The oviduct musculature of the cockroach Leucophaea maderae and its response to various neurotransmitters and hormones.код для вставкиСкачать
Archives of Insect Biochemistry and Physiology 167-178 (1984) The Oviduct Musculature of the Cockroach Leucophaea maderae and Its Response to Various Neurotransmitters and Hormones Benjamin J. Cook, G. Mark Holman, and Shirlee Meola Veterinary Toxicology and Entomology Research Laboratory, Agricultural Research Sewice, U S Department of Agriculture, P.O. Drawer GE, College Station, Texas The musculature of the oviduct consists of an outer, irregular layer of longitudinal muscle and an inner layer of circular muscle. The four basic modes of activity-compression, segmentation, peristalsis, and reverse peristalsis-were evident in the isolated oviduct. These spontaneous events often occurred in an organized sequence. In fact eggs could be transported down the lateral oviducts by this myogenic activity once the sphincter between the common oviduct and vagina was severed. Myographic recordings were made of only the contractions of the longitudinal muscles. M. The L-glutamate caused a distinct phasic contraction at 2.2 x response became larger and more complex as the concentration of the amino acid was increased. Acetylcholine (1.6 x l o w 5M) caused either a phasic or tonic response, or a combination thereof. By contrast, 5HT' and tyramine simply increased the frequency of small phasic contractions, although in some preparations both monoamines caused an inhibition. The ecdysones, a juvenile hormone analogue (1 x lop6 M), and prostaglandin E2 had no effect on oviduct activity. Initially high KCI solutions (162 mM) without C a + + induced a strong contraction but subsequent additions failed to do so. However, when a high KCI solution (158 mM) with 2 m M Ca+ was added to the preparation the responsewas partially restored.Also the potentcalcium antagonist Mn++ (2 mM) can suppress spontaneous activity. + Key words: oviduct, muscle, neurotransmitters, hormones, cockroach, f eucophaea maderae 'Abbreviations: 5-hydroxytryptamine = 5HT; scanning electron microscope = SEM. Acknowledgments: We thank Mrs. Tara Peterson for her illustrations and competent technical assistance. Received August 2,1983; accepted August 29,1983. Address reprint requests to B.J. Cook, USDA, ARS, VTERL, P.O. Drawer GE, College Station, TX 77841. @ 1984 Alan R. Liss, Inc. 168 Cook, Holman, and Meola INTRODUCTION Although the musculature of the oviduct has not received as much attention as other visceral muscles, it offers more possibilities for the study of physiological regulation. These muscles are controlled not only by innervation and hormones [1,2] in the hemolymph, but by male accessory gland secretions [3,4] as well. As yet no one has presented definitive evidence for a specific chemical that mediates any of these channels. However, a number of reports suggest the involvement of various neurotransmitters. The accessory gland and ejaculatory duct of several species of male moths contain a high titer of acetylcholine . Davey  reported a melanophortropic response from the skin of the frog after treatment with opaque accessory secretion from male Rhadnius prolixus. Such a reaction normally is attributed only to indolalkylamines. A tryptamine derivative also has been identified in the blood of ovipositing females of Schistocevca gveguriu . Several reports have shown that serotonin, 5-hydroxytryptamine, at low levels stimulates the oviduct in a number of insects. These amounts are M or less in M in Tubanus sulcifvons . Locusta rnigvutoria  and 2 X In an attempt to explore the three channels for regulation mentioned above, we initiated studies on the motile properties of oviduct muscles in the cockroach Leucophueu maderue and the response of these muscles to various ions, neurotransmitters, and hormones. MATERIALS AND METHODS Adult female L. maderue were collected from the stock colony 1-3 h after emergence and held with or without males from 24 h to 60 days. Saline for dissection and perfusion was of the following composition (in mh4): NaCl 156, KC1 2.7, CaC12 1.8, glucose 22. The pH was adjusted to 6.8. 5HT was used as the creatinine salt. This chemical and acetylcholine, glutamic acid, octopamine, and tyramine were all purchased from Sigma Chemical Co. The juvenile hormone analogue ZR5l5 (isopropyl=(2E,4E)-ll-methoxy-3,7,ll-trimethyl-2,4-dodecadienoate) was obtained from Zoecon, Inc. Ecdysone and 20-hydroxyecdysone were obtained from Simes Pharmaceutical (Milan, Italy) and Rohto Pharmaceutical Ltd (Osaka, Japan), respectively. Preparation of Oviducts for Myographic Recording Adult female cockroaches of varying age and reproductive state were decapitated and the legs and wings removed. A dorsal incision was then made from just anterior to the last abdominal sclerite through the pronotum. After opening the insect from the dorsal surface, the hindgut and the Malphigian tubules were removed to expose the lateral oviducts and ovaries beneath. Once the apical suspending ligaments and the lateral tracheal attachments of the ovaries were severed, it was possible to loosen the lateral oviduct from the side wall of the vagina by gently drawing the ovaries in a slightly anterior direction while cutting the remaining attachments. After both oviducts were freed in this manner, a dorsal patch of the vagina was cut out around the junction with the common oviduct. This released the Oviduct Musculature and Its Response to Drugs 169 preparation from the insect and a thread was tied about the junction of the vagina and the common oviduct. Another thread was fastened about both ovaries just above the lateral oviducts. The first thread was attached to a metal hook that could be lowered into a saline-filled muscle chamber by means of a micromanipulator; the second was fixed with wax to a balsawood lever that activated a Brush isotonic muscle transducer (model 33-03981). The balsa-wood beam was counterweighted to produce a tension of approximately 40-85 mg on the suspended organ. The saline was continuously oxygenated by pumping air through a hypodermic needle inserted through the rubber stopper at the bottom of the chamber. The saline could be changed with minimal disturbance to the preparation through a tube from the bottom of the chamber. The saline could be changed with minimal disturbance to the preparation through a tube from the bottom of the chamber. Myographs were recorded by simply connecting the transducer to a pen recorder. Preparation of Tissues for Scanning Electron Microscopy Oviducts were dissected out under saline, and then fixed for 2 h in a mixture of 3% glutaraldehyde, 2% paraformaldehyde, and 1%picric acid in 0.05 M phosphate buffer at pH 7.4. After five rinses in the phosphate buffer over a period of 1 h, specimens were placed for 2 h in phosphate buffer containing 1%osmium tetroxide, followed by 5-10 rinses in distilled water (for 1h). They were then dehydrated in an ascending series of concentrations of ethanol followed by three 15-min rinses in 100% acetone, and dried with liquid C 0 2 in a Denton critical-point drier. The dried specimens were mounted on scanning electron microscope stubs with silver conducting paint, coated with gold-palladium, and observed with a Cambridge Steroscan S4SEM at 10 kV. RESULTS Basic Structure and Muscle Networks The common oviduct in L. maderue is short, only one-fifth the length of the paired lateral projections that lead from it to connect with the ovaries (Fig. lA, B). Consequently, the lateral oviducts represent the main functional channel for egg transport in this insect. Scanning electron micrographs of the surface of these lateral ducts revealed an irregular patchwork of longitudinal and circular muscle fibers (Fig. 1C). The longitudinal fibers were superficial and clearly evident, while the circular muscles existed at a deeper level. Fiber branching and fine interconnecting protoplasmic bridges were conspicuous among the longitudinal fibers (Fig. 1D). Such an arrangement provides an obvious structural syncytium that may permit these fibers to function as a physiological unit. At the junction of the common oviduct and the vagina the reproductive canal is closed by a heavy folding of tissue which obstructs the passage of eggs in vitro. However, it is possible to draw eggs through this restriction with a pair of forceps. 170 Cook, Holman, and Meola Fig. 1. Anatomical features of the oviduct and i t s muscular topography. A) An extended view of the oviduct as it would appear in the muscle chamber. B) Dorsal view of the oviduct in situ. A large portion of the lateral oviduct lies against the ventral wall of the abdomen. ov, ovary; lo, lateral oviduct; co, common oviduct; vag, vagina. C ) Detail of the arrangement of circular and longitudinal muscles on the surface of the lateral oviduct. D) Another scanning electron micrograph showing the branching and variable size of longitudinal muscles on the lateral oviduct. Observed Movements and Myographic Recordings Spontaneous contractions were observed in 63% of the in situ preparations of the cockroach oviduct (n = 43) just after dissection. Connections to the central nervous system remained intact in these preparations, and the percentage of activity rose to 89% once the oviducts were perfused with saline solution. The isolated oviduct, like most visceral muscles, contracted in a spontaneous and rhythmic fashion without any neural input regardless of whether eggs were present. The often complex character of this motile activity could be resolved into the four basic categories previously described for insect visceral muscle . Although precise measurements of time could be made of both individual motile types (compression, segmentation, peristalsis, and reverse peristalsis) and of whole sequences by microscopic observation, it was not possible to render a completely quantitative treatment without cinematographic analysis. Nevertheless, the distinctive features of the various kinds of spontaneous activity can be effectively represented by the series of drawings in Figures 2 and 3. Oviduct Musculature and Its Response to Drugs 171 Compression was the dominant type of activity observed in the oviduct. This class of motility was caused by the localized contraction of longitudinal muscle fibers at some point on the lateral oviduct. These events had a duration of 2-7 sec and often showed a definite and organized sequence. A typical example is represented in Figure 2A. Compressions usually began near the common oviduct or in the pedicel just beneath the ovary, and progressed in either an anterior or posterior direction. If the contractions began just above the common oviduct, a series of sequential compressions would often proceed toward the pedicel; after a few seconds this progression would reverse direction. The overall effect of this sequence of activity imparted a complex oscillatory motion to the entire oviduct. Peristalsis was also evident in a number of preparations. This class of motile activity was caused by a localized contraction of circular muscles at some point along the oviduct, which then progressed as a wave in either a caudal or an anterior direction (reverse peristalsis). An example of this kind of activity/ together with compression, is shown in Figure 2B. Fig. 2. Drawings of a 40-sec sequence of compressions (A) and a 17-sec sequence of reverse peristalsis and compression (6) in two separate lateral oviducts. No eggs were found in either preparation. Beginning and ending frames are marked in the time elapsed from the observed procession. 172 Cook, Holman, and Meola Segmentation was evident in isolated oviducts that contained eggs. This kind of motility consisted of an annular constriction of the duct without progression. It had a duration of 3-5 sec. Segmentation appeared to provide a means of holding eggs in place while the adjacent duct wall was drawn across the egg surface by compression (Fig. 3A). This recurring sequence took 10-12 sec and was repeated about every 20 sec. The net result was a gradual movement of the egg down the lateral oviduct. Oviducts from insects that were in an active state of ovulation were generally filled with eggs shown in Figure 3B. Although the motile activity observed in such ducts kept a constant pressure on the muscular valve that separates the common oviduct from the vagina, no eggs were ever passed in Fig. 3. Drawings of motile events in egg-filled oviducts. A) A 20-sec sequence of compression and segmentation. B) Another example of compression and segmentation in various regions of the oviduct. C) A timed sequence of egg transport through the oviduct. Oviduct Musculature and Its Response to Drugs 173 vitro. However, once this valve was surgically removed eggs could be transported down the lateral oviduct and out of the common oviduct by a combination of the various motile patterns described (Fig. 3C). In spite of the various modes of activity just described, only the contractions of the longitudinal muscles (compression) could be directly recorded on myographs. Most of the preparations arranged for this kind of recording showed a simple phasic pattern of contraction that varied considerably between preparations. Age after adult emergence seemed to have little bearing on the character or frequency of spontaneous motile activity (Fig. 4, second line of records). Also, a careful comparison of oviduct activity from mated (n = 21) and unmated (n = 34) females showed no obvious change in pattern. Response to Various Neurotransmitters and Hormones Isolated oviducts have a threshold sensitivity to L-glutamic acid between 1x M and 4 x lop5 M (Table 1). The most characteristic response was a large initial phasic contraction followed by a series of contractions of declining amplitude. If the oviduct was exposed to increasing amounts of glutamate, a graded response was often observed (Fig. 5A1-3). Surprisingly, the oviduct was about as sensitive to acetylcholine (1to 4 X M) as to glutamate. Although there was an initial phasic contraction in the response, an evident tonic component was often present (Fig. 5B1-3). When the preparation was exposed to increasing amounts of acetylcholine, a graded response did not occur. In fact, there was often a decline at the higher concentrations. I I 10 1 Day 60Dayr Prep A 20 Prep B 40 Prep C Fig. 4. Myographic profiles of oviduct activity on various days after adult emergence. Second line of records are for three different preparations of the same age. Vertical calibration = 2 mm of tissue movement: horizontal time mark = 1 min. 174 Cook, Holman, and Meola TABLE 1. Response of the Oviduct of L. maderae to Various Neurotransmitters and Hormones Chemical No. of exueriments Inhibition Response Excitation None 4 12 14 2 3 3 1 31 17 4 10 14 6 6 2 Glutamic acid Acetylcholine Octopamine 15 17 7 Tyramine 5-H ydroxytryptamine Range of threshold 1.0to 4.0 x 10-5 1.0to 4.0 x 10-5 2.0 x ~ o - ~toM 2.0 x 10-'M 4.0 x 1 0 - 7 ~to 4.0 x lO-'M 1.2 x ~ o - ~toM 3.0 x lO-'M Maximum conc. tested Ecdysone 20-Hydroxyecdysone Juvenile hormone analog ZR 515 Prostoglandin E2 5 3 1.0 x 10-6M 1.0 x lO-'M 2 3 3 3.2 x 1 0 - 7 ~ 1.0 X lO-'M Octopamine, tyramine, and 5HT caused both inhibition and excitation of contractile activity (Table 1).Examples of inhibition with octopamine and tyramine are shown in Figure 5C and E, respectively. The excitation of the oviduct with 5HT caused an increase in the frequency of small phasic contractions (Fig. 5DI-2). On some preparations tyramine did the same thing (Fig. 5F), yet this excitation with tyramine could be suppressed by the addition of 5HT (Fig. 5G). Since ecdysone and the juvenile hormone are known to regulate the reproductive cycle in many insects, we wanted to test their effects on the muscles of the oviduct. Neither hormone, however, showed any effect at 1 x M. Prostaglandin E2 also showed no effect (Table 1). The Effects of Potassium, Calcium, and Manganese Ions The exposure of the isolated oviduct to high potassium solutions (158 mM K + plus 2 mM Ca") generally caused an immediate strong contraction that plateaued at 1-3 min (Fig. 6A-C) and then slowly dropped to a baseline tension in 5-12 min. Although a high potassium (162 mM) solution without Ca++ initially caused a substantial contraction of the oviduct (Fig. 6D1), a successive rinse in the same solution failed to evoke another contraction (Fig. 6D2). However, if the same oviduct was exposed to 158 mM KC1 with 2 mM Caf+ in it, the oviduct responded (Fig. 6D3). When 2 mM Mn++, a potent antagonist of the calcium ion, was added to the muscle spontaneous contractile activity was arrested (Fig. 6E). The fact that calcium can cause the depolarized muscles of the oviduct to contract and that the potent calcium antagonist Mn+ can suppress spontaneous activity offers good circumstantial evidence that Ca++ functions as an intracellular messenger for contraction in the oviduct. + Oviduct Musculature and I t s Response to Drugs A A 2x 1 O - ~ M 8x A lo-% 1x I O - ~ M A I 2~ 1 O - ~ M C 8 ~ 1 0 - ~ M D, A ~ x ~ o - ~ M E 175 F D1 2 x 1O - ~ M G Response of a Fig. 5. Responses of the isolated oviduct to various biogenic amines. preparation to increasing amounts of L-glutamate (arrows). The break between records indicates a saline rinse. B1-3) The effects of various concentrations of acetylcholine. C) Suppression of activity by 2 x lo-’ M octopamine. D1..*) Small phasic contractions caused by 5HT. E) Inhibition of activity with tyramine. F) Excitation with tyramine in another preparation. G ) Excitation with tyramine suppressed by 5HT. Vertical calibration = 2 m m of tissue movement; horizontal time mark = 1 min. 176 Cook, Holman, and Meola APB 4Jr JL A A A A A Fig. 6. Effect of high-potassium salines on oviduct contracture and its dependence on calcium. A-C) Three different responses to high potassium. D1-3)Calcium dependence of the potassium contracture (D,) 162 m M K + without C a + + added at arrow. D2)Same preparation 10 min later, 162 m M K + without C a + + added at arrow. D3) Response of the same preparation t o 158 m M K + with 2 m M Ca+ (arrow). E) Suppression of spontane cous activity of the oviduct with 2 m M M n + (arrow). Vertical calibration = 2 mm of tissue movement; horizontal time mark = 1 min. + + DISCUSSION It is possible to recognize the four basic motile patterns of compression, segmentation, peristalsis, and reverse peristalsis in the lateral oviducts of the cockroach, just as in the hindgut . Given such uniformity in motile forms between diverse organs, the question arises as to what function such apparently random and occasionally discontinuous activity can accomplish. Observations in the present study suggest that compression, segmentation, and peristalsis can assist in the transport of eggs down the lateral oviduct. In fact, the duct is generally kept full of ovulated eggs during the process, and the myogenic activity continues to press them into the short common oviduct. However, this activity alone is incapable of releasing the eggs from the common oviduct into the vagina in an in vitro preparation that has been deganglionated. But if the junction between these two structures is surgically removed, eggs will readily pass from the common oviduct by myogenic activity (Fig. 3C). The functional importance of myogenic activity is further emphasized by the fact that myotropins that regulate oviduct activity have been found in the neuroendocrine system of several insects [1,2,10]. Nevertheless, egg transport is not dependent on myogenic activity in the absolute sense because the muscles of the oviduct are innervated [ll]. Thus, neural impulses could activate the longitudinal muscles of the organ and cause sufficient compression to release all eggs from the ducts in several large contractions. Oviduct Musculature and Its Response to Drugs 177 In view of the chemical sensitivities detected in the present study, the prospects for chemical transmission at myoneural junctions in the oviduct look good. This is especially true for L-glutamate because it is active in micromolar amounts and gives a graded response to increasing levels of the amino acid. Furthermore, there is strong evidence that glutamate mediates the myoneural junctions in the hindgut of L. maderue [12,13]. Substances like acetylcholine and 5HT most probably originate from male gland secretions or the egg, and promote the transport of either the sperm or eggs. 5HT often caused an increase in small rhythmic contractions of the oviduct, an activity that could aid in the movement of sperm. However, not all preparations showed such an excitatory response; inhibition was evident in a number of instances. Moreover, preparations that were excitatory with tyramine often showed an inhibition to the subsequent addition of 5HT. A similar interaction between these same substances occurs in the visceral muscles of the locust . The variability in the contractile response of individual oviducts to high potassium solutions may reflect different levels in the intracellular calcium stores. The fact that 2 mM Ca++caused a partial restoration of the potassium contradure in depolarized muscles provides good circumstantial evidence that an inwardly directed calcium pulse may release cellular calcium from intracellular stores in the contraction process. The sensitivity of the oviduct to Mn++ is similar to the action of the same ion on the hindgut of L. maderue . In the hindgut, calcium-dependent action potentials were completely inhibited by 2 mM Mn++, along with all spontaneous contractile activity. Presumably the Mn+ + can compete for calcium sites on the muscle fiber membrane. LITERATURE CITED 1. Kriger FL, Davey KG: Ovarian motility in mated Rhodnius prolizus requires an intact cerebral neurosecretory system. Gen Comp Endocrinol48, 130 (1982). 2. 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