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\ 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 REFERENCES Arvanataki, A., and N. Chalazonitis. 1958. Configurations modales de l'activite, propres a differems neurons d'un meme centre. J. Physiol. (Paris) 50:122-125. Coggeshall, R. E. 1967. A light and electron microscope study of the abdominal ganglion of Aplysia californica. J. Neurophysiol. 30:1263-1287. Coggeshall, R. E. 1970. 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Studies on the neurosecretory control of egg laying in Aplysia. Amer. Zool. 12:513-519. Strumwasser, F. 1965. The demonstration and manipulation of a circadian rhythm in a single neuron, p. 442-462. In J. Aschoff [ed.], Circadian clocks. North Holland Publishing Co., Amsterdam. Strumwasser, F., J. W. Jacklet, and R. B. Alvarez. 1969. A seasonal rhythm in the neural extract induction of behavioral egg-laying in Aplysia. Comp. Biochem. Physiol. 29:197-206. Tauc, L. 1966. The activity of the mollusc neuron. Endeavor 25:39-44. Thompson, J. E., and A. Bebbington. 1969. Structure and function of the reproductive organs of three species of Aplysia (Gastropoda: Opisthobranchia). Malacologia 7:347-380. Toevs, L. A., and R. W. Brackenbury. 1969. Bag cell-specific proteins and humoral control of egg laying in Aplysia californica. Comp. Biochem. Physiol. 29:207-216.