- Email: [email protected]
Dispersal of Estradiol-17β from the Site of Injection in the Pectoral Muscles of Japanese Quail
Dispersal of Estradiol-170 from the Site of Injection in the Pectoral Muscles of Japanese Quail G. A. ROBINSON Department of Biomedical Sciences, University of Guelph, Guelph, Ontario, Canada NIG 2V/1 (Received for publication March 5, 1984)
1985 Poultry Science 64:1793-1797 INTRODUCTION
Estradiol-17/3 (E 2 ) has been used to stimulate vitellogenesis in males of several vitellogenic species (Ryffel, 1978; Tata and Smith, 1979). Vitellogenesis provides a combination of characteristics of interest to the molecular biologist; the molecule is synthesized in the liver in direct response to the presence of specific hormones, modified extensively following translation, transported from the site of synthesis, and in the female, cleaved in the ovary into two markedly different proteins (Deeley et al., 1975). For male Japanese quail, 16 /imol E 2 /100 g body weight, administered into the pectoral muscles, produced a maximum vitellogenic response without causing deaths (Gibbins and Robinson, 1982a). Vitellogenesis may occur because of a large quantity of E 2 flowing rapidly from the site of injection to the liver or because the E 2 flows slowly out into the body over several days and thereby applies a sustained hormonal pressure on the liver. In the present study, radiolabeled estradiol was used to ascertain how rapidly estradiol was transported from the site of injection. MATERIALS AND METHODS
Japanese quail, 3 to 4 months old, were kept under a photoperiod of 14 hr light and 10 hr darkness and provided with commercial game bird ration, water, and oyster shell fragments ad libitum. Male quail were given ethanolic solu-
tions of 16 /xmol E 2 labeled with 16-a-[ 1 2 S I]iodoestradiol/100 g, nine quail; 6 pmol [ 1 2 S I]E 2 /100 g, four quail; 16 /xmol estradiol/100 g labeled with 1 2 5 I — , four quail; or sodium iodide (Na[ 1 2 S I]), four quail. The ethanol used for making the solutions was first saturated (approximately .25 mM) with sodium thiosulfate ( N a 2 S 2 0 3 ) to maintain unbound 1 2 5 I in the iodide form. Labeling was .1 to 4 jLtCi [ 1 2 5 I ] / quail. The [ 1 2 5 I]E 2 and the Na 1 2 5 I were supplied by the Radiochemical Centre, Amersham, UK, and the unlabeled E 2 by the Sigma Chemical Co., St. Louis, MO. The feathers were plucked over the pectoral muscles of the right side. A black dot was made on the skin over the carinal apex and another black dot on the skin of the right side 12 mm lateral to the first dot. The 26-gauge needles were fitted with plastic spacers such that the solutions were delivered to a point in the muscles of each bird 5 mm from the skin surface. Before removing the needle, a slight lateral tension was applied to the skin so the skin slipped over the site of injection as the needle was withdrawn. Within 1 min of injection, the carinal apex was positioned over a reference mark made on the lead shielding 12 mm from the axis of the hole of a cylindrical collimator. The hole was 19 mm in diameter and 31 mm deep. An Nal scintillation probe was situated immediately below the hole. The face of the collimator was covered with a .1-mm thick sheet of acetate plastic to facilitate
1793
Downloaded from http://ps.oxfordjournals.org/ at Oakland University on June 2, 2015
ABSTRACT Exogenous estrogens, if given in sufficient quantity, stimulate vitellogenesis in the males of vitellogenic species. In the present study, ethanolic solutions of estradiol-17/3 (E 2 ), labeled 12S with 16-a-[ I]iodoestradiol (I 125 1] E 2 ) or sodium iodide (Na 125 1), were injected into the pectoral muscles of male Japanese quail. The rate of dispersal of the estradiol from the site of injection was measured in vivo during 4 days. The curves of radioactivity appeared to be diphasic. The dose percentages forming the second phase of these curves and the half-time for the second phase were: for 16 nmol E 2 ( [ I 2 5 I ] E 2 label)/100 g body weight, 84.6% and 27.6 hr ; for 6 pmol [ I 2 S I ] E 2 / 100 g, 20.0% and 17.2 hr ; for 16 jumol E, (Na 125 I label)/100 g, 6.7% and 99.0 hr, and for Na 1 2 5 1, 6.1% and 83.1 hr. Thus, in male quail the estradiol-induced stimulation of vitellogenesis apparently resulted from a continuing hormonal pressure on the liver during the period of study and not from a rapid flow of E 2 to the liver shortly after injection. (Key words: estradiol, iodoestradiol, Japanese quail, vitellogenesis)
1794
ROBINSON injected as described for the males. These females were presumed to have considerable amounts of endogenous estrogen so only the 6 pmol [ 1 2 S I]E 2 and the Na 1 2 5 I solutions were used. Eggs were collected each day and the yolk material suspended in sufficient detergent solution to make 5 ml of final volume. The 1 2 S I content of each yolk was expressed as a percent of the dose given. For the purpose of comparison, the curves obtained from data for in vivo measurements of 125 1 were presumed diphasic (Fig. 1). The first phase made a negligible contribution to the counting rates after 1 day. Thus, a linear regression was calculated from the data for each quail as, logarithm (In) (cmp/juCi of 125 1 given) vs. days, for the 1 to 4-day period. The program supplied by the manufacturer (Texas Instruments) of the calculator provided values for the product moment correlation coefficient, the fractional loss constant, and the intercept on the In (cpm//xCi) axis. The half-time value was then In 2/fractional loss constant. The intercept was expressed as a percentage of the counting rate at 1 min (= 100%). Means and standard errors were calculated for each treatment group for each parameter measured. For comparison of the percentage accumulations of 1 2 S I per
Because iodide accumulates in growing oocytes (Robinson, 1973), laying females were
Na' 2 5 l 10000
2
3
4
0
1 2
Days after injection 12S
in male male Japanese Japanese quail quail given given ethanolic ethanolic solution solution of: of: [['I2S I]E 2 16 pmol FIG. 1. In vivo measurement of II in 12S 125 I] iodoestradiol/100 g body weight, nine quail; trace [['"IJE I]E22, 6 pmol estradiol-170 labeled with 16a-[[125 125 2S [ 12S I]E 2 /100 g, four quail; E2 + Na'"l, 16 jumol estradiol/100 g labeled with 12S1I , four quail; Na , '" I, four quail. Means of measurements over the site of injection in the pectoral muscles of the right side. Standard error (SE) limits shown for Days 1, 2, 3, and 4. Other SE limits were approximately the same, numerically, as those shown. Counting rates were expressed as counts per minute per microcurie of 12SI injected.
Downloaded from http://ps.oxfordjournals.org/ at Oakland University on June 2, 2015
cleaning. The quail was then rolled laterally until the pectoral muscles of the right side were pressing on the acetate sheet and held thusly for .4 min while the radioactive counting rate was measured. This counting procedure was repeated at 1, 2, 3, 6, and 12 hr and at 1, 2, 3, and 4 days. Counting rates were corrected for background and nuclear decay and expressed as counts per min per microcurie of 1 2 5 I injected. The manufacturer's statement of /iCi/ml for the 16-a-[ 12s I]iodoestradiol preparation was accepted as the reference for these calculations. Upon completion of the in vivo counting at 4 days, the quail were killed and tissues excised and placed in gamma counting vials. Two other groups of quail provided data for 1 hr and 2 days. The thyroids were digested in 1 ml 20% sodium hydroxide (NaOH) solution, then made up to 5 ml with water. Other soft tissues were minced with sufficient water to make a final volume of 5 ml. Tibiotarsi were broken so the leg bones would not extend above the 5 ml mark on the vials. The interspaces among the bones were then filled to the 5 ml mark with water. Counting rates were corrected for resolving time errors of .8% and greater.
IODOESTRADIOL IN QUAIL
1795
TABLE 1. Regression analysts for the second phase of the diphasic curves obtained from in vivo counting over the site of injection of 12S / compounds in the pectoral muscles of male Japanese quail
Treatment 1
Number of quail
Product moment 2 correlation coefficient
Half-time2 second phase
Intercept 2 second phase
(hr)
(%)
16MmolE 2 ( [ I 2 5 I ] E 2 level)/100g 6pmol [ 1 2 s I ] E 2 / 1 0 0 g 16Mmol E 2 (Na 125 I label)/100g Na 1 2 5 I
9 4 4 4
- . 9 9 4 + .002 - . 9 8 4 ± .004 - . 7 6 9 ± .049 - . 8 9 1 ± .030
27.6+ 3.9 17.2+ 1.0*'3 99.0+30.0* 83.1 ± 8.2***
84.6 ± 8.6 20.0 ± 5.2*** 6.7 ± 1.7*** 6.1 ± 1.2***
' E j = Estradiol-17(3; [ 1 2 5 I ] E 2 = 16a-[ 125 I] iodoestradiol; Na 1 2 5 I = sodium iodide, carrier free. Solvent ethanol, .25 m/M for sodium thiosulfate. Dose .25 ml/100 g body weight; pectoral muscles.
3 P values, half-time and intercept, 16 /umol E 2 ( [ I 2 S I ] E 2 label) group vs. each of the other groups: *P<.05, ***P<.001.
mechanism had less E 2 to move. For the E 2 + Nal and the Nal curves, the 1 to 4-day portions comprised 6.7 and 6.1% of the doses given (Table 1). These portions represented either near attainment of equilibrium of the 1 2 5 I with body iodide levels, nonspecific binding of the label to tissues at the site of injection, or return RESULTS AND DISCUSSION of * 5 1 to the site of injection in the form of The curve constructed from the counting labeled hormones. Dispersal of iodide from the rates recorded for the site of injection of the 16 site of injection was apparently independent of jumol E 2 ( [ 1 2 5 I ] E 2 label)/100 g was diphasic the 16 /xmol/100 g load of E 2 . (Fig. 1). The first phase of the curve lasted Shapes of curves showing percentages in the approximately 6 hr. The rapid decline in counts in vitro tissues were, except for the thyroid, per minute per microcurie was attributed to approximately as seen for the in vivo counting, rapid transport of a portion of the label away so only maxima were listed in Table 2. Counts from the site of injection as the ethanol solvent for the pectoral muscles of the left side origiwas dissipated. The second phase of the curve nated from label transported by the plasma to represented a slow removal of the label from these muscles. Counts for the right side arose the site of injection. This phase comprised from label that never left the site of injection 84.6% of the E 2 (Table 1, Intercept %) and had plus label returning to the site from elsewhere a half-time (JVz) of approximately 27.6 hr. The in the body. The difference between right and slow removal of label was attributed to precipi- left was the percentage of the dose that did not tation in the pectoral muscles as the ethanol leave the injection site. For samples taken at 4 solvent was diluted with tissue fluid. Thus, in days, these differences were: 16 jumol E 2 studies where 16 /zmol E 2 /100 g doses were ( [ 1 2 5 I ] E 2 label)/100 g, 4.97%; 6 pmol [ 1 2 5 I] used to stimulate vitellogenesis in male quail, E 2 / 1 0 0 g, .5 32%; E 2 + Na 1 2 s I, .024%; and the precipitate provided a reservoir of E 2 that Na 1 2 s I, 007%. Apparently, deiodination of applied a continuing hormonal pressure to the [ 1 2 S I]E 2 was slight or nonexistent because liver for at least the 4 days required to get thyroidal accumulations of 1 2 S I were less than a maximum response. For the 6 pmol [ 1 2 5 I] 1% for the two [ 1 2 5 I ] E 2 groups (Table 2, E 2 /100 g group, the second component rep- Footnote 1). The manufacturer of the 16-aresented only 20.0% of the E 2 dose. This E 2 [ 12s I] iodoestradiol stated that less than 5% was carried away from the site of injection of the 1 2 S I in the commercial preparation was more rapidly, TV4 = 17.2 hr, than for the 16 present as iodide. The counting rates for the jumol/100 g dose, perhaps because the transport thyroids were attributed to this iodide. Some gram of tissue, as found at dissection, the means and standard errors were calculated for each tissue for each treatment group. Values for means were compared using Student's t test, unpaired data.
Downloaded from http://ps.oxfordjournals.org/ at Oakland University on June 2, 2015
2 Calculated from data for each quail, In (cpm/juCi) vs. days, for the 1 to 4-day period, then mean + SE for each treatment group. Presumed two-phase exponential function for which the first phase was negligible by 1 day after the injections.
.080 6.79 .135 .333 .030 .203 .050 39.7
± .0082 ± 1.22 ± .066 ± .057 ± .003 ± .024 + .007 ± 7 . 8 ( 2 days) .164 5.35 .104 1.14 .064 .373 .102 61.3
6 pmol 100 g
I]E2/
3
J .021**' i .63 i .024 i .21** i .007** i .067* i .017* i 9 . 0 ( 4 days)
I25
3.16 .753 .274 1.36 .990 2.16 .708 1452
± ± ± ± ± ± + ± 50
1 6 Mmol E 2 , N a 1 2 5 1 label 100 g
I compounds given into th
P values, each tissue, 16 Mmol E 2 ([ 1 2 5 I]E 2 label) group vs. each of the other groups: *P<.05, **P<.01, ***P<001.
Mean ± SE.
3
2
1 Quail dissected 1 hr, 2 days, or 4 days after injection of .25 ml ethanolic solution per 100 g body weight, 5 mm E 2 ), 6 quail at 1 hr, 6 at 2 days, 9 quail at 4 days. For other treatments, 4 quail at each of 1 hr, 2 days, and 4 day thyroids where the times of occurrence of maxim are in parentheses. Maxima for percent per thyroid were .586 ± .052 and 25.7 + 7.9 (2 days), respectively.
Plasma Pectoral muscles, right side Pectoral muscles, left side Liver Testes Kidneys F e m o r a + tibiotarsi Thyroid1
Tissue 2
16 jumol E 2 , [ , 2 S I ] E 2 label/ 100 g
TABLE 2. Maxima for percentage accumulations of dose per gram of tissue for
ded from http://ps.oxfordjournals.org/ at Oakland University on June 2, 2015
1797
IODOESTRADIOL IN QUAIL
1
1 !
m
m
40o a-
2. 30
Na,25| 7 quail 68 eggs
c
> '6S
O
-o 20 fe£
"5 10
Trace |_ ij E2 7 quail 65 eggs
da?" ' ' 1 1 I 1 1 1 1 2 3 4 5 6 7 8 9 10 Collection period FIG. 2. Deposition of 1 2 5 I in the yolks of eggs laid by producing Japanese quail subsequent to an intramuscular injection of: squares, 6 pmol [ 12S I] E 2 in ethanol/100 g body weight; circles, carrierfree sodium iodide (Na 12S I) in ethanol. Each datum point represents the mean for the cumulative total to date for percent of dose of 1 2 5 I , injected into the hen, present in the yolks of the eggs laid. Standard error (SE) limits are shown where such limits exceeded the size of the shapes used. Maxima and SE were: ( I 2 S I ] E a , .989 ± .051% for Day 3; N a m I , 15.00 + 1.17% for Day 3. Ten-day totals were: [ 125 I] E 2> 2.83 + .10%; Na 1 2 s I, 42.74 ± 2.52%. Sixty-seven eggs were laid by the [ I 2 5 I] E 2 group but 2 were found broken (Days 7 and 9), and 69 eggs were laid by the Na 1 2 5 I group with 1 lost on Day 10.
small a c c u m u l a t i o n of E 2 occurred for t h e livers ( 5 x plasma c o n c e n t r a t i o n ) a n d kidneys (2.5X) of quail given [ 1 2 5 1] E 2 (Table 2 ) . Testes and leg b o n e s of t h e [ 1 2 S I ] E 2 quail contained less 1 2 S I/g tissue t h a n did t h e plasma. T h e 1 2 5 I in t h e y o l k s of eggs laid b y t h e female quail was 4 2 . 7 % for t h e N a 1 2 5 I - i n j e c t e d quail b u t only 2 . 8 % for t h e [ 1 2 S I ] E 2 quail (Fig.
ACKNOWLEDGMENTS I t h a n k D . C. Wasnidge and F . F l o t o for their excellent assistance w i t h this project. This s t u d y was s u p p o r t e d b y t h e N a t u r a l Sciences a n d Engineering Council of Canada a n d b y t h e O n t a r i o Ministry of Agriculture a n d F o o d .
REFERENCES Deeley, R. G., K. P. Mullinix, W. Wetekam, H. M. Kronenberg, M. Meyers, J. D. Eldridge, and R. F. Goldberger, 1975. Vitellogenin synthesis in the avian liver. Vitellogenin is the precursor of the egg yolk phosphoproteins. J. Biol. Chem. 250: 9060-9066. Gibbins, A.M.V., and G. A. Robinson, 1982a. A comparison of diethylstilbestrol- and estradiol17 (3-induced vitellogenesis in quail. Poultry Sci. 61:1188-1193. Gibbins, A.M.V., and G. A. Robinson, 1982b. Comparative study of the physiology of vitellogenesis in Japanese quail. Comp. Biochem. Physiol. 72A:149-155. Robinson, G. A., 1973. The oocytes as major competitors for radioiodide in laying J apanese quail. Gen. Comp. Endocrinol. 21:123-128. Ryffel, G. U., 1978. Synthesis of vitellogenin, an alternative model for investigating hormoneinduced gene activation. Mol. Cell. Endocr. 12:237-246. Tata, J. R., and D. F. Smith, 1979. Vitellogenesis: a versatile model for hormonal regulation of gene expression. Recent Prog. Horm. Res. 35:47—95.
Downloaded from http://ps.oxfordjournals.org/ at Oakland University on June 2, 2015
2 ) . T h u s , for t h e trace dose of 6 p m o l / 1 0 0 g, t h e e x o g e n o u s E 2 did n o t transfer i n t o t h e growing o o c y t e s t o a n y m a r k e d e x t e n t . Again, little or n o 1 2 5 I was removed from t h e E 2 a n d recycled as iodide. Perhaps, h a d 1 6 jumol E 2 / 1 0 0 g doses been given t o females, m o r e [ 1 2 s l ] - l a b e l e d E 2 w o u l d have b e e n transferred i n t o t h e eggs. Such a dose caused cessation of laying in 4 or 5 days in a n o t h e r s t u d y (Gibbins and R o b i n s o n , 1 9 8 2 b ) ; t h e data w o u l d t h u s n o t cover t h e 10-day collection period of Figure 2 . Dispersal of 8 4 . 6 % of 16 /umol estradiol-17/3/ 1 0 0 g labeled w i t h 1 6 - a - [ 1 2 5 I ] iodoestradiol from t h e site of injection in t h e p e c t o r a l muscles of male Japanese quail was sufficiently slow (T'/a 2 7 . 6 days) t h a t b y t h e f o u r t h d a y 7% (in vivo m e a s u r e m e n t ) or 5% (in vitro meas u r e m e n t ) of t h e dose of E 2 was still present at t h e site of injection. T h u s , t h e p r e s u m e d s t i m u l a t i o n of vitellogenesis, as evinced b y a m a r k e d increase in t h e c o n c e n t r a t i o n s of p r o t e i n - b o u n d p h o s p h o r u s in t h e plasma in t h e male quail, a p p a r e n t l y resulted from a continuing h o r m o n a l pressure on t h e liver d u r i n g t h e period of s t u d y and n o t from a rapid flow of E 2 t o t h e liver shortly after injection.
1985 Poultry Science 64:1793-1797 INTRODUCTION
Estradiol-17/3 (E 2 ) has been used to stimulate vitellogenesis in males of several vitellogenic species (Ryffel, 1978; Tata and Smith, 1979). Vitellogenesis provides a combination of characteristics of interest to the molecular biologist; the molecule is synthesized in the liver in direct response to the presence of specific hormones, modified extensively following translation, transported from the site of synthesis, and in the female, cleaved in the ovary into two markedly different proteins (Deeley et al., 1975). For male Japanese quail, 16 /imol E 2 /100 g body weight, administered into the pectoral muscles, produced a maximum vitellogenic response without causing deaths (Gibbins and Robinson, 1982a). Vitellogenesis may occur because of a large quantity of E 2 flowing rapidly from the site of injection to the liver or because the E 2 flows slowly out into the body over several days and thereby applies a sustained hormonal pressure on the liver. In the present study, radiolabeled estradiol was used to ascertain how rapidly estradiol was transported from the site of injection. MATERIALS AND METHODS
Japanese quail, 3 to 4 months old, were kept under a photoperiod of 14 hr light and 10 hr darkness and provided with commercial game bird ration, water, and oyster shell fragments ad libitum. Male quail were given ethanolic solu-
tions of 16 /xmol E 2 labeled with 16-a-[ 1 2 S I]iodoestradiol/100 g, nine quail; 6 pmol [ 1 2 S I]E 2 /100 g, four quail; 16 /xmol estradiol/100 g labeled with 1 2 5 I — , four quail; or sodium iodide (Na[ 1 2 S I]), four quail. The ethanol used for making the solutions was first saturated (approximately .25 mM) with sodium thiosulfate ( N a 2 S 2 0 3 ) to maintain unbound 1 2 5 I in the iodide form. Labeling was .1 to 4 jLtCi [ 1 2 5 I ] / quail. The [ 1 2 5 I]E 2 and the Na 1 2 5 I were supplied by the Radiochemical Centre, Amersham, UK, and the unlabeled E 2 by the Sigma Chemical Co., St. Louis, MO. The feathers were plucked over the pectoral muscles of the right side. A black dot was made on the skin over the carinal apex and another black dot on the skin of the right side 12 mm lateral to the first dot. The 26-gauge needles were fitted with plastic spacers such that the solutions were delivered to a point in the muscles of each bird 5 mm from the skin surface. Before removing the needle, a slight lateral tension was applied to the skin so the skin slipped over the site of injection as the needle was withdrawn. Within 1 min of injection, the carinal apex was positioned over a reference mark made on the lead shielding 12 mm from the axis of the hole of a cylindrical collimator. The hole was 19 mm in diameter and 31 mm deep. An Nal scintillation probe was situated immediately below the hole. The face of the collimator was covered with a .1-mm thick sheet of acetate plastic to facilitate
1793
Downloaded from http://ps.oxfordjournals.org/ at Oakland University on June 2, 2015
ABSTRACT Exogenous estrogens, if given in sufficient quantity, stimulate vitellogenesis in the males of vitellogenic species. In the present study, ethanolic solutions of estradiol-17/3 (E 2 ), labeled 12S with 16-a-[ I]iodoestradiol (I 125 1] E 2 ) or sodium iodide (Na 125 1), were injected into the pectoral muscles of male Japanese quail. The rate of dispersal of the estradiol from the site of injection was measured in vivo during 4 days. The curves of radioactivity appeared to be diphasic. The dose percentages forming the second phase of these curves and the half-time for the second phase were: for 16 nmol E 2 ( [ I 2 5 I ] E 2 label)/100 g body weight, 84.6% and 27.6 hr ; for 6 pmol [ I 2 S I ] E 2 / 100 g, 20.0% and 17.2 hr ; for 16 jumol E, (Na 125 I label)/100 g, 6.7% and 99.0 hr, and for Na 1 2 5 1, 6.1% and 83.1 hr. Thus, in male quail the estradiol-induced stimulation of vitellogenesis apparently resulted from a continuing hormonal pressure on the liver during the period of study and not from a rapid flow of E 2 to the liver shortly after injection. (Key words: estradiol, iodoestradiol, Japanese quail, vitellogenesis)
1794
ROBINSON injected as described for the males. These females were presumed to have considerable amounts of endogenous estrogen so only the 6 pmol [ 1 2 S I]E 2 and the Na 1 2 5 I solutions were used. Eggs were collected each day and the yolk material suspended in sufficient detergent solution to make 5 ml of final volume. The 1 2 S I content of each yolk was expressed as a percent of the dose given. For the purpose of comparison, the curves obtained from data for in vivo measurements of 125 1 were presumed diphasic (Fig. 1). The first phase made a negligible contribution to the counting rates after 1 day. Thus, a linear regression was calculated from the data for each quail as, logarithm (In) (cmp/juCi of 125 1 given) vs. days, for the 1 to 4-day period. The program supplied by the manufacturer (Texas Instruments) of the calculator provided values for the product moment correlation coefficient, the fractional loss constant, and the intercept on the In (cpm//xCi) axis. The half-time value was then In 2/fractional loss constant. The intercept was expressed as a percentage of the counting rate at 1 min (= 100%). Means and standard errors were calculated for each treatment group for each parameter measured. For comparison of the percentage accumulations of 1 2 S I per
Because iodide accumulates in growing oocytes (Robinson, 1973), laying females were
Na' 2 5 l 10000
2
3
4
0
1 2
Days after injection 12S
in male male Japanese Japanese quail quail given given ethanolic ethanolic solution solution of: of: [['I2S I]E 2 16 pmol FIG. 1. In vivo measurement of II in 12S 125 I] iodoestradiol/100 g body weight, nine quail; trace [['"IJE I]E22, 6 pmol estradiol-170 labeled with 16a-[[125 125 2S [ 12S I]E 2 /100 g, four quail; E2 + Na'"l, 16 jumol estradiol/100 g labeled with 12S1I , four quail; Na , '" I, four quail. Means of measurements over the site of injection in the pectoral muscles of the right side. Standard error (SE) limits shown for Days 1, 2, 3, and 4. Other SE limits were approximately the same, numerically, as those shown. Counting rates were expressed as counts per minute per microcurie of 12SI injected.
Downloaded from http://ps.oxfordjournals.org/ at Oakland University on June 2, 2015
cleaning. The quail was then rolled laterally until the pectoral muscles of the right side were pressing on the acetate sheet and held thusly for .4 min while the radioactive counting rate was measured. This counting procedure was repeated at 1, 2, 3, 6, and 12 hr and at 1, 2, 3, and 4 days. Counting rates were corrected for background and nuclear decay and expressed as counts per min per microcurie of 1 2 5 I injected. The manufacturer's statement of /iCi/ml for the 16-a-[ 12s I]iodoestradiol preparation was accepted as the reference for these calculations. Upon completion of the in vivo counting at 4 days, the quail were killed and tissues excised and placed in gamma counting vials. Two other groups of quail provided data for 1 hr and 2 days. The thyroids were digested in 1 ml 20% sodium hydroxide (NaOH) solution, then made up to 5 ml with water. Other soft tissues were minced with sufficient water to make a final volume of 5 ml. Tibiotarsi were broken so the leg bones would not extend above the 5 ml mark on the vials. The interspaces among the bones were then filled to the 5 ml mark with water. Counting rates were corrected for resolving time errors of .8% and greater.
IODOESTRADIOL IN QUAIL
1795
TABLE 1. Regression analysts for the second phase of the diphasic curves obtained from in vivo counting over the site of injection of 12S / compounds in the pectoral muscles of male Japanese quail
Treatment 1
Number of quail
Product moment 2 correlation coefficient
Half-time2 second phase
Intercept 2 second phase
(hr)
(%)
16MmolE 2 ( [ I 2 5 I ] E 2 level)/100g 6pmol [ 1 2 s I ] E 2 / 1 0 0 g 16Mmol E 2 (Na 125 I label)/100g Na 1 2 5 I
9 4 4 4
- . 9 9 4 + .002 - . 9 8 4 ± .004 - . 7 6 9 ± .049 - . 8 9 1 ± .030
27.6+ 3.9 17.2+ 1.0*'3 99.0+30.0* 83.1 ± 8.2***
84.6 ± 8.6 20.0 ± 5.2*** 6.7 ± 1.7*** 6.1 ± 1.2***
' E j = Estradiol-17(3; [ 1 2 5 I ] E 2 = 16a-[ 125 I] iodoestradiol; Na 1 2 5 I = sodium iodide, carrier free. Solvent ethanol, .25 m/M for sodium thiosulfate. Dose .25 ml/100 g body weight; pectoral muscles.
3 P values, half-time and intercept, 16 /umol E 2 ( [ I 2 S I ] E 2 label) group vs. each of the other groups: *P<.05, ***P<.001.
mechanism had less E 2 to move. For the E 2 + Nal and the Nal curves, the 1 to 4-day portions comprised 6.7 and 6.1% of the doses given (Table 1). These portions represented either near attainment of equilibrium of the 1 2 5 I with body iodide levels, nonspecific binding of the label to tissues at the site of injection, or return RESULTS AND DISCUSSION of * 5 1 to the site of injection in the form of The curve constructed from the counting labeled hormones. Dispersal of iodide from the rates recorded for the site of injection of the 16 site of injection was apparently independent of jumol E 2 ( [ 1 2 5 I ] E 2 label)/100 g was diphasic the 16 /xmol/100 g load of E 2 . (Fig. 1). The first phase of the curve lasted Shapes of curves showing percentages in the approximately 6 hr. The rapid decline in counts in vitro tissues were, except for the thyroid, per minute per microcurie was attributed to approximately as seen for the in vivo counting, rapid transport of a portion of the label away so only maxima were listed in Table 2. Counts from the site of injection as the ethanol solvent for the pectoral muscles of the left side origiwas dissipated. The second phase of the curve nated from label transported by the plasma to represented a slow removal of the label from these muscles. Counts for the right side arose the site of injection. This phase comprised from label that never left the site of injection 84.6% of the E 2 (Table 1, Intercept %) and had plus label returning to the site from elsewhere a half-time (JVz) of approximately 27.6 hr. The in the body. The difference between right and slow removal of label was attributed to precipi- left was the percentage of the dose that did not tation in the pectoral muscles as the ethanol leave the injection site. For samples taken at 4 solvent was diluted with tissue fluid. Thus, in days, these differences were: 16 jumol E 2 studies where 16 /zmol E 2 /100 g doses were ( [ 1 2 5 I ] E 2 label)/100 g, 4.97%; 6 pmol [ 1 2 5 I] used to stimulate vitellogenesis in male quail, E 2 / 1 0 0 g, .5 32%; E 2 + Na 1 2 s I, .024%; and the precipitate provided a reservoir of E 2 that Na 1 2 s I, 007%. Apparently, deiodination of applied a continuing hormonal pressure to the [ 1 2 S I]E 2 was slight or nonexistent because liver for at least the 4 days required to get thyroidal accumulations of 1 2 S I were less than a maximum response. For the 6 pmol [ 1 2 5 I] 1% for the two [ 1 2 5 I ] E 2 groups (Table 2, E 2 /100 g group, the second component rep- Footnote 1). The manufacturer of the 16-aresented only 20.0% of the E 2 dose. This E 2 [ 12s I] iodoestradiol stated that less than 5% was carried away from the site of injection of the 1 2 S I in the commercial preparation was more rapidly, TV4 = 17.2 hr, than for the 16 present as iodide. The counting rates for the jumol/100 g dose, perhaps because the transport thyroids were attributed to this iodide. Some gram of tissue, as found at dissection, the means and standard errors were calculated for each tissue for each treatment group. Values for means were compared using Student's t test, unpaired data.
Downloaded from http://ps.oxfordjournals.org/ at Oakland University on June 2, 2015
2 Calculated from data for each quail, In (cpm/juCi) vs. days, for the 1 to 4-day period, then mean + SE for each treatment group. Presumed two-phase exponential function for which the first phase was negligible by 1 day after the injections.
.080 6.79 .135 .333 .030 .203 .050 39.7
± .0082 ± 1.22 ± .066 ± .057 ± .003 ± .024 + .007 ± 7 . 8 ( 2 days) .164 5.35 .104 1.14 .064 .373 .102 61.3
6 pmol 100 g
I]E2/
3
J .021**' i .63 i .024 i .21** i .007** i .067* i .017* i 9 . 0 ( 4 days)
I25
3.16 .753 .274 1.36 .990 2.16 .708 1452
± ± ± ± ± ± + ± 50
1 6 Mmol E 2 , N a 1 2 5 1 label 100 g
I compounds given into th
P values, each tissue, 16 Mmol E 2 ([ 1 2 5 I]E 2 label) group vs. each of the other groups: *P<.05, **P<.01, ***P<001.
Mean ± SE.
3
2
1 Quail dissected 1 hr, 2 days, or 4 days after injection of .25 ml ethanolic solution per 100 g body weight, 5 mm E 2 ), 6 quail at 1 hr, 6 at 2 days, 9 quail at 4 days. For other treatments, 4 quail at each of 1 hr, 2 days, and 4 day thyroids where the times of occurrence of maxim are in parentheses. Maxima for percent per thyroid were .586 ± .052 and 25.7 + 7.9 (2 days), respectively.
Plasma Pectoral muscles, right side Pectoral muscles, left side Liver Testes Kidneys F e m o r a + tibiotarsi Thyroid1
Tissue 2
16 jumol E 2 , [ , 2 S I ] E 2 label/ 100 g
TABLE 2. Maxima for percentage accumulations of dose per gram of tissue for
ded from http://ps.oxfordjournals.org/ at Oakland University on June 2, 2015
1797
IODOESTRADIOL IN QUAIL
1
1 !
m
m
40o a-
2. 30
Na,25| 7 quail 68 eggs
c
> '6S
O
-o 20 fe£
"5 10
Trace |_ ij E2 7 quail 65 eggs
da?" ' ' 1 1 I 1 1 1 1 2 3 4 5 6 7 8 9 10 Collection period FIG. 2. Deposition of 1 2 5 I in the yolks of eggs laid by producing Japanese quail subsequent to an intramuscular injection of: squares, 6 pmol [ 12S I] E 2 in ethanol/100 g body weight; circles, carrierfree sodium iodide (Na 12S I) in ethanol. Each datum point represents the mean for the cumulative total to date for percent of dose of 1 2 5 I , injected into the hen, present in the yolks of the eggs laid. Standard error (SE) limits are shown where such limits exceeded the size of the shapes used. Maxima and SE were: ( I 2 S I ] E a , .989 ± .051% for Day 3; N a m I , 15.00 + 1.17% for Day 3. Ten-day totals were: [ 125 I] E 2> 2.83 + .10%; Na 1 2 s I, 42.74 ± 2.52%. Sixty-seven eggs were laid by the [ I 2 5 I] E 2 group but 2 were found broken (Days 7 and 9), and 69 eggs were laid by the Na 1 2 5 I group with 1 lost on Day 10.
small a c c u m u l a t i o n of E 2 occurred for t h e livers ( 5 x plasma c o n c e n t r a t i o n ) a n d kidneys (2.5X) of quail given [ 1 2 5 1] E 2 (Table 2 ) . Testes and leg b o n e s of t h e [ 1 2 S I ] E 2 quail contained less 1 2 S I/g tissue t h a n did t h e plasma. T h e 1 2 5 I in t h e y o l k s of eggs laid b y t h e female quail was 4 2 . 7 % for t h e N a 1 2 5 I - i n j e c t e d quail b u t only 2 . 8 % for t h e [ 1 2 S I ] E 2 quail (Fig.
ACKNOWLEDGMENTS I t h a n k D . C. Wasnidge and F . F l o t o for their excellent assistance w i t h this project. This s t u d y was s u p p o r t e d b y t h e N a t u r a l Sciences a n d Engineering Council of Canada a n d b y t h e O n t a r i o Ministry of Agriculture a n d F o o d .
REFERENCES Deeley, R. G., K. P. Mullinix, W. Wetekam, H. M. Kronenberg, M. Meyers, J. D. Eldridge, and R. F. Goldberger, 1975. Vitellogenin synthesis in the avian liver. Vitellogenin is the precursor of the egg yolk phosphoproteins. J. Biol. Chem. 250: 9060-9066. Gibbins, A.M.V., and G. A. Robinson, 1982a. A comparison of diethylstilbestrol- and estradiol17 (3-induced vitellogenesis in quail. Poultry Sci. 61:1188-1193. Gibbins, A.M.V., and G. A. Robinson, 1982b. Comparative study of the physiology of vitellogenesis in Japanese quail. Comp. Biochem. Physiol. 72A:149-155. Robinson, G. A., 1973. The oocytes as major competitors for radioiodide in laying J apanese quail. Gen. Comp. Endocrinol. 21:123-128. Ryffel, G. U., 1978. Synthesis of vitellogenin, an alternative model for investigating hormoneinduced gene activation. Mol. Cell. Endocr. 12:237-246. Tata, J. R., and D. F. Smith, 1979. Vitellogenesis: a versatile model for hormonal regulation of gene expression. Recent Prog. Horm. Res. 35:47—95.
Downloaded from http://ps.oxfordjournals.org/ at Oakland University on June 2, 2015
2 ) . T h u s , for t h e trace dose of 6 p m o l / 1 0 0 g, t h e e x o g e n o u s E 2 did n o t transfer i n t o t h e growing o o c y t e s t o a n y m a r k e d e x t e n t . Again, little or n o 1 2 5 I was removed from t h e E 2 a n d recycled as iodide. Perhaps, h a d 1 6 jumol E 2 / 1 0 0 g doses been given t o females, m o r e [ 1 2 s l ] - l a b e l e d E 2 w o u l d have b e e n transferred i n t o t h e eggs. Such a dose caused cessation of laying in 4 or 5 days in a n o t h e r s t u d y (Gibbins and R o b i n s o n , 1 9 8 2 b ) ; t h e data w o u l d t h u s n o t cover t h e 10-day collection period of Figure 2 . Dispersal of 8 4 . 6 % of 16 /umol estradiol-17/3/ 1 0 0 g labeled w i t h 1 6 - a - [ 1 2 5 I ] iodoestradiol from t h e site of injection in t h e p e c t o r a l muscles of male Japanese quail was sufficiently slow (T'/a 2 7 . 6 days) t h a t b y t h e f o u r t h d a y 7% (in vivo m e a s u r e m e n t ) or 5% (in vitro meas u r e m e n t ) of t h e dose of E 2 was still present at t h e site of injection. T h u s , t h e p r e s u m e d s t i m u l a t i o n of vitellogenesis, as evinced b y a m a r k e d increase in t h e c o n c e n t r a t i o n s of p r o t e i n - b o u n d p h o s p h o r u s in t h e plasma in t h e male quail, a p p a r e n t l y resulted from a continuing h o r m o n a l pressure on t h e liver d u r i n g t h e period of s t u d y and n o t from a rapid flow of E 2 t o t h e liver shortly after injection.