GENERAL
AND
COMPARATIVE
27, 543-547 (1975)
ENDOCRINOLOGY
Plasma Testosterone
Levels in the Chick Embryo
JAMES E. WOODS, ROBERT M. SIMPSON, AND PATRICIA L. MOORE Department
of Biological Chicago,
Sciences, De Paul Illinois 60614
University,
Accepted August 8, 1975 The time when testosterone is first present in the blood of the chick embryo, as well as its plasma concentration throughout the embryonic period, was determined by means of immunofluorescence and radioimmunoassay. Testosterone is first present, in approximately equal amounts, in the blood of embryos of both sexes on day 5.5 of incubation. However, beginning on day 7.5, and continuing through day 17.5, there is a statistically significant difference between males and females in plasma testosterone concentrations. In both sexes a correlation was noted between the embryonic intervals of increasing plasma testosterone concentrations and that of Miillerian duct regression. Thus, the findings of this investigation are consonant with the view that androgens are the hormonal agents responsible for the regression of both Miillerian ducts in male and the right Miillerian duct in female embryos.
The hormonal regulation of Miillerian duct differentiation in the chick embryo is not fully understood. The results of numerous investigations indicate that testosterone or an allied androgen produced by the gonads may be involved in regression of both Miillerian ducts in male and the right Miillerian duct in female embryos (see review of literature in Romanoff, 1960; Burns, 1961; Hamilton and Teng, 1965; Stoll and Maraud, 1974; Price et al., 1975). If male sex hormones are the hormonal agents that regulate Miillerian duct regression in the chick, then their blood levels during embryonic development should reflect this role. Thus, it was the purpose of the present investigation to measure plasma testosterone levels of male and female chick embryos in order to determine if a correlation could be observed between testosterone concentration profiles and the time span of Miillerian duct regression. MATERIALS
the embryos at the time of blood drawing represents the time the eggs remained in the incubator. The presumptive sex of 3.5-5.5-day-old chick embryos was determined by microscopic examination of extraembryonic and amniotic epithelial cells for sex chromatin (Woods and Podczaski, 1974). Embryos of 6.5-17.5 days of incubation were sexed by the in situ microscopic examination of the gonads. Testosterone in the blood of very young embryos (days 3.5-6.5) was determined by an immunofluorescence assay while plasma testosterone levels of older embryos (days 7.5-17.5) were quantitated by means of radioimmunoassay. Immunofluorescence was used for the assay of blood testosterone levels of early embryos since in most cases the amount of this steroid hormone in the blood was below the sensitivity of the radioimmunoassay method. Also, it was difficult to obtain adequate blood aliquots from these embryos for use in radioimmunoassay. Blood was obtained at 24-hr intervals on days 3.5, 4.5, 5.5, and 6.5 of incubation and at 48-hr intervals on alternate days, beginning on day 7.5 and continuing until day 17.5. It was drawn from extraembryonic blood vessels by means of a heparinized l.O-cc Luer-Lok syringe fitted with a number 27 hypodermic needle. Samples for the immunofluorescence assay were used immediately, while those for radioimmunoassay were collected in heparinized capillary tubes, centrifuged, and the plasma stored at -20” until analyzed.
AND METHODS
All blood samples were obtained from white Leghorn eggs incubated at a temperature of 38 f 1.1” in a “Jamesway” forced-draft incubator. The age of 543 CopyriBht All rights
@ 1975 by Academic Press, Inc. of reproduction in any form reserved.
WOODS.
544 Testosterone
SIMPSON
Antiserum
The IgG fraction of a rabbit testosterone-3antiserum (T-3-IgG), prepared for us by the Cappel Laboratories, Inc., Downington, Pa., was used in both the immunofluorescence assay and the radioimmunoassay . Tritiated steroid binding studies have demonstrated that testosterone-3-antisera are highly specific for testosterone and dihydrotestosterone; they exhibit a small cross-reaction with all other androgens (< lo%), and a negligible cross-reaction with nonandrogens (i 0.01%) (see Woods and Podczaski. 1974). These findings have been confirmed in the present investigation (Table 1).
Zmmunojluorescence Assay Fluorescein isothiocyanate-labeled T-3-IgG was employed in the immunofluorescence assay. Its preparation and immunological specificity have been described previously (Woods and Podczaski, 1974). A blood smear preparation was made by drawing a clean microscope slide at a 45” angle across a drop of blood (0.04 ml) on another slide and allowing the preparation to air dry for 20 min. Then a drop of the fluorescein-labeled T-3-IgG was added, spread in a thin film, and allowed to remain in contact with the blood smear for 5 min. The excess fluid was drained off, the slides rinsed in six changes of cold 0.01 M phosphate-buffered isotonic saline, pH 7.0 (PBS), for 15 min, mounted with glycerol-saline solution, and examined with a fluorescence microscope. Microscopic observations were made with a Leitz Ortholux fluorescence microscope unit which consisted of an Osram HBO-200 mercury vapor lamp, a Leitz darkfield condenser. a Zeiss 40X planaTABLE
1
PERCENTAGE OF CROSS-REACTIVITY OF TESTOSTERONE ANTISERUM WITH VARIOUS STEROIDS
Steroid Testosterone Dihydrotestosterone Androstendiol Androstendione Dehydroepiandosterone Cholesterol Corticosterone Desoxycorticosterone 17&Estradiol Estrone Hydrocortisone Pregnenolone Pregnendione
Cross-reaction (%) 100 109 7.1 5.2
AND
COMPARATIVE
27, 543-547 (1975)
ENDOCRINOLOGY
Plasma Testosterone
Levels in the Chick Embryo
JAMES E. WOODS, ROBERT M. SIMPSON, AND PATRICIA L. MOORE Department
of Biological Chicago,
Sciences, De Paul Illinois 60614
University,
Accepted August 8, 1975 The time when testosterone is first present in the blood of the chick embryo, as well as its plasma concentration throughout the embryonic period, was determined by means of immunofluorescence and radioimmunoassay. Testosterone is first present, in approximately equal amounts, in the blood of embryos of both sexes on day 5.5 of incubation. However, beginning on day 7.5, and continuing through day 17.5, there is a statistically significant difference between males and females in plasma testosterone concentrations. In both sexes a correlation was noted between the embryonic intervals of increasing plasma testosterone concentrations and that of Miillerian duct regression. Thus, the findings of this investigation are consonant with the view that androgens are the hormonal agents responsible for the regression of both Miillerian ducts in male and the right Miillerian duct in female embryos.
The hormonal regulation of Miillerian duct differentiation in the chick embryo is not fully understood. The results of numerous investigations indicate that testosterone or an allied androgen produced by the gonads may be involved in regression of both Miillerian ducts in male and the right Miillerian duct in female embryos (see review of literature in Romanoff, 1960; Burns, 1961; Hamilton and Teng, 1965; Stoll and Maraud, 1974; Price et al., 1975). If male sex hormones are the hormonal agents that regulate Miillerian duct regression in the chick, then their blood levels during embryonic development should reflect this role. Thus, it was the purpose of the present investigation to measure plasma testosterone levels of male and female chick embryos in order to determine if a correlation could be observed between testosterone concentration profiles and the time span of Miillerian duct regression. MATERIALS
the embryos at the time of blood drawing represents the time the eggs remained in the incubator. The presumptive sex of 3.5-5.5-day-old chick embryos was determined by microscopic examination of extraembryonic and amniotic epithelial cells for sex chromatin (Woods and Podczaski, 1974). Embryos of 6.5-17.5 days of incubation were sexed by the in situ microscopic examination of the gonads. Testosterone in the blood of very young embryos (days 3.5-6.5) was determined by an immunofluorescence assay while plasma testosterone levels of older embryos (days 7.5-17.5) were quantitated by means of radioimmunoassay. Immunofluorescence was used for the assay of blood testosterone levels of early embryos since in most cases the amount of this steroid hormone in the blood was below the sensitivity of the radioimmunoassay method. Also, it was difficult to obtain adequate blood aliquots from these embryos for use in radioimmunoassay. Blood was obtained at 24-hr intervals on days 3.5, 4.5, 5.5, and 6.5 of incubation and at 48-hr intervals on alternate days, beginning on day 7.5 and continuing until day 17.5. It was drawn from extraembryonic blood vessels by means of a heparinized l.O-cc Luer-Lok syringe fitted with a number 27 hypodermic needle. Samples for the immunofluorescence assay were used immediately, while those for radioimmunoassay were collected in heparinized capillary tubes, centrifuged, and the plasma stored at -20” until analyzed.
AND METHODS
All blood samples were obtained from white Leghorn eggs incubated at a temperature of 38 f 1.1” in a “Jamesway” forced-draft incubator. The age of 543 CopyriBht All rights
@ 1975 by Academic Press, Inc. of reproduction in any form reserved.
WOODS.
544 Testosterone
SIMPSON
Antiserum
The IgG fraction of a rabbit testosterone-3antiserum (T-3-IgG), prepared for us by the Cappel Laboratories, Inc., Downington, Pa., was used in both the immunofluorescence assay and the radioimmunoassay . Tritiated steroid binding studies have demonstrated that testosterone-3-antisera are highly specific for testosterone and dihydrotestosterone; they exhibit a small cross-reaction with all other androgens (< lo%), and a negligible cross-reaction with nonandrogens (i 0.01%) (see Woods and Podczaski. 1974). These findings have been confirmed in the present investigation (Table 1).
Zmmunojluorescence Assay Fluorescein isothiocyanate-labeled T-3-IgG was employed in the immunofluorescence assay. Its preparation and immunological specificity have been described previously (Woods and Podczaski, 1974). A blood smear preparation was made by drawing a clean microscope slide at a 45” angle across a drop of blood (0.04 ml) on another slide and allowing the preparation to air dry for 20 min. Then a drop of the fluorescein-labeled T-3-IgG was added, spread in a thin film, and allowed to remain in contact with the blood smear for 5 min. The excess fluid was drained off, the slides rinsed in six changes of cold 0.01 M phosphate-buffered isotonic saline, pH 7.0 (PBS), for 15 min, mounted with glycerol-saline solution, and examined with a fluorescence microscope. Microscopic observations were made with a Leitz Ortholux fluorescence microscope unit which consisted of an Osram HBO-200 mercury vapor lamp, a Leitz darkfield condenser. a Zeiss 40X planaTABLE
1
PERCENTAGE OF CROSS-REACTIVITY OF TESTOSTERONE ANTISERUM WITH VARIOUS STEROIDS
Steroid Testosterone Dihydrotestosterone Androstendiol Androstendione Dehydroepiandosterone Cholesterol Corticosterone Desoxycorticosterone 17&Estradiol Estrone Hydrocortisone Pregnenolone Pregnendione
Cross-reaction (%) 100 109 7.1 5.2
