ZOOLOGICAL SCIENCE 4: 201-204 (1987) © 1987 Zoological Society of Japan

[ C O M M U N I C A T I O N ]

Prolactin Cells of Clarias batrachus in Response to Corpuscles of Stannius Extract Administration

SHYAM P. SRIVASTAV, KRISHNA SWARUP 1 and A J A I K. SRIVASTAV

Department of Zoology, University of Gorakhpur, Gorakhpur 273 009, India

ABSTRACT—Corpuscles of Stannius extract adminis­tration increased the activity of prolactin cells.

INTRODUCTION

Removal of corpuscles of Stannius (CS) causes a rise in serum calcium level [1-4] which is corrected by administration of CS extract [5-8]. This suggests that CS secrete hypocalcemic principle (s). So far, two hypocalcemic principles-'hypocal-cin' [9] and 'teleocalcin' [10] have been reported from the CS. Recently, a PTH-like substance (parathyrin) has been localized immunocytochemi-cally in the eel CS [11]. Extracts of CS have been found to be effective in inducing hypocalcemia in mammal [12], bird [13], amphibians [14] and intact fishes [2, 6, 9, 15, 16].

Induced hypocalcemia is met with an increased activity of parathyroid gland in tetrapods [17-19 ]. But in fishes, parathyroid gland is absent and in this group prolactin has specific hypercalcemic capacities [20-23]. There exists no earlier report regarding the response of administration of CS extract on prolactin cells in fish. In the present study, therefore, we have attempted to study such a response in a freshwater catfish, Clarias bat­rachus.

MATERIALS AND METHODS

Sixty adult male specimens of Clarias batrachus

(23-27 cm /80-120g) were collected locally during the first week of September and acclimatized to the laboratory conditions (temp. 23-25°C) for two weeks prior to use. They were then divided into two numerically equal groups-A and B. Group A: Control fish received intraperitoneally 1 ml/100g body weight of saline (0.6% NaCl solution; physiological saline) and were kept in tap water. Group B: Experimental fish received intraperi­toneally CS extract in a dosage of 5mg/ml/100g body weight and were maintained in tap water.

The CS used in this study were surgically removed from both sexes of adult Clarias bat­rachus (September-October is post-spawning period; in females during September the ovary displays large number of immature oocytes and unovulated mature oocytes in the process of resorption whereas in males the lobules show compactness, the interlobular septa become thick with numerous blood cells and the germ cells which are inactive during the period of functional maturity begin to divide). The glands were stored in ice and used almost immediately. Prior to use, the glands were weighed (weight of CS of average fish, 0.6mg) and homogenized in ice-cold saline (0.6% NaCl solution). The homogenate was centrifuged for 10min at 5,000rpm and the super­natant was collected. Final volume of the super­natant was made up so that every 1ml solution contained extract from 5mg of CS.

Six specimens from each group were anaesthe­tized with MS 222 at 1/2, 1, 2, 4 and 6hr following the injection and the blood samples were collected

Accepted September 22, 1986 Received May 19, 1986

1 To whom correspondence should be sent.

202 S. P. SRIVASTAV, K. SWARUP AND A. K. SRIVASTAV

by the sectioning of caudal peduncle. An initial sampling of blood (from 6 fish kept separately under similar conditions) was also taken before the start of the experiment (Zero hour). After clotting of the blood the sera were separated by centrifuga-tion at 3,500rpm and analysed for serum calcium level according to Trinder's [24] method. Pituita-ries were fixed in aqueous Bourn's fluid and Bouin-Hollande fixative. After routine processing in graded series of alcohol and clearing in xylene, tissues were embedded in paraffin wax. Serial sections were cut at 4-6 µm and stained according to Herlant tetrachrome and Heidenhain's azan techniques.

The nuclear diameter was measured with the aid of ocular micrometer. Each nucleus was measured along its long and short axes and mean value was calculated. From each group 300 nuclei were measured at every interval (Fifty nuclei were measured from each fish). The data are presented as mean±standard deviation of the mean. Differences in the serum calcium level and nuclear diameter among different groups were analysed by Student's t-test.

RESULTS

The serum calcium level, before the start of the experiment (Zero hour), was recorded as 9.77± 0.42mg/100ml.

In C. batrachus, CS extract administration evoked hypocalcemia after 1/2 hr which continued up to 1 hr. Thereafter, the serum calcium level increased progressively reaching the normal value after 4 and 6 hr of the initiation of the experiment (Fig. 1).

The prolactin cells of fish from group A (saline injected) showed no change in response to the treatment. The prolactin cells possessed indistinct boundaries, the nuclei, however, were distinct. The cytoplasm was noticed granular and azocarmi-nophilic and erythrosinophilic in nature (Fig. 2).

The first perceivable change in the prolactin cells of specimens of group B (CS extract-treated) was observed at 2 hr as the gland exhibited degranula-tion and increase in nuclear size (Fig. 1). These changes were pronounced and the nuclei became hyperchromatic after 4 hr of the treatment (Fig. 3).

Six hours after the initiation of the experiment, the prolactin cells showed recuperation and the nuclei had a tendency towards reduction in their size (Figs. 1 and 4).

DISCUSSION

In tetrapods, where parathyroids are the hyper-calcemic glands, there is an increase in nuclear size to meet the induced hypocalcemia [17-19]. In C. batrachus, CS extract induced hypocalcemia ren­ders prolactin cells to undergo similar changes (degranulation, increase in nuclear size and chro-maticity i .e . increased synthesis and release of the hypercalcemic factor) at hour 2 which is maximum at hour 4. This is evidently to meet the hypocalce-mic challenge and to restore the serum calcium

FIG. 1. Serum calcium level and nuclear diameter of prolactin cells after CS extract administration, a, b, c and d indicate significant differences compared with matching controls, P<0.05, P<0.02, P<0.01 and P<0.001, respectively.

203 Effects of CS Extract on Fish Prolactin Cells

level. Obviously, when the serum calcium level reaches normocalcemia at hour 6, the activity of these cells retards. These observations lend sup­

port to the suggestion of Pang et al. [7]"...the hypocalcemia caused by treatment with Stannius corpuscle homogenate might elicit the release of prolactin".

The present data suggest that calcium has a role in the regulation of prolactin secretion. Several reports agree with the present results showing that calcium levels in the blood and/or the environment are the main factors regulating the activity of prolactin cells [25-27]. Contrary to it, Olivereau and Olivereau [28] have suggested that sodium, and not calcium, plays a major role in the regulation of prolactin secretion. The reduced role of calcium in the regulation of prolactin cells has also been advocated by Ball et al. [29] and Olivereau et al. [30]. It has also been shown that the amount of prolactin released in vitro, by thtTilapia pituitary is not affected by the calcium concentration [31] but it is controlled by the osmotic pressure of the incubation medium [32].

ACKNOWLEDGMENT

The authors are thankful to M/S Sandoz Ltd., Basel for generous gift of MS 222 and UGC, New Delhi for financial assistance.

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FIG. 2. Prolactin cells of saline-injected C. batrachus showing granular cytoplasm and well marked nuclei. Herlant tetrachrome. X900.

FIG. 3. Prolactin ceils of C. batrachus showing hyper-chromatic nuclei and degranulation after 4 hr of CS extract administration. Herlant tetrachrome. X 900.

FIG. 4. Prolactin cells of C. batrachus showing recu­peration of secretory granules after 6hr of CS ex­tract administration. Herlant tetrachrome. X900.

204 S. P. SRIVASTAV, K. SWARUP AND A. K. SRIVASTAV

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