Indian Journal of Experimental Biology Vol. 39, July 2001 , pp. 629-635

Thymic structural changes in relation to seasonal cycle and testosterone administration in wall lizard Hemidactylus jlaviviridis (Ruppell)

B Hareramadas & U Rai * Department of Zoology, University of Delhi , Delhi 110007. India

Received 10 September 2000; revised 26 March 2001

Light microscopic and ultrastructural studies of thymus in wall lizard showed remarkable season dependent structural changes. In winter, the thymus was involuted and its cortico-medullary differentiation was not di stinct. Thymocytes were sparsely distributed. The epithelial cells exhibited atrophic features such as an appreciable decrease in the nuclear­cytoplasmic ratio and accordingly reduction in cell organelles. The reconstruction of thymus commenced during spring and it became fully developed with marked delineation of cortico-medullary regions during summer. The thymus was then densely populated with thymocytes and epithelial cells showed voluminous cytoplasm having numerous cell organelles. The thymus regression started again by the beginning of autumn. The results suggest that the thymic development in wall lizard have inverse relationship with the androgen level, as the testicular steroidogenic activity was seen maximum during winter and least in summer. This assumption gets support by castration and testosterone replacement experiments. Castration of lizards during winter resulted in profound development of thymus with an appreciable increase in thymocytes mainly in the cortex region . The cortex became delineated from the medulla. Following testosterone treatment , the thymus underwent regression and was comparable to testis-intact lizard' s thymus during winter season. After withdrawal of testosterone treatment, the thymus exhibited dense lymphoid and thymocyte population with a demarcation of cortico-medullary regions and sub-cortical region was regenerated .

Thymus is the primary lymphoid organ and plays an important role in the evolution of adaptive immunity i. e. the capability to react specifically against foreign substances by a specific process involving cellular proliferation and retention of a memory of that reaction which will result in heightened response upon being challenged 1.2_ It provides the microenvironment for the development of bone marrow-derived uncommitted lymphoid progenitor (stem) cells into mature T lymphocytes. It performs stringent T cell selection and maintains the tissue homeostasis by keeping T cells that can distinguish the self from non-self i. e. positive selection and by deleting any cells that are reactive against self antigens i.e. negative selection3

. The thymus in mammals become atrophied and impregnated with adipose tissue at puberty whereas in adult reptiles, like other ectotherms, the thymus undergoes seasonal changes. However, the effect of seasonal changes on thymic activity in reptiles presents a confusing picture. In some lizards, the thymus remain well developed and its cortico-medullary regions are well

*Correspondent author Fax: 7257524 E-mail: harib2k@yahou.co.in

differentiated during summer while gradual involution occurs in autumn and total regression during winter with the disappearance of cortico-medullary delineation4

-6

. In contrast, a profoundly involuted thymus has been reported during summer in snake Psammophis schokari7 and in turtle Mauremys caspica8

-10

• However, all these investigations except Leceta et al. 8 in Mauremis cas pica have dealt with light microscopic study.

The wall lizard Hemidactylus flaviviridis hibernates during winter to cope with the subsiding environmental temperatures. The survival rate of H. flaviviridis in captivity decreases drastically duri ng winter when they were taken out from their hideouts and kept in captivity while in summer no mortality occurs in captivity. Since the secretion of sex steroids . 'I . k b d d II " tn reptt es IS nown to e temperature epen ent · -and the circulating levels of sex steroids has been demonstrated to have an inverse relationship with thymic activity in mammals13

· 14

, the high mortality in winter may be presumed due to suppressive effect of sex steroids on thymus. Hence, the present study has been undertaken to ascertain the light microscopic and ultrastructural changes in the thymic structure of wall lizard H. flaviviridis in relation to seasonal changes and following testosterone administration .

630 INDIAN J EXP BIOL, JULY 200 I

·Materials and Methods The wall lizard H. flaviviridis is a seasonal breeder.

The spermatogenically active phase extends from November to April , quiescent phase from May to August and recrudescent phase from September to October 15

.

Adul t male lizards weigh ing 8 to I Og were purchased from a local supplier in February. After acclimating to the laboratory condition fo r a week, they were divided into four groups, each consisting of 5 li zards. Animals of the first three groups were castrated bilaterally under ether anesthesia, whereas the li zards of 4' 11 group were sham operated . After 7 days, the I" group received 111Jections of dihydrotestosterone (OJ-IT) subcutaneously on every alternate day at the dose of I 0 ng in 0.05mlllizard/day for 5 days. Each animal of the 2"d group was first administered wi th the above mentioned dose of OJ-IT for 5 days and subsequently the treatment was withdrawn .. The castrated li zards and sham operated li zards belonging to 3'd and 4111 groups respectively , received only veh icle (0.05 ml propylene glycol! li zard/day on alternate day fo r 5 days) of the

. b . sl 3 rd d 4'" hormone. The an1mal s elongmg to 1· , an groups were sacrificed with the use of chloroform a day after the last injection, whereas the li zards of 2"d group were sacrificed after 10 days of the withdrawal of the treatment. Their thymi were dissected out and processed for routine hi stological and electron

. . d' 16 17 T . . h I m1croscop1c stu 1es · . o 1nves t1 gate t e seasona changes in the thymic architecture, lizards were collected and examined during different seasons of the year i. e. su mmer, autumn , winter and spring.

Results The thymus in wall li zard H. flaviviridis is a

bilateral and bilobed organ located dorsoventral ly in

close assoc1at1on with carotid and jugular blood vessels, and vagus and hypoglossal nerves . It is delimited by a connective tissue capsule (Fig. 1) and differenti ated into outer cortex and inner medull a. Light microscopic and ultrastructural studies showed the predominance of thymocytes, which were identified by their dark and larger nuclei in relation to cytoplasm, in the cort ical region (Figs I and 2) . Besides thymocytes, reticular epithelial cell s, monocytes/macrophages, dendritic-like cell s and inter and intracellular epitheli al cysts were frequent ly seen in the medullary region. Many reticular epithelial cells extended stell ate cytoplasmic projections, which were joined together by desmosomes and fo rmed thymic parenchyma in both cortex and med ull a (Figs 3 and 4). These cel ls were charac terized by relati vely large nucleus with one or two prominent nucleoli . Numerous bundles of fine tonofilarnents and other cell organell es could be seen in their cytoplasm. Some of the epitheli al cel ls in medulla were seen to be involved in the format ion of intra (Fig. 5) and intercellular (Fig. 6) cysts. The wall of the intercellular cyst was made of both epithelial as well as non-epitheli al cel ls including thymocytes. The intracellular cysts showing microvilli towards lumen contained dense granules and degenerating cel ls (Fig. 5). Dendritic-like cells were seen intermingled with the thy mocytes in the medull ary region. Characteristically, these cell s estab li shed electron dense surface contacts with nei ghbouri ng thymocytes, but not with epitheli al cell s. The nucleus was irregul ar and eccentric. The presence of cell debri s in the cytoplasm of these cell s reflects their phagocy tic activ ity (Fig. 7). Macrophages were distingui shed by the presence of lysosomal and phagolysosomal type of inclusions, multi ves icular bodies, large number of vacuoles and vary ing sizes of electron dense granul es

Figs 1-12- (l) Electron micrograph of thymic cortex region encirc led by the connective ti ss ue capsule (CAP). Note the presence of numerous thymoeytcs (Th), and a few epithelial cell s (Ep) . X 3,250. (2) ultrastructure of thymocytcs (Th) with relati vely large nucleus (N) in relat ion to cytoplasm. Note the clumps of condensed chromat in along the periphery of inner nuclear membrane as we ll as in the nucleoplasm. X I 0,600. (3) a reticular epi thelial cell (Ep) extending cytoplasmic processes (arrows) which form epithelial reticulum in cortex as well as medulla. A prominent nucleolus (n) seem in the nucleus. Note the thymocytcs (Th) in the interstices of the epithelial reticulum. X 8,800. (4) an epithelial cell showi ng desmosomal connections (arrows) with neighbouring epithel ial cell s. X 8,000. (5) electron micrograph of an in tracellular epithelial cyst. Note the presence of numerous electron dense granu les (g), cell debris (arrows) and microvi lli (mv) projecting towards lumen in the cyst. X 6,500. (6) elect ron micrograph showing the participation of both epithelial as well as non-epithelial components including thymocytes (Th) in the formation of cystic wall of an intercellul ar epi thelial cyst. X 2, 100. (7) electron micrograph showing dendritic like cells (D). Note the electron dense surface contacts (arrows) wi th thymoeytes (Th). X 5,900. (8) ultras tructure of a macrophage showing numerous inclusions (In ), multi-vesicular bod ies (arrows), electron dense granules (g) and vacuoles (v) in it's cytoplasm. X 10,1 00. (9) histological preparati on of thymus in winter show ing the depletion of thymocytcs and ill­clefined cortex (c) nne! medullary (m) regions. X 1,200. (10) section of li zard' s thymus during spring. Commencemelll of thymic development along with differenti ati on of conico (c)-medul lary (m) zonati on can be seen. X 1,200. (II) hi stological prepara ti on of thymus during summer. Note the dense ly populated thymocytcs in concx (c) and medullary (m) regions. X 1,200. (12) sect ion of thymus showing depletion of thymocytes from both cortex (c) as we ll as medull~ (m) during autumn and absence of corti co-medullary delineation. X 1,200.

HARERAMADAS & RA I: TESTOSTERONE CONTROL OF LIZARD THYMUS

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632 INDIAN 1 EXP BIOL, JULY 2001

in their cytoplasm. The nucleus was elongated and often indented by the inclusions (Fig. 8).

Histological observations clearly showed that the thymus undergoes prominent season dependent changes. The light microscopic study showed that the thymus remai ned regressed with marked atrophy of lymphoid components in winter. Its cortico-medullary differentiation was less distinct (Fig. 9). The proliferation of thymocytes along with lymphoid components and epithel ial cells commenced in spring (Fig. 10). In summer, both the cortex and medulla were densely populated with thymocytes (Fig. 11) and the cortico-medullary differentiation became more prominent. At the onset of autumn, the commencement of regression of thymus was evident by marked depletion of thymocytes and lymphoid components (Fig. 12).

The ultrastructural observation of epithelial cells showed appreciable season dependent changes in thei r nuclear-cytoplasmic ratio and cell organelles. In summer, the epithelial cells contained numerous well developed cell organelles including bundles of tonofilaments, varying sizes of granules , ribosomes, rough and smooth endoplasmic reticulum and Golgi apparatus in the voluminous cytoplasm (Fig. 14). On the contrary, the nuclear-cytoplasmic ratio decreased markedly during winter. Condensation of chromatin material was also noticed (Fig. 13).

The thymus of reproductively active sham-operated control lizard was highly regressed, their cortico­medullary delineation was not di stinct and thymocytes were markedly depleted (Fig. 15). Castration resulted in thymic enlargement and cortico-medullary delineation . Considerable increase in number of thymocytes, macrophages and myoid cells was seen in both cortex and medulla, though thymocyte population was extremely rich in cortex, mainly in the sub-cortical region . The cortex was so

densely populated with thymocytes that the underlying epithelial network was not discernable (Fig. 16). The ultrastructural features of epithelial cell s in castrated lizard' s thymus resembled to those noted during summer with voluminous cytoplasm and numerous well developed cytoplasmic organelles (Fig. 19). The thymus of castrated lizards subjected to testosterone treatment underwent drastic regressive changes. The thymus got depleted off the thymocytes due to which the epithelial network become exposed (Fig. 17). Epithelial cells in the thymus of testosterone treated lizards (Fig. 20), like that in controls (Fig. 13), showed atrophic changes such as electron lucent scanty cytoplasm, condensed chromatin and cellular elongation. After the withdrawal of testosterone treatment, the thymus became densely populated. The cortico-medullary delineation became prominent. The regeneration of sub-cortical region with increased population of thymocytes was also seen (Fig. 18).

Discussion The thymus in reptiles, like other ectotherms,

displays marked season dependent species specific structural variations4

·5•18

'23

• In lizards Chalcides ocellatus4

, Mabuya quinquetaeniata, Uromastyx aegyptia5 and Sincus sincus6

, the gradual involution of thymus commences during autumn , followed by a total regression during winter, starts regenerating during spring and remains high ly developed during summer. The thymus of wall lizard H. flaviviridis also showed imil ar season dependen t changes. In contrast, the thymus in snake Psammophis schokari1

and turtle M. caspica8·9 has been reported to be

profoundly involuted with the depletion of thymocytes and lymphoid cells during summer.

Season dependent structural changes in thymus of wall lizard H. flaviviridis exhibit an inverse rel ationship with their seasonal testicul ar

Figs 13-20-(13) Ul trastructure of an epi thel ial cell during winter showing large nuc leus with prominent nucleol us (n) and scanty cytoplasm (Cy). X 9,600. (14) Electron micrograph of an epi thelial ce ll during summer wi th voluminous cytoplasm (Cy) with organelles like endoplasmic reticulum (arrow}, mitochondria (m), vacuoles and electron dense granules (g) . T he chromatin is comparatively less condensed as compared to that of winter (Fig. 13). X I 0,800. (15) histological preparat ion of control lizard's thymus during spermatogenically active phase. Note the feeble cortico (c)-medullary (m) demarcation. X 2,400. (16) section of thymus of a castrated lizard . Densely populated cortex (c) and medullary regions (m) with thymocytes can be seen. Besides, myoid cells (arrow head), dendritic like cells (arrows) and lymphoblasts (I) can also be noticed. X 2,400. (1 7) note the depletion of thymocytes and other lymphoid components from the castrated lizard 's thymus followi ng testosterone treatment and feebly demarcated cortico (c)-medullary tm) regions. X 2,400. (18) histological preparation of lizard ' s thymus following withdrawal of testosterone treatment. Both the cortex (c) as well as medulla ry (m) regions are densely populated with thymocytes and other lymphoid cells. Also notice the regeneration of sub-cortical region (sc). X 2,400. (19) ultrastructure of an epithelial cell of castrated lizard' s thymus. Note the decrease in nuclear-cytoplasmic ratio as compared to control, which is simi lar to that of winter (Fig. l 3). X 8,000. (20) electron micrograph of epi thelial cell in the castrated lizard ' s thymus after testosterone treatment. Notice the electron lucent scanty cytoplasm with less number of cell organelies. A predomi nant nucleolus (n) and clumps of condensed chromatin can also be seen in the nucleus. A cytoplasmic process (cp) forming desmosomal contact with neighbouring epithelial cells can also be seen. X 5,600.

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634 IN DIAN J EXP BIOL, JULY 200 I

steroidogen ic activity. The thymus remained involuted in winter when the testicular steroidogenic activity was high, whereas in summer the thymus was fully developed when the testicul ar steroidogenic acti vity was low as Leydig cells remained atrophied24

.

These observations support the contention of an inverse relationship between development of thymus and level of circulating testosterone. In mammals, there are evidences, which reveal that the androgenic hormone causes atrophy of thymus25

. The presence of androgen receptors in thymus25 further supports the view that sex hormones have direct effect on the thymus. Like mammals, the adm inistration of testosterone in turtle M. caspica26 has been reported to cause drastic thymic involution with marked reduction in lymphoid components. However, the epithelial structure of thymus was not affected by the treatment. In the present investigation the thymus was highly developed in castrated lizards with densely populated thymocytes mainly in the cortex and well defined cortico-medu ll ary regions. The testosterone treatment resulted in the regression of thymus and its demarcation in cortico-medullary regions became less prominent along with the depletion of thymocytes in the cortex. Unlike turtle M. caspica26

, the epithelial cell s in lizard' s thymus showed atrophic change. The involution of thymus in wall lizard was reversed and the same architecture reappeared following the withdrawal of androgens, as observed in castrated, vehicle injected animals. Similar result has been reported with the study on murine thymus23

.

In conclusion, it may be stated that the increase in testicular steroidogenic activity during. winter, which led to androgen production, would have caused the thymic involution as observed in case of testosterone treated lizards. Since the T cells are known to play crucial role in cell mediated immunity, the androgen induced suppression of thymic structure seems to be one of the important factors to cause mortality during winter.

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2 Zap:Ha A, Phyloge ny of fish im mune system, Bull Institute Pasteur, 81 (1983) 165 .

3 Elgert K D, Cell s and organs of immune system, in Immunology-understanding the immune system, (Wiley-Li ss Inc, New York), 1996,34.

4 Hussein M F, Badir N, El Ridi , R & Akcf N, Differenti al effect of seasonal variation on the lymphoid tissue of li zard Cha/cides ace/latus, Dev Camp /mmunol, 2 ( 1978) 297.

5 Hussein M F, Badir N, El Ridi R & Akef N, Effect of seasonal variation on the lymphoid ti ssues of li zards Mabuya quinquetaeniata and Uromastyx aegyptia, Dev Comp lmmunol, 2 ( 1978) 469.

6 Hussein M F, Badir N, El Ridi R & El Deeb S, Effect of seasonal variation in immune system of lizard Sincus sincus, J Exp Zoo/, 209 ( 1979) 91.

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8 Leceta J & Zapata A, Seasonal changes in the thymus and spleen of turtle M. caspica. A morphometrical and light microscopic study, Dev Comp lmmunol, 9 ( 1985) 653.

9 Leceta J, Villena A, Razaq uin B, Forfria J & Zapata A, Interdigi tating cells in the thymus of turtle M. caspica. Possible rel ation to macrophages, Cell Tissue Res, 238 ( 1984) 381.

10 Leceta J, Garrido E, Torroba M & Zapata A, Ultrastructural changes in the thymus of the turtle M. caspica in rel ati on to the seasonal cycle, Cell Tissue Res, 256 ( 1989) 213.

II Ando S, Panno M L, Ciarcia G, lmbeogno E, Beraldi E, Sisci D, Angelini F & Bolte V, Plasma sex hormone concentrations during the reproduction cycle in the male lizard Podarcis sicu/a sicula , J Reprod Fertil, 90 (1990) 353.

12 Naulleau G. Fleury F & Boissin J, Ann ual cyc les in plasma testosterone and thyoxi n in the male aspic viper Viper aspis (Reptili a, Viperidae) in relation to the sex ual cycle and hibernati on. Gen Camp Endocrino/, 65 ( 1987) 254.

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21 Plytycz B & Bigaj J, Seasonal cyclic changes in the thymus of adult frog Ran a temporaria, Thymus, 5 ( 1983) 327.

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