An Electrophoretic Study of LDH-Isozyme Distribution in Developing Embryonic and Adult Tissues of the Toad Bufo arabicus

Lactate (LDH) dehydrogenases were studied by electrophoresis to investigate the possibility of isozymic differences in different stages and organs of B. arabicus embryos, tadpoles and adults. The expression pattern of lactate dehydrogenase (LDH) isozymes was analysed using nondenaturing discontinuous polyacrylamide gel electrophoresis (PAGE). Five LDH isozymes were observed in five different adult tissues. The most cathodal isozyme, LDH-5 (A4), showed the strongest activity. All embryonic stages tested displayed the anodic isozymes first. Four LDH isozymes were present in the stages 1-16 (fertilized egg-tail bud stage). The first appearance of the fifth isozyme was detected in stage 17 when the muscular response started. This isozyme showed gradual increase in intensity from stage 17 to stage 43.


Introduction
or a range of animal groups, 'model' species are now well characterized in terms of the morphology and molecular biology of their embryonic and early larval stages.For anuran amphibians, the best characterized species is Xenopus laevis.However, if we are to understand the ways in which development has diverged within animal groups, and adapted to different environments (Gilbert, 2001), we need to examine both morphology and molecular expression in a variety of related species.
Molecular analysis provides additional information on species identity in areas where mixed genetic types are present (Simovich and Sassman, 1986).During cell differentiation, genes play an important role in metabolism, so multilocus isozymes constitute probes for investigating the mechanisms of spatial and temporal gene regulation in different stages of development (Philipp et al., 1979).Thus the study of isozymes provides considerable insight into the structure and function of metabolic enzymes.Lactate dehydrogenase (LDH) is considered a suitable system for studying several metabolic, genetic and ecological features (Coppes, 1990;Ropson et al., 1990;Almeida-Val et al., 1992).In most vertebrates, LDH is a tetrameric enzyme composed of two kinds of subunit polypeptides, A and B (Appella and Markert, 1961).LDH isozyme patterns are species and tissue specific and their expression patterns vary during development and several studies have been conducted on the electrophoretic distribution of LDH in amphibians (Mathews, 1975;Harintz and Diehl, 1999).This study aims to investigate the ontogeny of the enzyme lactate dehydrogenase (LDH) in some tissue of the Bufo arabicus embryos, tadpoles and adults.

Materials and methods
Embryos and tadpoles of stages 1-18, 21-25, 29, 31, 35, and 43 (Ba-Omar et al. 2004) were obtained by induced fertilization.Adult toads were killed by pithing and the following tissues were collected skeletal muscle, heart, liver, lung and stomach (Ambusaidi, 2002).The embryos and tadpoles were first washed in distilled water, then frozen in liquid nitrogen and stored at -70 ºC (Beis et al. 1992).The adult organs were dissected out, kept in ice, and washed with distilled water prior to the protein extract being prepared.
With slight modifications, the method of Markert et al. (1998) was used to analyse LDH isozymes.Thawed embryos and tadpoles were homogenized in 250-500 µl of sterile de-ionized water in an ice bath with a tissue tearor (Biospec products, USA) for 3-5 minutes depending on the sample size.The same procedure was used for adult organs using 1.0 ml of sterile de-ionized water.The homogenate was then centrifuged at low speed (X 2000g) in a microcentrifuge (Eppendorf 5402, Germany) for 15 minutes at 4º C. The supernatant was then taken and centrifuged at high speed (X 16000g) for 30 minutes at 4ºC.The resulting supernatant was removed for LDH analysis by electrophoresis on a polyacrylamide gel.
LDH isozymes were separated using non-denaturing discontinuous polyacrylamide gel electrophoresis following the method of Markert et al (1998).LDH isotrol (Sigma Co, USA) was used as a marker.LDH isozyme activity was visualized by incubating the gel at 37° C in the dark, in a reaction mixture specific for LDH, consisting of 20 ml 0.35 M Tris-HCl (pH 8.0), 80 ml de-ionized water, 2 ml nicotinamide adenine dinucleotide (Sigma Co, USA) (10 mg/ml), 2 ml nitro-blue tetrazolium (Sigma Co, USA) (10 mg/ml), 0.1 ml phenazine methosulphate (Sigma Co, USA) (10 mg/ml) and 2 ml of (3.0 M) lactic acid syrup (BDH Co, UK) until bands appeared after about 15 minutes.To determine whether any enzyme activity resulted from the activity of alcohol dehydrogenase, control gels were incubated in the absence of the substrate (lactate).

Results
The electrophoretic pattern of the LDH isozymes of the toad B. arabicus was distinctly different from those of the LDH isotrol standards from human tissue (Figure 1 lane m).The bands revealed by LDH specific stain were due to LDH activity.In the absence of a lactate substrate, no bands appeared.This confirms that the observed bands were produced by LDH activity and not by alcohol dehydrogenase or impurities (Figures 1-4).LDH-4 (A 3 B 1 )

Electrophoretic pattern of LDH in adult tissues
LDH activity was detected in all tissues examined.Five distinct bands appeared on the polyacrylamide gel and these were numbered from 1-5 in their order of migration from the cathode to the anode (Figure 1).The most cathodic band was the homotetramer isozyme LDH-5 (A 4 ) and the most anodic band was the homotetramer isozyme LDH-1 (B 4 ).The three intermediate heterotetramers from cathode to anode were LDH-4 (A 3 B 1 ), LDH-3 (A 2 B 2 ) and LDH-2 (A 1 B 3 ).All five isozymes LDH-5 to LDH-1 were observed in all adult tissues examined and the most cathodic isozyme (LDH-5 -A 4 ) showed the strongest activity.
In skeletal muscle and liver (Figure 1 lanes sm and li respectively) the most cathodal isozyme (LDH-5 -A 4 ) predominated and appeared as a very broad band, especially in skeletal muscle, followed by LDH-4 (A 3 B 1 ).The other isozymes [LDH-3 (A 2 B 2 ), LDH-2 (A 1 B 3 ) and LDH-1 (B 4 )] were only indicated by faint bands, especially in the skeletal muscle.The heart displayed all five bands with high intensity coloration (Figure 1 lane h).LDH-1 (B 4 ) was highly intense in the heart (Figure1 lane h) and secondly in the stomach (Figure 1

Discussion
The ontogeny of lactate dehydrogenase (LDH) expression in the toad B. arabicus, embryos, tadpoles and some adult organs, shown by electrophoresis gives a five-band pattern.The expression of LDH genes is not tissue-specific in B. arabicus since all five isozymes appeared in all organs examined, although the isozyme A 4 predominated in skeletal muscle and the liver.Similar observations were also reported by Hranitz and Diehl, (2000) in their study of B. woodhousii fowleri.The isozyme A 4 always predominated in skeletal muscle, confirming its metabolic role in reducing pyruvate during the anaerobic glycolysis.Vonwyl (1983), in a study of X. l. laevis, X. vestitus, X. borealis and X. wittei, claimed that the multibanded pattern of LDH isozymes or the presence of secondary isozymes facilitates the identification of the species and contributes strongly to a species specific pattern .
B. arabicus exhibits anodal bands in early embryonic stages (stages 1-16).Moyer et al., (1968), Kunz (1973) and Vonwyl (1983) showed that the species they studied are characterized by the appearance of anodal bands in oocytes and early embryonic stages.In B. arabicus, the first appearance of the fifth isozyme (LDH-5, A 4 ) was at stage 17, which coincides with the onset of muscular contraction.This can be explained by the synthesis of the A 4 homotetramer isozyme at this stage.According to Kunz (1973), the LDH pattern from the newly fertilized egg to early tail bud (stages 1-24) is the same and a change is observed at stage 25, when isozyme IX appears and this may be correlated with the onset of contractility.Moyer et al. (1968) indicated that clear evidence of the new isozyme was found around stage 19 when the heart starts beating.Basu et al., (1992) claimed that the initial predominance of B 4 over A 4 in fish up to the onset of muscular contraction (stage 17 in B. arabicus of ontogenesis) indicates that cell differentiation of the heart and kidney takes place earlier and retarded expression of A 4 may indicate that skeletal muscular cell differentiation and activity is somewhat delayed during ontogenesis.On the other hand, Goldberg et al., (1969) described the predominance of B subunits in early embryonic stages of lake and brook trout, with a gradual increase in the activity of A-containing isozymes as they develop.It seemed to be that LDH isozyme composition of unfertilized and cleavage embryos revealed that more B subunits than A subunits had been synthesized prior to or during these development stages.
It is possible that the synthesis of different isozymes is mediated by preformed stable mRNA during the process of oogenesis.Goldberg et al., (1969) indicated that at gastrulation, the increase in proportion of LDH-5 suggests that new mRNA synthesis takes place providing more A subunits for tetramer assembly at a time when the synthesis of many molecules is accelerated in embryos.
In B. arabicus, it is possible that new synthesis of mRNA takes place when muscular contraction starts (stage 17) which provides more A subunits for the synthesis of the homotetramer LDH-5 (A 4 ).

Figure 3 .
Figure 3. Electrophoretic patterns of LDH of B. arabicus embryos (stages 10-18).The numbers at the top refer to the developmental stages of B. arabicus embryos.Marker (m).