349
Changes in the pericardial cells of
Periplaneta americana induced by exposure to
homogenates of the corpus cardiacum
By K. G. DAVEY
(From the Zoological Laboratory, Downing St., Cambridge)
Summary
The pericardial cells of the cockroach, Periplaneta americana, undergo change
associated with secretion when they are exposed to homogenates of the corpus cardiacum. The cells become markedly larger, and the nuclei send out branches as far as
the cell border. Large secretory droplets appear as vacuoles first near the nucleus and
later throughout the cytoplasm. Smaller vacuoles come to occupy the area near the
periphery of the cell: these are considered to be the remains of the larger vacuoles
after they have discharged their contents. Prolonged exposure of the cells to the
homogenates in vitro leads to the cessation of these secretory activities, presumably
as a result of the exhaustion of metabolites concerned in the secretory process. The
cells also become more pronouncedly argentaffin as a result of exposure to the corpus
cardiacum. These changes are considered in the light of the discovery that the pericardial cells produce a pharmacologically active material from an inert precursor in
response to a secretion from the corpus cardiacum.
Introduction
T H E pericardial cells of insects, by virtue of their ability to take up particles
from the haemolymph, have long been described as excretory organs analogous
to the reticulo-endothelial system of vertebrates (Wigglesworth, 1953).
However, recent work (Davey, 1961 a, b) has revealed that the pericardial cells
of Periplaneta americana produce an o-dihydroxyindolalkylamine in response
to a peptide hormone released from the corpus cardiacum. Since the response
of the pericardial cells involves the production of the amine from an inert
precursor, rather than the release of a compound already present in the cells,
it should be possible to recognize changes associated with this secretion. It is
the purpose of this paper to describe some of these changes.
Materials and methods
The cockroaches used in this study were adult specimens of both sexes. In
order to obtain pericardial cells free from the influence of the corpus cardiacum, all the animals used in this study were decapitated at least 48 h
before any experiment. The method of decapitation and of preparing and
injecting extracts of the corpus cardiacum has already been described (Davey,
1961a). Where isolated hearts with their associated pericardial cells were
exposed to the hormone in vitro, the heart, pericardial cells, and alary muscles
were dissected away from the dorsal wall of the abdomen and immersed in
insect Ringer.
[Quart. J. micr. Sci., Vol. 103, pt. 3, pp. 349-58, 1962.]
3 so
Davey—Pericardial cells and corpus cardiacum of Periplaneta
For routine histological examination of the cells, hearts with their pericardial cells were subjected to Wigglesworth's (1957) osmium/ ethyl gallate
procedure, and sectioned in paraffin wax at 2 to 4 /x. The details of the histochemical tests can be found in Pearse (i960).
Results
The pericardial cells of Periplaneta are situated in the pericardial sinus
and can be divided into two groups. The first consists of large cells, roughly
spherical and about 30/x in diameter. These are closely packed together on
each side of the heart. The cells of the other group are somewhat smaller and
ovoid; they occur as strings of cells running between the fibres of the alary
muscles. Although only the cells of the first group are considered in this
paper, those of the second type undergo the same changes.
The non-secreting cell. Representative cells taken from preparations of
pericardial cells from animals decapitated 48 h previously are shown in fig. 1.
They are large (about 30 JU. in their largest diameter) and irregularly spherical.
The cells are usually binucleate, but individual cells have been observed with
up to 5 nuclei. The nuclei themselves are irregular in shape and show some
tendency to branch. Many of the cells contain a few large, clear vacuoles
confined to the area near the nucleus, but most of the cytoplasm displays
little differentiation.
The secreting cell. In order to determine the effect on the pericardial cells
of the factor from the corpus cardiacum, hearts, with their associated pericardial cells, were dissected from decapitated cockroaches and immersed in
insect Ringer containing ground-up corpora cardiaca. Other groups of pericardial cells were exposed to Ringer alone. At intervals cells from both
groups were fixed and sectioned. The pericardial cells which were exposed
to the Ringer alone showed no differences from the non-secreting cells
described above.
As little as 10 min after the exposure of the pericardial cells to the homogenates of corpora cardiaca, the pericardial cells exhibit distinct differences
from the controls. Many large vacuoles have appeared in the cytoplasm,
particularly in the area around the nucleus. The contents of these vacuoles
are not fixed by osmium. The nuclei are very distinctly branched, giving
the impression that the vacuoles nearest the nucleus are surrounded by extensions of the nucleus. A few smaller vacuoles are visible at the periphery
of the cell. Fig. 2 shows some typical cells taken from these preparations.
Thirty minutes after exposure to the hormone, the entire cell is taken up
with the larger, clear vacuoles except near the periphery, where a considerable
number of the small vacuoles are to be found. The nuclei remain extensively
branched. Fig. 3 shows cells at this stage.
After 2 h in the hormone, however, the cells present a picture not unlike
that presented by the controls (fig. 4). The nuclei are less branched and
few of the large vacuoles are seen. These cells differ from the controls in that
they contain a very large number of small vacuoles at the periphery.
Davey—Pericardial cells and corpus cardiacum of Periplaneta
351
F I G . 1. Drawings of typical pericardial cells, taken from preparations exposed to Ringer alone.
2421.3
Bb
352
Davey—Pericardial cells and corpus cardiacum of Periplaneta
FIG. Z. Drawings of typical pericardial cells, taken from preparations exposed to homogenates
of corpora cardiaca for 10 min. Note the branched nuclei and large vacuoles near the nuclei.
Davey—Pericardial cells and corpus cardiacum of Periplaneta
353
10/;
FIG. 3. Drawings of typical pericardial cells, taken from preparations exposed to homogenates
of corpora cardiaca for 30 min. The nuclei remain extensively branched and the large
vacuoles take up most of the cell. A few small vacuoles are visible near the periphery.
354 Davey—Pericardial cells and corpus cardiacum of Periplaneta
FIG. 4. Drawings of pericardial cells taken from preparations exposed to homogenates of
corpora cardiaca for 2 h. The nuclei are less branched, and there are only a few large vacuoles,
Many small vacuoles are visible near the periphery.
One of the most obvious changes brought about by exposure to the hormone involves differences in the size of the cells. The following figures
represent the means and standard deviations of the largest diameters, measured
in microns, of 30 cells taken after each of the treatments outlined above:
exposure
exposure
exposure
exposure
to Ringer alone for 10 min 29-5 i 5-1
to hormone for 10 min
34"4±6-o
to hormone for 30 min
28 -3 ± 5 "3
to hormone for 120 min
The means of the measurements of the cells exposed to hormone for 10 and
120 min are significantly different at the 1% level from the mean of the
measurements of the cells exposed to Ringer alone.
Similar results are obtained when cells treated with the hormone during life
Davey—Pencardial cells and corpus cardiacum of Periplaneta
355
are examined. To accomplish this, decapitated animals were injected either
with a homogenate of corpora cardiaca or, in the case of the controls, with
insect Ringer. Ninety minutes after injection, when the cells have been shown
to be secreting maximally (Davey, 1961a), the hearts with their pericardial
cells were dissected and fixed in osmium tetroxide solution. Histological
differences between control cells and those treated with hormone were
similar to those already described for cells treated in vitro. It should be noted
that cells taken from animals 90 min after injection with the hormone resemble
very closely the cells which have been exposed to the hormone in vitro for
only 30 min. Further, the mean diameter in microns of 100 cells from control
TABLE I
The proportion of pericardial cells exhibiting argentaffin granules from cockroaches injected with Ringer or homogenates of corpora cardiaca
Ringer
Staining batch
I
2
3
4
5
6
7
8
Hormone
/o positive
total cells
0/
/o positive
12
58
70
3O
15
29
124
117
118
119
162
40
119
100
total cells
3°
35
23
04
07
43
02
112
42
28
39
68
77
61
26
67
40
70
animals was 3O-8±S"3 as opposed to 35-4^4-6 for 100 cells from cockroaches
treated with hormone. This difference is significant at the 1 % level.
Histochemical changes. While no exhaustive study of the histochemical
reactions has been carried out, certain characteristics of the pericardial cells
are of interest. The active principle secreted by these cells is thought to be
an o-dihydroxyindolalkylamine, a relative of serotonin (Davey, 1961&). It is
therefore not surprising that the active cells are positive to Schmorl's reaction,
nor that they exhibit argentaffin granules.
The proportion of cells exhibiting argentaffin granules is greater in animals
exposed to the hormone from the corpus cardiacum. Table 1 shows the results of an experiment in which the proportion of argentaffin cells in control
animals is compared with that of cells from animals injected with hormone.
In order to reduce the variation which might arise from differences in the
staining procedure, the following protocol was observed. Decapitated animals
were injected with either Ringer or a homogenate of corpora cardiaca, and
their pericardial cells were removed and fixed in neutral formalin solution
90 min later. Paraffin sections were prepared, and the slides from a cockroach injected with Ringer were run through the staining procedure (Pearse,
i960) along with comparable slides from an animal injected with hormone.
In this way, the two animals comprising one staining batch could be
356
Davey—Pericardial cells and corpus cardiacum of Periplaneta
compared, but comparisons among staining batches might not be valid. Any
cell containing less than 10 argentaffin granules was considered to be negative.
In all but one case the percentage of argentaffin cells in the animals injected
with hormone was about double that in the controls. The argentaffin reaction
was abolished if the cells were fixed in dichromate or if cells fixed in formaldehyde solution were treated in 2% dichromate. The cells showed no sign of
a positive chromaffin reaction.
Discussion
It is clear from the foregoing description that pericardial cells undergo
secretory changes when they are exposed to homogenates of the corpora
cardiaca. The events described are interpreted in the following way. The
large secretory droplets are apparently products of the nucleus, because they
appear first near the nucleus, which shows evidence of secretory activity in
the many branches it sends into the cytoplasm. At the same time the cells
become considerably larger. The large vacuoles produced by the nucleus
presumably discharge their contents at the periphery of the cell, thereby
accounting for the progressive accumulation of the small vacuoles in this
area. Eventually the cells become smaller than those not exposed to the
hormone, and after 2 h they show signs of cessation of secretion. It is worth
noting that the pericardial cells become exhausted after about this length of
time (Davey, 1961a). On the other hand, the pericardial cells may not be so
easily exhausted during life, for judging by their large size they are secreting
go min after injection of the hormone. The physiological evidence supports
this supposition (Davey, 1961a). This is possibly a reflection of the greater
availability within the animal of the substrates necessary to maintain secretion.
While it is tempting to draw a parallel between the pericardial cells, which
produce an indolalkylamine, and the serotonin-producing cells to be found in
other animals, such a comparison should await a more extensive description of
the histochemistry of the pericardial cells. The fact that the secreting pericardial cells become more markedly argentaffin is, of course, simply a reflection of reducing substances in the cells. This is confirmed by their ability
to reduce ferricyanide in Schmorl's reaction. While this increased reducing
power may eventually help to throw some light on the reactions which produce the active amine from an inert precursor, it is of more immediate interest
in that it can assist in locating similar cells which must be present elsewhere.
The hormone from the corpus cardiacum also stimulates the contraction of
the muscles of the gut and Malpighian tubes (Cameron, 1953). Since the
hormone appears to be pharmacologically active only by virtue of its ability
to promote the secretion of active substances in or near the target organ, it
would be reasonable to suppose that cells in the gut or Malpighian tubes might
be argentaffin after treatment with the hormone. Preliminary studies on the
hind gut have indicated that certain cells of the epithelium of the hind gut
become strongly argentaffin after exposure to homogenates of the corpus
cardiacum.
Davey—Pericardial cells and corpus cardiacum of Periplaneta
357
Thus the 'myotropic hormone' of the corpus cardiacum first described by
Cameron (1953) is revealed as being a metabolic hormone which stimulates
cells other than muscles to produce a pharmacological agent. It may well be
that the primary target within the cell is the nucleus; certainly the secretion
which the hormone initiates seems to have its origin in the nucleus.
It is a pleasure to record my gratitude to Professor V. B. Wigglesworth for
his unfailing interest and enthusiasm. The author is a Research Fellow of
Gonville and Caius College, Cambridge.
References
CAMERON, M. L., 1953. 'Secretion of an o-diphenol in the corpus cardiacum of insects.'
Nature, 172, 349.
DAVEY, K. G., 1961a. 'The mode of action of the heart accelerating factor from the corpus
cardiacum of insects.' Gen. Comp. Endocrinol., I, 24.
19616. 'Substances controlling the rate of beating of the heart of Periplaneta.' In
preparation.
PEARSE, A. G. E., i960. Histochemistry. London (Churchill).
WIGGLESWORTH, V. B., 1953. The principles of insect physiology. London (Methuen).
1957- 'The use of osmium in the fixation and staining of tissues.' Proc. roy. Soc. B,
147, 185.