Sexual maturation and embryonic development in octopus: use of energy and antioxidant defence mechanisms using Octopus mimus as a model

Sexual maturation and reproduction influence the status of a number of physiological processes and consequently the ecology and behaviour of cephalopods. Using Octopus mimus as model species, the present study examined the changes in biochemical composition that take place during gonadal maturation of octopus females and its consequences in embryo and hatchlings characteristics, including energetic metabolites, digestive enzymes and antioxidant defence mechanisms. A total of 32 Octopus mimus adult females were sampled during ovarian maturation; biochemical composition (metabolites and digestive enzymes) of digestive gland (DG) and ovaries (only metabolites) were followed during physiological and functional maturation. Levels of protein (Prot), triacyl glycerol (TG), cholesterol (Chol), glucose (Glu) and glycogen (Gly) were evaluated. The activity of alkaline and acidic enzymes also was measured in DG. Simultaneously, groups of eggs coming from mature females were sampled along development, and metabolites (Prot, TG, Glu, Gly, TG, Chol), digestive enzymes activity (Lipases, alkaline and acidic), antioxidant defence mechanisms and radical oxygen species (ROS) were evaluated. This study shows that ovarium is a site for reserve of some nutrients for reproduction. Presumably, TG where stored at the beginning of the maturation processes followed by Chol, both at the same time were energetically supported by Glu, derived from Gly following gluconeogenic pathways. Nutrients and enzymes (metabolic, digestive and REDOX system) where maintained without significant changes and in a low activity during organogenesis. Our findings suggest that activity was not energetically costly; in contrast, during the embryo growth there was mobilization of nutrients and activation of the metabolic and digestive enzymes. Increments in consumption of yolk and glycogen, and reduction in molecules associated with oxidative stress allowed paralarvae to hatch with the antioxidant defence mechanisms ready to support ROS production.

(Legendre and Legendre, 1998). Data were square root (female data) or log- dividing between the standard deviation of each variable prior to analysis 3 2 0 (Legendre and Legendre, 1998).

2 1
A permutational multiple ANOVA was applied on the distance matrices to detect 3 2 2 differences amongst female octopuses in four different stages of gonadic permutations of raw data were used to generate the empirical F and t- functional maturity stage (Fig. 1A). At the end of maturation, total and 3 3 5 eviscerated weight resulted 3.2 and 2.7 higher than observed in immature maturity stages were 108, 135 and 2.55 times higher than in immature females, 3 4 0 respectively ( Fig 1B). As a consequence, increments in RSWI and OvwI were 3 4 1 observed along the maturity stages (Fig. 1C). when oocytes were send to reproductive coelom (Fig. 2). The hepatosomatic 3 4 8 index was reduced at the end of the maturation stage. Immature O. mimus females showed digestive gland glycogen levels 59% lower 3 5 3 than females at the beginning of the maturation process ( Fig. 1A almost all maturation process (p > 0.5; Fig 3B). Only females in late functional 3 6 9 maturation process had glucose levels significantly lower than in the rest of the immature females (0.5 mg/ml) than in late functional maturation condition (p < higher than in embryos and 1.3 times higher than observed in 2 d old 3 7 7 paralarvae (p < 0.002; Fig 3B). Digestive gland protein did not change during the female maturation process (p 3 8 0 > 0.05; Fig. 3C). In contrast, an increment of soluble protein was recorded in females and high at the end of the maturation process (p < 0.001; Fig. 3C).

8 3
There were no statistical differences in protein levels of embryos of stages I to (1.6 mg/ml) (p > 0.05). In contrast, TG values were high in females at the end of 3 9 4 physiological maturation and during the early maturation process (p < 0.001; stages I, IV, VII, and X to XVIII but low in stages II and IX (p < 0.01; Fig. 4A).

9 7
Values of TG recorded in 1 and 2 d old paralarvae resulted 1.5 times higher 3 9 8 than the maximum value recorded in embryos at stage X (p < 0.01; Fig. 4A). along the maturity stages (p > 0.05; Fig 5A). In contrast, a lower activity of  hatching. It is interesting to note that LPO and GSH levels start to be reduced 4 3 2 around stage XV to maintain a low level until hatch (p < 0.001; Fig 6). that the PCo1 explained 94.9 % of total variation in the data, with EBW, BW, OVW and RSW greatly contributing to ordination in the horizontal axis (Fig. 7A). The PCo2 explained only 4.2% of the total variation, with differences in DG The multiple ANOVA showed overall significant differences between stages of 4 4 5 gonadic maturation (Table 1). However, paired comparisons amongst centroids  comparison between centroids allowed for three distinct groups to be formed 4 5 7 based on ovary metabolites: immature; physiologically and early functionally 4 5 8 mature, and late functionally mature females (Table 2).

5 9
Ovary samples from immature females were high in glucose and cholesterol,  variation increased to 79% when the third PCo was considered. Glucose was 4 6 7 inversely correlated with glycogen, triacylglicerides and cholesterol, whilst coordinates. Significant differences between stages of gonadic maturation were 4 7 0 also detected by the general multiple MANOVA (Table 1). However, paired immature females and those in late functional maturity (Table 2).

7 3
Immature females had high concentrations of glucose and both acid and The first and second PCo explained 56.7% and 20% of total variation of  stages before organogenesis and towards the 2nd paralarvae, with paralarvae 4 9 7 having higher alkaline protease but lower acid protease activity than embryos. The multiple ANOVA showed significant differences between stages of 4 9 9 development (Table 1), and significant differences were detected between all 5 0 0 pairs of centroids except those representing the 1st and 2nd paralarvae ( Table   5  0  1 2). These results show four distinct groups of samples regarding enzyme larvae showed that the first and second PCo explained 68.7% and 12% of total 5 0 7 data variation (Fig. 8C). Lipid peroxidation and carboxylesterase were high differences throughout development (Table 1), paired tests amongst centroids 5 1 3 revealed significant differences only between extreme stages (Table 2). energy for egg production directly from food rather than from stored products in between both maturation stages. This indicates that storage reserves were not 5 3 0 transferred from tissue to tissue during ovaria maturation, which is consistent 5 3 1 with our findings. Moreover, our study suggests that during the maturation 5 3 2 process there was mobilization of nutrients at the DG and ovarium that where Hence the importance of those nutrients in the physiology of cephalopods. glycogenic pathways (Hochachka & Fields 1982) ( Fig. 9),as was previously  maturation. This suggests that glucose could be used as a source of energy at 5 5 8 the beginning of the complex processes involved in oocytes synthesis.

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However, a decrease of glucose levels was recorded during the functional is used only as a source of energy, why a reduction in glucose was recorded 5 6 2 when the vitellogenesis process was at its maximum level? It is possible that previous maturation stages, or alternatively, glucose was reduced to avoid its  Kunkel et al., 1987). It is therefore possible that a reduction of ovarium glucose 5 6 7 levels in octopus could be associated with mechanisms to avoid physiological there was no apparent mobilization of lipids from the DG to the ovarium. In  In the present study, high levels of TG and cholesterol were detected in that the ovary itself is a reserve site for nutrients that will be used at least at the The increment of TG observed after the maturation processes started, and the beginning by the formation of oocytes, which after growth will be transformed in process suggest that cholesterol was also stored in the yolk to be used by the As expected, the biochemical and physiological processes in embryos are 6 1 0 highly dynamics following two well identified developmental phases: Our results indicate that soluble proteins and amino acids may be used as a and that regulation of the gluconeogenic pathway works as a mechanism of the 6 2 3 glucose supply. Regulation thus appears to be coupled with the phases of 6 2 4 embryo development, without control until stage XI, and with control from stage 6 2 5 XII onwards (Fig. 10). Our findings also suggest that energetic demands of 6 2 6 embryos in the first phase of development were relatively low, without 6 2 7 significantly mobilization of energetic substrates and its associated enzymes 6 2 8 (Fig. 10). The molecules identified with redox stress and that were maintained in 6 2 9 the eggs without changes suggest that the antioxidant defence mechanisms 6 3 0 were inactive until the stage XIV. The reduction on soluble protein and glycogen, and the increment of lipases Ass UK 80:557-558. Research 93:106-117. biotransformation and antioxidant defense systems in multiple tissues. Aquatic Toxicology 1055:56-66. Rosas C, Sanchez A, Pascual C, Aguila y Elvira J, Maldonado T, and Roumbedakis K, Mascaró M, Martins ML, Gallardo P, Rosas C, and Pascual C. Sánchez-García A, Rodríguez-Fuentes G, Díaz F, Galindo-Sánchez C, Ortega  Noreña E, Caamal-Monsreal C, Pascual C, Estefanell J, and Gallardo P. on Octopus maya embryo and hatching quality. Zuñiga O, Olivarez A, Rojo M, Chimal ME, Díaz F, Uriarte I, and Rosas C. stages obtained in the present study and by Cortes et al. (1995). Physiological   Values as mean + SD. Different letters means statistical differences between  representing data in six multivariate sets of data obtained from female O. mimus 9 9 8 in different stages of gonadic maturation (Imm: immature, PhyMat: physiological 9 9 9 maturity, EarFuncMat: early functional maturity, and LatFuncMat: late functional 1 0 0 0 maturity), and from embryos and paralarvae in different stage of development 1 0 0 5