Ultrastructure of the optic gland of the squid Sepiotheutis sepioidea (Cephalopoda: Loliginidae).
The role of the 0G in the cephalopod life cycle is analogous to that of the hypophysis which controls sexual development in vertebrates (Wells & Wells, 1959, 1969, Wells 1960 and Wells et al. 1975). These glands also regulate protein synthesis in the ovaries, stimulate gonadal mitosis, mating behavior, feeding and longevity (Durchon & Richard 1967, OTIor & Wells 1973, Wells et al. 1975, Wodinsky 1977 and Mangold 1987). The OG hormone is species specific, not sex specific (Wells & Wells 1975), and glandular secretions have periodic oscillations associated with temperature and photoperiod (Defretin & Richard 1967 and Richard 1966, 1967 a, b, 1970). The glands are controlled by a reactive peptidergic inervation (Le Gall et al. 1988), and their activity varies during the life cycle, associated with the multiplication of reproductive cells (Koueta et al. 1995).
The fine structure of the gland suggests that it has several functions. Bjorkman (1963) suggested that the secretory cells had a high rate of synthesis. The presence of abundant mitochondriae implies that the gland may secrete a steroid hormone (Froesch 1979). In S. officinalis, however a peptidic like hormone was shown to be present in the OG and hemolymph (Koueta et al. 1992). The lipofuchsin granules have been related to the resorption of metabolic residues inside the secretory cells (Froesch & Mangold 1976, Mangold & Froesch 1977 and Froesch et al. 1987).
In this study the fine ultrastructure of the tropical squid Sepiotheutis sepioidea (Blainville, 1823) (Cephalopoda: Loliginidae) was examined in both sexes at different sexual maturity stages, using Transmission Electron Microscope (TEM) in order to compare secretory cell changes during the life cycle of this specie.
MATERIALS AND METHODS
206 individuals of S. sepioidea were caught (net mesh size 20 mm [empty set]) in Mochima National Park, Venezuela (10[degrees]20'22"-10[degrees]24'N; 64[degrees] 19'-64[degrees]22'W). They were acclimatized in tanks (1 500 L) with running sea water, and fed ad lib. for at least a week (Estacion Jose G. Hernandez, IDEA-Fundaciencias). Each specimen was weighed (body weight BW [+ or -] 0.05 g) and the dorsal mantle length (DML [+ or -] 0.1 cm) was measured before decapitation, followed by dissection of the OG, with the aid of a stereomicroscope. The OG was preserved by immersion as quoted below for light microscopy (LM) and TEM.
The mantle with the reproductive organs was preserved with 4% formalin (24 h), and then washed in running tap water and the reproductive organs were removed and measured ([+ or -] 0.05 cm; [+ or -] 0.0001 g). The sexual maturity stage was determined for each specimen as follows: juvenile (1), immature (11), in maturation (ID), mature (IV), and post-reproductive (V). This last was identified only in copulating males, and post-reproductive females were not captured. The sexual maturity stages were determined using the measurements of reproductive organs quoted for this species (Robaina & Voglar 1986, Voglar & Robaina 1987), and the female sexual maturity stages were estimated using the nidamental gland index (NGI = (Nidamental Gland length/Dorsal Mantle length)) (Durward et al. 1979). Gonadal index (GI) was estimated as the relationship between gonad weight and body weight, in the females the gonadal index was calculated using the formula (GI = (ovary weight/body weight) x 100), and in the males this was calculated as (GI = (testicule weight/body weight) x 100). An analysis of variance (ANOVA) was used to test for significant differences in the biometric parameters between the sexual maturity stages (females) and the dorsal mantle length intervals (males), these data were compared using an a posteriori Scheffe test (Sokal & Rohlf 1981).
For LM, samples including the optic lobe and the optic stalk were immersed in Gilson fixative (2-6 h), and then well washed in running tap water, dehydrated and embedded in paraffin. Paraffin sections (6 [micro]m) were stained with Hematoxilyn-Eosin (HE), Periodic acid Schift reaction (PAS) and Herlant tetrachromic stain (Humason 1979). LM sections were analyzed using an image analysis system (Javelin Smart Cam) adapted to a light microscope, with a digital micrometer.
For TEM, the brain was prefixed by immersion in either of the following fixatives (30 min.) with some drops of 0.1% (w/v)' methylene blue (vital stain)(Bern, H., pers. com.): 2.5% glutaraldehyde (GA) in O.lM phosphate buffer saline pH 7.8; 2.5 % GA in O.lM sodium cacodylate buffer pH 7.8; 3% GA, 20% sucrose (w/v), 0.5% Ca[Cl.sub.2], 1 % [K.sub.2][Cr.sub.2][O.sub.7], 0.1% picric acid in 0.1 M sodium cacodylate buffer (Evans at al. 1976). The brain was then washed in the same buffer, and the OG dissected from the cerebral tissue followed by one hour fixation in fresh fixative. The OG was washed, post-fixed (1% Os[O.sub.4], 90 min.), in bloc stained (2% uranyl acetate, 30 min.), and processed for conventional TEM. Thin sections were contrasted with uranyl acetate and lead citrate (Reynolds 1963), and observed in a H-600 TEM.
The OG is located over the optic stalk close to the optic lobe, it is covered by a fine connective layer, and has an elongate shape. The OG histology is well differentiated from the cerebral tissue, the OG cells are in a compact mass, whereas cerebral cells are larger, and form layers surrounding eosinophylic nerve fibers (Fig. 1). At the juvenile maturity stage the OG is almost undifferentiated from the cerebral tissue under a stereoscope, having a reddish coloration in individuals with a larger dorsal mantle length, mainly mature females. The female gonadal index (01) oscillated between 0.1-2.95 %, and the nidamental gland index between 0.9 -0.28 %, with significant differences between the sexual maturity stages (Table 1). The reproductive organs in the males showed a tendency to increase with dorsal mantle length, but the spermatophore number was associated with reproductive behaviour, GI oscillated between 0.22-1.38 % (Table 2). These data had significant differences (ANOVA, p < 0.001), but in an a posteriori Scheffe test the means did not differ (Table 3, 4).
Paraffin sections give a clear picture of OG organization. The secretory cells are the most numerous cells, they are ovoid with cytoplasmic processes (18.5-25.6 [micro]m), and the nucleus (9.11-11.38 [micro]m) is circular with a dense nucleoli, both showing a tendency to increase in size with the maturity stages (Table 1, 2). The blood vessels are prominently dispersed throughout the OG. Support cells are scarce, in some sections they are seen to be aggregated, with a characteristic round and basophilic nucleus (Figs. 1, 2, 3, 4).
At the ultrastructural level OG cytoarchitecture denotes a complex array of fine cell processes intermingled between the main cells, blood vessels, nerve cells and support cells (Figs. 5, 6). The main cell profile is oval with thin cytoplasmic processes, its organelles are very numerous giving a dense appearance. The nucleus is euchromatinic with scarce heterochromatin, the nucleoli have a clear pars amorpha. surrounding a darker nucleolonem (Figs. 5, 7). Ribosomes are numerous, and are found dispersed throughout the cell, associated with polysomes dispersed in the cytoplasm, rough endoplasmic reticulum, and covering clear cytoplasmic vesicles (Figs. 8, 10, 11). The rough endoplasmic reticulum cisterns are thin, and extend from the nuclear envelope to the cellular membrane (Figs. 8, 9, 19), at the secretory stage the endoplasmic reticulum cisterns are aggregated in swollen concentric saccules (Figs. 12, 13). The Golgi apparatus is active, it secretes clear and electrondense vesicles and there are at least two Golgi complexes in each cell associated with rough endoplasmic reticulum, and lipofuchsin granules. At the mature sexual stage (IV) the Golgi is very active secreting numerous electrondense granules (Fig. 12). Mitochondriae are elongated or oval with tubular cristae, they are very abundant in the 1, 11 and III stages, whilst in the IV stage there are few mitochondriae. These reductions could be associated with mitochondrial matrix desorganization and destruction by autophagic lysosornes (Figs. 8, 9, 12). Lipofuchsin granules are found from the third sexual maturitystage onwards, and are associated with the Golgi stacks, these profiles are electron dense with clear inclusions, and in the larger mantle length specimens they shown an increase in size (Figs. 7, 9, 12).
This gland is well vascularized with large blood vessels. The pericyte cells cover the endothelial cells, and have a double basal membrane. The cellular membranes are tightly joined in an irregular fashion, and there are collagen fibers in the cytoplasm. The cytoplasm of the endothelial cells is not easily identified because the lumen has an electrondense hemolyrnph which occupies nearly the entire space. Nevertheles the organelles are restricted mainly to the area close to the nucleus (Fig. 13, 14, 15).
The nerve cells are distributed between the principal cells and blood vessels in long axon bundles with terminal synaptic buttons with different kinds of neurosecretory vesicles. The axon bundles are tightly packed, they have long microtubules, and some microvesicles. The synaptic buttons are in contact with axon bundles, secretory cells and blood vessels, and there were no synaptic electron dense contacts identified. The neurosecretory vesicles were classified according to electron density and size in: small agranular (35-70 nm), agranular (90-150 nm), moderate to very dense (95 -130 nm) and dense with a smooth limiting membrane (90-170 nm) (Figs. 6, 11, 14, 16). Glial cells are intermingled between the OG cells, with long cytoplasmic processes, their cytoplasm is clear with a particulate material and few organelles, some mitochondriae, vacuoles and vesicles (Figs. 6, 11, 16).
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The morphological characteristics (mantle-length and reproductive organs) in males and females of S. sepioidea were good indicators of the sexual maturity stage, although sortie individuals reached the same maturity stage at different mantle length intervals. Robaina & Voglar (1986) and Voglar & Robaina (1987) found squids at different stages of sexual maturity with the same mantle lengths. In this study it was not possible to examine the OG of females during the spawning period or at the post-spawning stage, because their reproductive behavior shows care of eggs until hatching, this is followed by a short resting period in which the female broods most of the mature eggs and finally dies. Wodinsky (1977) quoted in Octopus humeelincki an apparently self destructive system of the OG which inhibits feeding in brooding females, preventing the predatory female from eating the eggs, and thus ensuring juvenile survival.
The OG shows a highly vascularized tissue and fine structure sections from S. Sepioidea were improved by modifying some combinations of fixatives and schedules. The fixer quoted by Evans et al. (1976) followed by extensive washings gave the best tissue preservation, while the aldehyde prepared in phosphate buffer saline with filtered sea water resulted in a bad preservation. Problems were also reported with O. vulgaris and S. officinalis OG fine structure by Defretin & Richard (1967) and Nishioka et al. (1970). In S. Sepioidea a hematonervous barrier built by the cellular wall, and gap junctions (zonula ocludens) between the endothelial cell and the perycite may halt the difussion of the fixative in the squid giant axon (Villegas & Villegas 1968). Villegas (pers. com.) obtained the best preservation by immersion instead of perfusion, this latter being the suggested procedure for delicate tissues.
The fine structure of the tropical squid S. sepioidea OG, showed the same cellular types described in other cephalopods, with some distinctive features. S. sepioidea secretory cells showed differences in organelle abundance between the sexual maturity stages: at the juvenile and immature stages there are numerous mitochondriae, endoplasmic reticulum cisterns are thin and dispersed in the cytoplasm, the Golgi apparatus has a curved profile and actively secretes granules of different densities, whilst in the IV maturation stage (females) and reproductive individuals (males) secretory cells are almost depleted of mitochondriae, endoplasmic reticulum cisterns are dilated and well developed in a concentric fashion, and the Golgi complex actively secretes electron dense granules which are dispersed in the cytoplasm. Some of these are close to the lipofuchsin granules and to the cellular membrane, suggesting a possible secretory mechanism through the blood vessels.
S. sepioidea OG secretory activity was demonstrated by the production of electron dense secretory granules in the sexually mature stage (Figs. 12, 13). However, the presence of clear vesicles, some of them associated with polysomes, could indicate the existence of other secretory processes in the life cycle, as seen by the extrusion of a clear vesicle close to a synaptic button (Fig. 11). Moreover, the cellular characteristics indicate active cells, with euchromatinic nuclei, an abundance of organelles, and an increase in lipofuchsin volume and cell size with dorsal mantle length.
Defretin & Richard (1967) demonstrated that when S. officinalis is kept in a dark photoperiod the OG secretory cells produce three kinds of secretory granules, suggesting that they are filled with the same product in different stages of synthesis. Octopus vulgaris OG enlargment occurred both in the nucleus and secretory cell cytoplasm after cerebral surgery (Wells 1964). Eledone cirrhosa OG enlargement was statisticaly associated with female gonad maturation, and it is suspected that this process occurs normally (Boyle & Thorpe 1984). Koueta et al. (1995) described cellular changes in S. officinalis OG histology during sexual maturation; and found that main, cell secretory activity was correlated with a peak of mitogenic activity in both sexes.
Lipofuchsin granules in the OG of some octopods are numerous, and measured up to several micrometers in diameter. Mangold & Froesch (1977) did not find a correlation between the size of lipofuchsin granules and sexual development in females. Lipofuchsin granules are implicated in the defense mechanism of O. vulgaris in the presence of high molecular weight molecules in the blood stream (Froesch & Mangold 1976, Froesch et al. 1978 and Froesch 1979). In S. sepiodea a lipofuchsin granule engulfing a degenerated mitochondriae was observed.
The presence of different types of neurosecretory microvesicles in the axon bundles and synaptic buttons in the OG of S. sepioidea indicates the existence of different stimuli controlling OG activity. These stimuli were reported as excitatory or inhibitory (Nishioka et al. 1966 and Froesch 1974). In the stellate ganglion of S. sepioidea the neurosecretory microvesicles are small and agranular (50-80 nm) (Castejon & Villegas 1964), in the stellate ganglion of Loligo vulgaris there are both small agranular vesicles (40-60 nm) and large agranular vesicles (average 65 nm) (Gervasio et al. 1971). In the OG of Loligo opalescens the more common granular vesicles have a looseJimiting membrane (80-160 nm), and a few axon-like processes containing dense vesicles (100-300 nm) with a smooth limiting membrane (Nishioka et al. 1970).
Field populations of S. sepioidea have a steady increase of the OG secretory cell diameter in both sexes. Sexual maturation in octopus was associated with a gross enlargment of the OG and a change in colour from cream to bright orange after optic nerve surgery (Wells & Wells 1959). In S. officinalis growth and sexual development was stimuled by a dark photoperiod and high temperature (Richard 1966, 1967 a, b, Defretin & Richard 1967). Likewise O. vulgaris mature individuals showed more intense histological reactions, and an increase in size in cytoplasm, nucleus and granules in the principal cells (Bonichon 1967). Eledone cirrhosa OG enlargment was statistically associated with the weight of the ovary and oviducal glands (Boyle & Thorpe 1984). Lipofuchsin granules and octopus body weight were well correlated, but the secretory mechanism was not demonstrated (Mangold & Froesch 1977).
I thank "Estacion Experimental Jose Gregorio Hernandez, Fundaciencias--IDEA", Jesus Rodriguez, Susan Tai, Gilma Hernandez de A., Frances Osborn and Osmar Nusetti.
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Instituto de Investigaciones en Biomedicina y Ciencias Aplicadas, Universidad de Oriente. Cumana, Sucre, Venezuela. Fax 58-93-514023. Tel. 58-93-514185. E-mail: firstname.lastname@example.org
Received. 9-II-1998. Corrected. 07-IV-1999 Accepted. 16-IV-1999.
TABLE 1 Biometric parameters of the female reproductive organs in the different sexual maturity stages (average [+ or -] standard deviation; N = sample size). The reproductive organs show a tendency for linear increase in size between the sexual maturity stages, including the diameter of the nucleus. Sample size is indicated below the mean and standard deviation. Sexual Maturity Stage I Dorsal Mantle length 71.43 [+ or -] 16.24 N=35 Body weight 33.90 [+ or -] 18.87 N=36 Ovary length 7.41 [+ or -] 3.02 N=31 Ovary weight 0.03 [+ or -] 0.02 N=34 Oviducal Gland length 11.59 [+ or -] 3.78 N=30 Nidamental Gland length 7.59 [+ or -] 1.29 N=22 Nidamental Gland Index % 0.09 [+ or -] 0.02 N=22 Gonadal Index % 0.07 [+ or -] 0.03 N=34 Nucleus diameter [micro]m 9.11 [+ or -] 1.11 Sexual Maturity Stage II Dorsal Mantle length 106.27 [+ or -] 12.07 N = 34 Body weight 84.73 [+ or -] 24.98 N=35 Ovary length 11.80 [+ or -] 3.69 N = 34 Ovary weight 0.09 [+ or -] 0.04 N=35 Oviducal Gland length 18.21 [+ or -] 4.88 N=33 Nidamental Gland length 11.99 [+ or -] 1.58 N=30 Nidamental Gland Index % 0.11 [+ or -] 0.014 N = 14 Gonadal Index % 0.11 [+ or -] 0.04 N=35 Nucleus diameter [micro]m 9.33 [+ or -] 1.12 Sexual Maturity Stage III Dorsal Mantle length 116.59 [+ or -] 12.07 N = 15 Body weight 115.95 [+ or -] 48.03 N=15 Ovary length 16.10 [+ or -] 5.18 N=15 Ovary weight 0.35 [+ or -] 0.30 N=15 Oviducal Gland length 24.63 [+ or -] 7.07 N =15 Nidamental Gland length 19.01 [+ or -] 3.09 N=13 Nidamental Gland Index % 0.16 [+ or -] 0.04 N=13 Gonadal Index % 0.34 [+ or -] 0.37 N=15 Nucleus diameter [micro]m 9.74 [+ or -] 1.50 Sexual Maturity Stage IV Dorsal Mantle length 135.80 [+ or -] 13.62 N= 17 Body weight 166.54 [+ or -] 43.37 N=17 Ovary length 36.01 [+ or -] 6.38 N=16 Ovary weight 4.06 [+ or -] 2.83 N=17 Oviducal Gland length 45.91 [+ or -] 6.61 N=14 Nidamental Gland length 37.74 [+ or -] 4.37 N = 17 Nidamental Gland Index % 0.28 [+ or -] 0.037 N=17 Gonadal Index % 2.95 [+ or -] 2.35 N=17 Nucleus diameter [micro]m 11.38 [+ or -] 1.24 TABLE 2 Biometric parameters of the male reproductive organs in the different sexual maturity stages. The individuals were grouped in mantle length intervals. The nucleus diameter was averaged at the fourth maturity stage. The oscillation of the espermatophores at the fourth maturity stage is associated with copulation. Sample size is indicated below the mean and standard deviation. Sexual Maturity II III Stage Mantle length 40-59 60 -79 interval Dorsal Mantle 53.14 [+ or -] 3.88 71.27 [+ or -] 5.66 length N=4 N=22 Body weight 16.13 [+ or -] 2.09 30.77 [+ or -] 6.11 N=4 N=29 Testicule 5.50 [+ or -] 1.96 11,53 [+ or -] 4.08 length N=4 N=23 Espermatophoric 4.42 [+ or -] 0.95 7.85 [+ or -] 2.46 Organ length N=3 N=24 Espermatophores -- 43 [+ or -] 50 N = 13 min.-max. 2 - 152 Espermatophore -- 4.78 [+ or -] 1.91 length N=6 Gonadal index % 0.22 [+ or -] 0.25 0.78 [+ or -] 0.63 N=3 N=23 Nucleus 9.25 [+ or -] 1.50 10.24 [+ or -] 1.19 diameter [micro]m Sexual Maturity IV IV Stage Mantle length 80-99 100-119 interval Dorsal Mantle 92.77 [+ or -] 4.78 11 0.48 [+ or -] 5.58 length N=29 N=29 Body weight 62.19 [+ or -] 13.98 89.56 [+ or -] 11.40 N=29 N=29 Testicule 20.85 [+ or -] 3.73 23.05 [+ or -] 2.35 length N=26 N=29 Espermatophoric 12.44 [+ or -] 1.66 13.35 [+ or -] 1.82 Organ length N=26 N=29 Espermatophores 143 [+ or -] 73 212 [+ or -] 87 N=24 N=28 min.-max. 40 - 279 47 - 367 Espermatophore 8.91 [+ or -] 1.06 10.46 [+ or -] 0.66 length N=23 N=29 Gonadal index % 1.53 [+ or -] 0.47 1.42 [+ or -] 0.24 N = 26 N=28 Nucleus 10.93 [+ or -] 1.12 -- diameter [micro]m Sexual Maturity IV IV Stage Mantle length 120-139 140-159 interval Dorsal Mantle 127.84 [+ or -] 4.62 145.67 [+ or -] 7.20 length N=15 N=5 Body weight 125.72 [+ or -] 14.31 176.50 [+ or -] 23.91 N=15 N=5 Testicule 27.89 [+ or -] 2.81 31.13 [+ or -] 6.28 length N=12 N=4 Espermatophoric 15.44 [+ or -] 2.54 18.65 [+ or -] 1.80 Organ length N=13 N=5 Espermatophores 223 [+ or -] 92 203 [+ or -] 44 N=13 N=5 min.-max. 94 - 376 155 - 264 Espermatophore 11.06 [+ or -] 0.39 12.37 [+ or -] 0.48 length N=13 N=5 Gonadal index % 1.46 [+ or -] 0.28 1.38 [+ or -] 0.18 N=12 N=5 Nucleus -- -- diameter [micro]m TABLE 3 Analysis of variance (ANOVA) and a posteriori Scheffe test of female morphometric parameters at the different sexual maturity stages. Variable ANOVA Fs Degrees of Freedom Dorsal Mantle length *** 91.729 3/97 Body weight *** 76.052 3/99 Ovary length *** 165.231 3/92 Ovary weight *** 55.319 3/97 Oviducal Gland length *** 141.538 3/88 Nidamental Gland length *** 585.428 3/81 Nidamental Gland Index *** 205.903 3/79 Gonadal index *** 40.749 3/97 Variable Scheffe Means Dorsal Mantle length 9.74 * Body weight 21.03 * Ovary length 3.05 * Ovary weight 0.79 I-III; IV Oviducal Gland length 3.78 * Nidamental Gland length Nidamental Gland Index 0.02 * Gonadal index 0.99 I-III; IV *: 0:01 < p < 0.5). ***: p < 0.001 TABLE 4 Analysis of variance (ANOVA) and a posteriori Scheffe test of the males morphometric parameters at the different sexual maturity stages. Variable ANOVA Fs Degrees of Freedom Dorsal Mantle *** 397.153 5/99 length Body weight *** 220.758 5/105 Testicule length *** 68.134 5/92 Espermatophores *** 11.736 5/78 Espermatophore *** 81.11 5/71 length Gonadal index *** 12.849 5/91 Variable Scheffe Means Dorsal Mantle 4.91 * length Body weight 11.38 * Testicule length 3.22 40-59; 60-79; 80-119; 120-139; 140-159. Espermatophores 72 60-79; 80-119; 100-119. Espermatophore 0.85 60-79; 80-99; length 100-139; 140-159. Gonadal index 0.60 40-79; 80-159. *: 0.01 < p < 0.5). ***: p < 0.001
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|Publication:||Revista de Biologia Tropical|
|Date:||Dec 1, 1999|
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