Progesterone in male white-crowned sparrows, Zonotrichia leucophrys gambelii

GENERAL

AND

COMPARATIVE

ENDOCRINOLOGY

37,

I-5

(1979)

Progesterone in Male White-Crowned Sparrows, Zonotrichia leucophrys gambelii BRIAN

R. MCCREERY

Department

AND DONALD

of Zoology, Seattle,

University

Washington

Accepted February

S. FARNER

of Washington, 98195

13, 1978

During the course of a photoperiodically induced cycle of testicular growth and regression in Zonorrichia leucophrys gambelii the concentration of progesterone in plasma was found to range from about 400 to about 1000 pg/ml but was at no time significantly different from that of controls held on constant short days (8L 16D). There were no apparent correlations between its concentration and those of luteinizing hormone, testosterone, or dihydrotestosterone, or with the phase of development of the testes, or with the onset of molt. The plasma levels of progesterone of four castrated males were within the ranges of those of the photostimulated birds and of the short-day controls, suggesting that at least the major source of plasma progesterone is extratesticular.

Progesterone has been reported, on the basis of seemingly reliable radioimmunoassay systems, in the plasma of the males of several non-passerine species of birds (Furr, 1973; Haase et al., 1976; Mashaly and Wentworth, 1974; Paulke, 1975; Silver et al., 1974). Although its occurrence in the plasma of males of passerine species is certainly to be expected, we are unaware of any previous demonstration thereof. In this communication we report its occurrence in the plasma of the male White-crowned Sparrow, Zonotrichia leucophrys gambelii, and examine its relationship to other hor,mones during the course of the cycle of development and function of the testis. MATERIALS

AND METHODS

Adult male White-crowned Sparrows were captured with Japanese mist nets at the Sunnyside Game Refuge, Yakima County, Washington, in March 1976. Eleven photosensitive birds, previously held on short days (8L 16D) for 9 weeks (22 September-24 November), were changed to a long-day regime (2OL 4D) to induce a cycle of testicular development and function. Eight others were retained on 8L 16D as unstimulated, short-day controls. Illumination for both groups was provided by Sylvania 40-W fluorescent lamps adjusted to provide an intensity of ca. 400

lux on the floors of the cages. Blood samples from individuals of both groups were taken prior to Day 0, on 16 October and 24 November, and then on Days 5, 15, 22, 29, 36, 43, 56, 67, 80, and 101 of photostimulation in the same order of rotation after the onset of the artificial day. These samples, 200-700 ~1, were collected in heparinized microhematocrit tubes from a wing vein. Plasma samples were stored at -20”. To be certain that normal testicular cycles were induced, immunoreactive luteinizing hormone (irLH), testosterone, and dihydrotestosterone (DHT) were assayed. Furthermore, laparotomies were performed at about 3week intervals to estimate testicular weight by comparison with a preserved series of testes of known weights. After removal of each blood sample the bird was inspected for molt of primary and secondary wing feathers. Four castrated White-crowned Sparrows, maintained in outdoor aviaries since capture at Sunnyside and removal of testes in the winter of 1970-1971, were sampled in late May 1977. These birds were the subject of a previous communication (Mattocks et al., 1976) on the effect of castration on the plasma level of luteinizing hormone. Assays were not begun until after all samples had been collected and frozen and were effected in a randomized order. A micromodification (Follett et al.. 1975) of the radioimmunoassay of Follett et al. (1972) was used for the determination of irLH. Androgens were measured by the method of Wingfield and Farner (1975). The separation and assay of progesterone was effected by a system developed in our laboratory for use on avian plasma (J. C. Wingfield, R. S.

Copyright 0 1979 by Academic Press. Inc. All rights of reproduction in any form reserved.

2

McCREERY

AND

Donham, B. R. McCreery, and D. S. Farner. manuscript in preparation) which uses a lyophilized antiserum produced in rabbits against 1lcr-hydroxyprogesterone-hemisuccinate conjugated with bovine serum albumin obtained through the courtesy of Doctor A. H. Surve (Endocrine and Chemistry Sections, Sandoz-Wander, Inc.. East Hanover, New Jersey). The only significant cross-reaction with this antiserum is with I lol- and 1I@hydroxyprogesterone (Surve ei al., 1976). For Celite microcolumn chromatography each dried extract is transferred in isooctane into a celite column (celite: propylene glycol: ethylene glycol, 6:1.5:1.5. w:v:v) with a celite water trap. The antiserum was reconstituted in 50 ml of PO,buffered saline solution with 0.1% gelatin (PBS-g) and stored in 0.2-ml aliquots at -20”. For use in assays, thawed aliquots were diluted to 1: 12,500 or 1:24,000 in PBS-g. Tritiated steroids, [1,2,6,7, 3H]progesterone, [ 1.2.6,7, 3H]dihydrotestosterone, and [ 1,2,6,7, “HI testosterone, were obtained from New England Nuclear (Los Angeles, Calif.) and stored in benzene:ethanol(9: I, v/v; 250 ~Ci/lOO ml) at 4”. Progesterone, 5a-dihydrotestosterone (17phydroxy-SLu-androStan-3-one), and testosterone were obtained from Sigma (St. Louis, MO.). Percentages of recovery of tritiated steroid hormones after extraction and chromatography were (mean 2 SE) 55.6 2 0.8 (n = 114) for progesterone, 88.6 2 0.8 (144) for dihydrotestosterone, and 84.2 ~?r 0.5 (187) for testosterone. Several subsamples from each blood sample were included in each assay. Only results from assays in which blanks gave measurements lower than the sensitivities of the standard curves (3.9 pg for dihydrotestosterone and testosterone: 15.6 pg for progesterone) were used. Two samples from a pool of drake plasma to which 50 and 100 pg. respectively, of dihydrotestosterone were added, were included in each assay to estimate interassay variation. Coefficients of variation were 14.5% for progesterone, 17.2% for dihydrotestosterone, and 14.0% for testosterone.

RESULTS AND DISCUSSION

The only unequivocal demonstrations of progesterone in the plasma of birds known to us are those of O’Malley et al. (1968) by gas-liquid chromatography and Furr and Pope (1970) by gas-liquid chromatography and mass spectrometry in the femal domestic fowl. However, because of the specificity of the antiserum and of the nature of the chromatographic system employed, we are of the view that no significant quantities of other steroid compounds have contrib-

FARNER

uted to the levels of progesterone reported in Table 1. Although there are some differences, the patterns of testicular development and regression and of the plasma levels of irLH, testosterone, and DHT, the responses of the male White-crowned Sparrows subjected to long days in this investigation (Table 1) are generally similar to those obtained in previous studies (Follett et al., 1975; Lam and Farner. 1976). In contrast with the photoperiodically induced increases in the plasma levels of irLH, testosterone, and DHT, there was at no time of sampling a significant difference in the plasma level of progesterone between the long-day birds and the short-day controls. In both groups there was a significant decrease by Day 29; in the long-day birds there was a further significant reduction between Days 43 and 101. At this time we can offer no explanation for these changes. Because each group of assays contained samples from all or most sample times and two samples from a pool of drake plasma, the downward trend in the levels of progesterone cannot be attributed to a timedependent artifact. It does seem clear that if there is any photoperiodic effect on the secretion of progesterone it is in no way similar to the effect on secretion of testosterone (Lam and Farner, 1976). By Day 69 four birds in the long-day group had begun to molt primary wing feathers; by Day 80 all 11 were molting these feathers. At Day 85 eight were molting secondary wing feathers. On Day 10 I all long-day birds were in intensive molt. No primary or secondary wing feathers were molted by the short-day controls. Superficially, at least, there appears to be no relation between the plasma levels of progesterone and the onset and course of molt. This is consistent with observations on wild White-crowned Sparrows under natural field conditions that progesterone levels are relatively low during the time of postnuptial molt (J. C. Wingfield, unpublished data).

0.2(19)

3.9 +

i.O(IO)

87.6 + 42.4(11)**

431.8 + 34.1(11)‘”

125.5 k 17.2(11)** -

1.6 f

-

46 f 6(3)9

715 + 213(9)‘** 494 + 90(10)*** 369 + 69(X)*** 138 k 27(7) II8 + 13(7)9 109 2 24(5)8

818 2 57(5)b.P 847 2 163(6)a,0 828 + 92(7)*.b 737 2 125(5P 633 + 124(5F 610 r 99(7)b,d 386 + 2q5P

-

I I I f q2ono 75 r 12(4)9 185 -c 47(9) 452 +115(10)***

-

Testosterone (p&W

5.wDHT tpg/mlY

-(0)7

-(OH -

-(0)6 -(0)6

210 -+ 62(4)6**** 125 c 28(5)6 I I2 f 27(4)7

76 + 8(6)26 -(0)6 99 + 19(2)8 191 z 18(3)6****

birds t2OL 4D)

82(25)“J’ 110(6)‘.” 138(6)’ 123(4F

907 i 1106 + 1183 -t 956 f

Progesterone (pg/mljb,”

Photosttmulated

TABLE

I

0.62 k

0.49 2

0.76 z 0.58 f

1.57 2 I.50 f 1.25 k

0.29 z 1.53 -c 1.34 -t 1.47 -c

0.06( 16) 0.23(5)’ 0.14(7)** 0.22(6)** o.l5(9)** 0.10(10)** 0.08(7)** 0.13(6)* 0.14(6) 0.09(7) 0.07(8)

Luteinking hormone (ir-LH) (ng/mV

SUBJECTED

OF PROGESTERONE

SPARROWS

LEVELS

AND

OTHER

2.1 2 0.2(6) -

1.8 + 0.2(8)

-

2.1 -r 0.2(8)

-

1.8 r O.?(B)

Weight of testes (mg)”

TO ~&HOUR

IN THE

453 + 87(3)b

-

622 z 89(4)b.C 678 f 18q2)*J’ 672 k 46(3)” MI6 + 63(3)* 556 k I 29(4)D.P

-

85 + 16(5) 92 (I)6

81 (I)5 72 + 11(2)6 51 + l(4)7 146 t 2(2)6 65 t 21(2)5

94 k 22(4)7

-CO)5

m(O)3 -

-w -(0)5

-(0)6 I00 + 19(3)5 -CO)5 -

93.6 + 6t217 -(0)4 -(0)6

I I3 f 20(6)7 IO1 r 32(4)8

x77 2 55(5)” 942 -r 65(2)‘.’ 587 z 161(3F’

(8L l6D)

5.a-DHT (p&W

controls

PLASMA

Progesterone (pg/mljb,“

Short-day

PHOTOPHASES

HORMONES DAILY

-

0.36 2 0.04(S)

0.31 z 0.03(7) -

0.30 f 0.07(4) 0.47 + 0.08(6) 0.30 + 0.04(6) 0.25 + 0.03(E) 0.25 + 0.03(7) 0.30 k 0.02(8) 0.3 I * 0.04(8)

-

ir-LH tng/ml)d

C Mean 2 SEM and n within parentheses. b Mean -+ SEM and n (number of samples with detectable hormone) within parentheses. Total number of samples, when greater than II, is outside parentheses. c Means with different superscripts (a-d) are significantly different (P < 0.05). d Steroid levels are for a pool of samples collected on I6 October and 24 November (44 days and 1 day prior to photostimulation. respectively) from all birds On 24 November testes weights were taken and samples from this day were also assayed for ir-LH. *P < 0.01 compared with controls. **P < 0.001 compared with controls. ***P < 0.01 compared with Day 0. ****P < 0.05 compared with Day 0.

o* 5 15 22 24 29 36 43 44 56 61 69 80 85 101

Day

Weight of testes (mg)”

-

AND

WHITE-CROWNED

DEVELOPMENT

OF MALE

TESTICULAR

w

4

McCREERY

The literature on a possible role of progesterone in the control of molt is based almost exclusively on experiments involving treatment with exogenous hormone. In this manner molt has been induced in the female domestic fowl (for reviews and discussion, see Assenmacher, 1958; Payne, 1972; and Voitkevich, 1962), as well as in Melopsittacus undulatus, Lonchra striata, and Coturrzix coturnix (Kobayashi, 1958). Kobayashi (1958), however, was unable to thus induce molt in 14 passerine species or in the domestic pigeon, and Wagner and Muller (1963) were also unable to do so with four passerine species, including Lonchura striata. We agree with Voitkevich (1962) and Payne (1972) that progesterone has little or no role in the control of molt and that the positive results obtained in experiments with a few species represent indirect effects. Blood samples taken from the four castrated White-crowned Sparrows on 22 May during the period of maximum plasma levels of irLH (Mattocks et al., 1976) contained no detectable testosterone, i.e., less than 40 pgIm1, indicating that no regeneration of the endocrine testis had occurred. The four samples contained 499, 611, 765, and 828 pg/ml progesterone, indicating that the testis is not the sole source of plasma progesterone if, indeed, it is a source at all. These results are consistent with those obtained with castrated male turkeys by Mashaly and Wentworth (1974) who raise the possibility of an adrenal origin of plasma progesterone in male birds. Among the reptiles, Highfill and Mead (1975) have reported an extragonadal source of progesterone in female garter snakes. On the other hand, Furr (1973) has concluded that the absence of detectable amounts of progesterone in the plasma of castrated cockerels makes it improbable that the adrenal cortex is a significant source of this hormone. Because progesterone has been reported from the testes of several species (e.g., Dehio et al., 1%7; Hahn and Cheng, 1%7; Hahn, 1970), it might be assumed that it is a

AND FARNER

source of some fraction of the progesterone in avian plasma. Although plausible, this assumption must be viewed with caution, not only because of its occurrence within normal ranges in the blood of castrated White-crowned Sparrows and turkeys, but also because it is apparently a precursor in the synthesis of testosterone (Fevold and Eik-Nes, 1963: Dorfman, 1969). Since, to our knowledge, these are the first published assays of progesterone in a passerine species, comparison with results reported for males of non-passerine species is of interest. Levels of plasma progesterone for male White-crowned Sparrows (Table 1) appear to be somewhat lower than those reported for Ring Doves (1270 ? 80 pg/ml: Silver et af., 1974), rather similar to those of the pigeon (580 2 80, for paired males; 900 2 120, for unpaired males; Haase et al., 1976) and perhaps male turkeys (630; range, 50-3180; Mashaly and Wentworth, 1974), but somewhat higher than those reported for cockerels (440 * 60; Furr, 1973). However, any generalization at this time concerning the levels of progesterone in the plasma of male birds would be premature. ACKNOWLEDGMENTS This investigation was supported in part by Grant No. BMS74-13933 from the National Science Foundation. We are grateful to Mr. P. W. Mattocks, Jr., for his assistance with the assay of luteinizing hormone and for the blood samples from castrated Whitecrowned Sparrows; Dr. John C. Wingfield and Mr. Richard S. Donham provided invaluable advice and assistance with the steroid assays. We are also grateful to Professor Brian K. Follett (Bristol) for providing us with chicken LH preparation and anti-chicken LH serum and to Dr. A. H. Surve (Sandoz, Inc., East Hanover, New Jersey) for anti-progesterone serum for the radioimmunoassays.

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IN

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