Naef (1923)30 initiated three periods of embryonic development characterized as i) formation of blastoderm, ii) gastrulation with the establishment of endoderm and mesoderm, and iii) organogenesis that ends when the embryo hatches31.In the present study, identifying the phase of blastoderm formation phase was difficult in O. maya embryos because the eggs were sampled until the female presumably finished spawning, seven days after the blastoderm formation had finished. Consequently, identifying the end of blastulation (stage VI-VII) was possible in a few days before the start of organogenesis and beyond until hatching30.In this sense, O. maya embryos were characterized in four general stages of development, 1)blastulation 2)organogenesis 3)activation and 4) growth.
To our knowledge, the exponential relationship between egg wet weight vs stage and embryo wet weight vs stage have been obtained for the first time. Interestingly, while the entire egg does not change within the stage until activation, more acute changes on embryos wet weight were recorded in the stage. The results indicate that as organogenesis develops, the changes in embryo weight are probably compensated by the small amounts of yolk consumed and the absorbed water, generating the idea that the egg weight does not change between blastulation (stages 6–8) to activation phase (stages 14–16). However, when the yolk and peri-vitelline fluid are removed and the weight of the embryo is measured, the real weight is obtained showing-as expected- a well-defined exponential curve. Thus, if the embryo entire egg is used to calculate oxygen consumption, very likely, an overestimation of metabolic rate should be obtained. Previous results obtained in O. vulgaris32, and O. mimus8 overestimated the oxygen consumption of embryos because the entire egg was used to express metabolic data by weight unit. The present study evaluated the standard metabolism of embryos using the TIMR method, where a low temperature (11°C) was used to depress the routine metabolism, as a proxy of standard metabolism and also a high temperature (30°C) was used to obtain the maximum metabolic rate. With those values, the thermal metabolic scope was calculated as the difference between maximum and low respiratory metabolism22. The results obtained showed that routine metabolism is close to the maximum metabolic rate, suggesting that embryo metabolism cannot increase higher than they have during their development when maintained in optimal temperature range (24–26°C). The results indicate that routine metabolic rate of O. maya embryos is probably the maxima possible, probably due to limitations imposed by the limited number of mitochondria, oxygen exchange rate between seawater and perivitelline liquid, and the possible role of the chorion as an oxygen-barrier. Other studies should be performed to understand (1) how mitochondria concentration changes with embryo development; (2) how oxygen flows through the chorion and temperature modulates the oxygen dissolved in perivitelline liquid of this octopus species. The results obtained in the present study show that the thermal metabolic scope (TMS) peaks when embryos reach the state of activation and organogenesis ends. Although a new peak was observed during the growth phase after a low TMS value was recorded, the downregulated processes could have been involved to limit the embryo energy demands during the last part of the growing phase.
A hypothesis could help to explain these results Marthy et al.(1976)33 described a natural tranquillizer in the perivitelline fluid of loliginid squid, which prevents premature hatching. Although that substance has not been identified in octopod species yet, possibly similar molecules could be acting in octopus embryos, provoking a reduction of the metabolic rate before hatch. Another hypothesis could be related with the dissolved oxygen availability at the end of the growth phase. Before hatch, an increment on standard oxygen consumption was observed indicating that the energy demands to satisfy the basal metabolism of embryos are at their maximum level. In contrast, a reduction in the routine metabolic rate and maximum oxygen consumption of embryos were observed also at the end of the embryo development, indicating that basal, routine and maximum metabolic rates have close values at this stage. In such circumstances, a reduction in dissolved oxygen in the perivitelline fluid at the end of the growth phase could be also expected due to the high oxygen demands of the embryo that cannot be satisfied by the oxygen in the perivitelline fluid limited by the chorion surface where they are enclosed. Without enough oxygen the reduction in the maximum metabolic rate observed could explain the reduction in the maximum metabolic rate at the end of development, resulting in a reduction in the thermal metabolic scope. Although the oxygen concentration in perivitelline fluid is not known, evidence indicates that brooding octopus females stimulate the embryos to hatch, expelling water directly to the eggs, probably in an attempt to maintain higher oxygen dissolved in the environment (To review see Villanueva and Normal et al., 2008)34.Although a relationship between the mechanical stimulation provided by brooding female on the egg mass and oxygen concentration in fluid perivitelline in O. maya embryos is not known, possibly females could be promoting a high oxygen dissolved level to prevent hypoxia and protect the embryos before hatch. The present study evaluated changes on some enzymes involved in antioxidant defense mechanism in the ovarium, unspawned eggs and along the development of O. maya embryos. Additionally, LPO and PO were evaluated in the same tissues to know how the relationship between the female and embryos is during yolk synthesis and embryo development (Fig. 4). In females, it is noteworthy that elevated LPO and PO values were observed in the ovarium (Ov), un-spawned eggs (USE), and embryo early developmental stages. Several studies have proposed a relationship between reproduction and oxidative stress35.Reproduction is an energetically costly process for females, increasing resource requirements, metabolism, and potentially ROS production35–37. It has been observed in Octopus mimus ROS produced during metabolic processes has been observed to occur during ovarian maturation and in part transferred to the egg, causing a maternal ROS load to the embryo17. This result coincides with those observed now in the present study indicating that during yolk formation, the female transfers an amount of peroxidized lipids to un-spawned eggs that must be neutralized during embryo development (Fig. 6).In general, in O. maya -as in other cephalopod species- biochemical and molecular processes are highly dynamic during embryonic development30,38,39.
In the present study a considerable increase of SOD, CAT and GsT was observed from the activation stage onwards, when the circulatory system is activated in stages XIV - XVI. That pattern was also observed in O. mimus embryos39 indicating that antioxidant defense mechanisms are also activated as a response against the embryo ROS production, resulting as the energy production increment to support the embryo growth phase. Catalase is one of the ROS detoxifying enzymes that can be found in the early embryonic development stages, observed that its activity remains practically stable until hatching in several species40–42. CAT and SOD are considered the first line of defense against ROS because they directly neutralize the oxygen singlets. In consequence, the embryos could need synthesizing both enzymes at the beginning of their development to maintain the balance between prooxidant-antioxidant homeostasis43, which explains the expression levels detected from the blastulation stage with an increase in organogenesis remaining stable until hatching. Superoxide dismutase (SOD) is a ubiquitous family of enzymes that efficiently catalyzes superoxide dismutase anion. To date, three superoxide dismutases (SODs) have been biochemically and molecularly characterized, whose structure and function are highly conserved for many species44.
SOD1 or CuZn-SOD is a copper- zinc-containing homodimer found almost exclusively in intracellular cytoplasmic spaces45 and SOD2 or Mn-SOD exists in the cell as a tetramer and is initially synthesized with a leader peptide, which directs this manganese-containing enzyme exclusively to the mitochondrial spaces46,47. The present study shows that SOD1 expression is current from the first development stage, while the activity of the enzyme starts during the organogenesis stage. Other studies observed that the specific activities of these enzymes follow a characteristic increase with development and growth. However, embryonic enzyme activities do not necessarily correlate precisely with mRNA levels. Abramov and Wells, (2011)40 and El-Hage and Singh, (1990)48 have suggested that mRNA specific to these genes may accumulate and not be immediately translated. Therefore, the increase in the expression of all the genes evaluated in the present study could represent a preparation of the embryo towards the activation stage, which metabolically is a critical one due to the beginning of the circulatory system functioning and embryo growth8,11,17. Although the HIF1A gene is a crucial mediator in response to hypoxic conditions, it is also induced by ROS49. Furthermore, HIF1A is essential for embryonic vascularization50,51 as it regulates the production of vascular endothelial growth factor A (VEGF-A)52. Therefore, at this stage, a high HIF1A expression is reflecting that the circulatory system development has begun. In O. maya embryos, vascularization and antioxidant response coincided during the organogenesis stage, indicating that the circulatory system and ROS neutralization are coordinated. It is essential to consider that female-inherited ROS could alter these processes, mainly when the parents are exposed to thermal stress39. In addition, while the circulatory system remains incomplete, O2 supply to developing embryonic cells could be diffusion limited, which could lead to a lack of energy. Although further studies are required to test this hypothesis, possibly in such circumstances, anaerobic metabolism could be activated to help maintain energy levels. HIF-1A controls almost all the aspects of anaerobic metabolism by regulating genes, such as glucose transporter 1 (GLUT-1) and most glycolytic enzymess53,54, suggesting that HIF1A high expression could be indicating that oxygen dissolve in the perivitelline liquid could be limited, provoking a reduction on high metabolism when the hatch process is closed. When the follicular cells start the process of yolk synthesis, peroxidized lipids and oxidized proteins are placed into the eggs probably as a by-product of the follicular cell metabolism. The results obtained in the present study showed that high levels of LPO and PO were recorded in the ovarian and the unspawned eggs. Moreover, high levels of GSH in unspawned eggs suggest that this antioxidant agent was packed as part of the molecules that are placed in the yolk to be used during embryo development. GSH is a molecule that can be a powerful antioxidant since it functions as a free radical scavenger if the enzymes GRx, GPx, GsT are present to catalyze the Glutathione oxide-reduction cycle, using NADPH within the glucose metabolism pathway55,56. Although how GSH is placed in octopus embryos is still unknown, recently vitello vesicles were recorded and isolated in chicken egg yolk filled with GSH57. When the vesicles were supplemented in pork embryo culture medium, an increment of GSH content and reduction of ROS generation in pork embryos maintained in vitro was recorded suggesting that a charge of GSH is placed in the egg
to help the embryo to neutralize the ROS inherited during the yolk synthesis57. Although until now there is no evidence of vitello vesicles in octopus yolk, a similar mechanism could be operating to transfer GSH to O. maya embryos, ensuring in this form that embryos can neutralize the inherited ROS. Previously, it has been observed in O. mimus and O. maya that although yolk consumption by embryos begins at the organogenesis stage, the highest rate of yolk consumption has been recorded in embryos in the activation stage onwards, suggesting during growth phase there are the highest energy demand9,58. Furthermore, in a detailed study of the embryonic development of O. mimus17 it was observed that the mobilization of reserves (glycogen, glucose, cholesterol, acyl glycerides, and proteins) from the yolk is significantly higher from the moment of activation of the circulatory system of the embryos until hatch, indicating that in the growth phase is when the embryo require higher quantity of metabolic energy. For this reason, it is possible to conclude that the most energetically costly stage is the activation stage, due to the synthesis of tissues, mobilization of nutrients, and consequently, the activation of organs and metabolic enzymes, all of these having repercussions on the respiratory metabolism and elimination of oxidative damage. In this sense, to answer the question, how hard is the octopus embryo’s life? The following hypotheses are put forward: 1) eggs were generated with relatively high levels of LPO and PO, indicating that a part of the maternal production of ROS during ovarian maturation was placed in the egg to be eliminated during embryo development (Fig. 6)6. ROS in O. maya eggs were controlled by the embryos during the growth phase of embryo. This was due to the activation of antioxidant defense mechanisms at stage XV, indicating the coupling between metabolic demands and the functioning of the antioxidant defense system against oxidative stress. This explains the increased expression of SOD and CAT enzymes during the organogenesis stage. Although the female transfers LPO and PO to the embryo in the yolk contents, the amount of GSH that is transferred to the embryos is sufficient to cope with them once the activity of GST, CAT, SOD enzymes is initiated. Preliminary expression analysis results during embryonic development (Publication in progress) indicate that in O. maya the glutathione system genes are overexpressed at the organogenesis stage indicating that, as with SOD and CAT, the glutathione system is in preparation for the neutralization of inherited peroxidized lipids. 2) When measuring HMR, stimulated by the increase in temperature (30°C) embryos maintain a metabolic rate very similar to their RMR measured at 24°C. This has important biological repercussions during development since, due to the multiple processes involved in organ formation and even the elimination of ROS transferred from the mother, this is reflected in a high metabolic rate even at their optimal developmental temperature. Therefore, even when stimulating a metabolic rate, it has a biological limitation caused by the processes involved in embryonic development. In this sense, an increase in temperature during embryonic development probably can modify the expression of these genes of the antioxidant system as well as other genes essential for proper embryonic development.