Download The Muscle Cells of the Follicle of the Ovotestis in Aplysia as the

\ M . ZOOLOGIST, 12:521-523 (1972).
The Muscle Cells of the Follicle of the Ovotestis in Aplysia
as the Probable Target Organ for Bag Cell Extract
RICHARD E. COGGKSHALL
Marine Biomedical Institute, Galveston, Texas 77550
SYNOPSIS. It has recently been demonstrated that activity o£ the bag cells, an identified
group of neurosecretory neurons in Aplysia, releases a substance that causes egg laying.
A cytologic analysis of the ovotestis, before and after administration of the bag cell
extract, suggests that one function ot the bag cell substance is to cause the small
muscle cells that surround each follicle of the ovotestis to contract, thus expelling
ripe oocytes from the ovotestis and beginning them on their journey through the
oviduct.
The nervous system of the marine gastropod, Aplysia, is relatively simple, easily
accessible, and contains many large neurons. For these reasons, many neurons in
this animal have been identified and individually studied (e.g., Arvanataki ano
Chalazonitis, 1958; Frazier et al., 1967;
Strumwasser, 1965; Tauc, 1966). Of particular interest for the present discussion is
an identified group of neurosecretory cells,
the bag cells, which were first described in
1967 by Frazier et' al. These cells are located in two clusters: at' the junctions of
(1) the right and (2) the left pleuroabdominal connectives with the abdominal
ganglion. Each bag cell sends out several
processes ("axons") some of which pass
directly into the fibrous sheath that surrounds the ganglion and the others wrap
around the axons in the connective before
turning to enter the sheath (Frazier et' al.,
1967). The processes end in the sheath
without innervating effector cells or other
neurons and are directly exposed to blood
which circulates throughout the interstices
of the sheath (Coggeshall, 1967). Granules
take shape in the Golgi complexes located
in the perikarya of the bag cells, and the
granules are found in great numbers in the
processes of these cells (Coggeshall, 1967;
Frazier et al., 1967). The granule-filled
neural processes that end blindly in a vascularized sheath presumably define a neuThis work was supported by grants XBO771I,
XB08109, XS 10161, and GM31574 from the National Institutes of Health.
rohaemal organ (Knowles and Carlisle,
1956).
The first clue to the function of the bag
cells came when Kupfermann (1967)
demonstrated that a crude extract of the
bag cells, upon injection into a second animal, caused the second animal to lay eggs.
Strumwasser et al. (1969) confirmed and
extended these observations, and in a parallel study, Toevs and Brackenbury (1969)
made progress in elucidating the active
compounds in bag cell extract. Kupfermann (1970; see also 1972) then made the
important' observation that if bag cells are
caused to fire in 1 ml of sea water
and then the fluid is collected and injected into a second animal, the second animal
lays eggs. From these morphological and
physiological findings, it is reasonable to
assume that active bag cells release a substance or substances into the circulation
and that this substance sets in motion a
chain of events that results in the deposition of an egg cordon.
A further step in the analysis of the
neuroendocrine control of egg laying in
Aplysia would be to gain at least a partial
understanding of the cytologic events that
underlie the deposition of the egg cordon
after injection of bag cell extract. Thus,
this study is an analysis of the normal maturation of the oocyte in the ovotestis followed by a description of the changes in
the ovotestis caused by injection of bag cell
extract.
The ovotestis in Aplysia is a follicular
521
522
RICHARD E. COGGESHALL
organ that produces both male and female
gametes (Eales, 1921). Each follicle opens
into a division of the ciliated small hermaphroditic duct. The oocytes develop in the
wall of each follicle and they can be
placed into one of five developmental
stages (Coggeshall, 1970). The exact cytology of the oocytes in each stage is not
relevant for the present discussion (the
stages are described in detail in Coggeshall, 1970). It is only necessary to understand that stage I oocytes are the youngest and smallest that can be clearly recognized, stage II and III oocytes are intermediate, and stage IV oocyt'es are regarded as
ripe or mature because they resemble the
oocytes found in the naturally deposited
egg cordons. Stage V oocytes are postmature and degenerating. All stages of
oocytes can be found in any ovotestis but
in the winter months the stage V oocytes
predominate and in summer months (the
egg laying season) stages I-IV predominate.
Oocytes in all stages of development are
surrounded by follicle (nurse) cells (Coggeshall, 1970). The intercellular gap between immature oocytes (Stages I-III) and
follicle cells is approximately 150 A and
occasional junctional complexes between
these two cell types can be seen. Thus, the
immature oocytes are closely bound to the
follicle wall. By contrast, t'he stage IV
oocyte is separated from the surrounding
follicle cell processes by a much wider distance of 1000-2000 A except for a few
places where the junctional specializations
maintain the 150 A interspace. The final
step, therefore, in the maturation of the
oocyte in Aplysia seems to be that the
oocyte separates from the follicle cells
(and the follicle wall) except for a few
places where simple junctional specializations hold follicle cells and oocytes together.
Another important feature of the structure of the ovotestis is that each follicle is
surrounded by t'he processes of small muscle cells (Thompson and Bebbington
19(")9; Coggeshall, 1970). These processes
are only 0.1-0.2 microns in diameter so
they are difficult to see with the light microscope, but electron microscopic examination demonstrates that1 they contain myofilaments similar to those in body wall
muscle (Coggeshall, 1970). From the disposition of the processes of these small
muscle cells, it is obvious that contraction
would reduce the volume of the follicle,
thus presumably shearing off the mature
oocytes and expelling loose follicle contents into the ciliated small hermaphroditic duct.
No nerve terminals have ever been seen
on the follicle muscle cells. This finding
contrasts with the ease with which nerve
endings can be found on other muscle
cells, for example, body wall or gut muscle, in this animal (Coggeshall, 1970).
It should be emphasized at this point
that the bag cell processes do not directly
innervate any structures in the ovotestis
(Frazier et al., 1967; Coggeshall, 1970).
Thus, the action of the bag cells cannot be
mediated by direct synaptic activation of
either the small muscle cells or of any
other cellular constituents of a follicle in
the ovotestis.
The appearance of the normal ovotestis,
as described above, can be compared with
the appearance of the ovotestis after bag
cell extract has been administered to an
animal. The principal cytologic change in
the treated animal is the absence of mature stage IV oocytes (Coggeshall, 1970).
There are no obvious cytologic changes in
follicle cells or immature oocytes. Another
point is the fact that many oocytes are
released from the ovotestis in less than one
minute after injection, and the released
oocytes can be seen in the dissecting microscope as they are transported down the
ciliated small hermaphroditic duct (Coggeshall, 1970). This short time interval is
much less than the 1-2 hours that elapse
from the time of injection until the appearance of the egg cordon. However, most
of the time from injection to appearance
of the cordon is spent as the oocytes pass
through the accessory mass and are placed
Aplysia OVOTESTIS AND BAG CELL EXTRACT
into capsules in the egg cordon (Coggeshall, 1972).
From the above findings, it would seem
reasonable to hypothesize that the small
muscle cells which surround each follicle
in the ovotestis are the target cells for the
action of the bag cell extract. The evidence for this suggestion is that (1) these
muscle cells are the obvious effector cells
in the ovotestis, (2) a direct neural innervation of either the muscle cells or any
other cells in the ovotestis has not been
seen, (3) the time course of the liberation
of the oocytes is so short (less than one
minute) that any release mechanism except muscular contraction is unlikely, (4)
there is no cytologic evidence for any process which might expel ripe oocytes except
for muscular contraction, and (5) the
mature oocytes are attached to the follicle
wall only by small simple junctions making them the most' likely cells to be expelled by muscular contraction. Thus, at
the present time, I believe that the bag
cells contain a substance or substances that
are released into the circulation of the
animal when the cells are active. This substance (s) is presumably transported to the
ovotestis and causes the small muscle cells
that surround each follicle to contract.
The contraction reduces the size of each
follicle, thus shearing off the ripe oocytes
from the follicle wall where they are held
to follicle cells only by a few simple junctions. The released oocytes are propelled
by the same contraction of follicle muscle
cells into the ciliated small hermaphroditic
duct and the cilia pass the oocytes
through the oviduct and accessory mass
where the oocytes are fertilized and placed
in the egg cordon. If this schema is correct, then the bag cell control of egg laying
in Aplysia has a remarkable similarity to
the posterior pituitary control of milk ejection in the mammalian breast.
523
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