15 and 20 mg/ml concentration ~were incubated for 2 h under the conditions previously described. Fig. 5 show the results obtained. It was found that the diffusion range increases with concentra-
24
J.
KOME N DER
and
EWA LESIAK-CY GANOWS KA
. ...":t ..
u
~
....!i'" • :t
....
~
•:t " :>
IS ...
10
Fig. 5. R elation of diffusion radius to enzyme concentration.
tion of the enzyme, measured by the diffusion radius. Both the mean radius values and the distribution of all results obtained in the analysis indicate an increase in the diffusion radius values with rise of enzyme concentration. This 'm akes possible comparison of various enzyme contents in incubated objects. d) Control with the use of proteolytic enzymes
Incubation of Sephadex beads containing collagenase and trypsin in 'a medium containing hyaluronic acid for 2 h did not lead to the appearance of the characteristic clear area around the objects (fig. 2b). This is one of the arguments proving the spe. cificity of hyaluronidase in this reaction.
Discussion Histoch emical localization of h yaluronidase seems to be an open questIOn. V er y good localization my be described by u se of immunohistochemicall'eaction, in this case h owever, not enzym e activity but other molecular properties of hyaluronidase are de termined. The substrate fIlm t ech-
Analysis of hyaluronidase diffusion for histochemical purposes
25
nique as well as determinatlOn of substrate depohmerizatlOn by tissue section enables the precise localization of this enzyme in the cells. So the trials based on other rules for hyaluronidase locali· zation are justified. I our opimon enzyme diffusion may be a proof of its 10calizatlOn in examined material. It is true that when we detect the diffused enzyme activity, it might be no longer present in examined tissue, but we are able to mdicate the source of diffusion without any doubts. And this is an indio rect eVidence of enzyme 10cahzatlOn. On the rule of diffusion of biological active substances another tests were already based e.g . .TERNE et al. (1963) test for detectlOn the cells containing hemolysine. The concluslOn may be darwn from the presented results that hyaluronidase diffusing from tissues IS an active enzyme. Its detection IS possible by finding areas not staining metachromati· cally, thus not contammg hyaluronic acid. An essential character of the method here presented is the stability of the preparations obtained. After 2 years of storage at room temperature they were still suitable for analYSIS. The negative control With proteases IS evidence of the specificity of the appearmg clear area. For elucidation of certain characteristlC features of the reaction artificial objects were used such as Sephadex G·I00 beads filled with hyaluronidase solution which could freely diffuse into the medium. This model did not fully correspond to the conditions of tissue hyaluronidase diffusion, but it allowed to establish optimal incubation conditions. In the present mvestigations hyaluronidase actiVity was measured m terms of the "diffusion radlUs". It is known from other studies (OSTROWSKI et al. 1972) that, under the given conditlOns, the value of the radius IS more convement than that of the surface area of the area analysed. It is also obvious that enzyme cliffuslOn could occur further than indicated by the diffusion radius, however m view of Its continuously dlmimshing concentration, Its activity may be undetectable. The artificial objects helped to establish that the diffusion radlUs is influenced by the size of the object, the duratlOn of incubation and the initial concentration of the enzyme. The effect of the size of the objects may be explained by the larger volume of the enzyme solution, which diffusing into the medium forms a wider diffusion zone. The mfluence of time and concentration on measurement results requires no comment, the more so as they are in agreement with the fundamental laws of diffusion (RAPOPORT 1965). Thus, it seems that detection and establishment of the range of diffusion of tissue substances may be convement for determining their localization in cells and tissues.
Literature GOULD, S. F., ami BERNSTEIN, M., The localization of bovine sperm hyaluronidase. Differentiation 11,123-132 (1975) . .TERNE, N. K., NORDIN, A. A., and HENRY, C., The agar plaque technique for recognizing antibody-producing cells, in B. Amos and H. KOPROWSKI: "Cell-Bound Antibodies", p 109, ViTistar Institute Press, Philadelphia 1963. KOMENDER, .T., GOIASZEWSKA, A., and MALCZEWSKA, H., Quantitative determination of hyaluronidase in tissue sectlOns. Histochemistry 35,219-225 (1973). OSTROWSKI, K., BLATON, 0., SKOPLN-SKA, E., GORSKI, A., and ZALESKA-RuTCZYNSKA, Z., Migration inhibition test based on the culture of spleen fragments: a Quantitative analysis. Folia Biologica IS, 117-126 (1972). RAPOPORT, S. M., Medizinische Biochemie. pp. lS-19. VEB Verlag Volk und Gesundheit. Berlin 1965. SZEMPLINSKA, H., SIERAKOWSKA, H., and SHUGAR, D., Histochemical localization of hyaluronidase and amylase by the substrate-film technique. Acta Biochim. Pol. 9, 239-244 (1962). Address: Doc. Dr . .T. KOMENDER, Department of Transplantology, Institute of Biostructure, Medical Academy, PL-02-004 Warsaw, Chalubinskiego 5.
Department of Transplantology, Institute of Biostructure, Medical Academy at \Varsaw, \Varsaw, Poland
Analysis of hyaluronidase diffusion for histochemical purposes By JANUSZ KOMENDER and
EWA
LESIAK-CYGANOWSKA
With 5 fIgures (Received July 13, 1978)
Summary Observations on hyaluronidase diffusion from natural and artificial objects to precipitated medium are described. The conclusions from this investigation are: (1) hyaluronidase diffusing from biological objects in a precipitated medium IS an active enzyme. (2) hyaluronidase diffusion may be utllized for localization of the enzyme. (3) certain parameters of the reaction hyaluronidase - hyaluronic acid may be measured WIth the use of the notion "dIffusion radius".
The known attempts of histochemical determination of hyaluronidase are based on the different methods. SZEMPLINSKA et al. (1962) used the substrate film technique. KOMENDER et al. (1973) determined hyaluronidase activity in histological section by incubation in the medium containing substrate, decreasing the substrate concentration was information concerning the activity of enzyme. GOULD and BERSTEIN (1975) localised precisely hyaluronidase in sperm by immunohistochemical reaction. In this paper a trial was undertaken to determine the localization of hyaluronidase by analizing the activity of the enzyme diffusing from the biological objects.
Material and methods Testes of mature rats of the Wlstar strain weighing about 200 g were used for the experiments, they were placed in 0.9 % NaCl at + 4 DC, cut into small pieces so as to obtain a suspension with fragments of the seminiferous tubules and single cells. This suspension was fixed In cold acetone and placed in a medium composed of gelatin 4.0 g hyaluronic acid 0.2 g, sodium chlorIde 8.7 gj 100 ml and 0.2 M acetate buffer pH = 4.0 up to 100 mI.
Course of exper,iment 1. The tubules and cell suspension were mixed with the incubation medium and smears about 1 mm thick were prepared. 2. The preparatlOn, were fIxed m 4 % buffered formahn at +4 DC. 3. After washing away the formalin preparations were incubated in 0.2 M acetate buffer pH = 4.0 at 37 DC.
21
Analysis of hyaluronidase diffusion for histochemical purpose 4. After incubation and washing of the preparations in 0.2 M acetate buffer pH were stamed with 0.5 % toluidmo blue at pH = 4.0 and 25°C for 30 mm. 5. Exce~s of dye was washed out with distilled water.
4.0 they
Sephadex preparation
+
I. Sephadex G·IOO beads were placed in hyaluromdase solutIOn 10 mg/ml and stored at 4°C for 24 h. 2. The Sephadex beads imbibed with hyaluromdase were placed m the above described medium. 3. Smears of the medium WIth the Sephadex beads were made on cover slip~. 4. The further procedure coinCIded with the course of the expenment (items 2 to 6). For checkmg the specificity of the reaction control tests were performed with Sephadex beads Imblded with trypsin or collagenase under conditions optImal for these enzymes.
Reagents 1. Hyaluronic acid (Koch & LIght) 2. Collagenase and Testicular Hyaluronidase (Sera and Vaccine Laboratories, 'Varsaw, Poland) 3. Trypsine (Difco) 4. Gelatine (Difco) 5. Sephadex G-IOO (Pharmacia) 6. Toluidme blue (BDH) 7. The remaining reagents: sodium chloride, acetic acid, sodium acetate, formalin, sodium phosphate were product of the Chemical Reagents Laboratories, Gliwice, Poland.
Measurements of diffusion radius The preparations containmg Sephadex beads or seminiferous tubules and single cells were mspected in microscope to observe diffusion of enzyme into the medIUm. If a clear, unstained area around them wa, observed, its diameter (SIze) was measured. The measurements were taken at several places on the same object, from the edge to the distinct border between the clear area and the background. Th,s diameter was named in paper "diffUSIOn radius". For each experimental point 50 measurements of the diffusion radIUS were taken. Mean values were calculated from data obtained and result was expresed in ftm. In order to obtain a better colour contrast during measurement of the preparations, a monochromatIC f,lter [A = 555 nm] was used. The total magnification of mICroscopic p,ctures was X 100.
Results Hyaluronidase diffusion from seminiferous tubules and single cells In material prepared according to the above described method clear areas could be seen, without metachromatic staining, around the seminiferons tubules (fig. I a, b, c). These light areas are sites where hyaluronic acid had been depolymerized by the enzyme diffusing from the cells. As seen in the microphotographs, the diffusion zone is not of equal dimensions, in spite of the uniform experiml:lntal procedure and the use of material from the same organ. Evaluation of free hyaluronidase diffusion from artificial objects For establishing optimal conditions for observation of hyaluronidase diffusion, Sephadex G-IOO beads imbibed hyaluronidase and incubated as described above
2~
J.
KOllIE N V E l t
a nd
EWA LESIAK · CYflAKO W RK A
1
2
Fig. 1. Seminifer ou s tubules (a , b) and group o f separate d cells (e ) from rat t estes , incubated as described in text. Around t h ese objects a cle ar, unstained area is visible. T olui d ine blue. x 100. F ig. 2. Sephadex G-IOO b eads filled with hyal uronidase (a) and with scnbeel in t ext. Toluidme blue . x lOO.
trip~ine
(b), incubated a s de -
were used (fig. 2) . With their aid several factores influencing the extent of diffusion could be ascertained. a) Dependence of diffusion radius on the size of the object
Advantage was taken of the differ ent size of the Sephadex beads (51,0 to 180,0 f-lm in diametre) to check whether the size of the object affect s the hyaluronidase diffusion radius. The results are shown diagram (fig. 3). The r adius length depends on dimensions of the analysed obj ect, it was therefore, decided to use in further studies beads with a diamet er exceeding 150 flm. b) Influence of incubation time on diffusion radius
In order t o establish the optimal incubation time the relation was checked between size o f the clear areas (diffusion radius) and the time of incubation of the pre-
Analysis of hyaluronidase chffusion for hIstochemical purposes
23
10
8
50 DIA,.,nCf~
100 OF SEPHAD£X
200
150 BEAD
JIm
Fig. 3. Relation 'of dIffusion radius to diameter of Sephadex beads. 6
E
-'
~
lJ)
::::>
a '<{
It:
"
<: 0
Vi
::::>
l:::
2i
2
<:
'<{ ~
~
30'
90
120' MINUTES
Fig. 4. Relation of diffusion radius to time of incubation. Curves were obtained in three separated experiments.
parations. The results are shown in fig. 4. It appeared that the diffusion radius length increases linearly with incubation time up to 90 min of incubation. Between 90 and 120 min the radius increase is rather slow, and after 120 min there is practically no more change. It was, therefore, considered that the optimal incubation time is 120 min. c) Dependence of diffusion radius on enzyme concentration
The Sephadex beads imbibed with enzyme of 2, 5, 10, 15 and 20 mg/ml concentration ~were incubated for 2 h under the conditions previously described. Fig. 5 show the results obtained. It was found that the diffusion range increases with concentra-
24
J.
KOME N DER
and
EWA LESIAK-CY GANOWS KA
. ...":t ..
u
~
....!i'" • :t
....
~
•:t " :>
IS ...
10
Fig. 5. R elation of diffusion radius to enzyme concentration.
tion of the enzyme, measured by the diffusion radius. Both the mean radius values and the distribution of all results obtained in the analysis indicate an increase in the diffusion radius values with rise of enzyme concentration. This 'm akes possible comparison of various enzyme contents in incubated objects. d) Control with the use of proteolytic enzymes
Incubation of Sephadex beads containing collagenase and trypsin in 'a medium containing hyaluronic acid for 2 h did not lead to the appearance of the characteristic clear area around the objects (fig. 2b). This is one of the arguments proving the spe. cificity of hyaluronidase in this reaction.
Discussion Histoch emical localization of h yaluronidase seems to be an open questIOn. V er y good localization my be described by u se of immunohistochemicall'eaction, in this case h owever, not enzym e activity but other molecular properties of hyaluronidase are de termined. The substrate fIlm t ech-
Analysis of hyaluronidase diffusion for histochemical purposes
25
nique as well as determinatlOn of substrate depohmerizatlOn by tissue section enables the precise localization of this enzyme in the cells. So the trials based on other rules for hyaluronidase locali· zation are justified. I our opimon enzyme diffusion may be a proof of its 10calizatlOn in examined material. It is true that when we detect the diffused enzyme activity, it might be no longer present in examined tissue, but we are able to mdicate the source of diffusion without any doubts. And this is an indio rect eVidence of enzyme 10cahzatlOn. On the rule of diffusion of biological active substances another tests were already based e.g . .TERNE et al. (1963) test for detectlOn the cells containing hemolysine. The concluslOn may be darwn from the presented results that hyaluronidase diffusing from tissues IS an active enzyme. Its detection IS possible by finding areas not staining metachromati· cally, thus not contammg hyaluronic acid. An essential character of the method here presented is the stability of the preparations obtained. After 2 years of storage at room temperature they were still suitable for analYSIS. The negative control With proteases IS evidence of the specificity of the appearmg clear area. For elucidation of certain characteristlC features of the reaction artificial objects were used such as Sephadex G·I00 beads filled with hyaluronidase solution which could freely diffuse into the medium. This model did not fully correspond to the conditions of tissue hyaluronidase diffusion, but it allowed to establish optimal incubation conditions. In the present mvestigations hyaluronidase actiVity was measured m terms of the "diffusion radlUs". It is known from other studies (OSTROWSKI et al. 1972) that, under the given conditlOns, the value of the radius IS more convement than that of the surface area of the area analysed. It is also obvious that enzyme cliffuslOn could occur further than indicated by the diffusion radius, however m view of Its continuously dlmimshing concentration, Its activity may be undetectable. The artificial objects helped to establish that the diffusion radlUs is influenced by the size of the object, the duratlOn of incubation and the initial concentration of the enzyme. The effect of the size of the objects may be explained by the larger volume of the enzyme solution, which diffusing into the medium forms a wider diffusion zone. The mfluence of time and concentration on measurement results requires no comment, the more so as they are in agreement with the fundamental laws of diffusion (RAPOPORT 1965). Thus, it seems that detection and establishment of the range of diffusion of tissue substances may be convement for determining their localization in cells and tissues.
Literature GOULD, S. F., ami BERNSTEIN, M., The localization of bovine sperm hyaluronidase. Differentiation 11,123-132 (1975) . .TERNE, N. K., NORDIN, A. A., and HENRY, C., The agar plaque technique for recognizing antibody-producing cells, in B. Amos and H. KOPROWSKI: "Cell-Bound Antibodies", p 109, ViTistar Institute Press, Philadelphia 1963. KOMENDER, .T., GOIASZEWSKA, A., and MALCZEWSKA, H., Quantitative determination of hyaluronidase in tissue sectlOns. Histochemistry 35,219-225 (1973). OSTROWSKI, K., BLATON, 0., SKOPLN-SKA, E., GORSKI, A., and ZALESKA-RuTCZYNSKA, Z., Migration inhibition test based on the culture of spleen fragments: a Quantitative analysis. Folia Biologica IS, 117-126 (1972). RAPOPORT, S. M., Medizinische Biochemie. pp. lS-19. VEB Verlag Volk und Gesundheit. Berlin 1965. SZEMPLINSKA, H., SIERAKOWSKA, H., and SHUGAR, D., Histochemical localization of hyaluronidase and amylase by the substrate-film technique. Acta Biochim. Pol. 9, 239-244 (1962). Address: Doc. Dr . .T. KOMENDER, Department of Transplantology, Institute of Biostructure, Medical Academy, PL-02-004 Warsaw, Chalubinskiego 5.