(Presented By Academician V.E.Sokolov 13 VII 1989)

UDK 611.86+812.86 MORPHOLOGY

Translated from Russian and published per authors´ request.
Source:"Doklady Akademii Nauk SSSR ,  1990.  Vol., 311   3"


Considerable role in the organization of animal behavior belongs to external metabolites that could carry meaningful message and perform chemo-communicative function. Special importance is gained by that chemical channel during the period of reproduction, while considerable load falls on oltactic analyzer and animals display high sensitivity to sexual pheromones /1/. It was supposed that olfactic sensitivity could be dependent on the degree of reproductive maturity of an animal /2/. Attempts to ascertain morphological restructuring in animal olfactic epithelium at different stages of their reproductive cycle and establish correlation between the structure of receptor neuron, its sensitivity, and implementation of adaptive behavior did not yield unambiguous results as yet /3, 4/.

In order to solve this important neurobiological problem, a necessity of selection of the most appropriate target spe-cies arose. In our opinion, studies of the ultra-structure of sensitive neurons of oltactic lining during different stages of the life cycle of an animal from that standpoint would serve the purpose of isolation of the structural foundation for changes in sensitivity of olfactic cells. We employed a natural model of olfaction participating in the formation of different phases of reproductive behavior in fishes. Earlier, behavioral experiments demonstrated that yellow-fin go-bies (Cottocomephorus grewingki, Dyb.), endemic to Lake Baikal, had increased sensitivity to sexual pherornones during the period of ripening and spawning of reproductive products. During the phase of parental behavior (forma-tion of set providing its viability ) such sensitivity was not observed /5/

For the investigation, male fishes at certain stages of their reproductive behavior were sampled, those who were easy to observe at the locations of their natural breeding bottoms, For morphological analysis, olfactic epithelium was sampled during spawning period (selection of nest, attraction of females, spawning of reproductive products), and during the phase of males switching to the protection of fecundated eggs. Twelve samples from each series were studied. Olfactic lining, fixed by standard procedure, was examined by means of optical and electron (scanning and transmission) microscopy.

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Olfactic receptor cells of yellow-fin goby were represented by flagellate and microvillar types, with quantitative pre-dominance of the former ( Fig. 1a; Fig. 2a, b). (Hover mouse to enlarge the picture.)

olfactic receptor cell

Figure I. Schematic representation of olfactic receptor cell at different phases of reproductive cycle of yellow-fin goby: a - interspawning period, b - active spawning phase, c - end of spawning season, d - phase of transition towards protection of nest; 1 - flagella; 2 - basal bodies; 3- microtubules; 4 - secretor vesicles.

Apex of flagellate cells was furnished with flagella the number of which varied between 5 and 8. Apical section of microvillar cells contained short ( 1-3 mkm), digitinform (finger-shaped) appendages without basal bodies and microtubular apparatus. In the pericarion of sensitive cells during in-terspawning period, sparse ribosomes were localized between arranged channels of reticule, insignificant part of which was assembled into polyribosomic complexes.

After the animals turned to spawning, there occurred a sharp increase in the number of ribosomes, both free and as-sociated into rosettes, in the cytoplasm of males. Regular pattern was observed in spatial organization of micro-tubules: in the upper sections of the cell body they formed parallel bundles that continued throughout the length of peripheral appendage, terminating near basal bodies of the clava. Indications of intensive nuclear-cytoplasmic inter-relations occurred. Hence, nuclear membrane had increased density of pores, through which an egress of granular component of nucleolus, tightly drawn together with nuclear membrane could occasionally be detected. Mitochon-dria with well-developed crests (ridges) were commonly located next to the pores (Fig. 1b). It could be supposed that the mentioned ultra-structural rearrangements reflected increased functional properties of olfactic cells.

During the phase of animals switching to parental behavior (protection of nest), the channels endoplasmic reticule would fragmentize in receptor cells. As this was taking place, cisternal cavity of those fragments usually looked ex-panded, as a result of which they developed a vacuole-like appearance. Cytoplasm preserved high density of polv-ribosomes (Fig. 1c). An important structural peculiarity of cell during that phase was the presence of unusually ex-panded peripheral appendage. Its diameter would exceed the dimensions characteristic of it during inter-spawning period by 1.5-2 times. The increase of diameter of peripheral appendage was observed throughout its length exclud-ing, as a rule, the zone of close contact with key cells located proximally from olfactic cilia. Hand in hand with mito-chondria and elements of smooth endoplasmic reticule, a large number of vesicles with light-colored content was contained throughout the length of peripheral appendage. Diameter of those vesicles varied between 0,1 and 0.5 mkm. Those vesicles were found in the body of receptor cells - close to a well developed Golgi apparatus. Distinct system of microtubules apparently contributed to their transportation from the origin into apical sections of the cell Fig. 2c.(Hover mouse to enlarge the picture.)

surface of olfactic epithelium

Figure 2. a - surface of olfactic epithelium (scanning electron microscopy) among the apexes of key cells of ciliated (CC) and secretor (SC) types, apical sections of receptor cells (RC) with flagella are visible, 3000 X; b- distal section of flagellated receptor cell, 15 000 X; c - apical section of receptor cell; flagella are absent, microtubules and secre-tor vesicles are visible. 15 000 X; d - apical section of receptor cell during the phase of transition to protection of nest and set; flagella are absent, a large number of vesicles is visible above basal bodies. b, c, d- basal bodies are marked with pointers 15 0000 X.

It was demonstrated in a series of ultra-thin mounts that olfactic cells, where intensive synthesis of vesicles and their transport through peripheral appendage occuÍred, had apex without flagella and microvilli. Scanning mi-croscopy confirmed that supposition. It is important to note that, during that period, special microtubes were defined in the matrix of olfactic apex that had distribution characteristic of flagella ((9 x 2) + 2). Such complexes of micro-tubes did not have common outer membrane, but each of them was associated with its basal body. During that pe-riod, the number of the newly-formed light-colored vesicles was not the same in different receptor cells. Thus, pe-ripheral appendage and the apex of 20% of cells were full of vesicles to such degree that they could be identified as secretor ones (Fig. 1d; Fig. 2d). Electron microscopy examination revealed that the vesicles were incorporated into surface membrane of matrix in order of their arrival, and, possibly, could discharge their content into olfactic mu-cus.As the vesicles accumulated, the shape of the apex changed. It grew in volume, while stretching in length, some-times exceeding is regular size 2 to 4 times. Along with that, it would often change its vertical orientation, bend to a position parallel to the plane of epithelium. Such cell preserved basal bodies of flagella at their original location, where they turned to be covered by large accumulation of secretor vesicles in the upper zones of apex. By morpho-logical features altered clava was strongly reminiscent of the forward section of growing axon - the cone of growth.

Thus, already by the end of spawning season, sensitive cells started losing flagella and microvilli, where, as claimed, receptor components were localized. The volume olfactic apex itself would increase due to incorporation of mem-brane material of vesicles into its apical section, probably displacing receptor zones located there earlier. Due of that, the specialized olfactic cell would apparently lose its ability for chemoreception.

Our data demonstrated that ultra-structural rearrangement in sensitive cells were directed at both activation of those cells for specific receptive function during sexual interaction of partners and emergency disengagement of that func-tion, which is necessary during the phase of males switching to the formation of fecundated set and protection of developing eggs.

The performed research demonstrated that the level of structural development of cells of olfaclic epithelium in fishes depended on the phase of their life cycle. In particular, during the spawning period, structural rearrangements would occur in receptor cells, directed at both the increase of preparedness of those cells for reception of sexual phero-mones, and further active changes terminating that peiod and needed to provide for the next entirely new pattern of reproductive behavior. Consequentlv, both the mechanisms of the central nervous system, and restructuring of pe-ripheral chemo-sensitive apparatus play considerable role in the change of phases of complex spawning behavior.