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Estrogen Sulfates in the Plasma of White Leghorn Laying Hens1,2
RESEARCH NOTES Estrogen Sulfates in the Plasma of White Leghorn Laying Hens1'2 C. P. W. TSANG, A. A. GRUNDER, and K. G. HOLLANDS Animal Research Centre, Ottawa, Ontario, K1A 0C6, Canada (Received for publication December 3, 1980) ABSTRACT Plasma samples from nine White Leghorn hens collected over six different intervals during the laying cycle contained high concentrations of estradiol-17/3-3-sulfate with an overall mean of 1.24 pmol/ml (464 pg/ml) versus .36 pmol/ml (98 pg/ml) for estradiol-17/3. In contrast, the mean concentration of estrone sulfate was only .59 pmol/ml (219 pg/ml) as compared to .41 pmol/ml (111 pg/ml) for estrone. (Key words: estrogen sulfates, estrogens, estradiol, estradiol sulfate, estrone sulfate, hens) 1981 Poultry Science 60:2548-2550
MATERIALS AND METHODS Nine White Leghorn layers of experimental Strain 1, which had been selected for high egg production at the Animal Research Centre, were caged individually, with feed and water provided ad libitum. The birds were 40 weeks of age and had been in a light regimen of 16L:8D with lights on at 0100 hr since they were 37 weeks of age. Room temperature was maintained at 13 C. During the study, the birds exhibited 24 to 26 hr laying cycles. Oviposition times were determined within 3 min by patrol. Blood (2 to 3 ml) was drawn from the brachial vein at 2.5, 5.75, 17, and 22 hr after oviposition (not the first egg of the clutch) and at 0 and 2.5 hr after the next oviposition. This collection schedule was chosen to reflect the reported fluctuations of the free estrogen levels at the various times of the laying cycle. Blood was chilled immediately and centrifuged within 1 hr to separate the plasma, which was stored at —20 C until assayed.
This note describes the results of radioimmunoassay measurements of plasma estradiol and estradiol-3-sulfate as well as estrone and estrone sulfate in laying hens.
1 Parts of the data were presented at the 69th Poultry Science Association Meeting, Purdue University, August, 1980. 2 Animal Research Centre Contribution No. 918.
Details of the separation and determination of estrogens have been described (Tsang, 1974; Tsang et al, 1975). Briefly, plasma (1 ml) was partitioned with benzene to remove the unconjugated estrogens. This was followed by extraction of the estrogen sulfates with tetrahydrofuran.-ethyl acetate (1:1). Estrone sulfate (EjS) and estradiol-17)3-3-sulfate (E^S) were hydrolyzed with 20% acetic acid in methanol and measured as the free compounds. Estrone ( E ^ and estradiol-17/3 (E 2 ), whether obtained directly from the benzene extraction or after hydrolysis of the sulfate fraction, were separated on Sephadex LH-20 columns prior to radioimmunoassay (Tsang and Hackett, 1979).
2548
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INTRODUCTION Several papers have described changes in plasma levels of unconjugated estrogens as determined by radioimmunoassay during the laying cycle (Peterson and Common, 1972; Senior, 1974; Lague et al, 1975; Shahabi et al, 1975; Shodono et al, 1975; Graber and Nalbandov, 1976). These studies have shown a major peak some 7 to 4 hr, and possible a minor peak approximately 22 hr before ovulation. Estrone sulfate has been demonstrated to be the major plasma estrogen in several mammalian species, i.e., sheep (Tsang, 1974, 1978), cows (Tsang et al, 1975), and pigs (Robertson and King, 1974). To our knowledge, the estrogen sulfates have never been determined in the plasma of hens. However, the earlier work of Mathur et al. (1969) demonstrated that radioactive estradiol-17)3 or estrone injected into mature hens was excreted principally as sulfoconjugates in the urine. Chan and Common (1974) also observed that an appreciable proportion of the labeled estrone injected into laying hens was present in plasma as the "bound" fraction of the estradiols, presumably monosulfates.
2549
RESEARCH NOTE
TABLE 1. Mean plasma estrogen concentrations in nine White Leghorn laying hens Hours before and after (+) oviposition
E2
E2S
E,
E,S
E 2 S/EP2
24-22 20-18 9-7 4-2 0 +3
.25* .40* .38* .54 .35* .24*
1.04* 1.24* 1.24* 1.58 1.39 .94*
.30* .46* .42* .59 .43* .26*
.52* .66* .64* .67 .62* .41*
4.8** 3.2 3.3 3.0 4.1 4.1
1.9** 1.5 1.6 1.2 1.5 1.8
Mean of above SE
.36 .02
1.24 .06
.41 .03
.59 .03
3.8 .4
1.6 .1
Molar ratio *-*i ^ ' *-*i
Molecular weights of E, = 270; E2 = 272; E, S (sodium salt) = 372; and E 2 S (sodium salt) = 374.
•Lower than the highest mean at 4 to 2 hr (P<.05). **Higher than the lowest ratio at 4 to 2 hr (P<.01).
Procedural losses of E j , E 2 , EjS, and E 2 S were corrected by adding the corresponding 3 Hlabeled compounds to every plasma sample prior to extraction. Synthesis of 3 H-labeled E 2 S has been described (Tsang and Hackett, 1979). The interassay coefficients of variation for measuring E i , E 2 , EjS, and E 2 S were 8%, 11%, 10%, and 10%, respectively. The accuracy of measuring known amounts of Elt E 2 , EjS, and E 2 S averaged 114%, 96%, 89%, and 85%, respectively. Statistical analyses were performed according to procedures for complete block-repeat measure designs (Gill, 1978b) and Dunnett's J-test (Gill, 1978a) with control equal to the time of expected highest level (4 to 2 hr before oviposition) for the estrogens or lowest level for E 2 S/E 2 and E J S / E J . RESULTS AND DISCUSSION
In contrast to E t S , the principle plasma estrogen in mammals, E 2 S was the most abundant estrogen in these"lien's and~a~ccounted for 48% of the total with an overall mean concentration of 1.24 pmol/ml (Table 1). The E2S~was present at about four times the concentration of E 2 while EiS was less than twice the concentrationof Hi. The concentrations of each of the four estrogens were higher at 4.2 hr before oviposition than at each of the other times except for E 2 S at oviposition. This experiment was not designed to study the detailed variation of the individual estrogens during the ovulatory cycle, but rather to establish the relative abun-
dancy of E 2 versus E 2 S, and E t versus EiS at certain key points of the cycle. Nevertheless, the general pattern of the estrogen profile was in reasonable accord with the literature. While the main source of the unconjugated estrogens is undoubtedly the ovary (Shahabi et al., 1975), the source of the estrogen sulfates is not known; Raud and Hobkirk (1968) indicated that the liyer, in particular, and the oviduct and vagina, to a lesser degree, were capable of converting Ex and E 2 to their 3-sulfates in vitro. It would be of interest to determine the estrogen sulfatase activity in these organs because the balance between the estrogen sulfatase and sulfokinase activity may be important in the regulation of estrogen action. This view is consistent with the observation that the molar ratios of both E 2 S/E 2 and EiS/Ei tended to reach minimum and maximum values at 4 to 2 hr, and 24 to 22 hr, respectively, before oviposition (Table 1). ACKNOWLEDGMENTS
The authors thank A. Burnett, P. Griffin, and J. W. Dickie for skillful technical help. We are most grateful to J. L. Gill, Professor of Biometry, Michigan State University, for kindly performing the statistical analysis of the data. REFERENCES Chan, A.H.-H., and R. H. Common, 1974. Identification of radioactive oestradiol-17a and oestradiol-
Downloaded from http://ps.oxfordjournals.org/ at Northern Arizona University on June 26, 2015
1
Picomole 1 per milliliter plasma
2550
TSANG ET AL. centrations of progesterone, oestrone, oestradiol170, and of oestrone sulfate in the pig at implantation, during pregnancy and at parturition. J. Reprod. Fertil. 4 0 : 1 3 3 - 1 4 1 . Senior, B. E., 1974. Changes in the concentrations of oestrone and oestradiol in the peripheral plasma of the domestic hen during the ovulatory cycle. Acta Endocrinol. 77:588-596. Shahabi, N. A., H. W. Norton, and A. V. Nalbandov, 1975. Steroid levels in follicles and the plasma of hens during the ovulatory cycle. Endocrinology 96:962-968. Shodono, M., T. Nakamura, Y. Tanabe, and K. Wakabayashi, 1975. Simultaneous determinations of oestradiol-17/3, progesterone and luteinizing hormone in the plasma during the ovulatory cycle of the hen. Acta Endocrinol. 78:565— 573. Tsang, C.P.W., 1974. Changes in plasma levels of estrone sulfate and estrone in the pregnant ewe around parturition. Steroids 23:855—868. Tsang, C.P.W., A. J. Hackett, and E. M. Turner, Jr., 1975. Plasma levels of estrone sulfate, estrone and estradiol-17/3 in the cow around parturition. Can. J. Anim. Sci. 55:509-512. Tsang, C.P.W., 1978. Plasma levels of estrone sulfate, free estrogens and progesterone in the pregnant ewe throughout gestation. Theriogenology 10: 97-110. Tsang, C.P.W., and A. J. Hackett, 1979. Metabolism of estrone sulfate in the pregnant sheep. Theriogenology 11:429-439.
Downloaded from http://ps.oxfordjournals.org/ at Northern Arizona University on June 26, 2015
17/3 in the plasma of the laying hen after injection of oestrone-4- 14 C. Comp. Biochem. Physiol. 49B:105-111. Gill, J. L., 1978a. Design and analysis of experiments in the animal and medical sciences. Vol. 1. Iowa State Univ. Press. Gill, J. L., 1978b. Design and analysis of experiments in the animal and medical sciences. Vol. 2. Iowa State Univ. Press. Graber, J. W., and A. V. Nalbandov, 1976. Peripheral estrogen levels during the laying cycle of the hen (Gallus domesticus). Biol. Reprod. 14:109-114. Lague, P. C , A. van Tienhoven, and F. J. Cunningham, 1975. Concentrations of estrogens, progesterone and LH during the ovulatory cycle of the laying chicken (Gallus domesticus). Biol. Reprod. 12: 590-598. Mathur, R. S., R. H. Common, D. C. Collins, and D. S. Layne, 1969. Steroid estrogen conjugates of hen's urine: formation in vivo of steroid estrogen monosulfates and disulfates from injected estradiol-17/3. Biochim. Biophys. Acta 176:394402. Peterson, A. J., and R. H. Common, 1972. Estrone and estradiol concentrations in peripheral plasma of laying hens as determined by radioimmunoassay. Can. J. Zool. 50:395-404. Raud, H. R., and R. Hobkirk, 1968. In vitro biosynthesis of steroid sulfates by cell-free preparations from tissues of the laying hen. Can. J. Biochem. 46:749-757. Robertson, H. A., and G. J. King, 1974. Plasma con-
MATERIALS AND METHODS Nine White Leghorn layers of experimental Strain 1, which had been selected for high egg production at the Animal Research Centre, were caged individually, with feed and water provided ad libitum. The birds were 40 weeks of age and had been in a light regimen of 16L:8D with lights on at 0100 hr since they were 37 weeks of age. Room temperature was maintained at 13 C. During the study, the birds exhibited 24 to 26 hr laying cycles. Oviposition times were determined within 3 min by patrol. Blood (2 to 3 ml) was drawn from the brachial vein at 2.5, 5.75, 17, and 22 hr after oviposition (not the first egg of the clutch) and at 0 and 2.5 hr after the next oviposition. This collection schedule was chosen to reflect the reported fluctuations of the free estrogen levels at the various times of the laying cycle. Blood was chilled immediately and centrifuged within 1 hr to separate the plasma, which was stored at —20 C until assayed.
This note describes the results of radioimmunoassay measurements of plasma estradiol and estradiol-3-sulfate as well as estrone and estrone sulfate in laying hens.
1 Parts of the data were presented at the 69th Poultry Science Association Meeting, Purdue University, August, 1980. 2 Animal Research Centre Contribution No. 918.
Details of the separation and determination of estrogens have been described (Tsang, 1974; Tsang et al, 1975). Briefly, plasma (1 ml) was partitioned with benzene to remove the unconjugated estrogens. This was followed by extraction of the estrogen sulfates with tetrahydrofuran.-ethyl acetate (1:1). Estrone sulfate (EjS) and estradiol-17)3-3-sulfate (E^S) were hydrolyzed with 20% acetic acid in methanol and measured as the free compounds. Estrone ( E ^ and estradiol-17/3 (E 2 ), whether obtained directly from the benzene extraction or after hydrolysis of the sulfate fraction, were separated on Sephadex LH-20 columns prior to radioimmunoassay (Tsang and Hackett, 1979).
2548
Downloaded from http://ps.oxfordjournals.org/ at Northern Arizona University on June 26, 2015
INTRODUCTION Several papers have described changes in plasma levels of unconjugated estrogens as determined by radioimmunoassay during the laying cycle (Peterson and Common, 1972; Senior, 1974; Lague et al, 1975; Shahabi et al, 1975; Shodono et al, 1975; Graber and Nalbandov, 1976). These studies have shown a major peak some 7 to 4 hr, and possible a minor peak approximately 22 hr before ovulation. Estrone sulfate has been demonstrated to be the major plasma estrogen in several mammalian species, i.e., sheep (Tsang, 1974, 1978), cows (Tsang et al, 1975), and pigs (Robertson and King, 1974). To our knowledge, the estrogen sulfates have never been determined in the plasma of hens. However, the earlier work of Mathur et al. (1969) demonstrated that radioactive estradiol-17)3 or estrone injected into mature hens was excreted principally as sulfoconjugates in the urine. Chan and Common (1974) also observed that an appreciable proportion of the labeled estrone injected into laying hens was present in plasma as the "bound" fraction of the estradiols, presumably monosulfates.
2549
RESEARCH NOTE
TABLE 1. Mean plasma estrogen concentrations in nine White Leghorn laying hens Hours before and after (+) oviposition
E2
E2S
E,
E,S
E 2 S/EP2
24-22 20-18 9-7 4-2 0 +3
.25* .40* .38* .54 .35* .24*
1.04* 1.24* 1.24* 1.58 1.39 .94*
.30* .46* .42* .59 .43* .26*
.52* .66* .64* .67 .62* .41*
4.8** 3.2 3.3 3.0 4.1 4.1
1.9** 1.5 1.6 1.2 1.5 1.8
Mean of above SE
.36 .02
1.24 .06
.41 .03
.59 .03
3.8 .4
1.6 .1
Molar ratio *-*i ^ ' *-*i
Molecular weights of E, = 270; E2 = 272; E, S (sodium salt) = 372; and E 2 S (sodium salt) = 374.
•Lower than the highest mean at 4 to 2 hr (P<.05). **Higher than the lowest ratio at 4 to 2 hr (P<.01).
Procedural losses of E j , E 2 , EjS, and E 2 S were corrected by adding the corresponding 3 Hlabeled compounds to every plasma sample prior to extraction. Synthesis of 3 H-labeled E 2 S has been described (Tsang and Hackett, 1979). The interassay coefficients of variation for measuring E i , E 2 , EjS, and E 2 S were 8%, 11%, 10%, and 10%, respectively. The accuracy of measuring known amounts of Elt E 2 , EjS, and E 2 S averaged 114%, 96%, 89%, and 85%, respectively. Statistical analyses were performed according to procedures for complete block-repeat measure designs (Gill, 1978b) and Dunnett's J-test (Gill, 1978a) with control equal to the time of expected highest level (4 to 2 hr before oviposition) for the estrogens or lowest level for E 2 S/E 2 and E J S / E J . RESULTS AND DISCUSSION
In contrast to E t S , the principle plasma estrogen in mammals, E 2 S was the most abundant estrogen in these"lien's and~a~ccounted for 48% of the total with an overall mean concentration of 1.24 pmol/ml (Table 1). The E2S~was present at about four times the concentration of E 2 while EiS was less than twice the concentrationof Hi. The concentrations of each of the four estrogens were higher at 4.2 hr before oviposition than at each of the other times except for E 2 S at oviposition. This experiment was not designed to study the detailed variation of the individual estrogens during the ovulatory cycle, but rather to establish the relative abun-
dancy of E 2 versus E 2 S, and E t versus EiS at certain key points of the cycle. Nevertheless, the general pattern of the estrogen profile was in reasonable accord with the literature. While the main source of the unconjugated estrogens is undoubtedly the ovary (Shahabi et al., 1975), the source of the estrogen sulfates is not known; Raud and Hobkirk (1968) indicated that the liyer, in particular, and the oviduct and vagina, to a lesser degree, were capable of converting Ex and E 2 to their 3-sulfates in vitro. It would be of interest to determine the estrogen sulfatase activity in these organs because the balance between the estrogen sulfatase and sulfokinase activity may be important in the regulation of estrogen action. This view is consistent with the observation that the molar ratios of both E 2 S/E 2 and EiS/Ei tended to reach minimum and maximum values at 4 to 2 hr, and 24 to 22 hr, respectively, before oviposition (Table 1). ACKNOWLEDGMENTS
The authors thank A. Burnett, P. Griffin, and J. W. Dickie for skillful technical help. We are most grateful to J. L. Gill, Professor of Biometry, Michigan State University, for kindly performing the statistical analysis of the data. REFERENCES Chan, A.H.-H., and R. H. Common, 1974. Identification of radioactive oestradiol-17a and oestradiol-
Downloaded from http://ps.oxfordjournals.org/ at Northern Arizona University on June 26, 2015
1
Picomole 1 per milliliter plasma
2550
TSANG ET AL. centrations of progesterone, oestrone, oestradiol170, and of oestrone sulfate in the pig at implantation, during pregnancy and at parturition. J. Reprod. Fertil. 4 0 : 1 3 3 - 1 4 1 . Senior, B. E., 1974. Changes in the concentrations of oestrone and oestradiol in the peripheral plasma of the domestic hen during the ovulatory cycle. Acta Endocrinol. 77:588-596. Shahabi, N. A., H. W. Norton, and A. V. Nalbandov, 1975. Steroid levels in follicles and the plasma of hens during the ovulatory cycle. Endocrinology 96:962-968. Shodono, M., T. Nakamura, Y. Tanabe, and K. Wakabayashi, 1975. Simultaneous determinations of oestradiol-17/3, progesterone and luteinizing hormone in the plasma during the ovulatory cycle of the hen. Acta Endocrinol. 78:565— 573. Tsang, C.P.W., 1974. Changes in plasma levels of estrone sulfate and estrone in the pregnant ewe around parturition. Steroids 23:855—868. Tsang, C.P.W., A. J. Hackett, and E. M. Turner, Jr., 1975. Plasma levels of estrone sulfate, estrone and estradiol-17/3 in the cow around parturition. Can. J. Anim. Sci. 55:509-512. Tsang, C.P.W., 1978. Plasma levels of estrone sulfate, free estrogens and progesterone in the pregnant ewe throughout gestation. Theriogenology 10: 97-110. Tsang, C.P.W., and A. J. Hackett, 1979. Metabolism of estrone sulfate in the pregnant sheep. Theriogenology 11:429-439.
Downloaded from http://ps.oxfordjournals.org/ at Northern Arizona University on June 26, 2015
17/3 in the plasma of the laying hen after injection of oestrone-4- 14 C. Comp. Biochem. Physiol. 49B:105-111. Gill, J. L., 1978a. Design and analysis of experiments in the animal and medical sciences. Vol. 1. Iowa State Univ. Press. Gill, J. L., 1978b. Design and analysis of experiments in the animal and medical sciences. Vol. 2. Iowa State Univ. Press. Graber, J. W., and A. V. Nalbandov, 1976. Peripheral estrogen levels during the laying cycle of the hen (Gallus domesticus). Biol. Reprod. 14:109-114. Lague, P. C , A. van Tienhoven, and F. J. Cunningham, 1975. Concentrations of estrogens, progesterone and LH during the ovulatory cycle of the laying chicken (Gallus domesticus). Biol. Reprod. 12: 590-598. Mathur, R. S., R. H. Common, D. C. Collins, and D. S. Layne, 1969. Steroid estrogen conjugates of hen's urine: formation in vivo of steroid estrogen monosulfates and disulfates from injected estradiol-17/3. Biochim. Biophys. Acta 176:394402. Peterson, A. J., and R. H. Common, 1972. Estrone and estradiol concentrations in peripheral plasma of laying hens as determined by radioimmunoassay. Can. J. Zool. 50:395-404. Raud, H. R., and R. Hobkirk, 1968. In vitro biosynthesis of steroid sulfates by cell-free preparations from tissues of the laying hen. Can. J. Biochem. 46:749-757. Robertson, H. A., and G. J. King, 1974. Plasma con-