GENERAL AND COMPARATIVE ENDOCRINOLOGY 48, l-6 (1982)

Ultimobranchial Gland of the Freshwater Snake Natrix piscator Schneider

RAMESH SINGH AND IBHA KAR

Department of Zoology, Banaras Hindu University, Varanasi 221 005, India

Accepted September 7, 1981

Natrix piscator possesses one pair of equal-sized ultimobranchial glands which are spheroidal, pinkish, or translucent yellow and measure 0.4-1.0 mm in diameter. They are bilaterally staggered in the thyro-thymic region (20-25 ventral scutes), the left one being anterior. The well-vascularized glandular parenchyma is surrounded by a connective tissue capsule and is composed of follicles with eosinophilic colloid and cell clumps. The gland showed seasonal histological changes. The ultimobranchial glands were seen to become enlarged and almost follicular in winter. Their follicles were filled up with dense stratified colloid, representing the storage of secretions. Ultimobranchialectomy caused significant hypercalcemia (P < 0.01) and hypophosphatemia (P < 0.01) in the male snakes.

Clark (1971b) stated in her review on the reptilian ultimobranchial gland that “Rep- tilian ultimobranchiai bodies are not well studied, probably because they are small, ill-defined glands which are often not visi- ble macroscopically.” Only a few his- tomorphological studies are available on the ultimobranchial gland of ophidians (Francescon, 1929; Watzka, 1933; Sehe, 1965; Yoshihara et al., 1979).

Extracts of the ultimobranchial glands of lizards, turtles, and snakes have been re- ported to produce hypocalcemia in white rats (Moseley et al., 1968; Clark, 1968; Copp and Parkes, T968; Uchiyama et al., 1978). But on the other hand, administra- tion of the mammalian calcitonin or ul- timobranchial extracts to reptiles has been without any effect on their blood calcium and phosphate levels (Clark, 1972). Ul- timobranchialectomy was performed earlier only in the lizard, Lacerta agilis (Sembrat and Drzewicki, 1936; Eggert, 1938), but no physiological tests were done. Thus, the physiological role of ultimobranchial gland has not, so far, been clarified in any reptil- ian species (Clark, 1971b). Therefore, further studies are necessary.

MATERIALS AND METHODS

Snakes were collected locally in the suburbs of the city of Varanasi, India. Animals were acclimated to the laboratory conditions (Singh and Kar, 1979).

More than 80 specimens were dissected under anesthesia with Nembutal (2.5 mg1100 g body wt) to locate the glands. As will be described later, Nutrix piscator has one pair of ultimobranchial glands, lo- cated in the thyro-thymic region (20-25 ventral scutes). A longitudinal incision was made on the mid- line in this region. Ultimobranchial glands were lo- cated with the help of a dissecting binocular micro- scope, and they were removed surgically. Special care was taken to avoid injury to the jugular vein and vas- cular supply of the thyroid gland. The incision was dusted with sulfadiazine powder and then sutured with surgical thread. The glands were removed in different seasons throughout the year by the above technique. They were fixed in Bouin’s fluid and sectioned serially at 5 pm by the routine paraftin method. Sections were stained with hematoxylin and eosin and subjected to histological examination.

An ultimobranchialectomy experiment, to study its effect on the concentration of serum calcium and phosphate, was performed on the adult male snakes in the months of June and July 1979. Females, having high blood calcium, perhaps due to estrogen secretion, were not used. A total of eight snakes were ultimo- branchialectomized by the above technique. Six sham- operated controls were given the same treatment, ex- cept that the glands were left intact.

Blood samples of the ultimobranchialectomized and sham-operated controls were collected with a

0016~6480432/090001-06$01.00/0 Copyright @ 1982 by Academic Press, Inc AlJ rights of reproduction in any form reserved.

2 SINGH AND KAR

hypodermic syringe from the postcaval vein at the time of operation and at the intervals of 1, 5, and 10 days postoperation, after making a small incision in the re- gion of ventral scutes 30-35. The incision was sutured as stated above. Serum calcium was analyzed by the method of Loureiro and Janz (1944). Serum inorganic phosphate was extracted by the method of Mukherjee et al. (1965) and estimated as P by the method of Mar- tin and Doty (1949). Student’s 1 test was used for the statistical evaluation.

RESULTS

All the specimens of the freshwater snake N. piscator were found to possess one pair of ultimobranchial glands. The approximate location of the glands is shown in Fig. 1. The glands were spheroidal in shape and pinkish or translucent yellow in color. The size of the gland varied (0.4-1.0 mm in di- ameter) in relation to the body size. How- ever, glands on either side of a specimen were equal in size. The glands were found to be markedly enlarged in size in winter; thus, it was somewhat easier to locate them in this season.

The normal histology of the gland is shown in Figs. 2 and 4. The glandular parenchyma was surrounded by a connec- tive tissue capsule. The well-vascularized glandular parenchyma was composed of compact cell clumps and follicles. The fol- licular lumen was generally ovoid and con- tained eosinophilic colloidal material. Fol- licular epithelium was simple. It was either stratified or cuboidal or low columnar. Ciliated and enlarged goblet cells were ab- sent. Within the limitations of the routine histological methods and light microscopy, the glandular parenchyma was found to be composed of a single type of cell. Nuclei were ovoid and had dense chromatin granules. The gland became almost follicu- lar in winter, and the amount of cell masses was seen to decrease during this period. Follicles were tilled up with dense stratified colloid, representing the storage of secre- tions (Fig. 3).

Ultimobranchialectomy was not fatal to N. piscator. No apparent pathological con- ditions were observed in ultimobran-

RPTG (L)

CA (L)

JV (L)

CPTG (L)

THYROID

:>ES T. )

SA (L)

PV

FIG. 1. Diagram showing the anatomical position of the pharyngeai endocrine glands of Natrix piscator. ANT., anterior; AZY., azygous vein; CA, carotid ar- tery; CPTG, caudal parathyroid gland; JV, jugular vein; (L), left; PA, pulmonary artery; PC, postcaval vein; POST., posterior; PV, pulmonary vein; (R), right; RPTG, rostral parathyroid gland; SA, systemic artery; THYM., thymus; UBB, ultimobranchiai body; VA, vertebral artery: VEST., vestigeal.

chialectomized snakes. They behaved like the sham-operated controls.

The effect of ultimobranchialectomy on the serum calcium concentration is present- ed in Fig. 5a. Normal serum calcium level in N. piscator averaged 10.41 & 0.38 mg/ 100 ml (mean -+ SE). Ultimobranchialec- tomy resulted in significant hypercalcemia (P < 0.01). The serum calcium concentra- tion was seen to rise steadily up to the 5th day after ultimobranchialectomy, but there- after, it declined slightly toward normal on the 10th day. Serum phosphate value de-

ULTIMOBRANCHIAL GLAND IN SNAKE 3

FIG. 2. Ultimobranchial gland of N. piscator showing compact cell clumps and follicles. C, Capsule; F, follicle. x57.

FIG. 3. Completely follicular ultimobranchial gland of N. piscator during winter. TG, Thyroid gland; UB, ultimobranchial gland. X57.

FIG. 4. An enlarged view of a part in Fig. 2. BC, Blood capillary; BS, blood sinusoids; CT, connective tissue stroma. X665.

creased significantly (P < 0.01) from the Ultimobranchialectomy also disturbed normal value of 3.57 + 0.17 to 1.81 + 0.15 the serum CaIZ’i ratio. The CaIPi value in- mg/lOO ml on the 5th day. This condition creased significantly by about 100% on the improved slightly to 2.05 + 0.21 mg/lOO ml 5th day of operation, but a slight normali- on the 10th day (Fig. 5b). zation in the condition was seen to start by

4 SINGH AND KAR

0

3 t-u-u-- 8.0 I- 6’0

4’0

E

DAYS AFTER ULTIMOBRANCHIALECTOMY

FIG. 5. Effect of ultimobranchiakctomy on serum calcium and inorganic phosphate in N. piscaror. 0, Sham-operated control; A, ultimobranchialectomized.

the 10th day after ultimobranchialectomy (Fig. 5~).

DISCUSSION

According to Francescon (1929), only the left gland is present in some species of snakes. “The ultimobranchial gland is characteristically present only on the left side of the neck in reptiles . . . ” (Clark, 1972). Our observations on N. piscator, along with those of Sehe (1965) and Yoshihara et al. (1979), however, indicate

that at least in snakes the gland is bilateral. The staggered location of the paired inter- nal organs is a characteristic feature of ophidian anatomy. This feature is apparent in N. piscutor as far as the ultimobranchial gland is concerned, and it has also been noted by Yoshihara et al. (1979) in some other ophidians. Similarity in the size of the ultimobranchial glands of either side, as we saw in N. piscator, has also been noted by Yoshihara et al. (1979), but these observa- tions are in contrast to those of Francescon (1929), Watzka (1933), and Sehe (1965).

The ultimobranchial gland of N. piscator has cell clumps and follicles, the typical histological feature of the gland in most of the reptiles (Watzka, 1933; Sehe, 1965; Clark, 1971b; Khairallah and Clark, 1971; Das and Swarup, 1975; Anderson and Capen, 1976; Yoshihara et al., 1979). The ciliated and goblet cells in the follicular ep- ithelium, as have been reported in some lizards and terrestrial snakes (Das and Swat-up, 1975; Anderson and Capen, 1976; Yoshihara et al., 1979), are absent in this species and in freshwater turtles (Clark, 1971b; Khairallah and Clark, 1971).

There seems to be some correlation be- tween the activity of the ultimobranchial gland and the physiological activity of N. piscutor. Seasonal changes were seen in the activity of the ultimobranchial gland of N. piscutor and were noticeable in the winter (the hibernation period of snake), when the gland seemed less active. Seasonal changes in the activity of the ultimobranchial glands of lizards have been reported by Eggert (1938) and Sehe (1965), but not by Das and Swarup (1975) and Anderson and Capen (1976).

Ultimobranchialectomy caused no mor- tality and no apparent pathology in N. pis- cutor. Sembrat and Drzewicki (1936) and Eggert (1938) have reported similar results in the lizard Lucertu ugilis.

No previous reports are available on the physiological effect of ultimobranchialec- tomy in reptiles, although some such

ULTIMOBRANCHIAL GLAND IN SNAKE 5

studies have been carried out on some other vertebrate groups. The hypercalcemic ef- fect of ultimobranchialectomy has been re- ported in teleosts (Chan, 1970; Fenwick, 1975). Ultimobranchialectomy also causes hypercalcemia and depletion of paraverte- bral lime sacs, the main site of calcium stor- age, in anuran tadpoles as well as adults, and their normal serum calcium concentra- tion can be restored again by implantation of the ultimobranchial gland (Robertson, 1968, 1969; Sasayama and Oguro, 1976; Sasayama, 1978). Garlich and Bryant (1975) have reported hypercalcemia and hypo- phosphatemia in ultimobranchialectomized cockerels, while Speers et al. (1970) have reported that such laying hens had consis- tently lower values of both serum calcium and phosphate. On the other hand, Brown et al. (1970) have reported that ultimobran- chialectomy in chickens is associated with no change in serum calcium, but is asso- ciated with a hypophosphatemic change.

Anderson and Capen (1976) have also suggested that this gland plays a role in the regulation of calcium level in reptiles, as this gland in Iguana iguana accumulates a large number of secretory granules in re- sponse to hypocalcemia and becomes less active. Hypercalcemia induces hyperactive changes in the ultimobranchial gland of lizards, Calotes versicolor (Dubewar and Suryawanshi, 1977) and Vromastix hard- wicki (Dubewar et al., 1978), but not in that of the monitor lizard, Varanus monitor (Das and Swarup, 1975).

The presence of hypocalcemic hormone (calcitonin) in the ultimobranchial glands of lizard, turtle, and snake has also been dem- onstrated in bioassays using white rats (Moseley et al., 1968; Clark, 1968; Copp and Parkes, 1968; Uchiyama et al., 1978). On the other hand, administration of mam- malian calcitonin or ultimobranchial extract has no effect on the blood calcium and phosphate levels in reptiles (Clark, 1968, 1971a, 1972; Dix et al., 1970; Uchiyama et al., 1978). Lack of this effect does not

prove that the hormone has no effect on calcium metabolism in reptiles (Clark, 1971b). Yarious probable reasons responsi- ble for the differences in the action of mammalian and reptilian calcitonin have been discussed by Clark (1971b).

Our present study indicates that the ul- timobranchial gland of N. piscator secretes a hypocalcemic and hyperphosphatemic factor. It is hypothesized that this factor might have a definite role in the regulation of calcium and phosphate metabolism in the reptilian body along with other glands, such as parathyroids.

ACKNOWLEDGMENTS The authors wish to express their thanks to Profes-

sor J. P. Thapliyal and Dr. B. Prasad for providing laboratory facilities. R. S. is indebted to CSIR for the award of SRF.

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6 SINGH AND KAR

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