Comp. Biochem. PhysioL, 1975, Vol. 50B, pp. 331 to 337. Pergamon Press. Printed in Great Britain
PROPERTIES OF TESTICULAR HYALURONIDASE OF THE HONEY BEE AND ORIENTAL HORNET: COMPARISON WITH INSECT VENOM AND MAMMALIAN HYALURONIDASES D. ALLALOUF1, A. BERI AND J. ISHAY~ 1Endocfinological Department of the Rogoff-Wellcome Medical Research Institute, Beilinson Hospital, Petah Tikva; and SDepartment of Physiology and Pharmacology, Sackler School of Medicine, Tel Aviv University, Israel
(Received 22 January 1974) Abstract--1. The hyaluronidase of crude extracts from testes of the honey bee (Apis mellifera) and of the oriental hornet (Vespa orientalis) was found to possess activity in depolymerizing hyaluronic acid and chondroitin-6-sulfate with pH optima 4.5-5.0. 2. With an increase in incubationtemperature from 37 to 56°C the enzyme showed a gradual increase in activity, in contrast with bovine and ovine testicular hyaluronidases which were gradulaly inactivated. 4. Preheating for 3 hr at 50°C did not affect activity. This stability was shared by the venom hyaluronidase from the same insects but not by the mammalian hyaluronidases which were almost completely inactivated.
INTRODUCTION
THE ~ , ~ A L I A ~ testicular hyaluronidase (hyalurohate glycanohydrolase E.C. 3.2.1.35) has been extensively studied. This enzyme in testes originates in the seminiferous epithelium of the mature testes and has been localized in the developing spermatides and mature spermatozoa (Mancini et al., 1964; Srivastava et aL, 1965). It is probably involved in the fertilization process, apparently in view of its capacity to exert a liquifying action on the viscous mucopolysaccharide gel which cements the cumulus oophorus cells around the eggs. Its precise role, however, is still uncertain (Mann, 1964; Srivastava et al., 1965). I n insects the insemination of eggs generally follows the same course as in mammalia. The extraordinary longevity, however, of insect spermatozoa as compared to the mammalian is worthy of special attention. In bees and hornets, for example, the supply of spermatozoa received by the successfully mated queens is generally sufficient to last for 1--4 years (Bischoff, 1927; Ruttner, 1968). During these long periods the spermatozoa, stored in the spermatheca, remain alive and active, suggesting either the existence of very favourable conditions in their environment or fundamental differences in the properties of some of their functional constituents. The study of enzymes associated with insect spermatoza appears therefore to be of biological importance. Apart from the testes certain insects possess a rich source of hyaluronidase: the venom sacs (Barker et al., 1966; Habermann, 1968; Allalouf et al., 1972), thus providing an opportunity for the comparison of
glycosaminoglycan-depolymerizingenzymes originating from two different insect tissues obviously involved in dissimilar physiological functions. The present work deals with the hyaluronidase of crude extracts from testes of the honey bee (Apis mellifera) and of the oriental hornet (Vespa orientalis) and with the comparison of some of its properties to bovine and ovine testicular hyaluronidases and to hyaluronidases from the venoms of the same insects. MATERIALS AND METHODS
Sources of hyaluronidase Testes were obtained from honey bees and oriental hornets 7-10 days old, either immobilized in the cold or which had been stored frozen, by incision through the abdomen and removal by the use of fine scissors and forceps. All manipulations were carried out in the cold and the collected preparations kept on ice. In the bee the separation of the testes with the seminal vesicles attached (in the functionally mature drone the latter contain most of the spermatozoa) could easily be done under magnifying lens. Attempts, however, to isolate the testes of the hornet, free of the mucus glands, were unsuccessful on account of the somewhat different anatomical structure of the reproductive system and we were compelled to use from this insect a combination of these organs. For convenience the preparations from both insects will be termed hereafter testes. Pools of testes from each species were homogenized in 0.1 M phosphate--citrate buffer pH5.0 using volumes determined in preliminary experiments to yield enzymatic activities in the extracts within convenient ranges for the assays. In a typical experiment 2"4 ml buffer were used for 1 g bee testes (representing 77 pairs) and 1.3 ml buffer for
331
D. ALLALOUF,A. BER AND J. ISHAY
332
1 g hornet testes (representing thirty pairs). After homogenization in a motor-driven glass-Teflon PotterElvehjem homogenizer, the suspensions were centrifuged in a Spinco Model L ultracentrifuge (Beckman) at 105,000 g for 1 hr. The high-speed supernatants were used for the determinations of enzymatic activities. It is appropriate to add here that the residues were devoid of activity, as will be discussed later. Venom sacs were obtained from bees and hornets by grasping the sting and lifting the sacs out of their beds. High-speed extracts were prepared from them as described for testes. Mammalian testicular hyaluronidases were commercial preparations from bovine and ovine testes (Sigma Chemical Co., Types IV and V respectively) labeled by the manufacturer to contain 750 and 1250 N F U per rag, respectively. These preparations were found to contain 80.9 and 84-9% protein by the Lowry et al. (1951) procedure using crystalline bovine albumin (Nutritional Biochemicals Co.) as the standard. The same method was used for the determination of protein in the extracts. Crude rat testicular extracts were prepared as described above using testes from adult albino rats (170-180 g), immediately after being killed.
N-acetylhexosamine was determined according to the method of Reissig et al. (1955) using as standards N-acetylglucosamine or N-acetylgalactosamine (Pfanstiehl Lab. Inc.). By this method N-acetylgalactosamine provides products whose chromogenicity is considerably lower than those from N-acetylglucosamine and this should be taken into consideration in Fig. 1 where optical densities are given. 2. Turbidimetric assay. The procedure described by di Ferrante (1956) was used. Aliquots from the extracts were incubated at 37°C with hyaluronic acid in 0"1 M acetate buffer pH5.0, 0-15 M NaCI as described in Table l. At the end of the incubation period the turbidity developing upon the addition of a 2-5% solution of cetyltrimethylammonium bromide (Fluka) in 2% NaOH was recorded at 400 nm. The measurement of turbidity was started immediately upon the addition of the quaternary ammonium compound and continued up to the maximal reading which was the value finally adopted. The fall in turbidity, which is a measure of the activity of the enzyme in depolymerizing the glycosaminoglycan substrate, was expressed as a percentage of the turbidity developed by the intact substrate. RESULTS AND DISCUSSION
Assays of hyaluronidase I. Release of N-acetylhexosamine-reacting products. Aliquots from the high-speed supernatants were incubated with hyaluronic acid (Sigma, unless otherwise stated) or chondroitin-6-sulfate from umbilical cord, kindly supplied by Dr. J. Cifonelli, University of Chicago. The composition of digestion mixtures and conditions of assay, carried out as described by Males & Turkington (1970), are given in the tables and legends to figures.
Extraction Males & Turkington (1970) recently reported that nearly all hyaluronidase activity from rat testes could be sedimented f r o m buffer homogenates by centrifugation at 105,000 g for 1 hr. This finding is in accordance with the assumption that hyaluronidase is of lysosomal origin in various tissues
Table 1. Hyaluronidase activity with time of A. mellifera and V. orientalis testicular extracts with hyaluronic acid as substrate. Comparison with venom sacs extracts and bovine and ovine testicular hyaluronidases Hyaluronidase activity Colorimetric assay N-acetylglucosamine (t~moles/mg protein)
Turbidimetric assay (% fall in turbidity)
Time (min) Preparation Testes, Apis Testes, Vespa Venom sacs, Apis Venom sacs, Vespa Testicular Hyaluronidase, bovine Testicular Hyaluronidase, ovine
30 0.035 2.38 1-10 0.81 2.33
Time (min)
60
120
180
Protein (tzg)
60
120
180
0.043 0"020 2-58 2.18 1.38 3"16
0.080 0.040 2.82 3.16 2.07 4"62
0.117 0-090 2.93 3.83 2.47 5-87
160 147 10 15 10 6
-7 90 39 78 78
7 15 93 68 90 80
25 27 93 71 91 86
The activity was assayed (1) by colorimetric measurement (Reissig et al., 1955) of N-acetyl-glucosamine-reacting products released from hyaluronic acid (supplied by D. Cifonelli) upon incubation at 37°C. The assay system consisted of 0.2 ml 0-1 M phosphate-citrate buffer pH 5.0, 0.05 ml highspeed homogenate supernatant and 0"05 ml substrate solution containing 300/~g hyaluronic acid and (2) by a turbidimetric method (di Ferrante, 1956). The assay system consisted of 0.5 ml substrate solution containing 200 tzg hyaluronic acid and 0.5 ml high-speed homogenate supematant containing amounts of protein given in the table, both in 0.1 M acetate buffer pH 5"0, 0.15 M NaCI. After incubation at 37°C 2 ml of a 2.5% solution of cetyl-trimethylammonium bromide in 2% NaOH were added and the maximal turbidity developing was recorded at 400 rim. The fall in turbidity is expressed as a percentage of the turbidity developing in the 200 t~g control substrate.
Testicular hyaluronidase of honey bee and oriental hornet (Vaes & Jacques, 1965; Hutterer, 1966; Aronson & Davidson, 1967; de Salegui et aL, 1967) and associated in the testes with the sperm acrosome which is considered to be a lysosome-like structure (Allison & Hartree, 1970). These authors included, therefore, a treatment of the crude homogenates with detergents, obtaining with two of them (eetyl-trimethylammonium bromide and Triton X-100) an increase in the specific activity of the supernatant and a good recovery. In initial experiments we examined the effect of the presence of detergents in the homogenization mixtures on the recovery of the hyaluronidase activity of the examined tissues in the high speed supernatants. Our results in the assay of the N-acetylglucosamine-reacting products from hyaluronic acid as substrate showed, however, negligible variations in the specific activities whether homogenizations were carried out in the presence or absence of 0.1% Triton X-100, cetyl-trimethylammonium bromide or sodium cholate. The completeness of the recovery of the enzyme in the supernatants was also documented by assaying the precipitates obtained upon centrifugation of homogenates in the buffer alone. To this end the pellets, suspended in buffer in the presence or absence of 0.1% Triton X-100, were assayed with hyaluronic acid as substrate. Except for the residue from the hornet venom s a c s - - a very rich source of hyaluronidase--which exhibited a weak activity, all the other tissue residues were completely inactive. It should also be noted that the addition of detergents to buffer homogenates supernatants prior to incubation with the substrate did not affect enzymatic activity. Likely interpretations for the lack of agreement on the effect of the presence of detergents between our recovery experiments and those of Males & Turkington (1970) should possibly be traced to the different nature of the tissues examined or to the different extraction procedures. F o r the reasons stated above the extraction with buffer alone was adopted in the present work. Table 1 presents hyaluronidase activities with time of representative insect testicular extracts as determined by both the colorimetric and turbidimetric methods using hyaluronic acid as substrate. In the same table are included for comparison the activities of the venom sacs extracts from the same insects and of commercial bovine and ovine hyaluronidases. Although there were some differences in the specific activities between various lots of tissues, the testicular extracts of both insects generally exhibited comparable activities by both assays. It is appropriate to add that the tissues from the two species were not identical since, as mentioned in Materials and Methods, it included in the hornet the mucus glands also and separate determinations made by us in extracts from testes and the mucus glands from the bee revealed a considerably higher activity in the glands. A study of this 12
333
phenomenon is currently in progress and will be published separately. Hyaluronidase activities of the testicular extracts were, on the basis of protein, considerably lower (1/100-1/60, depending on the lot) than of the homologous venom sacs extracts. To provide a comparison, there are given in Table 1 the activities of bovine and ovine testicular hyaluronidases labeled to contain 750 and 1250 N F U per mg corresponding to 927 and 1472 N F U per nag protein, respectively (see Materials and Methods). The specific activities,
2.0 1.5 I.O E 0.5 c
d
2.C
o w Z
"
,~
1.5
1.0
X
~ 0.5 ._1 t--
PROPERTIES OF TESTICULAR HYALURONIDASE OF THE HONEY BEE AND ORIENTAL HORNET: COMPARISON WITH INSECT VENOM AND MAMMALIAN HYALURONIDASES D. ALLALOUF1, A. BERI AND J. ISHAY~ 1Endocfinological Department of the Rogoff-Wellcome Medical Research Institute, Beilinson Hospital, Petah Tikva; and SDepartment of Physiology and Pharmacology, Sackler School of Medicine, Tel Aviv University, Israel
(Received 22 January 1974) Abstract--1. The hyaluronidase of crude extracts from testes of the honey bee (Apis mellifera) and of the oriental hornet (Vespa orientalis) was found to possess activity in depolymerizing hyaluronic acid and chondroitin-6-sulfate with pH optima 4.5-5.0. 2. With an increase in incubationtemperature from 37 to 56°C the enzyme showed a gradual increase in activity, in contrast with bovine and ovine testicular hyaluronidases which were gradulaly inactivated. 4. Preheating for 3 hr at 50°C did not affect activity. This stability was shared by the venom hyaluronidase from the same insects but not by the mammalian hyaluronidases which were almost completely inactivated.
INTRODUCTION
THE ~ , ~ A L I A ~ testicular hyaluronidase (hyalurohate glycanohydrolase E.C. 3.2.1.35) has been extensively studied. This enzyme in testes originates in the seminiferous epithelium of the mature testes and has been localized in the developing spermatides and mature spermatozoa (Mancini et al., 1964; Srivastava et aL, 1965). It is probably involved in the fertilization process, apparently in view of its capacity to exert a liquifying action on the viscous mucopolysaccharide gel which cements the cumulus oophorus cells around the eggs. Its precise role, however, is still uncertain (Mann, 1964; Srivastava et al., 1965). I n insects the insemination of eggs generally follows the same course as in mammalia. The extraordinary longevity, however, of insect spermatozoa as compared to the mammalian is worthy of special attention. In bees and hornets, for example, the supply of spermatozoa received by the successfully mated queens is generally sufficient to last for 1--4 years (Bischoff, 1927; Ruttner, 1968). During these long periods the spermatozoa, stored in the spermatheca, remain alive and active, suggesting either the existence of very favourable conditions in their environment or fundamental differences in the properties of some of their functional constituents. The study of enzymes associated with insect spermatoza appears therefore to be of biological importance. Apart from the testes certain insects possess a rich source of hyaluronidase: the venom sacs (Barker et al., 1966; Habermann, 1968; Allalouf et al., 1972), thus providing an opportunity for the comparison of
glycosaminoglycan-depolymerizingenzymes originating from two different insect tissues obviously involved in dissimilar physiological functions. The present work deals with the hyaluronidase of crude extracts from testes of the honey bee (Apis mellifera) and of the oriental hornet (Vespa orientalis) and with the comparison of some of its properties to bovine and ovine testicular hyaluronidases and to hyaluronidases from the venoms of the same insects. MATERIALS AND METHODS
Sources of hyaluronidase Testes were obtained from honey bees and oriental hornets 7-10 days old, either immobilized in the cold or which had been stored frozen, by incision through the abdomen and removal by the use of fine scissors and forceps. All manipulations were carried out in the cold and the collected preparations kept on ice. In the bee the separation of the testes with the seminal vesicles attached (in the functionally mature drone the latter contain most of the spermatozoa) could easily be done under magnifying lens. Attempts, however, to isolate the testes of the hornet, free of the mucus glands, were unsuccessful on account of the somewhat different anatomical structure of the reproductive system and we were compelled to use from this insect a combination of these organs. For convenience the preparations from both insects will be termed hereafter testes. Pools of testes from each species were homogenized in 0.1 M phosphate--citrate buffer pH5.0 using volumes determined in preliminary experiments to yield enzymatic activities in the extracts within convenient ranges for the assays. In a typical experiment 2"4 ml buffer were used for 1 g bee testes (representing 77 pairs) and 1.3 ml buffer for
331
D. ALLALOUF,A. BER AND J. ISHAY
332
1 g hornet testes (representing thirty pairs). After homogenization in a motor-driven glass-Teflon PotterElvehjem homogenizer, the suspensions were centrifuged in a Spinco Model L ultracentrifuge (Beckman) at 105,000 g for 1 hr. The high-speed supernatants were used for the determinations of enzymatic activities. It is appropriate to add here that the residues were devoid of activity, as will be discussed later. Venom sacs were obtained from bees and hornets by grasping the sting and lifting the sacs out of their beds. High-speed extracts were prepared from them as described for testes. Mammalian testicular hyaluronidases were commercial preparations from bovine and ovine testes (Sigma Chemical Co., Types IV and V respectively) labeled by the manufacturer to contain 750 and 1250 N F U per rag, respectively. These preparations were found to contain 80.9 and 84-9% protein by the Lowry et al. (1951) procedure using crystalline bovine albumin (Nutritional Biochemicals Co.) as the standard. The same method was used for the determination of protein in the extracts. Crude rat testicular extracts were prepared as described above using testes from adult albino rats (170-180 g), immediately after being killed.
N-acetylhexosamine was determined according to the method of Reissig et al. (1955) using as standards N-acetylglucosamine or N-acetylgalactosamine (Pfanstiehl Lab. Inc.). By this method N-acetylgalactosamine provides products whose chromogenicity is considerably lower than those from N-acetylglucosamine and this should be taken into consideration in Fig. 1 where optical densities are given. 2. Turbidimetric assay. The procedure described by di Ferrante (1956) was used. Aliquots from the extracts were incubated at 37°C with hyaluronic acid in 0"1 M acetate buffer pH5.0, 0-15 M NaCI as described in Table l. At the end of the incubation period the turbidity developing upon the addition of a 2-5% solution of cetyltrimethylammonium bromide (Fluka) in 2% NaOH was recorded at 400 nm. The measurement of turbidity was started immediately upon the addition of the quaternary ammonium compound and continued up to the maximal reading which was the value finally adopted. The fall in turbidity, which is a measure of the activity of the enzyme in depolymerizing the glycosaminoglycan substrate, was expressed as a percentage of the turbidity developed by the intact substrate. RESULTS AND DISCUSSION
Assays of hyaluronidase I. Release of N-acetylhexosamine-reacting products. Aliquots from the high-speed supernatants were incubated with hyaluronic acid (Sigma, unless otherwise stated) or chondroitin-6-sulfate from umbilical cord, kindly supplied by Dr. J. Cifonelli, University of Chicago. The composition of digestion mixtures and conditions of assay, carried out as described by Males & Turkington (1970), are given in the tables and legends to figures.
Extraction Males & Turkington (1970) recently reported that nearly all hyaluronidase activity from rat testes could be sedimented f r o m buffer homogenates by centrifugation at 105,000 g for 1 hr. This finding is in accordance with the assumption that hyaluronidase is of lysosomal origin in various tissues
Table 1. Hyaluronidase activity with time of A. mellifera and V. orientalis testicular extracts with hyaluronic acid as substrate. Comparison with venom sacs extracts and bovine and ovine testicular hyaluronidases Hyaluronidase activity Colorimetric assay N-acetylglucosamine (t~moles/mg protein)
Turbidimetric assay (% fall in turbidity)
Time (min) Preparation Testes, Apis Testes, Vespa Venom sacs, Apis Venom sacs, Vespa Testicular Hyaluronidase, bovine Testicular Hyaluronidase, ovine
30 0.035 2.38 1-10 0.81 2.33
Time (min)
60
120
180
Protein (tzg)
60
120
180
0.043 0"020 2-58 2.18 1.38 3"16
0.080 0.040 2.82 3.16 2.07 4"62
0.117 0-090 2.93 3.83 2.47 5-87
160 147 10 15 10 6
-7 90 39 78 78
7 15 93 68 90 80
25 27 93 71 91 86
The activity was assayed (1) by colorimetric measurement (Reissig et al., 1955) of N-acetyl-glucosamine-reacting products released from hyaluronic acid (supplied by D. Cifonelli) upon incubation at 37°C. The assay system consisted of 0.2 ml 0-1 M phosphate-citrate buffer pH 5.0, 0.05 ml highspeed homogenate supernatant and 0"05 ml substrate solution containing 300/~g hyaluronic acid and (2) by a turbidimetric method (di Ferrante, 1956). The assay system consisted of 0.5 ml substrate solution containing 200 tzg hyaluronic acid and 0.5 ml high-speed homogenate supematant containing amounts of protein given in the table, both in 0.1 M acetate buffer pH 5"0, 0.15 M NaCI. After incubation at 37°C 2 ml of a 2.5% solution of cetyl-trimethylammonium bromide in 2% NaOH were added and the maximal turbidity developing was recorded at 400 rim. The fall in turbidity is expressed as a percentage of the turbidity developing in the 200 t~g control substrate.
Testicular hyaluronidase of honey bee and oriental hornet (Vaes & Jacques, 1965; Hutterer, 1966; Aronson & Davidson, 1967; de Salegui et aL, 1967) and associated in the testes with the sperm acrosome which is considered to be a lysosome-like structure (Allison & Hartree, 1970). These authors included, therefore, a treatment of the crude homogenates with detergents, obtaining with two of them (eetyl-trimethylammonium bromide and Triton X-100) an increase in the specific activity of the supernatant and a good recovery. In initial experiments we examined the effect of the presence of detergents in the homogenization mixtures on the recovery of the hyaluronidase activity of the examined tissues in the high speed supernatants. Our results in the assay of the N-acetylglucosamine-reacting products from hyaluronic acid as substrate showed, however, negligible variations in the specific activities whether homogenizations were carried out in the presence or absence of 0.1% Triton X-100, cetyl-trimethylammonium bromide or sodium cholate. The completeness of the recovery of the enzyme in the supernatants was also documented by assaying the precipitates obtained upon centrifugation of homogenates in the buffer alone. To this end the pellets, suspended in buffer in the presence or absence of 0.1% Triton X-100, were assayed with hyaluronic acid as substrate. Except for the residue from the hornet venom s a c s - - a very rich source of hyaluronidase--which exhibited a weak activity, all the other tissue residues were completely inactive. It should also be noted that the addition of detergents to buffer homogenates supernatants prior to incubation with the substrate did not affect enzymatic activity. Likely interpretations for the lack of agreement on the effect of the presence of detergents between our recovery experiments and those of Males & Turkington (1970) should possibly be traced to the different nature of the tissues examined or to the different extraction procedures. F o r the reasons stated above the extraction with buffer alone was adopted in the present work. Table 1 presents hyaluronidase activities with time of representative insect testicular extracts as determined by both the colorimetric and turbidimetric methods using hyaluronic acid as substrate. In the same table are included for comparison the activities of the venom sacs extracts from the same insects and of commercial bovine and ovine hyaluronidases. Although there were some differences in the specific activities between various lots of tissues, the testicular extracts of both insects generally exhibited comparable activities by both assays. It is appropriate to add that the tissues from the two species were not identical since, as mentioned in Materials and Methods, it included in the hornet the mucus glands also and separate determinations made by us in extracts from testes and the mucus glands from the bee revealed a considerably higher activity in the glands. A study of this 12
333
phenomenon is currently in progress and will be published separately. Hyaluronidase activities of the testicular extracts were, on the basis of protein, considerably lower (1/100-1/60, depending on the lot) than of the homologous venom sacs extracts. To provide a comparison, there are given in Table 1 the activities of bovine and ovine testicular hyaluronidases labeled to contain 750 and 1250 N F U per mg corresponding to 927 and 1472 N F U per nag protein, respectively (see Materials and Methods). The specific activities,
2.0 1.5 I.O E 0.5 c
d
2.C
o w Z
"
,~
1.5
1.0
X
~ 0.5 ._1 t--
