BOLOGY OF REPRODUCTON 20, 905-909 (1979) Testosterone and Sex Related Physical Characteristics during the Maturation of the Male Japanese Quail (Coturnix coturnix japonica) MARY ANN OTTNGER1 and HOWARD J. BRNKLEY Department of Zoology, University of Maryland, College Park, Maryland 20742 ABSTRACT A series of experiments was conducted to examine hormonal and physical aspects of reproduction in the maturing and adult male Japanese Quail. Testicular secretion of testosterone was demonstrated in the adult male by the elevated testosterone concentration in the testicular vein (12 ng/ml serum) as compared to the testosterone concentration in the left ventricle (4.5 ng/ml serum). Testosterone concentrations in the peripheral circulation were found to vary diurnally with high concentrations in the morning and evening and lowered concentrations in the midafternoon. n the maturing male, testosterone concentrations, testicular weight and cloacal gland area remained low until Day 21 and rose rapidly thereafter. Spermatozoa were first detected in the testes on Day 26. By 30 days of age, spermatozoa were found in the testes and in the vasa deferentia in small quantities and by Day 35 there were large concentrations at both sites. There was a significant positive temporal correlation of changes in cloacal gland area, testes weight and circulating concentrations of testosterone. NTRODUCTON Testicular androgen is essential for the stimulation and maintenance of sex related morphology and behavior in the male Japanese Quail (Nagra et al., 1959; Beach and nman, 1965; Sachs, 1967; Adkins and Nock, 1976). Sexual behavior and sex related characters can be stimulated in castrates by administration of testosterone (Sachs, 1969; Adkins, 1977). Testicular stimulation that occurs with photoperiodic stimulation is associated with increased concentrations of peripheral LH, FSH and testosterone (Gibson et al., 1975; Follett, 1976; Follett and Davies, 1977). Furthermore, testosterone implantation in intact males reduces serum LH and FSH and in high doses maintains spermatogenesis (Brown and Follett, 1977, Desjardins and Turek, 1977). These experiments indicate that testosterone is a male gonadal hormone. However, they do not provide evidence of in vivo testosterone secretion by the testes. Serum testosterone concentration in the adult male quail is approximately 5.0 ng/ml plasma (Desjardins and Turek, 1977). During maturation, testicular development follows a period of rapid growth which occurs between 2 and 5 weeks of age (Yamoto, 1964; Mather and Wilson, 1964). Spermatogenesis was associated with a testicular weight of 1000 mg or an age of 36 days. ncreasing androgen concentrations may be expected with increasing testicular weight during maturation. A rise in serum testosterone from 0.59 ng/ml serum to approximately 3.0 ng/ml serum occurs with photoperiodic stimulation of somatically mature males (Follett, 1976). Reports for other avian and mammalian species also indicate that testosterone concentrations rise during maturation (Furr and Thomas, 1970; Schrocksnadel et al., 1971; August et al., 1972; Davidson, 1974). The objectives of these experiments were 1) to establish whether testosterone is secreted by quail testes in vivo and is therefore the appropriate hormone to monitor, 2) to investigate patterns of hormonal change during somatic maturation and associated changes in sex related traits and 3) to determine whether daily fluctuations occur in serum concentrations of the hormone in the adult. MATERALS AND METHODS Accepted November 8, 1978. Received Present May 31, address; 1978. Dept. Poultry Science, University of Maryland, College Park, MD 20742. Experimental Animals The original stock of Coturnix coturnix japonica was obtained from eggs provided by Dr. M. Schein, West Virginia University. Animals were maintained in 905
906 OTTNGER AND BRNKLEY a colony room with controlled temperature and lighting (16 h of light and 8 h of dark). Water and feed (Southern States Poultry Feed) were available ad libitum and the same caretakers fed and watered the animals daily. Experimental males were housed individually to avoid effects due to aggression betweeen males. Eggs were incubated in an automatic incubator (Marsh Rol-X, Marsh Farms, Garden Grove, CA). After hatching, which normally occurred on Day 18 of incubation, chicks were weighed, banded and transferred to a heated cage. Water and crushed grain were provided and the chicks were handled daily by the experimenter in order to accustom the animals to handling. After Day 14, animals were transferred to cages on the rack and housed either in breeding groups or individually in the case of experimental males. Measurement of Physical Characteristics Cloacal gland area. The cloacal gland area was calculated by a method similar to that of Sachs (1969). The area of the cloac was kept plucked to facilitate precise measurement. A mm ruler was used to measure the length and width of the surface of the cloaca and the product of these values was used as an estimate of the surface area of the gland. n these experiments, the length of the cloaca was used in the calculation rather than the distance from the vent to the edge of the cloaca used by Sachs (1969) in order to increase the sensitivity to changes in area. Testes wet weight. mmediately following sacrifice of the animal, the testes were removed from the body, cleared of surrounding membranes and weighed on a Mettler Analytic Balance (H54). Analysis of Testosterone Concentrations Sampling technique. With the exception of the first experiment, all animals were sacrificed by decapitation and the blood was collected in glass tubes. n the latter case, measurements were taken of body weight, in addition to testes weight and cloacal gland area. Validation of testosterone radioimmunoassay (RA). Testosterone concentrations were measured by a double antibody RA (l 25ll-testosterone and antibodies supplied by Micromedic Diagnostics, Horsham, PA). Accuracy of the assay was determined by evaluation of known quantities of testosterone in low testosterone quail serum. These estimated concentrations did not differ from the known concentrations (P>0.O1). Precision was calculated from 6 serum pools (coefficient of variability of within assay variation equaled 8.6%). The antiserum was somewhat cross reactive with dihydrotestosterone (30%) and to a negligible extent with other steroids. Finally, parallelism of serial dilutions of quail serum with the standard curve (P>0.O1) ensured no interference from other components of quail serum. Experiments and Specific Methods for Sexually Mature Male Male gonadal hormone. To determine whether testosterone is secreted by quail testes in vivo, a comparison was made of the testosterone concentrations in the effluent blood of the testes and in the general circulation. Twelve reproductively active adult males between 70 and 90 days of age were anesthetized (0.5 ml Equi-Thesin, Park Davis) and laparotomized between 0900 h and 1400 h EST. The left testis was exposed and the testicular vein, which is readily accessible on the surface of the testis, was clamped at its junction with the posterior vena cava to prevent dilution of the testicular venous blood with backflow from the general circulation. At least 0.5 ml of blood was collected directly from the vein. The second sample was taken by cardiac puncture of the left ventricle. Peripheral serum concentrations of testosterone were also measured in 14 adult males (between 60 and 90 days of age) that were nonreproductive due to prolonged exposure to a short photoperiod (6 h L;18 h D). Animals were tested for mating and for cloacal gland foam periodically to confirm the cessation of reproductive activity. Measurements were taken of testes wet weight and cloacal gland area. Diurnal rhythm in peripheral hormone concentration. A total of 28 adult males between 75 and 90 days of age were randomly assigned to 1 of 7 sampling times from 0800 h to 2300 h EST. Blood samples were taken over a period of 3 consecutive days. Testes wet weight and cloacal gland area were measured. The data were analyzed by one-way ANOVA and Student- Newman-Keuls test for significant differences among means. Testosterone, spermatogenesis and sex related morphology during maturation. A total of 75 animals taken from 5 hatches were sampled at 8 day intervals beginning with Day 1 and ending with Day 57. On TABLE 1. Testosterone concentration in testicular effluent and in serum from general circulation of same individuals. No. Source of serum Mean concentration of testosteronea (ng/ml serum ± SEM) 12 Testicular vein 12.23 ± 2.17 12 Left ventricle of heart 4.48 ± 1.35 General circulation, animals exposed to photo- 14 period of 6 h L:18 h D 0.07 ± 0.04 5All comparisons of means were significantly different (P<O.05).
TESTOSTERONE AND SEX RELATED PHYSCAL CHARACTERSTCS 907 20 0 OHOC 1. J T 4.48 ± 1.35 ng/ml serum, respectively (Table 1). The results of the present experiment 400 2000 0200 ventricular serum, 12.23 ± 2.17 ng/ml serum vs have shown that in addition to superior biological potency (Adkins, 1977) and testicular synthesis in vitro (Nakamura and Tanabe, 1972; Maung and Follett, 1976), testosterone is also secreted by the testes in vivo. This demonstration of the secretion of testosterone by the normal, intact male has provided direct evidence that testosterone is a male gonadal steroid in HOUR FG. 1. Fluctuations in the circulating concentra- Japanese Quail. tions of testosterone from 0800 h-2300 h. N = 28. Fourteen adult males maintained on a short photoperiod displayed no sexual behavior or cloacal gland function; testicular weight fell to 63.3 ± 37.0 mg and the mean circulating each sampling day, animals were sacrificed by decapi- concentration of testosterone was 0.07 ± 0.04 tation between 1100 h and 1300 h EST. The number of animals sampled varied with age due to the minimum ng/ml serum. This concentration was significantrequirement of 0.5 ml serum sample for each deter- ly lower (P<0.01) than the concentration of mination by RA. There were 5 replicates of this testosterone in either set of samples from the experiment with all animals in a replicate taken from a reproductively active individuals. single hatch. Measurements were taken of cloacal Testosterone concentrations found in animals gland area and testicular wet weight. n a separate experiment, 21 animals were randomly maintained on shortened photoperiod may be assigned to of 9 sampling days, between 20 and 36 considered minimal since animals in this state days of age. At the time of RA, serum was pooled had repressed testes and accessory sex structures and analyzed in one of the following categories: 1) and showed a complete lack of sexual behavior. animals with no spermatogenesis (approximately Day t is also possible that the serum testosterone 20) 2) animals with spermatozoa only in the testes (approximately Day 26), 3) animals with spermatozoa detected in nonreproductive males may be in the testes and in small quantities in the vasa defer- secreted from a nontesticular source such as the entia (approximately Day 30) and 4) animals with adrenal glands (Baird et al., 1969). large quantities of spermatozoa in the testes and in the Diurnal rhythm in testosterone concentravasa deferentia (approximately Day 35). These 4 pools tion. A definite pattern in hormonal concentrawere then assayed for testosterone. Smears taken from the vasa deferentia and from the testes were stained tions occurred within the interval of time (Berg s method, 1968) and the quantity of spermatozoa examined (Fig. 1). Mean concentrations of was categorized as previously mentioned, testosterone fell from 15.9 ± 2.5 ng/ml serum RESULTS AND DSCUSSON in the early morning to 8.9 ± 1.8 ng/ml serum at 1700 h. By 2000 h, concentrations had risen to 15.9 ± 5.9 ng/ml serum. There were significant differences (P<0.05) among the higher Sexually Mature Male (greater than 13 ng) and the lower (less than 11 Male gonadal steroid. Adult males had ng) concentrations (Table 2). significantly higher concentrations of testoster- These peripheral data show a daily rhythm one in the testicular venous serum than in the in the circulating concentrations of testosterone. TABLE 2. Student-Newman-Keuls test for significant differences among peripheral concentrations of testosterone between 0800 h and 2300 h, EST. Time of day (h) 0800 1100 1300 1500 1700 2100 2300 Mean testosterone concentrations 16.0O 14.ooa,c 1309c 1140b,c 902b,c ls.88a 16.8o (ng/m serum) a,b,cmens with the same superscript do not differ significantly. All other means differ significantly from each other (P<0.05).
908 OTTNGER AND BRNKLEY area in a similar manner and became enlarged DAY POSTHATCH FG. 2. Changes in testosterone, testes weight and cloacal gland area during the maturation of the male. Since no samples were taken from 2300 h to 0800 h, no conclusions can be made about a 24 h cycle in testosterone concentrations. However, these results indicate that testosterone concentrations are lowest in the early afternoon and highest in the early morning and evening. These data are consistent with reports for other species, including the domestic cockerel, man and the rhesus monkey (Resko and Rik- Ness, 1966; Rose et al., 1972; Goodman et al., 1974; Schanbacher et al., 1974). n all of these species, the testosterone concentrations were elevated during the nighttime or very early morning hours. Testosterone, Sex Related Morphology and Spermatogenesis during Maturation Testosterone concentrations, testicular weight and cloacal gland area all remained low in young animals and then rapidly increased (Fig. 2). Testosterone concentrations declined from 2.11 ± 0.30 ng/ml serum after Day 1, remained consistently low and then rose rapidly after Day 20 to a peak at Day 41. Testicular weight, which was low at hatching, increased rapidly after Day 25. The cloacal gland also changed its 2 and functional after Day 25. Linear correlation of these variables in time indicated a highly significant positive correlation of all combinations (Table 3). Spermatozoa were detected first on Day 26 for testicular samples and on Day 30 for samples from the vasa deferentia. By Day 35, there were large concentrations of sperm present in the vasa deferentia and in the testes. Testosterone concentrations in samples from these animals increased dramatically from 0.18 ± 0.10 ng/ml serum on Day 20 and 0.50 ± 0.16 ng/ml serum on Day 26 to 2.10 ± 0.60 ng/ml serum on Day 30. By Day 35, testosterone concentrations were approximately 9.00 ± 1.30 ng/ml serum. Between the time of the onset of spermatogenesis and full spermatogenesis, the cloacal gland area doubled, from 64 ± 5 mm2 to 170 ± 10 mm2. The mean testes weight was 256 ± 20 mg on Day 30 and 1202 ± 136 mg2 on Day 35. The components of the reproductive system developed in a sequence from the increase in testicular weight to the increase in testosterone concentrations. The concentration of testosterone attained at Day 41 may represent the achievement of maximal secretion of testosterone by the testes. The sequence of events during maturation resembles that observed by Follett (1976) with photically induced sexual maturation in somatically adult animals. The approximate age and testicular weight associated with full spermatogenic activity agrees with the observations of Mather and Wilson (1964). Testosterone concentrations rose dramatically between 26 and 35 days of age and, during this time, spermatogenic activity increased. Since spermatogenesis and the maintenance of testicular structure depend on testosterone (for review, see Hansson et al., 1976; Brown and Follett, 1977), these data may actually reflect a TABLE 3. Linear correlation of physical variables during maturation. Variables Correlation (r)a Testosterone concentration (ng/ml serum) vs cloacal gland area (mm2) 0.92 Testosterone concentration (ng/ml serum) vs testes weight (mg) 0.95 Cloacal gland area (mm2) vs testes weight (mg) 0.99 aall correlations significant (P<0.01).
TESTOSTERONE AND SEX RELATED PHYSCAL CHARACTERSTCS 909 dose dependent relationship between testosterone concentrations and spermatogenic activity. n summary, the documentation of testosterone secretion in vivo by testes of the male Japanese Quail has provided further evidence that testosterone is the male gonadal steroid. A diurnal rhythm in peripheral concentrations of testosterone was detected with elevated concentrations in the morning and evening and lowered concentrations at midday. Finally, these experiments have provided documentation of testostemne concentrations during maturation and evidence of a close relationship of androgen dependent cloacal gland function, testes weight and spermatogenic activity with serum concentration of testosterone. ACKNOWLEDGMENTS The authors would like to thank Dr. H. Opel for his assistance in the development of the surgical technique for the collection of blood samples from the testicular vein. The authors also thank D. Hoover for the use of the computer program for analysis of the RA data. Computer time was provided by Computer Science Center, University of Maryland. REFERENCES Adkins, E. K. (1977). Effects of diverse androgens on sexual behavior and morphology of castrated male quail. Horm. Behav. 8, 201-207. Adkins, E. K. and Nock, B. L. (1976). Behavioral responses to sex steroids of gonadectomized and sexually regressed quail. J. Endocrinol. 68, 49-5 5. August, C. P., Grumback, M. M. and Kaplan, S. L. (1972). Hormonal changes in puberty:. Correlation of plasma testosterone, LH, FSH, testicular size, and bone age with male pubertal development. J. Clin. Endocr. Metab. 34, 319-326. Baird, D. T., Uno, A. and Melby, J. C. (1969). Adrenal secretions of androgens and estrogens. J. Endocrinol. 45, 135-136. Beach, F. A. and nman, N. G. (1965). Effects of castration and androgen replacement on mating in male quail. Proc. Nat. Acad. Sci. 54, 1426-1431. Berg, L. (1968). Manual of Histologic Staining Methods of the Armed Forces nstitute of Pathology. (L. G. Luna, ed.). McGraw-Hill Book Co., New York. pp. 117-118. Brown, N. L. and Follett, B. K. (1977). Effects of androgens on the testes of intact and hypophysectomized Japanese Quail. Gen. Comp. Endocrinol. 33, 267-277. Davidson, J. M. (1974). Hypothalamic-pituitary regulation of puberty, evidence from animal experiments. n: Control of the Onset of Puberty. (M. M. Grumback, G. D. Grave, F. E. Meyer, eds.). John Wiley and Sons, London. pp. 79-102. Desjardins, C. and Turek, F. W. (1977). Effects of testosterone on spermatogenesis and luteinizing hormone release in Japanese Quail. Gen. Comp. Endocrinol. 33, 293-303. Follett, B. K. (1976). Plasma follicle stimulating hormone during photoperiodically induced sexual maturation in male Japanese Quail. J. Endocrinol. 69, 117-126. Follett, B. K. and Davies, D. T. (1977). The photoperiodic induction of gonadotropin release in quail: events during the first long day and their blockade by sodium pentobarbitone. J. Endocrinol. 72, 15. Furr, B. J. and Thomas, B. S. (1970). Estimation of testosterone in the plasma of the domestic fowl. J. Endocrinol. 48, xiii Abstr. Gibson, W. R., Follett, B. K. and Gledhill, B. (1975). Plasma levels of luteinizing hormone in gonadectomized Japanese Quail exposed to short or to long daylengths. J. Endocrinol. 64, 87-101. Goodman, R. L., Hotchkiss, J., Karch, F. J. and Knobil, E. (1974). Diurnal variations in serum testosterone concentration in the adult Rhesus monkey. Biol. Reprod. 11,624-630. Hansson, V., Calandra, R., Purvis, K., Ritzen, M. and French, F. (1976). Hormonal regulation of spermatogoenesis. Vita. Horm. 34, 187-214. Mather, F. B. and Wilson, W. 0. (1964). Postnatal testicular development in Japanese Quail. Poult. Sci. 43, 860-864. Maung, Z. W. and Follett, B. K. (1976). Effect of luteinizing hormone on testosterone release and cyclic AMP production from isolate interstitial cells of the quail testes. J. Endocrinol. 71, 98-99. Nagra, C. L., Meyer, R. K. and Bilstad, N. (1957). Cloacal glands in the Japanese Quail (Ccj): histogenesis and response to sex steroids. Anat. Record. 133,415. Nakamura, T. and Tanabe, V. (1972). Pathways for androgen synthesis in vitro by the testes of Japanese Quail (Ccj).J. Endocrinol. 55, 499-506. Resko, J. A. and Eik-Ness, K. B. (1966). Diurnal testosterone levels in peripheral plasma of human male subjects. J. Clin. Endocr. Metab. 26, 573-576. Rose, R. M., Gordon, R. P. and Bernstein,. S. (1972). Plasma testosterone in the male Rhesus: influence of sexual and social stimuli. Science 178, 643-644. Sachs, B. D. (1967). Photoperiodic control of the cloacal gland of the Japanese Quail. Science 157, 201-203. Sachs, B. D. (1969). Photoperiodic control of reproductive behavior and physiology of the male Japanese Quail (Ccj). Horm. Behav. 1(1), 7-24. Schanbacher, B. D., Gomes, W. R. and Van Demark, N. L. (1974). Dirunal rhythm in serum testosterone levels and thymidine uptake by testes in the domestic fowl. J. An. Sci. 38(6), 1245-1248. Schrocksnadel, H., Bator, A. and Frick, J. (1971). Plasma testosterone in cocks and hens. Steroids 18, 359-365. Vamoto, S. (1964). Morphological studies on the sexual maturation in the male Japanese Quail (Ccj).. Effect of continuous lighting and constant temperature on testicular growth. Tohoki. J. Agric. Res. 15, 241-257.