- Email: [email protected]
The prenatal development of the organ of Zuckerkandl in rats
Life Sciences, Vol. 41, pp. 1355-1359 Printed in the U.S.A.
Pergamon Journals
THE PRENATALDEVELOPMENT OF THE ORGANOF ZUCKERKANDL IN RATS L.L. Peters and B.G. Wood Department of Zoology University of Maine Orono, Maine 04401 (Received in final form July 6, 1987)
Summary The effect of maternal glucocorticoid depletion upon the fetal development of the organ of Zuckerkandl (OZ) in rats was determined. Maternal hypophysectomy at 13d8h gestation resulted in a f i f t y percent decrease in plasma corticosterone levels at 18d8h when compared to both sham operated and unoperated controls. No differences in the volume of the OZ among the three groups of animals were found. The chromaffinity of the OZ was decreased in the hypophysectomized and sham operated groups suggesting a stress-induced depletion of catecholamine stores. The data suggests that the OZ participates in fetal sympathoadrenal activity and that its development is independent of maternal corticosterone Liters. The Organ of Zuckerkandl (OZ), a mass of extra-adrenal chromaffin tissue, has been described in many species including the mouse, rat, guinea pig, and man (1-4). I t is located ventral to the abdominal aorta near the origins of the renal veins. In the rat, the organ f i r s t appears around day 15-16 of intrauterine l i f e , reaches peak size and chromaffinity at the time of birth, and thereafter degenerates. By the 15th postnatal day, i t has disappeared. During this time the adrenal medulla and s~nnpathetic nervous system reach full functional maturity. The OZ has been proposed to carry out the functions of these two systems in the fetus and newborn (5,6). Few studies on the OZ have been reported. Those that have appeared in the literature have focused upon aspects of its postnatal involution. No studies concerning the events leading to its appearance, differentiation, and function in utero have been done. In the early postnatal period, the size and chromaffinity of the OZ are increased and its degeneration delayed by glucocorticoid treatment (7). Such observations suggest a possible role for adrenal cortical hormones in the development and/or maintenance of this tissue in the fetus (8,9). Because the fetal pituitary-adrenal axis is not functional until day 18 of gestation in the rat (10,12), glucocorticoid hormones of maternal origin are implicated in the early differentiation of the OZ. This study was designed to determine the effects of glucocorticoid depletion by maternal hypophysectomy on the morphology and chromaffinity of the OZ during the fetal period.
0024-3205/87 $3.00 + .00 Copyright (c) 1987 Pergamon Journals Ltd.
1356
The Organ of Zuckerkandl
in Fetal Rats
Vol. 41, No. ii, 1987
Materials and Methods Sprague-Dawley rats weighing 200-250g were used. The animals were housed in single cages under controlled temperature (22-24°C) and lighting (lights on at 0700h and o f f at 1900h). Vaginal smears were taken daily. Females in proestrus were placed with males overnight. The presence of sperm in vaginal smears obtained the following morning confirmed coitus. Fertilization was assumed to occur at 010Oh. At 13d8h of gestation, mothers were anesthesized with Brevital (75 mg/kg BW) Heparinized blood (0.5 ml) was obtained from the retroorbital sinus, centrifuged, and the plasma stored at -20°C until assayed. The ventral surface of the pituitary was exposed by d r i l l i n g a hole in the basispheniod bone and cutting the dura. An 18 gauge needle was inserted, and the entire pituitary removed by aspiration. The procedure took 20-30 minutes to complete. Sham operations consisted of inserting the needle without aspiration. For unoperated controls, animals were anesthesized and allowed to recover undisturbed following procurement of blood from the retroorbital sinus. At 18d8h of pregnancy, the animals were again anesthesized and bled as above. Fetuses were removed and weighed. Heparinized blood was obtained from each fetus by decapitation and pooled. A retroperitoneal block of tissue extending from the adrenal glands to the lower pole of the kidneys was removed from each fetus and placed in 3.5% potassium dichromate: 40% formalin (9:1) for 24 hours. Tissues were dehydrated in graded alcohols, cleared, and embedded in paraffin. Serial sections were cut at 20~on an American Optical microtome, mounted, cleared in xylene, and coverslipped without further staining. The volume of the OZ in 2-3 fetuses from each l i t t e r was determined using a Zeiss Video Plan Image Analyzer. The area of the OZ in each section was traced directly from the slides at a magnification of lOOx. Volume determinations for fetuses of the same l i t t e r were averaged to obtain a final value. Corticosterone levels were determined by radioimmunoassay using materials obtained from Cambridge Medical Diagnostics ( B i l l e r i c a , MA). All results were analyzed by analysis of variance (ANOVA) and Tukey's Test for significance of difference between mean values. Results Both the hypophysectomized and sham operated animals recovered rapidly from the surgery and displayed normal behavior by the next day. At 18d8h, fetuses were seen to have developed normally in each group. The number of fetuses in the animals subjected to surgery did not d i f f e r from unoperated control animals. Fetuses of hypophysectomized animals, however, showed a significant (P
Vol. 41, No. 11, 1987
The Organ of Zuckerkandl in Fetal Rats
1357
TABLE I Fetal Weights in Control, Hypophysectomized and Sham Operated Rats at 18d8h of Gestation. Number of Number of Average Fetal GROUP Litters Fetuses per L i t t e r Weight , g CONTROL 5 12.8_+0.6 I . 29_+0.04 SHAM 6 11.8+0.9 1.20+0.03 HYPOPHYSECTOMY 5 12.8_+0.4 0.99_+0.04
E
I,~d8h
too
o,
(6)
OOoo .2
0
t
IBd8h
o
0
300
o E
200
o
(5) T
(6) T
(6) T
,oo
I1
0 Control
Shum
Hypophysectomy
FIG. 1 Maternal corticosterone levels at 13dSh (top panel) and 18d8h (bottom panel) of pregnancy. Values are means -+ SEM and the number of samples per group is shown in parentheses. m
E
(4)
cTM 3 0 0
o E =" 2 0 0 t/)
T
/
(4)
T
0
I
0.~--°~o I 0 0 u
0 Control
O
(5) ,, T
Sham
Hypophy-
sectomy
FIG. 2 Fetal corticosterone levels at 18d8h gestation. Values are means -+ SEM and the number of pooled samples per group is shown in parentheses.
1358
The Organ of Zuckerkandl in Fetal Rats
Vol. 41, No. Ii, 1987
No differences in the volume of the Organ of Zuckerkandl among the three groups of animals were found (Fig. 3). In all groups, the organ formed a compact, encapsulated structure extending from the level of the renal veins near the caudal end of the superior mesenteric ganglion to the lower poles of the kidneys. The intensity of the chromaffin reaction in both the hypophysectomized and sham operated animals was weaker than in the unoperated controls. The decrease in the chromaffinity of the Ol was most marked in the caudal regions of the organ, where numerous small vessels were apparent. E~
o_o
o'~
>
o z~
(5)
(6)
T
T
Control
Sham
(5) T
Hypophysectomy
FIG. 3
Volume of the Organ of Zuckerkandl in fetuses at 18d8h gestation. Values are means ± SEM and the number of samples per group is shown in parentheses. Discussion Within 8 hours after birth there is a marked decline in the functionality of the pituitary-adrenal-axis in the rat (13-16). During this time the OZ degenerates. The adrenal medulla, on the other hand, reaches full functional and morphological maturity during the early postnatal period (1,17). As cortical hormone levels are higher in the adrenal vein than in the peripheral circulation, i t is possible that these hormones are present in sufficient quantitites to support the adrenal medullary cells, but not the extra-adrenal chromaffin tissue, in the early postnatal period. Glucocorticoid injections in newborn rats prevent the disappearance of the OZ, supporting this hypothesis. In this study, maternal hypophysectomy at 13d8h of pregnancy produced a f i f t y percent decrease in corticosterone levels in maternal plasma at 18d8h. Prior to 18d8h, maternal glucocorticoids would be expected to be even lower as there is significant fetal ACTHrelease occurring on the 18th day (18). Despite this, the OZ developed normally. The prenatal differentiation of the OZ at day 15-16 of gestation in the rat, therefore, appears largely independent of maternal corticosterone, suggesting the events seen in the newborn are not related causally to maternal corticosteroid t i t e r . The decreased chromaffin reaction seen in the animals subjected to surgery most likely represents depletion of the catecholamine stores of the OZ in response to stress. I f so, this represents the f i r s t demonstration of a functional response of the extra-adrenal chromaffin tissue in the fetus. Studies of adrenal medullary cells in neonatal rats reveal that they are capable of secreting catecholamines by both neurogenic and non-neurogenic mechanisms depending upon the age of the animal (17). As the sympathetic nervous system is not functional in the fetus (19,20), one might speculate that the OZ releases catecholamines by a non-neurogenic mechanism. However, maternal stress induces
Vol. 41, No. ii, 1987
The Organ of Zuckerkandl in Fetal Rats
1359
premature onset of fetal sympathetic activity (21). One cannot rule out a possible neurogenic catecholamine-releasing mechanism in the OZ. The factors controlling both the development and function of the OZ in experimental animals may shed light on the etiology of extra-adrenal pheochromocytomas in human beings (22,23). Acknowledgements This research was supported in part by a grant from the American Heart Association, Maine a f f i l i a t e . REFERENCES I. 2. 3. 4. 5.
6. 7. 8. 9. i0. II. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.
M. R. G. P. DeGallardo, F. Freire, and J. H. Tramezzani, Acta Physiol. Latinoam. 24 290-304 (1974). R. E. Coupla-nd, J. Anat. Lond. 94 244-256 (1960). R. E. Coupland, J. Anat. Lond. 8~ 357-371 (1952). R. E. Coupland, J. Anat. Lond. ~'~ 455-464 (1953). G. B. West, D. M. Shepherd, and R. B. Hunter, Clin. Sci. 12 317-324 (1953). R. E. Coupland, C.Kent, and S. E. Kent, J. Endocr. 92 433-442 (1982). M. Lempinen, Acta Physiol, Scand. 62, Suppl. 231 (T~64). N. Ben-Jonathan, Life Sci. 23 39-7F4 (1978). J. Roffi and A. Jost, Science-" 154 275-277 (1966). J. P. Dupouy, H. Coffigny, and ~-~--.Magre, J. Endocr. 65 347-352 (1975). E. A. Petropoulos and C. Lau, J. Endocr. 59 183-184--(1973) A. Chatelain, J. Dupouy, and P. Allaume, En--~o. 106 1297-1303 (1980). A. Jost, Rec. Pros. Horm, Res. 22 541-574 (19~-~. A. Bartova, Gen. Comp. Endo. I0 ~35-239 (1968). A. Cohen, Horm, Metab. Res. ~--474-478 (1976). C. Walker, M. Perrin, W. Vale~ and C. R i v i e r , Endo. 118 1445-1451 (1986). F.J. Seidler and T. A. S l o t k i n , J. Physiol. 358 1-16 (1985). J. P. Dupouy and A. Chatelain, J. Endoc. I01 339-344 (1984). P.M. Gootman, N. M. Buckley, and N. Gootman, Reviews of Perinatal Medicine 3 1-72 (1979). 7. Pappano, Pharm, Rev. 29 3-33 (1977). P. G. Smith and E. M i l l s , - - L i f e Sci. 32 2497-2501 (1983). V. A. Kagali, M. A. Madoni, S. Bhitiya-l~ul, and H. H. Muller, NY State J. Med. 84 36-38 (1984). W. B. b-BER, Patholog~ Annual, S. C. Somers and P. P. Rosen, Eds. p. 103 Appleton-Century-crofts, Norwalk, CT. (1983).
Pergamon Journals
THE PRENATALDEVELOPMENT OF THE ORGANOF ZUCKERKANDL IN RATS L.L. Peters and B.G. Wood Department of Zoology University of Maine Orono, Maine 04401 (Received in final form July 6, 1987)
Summary The effect of maternal glucocorticoid depletion upon the fetal development of the organ of Zuckerkandl (OZ) in rats was determined. Maternal hypophysectomy at 13d8h gestation resulted in a f i f t y percent decrease in plasma corticosterone levels at 18d8h when compared to both sham operated and unoperated controls. No differences in the volume of the OZ among the three groups of animals were found. The chromaffinity of the OZ was decreased in the hypophysectomized and sham operated groups suggesting a stress-induced depletion of catecholamine stores. The data suggests that the OZ participates in fetal sympathoadrenal activity and that its development is independent of maternal corticosterone Liters. The Organ of Zuckerkandl (OZ), a mass of extra-adrenal chromaffin tissue, has been described in many species including the mouse, rat, guinea pig, and man (1-4). I t is located ventral to the abdominal aorta near the origins of the renal veins. In the rat, the organ f i r s t appears around day 15-16 of intrauterine l i f e , reaches peak size and chromaffinity at the time of birth, and thereafter degenerates. By the 15th postnatal day, i t has disappeared. During this time the adrenal medulla and s~nnpathetic nervous system reach full functional maturity. The OZ has been proposed to carry out the functions of these two systems in the fetus and newborn (5,6). Few studies on the OZ have been reported. Those that have appeared in the literature have focused upon aspects of its postnatal involution. No studies concerning the events leading to its appearance, differentiation, and function in utero have been done. In the early postnatal period, the size and chromaffinity of the OZ are increased and its degeneration delayed by glucocorticoid treatment (7). Such observations suggest a possible role for adrenal cortical hormones in the development and/or maintenance of this tissue in the fetus (8,9). Because the fetal pituitary-adrenal axis is not functional until day 18 of gestation in the rat (10,12), glucocorticoid hormones of maternal origin are implicated in the early differentiation of the OZ. This study was designed to determine the effects of glucocorticoid depletion by maternal hypophysectomy on the morphology and chromaffinity of the OZ during the fetal period.
0024-3205/87 $3.00 + .00 Copyright (c) 1987 Pergamon Journals Ltd.
1356
The Organ of Zuckerkandl
in Fetal Rats
Vol. 41, No. ii, 1987
Materials and Methods Sprague-Dawley rats weighing 200-250g were used. The animals were housed in single cages under controlled temperature (22-24°C) and lighting (lights on at 0700h and o f f at 1900h). Vaginal smears were taken daily. Females in proestrus were placed with males overnight. The presence of sperm in vaginal smears obtained the following morning confirmed coitus. Fertilization was assumed to occur at 010Oh. At 13d8h of gestation, mothers were anesthesized with Brevital (75 mg/kg BW) Heparinized blood (0.5 ml) was obtained from the retroorbital sinus, centrifuged, and the plasma stored at -20°C until assayed. The ventral surface of the pituitary was exposed by d r i l l i n g a hole in the basispheniod bone and cutting the dura. An 18 gauge needle was inserted, and the entire pituitary removed by aspiration. The procedure took 20-30 minutes to complete. Sham operations consisted of inserting the needle without aspiration. For unoperated controls, animals were anesthesized and allowed to recover undisturbed following procurement of blood from the retroorbital sinus. At 18d8h of pregnancy, the animals were again anesthesized and bled as above. Fetuses were removed and weighed. Heparinized blood was obtained from each fetus by decapitation and pooled. A retroperitoneal block of tissue extending from the adrenal glands to the lower pole of the kidneys was removed from each fetus and placed in 3.5% potassium dichromate: 40% formalin (9:1) for 24 hours. Tissues were dehydrated in graded alcohols, cleared, and embedded in paraffin. Serial sections were cut at 20~on an American Optical microtome, mounted, cleared in xylene, and coverslipped without further staining. The volume of the OZ in 2-3 fetuses from each l i t t e r was determined using a Zeiss Video Plan Image Analyzer. The area of the OZ in each section was traced directly from the slides at a magnification of lOOx. Volume determinations for fetuses of the same l i t t e r were averaged to obtain a final value. Corticosterone levels were determined by radioimmunoassay using materials obtained from Cambridge Medical Diagnostics ( B i l l e r i c a , MA). All results were analyzed by analysis of variance (ANOVA) and Tukey's Test for significance of difference between mean values. Results Both the hypophysectomized and sham operated animals recovered rapidly from the surgery and displayed normal behavior by the next day. At 18d8h, fetuses were seen to have developed normally in each group. The number of fetuses in the animals subjected to surgery did not d i f f e r from unoperated control animals. Fetuses of hypophysectomized animals, however, showed a significant (P
Vol. 41, No. 11, 1987
The Organ of Zuckerkandl in Fetal Rats
1357
TABLE I Fetal Weights in Control, Hypophysectomized and Sham Operated Rats at 18d8h of Gestation. Number of Number of Average Fetal GROUP Litters Fetuses per L i t t e r Weight , g CONTROL 5 12.8_+0.6 I . 29_+0.04 SHAM 6 11.8+0.9 1.20+0.03 HYPOPHYSECTOMY 5 12.8_+0.4 0.99_+0.04
E
I,~d8h
too
o,
(6)
OOoo .2
0
t
IBd8h
o
0
300
o E
200
o
(5) T
(6) T
(6) T
,oo
I1
0 Control
Shum
Hypophysectomy
FIG. 1 Maternal corticosterone levels at 13dSh (top panel) and 18d8h (bottom panel) of pregnancy. Values are means -+ SEM and the number of samples per group is shown in parentheses. m
E
(4)
cTM 3 0 0
o E =" 2 0 0 t/)
T
/
(4)
T
0
I
0.~--°~o I 0 0 u
0 Control
O
(5) ,, T
Sham
Hypophy-
sectomy
FIG. 2 Fetal corticosterone levels at 18d8h gestation. Values are means -+ SEM and the number of pooled samples per group is shown in parentheses.
1358
The Organ of Zuckerkandl in Fetal Rats
Vol. 41, No. Ii, 1987
No differences in the volume of the Organ of Zuckerkandl among the three groups of animals were found (Fig. 3). In all groups, the organ formed a compact, encapsulated structure extending from the level of the renal veins near the caudal end of the superior mesenteric ganglion to the lower poles of the kidneys. The intensity of the chromaffin reaction in both the hypophysectomized and sham operated animals was weaker than in the unoperated controls. The decrease in the chromaffinity of the Ol was most marked in the caudal regions of the organ, where numerous small vessels were apparent. E~
o_o
o'~
>
o z~
(5)
(6)
T
T
Control
Sham
(5) T
Hypophysectomy
FIG. 3
Volume of the Organ of Zuckerkandl in fetuses at 18d8h gestation. Values are means ± SEM and the number of samples per group is shown in parentheses. Discussion Within 8 hours after birth there is a marked decline in the functionality of the pituitary-adrenal-axis in the rat (13-16). During this time the OZ degenerates. The adrenal medulla, on the other hand, reaches full functional and morphological maturity during the early postnatal period (1,17). As cortical hormone levels are higher in the adrenal vein than in the peripheral circulation, i t is possible that these hormones are present in sufficient quantitites to support the adrenal medullary cells, but not the extra-adrenal chromaffin tissue, in the early postnatal period. Glucocorticoid injections in newborn rats prevent the disappearance of the OZ, supporting this hypothesis. In this study, maternal hypophysectomy at 13d8h of pregnancy produced a f i f t y percent decrease in corticosterone levels in maternal plasma at 18d8h. Prior to 18d8h, maternal glucocorticoids would be expected to be even lower as there is significant fetal ACTHrelease occurring on the 18th day (18). Despite this, the OZ developed normally. The prenatal differentiation of the OZ at day 15-16 of gestation in the rat, therefore, appears largely independent of maternal corticosterone, suggesting the events seen in the newborn are not related causally to maternal corticosteroid t i t e r . The decreased chromaffin reaction seen in the animals subjected to surgery most likely represents depletion of the catecholamine stores of the OZ in response to stress. I f so, this represents the f i r s t demonstration of a functional response of the extra-adrenal chromaffin tissue in the fetus. Studies of adrenal medullary cells in neonatal rats reveal that they are capable of secreting catecholamines by both neurogenic and non-neurogenic mechanisms depending upon the age of the animal (17). As the sympathetic nervous system is not functional in the fetus (19,20), one might speculate that the OZ releases catecholamines by a non-neurogenic mechanism. However, maternal stress induces
Vol. 41, No. ii, 1987
The Organ of Zuckerkandl in Fetal Rats
1359
premature onset of fetal sympathetic activity (21). One cannot rule out a possible neurogenic catecholamine-releasing mechanism in the OZ. The factors controlling both the development and function of the OZ in experimental animals may shed light on the etiology of extra-adrenal pheochromocytomas in human beings (22,23). Acknowledgements This research was supported in part by a grant from the American Heart Association, Maine a f f i l i a t e . REFERENCES I. 2. 3. 4. 5.
6. 7. 8. 9. i0. II. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.
M. R. G. P. DeGallardo, F. Freire, and J. H. Tramezzani, Acta Physiol. Latinoam. 24 290-304 (1974). R. E. Coupla-nd, J. Anat. Lond. 94 244-256 (1960). R. E. Coupland, J. Anat. Lond. 8~ 357-371 (1952). R. E. Coupland, J. Anat. Lond. ~'~ 455-464 (1953). G. B. West, D. M. Shepherd, and R. B. Hunter, Clin. Sci. 12 317-324 (1953). R. E. Coupland, C.Kent, and S. E. Kent, J. Endocr. 92 433-442 (1982). M. Lempinen, Acta Physiol, Scand. 62, Suppl. 231 (T~64). N. Ben-Jonathan, Life Sci. 23 39-7F4 (1978). J. Roffi and A. Jost, Science-" 154 275-277 (1966). J. P. Dupouy, H. Coffigny, and ~-~--.Magre, J. Endocr. 65 347-352 (1975). E. A. Petropoulos and C. Lau, J. Endocr. 59 183-184--(1973) A. Chatelain, J. Dupouy, and P. Allaume, En--~o. 106 1297-1303 (1980). A. Jost, Rec. Pros. Horm, Res. 22 541-574 (19~-~. A. Bartova, Gen. Comp. Endo. I0 ~35-239 (1968). A. Cohen, Horm, Metab. Res. ~--474-478 (1976). C. Walker, M. Perrin, W. Vale~ and C. R i v i e r , Endo. 118 1445-1451 (1986). F.J. Seidler and T. A. S l o t k i n , J. Physiol. 358 1-16 (1985). J. P. Dupouy and A. Chatelain, J. Endoc. I01 339-344 (1984). P.M. Gootman, N. M. Buckley, and N. Gootman, Reviews of Perinatal Medicine 3 1-72 (1979). 7. Pappano, Pharm, Rev. 29 3-33 (1977). P. G. Smith and E. M i l l s , - - L i f e Sci. 32 2497-2501 (1983). V. A. Kagali, M. A. Madoni, S. Bhitiya-l~ul, and H. H. Muller, NY State J. Med. 84 36-38 (1984). W. B. b-BER, Patholog~ Annual, S. C. Somers and P. P. Rosen, Eds. p. 103 Appleton-Century-crofts, Norwalk, CT. (1983).