GENERAL AND COMPARATIVE ENDOCRINOLOGY 51, 71-76 (1983)

Parathyroid Gland of the Freshwater Snake Natrix piscator Schneid,er

RAMESH SINGH AND IBHA KAR

Department of Zoology, Banaras Hindu University, Varanasi-221005, India

Accepted August 5, 1982

The structure and function of the parathyroid glands of freshwater snake, Natrix piscatuu, were studied. N. piscator has two pairs of parathyroid glands which are composed of cell cords of typical endocrine appearance, and follicles are absent. Parathyroidectomy was fol- lowed by significant (P < 0.001) hypocalcemia and hyperphosphatemia throughout the 5th week of experimental period. Following parathyroidectomy, calcium and phosphate concen- trations in bone were significantly (P < 0.05) increased after 5 weeks; however, these values in muscle were not significantly changed. Tetany was also observed in parathyraidec- tomized snakes. Histological structure and function of the glands do not appear to vary sea- sonally. Administration of parathyroid gland extract of this ophidian reptile into rats caused signilicant (P < 0.05) elevation of serum calcium.

Some recent functional studies confirm the importance of the parathyroid gland in calcium and phosphate metabolism in most groups of reptiles (Peters, 1941; Clark, 1965, 1971; Sidky, 1966; Clark et al., 1969; Dix et al., 1970; Oguro, 1970, 1972, 1975; Clark and Dantzler, 1972, 1975; Oguro and Tomisawa, 1972; Oguro et al., 1974; Du- bewar et al., 1975; Oguro and Sasayama, 1976).

Barring a few reports (Clark, 1965; Clark and Dantzler, 1972, 1975), all the studies mentioned above focused attention only on the changes in serum calcium and phos- phate levels following parathyroidectomy. In the only study found of the action of the parathyroid gland on kidney in ophidians, Clark and Dantzler (1972) have reported a change from the normal net tubular secre- tion to net tubular reabsorption of phos- phate, but no significant alteration in the reabsorption of calcium following parathy- roidectomy. Clark (1965) has reported no significa@t change, in the total calcium con- centration in bone of the turtle, following either parathyroidectomy or injection of parathyroid extract. No studies of the ef- fect of p$-athyroidectomy on bone calcium

and phosphate have been carried out in ophidians. The fact that removal of the parathyroid glands results in hypocalcemia, but does not alter reabsorption of calcium (Clark and Dantzler, 1972, 1975) indicates that the hormone might be important in ac- tion on bone.

The effect of total and partial parathy- roidectomy on serum calcium and phosphate (Singh and Kar, 1979a, b) in this species has been studied. The present investigation is a continuation of the above work in the same species. Its purpose is (1) to study the histo- morphology of the gland, (2) to determine the effects of parathyroidectomy on cal- cium and phosphate concentrations in the serum, bone, and muscle, and (3) to deter- mine the activity of the acid extract of para- thyroid gland of this ophidian reptile in a bioassay using white rats.

MATERIALS AND METHODS

Regular collection of snakes was made in the sub- urbs of Varanasi, India. Snakes were acclimatized to laboratory conditions for a period of 2 weeks before the parathyroidectomy experiments were performed on them. They were kept in cages containing water filled earthen bowls and were fed on small frogs.

For anatomical study, a total of 100 snakes were dis-

71 0016-6480183 $1 SO Copyright @ 1983 by Academic Press, Inc. AU rights of reproduction in zny form reserved.

72 SINGH AND KAR

sected throughout the year. For histological study, parathyroid glands were removed, fixed in Bouin’s fluid, paratfin embedded, sectioned at 5 p, and stained with hematoxylm and eosin. Following parathyroidec- tomy experiments were performed on the adult male snakes (average body wt 114.56 + 6.08 g).

Experiment I. This experiment was set up to investi- gate the effect of parathyroidectomy on calcium and phosphate concentrations in serum, bone, and muscle. Seventeen snakes were segregated into two experi- mental batches. Batch 1 consisted of five totally parathyroidectomized and four sham-operated con- trols; batch 2 consisted of four totally parathyroidecto- mized and four sham-operated controls. Parathyroid- ectomy procedures were the same as those previously described (Singh and Kar, 1979a). The glands were re- moved surgically. The caudal parathyroid glands were removed along with the anterior thymus lobes and were studied histologically to ensure the complete re- moval of the gland.

Blood samples were collected from the animals of batch 1 at the time of operation and at the lst, 3rd, 5th, and 7th day postoperation. Blood samples from the au- imals of batch 2 were collected at the time of operation and at the 2nd, 3rd, 4th, and 5th week postoperation. Animals were sacrificed after the 5th week. Bones (ribs) and longitudinal muscle strands, from either side of the vertebral column, were removed for mineral analysis. Bone and muscle were wet weighed and di- gested with 60% perchloric acid in separate Kjeldhal flasks, and made to volume with distilled water. Por- tions of the digested materials were used for estima- tion of total calcium and phosphate. Methods of cal- cium and phosphate estimation were the same as previously described (Singh and Kar, 1982).

Experiment II. This experiment was designed to in- vestigate any behavioral changes after parathyroidec- tomy, using three sham-operated controls and four to- tally parathyroidectomized snakes. Snakes were kept in individual cages and exercised from time to time to check for the appearance of tetany. Both parathyroid- ectomy experiments (I and II) were performed during the period between December and mid February (am- bient temperature 18.24 + 0.36”C) and were repeated during summer months, i.e., between May and June (ambient temperature 33.59 f 0.87”C).

Experiment ZZZ. This experiment was performed during the months of April and May to investigate the activity of acid extracts of parathyroid gland of Nutrix piscator in white rats. Methods were followed after Clark (1968). Rostral parathyroids were excised in 2.5 snakes of both sexes (101.00 -t 4.04 g body wt) and homogenized in glass homogenizers with ice-cold 0.1 N HCl. Homogenates were centrifuged at 5000 rpm for 30 min, and pellets were discarded. The supematants were frozen until use.

Nine male white rats (93.64 * 3.27 g body wt) were

acclimatized to the laboratory conditions for 2 weeks. They were fed on water-steeped grains of chick pea (Ciser arietinum), and water was given ad libitum. Rats were segregated into two batches and injected subcutaneously with 0.5 ml of 0.1 N HCI containing four parathyroid glands or 0.1 N HCl alone. They were sacrificed 1 hr after the injection, and blood was sam- pled from their hearts for analysis of serum calcium by the method used in Experiment I.

RESULTS

All the specimens were found to possess two pairs of parathyroid glands: parathyroid III (rostral) and parathyroid IV (caudal). The rostra1 parathyroids were located near the angle of jaws, and the caudal parathy- roids were found embedded in or attached to the posterior half of the anterior lobes of the thymus (Figs. 1, 2). The anatomical po- sition of the glands is shown diagrammati- cally in our previous observation (Singh and Kar, 1982). Accessory parathyroid tis- sue was found only in one female specimen in which there were two glands attached to the right anterior thymus (Fig. 2) and three glands attached to the left anterior thymus. The glands were spheroidal and pinkish. The size of the gland varied (0.5-2.0 mm) in relation to the body size. However, all the four glands of a specimen seemed equal in size. The parathyroid glands are encap- sulated and consist of a cord like cell ar- rangement; no follicles were present (Fig. 3). Only one cell type was seen (Fig. 4). The cell boundaries were generally inconspicu- ous, the cytoplasm was stained with eosin, and the nucleus had a prominent nucleolus and dense chromatin granules. There were no marked seasonal histological variations.

Normal serum calcium and phosphate levels in N. piscator averaged 10.41 5 0.38 and 3.57 ? 0.17 mg/lOO ml (mean ? SE), respectively. Normal CaIPi values aver- aged 3.08 f 0.46. Results of the parathyroi- dectomy experiments conducted in two dif- ferent seasons (winter and summer) did not differ from each other significantly, and so the data were compiled and presented in Fig. 5 and Tables 1 and 2. Total parathyroi-

SNAKE PARATHYROID GLAND 73

FIG. 1. A bilobed caudal parathyroid gland (CPTG) of N. piscator embedded in the thymus (TM). x60.

FIG. 2. Two caudal parathyroid glands (CmG) attached superficially to the thymus (TM) in an ex- ceptional female specimen of N. piscator. BC, blood capillaries; CT, connective tissue; x 60.

FIG. 3. Rostra1 parathyroid gland (RFTG) of Natrix piscator. C, capsule; N, nervous tissue; x 60. FIG. 4. The glandular parenchyma of the parathyroid ofN. piscator. C, capsule; arrowheads, nuclei

of the connective tissue fibrocytes; x 700.

74 SINGH AND KAR

WEEKS AFTER PAR*,“YROIDECTOw FIG. 5. Effect of parathyroidectomy on the levels of

serum (A), inorganic phosphate (B), and the Ca/Pi ratio (C) in N. piscator. FTXS, sham operated; PTX, parathyroidectomized.

dectomy caused significant (P < 0.001) hy- pocalcemia and hyperphosphatemia. No significant changes were observed in total calcium or phosphate levels in the muscle following parathyroidectomy (Table 1); however, these levels in bone increased sig- nificantly (Table 2). Parathyroidectomy did not cause any significant change in Ca/P ratio of bone.

Parathyroidectomy was not fatal to the animal. Tetany was observed (1) after 20 days in one out of the four snakes which were parathyroidectomized between De- cember and mid February, (2) in two out of four snakes which were parathyroidecto- mized between May and June. In one animal, it was observed after 33 days. Serum calcium was not analyzed in these snakes. Tetany was characterized by severe muscular spasms followed by bending of the head at a right angle to the neck; the body became kinked, stiff and coiled. The jaws were slightly skew, but there was no salivation. Sometimes the animal lay on its back. Ani- mals recovered to their normal state in about 2-3 min.

The effect of parathyroid gland extract treatment on serum calcium level of white rats is presented in Table 3. The parathy- roid gland extract of N. piscator caused sig- nificant elevation in serum calcium level in rats.

DISCUSSION

In ophidians, two pairs of parathyroid glands have been reported; although few re- ports before 1904 mentioned more than two pairs of parathyroids, but these gland like structures have not been definitely identi- lied as parathyroids (see Clark, 1970). Ac- cessory parathyroid tissue has been noted in some ophidians (Clark, 1971; Singh, 1981). In general, the location, morphol- ogy, and histology of the parathyroid glands of N. piscator compare favorably with those of reptilian species. Parathyroid follicles have been observed in some ophid- ians (Clark, 1971; Oguro, 1970, 1972). Only

TABLE 1 EFFECT OF PARATHYROIDECTOMY ON THE LEVEL OF MUSCLE CALCIUM AND PHOSPHATE IN

Natrix piscator, 5 WEEKS AFTER THE OPERATION

Nature of operation

Sham-operated control Parathyroidectomized

Ca P

(WlOO d @WOO d

8.35 + 0.40 222.38 r+ 8.72 8.67 + 0.55NS 213.30 k 8.14NS

CalP

0.037 2 0.0029 0.040 k 0.0031NS

SNAKE PARATHYROID GLAND 75

TABLE 2 EFFECT OF PARATHYROIDECTOMY ON THE LEVELS OF BONE CALCIUM AND PHOSPHATE IN

Natrix piscator, 5 WEEKS AFTER THE OPERATION

Nature of operation Ca WOO g) p (g/100 g) CdP

Sham-operated control Parathyroidectomized

* Difference significant P < 0.05.

10.41 + 0.40 5.33 2 0.58 2.09 t 0.26 13.00 +- 0.37” 6.93 t 0.50* 1.90 t O.ltP

the chief cells are identified in reptilian parathyroid; the oxyphil cells have been re- ported absent in them. The only exception is the ultrastructural observation on turtle (Clark and Kharaillah, 1972).

Total parathyroidectomy caused tetany, significant hypocalcemia, and hyperphos- phatemia in N. piscator. Thus, this species responds to parathyroidectomy in the same way as do other ophidians (Oguro, 1970, 1972; Clark, 1971). Clark and Dantzler (1972, 1975) have reported such changes following parathyroidectomy in four spe- cies of Nutrix (N. sipedon, N. rhombifera, N. cyclopion, and N. erythrogaster). The duration and characteristics of tetany were the same as reported in other reptilian species.

It is evident from histological as well as physiological observations that there was no significant seasonal variation in the physiological function of the parathyroid gland in N. piscator. This is in accord with the observations of Clark and Dantzler (1972, 1975).

TABLE 3 EFFECT OF PARATHYROID EXTRACT OF

Natrix piscator ON THE SERUM CALCIUM IN WHITE RATS

Nature of treatment Serum Ca

(mg/lOO ml f SE)

0.5 ml HCl (0.1 N) 11.87 t- 0.40 (control) (4)

0.5 ml parathyroid extract 13.55 f 0.53* in 0.1 N HCI = 4 glands/rat (5)

a Numbers in parentheses denote animal number. * Difference significant, P < 0.05.

It seems that parathyroid glands have no effect on calcium and phosphate content in the muscles of N. piscator. No such studies are available in other reptilian species. Parathyroidectomy caused significant ele- vation in the total calcitrm and phosphate content of bone in N. piscator. It is safe to suggest that the elevation of both calcium and phosphate levels in bone might be due to lack of resorption action of parathyroid hormone, as a result of ablation of the glands. This might also be responsible in part for decreased serum calcium, because parathyroidectomy does not alter the reab- sorption of calcium in kidney of snakes (Clark and Dantzler, 1972, 1975). Hypbcal- cemia in parathyroidectomized N. piscator might also be in part due to hypocalcemic action of ultimobranchiial calcitonin (Singh, 1981). Despite, the increased levels of bone phosphate after parathyroidectomy, serum phosphate values increased. This is un- doubtedly due to decreased excretion ,:of renal phosphate after, parathyroidectomy (Clark and Dantzler, 1972, 1975).

Crude extract of the parathyroid glands of N. piscator was effective in inducing hy- percalcemia in white rats. It was shown previously that mammalian parathyroid ex- tract will elevate serum calcium in snakes (Clark, 1971). Thus, parathyroid extract of either group (mammal and reptile) is eftec- tive in the other group. ‘However, this is not true for calcitonin (see Clark, 1972).

ACKNOWLEDGMENTS

We thank Professor I. P. Thapliyal and Dr. B. Pra- sad for providing laboratory facilities. One of us (RS) is indebted to C.S.I.R., New Delhi, for the award of a post doctoral fellowship.

76 SINGH AND KAR

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