Sex differences in plasma corticosterone levels in alligator (Alligator mississippiensis) embryos

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K.F. THE MEDLER JOURNAL AND V.A. OF EXPERIMENTAL LANCE ZOOLOGY 280:238–244 (1998)

Sex Differences in Plasma Corticosterone Levels in Alligator (Alligator mississippiensis) Embryos KATHRYN F. MEDLER1 AND VALENTINE A. LANCE2* 1 Department of Physiology and Zoology, Louisiana State University, Baton Rouge, Louisiana 70803. 2 Center for Reproduction of Endangered Species, Zoological Society of San Diego, San Diego, California 92112 ABSTRACT The sex of alligator embryos is determined by incubation temperature. Females are produced at temperatures between 29°C and 31°C and males at 33°C. As part of an ongoing study on the hormonal basis of sex determination in the alligator, we collected plasma and urogenital tissue from alligator embryos incubated at 30°C (females) and 33°C (males). Progesterone and corticosterone were determined by radioimmunoassay (RIA) in both plasma and urogenital tissue in embryos prior to the temperature-sensitive period (stage 17 in females and stage 20 in males) and at weekly intervals throughout the remainder of development until hatch. Corticosterone began increasing by stage 25 in both tissue and plasma of both sexes and continued to rise until hatching. Plasma progesterone on the other hand was very low throughout the second half of incubation in both sexes. Tissue levels of progesterone were low early in development and increased later in development. Plasma corticosterone values were significantly higher in female than in male embryos in the last week of incubation. However, by 3 weeks after hatch, plasma corticosterone levels had decreased significantly in both sexes and were not significantly different from one another. Plasma estradiol was significantly higher in female hatchlings than in male hatchlings. J. Exp. Zool. 280:238–244, 1998. © 1998 Wiley-Liss, Inc.

In chick embryos, corticosterone and its precursor, progesterone, have been identified in plasma from as early as day 9 of development. The level of progesterone in the blood increased dramatically from mid-development at day 11 to day 21 at hatching. Corticosterone showed a similar increase (Kalliecharan and Hall, ’74). There were no apparent sex differences for either progesterone or corticosterone in the plasma of chick embryos (Tanabe et al., ’86), but, in turkey embryos, Wentworth and Hussein (’85) found a significant sex difference in plasma corticosterone levels during and immediately after hatching. Plasma corticosterone levels in females were approximately twice those of males at this time but decreased shortly after hatching when the sex differences disappeared (Wentworth and Hussein, ’85). There are no similar data for reptiles. White and Thomas (’92) reported the corticosterone in the plasma of turtle embryos, Trachemys scripta, increased during the period of sex differentiation, from stage 15 to stage 21, but provided no data for later stages. They found detectable corticosterone levels apparent in male embryos by stage 17 of development, but not in female embryos until stage 19. By stage 21, circulating cor© 1998 WILEY-LISS, INC.

ticosterone was four times higher in females than in males (White and Thomas, ’92). As part of an ongoing study on the hormonal correlates of sex determination in the alligator (Lance and Bogart, ’94), we looked at steroid hormones in male and female embryos. Estradiol was not correlated with either sex or stage of development (Lance and Bogart, ’94). In this study, corticosterone and progesterone were measured in the plasma and urogenital tissue of American alligator (Alligator mississippiensis) embryos incubated at male- (33°C) and female- (30°C) producing temperatures from stage 17 in females and stage 20 in males (prior to the temperature-sensitive period) until hatching (stage 28). Progesterone, estradiol, and corticosterone were also measured in plasma from male and female alligators at 21 days after hatching.

V.A. Lance’s present address: Dept. of Physiology and Zoology, Louisiana State University, Baton Rouge, LA 70803. *Correspondence to: Dr. V.A. Lance, Center for Reproduction of Endangered Species, P.O. Box 551, San Diego, CA 92112. Received 23 September 1997; Accepted 5 November 1997

PLASMA CORTICOSTERONE IN ALLLIGATOR EMBRYOS

MATERIALS AND METHODS Animals Alligator eggs were collected shortly after oviposition from nests located on the Rockefeller Wildlife Refuge in Louisiana and transported to San Diego. Eggs were maintained on damp vermiculite in constant-temperature incubators at either 30°C (female producing) or 33°C (male producing). At these incubation temperatures, 100% female and 100% male hatchlings are produced (Lance and Bogart, ’92; Lang and Andrews, ’94). Three embryos from each incubation temperature were sacrificed at weekly intervals from day 23 until hatching (between days 62 and 75). A blood sample was collected using a glass capillary tube, and the urogenital tissue removed. From early embryos, less than 20 ãl of plasma was obtained. In some instances, pooled samples from two or three embryos were used, but by stage 24 and later it was possible to collect more than 200 ãl from each embryo. As it was not possible to dissect the gonad free of mesonephric tissue, the entire urogenital tract including the presumptive gonad, adrenal gland, and mesonephros was collected. To avoid bias in the results due to any possible circadian variation in hormone levels, all samples were collected between 9:00 A.M. and noon. Additional samples were obtained in subsequent experiments, and plasma from hatchlings at 3 weeks of age were also assayed for corticosteroid and estradiol. Embryos were staged according to Ferguson (’85). Plasma Radioimmunoassays (RIAs) were performed on all samples to measure progesterone and corticosterone. Extraction efficiencies for corticosterone and progesterone were estimated using recovery of 10,000 cpm of tritiated steroid added to a series of plasma samples (n = 10) and allowed to equilibrate overnight. Recovery for corticosterone was 91 ± 0.7%, and for progesterone, 89 ± 1.1%. Tritium-labeled steroids for progesterone and corticosterone assays were purchased from Dupont NEN Products, Boston, MA. The assay for corticosterone was similar to that described in Lance and Lauren ’84 with some modifications. Antibody specific for corticosterone (cross reactivity was 6.1% with desoxycorticosterone and < 0.5% with all other steroids tested) was purchased from ICN Biomedicals, Inc. (Costa Mesa, CA). For both corticosterone and progesterone assays, 20 to 50 ãl of plasma was extracted with 2 ml of ethyl ac-

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etate: n-hexane (3:2 v/v), the aqueous layer separated from the organic by snap freezing in a methanol dry ice slurry, and the organic phase dried under a stream of nitrogen at 38°C. The dried extract was reconstituted in 0.5 ml of 0.1 M PBS buffer (pH 7.0), 100 ãl of antibody at the working dilution and 100 ãl of radioactive corticosterone or progesterone in buffer added. The mixture was vortexed briefly, then held at 4°C overnight. Dextran charcoal was used to separate bound from free steroid, and radioactivity was estimated in a liquid scintillation counter. Progesterone antibody (cross reactivity: 13% with 5Ä-pregnan 3,20-dione, 5.2% with 5~-pregnan 3,20-dione, and less than 2% with all other steroids tested) was obtained from Dr. S.S.C. Yen (University of California, San Diego). Estradiol-17Ä was measured using a modification the 125I kit (Diagnostics Products Corp., Los Angeles, CA). The modifications included using extracted plasma reconstituted in PBS buffer, pH 7.0, using only 50 ãl of primary antibody instead of 100 ãl, and using dextran-charcoal instead of the second antibody to separate bound from free steroid. With this assay, a sensitivity of less than 1 pg/ assay tube was achieved. Plasma samples (50 or 100 ãl) were extracted with 2 ml of ethyl acetate: n-hexane (3:2), the extract dried under a stream of nitrogen at 37°C, and reconstituted in assay buffer. Pure crystalline corticosterone, progesterone, and estradiol for standards were obtained from Steraloids, Inc., NH. Urogenital tissue The entire urogenital tract including mesonephros, adrenal, and gonad was dissected from the embryo, plunged into liquid nitrogen, and stored at –20°C until assayed. Each tissue sample was then put into a 12 × 75 mm disposable glass tube with 1 ml PBS gel buffer (pH 7.0) and homogenized using a Branson sonifier cell disrupter, centrifuged to remove cell debris, and the supernatant retained for assay. For each assay, 100 ãl of the urogenital homogenate supernatant was added to 400 ãl PBS buffer and analyzed by radioimmunoassay. Statistics Results were analyzed using the Statview factorial ANOVA program with P(~ = 0.05) on an Apple Macintosh computer. RESULTS Duration of incubation ranged from 61 to 62 days for eggs incubated at 33°C (male-producing

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temperature) and from 73 to 75 days for eggs incubated at 30°C (female-producing temperature). Plasma A. Progesterone

Assay sensitivity was 3 pg. Intra-assay variation was 10%, and interassay variation was 14.7%. Plasma progesterone in male embryos ranged from less than 25 pg/ml to 3 ng/ml. Most values fell below 2 ng/ml. The highest values were found in early stages (21.5–22.5) but by stage 25 were less than 1 ng/ml. Progesterone values in the female were similar and ranged from less than 25 pg/ml to a single value of more than 8 ng/ml. Values were significantly higher from stages 20 to 23.5 than at later stages of incubation (P < 0.005), but there was considerable variation among samples. During the latter half of incubation, values dropped to less than 1 ng/ml in both sexes (Fig. 1). B. Corticosterone

Assay sensitivity was 15 pg. Intra-assay variation was 6.4%, and the inter-assay variation was 15%. Plasma corticosteroid levels of male embryos ranged from less than 50 pg/ml to more than 18 ng/ml (Fig. 2). Values were low throughout early development then gradually rose to significantly higher values (P < 0.001) by stage 25.5. Corticosterone levels continued to increase until hatching.

Plasma corticosterone levels of females ranged from less than 50 pg/ml to more than 29 ng/ml (Fig. 2). Values were low throughout early development until stage 24.5. After this point, the values were much higher and continued to increase through hatching. Corticosterone levels were significantly higher (P < 0.001) from stage 25 to hatching as compared to earlier values and were significantly higher than male values at the same stage. Three weeks after hatching, plasma corticosterone levels declined dramatically in both sexes, and sex-specific differences had disappeared. Mean plasma corticosterone in female hatchlings was 8.64 ± 1.49 ng/ml, n = 22; and mean plasma corticosterone in male hatchlings was 7.59 ± 1.51 ng/ ml, n = 33. Plasma estradiol was absent or below the sensitivity of the assay in male hatchlings, but, in female hatchlings, mean plasma estradiol was 19.15 ± 1.76 pg/ml, n = 24. Urogenital tissue A. Progesterone

Male tissue progesterone levels (Fig. 3) were low at the beginning of development and increased significantly (P < 0.001) from stage 24.5 until hatching. Progesterone levels in the female tissue were extremely variable. Levels were significantly higher (P < 0.002) from stage 22 through hatching as compared to earlier values (Fig. 3).

Fig. 1. Plasma progesterone in male alligator embryos (solid triangles) and female (open squares) alligator embryos during the latter half of incubation.

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Fig. 2. Plasma corticosterone in male alligator embryos (solid triangles) and female (open squares) alligator embryos during the latter half of incubation. Points represent means and standard errors of the mean of at least 10 samples per stage.

B. Corticosterone

Tissue corticosterone levels in males (Fig. 4) were very low early in development but began increasing from stage 22.5. From stage 24.5, steroid levels were significantly higher (P < 0.001) than earlier values. This pattern is comparable to plasma values for corticosterone, which also began increasing at this stage. Female tissue corticosterone levels were low early in the development but increased to significantly higher levels (P < 0.002) after stage 24 and remained high until hatching (Fig. 4). This pattern also corresponds to what was found in the plasma of female embryos. DISCUSSION The Crocodilia are more closely related to birds than they are to the other reptilian groups (Gauthier et al., ’88), and many aspects of their physiology are similar. It is not surprising therefore that plasma corticosterone concentrations in alligator embryos were in the same range as reported for chicken and turkey embryos. The major difference between the alligator and avian embryos was in the pattern of progesterone secretion. In chick embryos, plasma progesterone increases throughout the second half of incubation (Kalliecharan and Hall, ’74), whereas in alligator embryos, plasma progesterone remains low. Our initial analyses were unable to detect any sex differences in plasma estradiol (Lance and

Bogart, ’94) or plasma testosterone (Medler, ’92) in alligator embryos. The fact that alligator yolk contains very high levels of estradiol, androstenedione, and testosterone prior to the sex-determining period (Conley et al., ’97) forced us to discard samples contaminated with yolk. However, it is still not known if alligator yolk contains high levels of progesterone and corticosterone. In female alligator embryos, corticosterone was slightly higher than in males at stage 25. This difference continued during the remainder of incubation such that female values were approximately twice as high as males at hatch, similar to what has been reported in turkey embryos (Wentworth and Hussein, ’85). By 3 weeks after hatching these differences had disappeared. These sex-specific differences are similar to what has been seen in turkey embryos, but curiously not in chick embryos, which showed no sex differences (Wentworth and Hussein, ’85; Tanabe et al., ’86; Carsia et al., ’87). The pattern of sex differences in plasma corticosterone in Trachemys scripta embryos is different from that found in the alligator. In T. scripta, plasma corticosterone was higher in male embryos at an earlier stage than in female embryos, but by stage 21, when sex differentiation is complete, plasma corticosterone was higher in females than in males (White and Thomas, ’92), similar to what is seen in late-stage alligator embryos. In alligator embryos incubated at male-produc-

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Fig. 3. Progesterone levels in urogenital tissue of male (solid triangles) and female (open squares) alligator embryos during the latter half of incubation. Each point represents an individual observation.

ing temperatures, plasma progesterone levels were low throughout development. In alligator embryos at female-producing temperatures, plasma progesterone levels during the temperaturesensitive (stages 20–24) were higher than later in development, but still much lower than what has been reported for the chick embryo (Scanes et al., ’87). Desvages and Pieau (’91) showed that embryonic turtle (Emys orbicularis) gonads could metabolize progesterone to testosterone and estrogens during the sex-determining period. Low progesterone levels in the plasma of male embryos may indicate low hormonal production or a very rapid turnover of progesterone. In the urogenital tissue of both male and female alligator embryos, progesterone levels began to increase during the last third of development and remained high until hatching. This rise of progesterone in the tissue coincides with an increase of corticosterone found in the plasma and tissue. However, tissue progesterone levels were considerably lower than tissue corticosterone levels (Figs. 3, 4). In chick embryos, progesterone is converted by the adrenals into at least 12 metabolites. The main glucocorticoid produced is corticosterone (Marie, ’81; Gonzalez et al., ’83). Unlike alligator embryos in which plasma progesterone remained virtually undetectable during the latter third of incubation, chick embryos showed an

increase in plasma progesterone and plasma corticosterone during the latter half of incubation (Kalliecharan and Hall, ’74). Plasma progesterone in the chick embryo plasma was in the same concentration range or even higher than the corticosterone (Kalliecharan and Hall, ’74). Progesterone was found to be significantly higher in embryonic chick testes than in embryonic chick ovary from day 8 to day 12 of incubation, but no sex differences were seen in embryonic chick adrenal tissue. Plasma progesterone levels were low in both sexes through the middle of incubation but rose rapidly at hatching (Tanabe et al., ’86). Another study of chick embryos found a steady increase in adrenal progesterone and corticosterone during development, but a sudden sharp decrease in adrenal steroids on day 19, then a further increase on day 21 (Kalliecharan and Hall, ’76). The authors suggest that the sudden decrease on day 19 is due to maturation of the feedback system in the hypothalamus and the pituitary. No such change could be detected in the alligator tissues. In the alligator embryo, it was not possible to separate gonadal tissue from adrenal and mesonephros, but results are similar to those found in the chick adrenal tissue by Tanabe et al. (’86). Sex differences in embryonic alligator adrenal/gonadal tissue were not evident.

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Fig. 4. Corticosterone levels in urogenital tissue of male (solid triangles) and female (open squares) alligator embryos during the latter half of incubation. Each point represents an individual observation.

Corticosterone levels increased in the plasma and urogenital tissue of both male and female alligator embryos beginning at the last third of incubation (approx. stage 25) and continued to rise until hatching. This correlates with the trends seen in chick and turkey embryos. The role of corticosterone in embryonic birds is unknown, but it is believed to play a role similar to that of cortisol in mammalian embryos, in helping to prepare the embryo for the transition to life in air. Cortisol has been shown to be essential for lung maturation in mammalian fetuses (Challis and Brooks, ’89). In chicks, corticosterone has been shown to influence the growth, hydration, and lipid content of the embryonic chick lung, and proliferation of some or all types of pulmonary cells, and the biochemical maturation of the surfactant system (Hylka and Doneen, ’83). In a study of turkey embryos that were treated with a physiological dose of corticosterone, a significant increase in hatchability was found as well as a significant decrease in incubation period (Wentworth and Hussein, ’85). It has been shown in chicks that adrenal glucocorticoid secretion is controlled at three different levels: the adrenal itself, the pituitary, and the hypothalamus. The adrenal secretes glucocorticoids autonomously and maintains basal corti-

costeroid levels throughout development. The establishment of the pituitary control over the adrenal cortex occurs on day 14 of incubation (Hall, ’70), which coincides with increased mitotic activity in the adrenal cortex and higher plasma corticosteroid concentrations (Kalliecharan and Hall, ’74). Pituitary secretion of ACTH is responsible for the gradual increase in hormone levels during development, while the hypothalamus controls hormone secretion in response to stress (Wise and Frye, ’73; Woods and Thommes, ’84). Although the pituitary of the alligator is well developed by the latter third of incubation (Lance, unpubl.), there is no information available on pituitary function in the alligator embryo. Corticosterone concentrations increased in the plasma and urogenital tissue of both male and female alligator embryos during the last third of incubation (from about stage 25) and continued to increase until hatch. This pattern of corticosteroid secretion in alligator embryos is similar to that of chick and turkey embryos, and the range of values for alligators (approximately 10–20 ng/ ml) is also similar to those of birds (Wise and Frye, ’73; Kalliecharan and Hall, ’74; Scott et al., ’81; Wentworth and Hussein, ’85; Scanes et al., ’87). The reasons for these sex differences in birds and alligators remain unknown.

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ACKNOWLEDGMENTS Thanks to Dr. Ruth Elsey, Ted Joanen, and Larry McNease of the Louisiana Department of Wildlife and Fisheries, Rockefeller Wildlife Refuge, for their help in the collection of alligator eggs. Appreciation is also extended to Dr. Harriet Austin, University of Colorado, for donating samples collected in her laboratory. We would like to also thank Dr. Al Fivizzani, University of North Dakota, for advice on the modification of the estradiol assay. LITERATURE CITED Carsia, R.V., M.E. Morin, H.D. Rosen, and H. Weber (1987) Ontogenic corticosteroidogenesis of the domestic fowl: response of isolated adrenocortical cells. Proc. Soc. Exp. Biol. Med. 184:435–445. Challis, J.R.G., and A.N. Brooks (1989) Maturation and activation of hypothalamic-pituitary-adrenal function in fetal sheep. Endocrine Rev., 10:182–204. Conley, A.J., P. Elf, J.C. Corbin, S. Dubowsky, A. Fivizzani, and J.W. Lang (1997) Yolk steroids decline during sexual differentiation in the alligator. Gen. Comp. Endocrinol. 107:191–200. Desvages, G., and C. Pieau (1991) Steroid metabolism in gonads of turtle embryos as a function of the incubation temperature of eggs. J. Steroid Biochem. Mol. Biol. 39:203–213. Ferguson, M.W.J. (1985) The reproductive biology and embryology of crocodilians. In: Biology of the Reptilia. C. Gans, F. Billett, and F.A. Maderson, eds. Academic Press, London, Vol. 14, pp. 329–492. Gauthier, J., A.G. Kluge, and T. Rowe (1988) Amniote phylogeny and the importance of fossils. Cladistics 4:105–205. Gonzalez, C.B., E.N. Cozza, M.E.O. De Bedners, C.P. Lantos, and A. Aragones (1983) Progesterone and its reductive metabolism in steroidogenic tissues of the developing hen embryo. Gen. Comp. Endocrinol. 51:384–393. Hall, B.K. (1970) Response of the host embryonic chicks to grafts of additional adrenal gland. Can. J. Zool. 49:381–384. Hylka, V.W., and B.A. Doneen (1983) Ontogeny of embryonic chicken lung: effects of pituitary gland corticosterone, and other hormones upon pulmonary growth and synthesis of surfactant phospholipids. Gen. Comp. Endocrinol. 52:108–120. Kalliecharan, R., and B.K. Hall (1974) A developmental study of the levels of progesterone, corticosterone, cortisol, and cortisone circulating in plasma of chick embryos. Gen. Comp. Endocrinol. 24:364–372.

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