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Retinoid storage in the egg of reptiles and birds
Comparative Biochemistry and Physiology, Part B 157 (2010) 113–118
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Comparative Biochemistry and Physiology, Part B j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c b p b
Retinoid storage in the egg of reptiles and birds Toshiaki Irie a,⁎, Tamiko Sugimoto b, Nobuo Ueki b, Haruki Senoo c, Takaharu Seki d a
Laboratory of Biology, Department of General Education, Hakodate National College of Technology, Tokura-cho, Hakodate, Hokkaido 042-8501, Japan Hitec Co. Ltd., Sonezakishinchi, Kita-ku, Osaka 530-0002, Japan c Department of Cell Biology and Morphology, Akita University Graduate School of Medicine, Hondo, Akita 010-8543, Japan d Research and Development Center for Teacher Education, Osaka Kyoiku University, Kashiwara, Osaka 582-8582, Japan b
a r t i c l e
i n f o
Article history: Received 15 February 2010 Received in revised form 20 May 2010 Accepted 21 May 2010 Available online 27 May 2010 Keywords: Bird Egg Reptile Retinal Retinoid Retinol Vitamin A Yolk protein
a b s t r a c t Storage of retinal has been confirmed in eggs from a range of anamniotic vertebrates (teleosts and amphibians) and an ascidian, but the retinoid-storage state in eggs of oviparous amniotic vertebrates (reptiles and birds) has yet to be clarified in detail. We studied four reptilian and five avian species and found that retinal was commonly stored in their egg yolk. Furthermore, retinal was the major retinoid in reptilian eggs, with only low levels of retinol, whereas significant amounts of retinol as well as retinal were stored in avian eggs. In both reptilian and avian eggs, retinal was commonly bound to proteins, which were assumed to be homologous to the proteins that bind retinal in the eggs of anamniotic vertebrates. Despite the common storage state of retinal, retinol would be bound to different proteins. In the reptilian eggs, retinol was found in the yolk-granule fraction, which also contained retinal. However, retinol in avian eggs was found largely in the yolk-plasma fraction, separate from retinal. These results suggest that retinol storage in avian eggs acquired after the divergence of birds from the reptiles, while retinal storage was acquired before the appearance of the vertebrates, and has subsequently been conserved during evolution of oviparous vertebrates. © 2010 Elsevier Inc. All rights reserved.
1. Introduction Retinoids, derivatives of vitamin A, play two distinct roles, involving unrelated mechanisms, and derived from different evolutionary backgrounds. The function of 11-cis retinal (RAL) in vision is widely distributed in the animal kingdom. In contrast, the function of retinoic acid (RA) in regulating genes is commonly present in chordates, but RA function in non-chordate animals has been controversial (Shimeld, 1996; Duester, 2000; Marlétaz et al., 2006; Campo-Paysaa et al., 2008; Simões-Costa et al., 2008). Because vitamin A homeostasis is essential for the maintenance of normal physiological conditions, animals have evolved retinoid-storing mechanisms to prevent hypovitaminosis A. In adult vertebrates, the esterified product of retinol (ROL), retinylester (RE), is stored in the stellate cells, which are mostly localized in the liver (Blomhoff et al., 1990), although extrahepatic stellate cells also play an important role in RE storage in lower vertebrates (Wold et al., 2004; Yoshikawa et al., 2006). ROL and fatty acids are produced by hydrolysis of hepatic RE, and the resulting ROL
Abbreviations: RAL, retinal (vitamin A aldehyde); ROL, retinol (vitamin A alcohol); RE, retinylester (vitamin A ester); RBP4, serum retinol-binding protein; TTR, transthyretin. ⁎ Corresponding author. Tel./fax: + 81 138 59 6391. E-mail addresses: [email protected] (T. Irie), [email protected] (H. Senoo), [email protected] (T. Seki). 1096-4959/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpb.2010.05.009
then combines with serum ROL-binding protein (RBP4, formerly RBP), which is synthesized in the hepatocytes. In mammals and birds, the RBP4 associated with ROL (holo-RBP4) subsequently combines with transthyretin (TTR), a carrier protein of thyroid hormones. The holoRBP4–TTR complex is secreted into the blood, and then delivered to the cells via the circulation (Vogel et al., 1999). Retinoids in vertebrate eggs, however, are stored differently from those in adults. Plack et al. (1959) and Plack and Kon (1961) described the distribution of RAL in the eggs or ovaries in a wide range of oviparous vertebrates, but the biological significance of RAL in eggs has not been investigated. In our previous studies, we demonstrated that RAL was the major form of retinoid stored in amphibian and teleostean eggs (anamniotic vertebrates) (Seki et al., 1987; Irie et al., 1991, 2002; Irie and Seki, 2002), and was also the major form of retinoid stored in the eggs of the ascidian, Halocynthia roretzi (Urochordata) (Irie et al., 2003). We determined that RAL in the eggs of anamniotic chordates was commonly bound to lipovitellin 1 or its homologous proteins, by Schiff-base linkages (Irie et al., 1991, 2002, 2003; Irie and Seki, 2002). The common storage state of RAL in these diverse species suggests that the utilization of RAL for vitamin A storage in eggs was acquired before the appearance of vertebrates in chordate evolution (cf. Irie et al., 2004). In contrast to the eggs of anamniotic chordates, considerable amounts of ROL are known to be present in the yolks of chicken and quail eggs, though RAL has also been demonstrated in the eggs of poultry (Al-Hasani and Parrish, 1972; Joshi et al., 1973; Sivell et al.,
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1982; Dong and Zile, 1995). However, the content of RAL in avian eggs remains to be confirmed, as simple extractions with organic solvents are unable to dissociate RAL adequately from its binding protein because of the Schiff-base linkage. The retinoid-storage state in eggs of reptiles, which belong to the amniotic taxon together with birds and mammals, also remains to be determined. In the present study, we compared the retinoid storage strategies in the egg yolks of reptiles and birds. The results clearly showed that RAL was the major form of retinoid stored in the reptilian eggs, whereas ROL as well as RAL was stored in the avian eggs. Based on the results, we discuss the retinoid-storage state in eggs of oviparous amniotic vertebrates in comparison with that in anamniotic vertebrates, and in relation to vertebrate phylogeny. 2. Materials and methods 2.1. Eggs Chicken (Gallus gallus, order Galliformes) and Japanese quail (Coturnix japonica, order Galliformes) eggs were purchased from a supermarket in Osaka prefecture, Japan. An ostrich (Struthio camelus, supraorder Palaeognathae) egg was supplied from Akita Prefectural College of Agriculture (Akita, Japan). Ostrich and duck (Anas platyrhynchos domestica, order Anseriformes) eggs were purchased from Jonan Green System Co. Ltd. (Ibaraki, Japan). Common kestrel (Falco tinnunculus, order Falconiformes) eggs were supplied by Akita City Omoriyama Zoo (Akita, Japan). Red-eared slider (Trachemys scripta elegans, order Testudines) eggs were from Himeji City Aquarium (Hyogo, Japan), soft-shell turtle (Pelodiscus sinensis, order Testudines) eggs were from Ashida Kikaku Ltd. (Ishikawa, Japan), and Siamese crocodile (Crocodylus siamensis, order Crocodylia) eggs were from Koike Wani Souhonpo Co. Ltd. (Shizuoka, Japan). Loggerhead turtle (Caretta caretta, order Testudines) eggs were supplied by Yakushima Umigame-kan, NPO (Kagoshima, Japan), mediated by the Sea Turtle Association of Japan, NPO (Osaka, Japan). Because the loggerhead turtle is listed as endangered in the Red List, the turtle eggs were obtained with formal permission based on the regulations for sea turtle preservation of Kagoshima Prefecture, Japan. For all species, the eggshell was broken and the intact egg yolk was isolated from the albumen, after which the appropriate amount of yolk was collected. The yolk was suspended immediately in buffer (see below) for retinoid analysis, or stored in a deep freeze (−80 °C) until use. 2.2. Fractionation of the yolk and analysis of the yolk proteins All the reptilian egg yolks consisted of an aqueous part, which accounted for the majority of the yolk, and a small amount of floating lipids. No floating lipids were visible in the avian egg yolks. The floating lipids were obtained by layering an appropriate amount of n-hexane onto the yolk mass of the reptilian eggs and shaking gently. The lipids dissolved in the n-hexane layer were then collected using a Pasteur pipette. The aqueous part of the yolk was diluted 10-fold with 20 mM TrisHCl buffer (pH 7.4), suspended using a manually operated Teflon-pestle homogenizer, and centrifuged at 17,000 ×g for 20 min to separate the yolk-granule (precipitate) and yolk-plasma (supernatant) fractions. The precipitate was resuspended in the same buffer, and centrifuged again under the same conditions to remove the remaining yolk-plasma proteins from the yolk-granule fraction. Sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS– PAGE) was performed largely according to the procedure described by Laemmli (1970), using 1-mm thick 12.5% slab gels. The protein bands were visualized after staining with Coomassie Brilliant blue R-250. Precision Protein Standards (Bio-Rad Lab., CA, USA) were used as a molecular weight marker for SDS–PAGE.
Protein content was measured using the method of Lowry et al. (1951), with bovine serum albumin as the standard protein. 2.3. Retinoid extraction and analysis The samples (whole egg yolk or yolk fractions) were extracted with organic solvents using the oxime method (Suzuki and MakinoTasaka, 1983), following routine procedures (Irie et al., 1991). Briefly, each sample was treated with hydroxylamine hydrochloride (freshly neutralized NH2OH-HCl) and then extracted using dichloromethane and hexane. The oxime method results in good dissociation of the Schiff-base linkage between RAL and its binding protein. To confirm the Schiff-base linkage of RAL to proteins, the yolk suspension was treated with sodium borohydride (NaBH4) (Bownds and Wald, 1965) before extraction using the oxime method. High-performance liquid chromatography (HPLC) was performed as described previously (Irie and Seki, 2002). The HPLC system (PU2080, Jasco Corp., Tokyo, Japan or L-2130, Hitachi Ltd., Tokyo, Japan) was equipped with a 6 × 150-mm column of 3-µm silica gel (YMCPack SIL, YMC Co. Ltd., Kyoto, Japan), and was used at a flow rate of 2 mL/min. Absorbances were monitored using UV/visible detectors (dual wavelength, 875-UV and UV-970; Jasco Corp.) or a diode array detector (spectral surveillance, 300–400 nm, L-2450 Hitachi Ltd.). The eluent was 5% tert-butylmethyl ether, 0.04% ethanol and 25% benzene in n-hexane. Because RE isomers elute out in the void fraction under these eluent conditions, the fraction (retention time for 1.5–2.2 min) was collected, saponified to ROL with ethanolic KOH (Bridges and Alvarez, 1982), and rechromatographed under the same conditions as described above. RE isomers are detected as ROL isomers on the second round of HPLC. Extraction and analyses of retinoids were performed under dim red light to avoid photo-isomerization of retinoids during the experiments. 2.4. Gel chromatography of yolk-granule proteins Analysis of the protein-binding RAL was performed using the previously described method (Irie and Seki, 2002). The isolated yolkgranule proteins were solubilized in buffer containing 0.4 M NaCl. The solution was then centrifuged at 17,000 ×g for 20 min to remove any insoluble matter. The supernatant was treated with NaBH4, followed immediately by the addition of SDS (final concentration 2%) to form a retinyl–protein complex. The complex was stood on ice for 30 min and then dialyzed against 20 mM Tris-HCl buffer (pH 7.4) containing 0.1% SDS to avoid the formation of hydrogen gas bubbles due to any remaining NaBH4, during the chromatography. Mercaptoethanol (final concentration 5%) was then added and the sample was chromatographed using a Sephacryl S-300 HR column (2.5 × 86 cm) equilibrated with buffer containing 0.1% SDS and 0.02% dithiothreitol. The eluent was collected in 5-mL fractions and the absorbances of each fraction at 280 nm (proteins) and 330 nm (retinyl–protein complex) were measured. 3. Results 3.1. Amounts and composition of retinoids Fig. 1A1 and B1 show the HPLC chromatograms of extracts from the whole egg yolks of loggerhead turtles and Japanese quail. High levels of RAL were detected in the eggs of both reptilian and avian species. RAL was the major retinoid component in the turtle eggs, with a small amount of ROL, although considerable amounts of both ROL and RAL were present in the quail eggs. Table 1 shows the contents (μg/g yolk) and concentrations (ng/mg protein) of retinoids in the egg yolks. RAL, ROL and RE were present in varying amounts in the different species. Fig. 2 summarizes the retinoid compositions of the eggs examined in the present and
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Fig. 1. High-performance liquid chromatography analysis of retinoids in the whole egg yolk or yolk fractions from loggerhead turtles (A) and Japanese quail (B). The scale bar shows the absorbance at 360 nm. A1 and B1, extracts from whole egg yolk; A2 and B2, extracts from egg granule fractions; A3 and B3, extracts from egg plasma fractions. a and a′, syn and anti 11-cis retinaloximes; b and b′, syn and anti all-trans retinaloximes; c and c′, syn and anti all-trans 3,4-didehydroretinaloximes; d and d′, syn and anti 13-cis retinaloximes; e, all-trans retinol; f, 13-cis retinol.
previous studies. RAL was present in the eggs of every species of chordate examined, whereas storage of high levels of ROL was characteristic of avian eggs. RE was present in all the reptilian and avian eggs, but with considerable variation among species (Table 1, Fig. 2). The main RAL isomer detected in eggs was the all-trans form, with trace amounts of 11- and 13-cis isomers, depending on the species. The eggs of three reptilian species that lived in or near freshwater (red-eared slider, soft-shell turtle, Siamese crocodile) contained small amounts of 3,4-didehydroRAL (RAL2) isomers, whereas RAL2 isomers were scarcely detected in the eggs of loggerhead turtles (sea turtle). RAL2 isomers were not detected in any of the avian eggs examined. 3.2. Distribution and state of RAL and ROL in eggs Egg yolk suspensions were centrifuged to separate the yolkgranule and yolk-plasma fractions, and the retinoids in each fraction were then analyzed (Fig. 1A2, A3, B2, B3; Table 2). RAL was only
present in the yolk-granule fractions in both reptilian and avian eggs. These results suggest that the RAL in the eggs was bound to a yolkgranule protein in both reptiles and birds. No RAL was found in the yolk-granule or whole egg yolk extracts in any species after treatment with NaBH4, indicating that the egg RAL was bound to the proteins by Schiff-base linkages, similar to the situation in anamniotic chordates (Irie et al., 1991, 2003; Irie and Seki, 2002). The localization of ROL, however, differed between reptilian and avian eggs. A small amount of ROL was present in reptilian eggs and was mostly located in the yolk-granule fraction, where most of the RAL was also localized. ROL in avian eggs, in contrast, was found in the yolk-plasma fraction, where very little RAL was found. Thus, the distribution of ROL in egg yolk differed between reptiles and birds, in contrast to the common distribution of RAL. Most RE in the reptilian eggs was detected in the floating lipids, with little in the yolk-granule and plasma fractions. However, RE in avian eggs was detected in both the yolk-granule and the plasma fractions.
Table 1 Retinoid levels in egg yolks. Taxon
Species
n
Reptile
Loggerhead turtle Red-eared slider Soft-shell turtle Siamese crocodile Chicken Japanese quail Duck Common kestrel Ostrich
22 8 12 6 4 4 4 3 3
Bird
Retinoid content (μg/g yolk)
Retinoid concentration (ng/mg P)
RAL
ROL
RE
RAL
ROL
RE
2.36 ± 0.34 1.23 ± 0.52 1.46 ± 0.25 3.80 ± 0.19 3.25 ± 0.29 5.82 ± 0.42 2.99 ± 0.87 9.23 ± 1.29 1.96 ± 0.26
0.23 ± 0.05 0.28 ± 0.10 0.04 ± 0.03 0.36 ± 0.03 4.97 ± 0.72 8.96 ± 0.57 2.67 ± 1.01 4.15 ± 0.21 2.28 ± 0.38
0.32 ± 0.19 0.57 ± 0.34 0.13 ± 0.11 0.33 ± 0.04 1.04 ± 0.32 1.60 ± 0.28 0.34 ± 0.09 1.56 ± 0.52 0.77 ± 0.07
9.8 ± 2.1 4.9 ± 2.2 8.1 ± 1.5 19.9 ± 2.0 15.9 ± 2.3 25.7 ± 4.6 16.1 ± 5.1 58.6 ± 6.0 10.2 ± 2.3
0.97 ± 0.23 1.10 ± 0.43 0.20 ± 0.17 1.89 ± 0.17 24.4 ± 5.6 39.8 ± 8.6 14.5 ± 5.8 26.5 ± 1.9 11.6 ± 0.7
1.65 ± 0.63 2.29 ± 1.39 0.76 ± 0.65 1.54 ± 0.20 5.06 ± 1.70 7.05 ± 1.59 1.62 ± 0.45 9.61 ± 3.24 4.35 ± 0.41
Mean ± S.D.; n, number of eggs analyzed; mg P, mg of protein measured by Lowry's method; RAL, retinal; ROL, retinol; RE, retinylester.
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Fig. 2. Retinoid composition of whole egg yolk extract. 4) Seki et al., 1987; 5) Irie et al., 2002.
retinal;
retinylester; n, number of eggs analyzed 1) Irie et al., 2003; 2) Irie and Seki, 2002; 3) Irie et al., 1991;
retinol;
3.3. Analysis of the RAL-binding protein in quail eggs The protein components in the egg yolk fractions were analyzed using SDS–PAGE (quail yolk granules in Fig. 3B, e). High levels of yolkgranule and plasma proteins are present in avian eggs, as reported in the nutritional information provided for poultry eggs (Juneja and Kim, 1997), whereas the yolk-plasma fractions of reptilian eggs are poor in proteins (data not shown). The yolk-granule solution in NaCl-containing buffer (see Section 2.4) was treated with NaBH4 and SDS (Fig. 3B, f), and then chromatographed using Sephacryl S-300 (Fig. 3A). The chromatogram showed an absorption peak at 330 nm, which was derived from the retinyl–protein complex produced by NaBH4 treatment, and corresponded to the former part of the first protein peak indicated by absorption at 280 nm. Fig 3B, a–e shows the SDS–PAGE patterns of the fractions. Presumably, the first protein peak (Fig 3A, peak b) was heterogeneous; the peak would consist of several protein components with slightly different molecular weights. Although the protein components were not separated, RAL would be bound to the protein component with a highest molecular weight in the first protein peak of the chromatograph (Fig. 3A, peak a).
that RAL was the major storage retinoid in the eggs of several anamniotic vertebrates and a urochordate (Seki et al., 1987; Irie et al., 1991; Irie and Seki, 2002; Irie et al., 2002; 2003). Thus, RAL storage in eggs appears to be a common characteristic in oviparous chordates. RAL was found in the yolk-granule fraction in the eggs of every species examined in this study. In the poultry eggs, the yolk granules consist mostly of the high-density lipoproteins (lipovitellins) and phosvitin, which are vitellogenin-derived yolk proteins, whereas the yolk-plasma contains low-density lipoproteins and other minor protein components such as livetines, riboflavin- and biotin-binding proteins (Li-Chan et al., 1995). The results of our previous studies demonstrated that RAL was bound to lipovitellin 1 or its homologous protein in amphibian and teleostean eggs (Irie et al., 1991; Irie and Seki, 2002; Irie et al., 2002). The present study revealed that RAL in quail eggs was bound to RAL-binding proteins, which were assumed equivalent to those in amphibian and teleostean eggs, and that RAL in
4. Discussion The results of the present study demonstrate that RAL is stored in the egg yolks of both reptiles and birds. Our previous studies indicated Table 2 Retinoid levels in the egg yolk fractions. Species
Loggerhead turtle
Red-eared slider
Japanese quail
Fractions
Yolk granules Yolk-plasma Floating lipids Yolk granules Yolk-plasma Floating lipids Yolk granules Yolk-plasma
Retinoid content (μg/g yolk) RAL
ROL
RE
2.85 n.d. n.d. 1.04 n.d. n.d. 5.44 0.063
0.11 0.008 0.057 0.10 0.04 0.009 0.37 8.40
n.d. n.d. 0.24 n.d. n.d. 0.028 0.37 0.28
n.d., not detected; RAL, retinal; ROL, retinol; RE, retinylester.
Fig. 3. Gel chromatography of Japanese quail yolk-granule proteins treated with NaBH4, 2% SDS and 5% 2-mercaptoethanol, using a Sephacryl S-300 HR column (2.5 × 86 cm) equilibrated with 20 mM Tris-HCl buffer (pH 7.4) containing 0.1% SDS and 0.02% dithiothreitol (A), and SDS–polyacrylamide gel electrophoresis of the fractions and yolk-granule proteins (B). Absorbances at 280 nm (protein, ○) and 330 nm (retinyl product, ●, two-fold enlargement) of each fraction (5 mL) were measured after chromatography. a, Fr. 32; b, Fr. 36; c, Fr. 45; d, Fr. 58; e, yolk-granule proteins; f, yolkgranule proteins after treatment with NaBH4 and SDS; M, molecular weight markers.
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both reptilian and avian eggs was bound to proteins by Schiff-base linkages. Thus, not only RAL storage in eggs, but also its proteinbinding state, would be common throughout oviparous chordates. Vitellogenin is produced in the liver as a result of estrogen stimulation. RAL was detected in the blood plasma of estrogen-treated Xenopus laevis, and in the blood plasma fraction that contained vitellogenin (Azuma et al., 1993). Thus, egg RAL in oviparous vertebrates would be commonly derived from hepatic RE in the maternal liver, and transported to the oocytes via the blood circulation in association with vitellogenin. However, the details of these processes remain unclear. In contrast to the common storage of RAL in eggs of oviparous chordates, significant amounts of ROL, as well as RAL, were detected in avian eggs. High levels of ROL have previously been found in the eggs of several avian species (Royle, et al., 1999; Surai et al., 2000, 2001; Hargitai et al., 2006). Poultry eggs are known to be rich in vitamin A, which is derived from ROL. ROL storage in eggs therefore appears to be a common characteristic in birds. The amounts of ROL detected in chicken and quail eggs in the present study were similar to those reported in previous studies (Sivell et al., 1982; Watkins, 1995; Karadas et al., 2005). In contrast to the situation in avian eggs, only low levels of ROL were found in reptilian eggs. These results were consistent with those of Speake et al. (2001) and Thompson et al. (1999a, b) who found low vitamin A levels in reptilian eggs, due to the low or absent levels of ROL and RE. However, they did not quantify the amounts of RAL. These results imply that RAL is the main storage retinoid in reptilian eggs, as in the eggs of anamniotic chordates. Reptiles and birds are both amniotes. Although reptilian eggshells are often elastic, the morphological and functional characteristics of their eggs are similar, and the developmental features are virtually identical. However, the results of this study demonstrate that clear differences exist in terms of ROL storage. ROL in avian eggs was found mostly in the yolk-plasma fractions. Vieira et al. (1995) and Vieira (1998) reported that the specific TTRreceptor was present on the surface of the chicken oocytes, and that the holo-RBP4–TTR complex in the blood plasma was transported into oocytes mediated by the receptor. Involvement of the holo-RBP4–TTR complex in ROL-accumulating mechanisms in oocytes is suggested to be commonly distributed in birds. However, ROL in reptilian eggs was localized in the yolk-granule fractions, suggesting that ROL in these eggs may be derived from a vitellogenin-associated form, by nonspecific binding. TTR synthesis first appeared during vertebrate evolution in the choroid plexus of the stem reptile, but TTR synthesis in the liver subsequently evolved independently in birds, eutherians and some marsupial species (Harms et al., 1991; Schreiber and Richardson, 1997). TTR is barely detectable in the blood plasma of anamniotic vertebrates and reptiles (Harms, et al., 1991; Schreiber and Richardson, 1997; Power et al., 2000), though TTR genes are temporarily expressed in the liver in juvenile fish and in frog tadpoles (Richardson, 2009; Yamauchi and Ishihara, 2009). Richardson (2009) referred to the possibility that the maintenance of hepatic TTR synthesis in adulthood was related to homeothermy. ROL-accumulating mechanisms in eggs in poikilothermic vertebrates would therefore be insignificant, because of the absence of the holo-RBP4–TTR complex in the blood, despite the capacity of RBP4 from several poikilothermic vertebrates to bind to human TTR in vitro (Shidoji and Muto, 1977; Shidoji et al., 1979; Berni et al., 1992). It is likely that ROL accumulation into oocytes is mostly due to the mechanisms mediated by the TTR-receptor, and that the mechanisms were acquired after the acquisition of the holo-RBP–TTR complex in the blood by hepatic TTR synthesis. RE in the reptilian yolk was located mostly in the floating lipids. Our previous study also demonstrated that RE in teleostean eggs was localized in the lipid bodies (Irie and Seki, 2002). It is possible that the RE-accumulating mechanisms in reptilian oocytes are identical to those of teleosts, although these mechanisms remain unidentified.
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Fig. 4. Hypothetical scheme for acquisition of retinol-accumulating mechanisms into oocytes (bold lines) during amniote phylogeny, although there is some debate as to whether the squamates or turtles diverged first. The mechanisms are likely to have been acquired during the evolutionary process shown by the bold lines (between the crocodile–dinosaur split and the diversification of avian orders) in the Mesozoic era.
In conclusion, we propose that the mechanisms responsible for RAL accumulation into oocytes and the storage of RAL in eggs were acquired before the appearance of the vertebrates (cf. Irie et al., 2004), and that they have subsequently been conserved throughout the evolution of oviparous chordates. The mechanism of ROL accumulation into oocytes, however, which is mediated by the specific TTRreceptor, was acquired much later than that for RAL storage. It is widely believed that birds diverged from the Theropoda, a group of so-called dinosaurs. Crocodiles are the closest living reptilian relatives of birds (Iwabe et al., 2005). The mechanism of ROL storage found in avian eggs must therefore have been acquired during the period of evolution between the crocodile–dinosaur split and the diversification of the different avian orders (Fig. 4), in the Mesozoic era. To the best of our knowledge, retinoids have not been examined in mammalian eggs or oocytes. However, eutherians (placental mammals) have lost the capacity to synthesize vitellogenin during evolution. Because eutherians lack the RAL transporter, RAL is unlikely to be accumulated into eutherian oocytes. In the monotremes, however, the vitellogenin gene cluster is conserved in the platypus (Brawand et al., 2008; Babin, 2008), suggesting that RAL could possibly be incorporated into monotreme oocytes. No TTR was detected in monotreme blood (Schreiber and Richardson, 1997; Richardson, 2009), implying that ROL may not be accumulated into monotreme oocytes, and RAL would therefore be the major form of retinoid stored in monotreme eggs, as in reptilian eggs. Further studies are needed to test the validity of these predictions. Acknowledgments The authors are grateful to the following people for the donation of eggs (in order of supply date): Takeyoshi Tochimoto (Himeji City Aquarium, red-eared slider), Kazuo Ashida (Ashida Kikaku Ltd., softshell turtle), Yoshio Hamano (Akita Prefectural College of Agriculture, ostrich), Mamoru Komatsu (Akita City Omoriyama Zoo, common kestrel), Yoshimasa Matsuzawa (Sea Turtle Association of Japan, NPO, loggerhead turtle), Kazuyoshi Omuta (Yakushima Umigame-kan, NPO, loggerhead turtle), Katsuhiro Koike (Koike Wani Souhonpo Co. Ltd., Siamese crocodile). We would also like to thank Naosuke Kojima, Nobuyo Higashi-Kuwata, Kiwamu Yoshikawa and Mitsuru Sato (Akita University Graduate School of Medicine) for their cooperation and valuable discussions during these experiments. References Al-Hasani, S.M., Parrish, D.B., 1972. Forms of vitamin A and of carotenoids in tissues, blood serum and yolk of eggs from Coturnix coturnix japonica fed β-apo-carotenals. J. Nutr. 102, 1437–1440. Azuma, M., Irie, T., Seki, T., 1993. Retinals and retinols induced by estrogen in the blood plasma of Xenopus laevis. J. Exp. Biol. 178, 89–96.
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Comparative Biochemistry and Physiology, Part B j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c b p b
Retinoid storage in the egg of reptiles and birds Toshiaki Irie a,⁎, Tamiko Sugimoto b, Nobuo Ueki b, Haruki Senoo c, Takaharu Seki d a
Laboratory of Biology, Department of General Education, Hakodate National College of Technology, Tokura-cho, Hakodate, Hokkaido 042-8501, Japan Hitec Co. Ltd., Sonezakishinchi, Kita-ku, Osaka 530-0002, Japan c Department of Cell Biology and Morphology, Akita University Graduate School of Medicine, Hondo, Akita 010-8543, Japan d Research and Development Center for Teacher Education, Osaka Kyoiku University, Kashiwara, Osaka 582-8582, Japan b
a r t i c l e
i n f o
Article history: Received 15 February 2010 Received in revised form 20 May 2010 Accepted 21 May 2010 Available online 27 May 2010 Keywords: Bird Egg Reptile Retinal Retinoid Retinol Vitamin A Yolk protein
a b s t r a c t Storage of retinal has been confirmed in eggs from a range of anamniotic vertebrates (teleosts and amphibians) and an ascidian, but the retinoid-storage state in eggs of oviparous amniotic vertebrates (reptiles and birds) has yet to be clarified in detail. We studied four reptilian and five avian species and found that retinal was commonly stored in their egg yolk. Furthermore, retinal was the major retinoid in reptilian eggs, with only low levels of retinol, whereas significant amounts of retinol as well as retinal were stored in avian eggs. In both reptilian and avian eggs, retinal was commonly bound to proteins, which were assumed to be homologous to the proteins that bind retinal in the eggs of anamniotic vertebrates. Despite the common storage state of retinal, retinol would be bound to different proteins. In the reptilian eggs, retinol was found in the yolk-granule fraction, which also contained retinal. However, retinol in avian eggs was found largely in the yolk-plasma fraction, separate from retinal. These results suggest that retinol storage in avian eggs acquired after the divergence of birds from the reptiles, while retinal storage was acquired before the appearance of the vertebrates, and has subsequently been conserved during evolution of oviparous vertebrates. © 2010 Elsevier Inc. All rights reserved.
1. Introduction Retinoids, derivatives of vitamin A, play two distinct roles, involving unrelated mechanisms, and derived from different evolutionary backgrounds. The function of 11-cis retinal (RAL) in vision is widely distributed in the animal kingdom. In contrast, the function of retinoic acid (RA) in regulating genes is commonly present in chordates, but RA function in non-chordate animals has been controversial (Shimeld, 1996; Duester, 2000; Marlétaz et al., 2006; Campo-Paysaa et al., 2008; Simões-Costa et al., 2008). Because vitamin A homeostasis is essential for the maintenance of normal physiological conditions, animals have evolved retinoid-storing mechanisms to prevent hypovitaminosis A. In adult vertebrates, the esterified product of retinol (ROL), retinylester (RE), is stored in the stellate cells, which are mostly localized in the liver (Blomhoff et al., 1990), although extrahepatic stellate cells also play an important role in RE storage in lower vertebrates (Wold et al., 2004; Yoshikawa et al., 2006). ROL and fatty acids are produced by hydrolysis of hepatic RE, and the resulting ROL
Abbreviations: RAL, retinal (vitamin A aldehyde); ROL, retinol (vitamin A alcohol); RE, retinylester (vitamin A ester); RBP4, serum retinol-binding protein; TTR, transthyretin. ⁎ Corresponding author. Tel./fax: + 81 138 59 6391. E-mail addresses: [email protected] (T. Irie), [email protected] (H. Senoo), [email protected] (T. Seki). 1096-4959/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpb.2010.05.009
then combines with serum ROL-binding protein (RBP4, formerly RBP), which is synthesized in the hepatocytes. In mammals and birds, the RBP4 associated with ROL (holo-RBP4) subsequently combines with transthyretin (TTR), a carrier protein of thyroid hormones. The holoRBP4–TTR complex is secreted into the blood, and then delivered to the cells via the circulation (Vogel et al., 1999). Retinoids in vertebrate eggs, however, are stored differently from those in adults. Plack et al. (1959) and Plack and Kon (1961) described the distribution of RAL in the eggs or ovaries in a wide range of oviparous vertebrates, but the biological significance of RAL in eggs has not been investigated. In our previous studies, we demonstrated that RAL was the major form of retinoid stored in amphibian and teleostean eggs (anamniotic vertebrates) (Seki et al., 1987; Irie et al., 1991, 2002; Irie and Seki, 2002), and was also the major form of retinoid stored in the eggs of the ascidian, Halocynthia roretzi (Urochordata) (Irie et al., 2003). We determined that RAL in the eggs of anamniotic chordates was commonly bound to lipovitellin 1 or its homologous proteins, by Schiff-base linkages (Irie et al., 1991, 2002, 2003; Irie and Seki, 2002). The common storage state of RAL in these diverse species suggests that the utilization of RAL for vitamin A storage in eggs was acquired before the appearance of vertebrates in chordate evolution (cf. Irie et al., 2004). In contrast to the eggs of anamniotic chordates, considerable amounts of ROL are known to be present in the yolks of chicken and quail eggs, though RAL has also been demonstrated in the eggs of poultry (Al-Hasani and Parrish, 1972; Joshi et al., 1973; Sivell et al.,
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1982; Dong and Zile, 1995). However, the content of RAL in avian eggs remains to be confirmed, as simple extractions with organic solvents are unable to dissociate RAL adequately from its binding protein because of the Schiff-base linkage. The retinoid-storage state in eggs of reptiles, which belong to the amniotic taxon together with birds and mammals, also remains to be determined. In the present study, we compared the retinoid storage strategies in the egg yolks of reptiles and birds. The results clearly showed that RAL was the major form of retinoid stored in the reptilian eggs, whereas ROL as well as RAL was stored in the avian eggs. Based on the results, we discuss the retinoid-storage state in eggs of oviparous amniotic vertebrates in comparison with that in anamniotic vertebrates, and in relation to vertebrate phylogeny. 2. Materials and methods 2.1. Eggs Chicken (Gallus gallus, order Galliformes) and Japanese quail (Coturnix japonica, order Galliformes) eggs were purchased from a supermarket in Osaka prefecture, Japan. An ostrich (Struthio camelus, supraorder Palaeognathae) egg was supplied from Akita Prefectural College of Agriculture (Akita, Japan). Ostrich and duck (Anas platyrhynchos domestica, order Anseriformes) eggs were purchased from Jonan Green System Co. Ltd. (Ibaraki, Japan). Common kestrel (Falco tinnunculus, order Falconiformes) eggs were supplied by Akita City Omoriyama Zoo (Akita, Japan). Red-eared slider (Trachemys scripta elegans, order Testudines) eggs were from Himeji City Aquarium (Hyogo, Japan), soft-shell turtle (Pelodiscus sinensis, order Testudines) eggs were from Ashida Kikaku Ltd. (Ishikawa, Japan), and Siamese crocodile (Crocodylus siamensis, order Crocodylia) eggs were from Koike Wani Souhonpo Co. Ltd. (Shizuoka, Japan). Loggerhead turtle (Caretta caretta, order Testudines) eggs were supplied by Yakushima Umigame-kan, NPO (Kagoshima, Japan), mediated by the Sea Turtle Association of Japan, NPO (Osaka, Japan). Because the loggerhead turtle is listed as endangered in the Red List, the turtle eggs were obtained with formal permission based on the regulations for sea turtle preservation of Kagoshima Prefecture, Japan. For all species, the eggshell was broken and the intact egg yolk was isolated from the albumen, after which the appropriate amount of yolk was collected. The yolk was suspended immediately in buffer (see below) for retinoid analysis, or stored in a deep freeze (−80 °C) until use. 2.2. Fractionation of the yolk and analysis of the yolk proteins All the reptilian egg yolks consisted of an aqueous part, which accounted for the majority of the yolk, and a small amount of floating lipids. No floating lipids were visible in the avian egg yolks. The floating lipids were obtained by layering an appropriate amount of n-hexane onto the yolk mass of the reptilian eggs and shaking gently. The lipids dissolved in the n-hexane layer were then collected using a Pasteur pipette. The aqueous part of the yolk was diluted 10-fold with 20 mM TrisHCl buffer (pH 7.4), suspended using a manually operated Teflon-pestle homogenizer, and centrifuged at 17,000 ×g for 20 min to separate the yolk-granule (precipitate) and yolk-plasma (supernatant) fractions. The precipitate was resuspended in the same buffer, and centrifuged again under the same conditions to remove the remaining yolk-plasma proteins from the yolk-granule fraction. Sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS– PAGE) was performed largely according to the procedure described by Laemmli (1970), using 1-mm thick 12.5% slab gels. The protein bands were visualized after staining with Coomassie Brilliant blue R-250. Precision Protein Standards (Bio-Rad Lab., CA, USA) were used as a molecular weight marker for SDS–PAGE.
Protein content was measured using the method of Lowry et al. (1951), with bovine serum albumin as the standard protein. 2.3. Retinoid extraction and analysis The samples (whole egg yolk or yolk fractions) were extracted with organic solvents using the oxime method (Suzuki and MakinoTasaka, 1983), following routine procedures (Irie et al., 1991). Briefly, each sample was treated with hydroxylamine hydrochloride (freshly neutralized NH2OH-HCl) and then extracted using dichloromethane and hexane. The oxime method results in good dissociation of the Schiff-base linkage between RAL and its binding protein. To confirm the Schiff-base linkage of RAL to proteins, the yolk suspension was treated with sodium borohydride (NaBH4) (Bownds and Wald, 1965) before extraction using the oxime method. High-performance liquid chromatography (HPLC) was performed as described previously (Irie and Seki, 2002). The HPLC system (PU2080, Jasco Corp., Tokyo, Japan or L-2130, Hitachi Ltd., Tokyo, Japan) was equipped with a 6 × 150-mm column of 3-µm silica gel (YMCPack SIL, YMC Co. Ltd., Kyoto, Japan), and was used at a flow rate of 2 mL/min. Absorbances were monitored using UV/visible detectors (dual wavelength, 875-UV and UV-970; Jasco Corp.) or a diode array detector (spectral surveillance, 300–400 nm, L-2450 Hitachi Ltd.). The eluent was 5% tert-butylmethyl ether, 0.04% ethanol and 25% benzene in n-hexane. Because RE isomers elute out in the void fraction under these eluent conditions, the fraction (retention time for 1.5–2.2 min) was collected, saponified to ROL with ethanolic KOH (Bridges and Alvarez, 1982), and rechromatographed under the same conditions as described above. RE isomers are detected as ROL isomers on the second round of HPLC. Extraction and analyses of retinoids were performed under dim red light to avoid photo-isomerization of retinoids during the experiments. 2.4. Gel chromatography of yolk-granule proteins Analysis of the protein-binding RAL was performed using the previously described method (Irie and Seki, 2002). The isolated yolkgranule proteins were solubilized in buffer containing 0.4 M NaCl. The solution was then centrifuged at 17,000 ×g for 20 min to remove any insoluble matter. The supernatant was treated with NaBH4, followed immediately by the addition of SDS (final concentration 2%) to form a retinyl–protein complex. The complex was stood on ice for 30 min and then dialyzed against 20 mM Tris-HCl buffer (pH 7.4) containing 0.1% SDS to avoid the formation of hydrogen gas bubbles due to any remaining NaBH4, during the chromatography. Mercaptoethanol (final concentration 5%) was then added and the sample was chromatographed using a Sephacryl S-300 HR column (2.5 × 86 cm) equilibrated with buffer containing 0.1% SDS and 0.02% dithiothreitol. The eluent was collected in 5-mL fractions and the absorbances of each fraction at 280 nm (proteins) and 330 nm (retinyl–protein complex) were measured. 3. Results 3.1. Amounts and composition of retinoids Fig. 1A1 and B1 show the HPLC chromatograms of extracts from the whole egg yolks of loggerhead turtles and Japanese quail. High levels of RAL were detected in the eggs of both reptilian and avian species. RAL was the major retinoid component in the turtle eggs, with a small amount of ROL, although considerable amounts of both ROL and RAL were present in the quail eggs. Table 1 shows the contents (μg/g yolk) and concentrations (ng/mg protein) of retinoids in the egg yolks. RAL, ROL and RE were present in varying amounts in the different species. Fig. 2 summarizes the retinoid compositions of the eggs examined in the present and
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Fig. 1. High-performance liquid chromatography analysis of retinoids in the whole egg yolk or yolk fractions from loggerhead turtles (A) and Japanese quail (B). The scale bar shows the absorbance at 360 nm. A1 and B1, extracts from whole egg yolk; A2 and B2, extracts from egg granule fractions; A3 and B3, extracts from egg plasma fractions. a and a′, syn and anti 11-cis retinaloximes; b and b′, syn and anti all-trans retinaloximes; c and c′, syn and anti all-trans 3,4-didehydroretinaloximes; d and d′, syn and anti 13-cis retinaloximes; e, all-trans retinol; f, 13-cis retinol.
previous studies. RAL was present in the eggs of every species of chordate examined, whereas storage of high levels of ROL was characteristic of avian eggs. RE was present in all the reptilian and avian eggs, but with considerable variation among species (Table 1, Fig. 2). The main RAL isomer detected in eggs was the all-trans form, with trace amounts of 11- and 13-cis isomers, depending on the species. The eggs of three reptilian species that lived in or near freshwater (red-eared slider, soft-shell turtle, Siamese crocodile) contained small amounts of 3,4-didehydroRAL (RAL2) isomers, whereas RAL2 isomers were scarcely detected in the eggs of loggerhead turtles (sea turtle). RAL2 isomers were not detected in any of the avian eggs examined. 3.2. Distribution and state of RAL and ROL in eggs Egg yolk suspensions were centrifuged to separate the yolkgranule and yolk-plasma fractions, and the retinoids in each fraction were then analyzed (Fig. 1A2, A3, B2, B3; Table 2). RAL was only
present in the yolk-granule fractions in both reptilian and avian eggs. These results suggest that the RAL in the eggs was bound to a yolkgranule protein in both reptiles and birds. No RAL was found in the yolk-granule or whole egg yolk extracts in any species after treatment with NaBH4, indicating that the egg RAL was bound to the proteins by Schiff-base linkages, similar to the situation in anamniotic chordates (Irie et al., 1991, 2003; Irie and Seki, 2002). The localization of ROL, however, differed between reptilian and avian eggs. A small amount of ROL was present in reptilian eggs and was mostly located in the yolk-granule fraction, where most of the RAL was also localized. ROL in avian eggs, in contrast, was found in the yolk-plasma fraction, where very little RAL was found. Thus, the distribution of ROL in egg yolk differed between reptiles and birds, in contrast to the common distribution of RAL. Most RE in the reptilian eggs was detected in the floating lipids, with little in the yolk-granule and plasma fractions. However, RE in avian eggs was detected in both the yolk-granule and the plasma fractions.
Table 1 Retinoid levels in egg yolks. Taxon
Species
n
Reptile
Loggerhead turtle Red-eared slider Soft-shell turtle Siamese crocodile Chicken Japanese quail Duck Common kestrel Ostrich
22 8 12 6 4 4 4 3 3
Bird
Retinoid content (μg/g yolk)
Retinoid concentration (ng/mg P)
RAL
ROL
RE
RAL
ROL
RE
2.36 ± 0.34 1.23 ± 0.52 1.46 ± 0.25 3.80 ± 0.19 3.25 ± 0.29 5.82 ± 0.42 2.99 ± 0.87 9.23 ± 1.29 1.96 ± 0.26
0.23 ± 0.05 0.28 ± 0.10 0.04 ± 0.03 0.36 ± 0.03 4.97 ± 0.72 8.96 ± 0.57 2.67 ± 1.01 4.15 ± 0.21 2.28 ± 0.38
0.32 ± 0.19 0.57 ± 0.34 0.13 ± 0.11 0.33 ± 0.04 1.04 ± 0.32 1.60 ± 0.28 0.34 ± 0.09 1.56 ± 0.52 0.77 ± 0.07
9.8 ± 2.1 4.9 ± 2.2 8.1 ± 1.5 19.9 ± 2.0 15.9 ± 2.3 25.7 ± 4.6 16.1 ± 5.1 58.6 ± 6.0 10.2 ± 2.3
0.97 ± 0.23 1.10 ± 0.43 0.20 ± 0.17 1.89 ± 0.17 24.4 ± 5.6 39.8 ± 8.6 14.5 ± 5.8 26.5 ± 1.9 11.6 ± 0.7
1.65 ± 0.63 2.29 ± 1.39 0.76 ± 0.65 1.54 ± 0.20 5.06 ± 1.70 7.05 ± 1.59 1.62 ± 0.45 9.61 ± 3.24 4.35 ± 0.41
Mean ± S.D.; n, number of eggs analyzed; mg P, mg of protein measured by Lowry's method; RAL, retinal; ROL, retinol; RE, retinylester.
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Fig. 2. Retinoid composition of whole egg yolk extract. 4) Seki et al., 1987; 5) Irie et al., 2002.
retinal;
retinylester; n, number of eggs analyzed 1) Irie et al., 2003; 2) Irie and Seki, 2002; 3) Irie et al., 1991;
retinol;
3.3. Analysis of the RAL-binding protein in quail eggs The protein components in the egg yolk fractions were analyzed using SDS–PAGE (quail yolk granules in Fig. 3B, e). High levels of yolkgranule and plasma proteins are present in avian eggs, as reported in the nutritional information provided for poultry eggs (Juneja and Kim, 1997), whereas the yolk-plasma fractions of reptilian eggs are poor in proteins (data not shown). The yolk-granule solution in NaCl-containing buffer (see Section 2.4) was treated with NaBH4 and SDS (Fig. 3B, f), and then chromatographed using Sephacryl S-300 (Fig. 3A). The chromatogram showed an absorption peak at 330 nm, which was derived from the retinyl–protein complex produced by NaBH4 treatment, and corresponded to the former part of the first protein peak indicated by absorption at 280 nm. Fig 3B, a–e shows the SDS–PAGE patterns of the fractions. Presumably, the first protein peak (Fig 3A, peak b) was heterogeneous; the peak would consist of several protein components with slightly different molecular weights. Although the protein components were not separated, RAL would be bound to the protein component with a highest molecular weight in the first protein peak of the chromatograph (Fig. 3A, peak a).
that RAL was the major storage retinoid in the eggs of several anamniotic vertebrates and a urochordate (Seki et al., 1987; Irie et al., 1991; Irie and Seki, 2002; Irie et al., 2002; 2003). Thus, RAL storage in eggs appears to be a common characteristic in oviparous chordates. RAL was found in the yolk-granule fraction in the eggs of every species examined in this study. In the poultry eggs, the yolk granules consist mostly of the high-density lipoproteins (lipovitellins) and phosvitin, which are vitellogenin-derived yolk proteins, whereas the yolk-plasma contains low-density lipoproteins and other minor protein components such as livetines, riboflavin- and biotin-binding proteins (Li-Chan et al., 1995). The results of our previous studies demonstrated that RAL was bound to lipovitellin 1 or its homologous protein in amphibian and teleostean eggs (Irie et al., 1991; Irie and Seki, 2002; Irie et al., 2002). The present study revealed that RAL in quail eggs was bound to RAL-binding proteins, which were assumed equivalent to those in amphibian and teleostean eggs, and that RAL in
4. Discussion The results of the present study demonstrate that RAL is stored in the egg yolks of both reptiles and birds. Our previous studies indicated Table 2 Retinoid levels in the egg yolk fractions. Species
Loggerhead turtle
Red-eared slider
Japanese quail
Fractions
Yolk granules Yolk-plasma Floating lipids Yolk granules Yolk-plasma Floating lipids Yolk granules Yolk-plasma
Retinoid content (μg/g yolk) RAL
ROL
RE
2.85 n.d. n.d. 1.04 n.d. n.d. 5.44 0.063
0.11 0.008 0.057 0.10 0.04 0.009 0.37 8.40
n.d. n.d. 0.24 n.d. n.d. 0.028 0.37 0.28
n.d., not detected; RAL, retinal; ROL, retinol; RE, retinylester.
Fig. 3. Gel chromatography of Japanese quail yolk-granule proteins treated with NaBH4, 2% SDS and 5% 2-mercaptoethanol, using a Sephacryl S-300 HR column (2.5 × 86 cm) equilibrated with 20 mM Tris-HCl buffer (pH 7.4) containing 0.1% SDS and 0.02% dithiothreitol (A), and SDS–polyacrylamide gel electrophoresis of the fractions and yolk-granule proteins (B). Absorbances at 280 nm (protein, ○) and 330 nm (retinyl product, ●, two-fold enlargement) of each fraction (5 mL) were measured after chromatography. a, Fr. 32; b, Fr. 36; c, Fr. 45; d, Fr. 58; e, yolk-granule proteins; f, yolkgranule proteins after treatment with NaBH4 and SDS; M, molecular weight markers.
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both reptilian and avian eggs was bound to proteins by Schiff-base linkages. Thus, not only RAL storage in eggs, but also its proteinbinding state, would be common throughout oviparous chordates. Vitellogenin is produced in the liver as a result of estrogen stimulation. RAL was detected in the blood plasma of estrogen-treated Xenopus laevis, and in the blood plasma fraction that contained vitellogenin (Azuma et al., 1993). Thus, egg RAL in oviparous vertebrates would be commonly derived from hepatic RE in the maternal liver, and transported to the oocytes via the blood circulation in association with vitellogenin. However, the details of these processes remain unclear. In contrast to the common storage of RAL in eggs of oviparous chordates, significant amounts of ROL, as well as RAL, were detected in avian eggs. High levels of ROL have previously been found in the eggs of several avian species (Royle, et al., 1999; Surai et al., 2000, 2001; Hargitai et al., 2006). Poultry eggs are known to be rich in vitamin A, which is derived from ROL. ROL storage in eggs therefore appears to be a common characteristic in birds. The amounts of ROL detected in chicken and quail eggs in the present study were similar to those reported in previous studies (Sivell et al., 1982; Watkins, 1995; Karadas et al., 2005). In contrast to the situation in avian eggs, only low levels of ROL were found in reptilian eggs. These results were consistent with those of Speake et al. (2001) and Thompson et al. (1999a, b) who found low vitamin A levels in reptilian eggs, due to the low or absent levels of ROL and RE. However, they did not quantify the amounts of RAL. These results imply that RAL is the main storage retinoid in reptilian eggs, as in the eggs of anamniotic chordates. Reptiles and birds are both amniotes. Although reptilian eggshells are often elastic, the morphological and functional characteristics of their eggs are similar, and the developmental features are virtually identical. However, the results of this study demonstrate that clear differences exist in terms of ROL storage. ROL in avian eggs was found mostly in the yolk-plasma fractions. Vieira et al. (1995) and Vieira (1998) reported that the specific TTRreceptor was present on the surface of the chicken oocytes, and that the holo-RBP4–TTR complex in the blood plasma was transported into oocytes mediated by the receptor. Involvement of the holo-RBP4–TTR complex in ROL-accumulating mechanisms in oocytes is suggested to be commonly distributed in birds. However, ROL in reptilian eggs was localized in the yolk-granule fractions, suggesting that ROL in these eggs may be derived from a vitellogenin-associated form, by nonspecific binding. TTR synthesis first appeared during vertebrate evolution in the choroid plexus of the stem reptile, but TTR synthesis in the liver subsequently evolved independently in birds, eutherians and some marsupial species (Harms et al., 1991; Schreiber and Richardson, 1997). TTR is barely detectable in the blood plasma of anamniotic vertebrates and reptiles (Harms, et al., 1991; Schreiber and Richardson, 1997; Power et al., 2000), though TTR genes are temporarily expressed in the liver in juvenile fish and in frog tadpoles (Richardson, 2009; Yamauchi and Ishihara, 2009). Richardson (2009) referred to the possibility that the maintenance of hepatic TTR synthesis in adulthood was related to homeothermy. ROL-accumulating mechanisms in eggs in poikilothermic vertebrates would therefore be insignificant, because of the absence of the holo-RBP4–TTR complex in the blood, despite the capacity of RBP4 from several poikilothermic vertebrates to bind to human TTR in vitro (Shidoji and Muto, 1977; Shidoji et al., 1979; Berni et al., 1992). It is likely that ROL accumulation into oocytes is mostly due to the mechanisms mediated by the TTR-receptor, and that the mechanisms were acquired after the acquisition of the holo-RBP–TTR complex in the blood by hepatic TTR synthesis. RE in the reptilian yolk was located mostly in the floating lipids. Our previous study also demonstrated that RE in teleostean eggs was localized in the lipid bodies (Irie and Seki, 2002). It is possible that the RE-accumulating mechanisms in reptilian oocytes are identical to those of teleosts, although these mechanisms remain unidentified.
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Fig. 4. Hypothetical scheme for acquisition of retinol-accumulating mechanisms into oocytes (bold lines) during amniote phylogeny, although there is some debate as to whether the squamates or turtles diverged first. The mechanisms are likely to have been acquired during the evolutionary process shown by the bold lines (between the crocodile–dinosaur split and the diversification of avian orders) in the Mesozoic era.
In conclusion, we propose that the mechanisms responsible for RAL accumulation into oocytes and the storage of RAL in eggs were acquired before the appearance of the vertebrates (cf. Irie et al., 2004), and that they have subsequently been conserved throughout the evolution of oviparous chordates. The mechanism of ROL accumulation into oocytes, however, which is mediated by the specific TTRreceptor, was acquired much later than that for RAL storage. It is widely believed that birds diverged from the Theropoda, a group of so-called dinosaurs. Crocodiles are the closest living reptilian relatives of birds (Iwabe et al., 2005). The mechanism of ROL storage found in avian eggs must therefore have been acquired during the period of evolution between the crocodile–dinosaur split and the diversification of the different avian orders (Fig. 4), in the Mesozoic era. To the best of our knowledge, retinoids have not been examined in mammalian eggs or oocytes. However, eutherians (placental mammals) have lost the capacity to synthesize vitellogenin during evolution. Because eutherians lack the RAL transporter, RAL is unlikely to be accumulated into eutherian oocytes. In the monotremes, however, the vitellogenin gene cluster is conserved in the platypus (Brawand et al., 2008; Babin, 2008), suggesting that RAL could possibly be incorporated into monotreme oocytes. No TTR was detected in monotreme blood (Schreiber and Richardson, 1997; Richardson, 2009), implying that ROL may not be accumulated into monotreme oocytes, and RAL would therefore be the major form of retinoid stored in monotreme eggs, as in reptilian eggs. Further studies are needed to test the validity of these predictions. Acknowledgments The authors are grateful to the following people for the donation of eggs (in order of supply date): Takeyoshi Tochimoto (Himeji City Aquarium, red-eared slider), Kazuo Ashida (Ashida Kikaku Ltd., softshell turtle), Yoshio Hamano (Akita Prefectural College of Agriculture, ostrich), Mamoru Komatsu (Akita City Omoriyama Zoo, common kestrel), Yoshimasa Matsuzawa (Sea Turtle Association of Japan, NPO, loggerhead turtle), Kazuyoshi Omuta (Yakushima Umigame-kan, NPO, loggerhead turtle), Katsuhiro Koike (Koike Wani Souhonpo Co. Ltd., Siamese crocodile). We would also like to thank Naosuke Kojima, Nobuyo Higashi-Kuwata, Kiwamu Yoshikawa and Mitsuru Sato (Akita University Graduate School of Medicine) for their cooperation and valuable discussions during these experiments. References Al-Hasani, S.M., Parrish, D.B., 1972. Forms of vitamin A and of carotenoids in tissues, blood serum and yolk of eggs from Coturnix coturnix japonica fed β-apo-carotenals. J. Nutr. 102, 1437–1440. Azuma, M., Irie, T., Seki, T., 1993. Retinals and retinols induced by estrogen in the blood plasma of Xenopus laevis. J. Exp. Biol. 178, 89–96.
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