GENERAL

AND

COMPARATIVE

ENDOCRJNOLOGY

25,

323-331 (1975)

Control of the Interrenal Gland of the Freshwater Turtle Chrysemys picta in Viva and in Vitro1 GLORIA V. CALLARD Department

of Biology,

Tufts

University,

Medford,

Massachusetts

0215j2

Accepted October 8, 1974 Interrenal function was studied in the freshwater turtle Chrysemys picta by competitive protein-binding analysis of plasma corticosterone in vivo and corticosterone synthesis by interrenal cell suspensions in vitro. Although circulating levels of corticosterone in intact, unanesthetized turtles (0.22 PgllOO ml) are lower than in other vertebrates, hypophysectomy reduced and ACTH increased these levels. Synthetic ACTH,-,, was more potent in vivo than pACTH, and in vitro 10-1000 pg/ml gave a log dose increase in corticosterone production. Interrenal cell suspensions were also responsive to crude homogenates of turtle pituitary. Bleeding was a potent stimulus to interrenal secretion in vivo mediated in part via endogenous corticotropin release.

Morphologic studies indicate that the pituitary-inter-renal axis in reptiles is similar to that in other vertebrates (Wright and Chester-Jones, 1957; Callard and Willard, 1969; Callard and Chester-Jones, 197 l), and direct evidence for hypothalamic-pituitary-interrenal relationships was obtained in recent studies using competitive protein-binding assay for plasma corticosterone in lizards (Licht and Bradshaw, 1969; Daugherty and Callard, 1972; Callard, Chan, and Callard, 1973). In turtles, as in other reptiles, the major steroids synthesized from labeled precursors by inter-renal tissue in vitro are corticosterone and aldosterone (Macchi and Phillips, 1966; Sandor, Lamoureux, and Lanthier, 1964; Leloup-Hatey, 1968; Mehdi and Carballeira, 197 1). Corticosterone has also been measured by doubleisotope-dilution analysis in postcaval blood of Pseudemys (Nothstine et al., 1971). I Supported by a Tufts University Faculty Research Award. * Requests for reprints should be sent to Biology Dept., 2 Cummington St., Boston University, Boston, Mass. 02215.

However, experiments in vitro (Macchi and Phillips, 1966; Leloup-Hatey, 1968) and in vivo (Nothstine et al., 1971) failed to demonstrate interrenal responses to ACTH of either mammalian or chelonian origin or indicated that the turtle interrenal was relatively insensitive to these substances. The endocrine system and secretory controls in turtles may be particularly interesting from an evolutionary viewpoint, as Chelonia are thought to be the most primitive contemporary reptiles. This investigation reexamines the secretory response of the turtle interrenal in vivo and in vitro using the sensitive competitive protein-binding assay for corticosterone. MATERIALS

Animals Painted turtles (Chrysemys picta) 5-7 in. long, weighing 250-900 g were obtained from a commercial supplier (Nasco, Oshkosh, WI) between January and August 1973. Ten or fewer turtles were housed in concrete tanks measuring 30 X 36 in. Continuously running tap water maintained a level of about 3 in. An overhead heat lamp and rocks were provided to 323

Copyright @ 1975 by Academic Press, Inc. AU tights of reproduction in any form reserved.

AND METHODS

324

GLORIA

V. CALLARD

permit turtles to emerge from the water for “basking.” The temperature gradient in the tanks ranged from 15°C in the water to 33°C air temperature just beneath the heat lamps and 19°C air temperature at the periphery. No attempt was made to determine thermal preferences. Turtles were maintained under these conditions for 1-4 wk and fed ad lib lean ground beef fortified with bone meal and cod liver oil at weekly intervals except during experimentation.

Blood Collection Turtles were prepared for blood collection as follows: after immobilization in chipped ice, the heart was exposed by drilling a 5/16-in. hole in the plastron at the midline 1 cm posterior to the anterior margin of the abdominal scutes. A cork lubricated with antiointment (bacitracin-neomycin-polymixin, biotic Fougera, Hicksville, NY) sealed the hole until the start of the experiment and between sampling intervals. Turtles were returned to the holding tanks for 24-48 hr before the first sample was collected, and recovery, as indicated by swimming activity began immediately. At the time intervals indicated in the protocol, blood was removed by syringe directly from the ventricle of unanesthetized turtles with a minimum of disturbance. Samples were collected in heparinized tubes, centrifuged, and the plasma stored at - 15°C until analysis.

Hypophysectomy Turtles were hypophysectomized 24-48 hr prior to testing except as indicated in Expt. III. Sodium pentobarbital (Nembutal, 6 mg im) followed by immersion in chipped ice was found to be most satisfactory for anesthesia and recovery. A Plexiglas apparatus was constructed for holding the head firmly in position and jaws completely agape. A hole made in the basisphenoid bone with a dental drill exposed the anterior lobe which could be removed with watchmakers forceps and gentle suction. The cavity was plugged with Gelfoam absorbable sponge (Upjohn) and coated with antibiotic ointment. A check for hypophyseal remnants was carried out by inspection of the sella turcica with a dissecting microscope at autopsy or microscopic examination of stained sections cut from decalcified heads with the basisphenoid bone in situ.

Hormones Hormones were injected im in 0.05 ml 0.9% NaCl/lOO g body wt as follows: 8 IU porcine ACTH (Sigma); 2.5 or 5.0 pg ACTH,-,, (Synacthen, courtesy of Ciba, Summit, NJ); 1 mg x 3 days dexamethasone (Sigma). Synacthen (10, 100 and 1000 pg/ml incubate) was the corticotropin used in vitro.

Corticosterone Analysis Corticosterone was analyzed in 100 or 200 ~1 of plasma by a modification of Murphy’s competitive protein-binding method (Murphy, 1967; Daugherty and Callard, 1972). Corticosterone was read directly from a standard curve between 0.1 and 20 ng. Samples containing less than 0.1 ng were designated 0.

Protocol In Expt. I the response to porcine ACTH (8 IU/lOO g) was tested in individual turtles by collecting blood for corticosterone analysis at 0, 30, and 60 min after injection. Thus, three l-ml samples were collected from each animal. No more than 60 set was required for blood collection and turtles were returned to their tanks during sampling intervals. One group each of unoperated and hypophysectomized turtles were injected with vehicle alone to control for the effects of the blood collection procedure. Changes due to a reduction in blood volume were estimated by subjecting one group each of unoperated and hypophysectomized turtles to sham bleeding (cardiac puncture alone) at 0 and 30 min while blood for analysis was collected only at 60 min after vehicle injection. In Expt. II, plasma corticosterone was measured in groups of intact and hypophysectomized turtles after an injection of corticotropin. Porcine ACTH (8 IU/lOO g) was tested after 60 or 120 min while ACTH,..,, (2.5 or 5.0 pg/lOO g) was tested after 60 min only. Groups of unoperated and hypophysectomized turtles served as beginning controls and vehicle-injected controls. This experiment differed significantly from Expt. 1 in that a single blood sample only was collected from each animal. In Expt. III, one group of four hypophysectomized turtles was used to compare plasma corticosterone changes in response to bleeding before and after administration of dexamethasone (1 mg/lOO g X 3 days). One milliliter of blood was collected at 0, 30, and 60 min after an im injection of vehicle. The two parts of the experiment were separated by a I-wk interval. In Expt. IV, the effects of ACTH,..,, on corticosterone synthesis was tested in vitro. Interrenals were removed from 16 female turtles and a total of 79 1 mg used to prepare an interrenal cell suspension exactly as described by Sayers ef al. (1973) with the following changes: Krebs-Ringer bicarbonate glucose (KRBG) was modified to contain 0.65% NaCl and 100 mg/lOO ml glucose; glassware was not siliconized; trypsinized cells were filtered through nylon mesh before centrifuging; to each 0.9 ml of cell suspension containing 200,000 cells was added 10, 100, or 1000 pg Synacthen or 0.5, 2.0, or 4.0 mg of homol-

TURTLE

INTERRENAL

GLAND

IO

ogous pituitary homogenate in 0.1 ml vehicle or 0.1 ml vehicle alone; cells were incubated for 1 hr; incubates were frozen at - 15°C until assay of cotticosterone in 100~~1 samples by the competitive proteinbinding method. It should be noted that incubation and trypsinization was carried out at 37°C as in niammalian systems. The effects of incubation temperature on the turtle cell system will be reported elsewhere (Callard, 1975).

325

CONTROL

-Intact .----

Hypox

5

P”

Statistics Data were processed by single-factor analysis of variance. The significance of the difference between two means was determined using Student’s f test.

RESULTS Experiment I Table 1 summarizes plasma corticosterone changes in control and hypophysectomized turtles when blood was sampled 0, 30, and 60 min after ACTH or vehicle ,injection. Before treatment, the range for plasma corticosterone was 0.08-0.45 yg/lOO ml in unoperated turtles and O-O. 19 pg/lOO ml in hypophysectomized turtles. Comparison of all animals before injection shows a significant reduction from 0.22 + 0.04 to 0.06 & 0.02 pg/lOO ml plasma 24-48 hr after hypophysectomy (P < 0.01).

minufes

OF HYPOPHYSECTOMY

offer

1 60

ACTH

1. Effect of porcine ACTH (8 IU/lOO g) on plasma corticosterone of individual intact and hypophysectomized turtles. Blood was sampled from etih turtle at 0, 30, and 60 min after hormone injection im (Expt. I). FIG.

Corticosterone levels were higher 30 or 60 min after ACTH in both unoperated tid hypophysectomized turtles when compared to respective values at 0 min. Responses in individual turtles are depicted in Fig. 1. Despite considerable animal-to-animal variation, hypophysec-

TABLE EFFECTS

I 30

0

1

A&D C~RTICOTROPIN o~Chrystm)S (EXPT.

Corticosterone

ON PLASMA

CORTICOSTERONE

I)

(clg/lOO ml f SE)

(minutes postinjection) Treatment

II

Intact/sham Intact/vehicle Intact/ACTH Hypox/ACTH Hypoxlvehicle Hypox/sham

5 7 5 6 8 6

30

0 -

.22 .22 .03 .08

r .06 +- .07 2 .02 f .03 -

60

-

.57 1.54 .37 .24

+ k + 2 -

.22 .24 .14 .lO

.33 1.32 2.96 .54 1.15 .04

2 f 2 k k f

F; P*

.03 .35 .76 .16 .35 .02

5.85; 8.98; 4.42; 7.71;

Control of the interrenal gland of the freshwater turtle Chrysemys picta in vivo and in vitro.

GENERAL AND COMPARATIVE ENDOCRJNOLOGY 25, 323-331 (1975) Control of the Interrenal Gland of the Freshwater Turtle Chrysemys picta in Viva and in...
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