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The oviduct musculature of the cockroach Leucophaea maderae and its response to various neurotransmitters and hormones.

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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 [5]. Davey [4] 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 [6]. 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 [8].
Locusta rnigvutoria [7] 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 [9].
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 [9]. 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 [14].
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 [9]. 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. Nayar KK: Studies on the neurosecretory system of Iphifu limbutu StP1. V. Probable endocrine basis of oviposition in the female insect. Proc Indian Acad Sci 47, 233 (1958).
3. Davey KG: The migration of spermatozoa in the female of Rhodnius prolixus Stal. J Exp
Biol35, 694 (1958).
4. Davey KG: A pharmacologically active agent in the reproductive system of insects. Can J
Zoo1 38, 39 (1960).
5. Schachter M: Acetylcholine in non-nervous tissues of insects. In: Comparative Neurochemistry. Richter D, ed. Macmillan, New York, pp 341-345 (1964).
6. Highnam KC: Variation in neurosecretory activity during oocyte development in Schisfocercu greguriu. J Endocrinol24, 4 (1962).
7. Chalaye D: Neurosecretions au niveau de la chaine nerveuse ventrale de Locustu rnigruforiu
rnigruforioides (R. et F.): Etude histologique, histochimique, ultrastructurale et experimentale. These Univ Paris, No. CNRS A. 0. 9450 (1974).
8. Cook BJ, Meola S: The oviduct musculature of the horsefly, Tabunus sulcifrons, and its
response to 5-hydroxytryptamine and proctolin. Physiol Entomol3, 273 (1978).
9. Cook BJ, Reinecke JP: Visceral muscles and myogenic activity in the hindgut of the
cockroach, LRucophuea maderue. J Comp Physiol84,95 (1973).
10. Girardie A, Lafon-Cazal M: Controle endocrine des contractions de I'oviducte isole de
Locustu migrntoriu migrutorioides. C R Hebd Seanc Acad Sci, Paris 274, 2208 (1972).
178
Cook, Holman, and Meola
11. Engelmann F: Die innervation der genital- und postgenitalsegmente bei weibchen der
schabe Leucophaea maderue. Zoo1 Jb Anat 82, l(1963).
12. Holman GM,Cook BJ: Pharmacological properties of excitatory neuromuscular transmission in the hindgut of the cockroach, Leucophaea maderue. J Insect Physiol 26, 1891 (1970).
13. Cook BJ, Holman GM: The neural control of muscular activity in the hindgut of the
cockroach Leucophaea maderae: Prospects of its chemical mediation. Comp Biochem Physiol
50C, 137 (1975).
14. Huddart H, Oldfield AC: Spontaneous activity of foregut and hindgut visceral muscle of
the locust, Locusta rnigratoriu. 11. The effect of biogenic amines. Comp Biochem Physiol
73C, 303 (1982).
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