System for supply of free arginine in the spermatophore of Bombyx mori

Insect Biochem. Vol. 17, No. 2, pp. 317-322, 1987 Printed in Great Britain. All rights reserved

0020-1790/87 $3.00+0.00 Copyrisht © 1987PergamonJournalsLtd

SYSTEM FOR SUPPLY OF FREE ARGININE IN THE SPERMATOPHORE OF B O M B Y X M O R I ARGININE-LIBERATING ACTIVITIES OF CONTENTS OF MALE REPRODUCTIVE GLANDS HIROKO KASUGA, TO~HIROAIGAKI and MINORU OSANAI* Department of Biology, Tokyo Metropolitan Institute of Gerontology, Sakaecho 35-2, Itabashi-ku, Tokyo, 173, Japan (Received 3 February 1986; revised and accepted 29 April 1986)

Abstract--The mechanism of supply of free arglnine in the Bombyx mori spermatophore was analyzed by examining the arginine-liberating activities of the contents of male reproductive glands, which are constituents of the spermatophore formed during ejaculation. On autolysis, only the contents of glandula (g.) prostatica showed significant production of only free arginine. On incubation in combination with the contents of the g. prostatica, arginine liberation from the vesicula (v.) seminalis contents was highest. Similarly, the contents of the g. spermatophorae also showed arginine liberation, but those of the g. lacteola showed generalized liberation of various amino acids. Following digestion of these preparations with bovine trypsin, the v. seminalis, g. spermatopborae and g. prostatica liberated free lysine as well as arginine, while those of the g. lacteola liberated various amino acids. These findings strongly suggest that a specificproteolytic system for production of free arginine is active in the spermatopbore. That is, an endopeptidase derived from the g. prostatica specificallycleaves protein at the C-terminal side of arginine residues, and then exopeptidases derived from the v. seminalis, g. spermatophorae and g. prostatica liberate free arginine from the peptides produced by the endopeptidase. Key Word Index: Arginine-liberating activity, spermatophore, glandula prostatica, vesicula seminalis, protease, Bombyx mori

INTRODUCTION During copulation of the silkworm, Bombyx mori, the "male reproductive content," which is a general term for the secretions of the glandula (g.) pellucida, g. lacteola, g. spermatophorae, g. alba and g. prostatica and seminal fluid in the vesicula (v.) seminalis, is conveyed to the bursa copulatrix to form the spermatophore and its accessory materials (0mura, 1938). The silkworm spermatophore is not a mere repository or transport vehicle for spermatozoa (Mann, 1984), but a reactor for spermatozoan maturation (Katsuno, 1977; Kasuga et aL, 1985). Maturation proceeds with time from acquisition of motility of anucleated apyrene spermatozoa and the breakdown of eupyrene bundles to the separation of individual eupyrene spermatozoa that fertilize eggs and their partial acquisition of motility, which is completed later in the spermatheca. Our previous series of studies (Osanai and Aigaki, 1984; Aigaki and Osanai, 1985; Kasuga et al., 1985; Osanai et aL, 1986, 1987) demonstrated a unique energy-yielding pathway, in which arginine degradation is coupled with a glycolytic cascade in the spermatophore of Bombyx. In an early stage of spermatophore formation, only arglnine increases, and then with time it is metabolized to 2-oxoglutarate via ornithine and glutamate, resulting in aoeurnu*To w h o m correspondence should be addressed.

317

lation of much alanine. 2-Oxoglutarate was shown to function both as a preferable energy substrate and as a stimulator of pyruvate oxidation in the spermatophore (Osanai et aL, 1987). The arginine degradation cascade must be triggered by a free arginine supply after ejaculation, because the silkworm spermatophore shows very high arginase activity (Osanal and Aigaki, 1984; Aigaki and Osanai, 1985; Osanai et aL, 1986). The amount of free arginine is very low throughout the male reproductive tract before mating (Osanai et aL, 1986). Thus free arginine in the spermatophore must be supplied de novo by reactions during spermatophore formation. Since arginine is an indispensable amino acid for the silkworm (Inokuchi, 1969), arginine is probably supplied from proteins. In fact, the digestion of proteinaeeous envelopes of eupyrene bundles and the increase in various amino acids with time after spermatophore formation (Kasuga et al., 1985; Osanai et aL, 1986) strongly suggest the presence of proteolytic activity in the spermatophore. In this connection, it is noteworthy that sperm activation can be induced by addition of some proteases to immotile spermatozoa of saturniid moths (Shepherd, 1974). In the present study, we examine the mechanisms of free arglnine supply in the silkworm spermatophore by analyzing the arglnine-liberating activities of the contents of various male reproductive glands, which form the sperrnatophore during ejaculation.

HIROKO KASUGAel al.

318 MATERIALS AND METHODS

Silkworm An F I hybrid, J124 x Ch124, of the silkworm, Bombyx mori, was supplied by Dr M. Kusuno, Director of the Tokyo Metropolitan Sericultural Station, Akigawa, and reared on mulberry leaves at 25°C. The sexes were separated at the pupal stage. Moths were kept in an incubator at 25°C. Fuyo × Tokai hybrids, were supplied by Dr T. Ohoka, Department of Biology, Faculty of Science, Tokyo Metropolitan University, Tokyo, were used in some experiments, but no marked differences between results with these two hybrids were observed.

Preparation of the contents Of male reproductive glands Three male reproductive ducts, the g. lacteola, g. spermatophorae, and g. prostatica, and the v. seminalis (Fig. 1), the contents of which form the spermatophore, were isolated separately from unmated male moths. The contents of the above ducts from two insects each were suspended in 50/~1 of the silkworm Ringer (Narahashi, 1963), because very small amounts of material were obtained from one insect. However, the contents of one v. seminalis suspended in 50/~1 of Ringer were sufficientfor a single determination. The determination was repeated at least three times.

Digestion of the contents of male reproductive glands The contents of each gland alone or in combination with those of other glands or with bovine trypsin (type I, Sigma Chemical Co., St Louis, Missouri, U.S.A.) were digested by incubation in a plastic micro-centrifuge tube (1.5 ml) for 10 min at 25°C. The reaction was stopped by adding cold ethanol to a final concentration of 75%. The mixtures were homogenized, and then the 75% ethanol extracts of each sample were analyzed on an amino acid autoanalyzer (Hitachi 835, Tokyo), as described by Osanai et al. (1986).

levels of the contents of the v. seminalis (51.5 nmol/ gland), g. spermatophorae (3.4 nmol/gland) and g. lacteola (3.0 nmol/gland) increased to 696, 283 and 97% of the original levels [7.4, 0.6 and 1.9 nmol/ gland, respectively (Osanai et al., 1986)]. Unlike the contents of other glands, those of the g. lacteola also yielded various other amino acids. A conspicuous decrease in the glycine level was observed in the contents of the v. seminalis, calculated as the difference from the level produced during autolysis when the glycine level increased markedly (Table 1). These results indicate that liberation of free arginine was enhanced by incubation of the contents of other male reproductive glands with those of the g. prostatica.

Effects on the amino acid pools of the contents oJ various male reproductive glands following their incubation with trypsin In studies on the properties of the arginineliberating system, we incubated the glandular contents with bovine trypsin (0.1 mg/ml), which is an endopeptidase cleaving at only the C-terminal sides of arginine and lysine residues of proteins (Table 3). The amino acid levels of all the secretions increased to a greater or lesser degree during incubation with trypsin. Some changes were very similar to those observed on incubation with the contents of the g. prostatica. The increases in the g. lacteola secretion were very different from those in the other secretions in that various amino acids were liberated. A similar tendency was observed in the v. seminalis secretion, but the increases of each amino acid per tissue weight

RESULTS

Effects of autolysis on amino acid pools in the contents of male reproductive glands For examination of possible autolytic liberation of free arginine in the silkworm spermatophore, the contents of each male reproductive gland, which are constituents of the spermatophore, were incubated for 10 min at 25°C, and then examined for quantitative changes in their amino acid pools. The amount of free arginine increased significantly only in the g. prostatica secretion (Fig. 2). There were no marked changes in free amino acids in the contents of the other three glands, except for an increase in glycine in that of the v. seminalis (Table 1). Under these experimental conditions, free arginine in the g. prostatica secretion (8.4nmol/gland) increased to 255% of the original amount (3.3 nmol/gland, Osanai et al., 1986). These findings indicate that the g. prostatica secretion contains an arginine-liberating system.

g. I ac teola

v.semi nal is

g. s p e r m a t o p h o r a e

g. p r o s t a t i c a

Effects on the amino acid pools of the contents of other glands following their incubation with the g. prostatica secretion Next we examined arginine liberation on incubation of the contents of the other gland with the g. prostatica secretion. As shown in Table 2, under these conditions, the contents of all the glands liberated free arginine, but the liberation was much greater in the contents of the v. seminalis than in those of the g. lacteola or g. spermatophorae: the free arginine

Fig. 1. Male reproductive system of the silkworm, Bomby:~ mori drawn schematically. Only the three ducts and v. seminalis, whose contents were used in this work, are indicated.

Arginine supply system in spermatophore

319

(A)

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GinI Thr

Glu

Gly

Tyr Leu

VO[ i

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i

i

Arg

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i

i

(B)

~ 0

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2'0

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I

40

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i

70

i

8O

Retention lime (rnin) Fig. 2. Elution profiles of amino acids of the g. prostatica secretion. (A) After incubated for 10 min at 25°C. (B) Not incubated.

of g. lacteola were generally much higher than those in the v. seminalis secretion; namely, they were higher than those in the other three glands. In the other glands, the increases in arginine and lysine were greater than those in other amino acids. The ratio of lysine/arginine increase was very small in the contents of the g. lacteola (0.27), while high in those of the g. prostatica (0.72) and intermediate in the other two glandular secretions (about 0.4).

The digest of the secretion of the v. seminalis, which is the largest gland, was compared with that of the g. prostatica secretion (Fig. 3). Figure 3A shows the chromatographic elution profile of amino acids in the non-incubated contents of the v. seminalis. The amounts of basic amino acids were low. Hardly any difference was found between this profile and that after incubation, except for an increase in the peak of glycine (Fig. 3B). However, a large peak of arginine

Table 1. Differences in mean amino acid levels of incubated samples from the initial levels shown in Table 2 of a previous paper (Osanai et al., 1986) Amino acid (nmol) Asp Gin + Thr Ser Glu Gly Ala Val Met lie Leu Tyr Phe Orn Lys His Arg Pro

g. lacteola (2.5 mg)* 0.3 4.4 4.1 -0.2 1.4 3.3 - 0. I 0.4 0.0 0.0 0.8 0.1 0.1 0.4 0.3 - 0.1 0.0

v. seminalis (11.3 rag)* -7.5 1.4 -2.8 5.0 22.4 1.8 1.8 0.0 0.1 0.0 - 1.6 -0.5 -0.5 0.6 - 1.4 - 0.2 0.0

g. spermatophorae (1.0 mg)* -0.2 2.3 1.9 0.5 1.9 0.4 0.2 0. I 0.0 - 0. I 0.2 -0.6 -0.2 0.6 -0.2 0.4 0.0

Values are means for three samples and are given per insect. *Mean tissue weight in seven determinations (Osanai et al., 1986).

g. prostatica (1.6 mg)* 0.6 1.9 - 1.5 -0.4 -0.2 0.6 0.8 0.2 0.0 0.0 0. I 0.0 -0.1 0. I -0.3 8.4 0. l

H1ROKO KASUGA et al.

320

Table 2. Changes in amino acid levels on incubation with g. prostatica secretion Amino acid (nmol)

g. lacteola (2.5 mg)*

v. seminalis (11.3 mg)*

g. spermatophorae (1.0 mg)*

Asp Gin + Thr Set Glu Gly Ala Val Met lie Leu Tyr Phe Orn Lys His Arg Pro

2.6 13.3 21.4 5.3 7.5 3.7 0.7 0.4 1.1 1.7 0.6 0.1 3.0 5.5 -0.5 3.0 2.1

- 1.2 -4.4 -3.4 7.7 - 29.5 - 1.1 -2.2 0.1 1.0 0.7 1.8 0.7 0.5 2.1 0.1 51.5 1.5

-0.9 -6.7 -6.9 -2.1 - 4.9

Arg increase %

97

696

-

1.5

-0.3 -0.8 0.0 0.6 -0.9 -0.5 0.0 - 1.0 0.3 3.4 0.4 283

Changes were calculated as follows: (mean amino acid level of a combination of the secretion of the test gland and that of the g. prostatica) - (level after incubation of the test secretion alone)- (level after incubation of the g. prostatica secretion alone). Values per insect are shown and are means for three samples. *Mean tissue weight in seven determinations (Osanai et al., 1986). Table 3. Changes in amino acid levels on incubation with trypsin Amino acid (nmol) Asp Gln+ Thr Ser Glu Gly Ala Val Met Ile Leu Tyr Phe Orn Lys His Arg Pro Lys/Arg

g. lacteola (2.5 mg)* 3.1 3.8 I 1.0 5.0 3.6 11.7 2.8 4.2 3.6 4.4 6.2 5.1 2.6 7.0 0.1 26.1 3.6 0.27

v. seminalis (I 1.3 mg)* 6.0 2.9 - 0.8 0.4 - 28. I 8.8 2.9 2.8 4.2 7.5 4.8 5.5 1.1 25.5 0.5 65.8 4.4 0.39

g. spermatophorae (1.0 mg)* 0.1 0.5 -0.7 -0.1 -0.7 0.8 0.7 0.2 0.2 0.9 0.3 0.7 0.1 1.6 0.0 3.8 0.5 0.40

g. prostatica (1.6 mg)* 0.1 4.1 2.9 -3.4 -0.6 1.2 1.2 0.6 0.7 1.3 0.1 1.0 0.0 6.2 -0.8 8.6 0.3 0.72

Changes were calculated as follows: (mean ammo acid level after incubation of the test secretion with 0.1 mg/ml bovine trypsin) - (level after incubation of the test secretion alone). Values per insect are shown and are means for triplicate determitiations in each case. *Mean tissue weight in seven determinations (Osanai et al., 1986).

was f o u n d in t h e profile after i n c u b a t i o n o f the c o n t e n t s with t h e g. p r o s t a t i c a secretion (Fig. 3C), while m a j o r p e a k s o f arginine a n d lysine a p p e a r e d o n i n c u b a t i o n with t r y p s i n (Fig. 3D). T h e s e t w o a m i n o acids c o i n c i d e d exactly with the m a i n Ct e r m i n a l a m i n o acids o f p e p t i d e s p r o d u c e d b y t r y p s i n treatment. DISCUSSION

B o m b y x s p e r m a t o z o a are k n o w n to u n d e r g o a m a t u r a t i o n p r o c e s s in the s p e r m a t o p h o r e f o r m e d by t r a n s f e r o f t h e m a l e r e p r o d u c t i v e c o n t e n t s to t h e female at e j a c u l a t i o n ( K a s u g a et al., 1985). A specific e n e r g y - y i e l d i n g p a t h w a y , a r g i n i n e d e g r a d a t i o n coupled with glycolysis, h a s been d e m o n s t r a t e d in t h e

s p e r m a t o p h o r e o f this insect ( O s a n a i et al., 1986, 1987). In this m e t a b o l i c p a t h w a y , f o r m a t i o n o f glyc o g e n , a n i m p o r t a n t e n e r g y source, a n d its d i s a p p e a r a n c e with time have been r e c o g n i z e d in t h e m a l e r e p r o d u c t i v e tracts a n d s p e r m a t o p h o r e o f the silkw o r m , B o m b y x mori ( K a s u g a et al., 1985) a n d ano t h e r l e p i d o p t e r a n , M a n d u c a s e x t a ( S h e p h e r d et al., 1981). H o w e v e r , n o i n f o r m a t i o n is available a b o u t t h e m e c h a n i s m o f s u p p l y o f free arginine, a n o t h e r s o u r c e o f energy in the s p e r m a t o p h o r e . T h e r e are t h r e e possible m e c h a n i s m s o f free arginine p r o d u c t i o n by p r o t e o l y s i s in the s i l k w o r m spermatophore: the contents of male reproductive g l a n d s m a y c o n t a i n either (1) a n a r g i n i n e - r i c h p r o t e i n with protease(s), o r (2) p r o t e i n s a n d a specific p r o tease s y s t e m c o n s i s t i n g o f a n e n d o p e p t i d a s e a n d an

Arginine supply system in spermatophore

321

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v _'2

Q

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322

HIROKO KASUGAet al.

exopeptidase necessary for specific production of arginine, or (3) both an arginine-rich protein and a specific protease system. In the present study we examine mainly the second possibility by studies on the contents of reproductive glands isolated from unmated male silkmoths. During autolysis of the contents of each gland, only those of the g. prostatica, the most distal male reproductive gland, showed significant production of free arginine. Moreover, arginine liberation was enhanced by incubation of the secretion of the g. prostatica with the contents of other reproductive glands, especially those of the v. seminalis. Previously we reported (Osanai et al., 1986), that the weights of the g. iacteola, v. seminalis, g. spermatophorae and g. prostatica of male silkmoths were 2.5, 11.3, 1,0 and 1.6 mg, respectively. Therefore, although these weights do not necessarily reflect the exact volumes of their contents, it is probable that the amount of arginine liberated from the contents of the v. seminalis was large, while that liberated from the contents of the g. spermatophorae and g. prostatica were very small. Since the contents of the v. seminalis are the main component of the spermatophore (Kasuga et al., 1985), they must be the main source of substrates for the proteolytic enzymes. On their incubation with the g. prostatica secretion, arginine was the main amino acid liberated, whereas on their incubation with trypsin both arginine and lysine were liberated. This finding suggests that the endopeptidase in the g. prostatica secretion cleaves only at the C-sides of arginine residues, like the enzyme found in mouse submaxillary gland (Schenkein et al., 1977). From these findings it is concluded that (1) the contents of the v. seminalis and g. spermatophorae contain little or no endopeptidase activity, (2) the g. prostatica contains at least an endopeptidase that cleaves proteins specifically at the C-sides of arginine residues, (3) the g. lacteola contains proteolytic enzymes yielding various amino acids and (4) all the contents contain exopeptidase(s). Studies on the endopeptidase and exopeptidase(s) in the male reproductive contents are now in progress. Since rapid increase in free arginine begins during transfer of the male reproductive secretion to the female in the early stage of spermatophore formation (Osanai et al., 1986), arginine liberation must start on mixing of the specific endopeptidase, exopeptidase(s) and substrate proteins, which are originally localized separately in the male reproductive glands. As a result, much free arginine is supplied in the spermatophore. This proteolytic system does not appear to require arginine-rich proteins as specific substrates, since it can produce arginine from the secretions of all the male reproductive glands owing to its specificity and high activity. However, the efficiency of arginine liberation would be increased, if arginine-rich proteins were available. In Musca domestica an argininerich protein was found in the ejaculatory ducts

(Leopold, 1970). Possibly there is a similar protein in the male reproductive system of Bombyx. Acknowledgements--The authors express their appreciation

to Director M. Kusuno, Tokyo Metropolitan Sericulture Station, Akigawa, and Dr T. Ohoka, Department of Biology, Faculty of Science, Tokyo Metropolitan University, Tokyo for supplying silkworms. They also thank Dr Nomura, Department of Biochemistry, Tokyo Metropolitan Institute of Gerontology, Tokyo for help in amino acid analysis. REFERENCES

Aigaki T. and Osanai M. (1985) Arginase activity in the silkworm, Bombyx mori: developmental profiles, tissue distribution and physiological role. J. comp. Physiol. B155, 653~57. Inokuchi T. (1969) Nutritional studies of amino acids in the silkworm, Bombyx mori. II. Effect of several amino acids on proline and arginine requirements. Bull. Sericult. exp. Sta. 23, 389-500. Kasuga H., Osanai M., Yonezawa Y. and Aigaki T. (1985) An energy supply device for spermatozoa in spermatophore of the silkworm. Devl Growth Differ. 27, 514. Katsuno S. (1977) Studies on eupyrene and apyrene spermatozoa in the silkworm, Bombyx mori L. (Lepidoptera: Bombycidae). V. The factor related to the separation of eupyrene sperm bundles. Appl. ent. Zool. 12, 370-371. Leopold R. A. (1970) Cytological and cytochemical studies on the ejaculatory duct and accessory secretion in Musca domestica. J. Insect Physiol. 16, 1859-1872. Mann T. (1984) Spermatophores. Development, Structure, Biochemical Attributes and Role in the Transfer of Spermatophore. Springer, Berlin.

Narahashi T. (1963) The properties of insect axons. In Advances in Insect Physiology (Edited by Beament J. W. L., Treherne J. E. and Wigglesworth V. B,), Vol. 1, pp. 175-256. Academic Press, London. Omura S. (1938) Studies on the reproductive systems of the male of Bombyx mori. II. Post-testicular organs and post-testicular behavior of the spermatozoa. J. Fac. Agric. Hokkaido Imp. Univ. 40, 129-170. Osanai M. and Aigaki T. (1984) Sex and strain differences in arginase activity of the adult silkworm, Bombyx mori. J. sericult. Sci. Jpn 53, 519-526. Osanai M., Aigaki T., Kasuga H. and Yonezawa Y. (1986) Role of arginase transferred from the vesicula seminalis during mating and changes in amino acid pools of the spermatophore after ejaculation in the silkworm, Bombyx mori. Insect Biochem. 16, 879-885. Osanai M., Aigaki T. and Kasuga H. (1987) Energy metabolism in the spermatophore of the silkmoth, Bombyx mori, associated with accumulation of alanine derived from arginine. Insect Biochem. 17, 71-75. Scbenkein I., Levy M., Franklin E. C. and Frangione B. (1977) Proteolytic enzymes from the mouse submaxillary gland. Specificity restricted to arginine residues. Archs Biochem. Biophys. 182, 64-70. Shepherd J. G. (1974) Sperm activation in saturniid moths: some aspects of the mechanism of activation. J. Insect Physiol. 20, 2321-2328. Shepherd J. G., Turner P. M. and Wilson K. A. (1981) Metabolism of moth spermatozoa. In Advances in Invertebrate Reproduction (Edited by Clark W. H. and Adams T. S.), p. 315. Elsevier/North Holland, Amsterdam.