_self">Figure 4(a) and Figure 4(b)).
10) PAS Reaction
PAS-technique revealed an intensive amount of glycogen in early stage (46) of development (Figure 5(a)), negatively reaction at stages 48 a 66 (Figure 5(b) and Figure 5(d)) and moderate amount of glycogen at stage 52
Figure 3. Sections of the liver of Bufo regularis tadpoles showing (a) stage 54, (b) stage 55, (c) stage 64, (d) stage 66, blood sinusoids (bs), blood vessel (bv), degeneration (de), eccentric nuclei (ec), endothelial cells (en), hepatocytes (h), kupffer cells (kc), leucocytes (le), lipid droplets (l), pyknosis (p), (H & E, X 400 and scale bar = 25 µm).
Figure 4. Sections of the liver of Bufo regularis tadpoles (stage 66) showing positively stained connective tissue, apoptotic nuclei (ap), bile duct (bd), bile ductiulies (bds), blood sinusoids (bs), hepatocytes (h), hepatic acini (ha), Kupffer cells (kc), large blood vessel (lbv), melanomacrophage (m) (Milligan’s trichrome stain, (a) X 100, (b) X 400 and scale bar = 25 µm).
Figure 5. Sections of the liver of Bufo regularis tadpoles showing a huge amount of glycogen in (a) stage 46, negatively reaction in (b) stage 48 and (d) stage 66, moderately amount of glycogen in (c) stage 52, and positively stained nuclei in (b). Bile duct (bd), blood vessel (bv), nucleus (n) (PAS reaction without diastase enzyme, X 400 and scale bar = 25 µm).
(Figure 5(c)). In comparison between sections 5b and 5d the investigators noticed positively stained nuclei at stage 48 (Figure 5(b)) and negatively stained nuclei at stage 66 which might be due to apoptosis.
3.2.1 Back Body Skin
1) Stage 44
The skin consists of two layers epidermis and dermis; the first layer (epidermis) consisted of only one layer of cells based on the basement membrane. The basement membrane is too thin to be seen by light microscope. Some cells are oval in shape with oval nuclei and others are polygonal in shape with rounded nuclei. Epidermal melanophores appeared at this layer. The dermis consisted of collagenous layer appeared underneath the basement membrane separating the dermis from epidermis, loosely connective tissue, dermal melanophores and fibroblast cells (Figure 6(a)).
2) Stage 48
The epidermis at this stage consisted of two types of cells: polygonal and skin cells. Epidermal melanin pigments dispersed within the epithelial cells. No drastic changes were noticed in the dermis at this stage apart from the increase in the dermal layer which contains blood capillary and dermal melanophores with dispersed melanin pigments within their processes (Figure 6(b)).
3) Stage 54
Apical layer of the epiderms contains micro processes which contains secretion, dermal layer of the skin more thick and contains loosely connective tissue with all contents as shown in (Figure 6(c)). Semithin section of the skin at stage 54 stained with Toluidine blue showing the epidermal layer cells of two types polygonal cells with faintly stained nuclei interposed with triangular cells (skein cells) and also divided cells were noticed. The dermis consists of loose connective tissue separated from the epidermis by a collagen layer. Fibroblast found underneath this layer in contact with it. Blood capillaries, pigmented melanophores processes were seen (Figure 6(e)).
4) Stage 55
Epidermal layer of the skin contains two types of cells, the first one polygonal cells with oval nuclei and slightly basophilic cytoplasm, and the second one is skein cells (triangular cells) with dark triangular nuclei and dark basophilic cytoplasm, underneath this layer there was a collagenous fibers which is attached with fibroblast
Figure 6. Sections and semithin section of the back body skin of Bufo regularis tadpoles stage 44, (b) stage 48, (c) and (e) stage 54, (d) stage 55 showing, blood vessel (bv), collagenous fiber (co), dermal layer (d), dermal melanophores (dmp), epidermal melanophores (emp), fibroblast cells (fb), mitotic figure (mf), mucous secretion (ms), nucleus (n), polygonal cells (po), skein cells (sk), (a)-(d), H & E. X 1000 and scale bar = 10 µm) ((e), Toluidine blue. X 1000 and scale bar = 10 mm).
cells of the dermal layer, dermal melanophores also appeared in the dermal layer. This layer consisted of loose connective tissue with collagenous bundles and contains different types of connective tissue cells, blood and lymphatic vessels also shown in this layer (Figure 6(d)).
5) Stage 64
Epidermal layer appeared as stratified columnar epithelial tissue which contains basally located columnar cells with oval nuclei at the base (germinal layer). This layer is separated from the dermis by a basement membrane, too thin to be resolved by light microscope, polygonal cells with rounded nuclei in the middle, but in the outer surface columnar cells with oval nuclei; also skein cells appeared in the outer layer of the skin. Epidermal melanophores invaded the cells of this layer. Dermal layer contains two types of glands (mucous and poison glands). Mucous glands lined with cubic cells with rounded nuclei and contain mucous secretions. The poisonous glands are large and distinguished by their wall being formed of a syncytium, that is to say the cell membrane between the cells which compose it has disappeared so that their nuclei appeared in a continuous mass of protoplasm. Dermal layer also contains loosely and compact connective tissue. Collagenous fibers began to appear in compact layer at this stage, this layer contained different types of connective tissue cells and melanophores. Blood and lymph vessel also appeared as shown in (Figure 7(a) and Figure 7(b)).
6) Stage 66
Epidermal layer consists of stratified squamous epithelial tissue based on the basement membrane; dermal layer contains mucous and poisonous glands which are open by tube in the surface to excrete their secretion. Flattened faintly stained fibroblast cells with their flattened nuclei are aggregated underneath the basement membrane. Wide lymph vessel also appeared in the dermal layer. Thickness of epidermal and dermal layer increased (Figure 7(c) and Figure 7(d)).
7) Milligan’s Trichrome Stain
Stage 54, as shown in Figure 8(a), section of skin showed dermal layer contains faintly staining collagenous fibers.
Stage 55, as shown in Figure 8(b), collagenous fibers are highly stained with Milligan’s trichrome stain.
Stage 64 and stage 66 are presented in Figure 8(c) and Figure 8(d) showing collagenous fibers strongly stained with Milligan’s trichrome stain, also epithelial tissue which lined the glands stained with Milligan’s trichrome. Connective tissue and glandular granules stained in light green by these stain. The granules stained light green
Figure 7. Sections of the back body skin of Bufo regularis tadpoles (a) & (b) stage 64, (c) and (d) stage 66 showing, collagenous fibers (co), dermal melanophores (dmp), epidermal melanophores (emp), fibroblast cells (fb), dermal layer (d), mitotic figure (mf), mucous gland (mg), poisonous gland (pg) and stratified squmous epithelial tissue (st). H & E, (a) and (c), X 400, scale bar = 50 µm), (b) and (d), X 1000, scale bar = 10 µm.
also by this stain. Mucous glands negatively stained.
8) PAS Reaction
Section staining with PAS reaction: collagen layer, granules in the lumen of the poisonous gland, also around these glands and the connective tissues strongly stained with PAS (Figure 9).
3.2.2. Tail Skin
Comparing the histological structure of the tail skin at stage 44 with that at stage 64 we noticed that the epidermal layers in both consisted of one layer only. Stage 44 is as described previously in the back body skin. The epidermis was noticed to be consisted of only one layer of cells based on the basement membrane. The basement membrane is too thin to be resolved by light microscope. Some cells are oval in shape with oval nuclei and others are polygonal in shape with rounded nuclei (Figure 10(a)).
In histological structure of the tail skin at stage 64 the epidermis still also consisted of one layer of cells, this layer suffering from histolysis (Figure 10(b)).
Figure 8. Sections of the back body skin of Bufo regularis tadpoles showing (a) stage 54, (b) stage 55, (c) stage 64, (d) stage 66, compact layer (cl), collagenous fibers (co), dermal melanophores (dmp), epidermal melanophores (emp), mucous secretion (ms), mucous gland (mg), poisonous gland (pg), polygonal cells (po), spongy layer (sl), stratified squamous epithelial tissue (st) (Milligan’s trichrome stain, X 400 and scale bar = 50 µm).
Figure 9. Section of the back body skin of Bufo regularis tadpoles (stage 66) showing positively stained granules with lumen. Collagenous fibers (co), dermal melanophores (dmp), epidermal melanophores (emp), poisonous gland (pg), stratified squmous epithelial tissue (st) (PAS reaction and H, X 400 and scale bar = 50 µm).
Figure 10. Sections of the tail skin of Bufo regularis tadpoles (a) Stage 44 showing epidermis and collagen layer, (b) Stage 64 showing break down and disintegration epithelial layer, as well as disappearance of the collagenous layer underneath the epidermis; dermal melanophores (dmp), epidermal melanophores (emp), polygonal cells (po), skein cells (sk) (H & E, X 1000 and scale bar = 10 µm).
In the present investigation the histogenesis of the liver of Bufo regularis tadpoles was studied from stage 44 (the beginning of feeding) up to metamorphosis climax by using the light microscope. The investigators noticed that the hepatocyte increased in size with the increase of the tadpole in age. The young tadpole had well developed hepatocytes. Many vacuoles with different sizes field the hepatocytes later and then these vacuoles fused together. Large amount of glycogen were observed in the liver cells during the active period of feeding stage (46), of the tadpoles and this amount dropped at stage (54) and lipid droplets were noticed in many hepatocytes. Olurin et al. stated that the formation of vacuoles in hepatocytes is due to the degeneration of cell membranes  and an imbalance between the rate of synthesis and utilization of substances in cells  . In contrast, Crawshaw and Winkle mentioned that hepatocyte vacuolation representing fat and glycogen stores is a common histological finding in amphibians, and in many cases cannot be considered a pathological case  . Hepatic lipidosis is likely a consequence of the mobilization of fat depots in response to anorexia or starvation.
Griffiths stated that the transformation of the larval stomach into the adult organ takes place in a period between 7 and 10 days  . Dodd and Dodd (1976) noticed that during this period the animal stops to ingest food and keeps fasting  . Their findings were in coordination with the present investigation. Where we noticed moderate amount of glycogen at stage 64 and then suddenly declined at stage 66. This may be in a synchrony with the emission of the forelimbs.
The number of melanomacrophages and Kuppfer cells decreased as development progressed. The number of pyknotic nuclei of the hepatocytes, macrophages and lymphocytes increased within the blood sinusoids near the hepatocytes as development progressed. In the present work dilatation in blood sinusoids was noticed. Sinusoidal dialation in the liver is attributed to the impairment of outflow of the hepatic veins  .
 found the visual cells of developing tadpole contain glycogen in their nuclei which indicates pathological degeneration. In the present study the results coincide with the observations of  , where glycogen granuules were seen in some nuclei of developing hepatocytes and disappeared at the commencement of metamorphosis. These nuclei may be considered in their way for apoptosis.
The histological transformation of amphibian liver during metamorphosis was studied by using the light and EM microscopes and DNA fragmentation in the tadpoles of Bufo regularis, Bufo viridis, Rana bedriagae and Ptychadena mascareniesis by  . They found in their study, the frequency of cell death was high variable depending on the developmental stage and the species of anuran amphibians used. Furthermore, apoptosis occurred in some species as a single wave; whereas at least two distinct waves could be detected in other species. In contrast, programmed cell death (apoptosis) in anuran amphibian embryos starts at the onset of gastrulation and then continues during gastrulation and neurulation  . During these early stages, apoptosis seems to occur without obvious peaks in time or space  .  mentioned that deletion of hepatic cells during embryonic development and spontaneous metamorphosis of the four tested species of anuran amphibians is accomplished by ultrastructurally typical apoptosis. The morphological appearance being essentially the same as those described in vertebrate embryos  - . The phenomenon involves a stereo typed sequence of changes and begins with peripheral aggregation of nuclear chromatin. This is followed by nuclear fragmentation and cytoplasmic condensation associated with prolific budding to produce membrane-bound apoptotic bodies of varying sizes. As is the case in other solid tissues, the actual processes of budding is rarely observed, possibly viable cells engulf the condensing ones   - .
During metamorphosis and particularly the climax, severe cell death could be observed in the liver of the anuran species. Interestingly, cell death started before the tail desorption and it was observable over a long period from stage 58 (beginning of climax and tail regression), cell death in the liver was increasingly observed   and  found heavy cell death affecting the liver prior to climax (stages 54 to 58). This observation fits with studies of hepatic resident macrophage activity   - . Macrophages initially appeared at early climax, increased rapidly in number during muscle regression and reach peak density at late climax    and these results are similar to our observation, where we observed many macrophages in the sinusoidal lumen near the hepatic cells at stage 54 as shown in the semithin sections.
In the present work haematopiotic tissues were noticed within the blood sinusoids and underneath the hepatic capsule. The liver plays an important role in hematopoiesis in the amphibian embryo. Histiocytic and erythroblastic cell types predominate, but lymphocytic and granulocytic precursors are also present. After metamorphosis, other organs such as the bone marrow take over this role, but residual activity may be seen in adult amphibians, particularly beneath the liver capsule. The liver also has a significant immune function. In addition to leukocytopoiesis, the large population of melanomacrophages and the related Kupffer cells that line the sinusoids act both as phagocytes and in antigen recognition  . In the postembryonic stages, they scavenge embryonic erythrocytes  - . Melanin is a complex polymer that can absorb and neutralize free radicals and other toxic agents   . Melanomacrophages are focal accumulations of pigmented macrophages in liver, spleen and more rarely in kidneys of fishes  and also in liver of frogs  . These may contain four types of brown to black pigments, namely melanin, lipofuscin, ceroid, and hemosiderin/ferritin. Melanomacrophage proliferation has been associated with several natural factors, such as normal aging, starvation, and infectious diseases  . Hepatic melanomacrophages appear to increase in number also with antigenic stimulation, and with other factors causing tissue breakdown  . Increased density of these macrophages has also been observed in experimentally treated fish  , amphibians  , and naturally polluted frogs  .
Amphibian metamorphosis provides an excellent model for studying the physiological cell death as in keratinization or terminal differentiation of body epidermis (diffpoptosis) and tail tissue regression serves as a model of programmed cell death (PCD) (apoptosis)  . Additionally, amphibian metamorphosing skin is one of the best studied examples of an organ that remodels in response to thyroid hormones. Modification of ionic calcium concentration also markedly alters the pattern of proliferation and differentiation in amphibian epidermal cells in vitro  . Remodeling of existing organs is accomplished by the death of larval cells and the growth and differentiation of adult stem cells   .
Amphibian epidermis changes its nature during metamorphosis from a noncornified bilayered epidermis to a stratified epidermis with actively dividing basal cells and a keratinized outer layer  .
The epidermis is one of many amphibian tadpole tissues that are remodeled at metamorphosis   . The tadpole epidermis consists of two to three cell layers in contact with a cellular collagen lamella. The premetamorphic dermis consists of the collagen lamella, an adjacent single layer of subepithelial fibroblasts, and melanophores  . These results are in accordance with the present work. As thyroid hormone (TH) concentration raises in the tadpole during metamorphic climax the outer two cell layers of the epidermis are thought to die by apoptosis  . The basal cells of the tadpole epidermis are progenitors for the adult frog epidermis. At metamorphosis the subepithelial fibroblasts secrete proteolytic enzymes such as collagenase-3 and invade the collagen lamella, the dermis thickens, and adult skin glands form  .
In the present work we observed that the epidermis of tadpole back body skin consisted of one layer at stage 44 and the tail epidermis at stage 64 consisted only of a disintegrated one layer.  mentioned that during metamorphosis the tissue develops adult skin in the body region and histolyzes larval skin in the tail region. These results are in accordance with the present work.
    stated that at early stages of development, there are no differences between the tail and body epidermis. As metamorphosis progresses at late stages, basal cells appeared in the body epidermis which proliferate and differentiate into germinative basal cells of the adult-type of keratinized epidermis. These results are in agreement with the present work.
In the present work using light microscope, we noticed two types of cells forming the body skin in the tadpoles. These cells were skein and polygonal cells rested on the basement membrane.  describe skein cells as the cells which have large intracellular bundles of tonofilaments known as Figure of Eberth’ which are connected by numerous microvilli.
Tail dermis probably loses the ability to develop the normal structure of epidermis-dermis junction during metamorphosis  spontaneous metamorphosis, the initial sign of degeneration of tail skin is detected in the dermal components, followed by degeneration in the epidermis  . This result is in agreement with the present investigation, where we noticed that there was no any componants of the connective tissues in the late stage of development of the tail dermis such as collagene layer underneath the epidermis, fibroblast or blood vessels as those observed in the epidermis of the body skin.
In the present work we noticed at stage 64 two types of glands, mucous and poisonous glands. In agreement with  described two types of glands in the amphibian skin, both of them alveolar (or acinar): mucous glands, and granular or serous glands. Other types have been described in certain anuran species  .
Genesis of the cutaneous glands has been studied in the anurans   . These glands originated from the epidermis. In the basal portion of this tissue, there arose groups of cells, generated perhaps through the division of a single precursor cell  . These groups form the gland primordia or gland rudiments   . The cells of these primordia then divide, migrate in the direction of the stratum spongiosum of the dermis  and differentiate in the cell layers of the epithelial portion of the gland alveolus   .
The granular glands produce a toxic or repellent secretion with an effect on various vertebrate species; this is one of the principal elements in amphibian defense. In the course of the secretory cycle, these cells usually lose their limits and turn into a syncytium   . In anuran species, the granular glands, in the present work named poisonous glands, possess secretory layer, syncytial in its nature  . In various species of apodans and urodelans, the secretory layer consists of individualized cells   at least at the beginning of the secretory cycle   . The granular glands are generally made up of a gland alveolus formed by a secretory layer covered externally by a myoepithelial layer. This contractile layer is related with the extrusion of glandular products   .
In conclusion, the result obtained from this study indicates that the amphibian metamorphosis is a complex process that has been speculated to involve histogenesis in most of body organs especially liver and skin as target organs in the present investigation. Histochemical studies display different values during amphibian development and can be used as an investigation tool for the evaluation of the histological changes of the Egyptian toad during metamorphosis.
This research carried out at Zoology Department, Faculty of science, Assiut University, Assiut, Egypt and funded by the Ministry Higher Education from Libyan Government.