- Email: [email protected]
Plasma T3 and T4 levels in a snake, Elaphe taeniura
Camp. Biochem. Physiol. Vol. 107A, No. 1, Pp. 107-I 12, 1994 Printed in Great Britain
0300-%29/94 S6.00 + 0.00 0 1993 Pergamon Press Ltd
Plasma T3 and T4 levels in a snake, Elaphe taeniura K. W. Chiu and K. Y. Lam Department
of Biology, The Chinese University of Hong Kong, Hong Kong
The plasma thyroid hormones (TH) in the snake, Elaphe tueniura, were studied using RIA. Samples of plasma were obtained from untreated snakes in March, May and September as well as thyroidectomized snakes in May. Scale biopsies were taken from each snake in order to see if there were any correlations of plasma T3 and T4 levels and epidermal changes during the sloughing cycle. Plasma samples were also taken from another snake species prvaS korrus and a chelonia sp. Clcmn?ys mufica for comparison. Data showed that the plasma T3 and T4 levels of these reptilian species were very low (less than 1 nmol/l) and there were extensive individual variations (from below detection to 0.910 q mol/l). There were seasonal variations in these levels in E. iueniuru. One week after thyroidectomy, the plasma levels of T3 and T4 could not be detected, suggesting that the thyroid gland is the only source of thyroid hormones in the snake. There was no correlation between mean plasma T3 and T4 levels during the resting and renewal phase. The extreme low level of plasma T4 preceding, and its absence following thyroidectomy which always precipitates, the renewal phase provide circumstantial support to the inhibitory role of TH on epidermal proliferation/diierentiation in sloughing. Key words: T3; T4; Eluphe tueniura; Sloughing cycle; Thyroidectomy. Comp. Biochem. Physiol. 107A, 107-112, 1994.
Introduction In the mammalian epidermis, the outermost cell layers (stratum comeum) are dead and are continually being shed and replaced by the differentiation of the daughter cells of the deepest epidermal layer, the stratum germinativum (Flaxman et al., 1968; Irish et al., 1988; Thibodeau, 1990). In squamate reptiles, in contrast, the epidermis shows periodic synchronous production, differentiation and subsequent sloughing of an entire “epidermal generation” of cells (Maderson, 1965, 1985; Flaxman et al., 1968; Landmann, 1979, 1986; Walker, 1987; Hildebrand, 1988; Irish et al., 1988). The thyroid gland is involved in the sloughing cycle in these squamates (Maderson et al., 1970a). In lizards, hypothyroidism decreases and hyperthyroidism increases the sloughing frequency (SF) (Chiu, 1982; Chiu et al., 1986). The hormonal effect on sloughing in the lizard is through general metabolic changes and SF is merely a reflection of the general metabolic
status of the animal. In snakes, the relation of the thyroid status to SF appears the reverse. Thyroidectomy (TX) increases the SF of snakes while TH injections inhibit the sloughing in a number of species of snakes (Schaeffer, 1933; Chiu and Lynn, 1970, 1971; Chiu et al., 1983). In TX snakes, cellular proliferation from stratum germinativum seems to occur continuously and the epidermal histology is always in the renewal phase. T3 injection, on the other hand, inhibits the epidermal cellular proliferation and delays indefinitely the onset of the renewal phase in intact and TX snakes. Chiu et al. (1983) suggested that snake sloughing is directly controlled by the circulating TH levels. In early 1970, Maderson et al. (1970b) observed that in the snake, the new inner epidermal generation differentiates during a period of lowest thyroid gland activity, and gland activity is the highest around sloughing judging by the thyroidal follicular cell height. There is no direct evidence on the relation between TH and sloughing. To date, there are few reports on the circulating thyroid hormone (TH) levels in the snake let alone the correlation on the plasma TH levels with any known physiological
Correspondence ro: K. W. Chiu, Department of Biology, The Chinese University of Hong Kong, Hong Kong. Fax. 852-603-5123. Received 1 March 1993; accepted 31 March 1993. 107
108
K. W. Chiu and K. Y. Lam
processes. The problem is the extreme low level and variability (Chiu, 1982; Chiu and Wong, 1984). The present study was another attempt to determine the plasma TH levels in the striped racer snake, Elaphe taeniura. Factors such as season, thyroid gland activity, skin shedding which might be related to TH levels are taken into consideration.
Materials and Methods Season
In March, May and September, batches of mature, male snakes, Elaphe taeniura, were purchased and killed immediately by decapitation. Blood samples were collected at once into heparinized tubes. Of those batches of snakes in May and September six and seven snakes were in the cloudy eye phase (see below). For comparison, six males colubrid snakes of another species (Ptyas korros) and seven chelonians (Clemmys mutica) were also obtained in May. Their plasma T3 and T4 were also determined in a similar manner. Additionally in view of the low T3 titer and its absence in many blood samples, the plasma T3 of SD rats was performed as well as a check to the T3 RIA methodology. Blood samples were centrifuged at about 2000 rpm (900 g) at room temperature. The clear supernatant (plasma) were collected and stored at -20°C for RIA measurement of plasma T3 and T4 levels. Thyroid gland activity
In May, 12 striped racer snakes (Elaphe taenilocally and were kept in the animal house with a 12-hr photoperiod. The room temperature was about 20-25°C. The animals were provided with water but were not fed, since experience shows that snakes eat very infrequently and regurgitate if force fed. All the snakes (12) were thyroidectomized as described (Chiu and Lynn, 1970). Seven of the TX animals were killed after 7 days while the remaining animals were killed about 16 days after TX. Blood samples were taken from individual snakes and prepared for measurement of T3 and T4 levels. Remnants of the thyroid gland were checked in these animals. ura) were purchased
Sloughing
Snakes slough (skin-shedding) periodically. During the sloughing cycle, the epidermis undergoes cyclic changes. Based on these changes, Maderson (1965) has divided the sloughing cycle into two phases: resting phase (in which the skin consists of one epidermal generation) and renewal phase (in which the skin consists of two epidermal generations), at the end of the latter phase, sloughing occurs
wherein the outer epidermal generation is lost and the skin returns to the resting phase once more. Detailed histological studies showed that there are six epidermal conditions (stages l-6) during the cycle: the skin is in stage 1 during the resting phase and in stages 2-6 during the renewal phase. During the later part of the renewal phase, the snakes are characterised by their spectacles being cloudy, “cloudy eye” phase (Maderson, 1965). To study possible relationship between sloughing and thyroid gland, a scale sample was taken from all snakes in the above studies at the time of killing when a blood sample was collected. The scale sample was subsequently processed for histological examination to determine the epidermal stages/resting or renewal phase of the sloughing cycle (Maderson, 1965). The correlation, if any, between the epidermal stage and the TH level was sought. RIA of plasma thyroid hormones Preparation of hormone free
serum (HFS).
Norit A activated charcoal (Serva, Germany) (20g) and 2 g dextran T-70 (Pharmacia, Sweden) were mixed in 100 ml 0.5 M sodium phosphate buffer, pH 7, with stirring for 10 min at room temperature. After centrifugation at 2000 t-pm (900g) for 5 min, the supernatant was discarded and the charcoal was then added to 100 ml serum from a pool of serum of untreated and presumably “euthyroid” striped racer snakes. The serum was incubated overnight at 4°C with stirring. The resulting slurry was centrifuged three times in a 52-21 centrifuge (Beckman, U.S.A.) at 9000rpm (10,OOOg) at 4°C to remove the charcoal. The HFS was then stored at -20°C until use. Bu@r. A solution of 0.8 M barbital sodium buffer titrated with 1 N HCl to pH 8.4 was enriched with 8 g/l BSA and 200 mg/l thimerosal. All the reagents required were diluted with this buffer. Assay of plasma T3. The plasma T3 level in the blood serum was determined by a double antibody RIA in 1.5 ml microcentrifuge tubes. Various reagents were added to yield a final volume of 700 ~1: (1) 200 ~1 sample, (2) 100~1 2% normal rabbit serum (NRS), (3) 100 ~1 ANS (8-anilino-l-naphthalenesulfonic acid, 120 mg/ 100 ml, Serva), (4) 100 ~1 of a 1:7500 dilution of rabbit anti-T3-BSA antiserum (first antibody), (5) 100 ~1, “‘I-T3 approximately 20,000 cpm (tracer specific activity: > 1.2 pCi/pg, Amersham). Since the plasma T3 level of snakes is so low that 200 ~1 samples were used for the assay, 200 ~1 of HFS were added to all the tubes of standard curve determination. The standard curve was made up of 100 ,Y1of various dilutions
Snake plasma T3 and T4
of T3 (free acid, Henning, Berlin), i.e. 0.0803.0 nmol/l L-T3, were employed for a six-point standard curve with the addition of the above reagents. The total volume was adjusted to 700 ,ul by the buffer. The standard curve and samples were assayed in duplicate. The tubes were then mixed and incubated overnight at room temperature. To precipitate ]*‘I-T3 bound to antibody, 200 ~1 of a pre-titered goat anti-rabbit rglobulin (second anti-body, 1: 40, Antibodies Inc., CA, U.S.A.) with 10% polyethyleneglycol (PEG, Merck, Mol. wt 6000) was added to each tube after the tubes had been ice-cooled for an hour. The tubes were then incubated at 4°C for another 30min. To take off the assays, the tubes were centrifuged in a Sigma 2MK microcentrifuge (Sigma Laborzentrifugen GmbH, Germany) at 9000 rpm (7500 g) for 5 min at 4°C. The supernatant fluids were aspirated and the pellets were washed once with ice-cold Tris-HCl buffer without BSA, pH 8.6. After centrifugation for another 5 min, the supernatant was discarded and the pellets in the tubes were ready for counting in a gamma-counter (Kontron, Switzerland). The results were expressed as the percentage of added label found in the pellet (%B) corrected for nonspecific binding calculated from data for tubes which contained 100 ~1 buffer in place of the first antibody. The sensitivity of the T3 RIA was 0.080 nmol/l T3, and the intra-assay coefficient of variation was less than 6%. Assay of plasma T4. The antibody used in plasma T4 assay was solid phase antibody (Corning, U.S.A.). In 1.5 ml microcentrifuge tubes, the following reagents were added and the final volumes were adjusted to 450 ~1 with buffer: (1) 100~1 sample, (2) 100~1 ANS ~oin~/lOO ml, Serva), (3) 50 ~1 antibody, (4) ‘*‘I-T4 approximately 20,000 cpm (> l.Z;Ci/pg, Amersham).
109
One hundred microliters of various dilutions of T4 (free acid, Henning, Berlin), i.e. 0.025-1.0 nmol/l L-T4, were employed for a six-point standard curve, 100 ~1 HFS was added to all the tubes of standard curve determination. The standard and samples were assayed in duplicate. After incubation at room temperature overnight, the tubes were centrifuged in a Sigma 2MK microcentrifuge at 15°C at 9000 rpm (7500g) for 5 min. The supematant was aspirated and the pellets were washed once with 1 ml barbital sodium buffer pH 8.6 without BSA. After centrifugation for another 5 min, the supematant was discarded and the pellets in the tubes were ready for counting in a gammacounter (Kontron, Switzerland). The lowest detection limit was 0.025 nmol/l. In order to ensure the accuracy in each measurement, duplicate quality control tubes with known amount of T3/T4 were added before and after the sample tubes in every assay. All plasma TH values in a group, e.g. months of year, epidermal stages, phases of the sloughing cycle, are presented as mean + SEM. Student’s t-test was used to compare differences between groups. The level of significance was P < 0.05.
Results The plasma T3 and T4 levels in Elaphe, Ptyas and Clemmys are shown in Table 1. The mean values were very low, less than 0.4 nmol/l in these reptilian species. There were plasma samples in which T4 and/or T3 levels were below the detection limit of the present technique. For plasma T3, there was at least a 4-fold differences between the snake and the rat (1.021 f 0.042 nmol/l, n = 21). Except Ptyas, plasma T3 was lower than plasma T4. Seasonal variations were obvious in E. taeniura, but the
Table 1. The plasma T3 and T4 levels (nmol/l) in some reptilian species Animal A. E. B. E. C. E. D. P. E. C.
taeniura (n = 6, March) taeniura (n = 13, May) taeniura (n = 26, Sept.)
korros (n = 6, May) mutica (n = 7, May)
T3 *0.385 k 0.179 0.254 + 0.0721 to.015 & 0.040$ 0.200 f 0.040~~ 0.068 + 0.0098
n: Number of animals
*Mean & SEM tsignificantly lower than Group A and B, P < 0.05. SThree out of 13 samples below detection limit. $Twenty-three out of 26 samples below detection limit. IlOne out of six samples below detection limit. l/Five out of seven samples below detection limit. **Significantly higher than Group A, P < 0.05. TtSignificantly lower than Group B, P c 0.05. $#One out of 13 samples below detection limit. &Thirteen out of 26 samples below detection limit.
I-4 0.092 + **0.282 + tto.054 * 0.096 + 0.11 I +
0.035 0.057$$ 0.014 0.017 0.019
K. W. Chiu and K. Y. Lam
110 IOc
Table 3. The plasma T3 and T4 levels (nmol/l) during the sloughing cycle of h%zjrheIaeniura in September
A
T3 -
Epidermal condition Resting phase (18)
. .
late 1 (6) Renewal phase (8) 2(l) 5 (6) 6(l)
00’
L
;
Nan-8901 intact (N43)
-
1w
;
;
TX after
I week (N=7)
-
;
‘00
TX after 2 weeks (N=5)
Fig. 1. Plasma T3 and T4 level of Elaphe taeniura in May. TX: Thyroidectomy.
patterns differed between T3 and T4. The plasma T3 and T4 levels were higher in May compared with those in September. There were also extensive variations (Fig. 1). Tables 2 and 3 show the plasma T3 and T4 levels with reference to the two phases of the sloughing cycle and the epidermal stages of untreated and TX snakes in May and September, respectively. The plasma levels in both T3 and T4 of the normal untreated snakes show no significant difference in the resting and renewal phases. Nor was there any significant difference in either T3 and T4 level in snakes among different epidermal stages, except stage 3 where no sample was available. However, when stage 1 was further divided into a late stage 1, the plasma T4 level showed a significant reduction compared with those in the rest (earlier) of the stage 1 (Table 3). After TX, both the plasma T3 and T4 levels of the animals were undetectable, with isolated
0.049 * 0.030* $0.065;0.039* -
Epidermal condition
1(2)
2 (1) 3 (4) TX snakes (16 days after TX) 4 (5)
0.037 f 0.013* to.062 f 0.019* 0.001 f 0.001* 0.079 f 0.037’ @I.l05f0.045* -
Parentheses refer to number of animals in that skin stage. *Some of the values are below the detection limit. -Below detection limit. tSignificantly higher than stage late 1, P c 0.05. $Significantly higher than stage late 1, P cO.05. @ignificantly higher than stage late 1, P < 0.05.
specimens being exception (Fig. 1). Of all TX snakes, five were already in early renewal phase in 1 week while all were in mid-renewal phase in 2 weeks (Table 2).
Discussion The plasma TH levels in a snake are very low and very variable. Despite numerous attempts, the highest plasma T4 level obtained for an untreated Elaphe, and indeed other species never exceed 1 nmol/l (0.077 pg/dl). This is in accord with earlier data from Naja naja, Ptyas korros, P. mucosa, E. taeniura, E. radiata and Hydrophis cyanocinctus (Chiu, 1982; Chiu and Wong, 1984) and those in the lower range of plasma T4 values in Naja naja (Bona Gallo et al., 1980). The mean plasma T4 level in the snake is the lowest among the reptilian species reported using RIA (e.g. Cnemidophorus: Sellers et al., 1982; Calotes: Kar and ChandolaSaklani, 1985; Sceloporus : John-Alder and Joos, 1991) and perhaps among the vertebrates
Table 2. The plasma T3 and T4 levels (nmol/l) during the sloughing cycle of Elaphe taeniura in May. Untreated (intact)* Resting phase (6) (stage 1) Renewal phase (7) 4 (2) 5 (4) 6(l) TX snakes (7 days after TX)
T4
T3
T4
0.308 & 0.152t
0.222 * 0.0797
0.208 f 0.0747 0.328 0.163 f 0.096t 0.150
0.335 & 0.094 0.394 0.356 k 0.141 0.133
-
0.059
-
Parentheses refer to number of animals in that epidermal condition. *Both the plasma T3 and T4 levels are significantly higher than those of the TX snakes, P < 0.05. tSome are below the detection limit. -Below detection limit.
Snake plasma T3 and T4
(Bufo : Rosenkilde and Jorgensen, 1977; Rana: Mondou and Kaltenbach, 1979; Notophthalmus: Liversage et al., 1988; Oncorhynchus: Leatherland et al., 1989; Salmo: McCormick and Saunders, 1990). The present results also suggest that this is also true with the plasma T3 level, although there are even fewer available reports on plasma T3 (e.g. Calotes: Kar and Chandola-Saklani, 1985; Suncus: Tsukahara et al., 1990; Sceloporus: John-Alder and Joos, 1991). Of particular interest is the present finding that plasma T3 can be equal to, or even exceed that of T4. Licht et al. (1990) in a recent study on juvenile turtles discussed at length the variation of plasma T4 and factors involved such as age, sex, environment, “stress” associated with handling and various manipulations (surgical and chemical). Extensive individual variation in circulatory plasma TH in snakes had been previously noted (Chiu and Wong, 1984). In view of the design of the present study, many of these factors are excluded (sex? only males used, age? only mature ones used, surgical nor chemical treatment? untreated were considered). The snakes were bought from the snake shop, decapitated and bled. The only obvious variable was that of seasonal, i.e. environment. However, in the present study, a physiological process, i.e. sloughing may contribute to the variation in the plasma TH levels in snakes (see below). Blood collected from the striped racer snake (E. taeniura) in different months of the year had different plasma TH levels. This is in accord with the changes in plasma T4 levels in N. naja which ranged from 0.1 to 1.5 pg/dl or 1.29 to 19.30 nmol/l (Bona-Gall0 et al., 1980). The plasma T4 level of E. taeniura collected is highest in May (less than l/20 of the concentration in Naja!), while the plasma T3 level is highest in March. The plasma T3 and T4 levels are lowest in September. This seasonal variation of TH may be in part due to variation in the secretion from the gland and the deiodinating activities of peripheral organs (Wong et al., 1993). The total plasma TH (T3 + T4) is highest in May and lowest in September. The functional significance of this phenomenon is perhaps to support an increase in the activity of the animal for finding food and mate in spring and to reduce its activity for conserving food in the cold season. After TX, both the plasma T3 and T4 levels were absent. This occurred one week after surgery, suggesting a fairly rapid turnover of TH. It has been reported that the in vitro deiodinating activity of the pancreas was also reduced following TX (Lam, 1992). It might be argued in snakes the thyroid gland is the
111
only source of THs. It is interesting to note the rate of epidermal development following TX in Elaphe was similar to that of other snake species adjudged by the epidermal stages in scale samples obtained 7 and 16 days after surgery. The germinal layer entered into the early renewal phase (stages 2 and 3) within 1 week and the epidermal differentiation was in the middle of renewal phase (stage 4) in 2 weeks’ time. This is indicative of a correlation of the thyroid gland activity with the occurrence of the renewal phase (stages 2-6). In an attempt to correlate the plasma TH levels, there is, however, no difference in both plasma T3 and T4 in snakes between the resting and renewal phases of the sloughing cycle nor is there any significant difference between the stage 1 epidermal condition (resting phase) and stages 3-5. There was no sample in the untreated snakes for stage 3 and only one sample for stage 2. It is, therefore, impossible to comment specifically on the plasma T3 and T4 during these stages in the sloughing cycle. In the September samples, however, plasma T4 in stages 1 and 5 were significantly higher than that in stage late 1. Since both T3 and T4 have been shown effective in inhibiting the onset of the renewal phase, and TX invariably precipitates sloughing, it has been argued that snake sloughing is directly controlled by the circulating TH levels (Chiu et al., 1983). THs act directly on the epidermis to inhibit its proliferation and/or differentiation and hence the onset of the renewal phase. One would expect a drop in TH levels at the end of the resting phase prior to the renewal phase, to induce/permit cell proliferation and differentiation, namely a drop in TH level at the end of stage 1 or at the beginning of stage 2 is expected. The present data indeed showed the plasma T4 level dropped sharply in late 1 epidermal condition. Furthermore, TX led to a precipitous fall in the TH within 7 days and in these TX snakes, the renewal phase was observed. These findings strongly indicate that low TH levels trigger the cellular proliferation and differentiation from the stratum germinativum, and support the hypothesis of Maderson et al. (1970b) on the initiation of the renewal phase. In summary, the present data suggest that the low level of, and their extensive variations in, plasma T4 and T3 levels in snakes might be related to the epidermal proliferation/ differentiation in the sloughing cycle in the snake, apart from the seasonal activity of the gland (Bona-Gall0 et al., 1980) and different extra-thyroidal T4 and T3 conversion (Kar and Chandola-Saklana, 1985; Lam, 1992; Wong et al., 1993) in snakes.
K. W. Chiu anId K. Y. Lam
112
References Bona-Gal10 A., Licht P., Mackenzie D. S. and Lofts B. (1980) Annual cycles in pituitary and plasma gonadotropin, gonadal steroids, and thyroidal activity in the Chinese cobra Naja naja. Gen. camp. Endocrinol. 42, 477-493.
Chiu K. W. (1982) Thyroid function in the squamate reptiles. In Phylogenic Aspects of Thyroid Hormone Actions (Edited by Suzuki S.), pp. 107-122. Centre for Academic Publications, Tokyo. Chiu K. W. and Lynn W. G. (1970) The role of the thyroid in skin-shedding in the shovel-nosed snake Chionactis occipitalis. Gen.-camp. Endocrinol. 14, 467-474. Chiu K. W. and Lvnn W. G. (1971) Further observations
on the role of thd thyroid in ‘skin-shedding in the shovelnosed snake Chionactis occipitalis. Gen. camp. Endocrinol. 17, 508-5 11. Chiu K. W. and Wong C. C. (1984) 7th International Congress of Endocrinology, p. 348. Excepta Medica,
Amsterdam. Chiu K. W., Leung M. S. and Maderson P. F. A. (1983) Thyroid and skin-shedding in the rat snake (Ptyas korros). J. exp. Zool. 225, 407-410. Chiu K. W., Sham J. S. K., Maderson P. F. A. and Zucker
A. H. (1986) Interaction between thermal environments and hormones affecting skin-shedding frequency in the tokay (Gekko gecko) (Gekkonidae, Lacertilia). Comp. Biochem. Physiol. 84A, 345-35 1. Flaxman B. A., Maderson P. F. A., Sxabo G. and Roth S. I. (1968) Control of cell differentiation in lizard epidermis in vitro. Dev. Biol. 18, 354-374. Hildenbrand M. (1988) Integument and its derivatives. In AMI@ of fertebiate SGucture. 3rd Edn (Edited by Hildebrand M.). DD. 89-105. Wilev. New York. Irish F. J., Williams E. E. and Seling E. (1988) Scanning electron microscopy of changes in epidermal structure occurring during the shedding cycle in squamate reptiles. J. Morph. 197, 105-126. John-Alder H. B. and Joos B. (1991) Interactive effects of thyroxine and experimental location on running endurance, tissue masses, and enzyme activities in captive versus field-active lizards (Sceloporus undulatus). Gen. camp. Endocrinol. 81, 120-132. Kar A. and Chandola-Saklani A. (1985) Circulating thyroid hormone concentrations in relation to seasonal events in the male Indian garden lizard, Calotes uersicolor. Gen. camp. Endocrinol. 60, 1419. Lam K. Y. (1992) The effects of thyroid hormone on the sloughing process of Elaphe taeniura. M.Phil. thesis. The Chinese University of Hong Kong. Landmann L. (1979) Keratin fonnation and barrier mechanisms in the epidermis of Natrix natrix (reptilia Serpentes): an ultrastructural study. J. Morphol. 162, 93-126. Landmann L. (1986) Epidermis and dermis. In Biology of the Integument Vol 2 (Edited by Bemiter-Hahn J., Matoltsy A. G. and Richards K. S.), pp. 50-187. Springer, Berlin. Leatherland J. F., Down N. E., Donaldson E. M. and Dye I.
.
.
H. M. (1989) Changes in plasma thyroid hormone levels in pink salmon, Oncorhynchus go&.&a, during their spawning migration in the Fraser River (Canada). J. Fish Biol. 35, 199-205.
Licht P., Denver R. J. and Stamper D. L. (1990) Relation of plasma thyroxine binding to thyroidal activity and determination of thyroxine binding proteins in a turtle, Pseu&mys scripta. Gem. camp. Endocrinol. 80, 238-256.
Liversage R. A., Foty R. A. and McLaughlin D. S. (1988) Immunoreactive thyroxine levels in the serum of thyroidectomixed adult newts, Notophthalmus viridpscens. Can. J. Zool. 66, 1900-1903.
Maderson P. F. A. (1965) Histological changes in the epidermis of snakes during the sloughing cycle. J. Zool. (Land). 146, 98-l 13. Maderson P. F. A. (1985) Some developmental problems of the reptilian integument. In Biology o>the Repiiles. Vol II. (Edited bv Gans C.. Billett F. and Maderson P. F. A.). pp. 523-598. John Wiley & Sons, New York. ” Maderson P. F. A., Chiu K. W. and Phillips J. G. (197Oa) Endocrine-epidermal relationships in squamate reptiles. Mem. Sot. Endocrinol. 18, 259-284. Maderson P. F. A., Chiu K. W. and Phillips J. G. (1970b) Changes in the epidermal histology during the sloughing cycle in the rat snake Ptyas korros Schlegel, with correlated observations on the thyroid gland. Biol. Bull. 139, 304-321.
McCormick S. D. and Saunders R. L. (1990) Influence of ration level and salinity on circulating thyroid hormones in juvenile Atlantic salmon (Salmo salar). Gen. camp. Endocrinol. 78, 224-230.
Mondou P. M. and Kaltenbach J. (1979) Thyroxine concentrations in blood serum and pericardial fluid of metamorphosing tadpoles and of adult frogs. Gen. camp. Endocrinol. 39, 343-349.
Rosenkilde P. and Jorgensen I. (1977) Determination of serum thyroxine in two species of toads: variation with season. Gen. Comp. Endocrinol. 33, 566-573. Schaeffer W. H. (1933) Hypophysectomy and thyroidectomy of snakes. Proc. exp. Biol. Med. 38, 1363-1365. Sellers J. C., Wit C. L., Ganjam V. K., Etheridge K. A. and Ragland I. M. (1982) Seasonal plasma T4 titers in the hibernating lizard. Cnemidophorus sexlineatus. Gen. camp. Endocrinol. 46, 24-28.
Thibodeau G. A. (1990) The skin and its appendages. In Textbook of Anatomy and Physiology. 13th Edn (Edited by Hudlin H. C., Kocher L. and Genz J.), pp. 113-132. Times Mirror/Mosby College, St Louis. Tsukahara F., Muraki T. and Nomoto T. (1990) Serum concentrations of thyroid hormones and activity of iodothyronine deiodinase in peripheral tissues of the house musk shrew, Suncus murinus. J. Endocrinol. 125, 117-122. Walker W. F. (1987) Functional anatomy of vertebrates: an evolutionary nersnective. (Edited by Walker W. F.) pp. 123-137. Saunders, Philadelphia. _ Wong C. C., Lam K. Y. and Chiu K. W. (1993) The extrathyroidal conversion of T4 to T3 in EIaphe taeniura. J. camp. Physiol. B 163, 212-218.
0300-%29/94 S6.00 + 0.00 0 1993 Pergamon Press Ltd
Plasma T3 and T4 levels in a snake, Elaphe taeniura K. W. Chiu and K. Y. Lam Department
of Biology, The Chinese University of Hong Kong, Hong Kong
The plasma thyroid hormones (TH) in the snake, Elaphe tueniura, were studied using RIA. Samples of plasma were obtained from untreated snakes in March, May and September as well as thyroidectomized snakes in May. Scale biopsies were taken from each snake in order to see if there were any correlations of plasma T3 and T4 levels and epidermal changes during the sloughing cycle. Plasma samples were also taken from another snake species prvaS korrus and a chelonia sp. Clcmn?ys mufica for comparison. Data showed that the plasma T3 and T4 levels of these reptilian species were very low (less than 1 nmol/l) and there were extensive individual variations (from below detection to 0.910 q mol/l). There were seasonal variations in these levels in E. iueniuru. One week after thyroidectomy, the plasma levels of T3 and T4 could not be detected, suggesting that the thyroid gland is the only source of thyroid hormones in the snake. There was no correlation between mean plasma T3 and T4 levels during the resting and renewal phase. The extreme low level of plasma T4 preceding, and its absence following thyroidectomy which always precipitates, the renewal phase provide circumstantial support to the inhibitory role of TH on epidermal proliferation/diierentiation in sloughing. Key words: T3; T4; Eluphe tueniura; Sloughing cycle; Thyroidectomy. Comp. Biochem. Physiol. 107A, 107-112, 1994.
Introduction In the mammalian epidermis, the outermost cell layers (stratum comeum) are dead and are continually being shed and replaced by the differentiation of the daughter cells of the deepest epidermal layer, the stratum germinativum (Flaxman et al., 1968; Irish et al., 1988; Thibodeau, 1990). In squamate reptiles, in contrast, the epidermis shows periodic synchronous production, differentiation and subsequent sloughing of an entire “epidermal generation” of cells (Maderson, 1965, 1985; Flaxman et al., 1968; Landmann, 1979, 1986; Walker, 1987; Hildebrand, 1988; Irish et al., 1988). The thyroid gland is involved in the sloughing cycle in these squamates (Maderson et al., 1970a). In lizards, hypothyroidism decreases and hyperthyroidism increases the sloughing frequency (SF) (Chiu, 1982; Chiu et al., 1986). The hormonal effect on sloughing in the lizard is through general metabolic changes and SF is merely a reflection of the general metabolic
status of the animal. In snakes, the relation of the thyroid status to SF appears the reverse. Thyroidectomy (TX) increases the SF of snakes while TH injections inhibit the sloughing in a number of species of snakes (Schaeffer, 1933; Chiu and Lynn, 1970, 1971; Chiu et al., 1983). In TX snakes, cellular proliferation from stratum germinativum seems to occur continuously and the epidermal histology is always in the renewal phase. T3 injection, on the other hand, inhibits the epidermal cellular proliferation and delays indefinitely the onset of the renewal phase in intact and TX snakes. Chiu et al. (1983) suggested that snake sloughing is directly controlled by the circulating TH levels. In early 1970, Maderson et al. (1970b) observed that in the snake, the new inner epidermal generation differentiates during a period of lowest thyroid gland activity, and gland activity is the highest around sloughing judging by the thyroidal follicular cell height. There is no direct evidence on the relation between TH and sloughing. To date, there are few reports on the circulating thyroid hormone (TH) levels in the snake let alone the correlation on the plasma TH levels with any known physiological
Correspondence ro: K. W. Chiu, Department of Biology, The Chinese University of Hong Kong, Hong Kong. Fax. 852-603-5123. Received 1 March 1993; accepted 31 March 1993. 107
108
K. W. Chiu and K. Y. Lam
processes. The problem is the extreme low level and variability (Chiu, 1982; Chiu and Wong, 1984). The present study was another attempt to determine the plasma TH levels in the striped racer snake, Elaphe taeniura. Factors such as season, thyroid gland activity, skin shedding which might be related to TH levels are taken into consideration.
Materials and Methods Season
In March, May and September, batches of mature, male snakes, Elaphe taeniura, were purchased and killed immediately by decapitation. Blood samples were collected at once into heparinized tubes. Of those batches of snakes in May and September six and seven snakes were in the cloudy eye phase (see below). For comparison, six males colubrid snakes of another species (Ptyas korros) and seven chelonians (Clemmys mutica) were also obtained in May. Their plasma T3 and T4 were also determined in a similar manner. Additionally in view of the low T3 titer and its absence in many blood samples, the plasma T3 of SD rats was performed as well as a check to the T3 RIA methodology. Blood samples were centrifuged at about 2000 rpm (900 g) at room temperature. The clear supernatant (plasma) were collected and stored at -20°C for RIA measurement of plasma T3 and T4 levels. Thyroid gland activity
In May, 12 striped racer snakes (Elaphe taenilocally and were kept in the animal house with a 12-hr photoperiod. The room temperature was about 20-25°C. The animals were provided with water but were not fed, since experience shows that snakes eat very infrequently and regurgitate if force fed. All the snakes (12) were thyroidectomized as described (Chiu and Lynn, 1970). Seven of the TX animals were killed after 7 days while the remaining animals were killed about 16 days after TX. Blood samples were taken from individual snakes and prepared for measurement of T3 and T4 levels. Remnants of the thyroid gland were checked in these animals. ura) were purchased
Sloughing
Snakes slough (skin-shedding) periodically. During the sloughing cycle, the epidermis undergoes cyclic changes. Based on these changes, Maderson (1965) has divided the sloughing cycle into two phases: resting phase (in which the skin consists of one epidermal generation) and renewal phase (in which the skin consists of two epidermal generations), at the end of the latter phase, sloughing occurs
wherein the outer epidermal generation is lost and the skin returns to the resting phase once more. Detailed histological studies showed that there are six epidermal conditions (stages l-6) during the cycle: the skin is in stage 1 during the resting phase and in stages 2-6 during the renewal phase. During the later part of the renewal phase, the snakes are characterised by their spectacles being cloudy, “cloudy eye” phase (Maderson, 1965). To study possible relationship between sloughing and thyroid gland, a scale sample was taken from all snakes in the above studies at the time of killing when a blood sample was collected. The scale sample was subsequently processed for histological examination to determine the epidermal stages/resting or renewal phase of the sloughing cycle (Maderson, 1965). The correlation, if any, between the epidermal stage and the TH level was sought. RIA of plasma thyroid hormones Preparation of hormone free
serum (HFS).
Norit A activated charcoal (Serva, Germany) (20g) and 2 g dextran T-70 (Pharmacia, Sweden) were mixed in 100 ml 0.5 M sodium phosphate buffer, pH 7, with stirring for 10 min at room temperature. After centrifugation at 2000 t-pm (900g) for 5 min, the supernatant was discarded and the charcoal was then added to 100 ml serum from a pool of serum of untreated and presumably “euthyroid” striped racer snakes. The serum was incubated overnight at 4°C with stirring. The resulting slurry was centrifuged three times in a 52-21 centrifuge (Beckman, U.S.A.) at 9000rpm (10,OOOg) at 4°C to remove the charcoal. The HFS was then stored at -20°C until use. Bu@r. A solution of 0.8 M barbital sodium buffer titrated with 1 N HCl to pH 8.4 was enriched with 8 g/l BSA and 200 mg/l thimerosal. All the reagents required were diluted with this buffer. Assay of plasma T3. The plasma T3 level in the blood serum was determined by a double antibody RIA in 1.5 ml microcentrifuge tubes. Various reagents were added to yield a final volume of 700 ~1: (1) 200 ~1 sample, (2) 100~1 2% normal rabbit serum (NRS), (3) 100 ~1 ANS (8-anilino-l-naphthalenesulfonic acid, 120 mg/ 100 ml, Serva), (4) 100 ~1 of a 1:7500 dilution of rabbit anti-T3-BSA antiserum (first antibody), (5) 100 ~1, “‘I-T3 approximately 20,000 cpm (tracer specific activity: > 1.2 pCi/pg, Amersham). Since the plasma T3 level of snakes is so low that 200 ~1 samples were used for the assay, 200 ~1 of HFS were added to all the tubes of standard curve determination. The standard curve was made up of 100 ,Y1of various dilutions
Snake plasma T3 and T4
of T3 (free acid, Henning, Berlin), i.e. 0.0803.0 nmol/l L-T3, were employed for a six-point standard curve with the addition of the above reagents. The total volume was adjusted to 700 ,ul by the buffer. The standard curve and samples were assayed in duplicate. The tubes were then mixed and incubated overnight at room temperature. To precipitate ]*‘I-T3 bound to antibody, 200 ~1 of a pre-titered goat anti-rabbit rglobulin (second anti-body, 1: 40, Antibodies Inc., CA, U.S.A.) with 10% polyethyleneglycol (PEG, Merck, Mol. wt 6000) was added to each tube after the tubes had been ice-cooled for an hour. The tubes were then incubated at 4°C for another 30min. To take off the assays, the tubes were centrifuged in a Sigma 2MK microcentrifuge (Sigma Laborzentrifugen GmbH, Germany) at 9000 rpm (7500 g) for 5 min at 4°C. The supernatant fluids were aspirated and the pellets were washed once with ice-cold Tris-HCl buffer without BSA, pH 8.6. After centrifugation for another 5 min, the supernatant was discarded and the pellets in the tubes were ready for counting in a gamma-counter (Kontron, Switzerland). The results were expressed as the percentage of added label found in the pellet (%B) corrected for nonspecific binding calculated from data for tubes which contained 100 ~1 buffer in place of the first antibody. The sensitivity of the T3 RIA was 0.080 nmol/l T3, and the intra-assay coefficient of variation was less than 6%. Assay of plasma T4. The antibody used in plasma T4 assay was solid phase antibody (Corning, U.S.A.). In 1.5 ml microcentrifuge tubes, the following reagents were added and the final volumes were adjusted to 450 ~1 with buffer: (1) 100~1 sample, (2) 100~1 ANS ~oin~/lOO ml, Serva), (3) 50 ~1 antibody, (4) ‘*‘I-T4 approximately 20,000 cpm (> l.Z;Ci/pg, Amersham).
109
One hundred microliters of various dilutions of T4 (free acid, Henning, Berlin), i.e. 0.025-1.0 nmol/l L-T4, were employed for a six-point standard curve, 100 ~1 HFS was added to all the tubes of standard curve determination. The standard and samples were assayed in duplicate. After incubation at room temperature overnight, the tubes were centrifuged in a Sigma 2MK microcentrifuge at 15°C at 9000 rpm (7500g) for 5 min. The supematant was aspirated and the pellets were washed once with 1 ml barbital sodium buffer pH 8.6 without BSA. After centrifugation for another 5 min, the supematant was discarded and the pellets in the tubes were ready for counting in a gammacounter (Kontron, Switzerland). The lowest detection limit was 0.025 nmol/l. In order to ensure the accuracy in each measurement, duplicate quality control tubes with known amount of T3/T4 were added before and after the sample tubes in every assay. All plasma TH values in a group, e.g. months of year, epidermal stages, phases of the sloughing cycle, are presented as mean + SEM. Student’s t-test was used to compare differences between groups. The level of significance was P < 0.05.
Results The plasma T3 and T4 levels in Elaphe, Ptyas and Clemmys are shown in Table 1. The mean values were very low, less than 0.4 nmol/l in these reptilian species. There were plasma samples in which T4 and/or T3 levels were below the detection limit of the present technique. For plasma T3, there was at least a 4-fold differences between the snake and the rat (1.021 f 0.042 nmol/l, n = 21). Except Ptyas, plasma T3 was lower than plasma T4. Seasonal variations were obvious in E. taeniura, but the
Table 1. The plasma T3 and T4 levels (nmol/l) in some reptilian species Animal A. E. B. E. C. E. D. P. E. C.
taeniura (n = 6, March) taeniura (n = 13, May) taeniura (n = 26, Sept.)
korros (n = 6, May) mutica (n = 7, May)
T3 *0.385 k 0.179 0.254 + 0.0721 to.015 & 0.040$ 0.200 f 0.040~~ 0.068 + 0.0098
n: Number of animals
*Mean & SEM tsignificantly lower than Group A and B, P < 0.05. SThree out of 13 samples below detection limit. $Twenty-three out of 26 samples below detection limit. IlOne out of six samples below detection limit. l/Five out of seven samples below detection limit. **Significantly higher than Group A, P < 0.05. TtSignificantly lower than Group B, P c 0.05. $#One out of 13 samples below detection limit. &Thirteen out of 26 samples below detection limit.
I-4 0.092 + **0.282 + tto.054 * 0.096 + 0.11 I +
0.035 0.057$$ 0.014 0.017 0.019
K. W. Chiu and K. Y. Lam
110 IOc
Table 3. The plasma T3 and T4 levels (nmol/l) during the sloughing cycle of h%zjrheIaeniura in September
A
T3 -
Epidermal condition Resting phase (18)
. .
late 1 (6) Renewal phase (8) 2(l) 5 (6) 6(l)
00’
L
;
Nan-8901 intact (N43)
-
1w
;
;
TX after
I week (N=7)
-
;
‘00
TX after 2 weeks (N=5)
Fig. 1. Plasma T3 and T4 level of Elaphe taeniura in May. TX: Thyroidectomy.
patterns differed between T3 and T4. The plasma T3 and T4 levels were higher in May compared with those in September. There were also extensive variations (Fig. 1). Tables 2 and 3 show the plasma T3 and T4 levels with reference to the two phases of the sloughing cycle and the epidermal stages of untreated and TX snakes in May and September, respectively. The plasma levels in both T3 and T4 of the normal untreated snakes show no significant difference in the resting and renewal phases. Nor was there any significant difference in either T3 and T4 level in snakes among different epidermal stages, except stage 3 where no sample was available. However, when stage 1 was further divided into a late stage 1, the plasma T4 level showed a significant reduction compared with those in the rest (earlier) of the stage 1 (Table 3). After TX, both the plasma T3 and T4 levels of the animals were undetectable, with isolated
0.049 * 0.030* $0.065;0.039* -
Epidermal condition
1(2)
2 (1) 3 (4) TX snakes (16 days after TX) 4 (5)
0.037 f 0.013* to.062 f 0.019* 0.001 f 0.001* 0.079 f 0.037’ @I.l05f0.045* -
Parentheses refer to number of animals in that skin stage. *Some of the values are below the detection limit. -Below detection limit. tSignificantly higher than stage late 1, P c 0.05. $Significantly higher than stage late 1, P cO.05. @ignificantly higher than stage late 1, P < 0.05.
specimens being exception (Fig. 1). Of all TX snakes, five were already in early renewal phase in 1 week while all were in mid-renewal phase in 2 weeks (Table 2).
Discussion The plasma TH levels in a snake are very low and very variable. Despite numerous attempts, the highest plasma T4 level obtained for an untreated Elaphe, and indeed other species never exceed 1 nmol/l (0.077 pg/dl). This is in accord with earlier data from Naja naja, Ptyas korros, P. mucosa, E. taeniura, E. radiata and Hydrophis cyanocinctus (Chiu, 1982; Chiu and Wong, 1984) and those in the lower range of plasma T4 values in Naja naja (Bona Gallo et al., 1980). The mean plasma T4 level in the snake is the lowest among the reptilian species reported using RIA (e.g. Cnemidophorus: Sellers et al., 1982; Calotes: Kar and ChandolaSaklani, 1985; Sceloporus : John-Alder and Joos, 1991) and perhaps among the vertebrates
Table 2. The plasma T3 and T4 levels (nmol/l) during the sloughing cycle of Elaphe taeniura in May. Untreated (intact)* Resting phase (6) (stage 1) Renewal phase (7) 4 (2) 5 (4) 6(l) TX snakes (7 days after TX)
T4
T3
T4
0.308 & 0.152t
0.222 * 0.0797
0.208 f 0.0747 0.328 0.163 f 0.096t 0.150
0.335 & 0.094 0.394 0.356 k 0.141 0.133
-
0.059
-
Parentheses refer to number of animals in that epidermal condition. *Both the plasma T3 and T4 levels are significantly higher than those of the TX snakes, P < 0.05. tSome are below the detection limit. -Below detection limit.
Snake plasma T3 and T4
(Bufo : Rosenkilde and Jorgensen, 1977; Rana: Mondou and Kaltenbach, 1979; Notophthalmus: Liversage et al., 1988; Oncorhynchus: Leatherland et al., 1989; Salmo: McCormick and Saunders, 1990). The present results also suggest that this is also true with the plasma T3 level, although there are even fewer available reports on plasma T3 (e.g. Calotes: Kar and Chandola-Saklani, 1985; Suncus: Tsukahara et al., 1990; Sceloporus: John-Alder and Joos, 1991). Of particular interest is the present finding that plasma T3 can be equal to, or even exceed that of T4. Licht et al. (1990) in a recent study on juvenile turtles discussed at length the variation of plasma T4 and factors involved such as age, sex, environment, “stress” associated with handling and various manipulations (surgical and chemical). Extensive individual variation in circulatory plasma TH in snakes had been previously noted (Chiu and Wong, 1984). In view of the design of the present study, many of these factors are excluded (sex? only males used, age? only mature ones used, surgical nor chemical treatment? untreated were considered). The snakes were bought from the snake shop, decapitated and bled. The only obvious variable was that of seasonal, i.e. environment. However, in the present study, a physiological process, i.e. sloughing may contribute to the variation in the plasma TH levels in snakes (see below). Blood collected from the striped racer snake (E. taeniura) in different months of the year had different plasma TH levels. This is in accord with the changes in plasma T4 levels in N. naja which ranged from 0.1 to 1.5 pg/dl or 1.29 to 19.30 nmol/l (Bona-Gall0 et al., 1980). The plasma T4 level of E. taeniura collected is highest in May (less than l/20 of the concentration in Naja!), while the plasma T3 level is highest in March. The plasma T3 and T4 levels are lowest in September. This seasonal variation of TH may be in part due to variation in the secretion from the gland and the deiodinating activities of peripheral organs (Wong et al., 1993). The total plasma TH (T3 + T4) is highest in May and lowest in September. The functional significance of this phenomenon is perhaps to support an increase in the activity of the animal for finding food and mate in spring and to reduce its activity for conserving food in the cold season. After TX, both the plasma T3 and T4 levels were absent. This occurred one week after surgery, suggesting a fairly rapid turnover of TH. It has been reported that the in vitro deiodinating activity of the pancreas was also reduced following TX (Lam, 1992). It might be argued in snakes the thyroid gland is the
111
only source of THs. It is interesting to note the rate of epidermal development following TX in Elaphe was similar to that of other snake species adjudged by the epidermal stages in scale samples obtained 7 and 16 days after surgery. The germinal layer entered into the early renewal phase (stages 2 and 3) within 1 week and the epidermal differentiation was in the middle of renewal phase (stage 4) in 2 weeks’ time. This is indicative of a correlation of the thyroid gland activity with the occurrence of the renewal phase (stages 2-6). In an attempt to correlate the plasma TH levels, there is, however, no difference in both plasma T3 and T4 in snakes between the resting and renewal phases of the sloughing cycle nor is there any significant difference between the stage 1 epidermal condition (resting phase) and stages 3-5. There was no sample in the untreated snakes for stage 3 and only one sample for stage 2. It is, therefore, impossible to comment specifically on the plasma T3 and T4 during these stages in the sloughing cycle. In the September samples, however, plasma T4 in stages 1 and 5 were significantly higher than that in stage late 1. Since both T3 and T4 have been shown effective in inhibiting the onset of the renewal phase, and TX invariably precipitates sloughing, it has been argued that snake sloughing is directly controlled by the circulating TH levels (Chiu et al., 1983). THs act directly on the epidermis to inhibit its proliferation and/or differentiation and hence the onset of the renewal phase. One would expect a drop in TH levels at the end of the resting phase prior to the renewal phase, to induce/permit cell proliferation and differentiation, namely a drop in TH level at the end of stage 1 or at the beginning of stage 2 is expected. The present data indeed showed the plasma T4 level dropped sharply in late 1 epidermal condition. Furthermore, TX led to a precipitous fall in the TH within 7 days and in these TX snakes, the renewal phase was observed. These findings strongly indicate that low TH levels trigger the cellular proliferation and differentiation from the stratum germinativum, and support the hypothesis of Maderson et al. (1970b) on the initiation of the renewal phase. In summary, the present data suggest that the low level of, and their extensive variations in, plasma T4 and T3 levels in snakes might be related to the epidermal proliferation/ differentiation in the sloughing cycle in the snake, apart from the seasonal activity of the gland (Bona-Gall0 et al., 1980) and different extra-thyroidal T4 and T3 conversion (Kar and Chandola-Saklana, 1985; Lam, 1992; Wong et al., 1993) in snakes.
K. W. Chiu anId K. Y. Lam
112
References Bona-Gal10 A., Licht P., Mackenzie D. S. and Lofts B. (1980) Annual cycles in pituitary and plasma gonadotropin, gonadal steroids, and thyroidal activity in the Chinese cobra Naja naja. Gen. camp. Endocrinol. 42, 477-493.
Chiu K. W. (1982) Thyroid function in the squamate reptiles. In Phylogenic Aspects of Thyroid Hormone Actions (Edited by Suzuki S.), pp. 107-122. Centre for Academic Publications, Tokyo. Chiu K. W. and Lynn W. G. (1970) The role of the thyroid in skin-shedding in the shovel-nosed snake Chionactis occipitalis. Gen.-camp. Endocrinol. 14, 467-474. Chiu K. W. and Lvnn W. G. (1971) Further observations
on the role of thd thyroid in ‘skin-shedding in the shovelnosed snake Chionactis occipitalis. Gen. camp. Endocrinol. 17, 508-5 11. Chiu K. W. and Wong C. C. (1984) 7th International Congress of Endocrinology, p. 348. Excepta Medica,
Amsterdam. Chiu K. W., Leung M. S. and Maderson P. F. A. (1983) Thyroid and skin-shedding in the rat snake (Ptyas korros). J. exp. Zool. 225, 407-410. Chiu K. W., Sham J. S. K., Maderson P. F. A. and Zucker
A. H. (1986) Interaction between thermal environments and hormones affecting skin-shedding frequency in the tokay (Gekko gecko) (Gekkonidae, Lacertilia). Comp. Biochem. Physiol. 84A, 345-35 1. Flaxman B. A., Maderson P. F. A., Sxabo G. and Roth S. I. (1968) Control of cell differentiation in lizard epidermis in vitro. Dev. Biol. 18, 354-374. Hildenbrand M. (1988) Integument and its derivatives. In AMI@ of fertebiate SGucture. 3rd Edn (Edited by Hildebrand M.). DD. 89-105. Wilev. New York. Irish F. J., Williams E. E. and Seling E. (1988) Scanning electron microscopy of changes in epidermal structure occurring during the shedding cycle in squamate reptiles. J. Morph. 197, 105-126. John-Alder H. B. and Joos B. (1991) Interactive effects of thyroxine and experimental location on running endurance, tissue masses, and enzyme activities in captive versus field-active lizards (Sceloporus undulatus). Gen. camp. Endocrinol. 81, 120-132. Kar A. and Chandola-Saklani A. (1985) Circulating thyroid hormone concentrations in relation to seasonal events in the male Indian garden lizard, Calotes uersicolor. Gen. camp. Endocrinol. 60, 1419. Lam K. Y. (1992) The effects of thyroid hormone on the sloughing process of Elaphe taeniura. M.Phil. thesis. The Chinese University of Hong Kong. Landmann L. (1979) Keratin fonnation and barrier mechanisms in the epidermis of Natrix natrix (reptilia Serpentes): an ultrastructural study. J. Morphol. 162, 93-126. Landmann L. (1986) Epidermis and dermis. In Biology of the Integument Vol 2 (Edited by Bemiter-Hahn J., Matoltsy A. G. and Richards K. S.), pp. 50-187. Springer, Berlin. Leatherland J. F., Down N. E., Donaldson E. M. and Dye I.
.
.
H. M. (1989) Changes in plasma thyroid hormone levels in pink salmon, Oncorhynchus go&.&a, during their spawning migration in the Fraser River (Canada). J. Fish Biol. 35, 199-205.
Licht P., Denver R. J. and Stamper D. L. (1990) Relation of plasma thyroxine binding to thyroidal activity and determination of thyroxine binding proteins in a turtle, Pseu&mys scripta. Gem. camp. Endocrinol. 80, 238-256.
Liversage R. A., Foty R. A. and McLaughlin D. S. (1988) Immunoreactive thyroxine levels in the serum of thyroidectomixed adult newts, Notophthalmus viridpscens. Can. J. Zool. 66, 1900-1903.
Maderson P. F. A. (1965) Histological changes in the epidermis of snakes during the sloughing cycle. J. Zool. (Land). 146, 98-l 13. Maderson P. F. A. (1985) Some developmental problems of the reptilian integument. In Biology o>the Repiiles. Vol II. (Edited bv Gans C.. Billett F. and Maderson P. F. A.). pp. 523-598. John Wiley & Sons, New York. ” Maderson P. F. A., Chiu K. W. and Phillips J. G. (197Oa) Endocrine-epidermal relationships in squamate reptiles. Mem. Sot. Endocrinol. 18, 259-284. Maderson P. F. A., Chiu K. W. and Phillips J. G. (1970b) Changes in the epidermal histology during the sloughing cycle in the rat snake Ptyas korros Schlegel, with correlated observations on the thyroid gland. Biol. Bull. 139, 304-321.
McCormick S. D. and Saunders R. L. (1990) Influence of ration level and salinity on circulating thyroid hormones in juvenile Atlantic salmon (Salmo salar). Gen. camp. Endocrinol. 78, 224-230.
Mondou P. M. and Kaltenbach J. (1979) Thyroxine concentrations in blood serum and pericardial fluid of metamorphosing tadpoles and of adult frogs. Gen. camp. Endocrinol. 39, 343-349.
Rosenkilde P. and Jorgensen I. (1977) Determination of serum thyroxine in two species of toads: variation with season. Gen. Comp. Endocrinol. 33, 566-573. Schaeffer W. H. (1933) Hypophysectomy and thyroidectomy of snakes. Proc. exp. Biol. Med. 38, 1363-1365. Sellers J. C., Wit C. L., Ganjam V. K., Etheridge K. A. and Ragland I. M. (1982) Seasonal plasma T4 titers in the hibernating lizard. Cnemidophorus sexlineatus. Gen. camp. Endocrinol. 46, 24-28.
Thibodeau G. A. (1990) The skin and its appendages. In Textbook of Anatomy and Physiology. 13th Edn (Edited by Hudlin H. C., Kocher L. and Genz J.), pp. 113-132. Times Mirror/Mosby College, St Louis. Tsukahara F., Muraki T. and Nomoto T. (1990) Serum concentrations of thyroid hormones and activity of iodothyronine deiodinase in peripheral tissues of the house musk shrew, Suncus murinus. J. Endocrinol. 125, 117-122. Walker W. F. (1987) Functional anatomy of vertebrates: an evolutionary nersnective. (Edited by Walker W. F.) pp. 123-137. Saunders, Philadelphia. _ Wong C. C., Lam K. Y. and Chiu K. W. (1993) The extrathyroidal conversion of T4 to T3 in EIaphe taeniura. J. camp. Physiol. B 163, 212-218.