Methionine-enkephalin immunoreactivity in the gonads and nervous system of two insect species: Locusta migratoria and Sarcophaga bullata

GENERAL

AND

COMPARATIVE

69, 1-12 (1988)

ENDOCRINOLOGY

Methionine-Enkephalin lmmunoreactivity in the Gonads and System af Two Insect Species: Lscusta migraforia an Sarcophaga EIL~ANE SCHQOFS,SYLVAIN Zoological

institute

bullafa

SCHROOTEN,RQGERHUYBRECHTS,AND ARNOLDDELOOF

of the University,

Naamsestraat

59, 3000 Leuven,

Belgium

AcceptedMarch 8, 1987 Methionine(met)-enkephalin immunoreactivity as visualized by the peroxidase-antiperoxidase procedure, is present in spermatogonia, spermatocytes, spermatids, and young ovarian follicles of Locusta (panoistic type) and Sarcophaga (polytrophic type). Follicle cells and mature spermatozoa are always immunonegative as are locust vitellogenic follicles. In oocytes and in trophocytes, the met-enkephalin-like material first appears around the nucleus and is then dispersed throughout the cytoplasm. Later, it is present only in the periphery. In the ovary of both insects, no immunoreactivity is found with antisera against adrenocorticotrophic hormone, melanophore stimulating hormone, P-endorphin, corticotropin releasing factor. or leucine-enkephalin. All these antisera yield a positive reaction when applied to the central nervous system as does the met-enkephalin antiserum. This study indicates that the met-enkephalin-like peptide may play a role in reproductive physiology. Q 1988 Academic Press. Inc.

Enkephalins were first discovered in higher vertebrates by Hughes (1975) and have now been found in most vertebrates (Udenfriend and KiTpatrick, 1983) and in some invertebrates (Greenberg and Price, 1983). In insects in particular, met-enkephalin immunoreactivity has been found in the central nervous system of Peripianeta americana (Verhaert and De Loof, 1984), drosophila melanogaster (Pages et al.) 1983), Loccssta migratoria (Remy and Dubois, 1981; Romoeuf and RCmy, 1984; Gras et al., 1978), Leucophaea maderae (Hansen et al., 19521, and Eristalis aeneus (El Salhy et al., 1983). Peptides of the enkephahn family are also detected in various nonnervous tissues of vertebrates (Petraglia et al., 198.5). Recently, there is increasing evidence that these substances are widely distributed throughout the body of invertebrates as well. Met-enkephalin immunoreactivity has already been demonstrated in the gut of

Periplaneta americana (Schols et al., 1987) and of Aeshna cyanea (Andries and Tramu, 1985), in the ovary of the protochordate Ciona intestinalis (Georges and 1984), and in the hermaphroditic gonad a the snail Helix aspersa (Marchand Dubois, 1985, 1986). Recently, the presence of vertebratetype steroid hormones has been demonstrated in insect gonads (De Eoof and De Clerck, 1986). We report here that peptide hormones such as m~thioni~e-enkepha~~~ are present not only in the central nervous system, but also in the ovaries and testes of two insect species, the migratory locust and the grey fleshfly. The similarity of t docrine system in vertebrates and invertebrates, with regard to the rn~t-e~~k~~~a~~~ substance rn the gonads, is discussed. MATERIALS Animals toria R

and preparation

AND METW of tissues. Larusta migrab&/rata Parkerwe!-e

and F and Sarcophapa

1

001~6-6480188 $1.50 Copyright All rights

0 1988 by Academic Press. inc. of reproduciion in any farx reserved.

2

SCHOOFS ET AL.

reared under laboratory conditions as described by Materials and Methods result in entirely Ashby (1972) and by Huybrechts and De Loof (1981). negative immunoreactions. After preadFor tissue sections, ovaries and testes of adult flies sorption of the met-enkephalin antiserum and locusts were microdissected while immersed in with the Sepharose 4B-met-enkephalin 10% Bouin Hollande’s fixative. Brains, corpora cardiimmunoreactivity aca-corpora allata, and ventral nerve cords of adult f beads, all met-enkephalin and larval (stages I, II, III, IV, and V) locusts and is completely abolished on complete series brains of adult flies were dissected and fixed in situ in of Locusta and Sarcophaga ovary and the same fixative solution. After fixation, all tissues brain sections. were washed in water, dehydrated in ethanols, cleared Moreover, preadsorption of the enkephin xylene, and embedded in Paraplast. After the tissues were sectioned at 4 pm and rehydrated, the sec- alin antiserum with the control Sepharose tions were processed with the immunohistochemical 4B beads (without bound met-enkephalin procedure. peptide) does not alter the staining pattern Zrnmunohistochemistry. The met-enkephalin antiseof met-enkephalin immunoreactivity. We rum was prepared according to a previously described illustrate here the specificity of the metmethod (Verhaert and De Loof, 1985). A second anenkephalin antiserum, by demonstrating tiserum, commercially purchased from UCB (Belgium), was applied as well. The met-enkephalin-like sections, treated either with the diluted substance was demonstrated by means of the peroxiantibody or with the antibody preadsorbed dase-antiperoxidase method as used by Vandesande with met-enkephalin. The reaction is comet al. (1981). pletely inhibited after preadsorption A working dilution of Ii1000 appeared to be (Fig. 1). appropriate for an optimal immunoreaction. Method specificity was controlled by the application Both met-enkephalin antisera used label of a nonimmune rabbit antiserum (serially diluted from the very same structures in both gonads l/l00 to 1110000) as well as by the processing of a and nervous system, indicating that the relseries of immunocytochemical stainings in which the evant peptide has a considerable degree of various steps are one by one omitted from the regular similarity to met-enkephalin. staining sequence. To reduce background staining, caused by nonimmunological adsorption of either primary or secondary Gonads antibodies, all tissue sections were preincubated with a preimmune goat serum. (a) Locusta migratoria. The ovary of LoSerum specificity was examined by means of solid custa is of the panoistic type. In vitellophase adsorption. For this purpose, 3 mg synthetic met-enkephalin was covalently bound to 0.5 g CNBR genie females, each ovariole may contain a activated Sepharose 4B beads (Pharmacia). After ? number of follicles in various developmenovernight incubation (end-over-end mixing at room * tal stages. temperature) of 2 ml 11250 met-enkephalin antiserum, The cytoplasm of the previtellogenic antibodies were removed from the gel by suction oocyte is positively labeled with the metthrough a sintered glass filter covered with a Gelman filter membrane (0.45urn-diameter). The filtered enkephalin antiserum. The follicle cells are preadsorbed met-enkephalin antiserum was applied as not immunoreactive. Small-sized oocytes primary antiserum in the peroxidase-antiperoxidasein the lampbrush chromosome stage show (PAP) staining procedure. strong positive reaction in numerous denseThe same solid phase adsorption procedure was ly packed granules that are located in the followed with a CNBR-Sepharose control gel (without bound metenkephalin peptide) to examine if the preadcytoplasm near the nucleus (Fig. 2). sorption with met-enkephalin was due to the As the oocytes increase in size, the imcharacteristics of the gel itself.

RESULTS

Both method and serum specificity controls are conclusive. The method specificity tests 1 and 2 as described under

munoreactive granules become distributed throughout the cytoplasm (Fig. 3). In previtellogenic medium-sized oocytes, most of the dense immunoreactive granules are present in the periphery of the cytoplasm, and only a few granules can be observed

FIGS. l-4. Cross-sections through locust ovarian follicles. The immunoreaction is completely inhibited after preadsorption of the met-enkephalin antiserum (Fig. 1, x400). In small sized previtellogenie oocytes, a dense ring of intensively stained granules can be seen around the nucleus (Fig. 2, x312). In medium-sized oocytes, the met-enkephalin immunoreactive granules are dispersed throughout the cytoplasm (Fig. 3, x400). An intense immunoreaction is located in the periphery of the previtellogenic nearly full-sized oocyte (Fig. 4, arrow). The vitellogenic oocyte is immunonegative to the met-enkephalin antiserum (Fig. 4, upper part, x312). The follicle cells (F) are immunonegative.

FIGS. 5, 8. Cross sections through the testis of Locusta migvatoria (Fig. 5, x400) and Sarcophaga (Fig. 8, x400) show met-enkephalin immunoractive spermatogonia (SG), spermatocytes (SC), spermatids (ST). and young spermatozoa (SP). FIGS. 6, 7. Cross-sections through the ovarian follicle of Sarcophaga bullata showing metenkephalin-like material around the nucleus of the oocyte (0) and the trophocytes (TR) of the previtellogenic follicle (Fig. 6, x62.5) and in a small layer of ooplasm (arrow) in the vitellogenic follicle (Fig. 7, x400). The follicle cells (F) are immunonegative. bullata

4

MET-ENKEPHALIN

IN

INSECT

near the nucleus (Fig. 4). In nearly full-sized previtellogenic oocytes, all immunoreactive material is located in the periphery of the cytoplasm. From the onset of vitellogenin uptake on, all oocytes become immunonegative to the met-enkephalin antiserum. cells of the spermatogenic cell line, except the mature spermatozoa are metenkephalin immunoreactive (Fig. 5). In the spermatids, a dense ring of immunoreactive cytoplasm can be seen around the nucleus. In the spermatogonia and the spermatocytes, the immunoreactive material is organized in intensively labeled granules, located all over the cytoplasm. (b) Sarcophaga i$ullata. The follicles present in the polytrophic ovary of Sarcophaga &data consist of 15 nurse cells and one oocyte that are surrounded by a layer of mesodermal follicle ceils. All cells of the germ cell line, the trophocytes and the oocyte, show met-enkephalin immunoreactivity in both previtellogenic (Fig. 6) and vitellogenic follicles (Fig. 7). In the trophocytes, the distribution of the metenkephalin immunoreactive granules is, like in the locust oocyte, also dependent on the stage of development of the follicle. In the previtellogenic stage, the immunoreactive granules are located near the nucleus, whereas in the vitellogenic stage 4C, they are situated in the periphery of the cell cytoplasm. In the previtellogenic oocyte, metenkephalin immunoreactivity is present around the nucleus (Fig. 6) whereas in the vitellogenic oocyte, it can be observed in a thin layer of ooplasm between the follicular cell layer and the yolk mass (Fig. 7). No met-enkephalin immunoreactivity is found in the follicle cells. In the testes, the situation is comparable with the locust male gonads. All spermatogenie cells, except the mature spermatozoa, are immunopositive (Fig. 8). In the ovary of both Locusta and Sarcophaga, no immunoreactivity is found with antisera against adrenocorticotrophic

GONADS

AND

5

CNS

hormone (ACTH), CPMSEI (rne~a~o~~~~~~ stimulating hormone), P-MS phin, CRF (corticotropin releasing factor)? and leucine-enkephalin. System Locusta migratoria

Nervoafs

(Fig. 9). In t

FIG. 9. Schematic representation of brain, corporacardiaca-corpora ailata complex and ventral nerve cord of Locusta migratoria, showing met-e~ke~bai~~ immunoreactive neurons (closed circles j and fibers (dots). AG,,, abdominal ganglia; CC-CA, corpora cardiaca-corpora ailata; DEU deuterocerebrom; MESO, mesothoracic ganglion; META, metathoracic ganglion; OC, ocetlus; OL, optic Lobe; PRO, prothoracic ganglion; PROTO, protocerebrum; SOG, subesophageal ganglion; TRITO, tritocerebrum.

6

SCHOOFS ET AL.

intercerebralis, a great number of cells are immunoreactive to the met-enkephalin antiserum, which exhibit a medium-to-dense immunoreactivity (Fig. 10). Positively stained cell bodies are also observed at the base of the eyestalks. In the deuterocerebrum, at least five immunopositive perikarya are visible and the tritocerebrum shows about 15 immunoreactive cell bodies (Fig. 11). Many immunopositive nerve fibers are homogeneously distributed throughout the cerebral neuropil. About 12 large perikarya are observed in the subesophageal ganglion (Fig. 12). A strong labeling of fibers is obtained in the corpora cardiaca (Fig. 13). In the ventral nerve cord, each thoracal and abdominal ganglion revealed a great number of positively stained cell bodies and nerve fibers (Fig. 14).

No difference in staining pattern can be observed between male and female insects or between the different larval stages. Sarcophaga bullata (Fig. 15). In the fly, numerous neuronal cell bodies and fibers staining for met-enkephalin are localized in the brain. A great number of immunoreactive neurons are observed in the ventral part of the subesophageal ganglion. In these large perikarya, the whole cytoplasm contains fine, intensively stained granules, while the nucleus is negative (Fig. 16). Another cell group, containing metenkephalin immunopositivity, is located at the basis of each optic lobe (Fig. 17). In the ventral part of the optic lobes, only a few large cell bodies exhibit immunoreactivity, while dorsally, an intense staining reaction is observed in a large number of smaller neurons. In the pars intercerebralis, a few

immunoreactive cells are occasionally found. In the tritocercbrum, a group of cells, dorsally located with respect to the esophageal foramen, also displays metenkephalin immunoreactivity. DISCUSSION

Our study demonstrates for the first time the presence of a met-enkephalin-like compound in the gonads of insects by means of immunocytochemistry. In Locusta migratoria and Sarcophaga bullata, the distribution of the met-enkephalinimmunopositive granules in the cell cytoplasm is dependent on the stage of development of the ovarian follicles. In this respect, a striking comparison can be made between our immunohistological data in the ovarian follicle of Locusta and Sarcophaga on one hand and the data of Georges and Dubois (1984) as to met-enkephalin immunoreactivity in the protochordate Ciona intestinalis on the other hand. These researchers detected the very same variation in the distribution of metenkephalin-like material according to the size of the oocytes. The disappearance of the immunoreactivity to the metenkephalin antiserum in the older stages may indicate that the met-enkephalin-like substance is destroyed, modified, or released. The present observations are also in agreement with the recently published immunohistochemical data of Marchand and Dubois (1986), reporting the occurrence of a met-enkephalin-like peptide in all but the oldest cells of different spermatogenic and oogenic stages of Helix aspersa. However,

FIGS. 10-12. Cross sections through the brain of Locusta migrutoriu showing met-enkephalin immunoreactivity in a group of neurosecretory cells in the pars intercerebralis (Fig. 10, x320), in the tritocerebrum (Fig. 11, x320), and in the subesophageal ganglion (Fig. 12, x320). FIG. 13. Met-enkephalin immunoreactive nerve fibers in the corpora cardiaca of Locusta migratoriu (x320). FIG. 14. Cross section through the metathoracic ganglion of Locusta containing a group of large granulated immunoreactive cells (x312).

MET-ENKEPHALIN

IN

INSECT

GONADS

AND

CNS

8

SCHOOFS ET AL.

FIG. 15. Schematic representation of the brain and subesophageal ganglion of Sarco~huga bullnra showing met-enkephalin-like neurons. OL, optic lobe: OF, esophageal foramen; PI, pars intercerebralis; PL, pars lateralis; SOG, subesophageal ganglion. FIGS. 16-17. PAP-stainings in the nervous sytem of Sarcoplzaga bullata showing met-enkephalin immunoreactive cells in the subesophageal ganglion (Fig. 16, x800) and at the basis of the optic lobes (Fig. 17, x625).

MET-ENKEPHALIN

IN

INSECT

our findings do not entirely parallel the observations in Helix. Indeed, these authors also demonstrated immunoreactivity toward antibodies raised against other fragments of the proopiomelanocortin (POMC)-molecule, such as ACTH,-Z,, a-MSH. and p-MSH. In the gonads of Locusta and Surco~~haga, however, no immunoreactivity is found with antisera against these POMC-derived peptides. Nevertheless, these antisera yield positive results when applied to the central nervous system (&hoofs et ul., 19871, as does the metenkephahn antiserum. The distribution of the met-enkephalinlike cells in our study does not quite parallel the results obtained by RCmy and Dubois (1981) reporting a met-enkephalin-like peptide in the brain of adult and even embryonal locusts (Romoeuf and Wemy, 1984). We observe a histological difference concerning the number of immunoreactive cells. Indeed these authors found only four cells, located in the locust protocerebrum and at the base of the optic lobes by means of the indirect immunofluorescent technique V y the use of the PAP-method. we observed met-enkephaiin immunoreactive material in all cerebral ganglia, at the base of the optic lobes, nn the subesophageal ganglion, and in the corpora cardiaca. With the immunohistological technique as used by emy and Dubois, no immunofiuorescence could be detected in the latter organ. Differences in the properties of the antisera or in the immunocytochemical methods used may offer a possible explanation for this discrepancy. Nevertheless, our data correspond very well with the quantitative results of Gros eil al. (19X), who demonstrated the presence of met-enkephalin-like substances by means of radioimmunoassay in the locust optic lobes, cerebral ganglia, the subesophageal ganglion, and in the corpora cardiaca. 4n vertebrates, met-enkephalin originates from a precursor molecule, called proenkephalin (Rossier el al., 1986). On the

GONADS

AND

CNS

basis of our imm~noh~stoc~emica~ s we are not able to draw conc~~~~o~s the presence of the enkephalinas part of a larger molecule. T Leung and Stefano (1983) is, to edge, thus far the only one to ~em~n$trate in the mollusc rl/pydlus eduiis, by isolation and amino acid sequencing, the met- and leu-enkephalin a kephalin-Arg6-Phe’, the latter of suggested to present a “pro-en type” compound. Pages et al. (1983) noticed rnetb~~~~~eand ieucine-enkephalin Immune ctivity in the central nervous system in the gonads of Drosophila melanogaskv-. ‘The fact that met-cnke~ba~~n immunor is found In the gonads of three insect species and in other ~nvert~b~te~ as well suggests that we are dealing with a generai biological process. In vertebrates, endogenous peptides, i.e., ~-e~do~~~~~ met-enkephalin have been detected in era1 peripheral tissues such as the pancreas, gut, gonads, and placenta ~~etrag~~a et a/., 1985). The presence of ~rnrn~~oreac~~ve @-endorphin has been described in ovarian homogenates from (kim et ai., 1983), rats (Tsong et ai., 1 mice (Shaha et ai.) 1984), and in extra rat testis (Sharp ef al., 1980). The presence of met-enk immunoreactive material has been strated in mammalian ovaries al., 1981) and spermatozoa (Sastry eZ ai., 1982). Increasing CQ~ce~tratio~s In dev oping follicles (Lim el al., 1983) and t presence of opiat viridis

oocytes

i

suggests that the have important reguiatory fn~~t~ons in the reproductive system. The present observation that metenkephalin reaction products first appear around the nuclei, then in the periphery of the cells, and subsequently ~~sa~~ear indicates that their presence related to local synthesis a The function of the met-en

10

SCHOOFS ET AL.

substances in the insect gonads is speculative. It can be suggested that they may affect reproductive physiology by acting at several levels in the reproductive neuroendocrine system. Furthermore, their presence in the previtellogenic oocytes of both insects suggests the involvement of the met-enkephalin-like substances in the developmental regulation of ovarian function. A possible control in sexual maturation as considered by Georges and Dubois (1984) in Ciona and by Marchand and Dubois (1986) in Helix can also be suggested for Locusta and Sarcophaga. Opioid peptides increase dopamine concentrations in certain ganglia of the marine mollusc Mytilus edulis (Stefano et al., 1981), the freshwater mollusc Anodonta cygnea (Stefano and Hiripi, 1979), and the land snail Helix pomatia (Stefano et al., 1980). The demonstration of high affinity binding sites for Dala-met-enkephalin-amide, a synthetic analogue of met-enkephalin, in both brain and midgut of the insect Leucophaea maderae (Stefano and Scharrer, 1981; Stefano et al., 1982) argues in favor of the presence of an active opioid system in insects as well. These authors postulated that opiate receptors and their endogenous effecters play a prominent role in regulation of transmitter release in invertebrates. This contrasts with the suggestions of Simantov et al. (1976), who presented that morphinometic substances occur only in vertebrates. In vertebrates, the endogenous opiate peptides, the enkephalins, and the endorphins are involved in many biological processes: stress, gastro-intestinal functions, eating, drinking, transit of blood, thermoregulation, and modulation of learning and memory, of cardiovascular responses, and of respiratory effects (Olson et al., 1984). Further investigation is needed to elucidate the physiological role of the metenkephalin-like peptide in insects. The large amount of met-enkephalin immunoreactive material present in the go-

nads offers great perspectives in the purification of this substance. In conclusion, the present study provides support for the view that peptides of the enkephalin family are widely distributed through the animal kingdom (Scharrer, 1978). ACKNOWLEDGMENTS We gratefully thank Mr. J. Gijbels for preparing the primary antiserum and Mrs. J. Puttemans for photography. Liliane Schoofs gratefully acknowledges research grants from the IWONL of Belgium.

REFERENCES Andries, J. C., and Tramu, G. (1985). Distribution patterns of mammalian-like peptide immunoreactive cells in the midgut of Aeshna cyanea (Insecta, Odonata). Experientia 41, 500-503. Ashby, G. J. (1972). Locusts. In “The U.F.A.W. Handbook on the Care and Management of Laboratory Animals” (U.F.A.W., Ed.), pp. 582-587. Livingstone, Edinburgh/London. Balbalkin, G. Y., and Yakovleva, T. V., Nikitina, L. A., Korobov, N. V., Vinogradov, V. A., and Titov, M. I. (1983). Opiate binding sites and endogenous opioids in Bufo viridis oocytes. Biothem. Biophys. Res. Commun. 117, 718-724. De Loof, A., and De Clerck, D. (1986). Vertebratetype steroids in arthropods: Identification, concentrations and possible functions. Adv. Invert. Reprod. 4, 117-123. El Salhy, M., Falkmer, S., Kramer, K. J., and Speirs, R. D. (1983). Immunohistochemical investigation of neuropeptides in the brain, corpora cardiaca and corpora allata of an adult lepidopteran insect Manduca Sexta L. Cell Tiss. Res. 232, 295-317. Georges, D., and Dubois, M. P. (1984). Methionineenkephalin-like immunoreactivity in the nervous ganglion and the ovary of a protochordate, Ciona intestinalis. Cell Tiss. Res. 236, 165-170. Greenberg, M. J., and Price, D. A. (1983). Invertebrate neuropeptides: Native and Naturalized. Anna. Rev. Physiol. 45, 271-288. Gros, C., Lefon-Cazal, M., and Dray, F. (1978). Presence de substances, immunoreactivement apparentees aux enkephalins chez un insecte, Locusta migratoria.

C.R.

Acad.

Sci. Paris

278, 647-650.

Hansen, H. L., Hansen, G. N., and Scharrer, B. (1982). Immunoreactive material resembling vertebrate neuropeptides in the corpus cardiacum

MET-ENKEPHALIN

IN INSECT

and corpus allatum of the insect Leucophaea madereae. Cell Tiss. Res. 225, 319-329. Hughes, J. (1975). Isolation of an endogenous compound from the brain with pharmacological properties similar to morphine. Brain Res. 88, 295-308. Huybrechts, R., and De Loof, A. (1981). Effect of ecdysterone on vitellogenin concentration in haemolymph of male and female Sarcophaga. Znt. J. Invert. Reprod. 3, 157-168. Leung, M., and Stefano, G. B. (1983). Isolation of molluscan opioid peptides. Life Sci. 33, 77-80. Lim, A. T., Lolait, S., Barlow, J. W., Wai Sum, U., Zois, I., Toh, B. H., and Funder, J. W. (1983). Immunoreactive B-endorphin in sheep ovary. Nature (London) 303, 709. Marchand, C. R., and Dubois, M. P. (1985). Mise en evidence par immunocytologie de sites antigbniques fixant des anticorps anti-8-MSH et anti-methionine-enkephaline dans l’ovotestis de 1’Escargot adulte Petit-Gris (Helix aspersa). C.R. Acad. Sci. Paris 301, 228-233. Marchand, C. R., and Dubois, M. P. (1986). Immunoreactivity of the hermaphroditic gonad of the snail He/ix aspersa Miiller towards antibodies raised to fragments of pre-proopiomelanocortin. Cell Tiss. Res. 245, 337-341. Olson, 6. A., Olson, R. D., and Kastin, A. J. (1984). Endogenous opiates: 1983. Peptides 5, 975-992. Pages, M., Jimenez, F., Ferrus, A., Peralta, E., Ramirez, G., and Gelpi, E. (1983). Enkephalinlike immunoreactivity in Drosophila melanogaster. Neuropeptides 46, 87-98. Petraglia, F., Segre, A., Facchinetti, F., Campanini, D., Ruspa, M., and Genazzani, A. R. (1985). B-endorphin and met-enkephalin in peritoneal and ovarian follicular fluids of fertile and postmenopausal women. Fertil. Steril. 44, 615-621. Remy, C., and Dubois, M. P. (1981). Immunohistochemical evidence of methionine enkephalin-like material in the brain of the migratory locust. Cell Tiss. Res. 218, 271-278. Romoeuf, M., and RCmy, C. (1984). Early immunohistochemical detection of somatostatin- and methionine-enkephalin-like neuropeptides in the brain of the migratory locust embryo. Cell Tiss. Res. 236, 289-292. Rossier, J., Trifaro, J. H., Lewis, R. V., Lee, R. W. H., Stern, A., Kimura, S., Stein, S., and Udenfriend, S. (1980). Studies with [‘?S]methionine indicate that the 22,000 dalton (Met)enkephalin containing protein in chromaftin cells is a precursor of (Met)enkephalin. Proc. Natl. Acad. Sri. USA 77, 6889-6891. Sastry, B. V. R., Janson, V. E., Owens, L. K., and Tayeb, 0. S. (1982) Enkephalin- and substance P-like immunoreactivities of mammalian sperm

GONADS

AND

CNS

31

and accessory sex glands. Biochem. Pkarmacol. 31, 351%3522.

Scharrer, B. (1978). Peptidergic neurons: Facts and trends. Gen. Comp. Endocrinol. 34, 50-62. Schols, D., Verhaert, P., Huybrechts, R., Vaudry, II. i Jtgou, S., and De Loof, A. (1987). Immnnocytochemical demonstration of proopiomelanocortinand other opioid-related substances and a CRFlike peptide in the gut of the American cockroach, Periplaneta americana L. Histochemistry 86, 345-35 1. Schoofs, L., Jegou, S., Vaudry, H., Verhaert, P., and De Loof, A. (1987). Localization of melanotropinlike peptides in the central nervous system of two insect species, the migratory locust, Locusta tnigratoria and the fleshfly, Sarcophaga buliata. Cell Tiss. Res. 248, 25-31. Shaha, C., Margioris, A., Liotta, A. S.. Krieger, D. T., and Bardin, C. W. (1984). Demonstration of immunoreactive 8-endorphin and y,-melanocyte stimulation hormone-related peptides in the ovaries of neonatal, cyclic and pregnant mice. Endocrinology 115, 378. Sharp, B., Pekary, A. E., Mayer, N. V., and Hershman, J. M. (1980). 8-endorphin in male reproductive organs. Biochem. Biophys. Res. Commun. 95, 618-623. Simantov, R., Goodman, R., Aposhian, D., and Snyder, S. H. (1976). Phylogenetic distribution of a morphine-like peptide “enkephalin.” Brain Res. 111, 204-211. Stefano, G. B., Hall, B., Makman, M. H., and Dvorkin, B. (1981). Opioid inhibition of dopamine release from nervous tissue of Mytilus edulis and Octopus bimacuiatus. Science 213, 928-930. Stefano, G. B., and Hiripi, L. (1979). Methionineenkephalin and morphin alter monoamine and cyclic nucleotide levels in the cerebral ganglia of the freshwater bivalve Anodonta cygnea. Life Sci. 25, 291-298. Stefano, G. B., Rozsa, K. S., and Hiripi, L. (1980). Actions of methionine-enkephalin and morphin on single neuronal activity in Helix pomatia &. Comp. Biochem. Physiol. 46, 193-198. Stefano, G. B., and Scharrer, B. (1981). High affinity binding of an enkephalin analog in the cerebral ganglion of the insect Leucophaea maderae (Blatteria). Brain Res. 225, 107-114. Stefano, G. B., Scharrer, B., and Assanah, P. (1982). Demonstration, characterization and localization of opioid binding sites in the midgut of the insect Leucophaea maderae (Blatteria). Bruin Res. 253, 205-212.

Tsong, S. D., Phillips, M. D., Halmi, N., Krieger, D. T., and Bardin, C. W. (1982). ~-e~do~~in is present in the male reproductive tract of five species. Biol. Reprod. 27, 755. Udenfriend, S., and Kilpatrick, D. L. (1983). Bio-

12

SCHOOFS ET AL.

chemistry of the enkephalins and enkephalin containing peptides. Arch. Biochem. Biophys. 222, 30%322. Vandesande, F. (1983). Immunohistochemical double staining techniques. In “Neuroimmunocytochemistry” (E. Cuello, Ed.), pp. 257-272. Wiley, New York. Verhaert, P., and De Loof, A. (1985). Immunocytochemical localization of a methionine-enkephalin

resembling neuropeptide in the central nervous system of the american cockroach Z’eriplunetu americana L. J. Camp. Neurol. 239, M-61. Wenger, T., Leonardelli, O., and Tramu, G. (1981). Enkephalins in the adult rat ovary. Presented at the International Symposium on “Pituitary Hormones and Related Peptides: From Cell Biology to Clinical Application. San Marino, Republic of San Marino, May 25 to 27.” pp. 122. Abstract 97.