Serum Progesterone and Estrogens in Elephants EDWARD D. PLOTKA, ULYSSES S. SEAL, EARL E. SCHOBERT, AND GEORGE C. SCHMOLLER Marshfield Medical Foundation, Inc., Marshfield, Wisconsin 54449; Department of Biochemistry, University of Minnesota and Veterans Administration Hospital, Minneapolis, Minnesota 55417; Busch Gardens, Tampa, Florida 33612; Marshfield Medical Foundation, Inc., Marshfield, Wisconsin 54449 ABSTRACT. Serum progesterone and estrogens were measured by radioimmunoassay in the serum of immature, mature, and pregnant African and Asian elephants. Progesterone was elevated from 26 to 215 pg/ml in nonpregnant animals and up to 480
pg/ml in late pregnancy animals. No relationship to reproductive state was evident for the low levels of estrogens which ranged from 9 to 37 pg/ml (Endocrinology 97: 485,1975)
DGAR reported in 1953 (1) that he had E detected progesterone in the corpus luteum [CL] of the African elephant. Since that time,
steroids by two incubations with charcoal at 45 C. The standards and their recoveries given as X ± SE were: 25 pg/ml, 26.4 ± 2.2 pg/ml (N = 6); 50 pg/ml, 51.4 ± 1.8 pg/ml (N = 6), and 100 pg/ml, 93.0 ± 6.8 pg/ml (N = 6). The stripped plasma yielded a value of 14.7 ±1.3 pg/ml (N = 6) which was subtracted from the recovery values. Distilled water gave 2.7 ± 1.0 pg/ml (N = 6). Estrogen was assayed by the method described by Plotka et al.* with antibody kindly supplied by Drs. Steven Tillson and Burton Caldwell. The estrogen assay has a sensitivity of 10 pg and a coefficient of variation at 10 pg of 20%. This antiserum has a cross-reaction with estriol of 4% and with estrone of 22%. The accuracy of the estrogen assay also was estimated by measurements of standards in stripped plasma. The standards and their recoveries given as X ± SE were: 10 pg/ml, 9.4 ± 1.3 pg/ml (N = 9); 25 pg/ml, 27.6 ± 2.1 pg/ml (N = 11); 50 pg/ml, 45.0 ±2.6 pg/ml (N = 11), and 100 pg/ml, 93.5 ± 6.5 pg/ml (N = 11). The stripped plasma yielded a value of 11.0 ± 1.0 pg/ml (N = 8) and distilled water gave 6.1 ± 0.8 pg/ml (N = 3).
several reports have demonstrated that only very small quantities of progesterone are present in elephant CL tissue (2-5). Hanks and Short (6) reported that very little progesterone was secreted by elephant CL and they could not detect the hormone in peripheral blood even during gestation. We wish to report on progesterone and estrogen concentrations in peripheral blood plasma of nonpregnant and pregnant African (Loxodonta africana) and Asian (Elaphus maximus) elephants held in captivity.
Materials and Methods Ten samples of blood were collected from the ear vein of nine standing female elephants at various times in their reproductive cycle. Progesterone was assayed by radioimmunoassay (RIA)*. Antibody for the progesterone assay was raised in rabbits to lla-OH progesterone-succinylbovine serum albumin. The progesterone assay had a sensitivity of 10 pg and a coefficient of variation at 10 pg of 17%. The cross reaction of this antiserum with other steroids was found to be: lla-hydroxyprogesterone 15.3%, 17a-hydroxyprogesterone 3.5%, 20a-hydroxyprogesterone 3.8%, cortisol 0.03%, corticosterone 0.9%, 5/3-pregnane-3, 20-dione 7.0%, 5apregnane-3, 20-dione 9.0%, androstenedione 0.97%, and estrone 0.18%. The accuracy of the progesterone assay was established by measurements of standards in plasma which had been stripped of endogenous Received January 20, 1975. Supported by US PHS, NIAMDD Research Grant 5-R01-AM-11376. • Plotka, E. D., C. W. Foley, D. M. Witherspoon, and G. C. Schmoller, Am J Vet Res (submitted for publication).
Results and Discussion Progesterone levels in peripheral serum ranged from 26 pg/ml to 482 pg/ml in our elephants depending on the stage of the reproductive cycle (Table 1). Progesterone showed significant changes when immature, mature nonpregnant, and mature pregnant animals were compared. Estrogens were assayed in nine elephants and ranged from 9 to 37 pg/ml (Table 1). There was no distinct relationship between state of reproduction and estrogen level. Hanks and Short (6) have suggested that for the African elephant there appears to be a critical mass of luteal tissue that needs to accumulate before conception can occur. They also reported that very little progesterone was secreted by the elephant CL, since they could 485
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Endo • 1975 Vol 97 • No 2
NOTES AND COMMENTS
486
TABLE 1. Reproductive status and hormone levels in captive African and Asian elephants Hormone level (pg/ml) Identification
Date
Source
Type
Progesterone
Estrogen
Tinkerbell
8-26-74
Busch Gardens
African
69
9
Dolly Elka
8-26-74 8-26-74
Busch Gardens Busch Gardens
African African
208
12
215
16
Hussey
8-26-74
Busch Gardens
African
416
11
Pregnant (approximately 14 months)
Be Be Susie
8-26-74 9-9-74
Busch Gardens
482
20
Pregnant (200 lb fetus at autopsy)
Ringling Bros.
African Asian
51
26
7 or 8 yr—immature
Susie Luci
9-11-74 10-6-67
Ringling Bros.
Asian
26 195
37 26
Not pregnant—adult
Belle
4-26-72
Portland Zoo
Asian
153
30
Not pregnant—adult
Tuy Hoa
4-27-72
Portland Zoo
Asian
263
26
Delivered July 19, 1972
not detect progesterone in peripheral blood plasma by their gas chromatographic procedure. The more sensitive RIA procedure confirms that progesterone is very low. However, we can show differences between immature, mature non-pregnant, and pregnant females. Our data disagree with those of Hanks and Short (6) to the extent that we found levels of 400 to 500 pg/ml whereas they reported less than 200 pg/ml for pregnant elephants. Serum from three animals was assayed by a competitive binding technique (7) as well as by RIA and the two assays agreed remarkably well with two of the sera (180 vs 160, 180 vs 180, and 160 vs 270 pg/ml) despite the use of the competitive binding assay at its limit of sensitivity. Nevertheless, the elephant must be extremely sensitive to progesterone, as Hanks and Short (6) suggested, if the corpora lutea are necessary for endometrial development. These low levels of progesterone are similar only to those seen in tammar wallabys. Lemon (8) reported levels of progesterone of 129 ± 72 pg/ml during the estrous cycle and 266 ± 57 pg/ml during pregnancy in this marsupial. Although these data on the elephant do not establish a function for progesterone during pregnancy in this species, the increase during pregnancy favors a typical mammalian role. The lack of a distinct relationship between state of reproduction and estrogen level suggests that either the ovaries or corpora lutea
Time of reproductive cycle Immature female 5 yr old Not pregnant (autopsy)—adult Not pregnant (cycled)—adult
or both secrete very little hormone in relation to other species and that the placenta secretes little or no hormone. Since Smith et al. (4) could only isolate small amounts of progesterone (0.18 jug/g) from a large quantity of luteal tissue taken from a pregnant animal, it is likely that the CL has limited steroid secreting capacity. However, since no one has measured the contribution of steroids to either maternal or fetal elephant blood by the placenta, its contribution to circulating levels cannot be assessed. This apparent lack of correlation of estrogen level with stage of reproduction can be attributed to different causes; (1) the major estrogen is not estradiol and thus we are underestimating the amount of active estrogen because of the limited cross reactivity with our antibody, (2) the radioimmunoassay is not sensitive enough to detect the differences in concentration at different states of reproduction, (3) the elephant does not need estrogen for estrus or pregnancy, (4) receptors in target tissues have a high affinity for the hormone and can be saturated at these low levels of circulating hormone, or (5) lack of specific serum binding proteins. Which, if any, of these factors is responsible for our results remains to be elucidated.
Acknowledgments The authors extend their sincere thanks to Dr. J. Y. Henderson of Ringling Bros.-Barnum and Bailey Circus, Dr. P. Ogilvie of the Portland, Oregon Zoo, and
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 June 2015. at 11:27 For personal use only. No other uses without permission. . All rights reserved.
NOTES AND COMMENTS Dr. Lee Simmons, Omaha Zoo, for assistance in sample collection.
References 1. Edgar, D. G.J Endocrinol 10: 54, 1953. 2. Short, R. V., and I. O. Buss,7 Reprod Fertil 9: 61, 1965. 3. , East Afr WldlfJ 4: 56, 1966.
487
4. Smith, J. G., J. Hanks, and R. V. Short, J Reprod Fertil 20: 111, 1969. 5. Ogle, T. F., H. H. Braach, and I. O. Buss, Anat Rec 175: 707, 1973. 6. Hanks, J., and R. V. Short, J Reprod Fertil 29: 79, 1972. 7. Plotka, E. D., D. M. Witherspoon, and C. W. Foley, Am J Vet Res 33: 917, 1972. 8. Lemon, M.J Endocrinol 55: 63, 1972.
Masculine-like Hypothalamic-Pituitary Axis in the Androgen-Insensitive Genetically Male Rat Pseudohermaphrodite BERNARD H. SHAPIRO,1 ALLEN S. GOLDMAN,1-2 AND JAN-AKE GUSTAFSSON3 1
Division of Experimental Pathology, Children's Hospital of Philadelphia, and the Department of Pediatrics, University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania 19104; and Kemiska Institution of the Karolinska Institutet, Stockholm, Sweden ABSTRACT. The hypothalamic-pituitary sex of the androgen insensitive, genetically male rat pseudohermaphrodite was studied by examining its vaginal cytology, response to ovarian transplants and urinary steroidal excretion patterns. More than half the pseudohermaphrodites studied were in constant vaginal estrus, while the remaining rats displayed either persistent diestrus or irregular cyclicity tending towards lengthened estrus. Following gonadectomy and ovarian transplantation, normal females displayed regular 4-day cycles while pseudohermaphrodites remained in constant vaginal estrus. In pseudohermaphrodites with ovarian transplants, only C19 steroids were detected in the urine while females excreted both C2i and C19
steroids. Indicative of the urinary findings, transplants in females had corpora lutea and maturing follicles while grafts from pseudohermaphrodites and males contained follicular cysts and luteinized theca. In addition, distribution and activity of histochemical 3/3-hydroxy-A5 steroid oxidoreductase were similar in the grafts from pseudohermaphrodites and males, but unlike the females. Although previous reports have shown that much of the sex-dependent differentiation of the genetic male rat pseudohermaphrodite is phenotypically female, our results suggest that the phenotype of the hypothalamic-pituitary axis of this animal is, at least in certain respects, male. (Endocrinology 97: 487, 1975)
TANLEY et al. (1) have described a herediS tary form of pseudohermaphrodism in the rat in which the animal is a genetic male with
Furthermore, the pseudohermaphrodite has a feminine pattern of urinary corticosteroid excretion (1,2) and hepatic steroid metabolism (3). Although the animals exhibit no sexual behavior (1) they do have a feminine saccharin preference (4). The development of a female phenotype in the pseudohermaphrodite is due to an absence of androgen dependent differentiation. The primary genetic defect of the male rat pseudohermaphrodite is apparently the inherited end organ insensitivity to androgens reflected at the cellular level by deficient target organ androgen binding proteins (5). Although characterized as a phenotypic female, recent reports have suggested some degree of masculine differentiation in the genetically male rat pseudohermaphrodite, and in certain respects the rat may properly be classified as a phenotypic intersex. Its preputia] gland (6) and body weight (1,7) are intermediate
an XY karyotype and a chromatin negative nuclear sex, but possesses a feminine phenotype. These rats have inguinal testes with well-developed Leydig cells and germinal arrest, no male reproductive structures of wolffian duct origin, no mullerian duct derivatives, a short vagina ending in a blind cul de sac, and a feminine number and positioning of nipples. Received July 15, 1974. Recipient of Career Development Award (HD13,628) from the U.S.P.H.S. This work was aided by a contract NOl-HD-3-2746 and by research grants from the U.S.P.H.S. (HD4683), the Rockefeller Foundation, The Swedish Medical Research Council (no. 13X-2819), and The NATO Research Programme. 2
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pg/ml in late pregnancy animals. No relationship to reproductive state was evident for the low levels of estrogens which ranged from 9 to 37 pg/ml (Endocrinology 97: 485,1975)
DGAR reported in 1953 (1) that he had E detected progesterone in the corpus luteum [CL] of the African elephant. Since that time,
steroids by two incubations with charcoal at 45 C. The standards and their recoveries given as X ± SE were: 25 pg/ml, 26.4 ± 2.2 pg/ml (N = 6); 50 pg/ml, 51.4 ± 1.8 pg/ml (N = 6), and 100 pg/ml, 93.0 ± 6.8 pg/ml (N = 6). The stripped plasma yielded a value of 14.7 ±1.3 pg/ml (N = 6) which was subtracted from the recovery values. Distilled water gave 2.7 ± 1.0 pg/ml (N = 6). Estrogen was assayed by the method described by Plotka et al.* with antibody kindly supplied by Drs. Steven Tillson and Burton Caldwell. The estrogen assay has a sensitivity of 10 pg and a coefficient of variation at 10 pg of 20%. This antiserum has a cross-reaction with estriol of 4% and with estrone of 22%. The accuracy of the estrogen assay also was estimated by measurements of standards in stripped plasma. The standards and their recoveries given as X ± SE were: 10 pg/ml, 9.4 ± 1.3 pg/ml (N = 9); 25 pg/ml, 27.6 ± 2.1 pg/ml (N = 11); 50 pg/ml, 45.0 ±2.6 pg/ml (N = 11), and 100 pg/ml, 93.5 ± 6.5 pg/ml (N = 11). The stripped plasma yielded a value of 11.0 ± 1.0 pg/ml (N = 8) and distilled water gave 6.1 ± 0.8 pg/ml (N = 3).
several reports have demonstrated that only very small quantities of progesterone are present in elephant CL tissue (2-5). Hanks and Short (6) reported that very little progesterone was secreted by elephant CL and they could not detect the hormone in peripheral blood even during gestation. We wish to report on progesterone and estrogen concentrations in peripheral blood plasma of nonpregnant and pregnant African (Loxodonta africana) and Asian (Elaphus maximus) elephants held in captivity.
Materials and Methods Ten samples of blood were collected from the ear vein of nine standing female elephants at various times in their reproductive cycle. Progesterone was assayed by radioimmunoassay (RIA)*. Antibody for the progesterone assay was raised in rabbits to lla-OH progesterone-succinylbovine serum albumin. The progesterone assay had a sensitivity of 10 pg and a coefficient of variation at 10 pg of 17%. The cross reaction of this antiserum with other steroids was found to be: lla-hydroxyprogesterone 15.3%, 17a-hydroxyprogesterone 3.5%, 20a-hydroxyprogesterone 3.8%, cortisol 0.03%, corticosterone 0.9%, 5/3-pregnane-3, 20-dione 7.0%, 5apregnane-3, 20-dione 9.0%, androstenedione 0.97%, and estrone 0.18%. The accuracy of the progesterone assay was established by measurements of standards in plasma which had been stripped of endogenous Received January 20, 1975. Supported by US PHS, NIAMDD Research Grant 5-R01-AM-11376. • Plotka, E. D., C. W. Foley, D. M. Witherspoon, and G. C. Schmoller, Am J Vet Res (submitted for publication).
Results and Discussion Progesterone levels in peripheral serum ranged from 26 pg/ml to 482 pg/ml in our elephants depending on the stage of the reproductive cycle (Table 1). Progesterone showed significant changes when immature, mature nonpregnant, and mature pregnant animals were compared. Estrogens were assayed in nine elephants and ranged from 9 to 37 pg/ml (Table 1). There was no distinct relationship between state of reproduction and estrogen level. Hanks and Short (6) have suggested that for the African elephant there appears to be a critical mass of luteal tissue that needs to accumulate before conception can occur. They also reported that very little progesterone was secreted by the elephant CL, since they could 485
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 June 2015. at 11:27 For personal use only. No other uses without permission. . All rights reserved.
Endo • 1975 Vol 97 • No 2
NOTES AND COMMENTS
486
TABLE 1. Reproductive status and hormone levels in captive African and Asian elephants Hormone level (pg/ml) Identification
Date
Source
Type
Progesterone
Estrogen
Tinkerbell
8-26-74
Busch Gardens
African
69
9
Dolly Elka
8-26-74 8-26-74
Busch Gardens Busch Gardens
African African
208
12
215
16
Hussey
8-26-74
Busch Gardens
African
416
11
Pregnant (approximately 14 months)
Be Be Susie
8-26-74 9-9-74
Busch Gardens
482
20
Pregnant (200 lb fetus at autopsy)
Ringling Bros.
African Asian
51
26
7 or 8 yr—immature
Susie Luci
9-11-74 10-6-67
Ringling Bros.
Asian
26 195
37 26
Not pregnant—adult
Belle
4-26-72
Portland Zoo
Asian
153
30
Not pregnant—adult
Tuy Hoa
4-27-72
Portland Zoo
Asian
263
26
Delivered July 19, 1972
not detect progesterone in peripheral blood plasma by their gas chromatographic procedure. The more sensitive RIA procedure confirms that progesterone is very low. However, we can show differences between immature, mature non-pregnant, and pregnant females. Our data disagree with those of Hanks and Short (6) to the extent that we found levels of 400 to 500 pg/ml whereas they reported less than 200 pg/ml for pregnant elephants. Serum from three animals was assayed by a competitive binding technique (7) as well as by RIA and the two assays agreed remarkably well with two of the sera (180 vs 160, 180 vs 180, and 160 vs 270 pg/ml) despite the use of the competitive binding assay at its limit of sensitivity. Nevertheless, the elephant must be extremely sensitive to progesterone, as Hanks and Short (6) suggested, if the corpora lutea are necessary for endometrial development. These low levels of progesterone are similar only to those seen in tammar wallabys. Lemon (8) reported levels of progesterone of 129 ± 72 pg/ml during the estrous cycle and 266 ± 57 pg/ml during pregnancy in this marsupial. Although these data on the elephant do not establish a function for progesterone during pregnancy in this species, the increase during pregnancy favors a typical mammalian role. The lack of a distinct relationship between state of reproduction and estrogen level suggests that either the ovaries or corpora lutea
Time of reproductive cycle Immature female 5 yr old Not pregnant (autopsy)—adult Not pregnant (cycled)—adult
or both secrete very little hormone in relation to other species and that the placenta secretes little or no hormone. Since Smith et al. (4) could only isolate small amounts of progesterone (0.18 jug/g) from a large quantity of luteal tissue taken from a pregnant animal, it is likely that the CL has limited steroid secreting capacity. However, since no one has measured the contribution of steroids to either maternal or fetal elephant blood by the placenta, its contribution to circulating levels cannot be assessed. This apparent lack of correlation of estrogen level with stage of reproduction can be attributed to different causes; (1) the major estrogen is not estradiol and thus we are underestimating the amount of active estrogen because of the limited cross reactivity with our antibody, (2) the radioimmunoassay is not sensitive enough to detect the differences in concentration at different states of reproduction, (3) the elephant does not need estrogen for estrus or pregnancy, (4) receptors in target tissues have a high affinity for the hormone and can be saturated at these low levels of circulating hormone, or (5) lack of specific serum binding proteins. Which, if any, of these factors is responsible for our results remains to be elucidated.
Acknowledgments The authors extend their sincere thanks to Dr. J. Y. Henderson of Ringling Bros.-Barnum and Bailey Circus, Dr. P. Ogilvie of the Portland, Oregon Zoo, and
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 June 2015. at 11:27 For personal use only. No other uses without permission. . All rights reserved.
NOTES AND COMMENTS Dr. Lee Simmons, Omaha Zoo, for assistance in sample collection.
References 1. Edgar, D. G.J Endocrinol 10: 54, 1953. 2. Short, R. V., and I. O. Buss,7 Reprod Fertil 9: 61, 1965. 3. , East Afr WldlfJ 4: 56, 1966.
487
4. Smith, J. G., J. Hanks, and R. V. Short, J Reprod Fertil 20: 111, 1969. 5. Ogle, T. F., H. H. Braach, and I. O. Buss, Anat Rec 175: 707, 1973. 6. Hanks, J., and R. V. Short, J Reprod Fertil 29: 79, 1972. 7. Plotka, E. D., D. M. Witherspoon, and C. W. Foley, Am J Vet Res 33: 917, 1972. 8. Lemon, M.J Endocrinol 55: 63, 1972.
Masculine-like Hypothalamic-Pituitary Axis in the Androgen-Insensitive Genetically Male Rat Pseudohermaphrodite BERNARD H. SHAPIRO,1 ALLEN S. GOLDMAN,1-2 AND JAN-AKE GUSTAFSSON3 1
Division of Experimental Pathology, Children's Hospital of Philadelphia, and the Department of Pediatrics, University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania 19104; and Kemiska Institution of the Karolinska Institutet, Stockholm, Sweden ABSTRACT. The hypothalamic-pituitary sex of the androgen insensitive, genetically male rat pseudohermaphrodite was studied by examining its vaginal cytology, response to ovarian transplants and urinary steroidal excretion patterns. More than half the pseudohermaphrodites studied were in constant vaginal estrus, while the remaining rats displayed either persistent diestrus or irregular cyclicity tending towards lengthened estrus. Following gonadectomy and ovarian transplantation, normal females displayed regular 4-day cycles while pseudohermaphrodites remained in constant vaginal estrus. In pseudohermaphrodites with ovarian transplants, only C19 steroids were detected in the urine while females excreted both C2i and C19
steroids. Indicative of the urinary findings, transplants in females had corpora lutea and maturing follicles while grafts from pseudohermaphrodites and males contained follicular cysts and luteinized theca. In addition, distribution and activity of histochemical 3/3-hydroxy-A5 steroid oxidoreductase were similar in the grafts from pseudohermaphrodites and males, but unlike the females. Although previous reports have shown that much of the sex-dependent differentiation of the genetic male rat pseudohermaphrodite is phenotypically female, our results suggest that the phenotype of the hypothalamic-pituitary axis of this animal is, at least in certain respects, male. (Endocrinology 97: 487, 1975)
TANLEY et al. (1) have described a herediS tary form of pseudohermaphrodism in the rat in which the animal is a genetic male with
Furthermore, the pseudohermaphrodite has a feminine pattern of urinary corticosteroid excretion (1,2) and hepatic steroid metabolism (3). Although the animals exhibit no sexual behavior (1) they do have a feminine saccharin preference (4). The development of a female phenotype in the pseudohermaphrodite is due to an absence of androgen dependent differentiation. The primary genetic defect of the male rat pseudohermaphrodite is apparently the inherited end organ insensitivity to androgens reflected at the cellular level by deficient target organ androgen binding proteins (5). Although characterized as a phenotypic female, recent reports have suggested some degree of masculine differentiation in the genetically male rat pseudohermaphrodite, and in certain respects the rat may properly be classified as a phenotypic intersex. Its preputia] gland (6) and body weight (1,7) are intermediate
an XY karyotype and a chromatin negative nuclear sex, but possesses a feminine phenotype. These rats have inguinal testes with well-developed Leydig cells and germinal arrest, no male reproductive structures of wolffian duct origin, no mullerian duct derivatives, a short vagina ending in a blind cul de sac, and a feminine number and positioning of nipples. Received July 15, 1974. Recipient of Career Development Award (HD13,628) from the U.S.P.H.S. This work was aided by a contract NOl-HD-3-2746 and by research grants from the U.S.P.H.S. (HD4683), the Rockefeller Foundation, The Swedish Medical Research Council (no. 13X-2819), and The NATO Research Programme. 2
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