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The effects of estradiol on plasma calcium and femoral bone structure in alligators (Alligator mississippiensis)
Camp. Biockm.
Vol.
Physiol.
84A, No. 1, pp. 107-110,
1986
0300-9629/86$3.00+ 0.00 0 1986Pergamon Press Ltd
Printed in Great Britain
THE EFFECTS OF ESTRADIOL ON PLASMA CALCIUM AND FEMORAL BONE STRUCTURE IN ALLIGATORS (ALLIGATOR
MISSISSIPPIENSIS)
RUTH M. ELSEX*and CAROLE S. WINK? *Louisiana Department of Wildlife and Fisheries, Rockefeller Wildlife Refuge, Grand Chenier, LA 70643, USA. TDepartment of Anatomy, Louisiana State University Medical Center, 1901 Perdido Street, New Orleans, LA 70112, USA. Telephone: (504) 582-5718 (Received 3 September 1985) Abstract-l. The effects of exogenous estradiol on plasma calcium and femoral bone structure were studied in young male and female alligators. 2. Males and females responded in the same manner to estrogen treatment. 3. Eight days after the initial injection, plasma calcium was significantly greater in experimentals than in controls. 4. No changes in femoral bone structure were observed.
INTRODUCDON
Birds and many reptiles are egg-layers. In birds, estrogen-dependent hypercalcemia pre-ovulatory, and medullary bone proliferation are followed by resorption of this endosteal bone which can be correlated with formation of the calcareous eggshell (Simkiss, 1967; Taylor et al., 1971). Estrogen treatment elicits hypercalcemia and endosteal bone formation in birds (Bloom et al., 1941), and hypercalcemia without endosteal bone formation in lower reptiles, such as turtles (Simkiss, 1961; Magliola, 1984). Turtles, apparently, have not evolved the skeleton-sparing mechanism of pre-ovulatory bone formation to form eggshells; they resorb structural bone during eggshell formation (Edgren, 1960; Simkiss, 1961; Suzuki, 1963; Magliola, 1984). It has been shown that breeding female alligators, like other egg-laying vertebrates, exhibit a pre-ovulatory hypercalcemia associated with eggshell formation (Lance et al., 1983). Recent studies in our laboratory have indicated that the alligator also resorbs structural bone during eggshell formation (Elsey and Wink, 1985). It is not known, however, how the skeleton and plasma calcium levels of alligators respond to exogenous estrogen treatment. Alligators, which are closely related to birds in evolution, may, like birds, develop hypercalcemia and form endosteal bone in response to estrogen treatment in addition to resorbing structural bone during eggshell formation, as do turtles. Therefore, the purpose of this study was to determine the effects of estrogen treatment on plasma calcium levels and femoral bone morphology of the alligator. MATERIALS AND
METHODS
Ten young alligators (Alligator miskwippiensis) (3.5 years old) were obtained fr.om -a commercial alligator farm (Sauros Inc.. Bell City, LA). These alligators had been hatched from artificial& incubated alligator eggs collected from Rockefeller Wildlife Refuge (Joanen and McNease,
1977). The animals were sexed by palpating within the cloaca and noting the presence or absence of a penis. They were then divided into two groups, with three females and two males in each group. They were maintained at Rockefeller Wildlife Refuge, Grand Chenier, LA, in controlled environmental chambers (Joanen and McNease, 1977) and fed a diet of ground nutria (Myocostor coypus). An initial 5 ml blood sample was taken from each animal by cardiac puncture with a heparinized syringe. One group (three females, two males) was injected with 5 mg estradiol dissolved in 1 ml sesame oil (experimental). The other group of animals (three females, two males) was injected with 1 ml sesame oil only (controls). Injections were given intramuscularly at three sites (tail and each lower extremity). Every 8 days for the next month, blood samples were taken from each animal, and each animal was re-injected with estradiol, or vehicle, as above. Blood samples were immediately centrifuged and the plasma was removed and frozen for later assay. At the end of 1 month (after four injections) the animals were killed by a blow to the head and the spinal cords severed. Alligators were weighed and body lengths were measured. Necropsies were performed, and livers, ovaries, oviducts, testes and penes were removed and weighed. Femora were also removed and frozen for later analysis. Femora were chosen because Ferguson (1982) showed that the femur undergoes less remodeling during aging than other bones sampled (tibia, fibula, humerus, radius, ulna, mandible, vertebrae, frontal, maxilla, palatine, pterygoid, nasal, jugal, ribs and dorsal neck scutes). Phma calcium &terminations Plasma calcium levels were determined by atomic absorption with a 305B Perkin-Elmer atomic absorption spectrophotometer. Femur robusticity index Femora were thawed and placed in an 85°C water bath for 1 week to remove soft tissue. They were then defatted and dehydrated in a series of alcohols and ethers and finally dried in a vacuum oven as described previously (Wink and Felts, 1980). They were weighed, and lengths were measured with vernier calipers. Bone robustness was determined using the formula: bone length robusticity index = 3 (bone weight)’ 107
108
Rull~ M.
EL~EY
and
This robusticity or ponderal index was first proposed by Riesenfeld (1972) and has been used to indicate the robustness or density of a bone as a whole (Riesenfeld, 1975, 1981; Simon, 1984). The higher the index number, the less dense the bone; the lower the number, the more robust or denser the bone. Femoral morphome&ics
Sections of bone 2cm long were cut from the middle of each dried femoral shaft and embedded in Ward’s Bioplastic. Cross sections of 200 pm thickness were cut from each piece using a Bronwill Hard Tissue Cutting Machine and a diamond saw. Microradiographs of the femoral cross sections were made with a Faxitron X-ray machine and Kodak high resolution plates (649-O).The microradiographs were processed routinely, projected with a microprojector and drawn at 20 x magnification. Drawings of bone sections were analyzed with a Numonics Electronic Calculator for total area, area occupied by bone (rnm2), percentage area occupied by bone, area occupied by marrow cavity (nur?) and percentage area occupied by marrow cavity. Total area was obtained by marking around the perimeter of the enlarged drawing of each bone section. Area occupied by bone was obtained by marking around bone in the section (excluding marrow cavity and “holes” or radioluccnt areas in the bone). Area of marrow cavity was obtained by marking around the marrow cavity of each section. The percentage areas occupied are obtained using: percentage area occupied by bone =
area occupied by bone total area
of marrow cavity percentage area of marrow cavity = total area Data were analyzed by Student’s f-test. area
RESULTS There were no significant differences in body weight, body length, liver weight or gonad weight between the sexes (male and female control animals or male and female experimental animals). There were no significant differences in body weight, body length or gonad weight between experimentals and controls (Table 1). Oviduct weight was significantly greater in estrogen-treated females than in control females and liver weight was significantly greater in experimentals than in controls (Table 1). There were no significant differences in femur robusticity indices, percentage area occupied by bone or percentage area
CAROLE S. WINK
of marrow cavity between the groups (Table 2, Fig. 1). Figure 2 shows that at the beginning of the experiment (0 weeks) there were no significant differences in plasma calcium levels between experimentals and controls. One week after the initial injection, plasma calcium levels were significantly greater (P c 0.02) in experimentals when compared to controls; after 2 weeks, P < 0.001, after 3 weeks, P < 0.001 and after 4 weeks, P < 0.01. DISCUSSION
It is well known that in egg-laying vertebrates the developing ovarian follicle secretes estradiol, which stimulates the liver to synthesize vitellogenin, a calcium-binding lipophosphoprotein (Ho et al., 1982). Similarly, estradiol injections caused synthesis of vitellogenin in a croeodilian, Caiman latirostris, (Van Brunt and Menzies, 1971). In the present study estrogen treatment induced liver hypertrophy in male and female alligators as it did in snakes (Dessauer and Fox, 1959) and lizards (Callard et al., 1972; Callard and Klotz, 1973; Hahn, 1967). This liver hypertrophy was probably secondary to vitellogenin synthesis. Our study also confirms the findings of Forbes (1938), who observed an extreme hypertrophy of the oviducts of female alligators injected with estrogen, and no differences in the penes of estrogentreated males when compared to those of control males. Histological studies (Forbes, 1938) of the gonads from estrogen-treated animals showed hypertrophy of both ovarian and testicular cortiees with no changes seen in the medullas. Gonad weights in the experimental alligators of our study were greater (although not significantly) than in the controls, which may have reflected cortical hypertrophy with no increase in the medulla. Prosser and Suzuki (1968) injected another crocodilian, Caiman sclerops, with estrogen and observed a gradual hypercaleemic response that did not hecome significant until 3 weeks after treatment was hegun. Also, there were no mo~holo~~al changes in the bones. However, the investigators used hatchling and young juvenile caimans for their studies and noted that the immaturity of the animals may have heen responsible for the low response to estrogen
Table 1. Effects of estrogen on body weight and length, weight of gonads, penis, oviduct, liver and liver weight/body weight (mean f SD) Group
N
Experimental (Bstrogen-treated)
5
Control
5
Body weight (kg)
Body length (cm)
7.81 I 0.85
132.33 I3.73 135.38 -i. 2.19
7.69 F 0.79
*Significantly different from controls, (x) = number of animals.
P
Liver weight LivQr weight Gonad weight Penis weight Oviduct weight Body weight (g) (9) (g) (8) 4.70 & 1.6’7 5.80 + 0.85 (2) 204.40 & 58.10, (3) 136.18 L-26.80’ 1.73 f 0.23* 3.56 +
1.27 7.45 + 6.86 (2)
9.83 * 4.70 (3)
90.7+15.20
< 0.01.
Table 2. Effects of estrogen on femoral robusticity index, total area of femoral microradie~aphs, and percentage area occupied by marrow cavity and bone (mean + SD) Mid-shaft cross section x 20 Group Experimental (estrogen-treaty)
N
Robusticity index
Total Area mm*
% Marrow cavity
5
4.07 f0.13
282.25 + 14.25
6.04 f 2.03
% Bone __.__ 91.40* 1.76
Control
5
4.00*0.13
293.45 _+27.46
5.43 +2.15
91.48 f 2.49
1.17+0.09
Effects of estrogen
Fig. 1. Microradiograph and (B) control
on alligators
(4 x magnification) of femoral cross sections from (A) estrogen-treated female female. Note similarity of marrow cavities and endosteal resorption areas.
treatment. Budy ef al. (1952) also attributed lack of endosteal bone formation, after estrogen treatment, to age in newborn rats. In the present study, we used older (3.5 years old) sexually immature alligators, and the hypercalcemic response was much more rapid (at 8 days). Eight days was the earliest time we sampled blood after the initial injection; there may have been a significant difference in calcium levels between experimentals and controls before 8 days. Suzuki and Prosser (1968) reported significant differences in serum calcium between estrogen-treated adult lizards and controls after 5 days.
We could see no evidence of medullary bone proliferation in the experimental alligators. Marrow cavity areas were the same in both groups and no “extra” bone filled in resorption areas of the endosteal bone. It is unlikely that there was medullary bone proliferation in the femur at any other site than the one we sampled at midshaft, since the robusticity indices of the femora did not differ between the groups. In conclusion, it appears that the alligator, like lower reptiles, such as turtles, lizards and snakes, responds to estrogen treatment with hypercalcemia, and, unlike the bird, has not evolved the skeletonsparing mechanism of estrogen-dependent, preovulatory medullary bone proliferation for the formation of egg shells. Acknowledgernears-The authors would like to thank Ted Joanen and Larry McNease of the Louisiana Department of Wildlife and Fisheries for assistance in maintenance and sampling of the alligators. We are grateful to Robert Perkins for the alligators used in this project. Appreciation is extended to Dr Valentine Lance for his advice and suggestions throughout this project and to Deborah Wilson for help with the calcium assays. This project was funded in part by the Louisiana Department of Wildlife and Fisheries.
0
2
1
3
I
WEEKS
Fig. 2. Response of plasma calcium levels in Alligaror mississippiends after estradiol injections. Hatched line represents experimental (estradiol-injected) alligators, solid line represents control alligators (in each group N = 5). Experimentals were significantly different from controls by 1 week (P < 0.02); after 2 and 3 weeks (P < 0.001); and after 4 weeks (P < 0.01).
REFERENCES Bloom W., Bloom M. and McLean F. C. (1941) Calcification and ossification, medullary bone changes in the reproductive cycle of the female pigeon. Amt. Rec. 81, 443415. Budy A. M., Urist M. R. and McLean F. C. (1952) The effect of estrogens on the growth apparatus of the bones of immature rats. Am. J. Path. 28, 1143-1167.
110
RUTH M. ELSEYand CAROLES. WINK
Callard I. P.. DooMtIe J. and Banks W. L. (1972) Recent studies On the Coned Of the redtihan ovarian cvcle. Gen. camp. Adocr. Suppl. 3, 65-75: Callard I. P. and Kletx K. L. (19731 Sensitivitv of parameters of estrogen action‘ in ’ the ing&nid lizard, Bipmsaurus domalk Gen. camp. Enabcr. 21, 314-321. Dessauer H. C. and Fox W. (1959) Changes in ovarian follicle composition with plasma levels of snakes during estrus. Am. J. Physioi. 197, 360-366. Edgren R. A. (1960) A seasonal change in bone densitv in female musk turtles, Sternotherusodoratur (Latreiile). Comn. Biochem. Phvsiol. 1. 213-217. Elsey R. M. and Wink C. S. (1985) Femoral bone as a possible source of calcium for eggshell deposition in Alligator mississippiensb. Anat. Rec. 211, 57A. Ferguson M. W, J. (1982) The structure and development of the palate in Alligator mL&.ssippiensis. Doctoral dissertation, Queens University, Belfast. Forbes T. R. (1938) Studies on the reproductive system of the alliaator. II. The effects of uroloneed in&ions of oestronc in the immature alligator. Jy exp.- ZooI. 78, 335-367. Hahn W. E. (1967) Estradiol-induced vitellogenesis and concomitant ‘fat t&bilixation in the lizard Ura stansburiana. Comp. 3iachem. Physiol. 23, 03-93. Ho S. M., Kleis S., McPherson R., Heisermann G. J. and Callard I. P. (lM12) Remtlation of vitellonenesis in ren1 tiles. Herpetologica’3& -&HO. Joanen T. and McNease L. (1977) Artificial incubation of alligator eggs and post hatching culture in controlled environmental chambers. Proc. world Muric. Meet. 8, 483-490. Lance V., Joanen T. and McNease L. (1983) Selenium, vitamin E, and trace elements in plasma of wild and fan&eared alligators during the reproductive cycle. Can. J. 2001. 61, 1744-1751. Magliola L. (1984) The effects of estrogen on skeletal calcium metabolism and on plasma parameters of vitel-
logenesis in the male, three-toed box turtle (Terrapene carokna trknqnis). Gen. camp. &a&r. !H, 162170. Presser R. L. and Suzuki H. K. (1968) The effects of estradiol valerate on the serum and bone of hatching and juvenile caiman crocodiles (C&tan scierops). Comp. Bio them. Physiol. X$529-534. Riesenfeld A. (1972) Metatarsal rob&city in bipedal rats. Am. J. Phys. Anthrop. 36, 229-234. Rieaenfeld A. (1975) Endocrine control of skeletal robusticity. Actu Anat. 91, 481499. Riesenfeld A. (198 1) Age changes of bone size and mass in two strains of senescent rats. Acta Anat. 109, 64-69. Simkiss K. (1961) The influence of large doses of oeatrogens upon the structure of the bones of some reptiles. Nature 190, 1217-1218. Simkiss K. (1967) Calcium in Reproductive Physiology. Rheinhold, New York. Simon M. R. (1984) The rat as an animal model for the study of senile idiopathic osteoporosis. Acta Anat. 119, 248-250. Suzuki H. K. (1963) Studies on the ossesus system of the slider turtle. Ann. NY Acad. Sci. 109, 351-410. Suzuki H. K. and Roamer R. L. (1%8) The effects of estradiol valerate upon the serum and bone of the lizard, Scefoparus cyanogenys. Proc. Sot. exp. Bfoi. med. 127, 4-7. Taylor T. G., Simkiss K. and Stringer D. A. (1971) The skeleton: Its structure and metabolism. In Physiology and Biochemistry of the Domestic Fowl (Ed&cd by Bell D. J. and Freeman B. M.), Vol. 2, pp. 621-640. Academic Press, New York. Van Brunt J. F. and Menxies R. A. (1971) Synthesis of serum calcium-binding protein lipophosphoprotein complex by South American alligators (Caimrm latirostris) in response to 17/J-estradiol. Fedn Proc. Fedn Am. Sots exp. Biol. 30, 1160. Wink C. S. and Felts W. J. L. (1980) Effect of castration on the bone structure of male rats: a model of osteoporosis. Cal@ Tissue Int. 32, 77-82.
Vol.
Physiol.
84A, No. 1, pp. 107-110,
1986
0300-9629/86$3.00+ 0.00 0 1986Pergamon Press Ltd
Printed in Great Britain
THE EFFECTS OF ESTRADIOL ON PLASMA CALCIUM AND FEMORAL BONE STRUCTURE IN ALLIGATORS (ALLIGATOR
MISSISSIPPIENSIS)
RUTH M. ELSEX*and CAROLE S. WINK? *Louisiana Department of Wildlife and Fisheries, Rockefeller Wildlife Refuge, Grand Chenier, LA 70643, USA. TDepartment of Anatomy, Louisiana State University Medical Center, 1901 Perdido Street, New Orleans, LA 70112, USA. Telephone: (504) 582-5718 (Received 3 September 1985) Abstract-l. The effects of exogenous estradiol on plasma calcium and femoral bone structure were studied in young male and female alligators. 2. Males and females responded in the same manner to estrogen treatment. 3. Eight days after the initial injection, plasma calcium was significantly greater in experimentals than in controls. 4. No changes in femoral bone structure were observed.
INTRODUCDON
Birds and many reptiles are egg-layers. In birds, estrogen-dependent hypercalcemia pre-ovulatory, and medullary bone proliferation are followed by resorption of this endosteal bone which can be correlated with formation of the calcareous eggshell (Simkiss, 1967; Taylor et al., 1971). Estrogen treatment elicits hypercalcemia and endosteal bone formation in birds (Bloom et al., 1941), and hypercalcemia without endosteal bone formation in lower reptiles, such as turtles (Simkiss, 1961; Magliola, 1984). Turtles, apparently, have not evolved the skeleton-sparing mechanism of pre-ovulatory bone formation to form eggshells; they resorb structural bone during eggshell formation (Edgren, 1960; Simkiss, 1961; Suzuki, 1963; Magliola, 1984). It has been shown that breeding female alligators, like other egg-laying vertebrates, exhibit a pre-ovulatory hypercalcemia associated with eggshell formation (Lance et al., 1983). Recent studies in our laboratory have indicated that the alligator also resorbs structural bone during eggshell formation (Elsey and Wink, 1985). It is not known, however, how the skeleton and plasma calcium levels of alligators respond to exogenous estrogen treatment. Alligators, which are closely related to birds in evolution, may, like birds, develop hypercalcemia and form endosteal bone in response to estrogen treatment in addition to resorbing structural bone during eggshell formation, as do turtles. Therefore, the purpose of this study was to determine the effects of estrogen treatment on plasma calcium levels and femoral bone morphology of the alligator. MATERIALS AND
METHODS
Ten young alligators (Alligator miskwippiensis) (3.5 years old) were obtained fr.om -a commercial alligator farm (Sauros Inc.. Bell City, LA). These alligators had been hatched from artificial& incubated alligator eggs collected from Rockefeller Wildlife Refuge (Joanen and McNease,
1977). The animals were sexed by palpating within the cloaca and noting the presence or absence of a penis. They were then divided into two groups, with three females and two males in each group. They were maintained at Rockefeller Wildlife Refuge, Grand Chenier, LA, in controlled environmental chambers (Joanen and McNease, 1977) and fed a diet of ground nutria (Myocostor coypus). An initial 5 ml blood sample was taken from each animal by cardiac puncture with a heparinized syringe. One group (three females, two males) was injected with 5 mg estradiol dissolved in 1 ml sesame oil (experimental). The other group of animals (three females, two males) was injected with 1 ml sesame oil only (controls). Injections were given intramuscularly at three sites (tail and each lower extremity). Every 8 days for the next month, blood samples were taken from each animal, and each animal was re-injected with estradiol, or vehicle, as above. Blood samples were immediately centrifuged and the plasma was removed and frozen for later assay. At the end of 1 month (after four injections) the animals were killed by a blow to the head and the spinal cords severed. Alligators were weighed and body lengths were measured. Necropsies were performed, and livers, ovaries, oviducts, testes and penes were removed and weighed. Femora were also removed and frozen for later analysis. Femora were chosen because Ferguson (1982) showed that the femur undergoes less remodeling during aging than other bones sampled (tibia, fibula, humerus, radius, ulna, mandible, vertebrae, frontal, maxilla, palatine, pterygoid, nasal, jugal, ribs and dorsal neck scutes). Phma calcium &terminations Plasma calcium levels were determined by atomic absorption with a 305B Perkin-Elmer atomic absorption spectrophotometer. Femur robusticity index Femora were thawed and placed in an 85°C water bath for 1 week to remove soft tissue. They were then defatted and dehydrated in a series of alcohols and ethers and finally dried in a vacuum oven as described previously (Wink and Felts, 1980). They were weighed, and lengths were measured with vernier calipers. Bone robustness was determined using the formula: bone length robusticity index = 3 (bone weight)’ 107
108
Rull~ M.
EL~EY
and
This robusticity or ponderal index was first proposed by Riesenfeld (1972) and has been used to indicate the robustness or density of a bone as a whole (Riesenfeld, 1975, 1981; Simon, 1984). The higher the index number, the less dense the bone; the lower the number, the more robust or denser the bone. Femoral morphome&ics
Sections of bone 2cm long were cut from the middle of each dried femoral shaft and embedded in Ward’s Bioplastic. Cross sections of 200 pm thickness were cut from each piece using a Bronwill Hard Tissue Cutting Machine and a diamond saw. Microradiographs of the femoral cross sections were made with a Faxitron X-ray machine and Kodak high resolution plates (649-O).The microradiographs were processed routinely, projected with a microprojector and drawn at 20 x magnification. Drawings of bone sections were analyzed with a Numonics Electronic Calculator for total area, area occupied by bone (rnm2), percentage area occupied by bone, area occupied by marrow cavity (nur?) and percentage area occupied by marrow cavity. Total area was obtained by marking around the perimeter of the enlarged drawing of each bone section. Area occupied by bone was obtained by marking around bone in the section (excluding marrow cavity and “holes” or radioluccnt areas in the bone). Area of marrow cavity was obtained by marking around the marrow cavity of each section. The percentage areas occupied are obtained using: percentage area occupied by bone =
area occupied by bone total area
of marrow cavity percentage area of marrow cavity = total area Data were analyzed by Student’s f-test. area
RESULTS There were no significant differences in body weight, body length, liver weight or gonad weight between the sexes (male and female control animals or male and female experimental animals). There were no significant differences in body weight, body length or gonad weight between experimentals and controls (Table 1). Oviduct weight was significantly greater in estrogen-treated females than in control females and liver weight was significantly greater in experimentals than in controls (Table 1). There were no significant differences in femur robusticity indices, percentage area occupied by bone or percentage area
CAROLE S. WINK
of marrow cavity between the groups (Table 2, Fig. 1). Figure 2 shows that at the beginning of the experiment (0 weeks) there were no significant differences in plasma calcium levels between experimentals and controls. One week after the initial injection, plasma calcium levels were significantly greater (P c 0.02) in experimentals when compared to controls; after 2 weeks, P < 0.001, after 3 weeks, P < 0.001 and after 4 weeks, P < 0.01. DISCUSSION
It is well known that in egg-laying vertebrates the developing ovarian follicle secretes estradiol, which stimulates the liver to synthesize vitellogenin, a calcium-binding lipophosphoprotein (Ho et al., 1982). Similarly, estradiol injections caused synthesis of vitellogenin in a croeodilian, Caiman latirostris, (Van Brunt and Menzies, 1971). In the present study estrogen treatment induced liver hypertrophy in male and female alligators as it did in snakes (Dessauer and Fox, 1959) and lizards (Callard et al., 1972; Callard and Klotz, 1973; Hahn, 1967). This liver hypertrophy was probably secondary to vitellogenin synthesis. Our study also confirms the findings of Forbes (1938), who observed an extreme hypertrophy of the oviducts of female alligators injected with estrogen, and no differences in the penes of estrogentreated males when compared to those of control males. Histological studies (Forbes, 1938) of the gonads from estrogen-treated animals showed hypertrophy of both ovarian and testicular cortiees with no changes seen in the medullas. Gonad weights in the experimental alligators of our study were greater (although not significantly) than in the controls, which may have reflected cortical hypertrophy with no increase in the medulla. Prosser and Suzuki (1968) injected another crocodilian, Caiman sclerops, with estrogen and observed a gradual hypercaleemic response that did not hecome significant until 3 weeks after treatment was hegun. Also, there were no mo~holo~~al changes in the bones. However, the investigators used hatchling and young juvenile caimans for their studies and noted that the immaturity of the animals may have heen responsible for the low response to estrogen
Table 1. Effects of estrogen on body weight and length, weight of gonads, penis, oviduct, liver and liver weight/body weight (mean f SD) Group
N
Experimental (Bstrogen-treated)
5
Control
5
Body weight (kg)
Body length (cm)
7.81 I 0.85
132.33 I3.73 135.38 -i. 2.19
7.69 F 0.79
*Significantly different from controls, (x) = number of animals.
P
Liver weight LivQr weight Gonad weight Penis weight Oviduct weight Body weight (g) (9) (g) (8) 4.70 & 1.6’7 5.80 + 0.85 (2) 204.40 & 58.10, (3) 136.18 L-26.80’ 1.73 f 0.23* 3.56 +
1.27 7.45 + 6.86 (2)
9.83 * 4.70 (3)
90.7+15.20
< 0.01.
Table 2. Effects of estrogen on femoral robusticity index, total area of femoral microradie~aphs, and percentage area occupied by marrow cavity and bone (mean + SD) Mid-shaft cross section x 20 Group Experimental (estrogen-treaty)
N
Robusticity index
Total Area mm*
% Marrow cavity
5
4.07 f0.13
282.25 + 14.25
6.04 f 2.03
% Bone __.__ 91.40* 1.76
Control
5
4.00*0.13
293.45 _+27.46
5.43 +2.15
91.48 f 2.49
1.17+0.09
Effects of estrogen
Fig. 1. Microradiograph and (B) control
on alligators
(4 x magnification) of femoral cross sections from (A) estrogen-treated female female. Note similarity of marrow cavities and endosteal resorption areas.
treatment. Budy ef al. (1952) also attributed lack of endosteal bone formation, after estrogen treatment, to age in newborn rats. In the present study, we used older (3.5 years old) sexually immature alligators, and the hypercalcemic response was much more rapid (at 8 days). Eight days was the earliest time we sampled blood after the initial injection; there may have been a significant difference in calcium levels between experimentals and controls before 8 days. Suzuki and Prosser (1968) reported significant differences in serum calcium between estrogen-treated adult lizards and controls after 5 days.
We could see no evidence of medullary bone proliferation in the experimental alligators. Marrow cavity areas were the same in both groups and no “extra” bone filled in resorption areas of the endosteal bone. It is unlikely that there was medullary bone proliferation in the femur at any other site than the one we sampled at midshaft, since the robusticity indices of the femora did not differ between the groups. In conclusion, it appears that the alligator, like lower reptiles, such as turtles, lizards and snakes, responds to estrogen treatment with hypercalcemia, and, unlike the bird, has not evolved the skeletonsparing mechanism of estrogen-dependent, preovulatory medullary bone proliferation for the formation of egg shells. Acknowledgernears-The authors would like to thank Ted Joanen and Larry McNease of the Louisiana Department of Wildlife and Fisheries for assistance in maintenance and sampling of the alligators. We are grateful to Robert Perkins for the alligators used in this project. Appreciation is extended to Dr Valentine Lance for his advice and suggestions throughout this project and to Deborah Wilson for help with the calcium assays. This project was funded in part by the Louisiana Department of Wildlife and Fisheries.
0
2
1
3
I
WEEKS
Fig. 2. Response of plasma calcium levels in Alligaror mississippiends after estradiol injections. Hatched line represents experimental (estradiol-injected) alligators, solid line represents control alligators (in each group N = 5). Experimentals were significantly different from controls by 1 week (P < 0.02); after 2 and 3 weeks (P < 0.001); and after 4 weeks (P < 0.01).
REFERENCES Bloom W., Bloom M. and McLean F. C. (1941) Calcification and ossification, medullary bone changes in the reproductive cycle of the female pigeon. Amt. Rec. 81, 443415. Budy A. M., Urist M. R. and McLean F. C. (1952) The effect of estrogens on the growth apparatus of the bones of immature rats. Am. J. Path. 28, 1143-1167.
110
RUTH M. ELSEYand CAROLES. WINK
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