- Email: [email protected]
The annual reproductive cycle in mallards
.I. steroid
Printed
Biochem.
in Great
Vol. 19. No. I. PP. 731-737. 1983 Britain. All rights reserved
0022-473 l/83 $3.00 + 0.00 Copyright 0 1983 Pergamon Press Ltd
THE ANNUAL REPRODUCTIVE IN MALLARDS
CYCLE
EBERHARD HAASE
Institut
fllr Haustierkunde Christian-Albrechts-Universitlt D 2300 Kiel, Fed. Rep. Germany
SUMMARY
In wild mallard drakes plasma FSH and LH levels were elevated at the height of the breeding season in spring, decreased towards the end of the reproductive phase and were low during the time of photorefractoriness in summer, when the testes were regressed. In contrast plasma prolactin concentrations increased only towards the end of the breeding season and were at their annual height during the summer. Castration of photorefractory mallards caused a steep rise of plasma LH levels indicating that the testes were involved in the maintenance of the low LH concentrations during the refractory period. In these drakes LH titers remained high throughout the year and, thus, in the absence of the testes the birds did not become photorefractory. In photorefractory drakes removal of 90% of the testicular tissue not only increased plasma LH levels but induced complete spermatogenesis in the testicular remains within 6 weeks. Therefore it seems that photorefractoriness in mallards is due to an increased sensitivity of the hypothalamo-hypophyseal system to the negative feedback action of testicular androgens. After injection of tritiated testosterone the hypothalamus, anterior pituitary and testes. but not other organs studied, took up more radioactivity during the refractory phase than at the height of the breeding season. It is speculated that the increased sensitivity of the hypothalamo-hypophyseal unit to the negative androgenic feedback during photorefractoriness depends on an increased number of androgen receptors in these structures which, again, could be due to the elevated prolactin levels during this phase of the annual cycle.
INTRODUCTION
changes of plasma concentrations of gonadotropins and prolactin and to compare these patterns with the spermatogenetic and androgenic activity of the testes [ 121. The seasonal pattern of plasma LH was characterized by high titers during the reproductive phase in the spring, a steep decrease towards the end of this phase, low levels during the period of photorefractoriness and a second annual peak in the autumn (Fig. 1). These data confirm earlier findings in our laboratory [ll, 121 and the results of Donham[6]. The seasonal LH pattern was paralleled by seasonal fluctuations in the plasma testosterone levels and the concentrations of the two hormones were linearly correlated in plasma samples taken at monthly intervals [14]. Only the first annual LH peak was associated with corresponding testicular size changes whereas during the autumnal LH peak the testes remained regressed and the birds sterile. Plasma FSH levels in our drakes increased steeply during the late winter and early spring and culminated before maximal testicular size was reached (Fig. I). The finding that plasma FSH levels in seasonal breeding birds are high during the phase of rapid testicular growth was also described for the Japanese quail [l5], the rook [16], and the whitecrowned sparrow [17]. It suggests that FSH is involved in the control of avian spermatogenesis and this suggestion was experimentally confirmed by the results of FSH and/or androgen treatments of intact and hypophysectomized quail [ 18, 191. At the height of the breeding season plasma FSH levels in our
Anas platyrhynchos, with its nominate subspecies A. p. phtyrhynchos is widely distributed in Europe, Asia and North America. In adaptation to seasonal changes in its natural environment the wild mallard has evolved an annual reproductive cycle. From laying dates, testicular weight changes and histological investigations of the testes it is known that germ cell production in the two sexes is essentially restricted to the rather short period from March to June both in the wild and in captivity [l-6]. A decisive environmental factor controlling the reproductive cycle of mallards is the photoperiod [7-93. During the autumn and winter the birds become photosensitive and in this state they respond to the increased photoperiods of the spring with gonadal growth. Germ cell production ceases, however, in late spring or early summer though the birds experience photoperiods which are longer than those which originally stimulated breeding in the spring. Such birds are termed “photorefractory” which-in contrast to hamsters-means that reproductive activity in mallards (and other birds) is not maintained by long days. Physiological results and hypotheses on the endocrine control mechanisms of the male mallards reproductive cycle were compiled a few years ago [ 10, 1 l] and in this paper I would like to focus on more recent and mostly unpublished findings. The wild mallard,
GONADOTROPINS Our
goal
S.B.19-l(c+B
was to gather
AND PROLACTIN information
on the seasonal 731
732
tern
,*7*
FMAMJJASONDJFMAMJJAS
Fig. I. Seasonal variations in plasma prolactin and gonadotropin concentrations in 7 adult wild mallard drakes kept in an outdoor aviary in Kiel (54 N). The blood samples were taken at intervals of 3 weeks, RIA determinations were carried out according to the methods of McNeilly et al.[4S] for prolactin, of Scanes et al.[46] for FSH. and of Follett CJ~(11.[47] for LH. Data on testes weights (O- --0) recorded from different drakes during the lasl years arc included for comparison. Hatched vertical bars indicate normal molt periods. drakes had begun to decrease. The decline continued towards the onset of the refractory phase and the plasma FSH concentrations remained low during the rest of the year. In rooks and White-crowned sparrows, too, the fall in plasma FSH preceded testicular regression and photorefractory birds had low FSH levels [ 16, 171. In male black swans FSH levels were highest at the start of the nesting period and declined during incubation [ZO]. Plasma prolactin levels in our drakes began to rise later than the gonadotropins. High values were reached during late phases of the reproductive period when the gonadotropin concentrations had begun to fall. Prolactin titers were at their highest at the end of the male reproductive cycle and during the refractory phase. They decreased during the summer and had reached moderate to low levels in the ~~utumn when LH levels began to rise again (Fig. 1). There was, however, no statistically significant correlation between LH and prolactin levels through the observation period. High plasma prolactin concentrations towards the end of the breeding cycle have been found in the
males of some other birds species, too, (rook [16], barheaded goose [Zl], black swan [20]). In female birds LH drops and prolactin levels rise when the birds cease to lay and start incubation (chicken [22], ruffed turkey [23,24], grouse [25], barheaded goose [21], wild mallard [26], black swan [20]) and the role of prolactin for the induction of broodiness has been repeatedly discussed in these papers. Since mallard drakes, however, do not participate in incubation and leading one would expect other functions of increased prolactin in these birds. The reciprocat changes at the end of the breeding season in the plasma gonadotropin and prolactin concentrations in our mallards and other avian species (uide supra) make it especially tempting to speculate on the role of prolactin in relation to gonadal regression and photorefractoriness. It has long been known that prolactin injections in sexually mature roosters and pigeons reduced testis weight, size of androgen dependent secondary sex characters and male behaviour. Since these effects could be prevented if FSH or androgens were injected along with prolactin it was thought that the gonad-inhibiting action of prolactin was due to
733
Annual reproductive cycle in mallards blocking the secretion of gonadotropins from the pituitary [27,28]. Moreover, in photosensitive whitecrowned sparrows photoperiodic stimulation of gonada1 recrudescence was reduced if the birds received prolactin injections [29] and thus, under the influence of prolactin, they reacted like photorefractory males. Our findings support the idea of a prolactin mediated gonadal regression via an interference with the secretion of gonadotropins. The exact mechanism of this action remains to be elucidated. CASTRATION
The effects of castration on pituitary and plasma gonadotropin levels in different avian species have yielded conflicting arguments with respect to the role of the testes in the control of avian reproductive cycles and photorefractoriness [30]. Castration of photosensitive domestic mallard drakes caused an increase in plasma LH and FSH levels. This increase could be prevented by injections of testosterone propionate[33,32] indicating that the testes were involved in the regulation of gonadotropin levels via negative androgen feedback. We have castrated photorefractory wild mallard drakes to find out whether the testes contribute to the suppression of the plasma LH concentration typical for the refractory period. The drakes (n = 8) were castrated during the late summer when their gonads were completely regressed (combined testicular weight 0.3 g) and plasma LH levels were low (1.3 ng/ml). Four to six weeks after castration LH concentrations had increased to 23 @ml showing that the gonadal feedback was involved in the maintenance of low LH levels during the refractory period [30]. Similar results were obtained in red grouse[33] and willow ptarmigan [34]. These findings, together with the occurrence of high LH and androgen levels during the reproductive period and low LH and androgen levels during the refractory phase, suggest that the change from photosensitivity to photorefractoriness depends
on an increased sensitivity of the hypothalamo-hypophyseal system to the negative feedback action of testicular hormones. Earlier studies are consistent with this view of an increased sensitivity. Thus, in the Pekin drake unilateral castration at the onset of seasonal breeding resulted in an accelerated growth of the remaining testis. If, however, one testis was removed when the birds were becoming refractory, the remaining testis showed no or little increase in size [37]. It seems that the sensitivity of the hypothalame-pituitary unit had increased suficiently for the secretion of the remaining testis to suppress plasma gonadotropin levels. In other studies on Pekin drakes it was found that the dose of exogenous testosterone required to inhibit gonadal growth and induce testicular regression decreased during the course of the breeding season [38]. In our castrated drakes the seasonal pattern of plasma LH concentrations characteristic of intact drakes (Figs I and 2) was not found. Instead, average LH levels remained high during the 16 months sampling period following castration. Interestingly, at the end of the breeding season no significant drop in the LH levels of the castrates occurred (Fig. 2). Similar findings were obtained in castrated Pekin drakes [35], but the data from domesticated drakes are difficult to interpret because some of these birds may not become refractory or exhibit a very brief period of photorefractoriness[36]. Our results in the wild mallards indicate that the LH secreting mechanism had not become photorefractory in long-term castrated drakes and that the testes are involved in the induction of photorefractoriness. INCOMPLETE
CASTRATION
The findings of the castration experiment could be explained in different ways. I have tried to verify my interpretation by using it as basis for predictions which could be experimentally tested. If decreased gonadotropin levels and testicular regression at the
30 l-
1111111111111 FMAMJJASONDJFMAYJJASONDJF
1
I
I
I
8
1
I
Fig. 2. Plasma LH levels of 10 castrated wild mallard drakes (X i SEM) sampled from Oct. 1977 to Jan. 1979. For comparison data on intact drakes from other years (“controls”) arc included.
end of the breeding season are due to an increased sensitivity of the hypitthrtlamo-hypophysenl system to the negative feedback action of testicular hormones, then incomplete removal of the testes during the refractory period should not only elevate the titer of plasma immunoreactive gonadotropins but also induce recrudescence of the testicular remains. Wild mallard drakes were incompletely castrated during the refractory period in early August lOXI. About 90:‘;, of the testicular tissue were removed. The results are shown in Table 1 and Fig. 3. After h weeks under
naturat daylength the testicular remains had increased in wright 9.3 times. The controls were I~~~~lrotomized by mid September ;tnd transferred indoors fa ;t fang day photoperiod (I 61, 8D) to find out whether during the course of the experiment the birds had regained their photosensitivity (Table 1). Only in one of the drakes did 6 weeks of long photoperiods induce spermatogenetic recrudescence. Mean testicular size of ali birds slightly decreased. The photostimulation test thus proved that the findings after incomplete castration were not due to the regain of photosensi-
Fig. 3. ~~~t~rnjcr~g~~~~ of testicular tissues from a ~~~t~re~r~~ct~r~ wild tmillard drake which WBS incompletely castrated. {a) Tissue at the time of castration un Aug. 4th. Narro% tubules and PASposit&e inclusions in the lining epithetium. (b) Tissue from the same bird on Sept. i&h after 6 weeks of incomplete castration. Tubules are widened, cleared from degenerating mateM. and ~perrn~~t~g~~et~c recrudescence has occurred. (a) and (b) at same magnification. PAS Ehrlich‘s hemtltoxylin.
Annual Table 2. Androgen Hypoth.
Date Apr. 18
61 f
June 25
uptake
by wild mallard
Ant. pit.
11*
reproductive
tissues 30 min after i.v. injection
Testis
Pect. m.
Cerebel.
114 + 7 (5)
12 + 11 (4) 65 + 3 (5)
135 F 9*
215 2 15
995 4 236
(5)
(5)
(5)
(5)
80 + 4*
331 f 29*
128 * 8*
179 + 38
678 & 124
117 * 14
(5)
(5)
(5)
(3)
(5)
significance
(r-test, P < 0.05) between
of the drakes during the course of the experiment but resulted from the ablation of testicular tissue. tivity
ANDROCEN
UPTAKE
The evidence so far presented for an increased sensitivity of the hypothalamo-hypophyseal system to the negative testicular feedback during photorefractoriness provides no information as to the chemical nature of the feedback substance(s), However, data from other laboratories point to testosterone and/or its metabolites as promising candidates. As mentioned above testosterone propionate injections can prevent the post-castrational rise of plasma gonadotropins in domestic drakes [3 1,321 and can induce testicular regression in drakes with increasing sensitivity of the system at the time of seasonal testicular regression [38 J. Microimplants of testosterone crystals in the preoptic region or the ventromedial nucleus of the hypothalamus of drakes inhibited testicular growth or induced testicular regression [39]. Moreover, the steroid seems to act at the level of the anterior pituitary by changing its responsiveness to GnRH [40,41]. I have tried to find out whether during different phases of the reproductive cycle the hypothalamohypophyseal system and other organs of drakes varied in the uptake of labelled testosterone. On April 18th and June 29th wild mallard drakes were i.v. injected with [ 1,2,6,7-3H]-testosterone (NEN, SA 98.8 Cijmmol; 80 &i/kg body weight) and sacrified after 30min. Different organs were quickly removed, weighed and dissolved in Soluene (Packard). Uptake of testosterone and its met~~bolites was expressed in d.p.m./mg tissue. The results are shown in Table 2. Interestingly the structures of the hypothalamo-pituitary-testicular axis but not the other organs significantly differed in the uptake of radioactivity between the two phases of the breeding cycle. During photorefractoriness, when the hypothalamohypophyseal system was shown to be more sensitive to the testicular feedback it took up more labelled androgen than during the reproductive phase. Thus, the increased sensitivity to the testicular feedback seems to be correlated to an increased ability to take up androgens. The increased androgen uptake could be due to an augmented number of androgen recepUnfortunately,
Liver
229 + 23*
x & SEM (n); *statistical
scarcely
Plasma
of [3H]-testosterone
(4) (5)
tors.
735
cycle in mallards
androgen
receptors
in birds
have
been studied. They occur in the pituitary
the 2 dates.
gland of chickens [42] but data on the hypothalamus are lacking. In this situation it means entering upon soft ground to propose a model on the endocrine mechanism controlling photorefractoriness in wild mallards. The model is consistent with the findings reported here and may serve as a working hypothesis: During the annual gonadal cycle of mallard drakes the termination of the reproductive phase is caused by an increased sensitivity of the hypothalamo-hypophyseal system to the negative androgenic feedback of the testes. This results in a reduced gonadotropin secretion and testicular regression. The increased sensitivity of the hypothalamo-hypophyseal system is due to an augmented number of androgen receptors in these structures. A possible reason for the augmented number of androgen receptors could lie in the increased plasma prolactin concentrations at the end of the reproductive phase and during refractoriness. In mammals prolactin can stimulate the uptake of labelled androgens into male accessory sex glands [43] and it seems to increase the amount of androgen receptor complex in androgen responsive tissue [44]. Acknowledl/mmrrrts-This work was supported by the Deutsche Forschungsgemeinschaft SPP “Mechanismen biologischer Uhren” Ha 82017. I wish to thank Mr R. Talbot, W. Kochling, and R. Schmedemann for RIA work. Dr E. Paulke performed the studies on [‘Hltestosterone uptake. Professor Nieschlag kindly provided anti-serum for the testosterone assay. REFERENCES
1. HGhn E. 0.: Seasonal behaviour and seasonal changes in the gonads and adrenals of the mallard. Proc. Zool. sot. L&f. 117 (1947) 281-304. 2. Johnson 0. W.: Reoroductive cvcle of the mallard duck. Tlze Cordor 63‘(1961) 351-364. 3. Johnson 0. W.: Quantitative features of spermatogenesis in the mallard. Titr Auk 83 (1966) 233-239. 4. Bauer K. M. and Glutz von Blotzheim U. N.: Handbuch der Vogel Mitteleuropas. Vol. 2 Anserifbrrnes (1. Teil). Akademische Verlagsgesellschaft. Frankfurt a.M. (1968). 5. Paulke E.: Hormonphysiologische Untersuchungen zum Fortpflanzungszyklus von Wild- und Hauserpeln (Anus piatyrhyt~chos L. und A. plutyrhywhos J dam. Khuki Campbell). Dissertation, Univ. Kiel (1975). 6. Donham R. S.: Annual cycle of plasma luteinizing hormone and sex hormones in male and femaie mallards (Anus ~~ur~r~~~~~~r~~~)~Bini. ~~prffd. 21 (1979) 1273-1285. 7. Lofts B. and Coombs C. J. F.: Photoperiodism and the testicular refractory period in the mallard. d. Zool., Lnnd. 146 (1965) 44-54.
x. Lolis B. and Murton R K.. ~l~~~t(~perl~~di~and physio-
9.
IO.
11.
12.
13.
14.
15.
16.
17.
IX.
19.
20.
21.
22.
23.
logical adaptations regulating ;ivian breeding cycles and their ecological significance. J. Zoo/.. i*c&. 155 (1968)327 393. Storey C. R. and Nicholls T. J.: Observations on the regulation of sexual quiescence in juvenile and adult male mallard (A~~trs pltrr~~h?,rti,ht,s). J. Zoo/.. Lend. 184 (197X) IX1 -1X6. Haase E.: Hormonphysiologische Untersuchungen zum Jahreszyklus der Fortpflanzung an Stock- und Hauserpeln. Ahhdl. cl.tl. G‘&. tl. b’o&kd. 6 (1979) 3 1% 317. Haose E. and Paulke E.: The control of the annual gonadal cycle of wild mallard drakes: Some endocrinological aspects. In Acta XVII Congressus Internationalis Ornithologici (Edited by R. Niihring). Verlag der Deutschen Ornithoiogen-Ges~llsch~~ft, Serlin, Vol. I (19X0) pp. 453 457. Haase E.. Paulke E. and Sharp P. J.: Seasonal changes of plasma gonadotropins and prolactin in wild mallard drakes. Unpublished data. Haase E.. Sharp P. J. and Paulke E.: Annual cycle of plasma luteinizing hormone concentrations in wild mallard drakes. J. c\-p. Zoo/. 194 (I 975) 553-55X. Paulkc E. and Haase E.: A comparison of seasonal changes in the concentrations of rtndrogens in the peripheral blood of wild and domestic ducks. Gc,n. L’oI~~. Erir/r>c~r.34 ( 197X) 38 I 390. Follett B. K.: Plasma follicle-stimul~~ting hormone during photoperiodi~~~ily induced scsusi rn~~tllr~~tion in male Japanese quail. .I. Emfoct. 69 (1976)I 17~ 126. Lincoln G. A.. Racey P. A.. Sharp P. J. and Klandorf H.: Endocrine changes associated with spring and autumn sexuality of the rook. Corrrrs ,f~u~qilq~c.~. J. Zoo/.. LO!lii. 190 (I 980) 137--l 53. Wingfield J. C.. Follett 3. K.. Matt K. S. and Farner D. S.: Effect of day length on plasma FSH and LH in castrated and intact white-crowned sparrows. Grtl. corup, I%doc.r. 42 ( 1980) 464-470. Brown N. L.. Boyle J.-D.. Scanes C. G. and Follett B. K.: Chicken gonadotropins: Their effects on the testes of immature and hypophqsectomized Japanese yuail. Ceil Tissue, Re. 156 (1975) 499 520. Brown N. L. and Follett B. K.: Effects of androgen on the testes of intact and hyp[~physe~tomized Japanese quail. Gen. riritlp. ~~~~~~~(,~. 33 ( 1977) 167-277. Goldsmith A. R.: The Australian black swan (C?:tijm.s atrnrus): Prolactin and gonadotrophin secretion during breeding including incubation. &I. contp. E&ocv. 46 (1981) 45X-462. Dittami J. P.: Seasonal changes in the behaviour and plasma titers of various hormones in barheaded geese. All,st,r i,ldiclrs. 2. TicqJ”~&~/. 5s ( I98 I ) 2X9-324. Sharp P. J.. Scnnes C. C.. Williams J. B.. Harvey S. and Chadwick A.: Variation in concentrations of prolactin, luteinizing hormone, growth hormone and progesterone in the plasma of broody bantams (Crrilus tlontc+ rinrp. E/t&r. 41 (1980) 92-
100. J. A. and Opel H.: Turkey prolactin: Vali24. Proudman datlon of :I radioimmunoassav and measurement of changes associated with broodiness. Biol. Reprorl. 25 ( 19XI ) 573%5X0. A. L.: Plasma 25. Etches R. J.. Garbutt A. :md Middleton concentrations of prolnctin and the incubation in the rutfed grouse (EOII(I.S~I t&v/ilc,t). C‘
27. A. V.. Hochha&er M. and Dugas M.: A 28. Naibandov study of the effect of prolactin on broodiness and on cock testes. E~~tlocrinolo~q~ 36 (1945) 25 I -75X. 29. Bailey R. E.: Inhibition with prolactin of light-induced gonad increase in white-crowned sparrows. Tl~r CO/Idor 52 ( 1950) 247 257. 30. Haase E., Sharp P. J. and Paulkc E.: The effects of castration on the seasonal pattern of plasma LH concentrations in wild mallard drakes. Gcarl. cct,,,p. Ot&rc~r. 46 (1982) 113-l 15. I.: Effect 31. Jaliageas M.. Follett B. K. and Assenmacher of castration and:‘or testosterone ~~drninistr~ition on plasma LH and testosterone ~oncentr~itions in the duck. &,I. corttp. E&ocr. 29 ( 1976) 171. J.. Heyns W. and Hendrick 32. Dcviche P.. Balthuzart J.-C.: Endocrine effects of castration followed by androgen replacement and ACTH injections in the male domestic duck ( 4r1us p/trt!,rh?,r~c,ho.s). &II. c’o,np. Eltrlocr. 41 ( 19x0) 5.1-61. 33. Sharp P. J. and Moss R.: The effects of castration on concentrations of luteinizing hormone in the plasma of photorefractory red grouse (La~Jopus luqoptts ,swtims). GUI. w,~~p. Etrhcr. 32 ( 1977) 2X9-293. 34. Stokkan K.-A. nnd Sharp P. J.: The roles of daylength and the testes in the regulation of plasma LH levels in ph~~t(~sensitive and photorefr~ctory willow pt~~rrni~~~n (~~/~~(~pi~,~ ~~~~J~~p~~.s hJoptc\)_ Germ.ccimp. E/idow. 41 ( 1980) 520&5’6. I.: Further evidence for 35. Jail;tgeas M. and Assenmacher rcciprocnl internctions between the annual sexual and thyroid cycles in male Pekin ducks. &,I. c’or,tp. Emlocr. 37 ( 1979) 44-5 I, 36. Hanse E.. Sharp P. J. and Paulke E.: Seasonal changes in plasma LH levels in domestic ducks. J. Keprotl. Ferf. 44 (1975) 591-594. 37. Benoit J., Assenmacher I. and Walter F. X.: Rksponses du mkchanisms gonado-stimulant d I’&lairement artificiel et de Ia prChypophyse aux castration bilaterale et unil;it&ale. chez le canard domestique male, ;tu tours de la @iode de rbgression testiculare saisonni&c. C.R. SIC. Bid. 144 (1950) 573-577. F.: R~tro~iction des st&oides sexuels sur les 38. Gognn fonctions gonadotrops de I‘oiseau. In ‘~~uroeilctocrirrctlog/tit>(Edited by J. Benoit and C. Kordon). C.N.R.S.. Paris (lY70) pp. 352-362. B la testosterone 39. Gogan F.: SensibilitC hypothalamique cher le canard. &II. co,np. E!~riocr. 11 (1970) 3 16. 327. 40. Davies D. T. and Bicknell R. J.: The effect of testosterone on the responsiveness of the quail’s pituitary to luteinizing hormone-releasing hormone (LH---RH) during photoperiodicnlly induced testicular growth. Grrl. wmp, Emloc~r. 30 ( 1976) 4X7-499. J., Willems J. and Hendrick J. C.: Changes 41. Balthazart in pituituq responsiveness to luteinizing hormonereleasing hormone during an anno; cycle in the domestic duck. .~/Ic~.s~~~~~~,~~~~~i~~?~).~ L. J. cup. Zoo/. 21 I
(19X0)113.-1’3. M.. Inagami M. and Kawash52. Tanaka K.. Kamiyoshi ima M.: Presence of a testosterone receptor in the pituitary of the male chicken. Proc. XVI Wnfftl’.s Poltll. Ci>il(/“.. Vol. 7 ( 1978) 1I77 I I x3. J. A.. Mawhinney M. G. and 43. Zepp E,. A.. Thomas Lloyd J. W.: Differential response to either bovine or ovine prolnctin on testosterone-‘H (T-Ha) metabolism of various lobes of the rat prostate gland. Plz~rrr~tcccolo(/iSl 1s (I 973) 256. 44. Bardin C. W.: Pituitary--testicular axis. In Rrprdwtiro El~dot,~r,~cl/o~~~ (Edited by S. S. <‘. Yen and R. R. Jaffe). Saunders. Philadelphia f 197X) p_ 123, pp. I lo- 175.
Annual reproductive cycle in mallards 45. McNeilly A., Etches R. and Friesen H.: A herologous radioimmunoassay for avian prolactin: Application the measurement of prolactin in the turkey. Acta Endocr., Copetzh. 89 (1978) 60-69. 46. Scanes C. G., Godden P. M. M. and Sharp P. J.: A
homologous
radioimmunoassay
for chicken follicle-
737
stimulating hormone: observations on the ovulatory cycle. J. Endow. 73 ( 1977) 473-48 1. 47. Follett B. K., Scanes C. G. and Cunningham F. J.: A radioimmunoassay for avian iuteinizing hormone. .f. Endocr. 52 (1972) 359-378.
Printed
Biochem.
in Great
Vol. 19. No. I. PP. 731-737. 1983 Britain. All rights reserved
0022-473 l/83 $3.00 + 0.00 Copyright 0 1983 Pergamon Press Ltd
THE ANNUAL REPRODUCTIVE IN MALLARDS
CYCLE
EBERHARD HAASE
Institut
fllr Haustierkunde Christian-Albrechts-Universitlt D 2300 Kiel, Fed. Rep. Germany
SUMMARY
In wild mallard drakes plasma FSH and LH levels were elevated at the height of the breeding season in spring, decreased towards the end of the reproductive phase and were low during the time of photorefractoriness in summer, when the testes were regressed. In contrast plasma prolactin concentrations increased only towards the end of the breeding season and were at their annual height during the summer. Castration of photorefractory mallards caused a steep rise of plasma LH levels indicating that the testes were involved in the maintenance of the low LH concentrations during the refractory period. In these drakes LH titers remained high throughout the year and, thus, in the absence of the testes the birds did not become photorefractory. In photorefractory drakes removal of 90% of the testicular tissue not only increased plasma LH levels but induced complete spermatogenesis in the testicular remains within 6 weeks. Therefore it seems that photorefractoriness in mallards is due to an increased sensitivity of the hypothalamo-hypophyseal system to the negative feedback action of testicular androgens. After injection of tritiated testosterone the hypothalamus, anterior pituitary and testes. but not other organs studied, took up more radioactivity during the refractory phase than at the height of the breeding season. It is speculated that the increased sensitivity of the hypothalamo-hypophyseal unit to the negative androgenic feedback during photorefractoriness depends on an increased number of androgen receptors in these structures which, again, could be due to the elevated prolactin levels during this phase of the annual cycle.
INTRODUCTION
changes of plasma concentrations of gonadotropins and prolactin and to compare these patterns with the spermatogenetic and androgenic activity of the testes [ 121. The seasonal pattern of plasma LH was characterized by high titers during the reproductive phase in the spring, a steep decrease towards the end of this phase, low levels during the period of photorefractoriness and a second annual peak in the autumn (Fig. 1). These data confirm earlier findings in our laboratory [ll, 121 and the results of Donham[6]. The seasonal LH pattern was paralleled by seasonal fluctuations in the plasma testosterone levels and the concentrations of the two hormones were linearly correlated in plasma samples taken at monthly intervals [14]. Only the first annual LH peak was associated with corresponding testicular size changes whereas during the autumnal LH peak the testes remained regressed and the birds sterile. Plasma FSH levels in our drakes increased steeply during the late winter and early spring and culminated before maximal testicular size was reached (Fig. I). The finding that plasma FSH levels in seasonal breeding birds are high during the phase of rapid testicular growth was also described for the Japanese quail [l5], the rook [16], and the whitecrowned sparrow [17]. It suggests that FSH is involved in the control of avian spermatogenesis and this suggestion was experimentally confirmed by the results of FSH and/or androgen treatments of intact and hypophysectomized quail [ 18, 191. At the height of the breeding season plasma FSH levels in our
Anas platyrhynchos, with its nominate subspecies A. p. phtyrhynchos is widely distributed in Europe, Asia and North America. In adaptation to seasonal changes in its natural environment the wild mallard has evolved an annual reproductive cycle. From laying dates, testicular weight changes and histological investigations of the testes it is known that germ cell production in the two sexes is essentially restricted to the rather short period from March to June both in the wild and in captivity [l-6]. A decisive environmental factor controlling the reproductive cycle of mallards is the photoperiod [7-93. During the autumn and winter the birds become photosensitive and in this state they respond to the increased photoperiods of the spring with gonadal growth. Germ cell production ceases, however, in late spring or early summer though the birds experience photoperiods which are longer than those which originally stimulated breeding in the spring. Such birds are termed “photorefractory” which-in contrast to hamsters-means that reproductive activity in mallards (and other birds) is not maintained by long days. Physiological results and hypotheses on the endocrine control mechanisms of the male mallards reproductive cycle were compiled a few years ago [ 10, 1 l] and in this paper I would like to focus on more recent and mostly unpublished findings. The wild mallard,
GONADOTROPINS Our
goal
S.B.19-l(c+B
was to gather
AND PROLACTIN information
on the seasonal 731
732
tern
,*7*
FMAMJJASONDJFMAMJJAS
Fig. I. Seasonal variations in plasma prolactin and gonadotropin concentrations in 7 adult wild mallard drakes kept in an outdoor aviary in Kiel (54 N). The blood samples were taken at intervals of 3 weeks, RIA determinations were carried out according to the methods of McNeilly et al.[4S] for prolactin, of Scanes et al.[46] for FSH. and of Follett CJ~(11.[47] for LH. Data on testes weights (O- --0) recorded from different drakes during the lasl years arc included for comparison. Hatched vertical bars indicate normal molt periods. drakes had begun to decrease. The decline continued towards the onset of the refractory phase and the plasma FSH concentrations remained low during the rest of the year. In rooks and White-crowned sparrows, too, the fall in plasma FSH preceded testicular regression and photorefractory birds had low FSH levels [ 16, 171. In male black swans FSH levels were highest at the start of the nesting period and declined during incubation [ZO]. Plasma prolactin levels in our drakes began to rise later than the gonadotropins. High values were reached during late phases of the reproductive period when the gonadotropin concentrations had begun to fall. Prolactin titers were at their highest at the end of the male reproductive cycle and during the refractory phase. They decreased during the summer and had reached moderate to low levels in the ~~utumn when LH levels began to rise again (Fig. 1). There was, however, no statistically significant correlation between LH and prolactin levels through the observation period. High plasma prolactin concentrations towards the end of the breeding cycle have been found in the
males of some other birds species, too, (rook [16], barheaded goose [Zl], black swan [20]). In female birds LH drops and prolactin levels rise when the birds cease to lay and start incubation (chicken [22], ruffed turkey [23,24], grouse [25], barheaded goose [21], wild mallard [26], black swan [20]) and the role of prolactin for the induction of broodiness has been repeatedly discussed in these papers. Since mallard drakes, however, do not participate in incubation and leading one would expect other functions of increased prolactin in these birds. The reciprocat changes at the end of the breeding season in the plasma gonadotropin and prolactin concentrations in our mallards and other avian species (uide supra) make it especially tempting to speculate on the role of prolactin in relation to gonadal regression and photorefractoriness. It has long been known that prolactin injections in sexually mature roosters and pigeons reduced testis weight, size of androgen dependent secondary sex characters and male behaviour. Since these effects could be prevented if FSH or androgens were injected along with prolactin it was thought that the gonad-inhibiting action of prolactin was due to
733
Annual reproductive cycle in mallards blocking the secretion of gonadotropins from the pituitary [27,28]. Moreover, in photosensitive whitecrowned sparrows photoperiodic stimulation of gonada1 recrudescence was reduced if the birds received prolactin injections [29] and thus, under the influence of prolactin, they reacted like photorefractory males. Our findings support the idea of a prolactin mediated gonadal regression via an interference with the secretion of gonadotropins. The exact mechanism of this action remains to be elucidated. CASTRATION
The effects of castration on pituitary and plasma gonadotropin levels in different avian species have yielded conflicting arguments with respect to the role of the testes in the control of avian reproductive cycles and photorefractoriness [30]. Castration of photosensitive domestic mallard drakes caused an increase in plasma LH and FSH levels. This increase could be prevented by injections of testosterone propionate[33,32] indicating that the testes were involved in the regulation of gonadotropin levels via negative androgen feedback. We have castrated photorefractory wild mallard drakes to find out whether the testes contribute to the suppression of the plasma LH concentration typical for the refractory period. The drakes (n = 8) were castrated during the late summer when their gonads were completely regressed (combined testicular weight 0.3 g) and plasma LH levels were low (1.3 ng/ml). Four to six weeks after castration LH concentrations had increased to 23 @ml showing that the gonadal feedback was involved in the maintenance of low LH levels during the refractory period [30]. Similar results were obtained in red grouse[33] and willow ptarmigan [34]. These findings, together with the occurrence of high LH and androgen levels during the reproductive period and low LH and androgen levels during the refractory phase, suggest that the change from photosensitivity to photorefractoriness depends
on an increased sensitivity of the hypothalamo-hypophyseal system to the negative feedback action of testicular hormones. Earlier studies are consistent with this view of an increased sensitivity. Thus, in the Pekin drake unilateral castration at the onset of seasonal breeding resulted in an accelerated growth of the remaining testis. If, however, one testis was removed when the birds were becoming refractory, the remaining testis showed no or little increase in size [37]. It seems that the sensitivity of the hypothalame-pituitary unit had increased suficiently for the secretion of the remaining testis to suppress plasma gonadotropin levels. In other studies on Pekin drakes it was found that the dose of exogenous testosterone required to inhibit gonadal growth and induce testicular regression decreased during the course of the breeding season [38]. In our castrated drakes the seasonal pattern of plasma LH concentrations characteristic of intact drakes (Figs I and 2) was not found. Instead, average LH levels remained high during the 16 months sampling period following castration. Interestingly, at the end of the breeding season no significant drop in the LH levels of the castrates occurred (Fig. 2). Similar findings were obtained in castrated Pekin drakes [35], but the data from domesticated drakes are difficult to interpret because some of these birds may not become refractory or exhibit a very brief period of photorefractoriness[36]. Our results in the wild mallards indicate that the LH secreting mechanism had not become photorefractory in long-term castrated drakes and that the testes are involved in the induction of photorefractoriness. INCOMPLETE
CASTRATION
The findings of the castration experiment could be explained in different ways. I have tried to verify my interpretation by using it as basis for predictions which could be experimentally tested. If decreased gonadotropin levels and testicular regression at the
30 l-
1111111111111 FMAMJJASONDJFMAYJJASONDJF
1
I
I
I
8
1
I
Fig. 2. Plasma LH levels of 10 castrated wild mallard drakes (X i SEM) sampled from Oct. 1977 to Jan. 1979. For comparison data on intact drakes from other years (“controls”) arc included.
end of the breeding season are due to an increased sensitivity of the hypitthrtlamo-hypophysenl system to the negative feedback action of testicular hormones, then incomplete removal of the testes during the refractory period should not only elevate the titer of plasma immunoreactive gonadotropins but also induce recrudescence of the testicular remains. Wild mallard drakes were incompletely castrated during the refractory period in early August lOXI. About 90:‘;, of the testicular tissue were removed. The results are shown in Table 1 and Fig. 3. After h weeks under
naturat daylength the testicular remains had increased in wright 9.3 times. The controls were I~~~~lrotomized by mid September ;tnd transferred indoors fa ;t fang day photoperiod (I 61, 8D) to find out whether during the course of the experiment the birds had regained their photosensitivity (Table 1). Only in one of the drakes did 6 weeks of long photoperiods induce spermatogenetic recrudescence. Mean testicular size of ali birds slightly decreased. The photostimulation test thus proved that the findings after incomplete castration were not due to the regain of photosensi-
Fig. 3. ~~~t~rnjcr~g~~~~ of testicular tissues from a ~~~t~re~r~~ct~r~ wild tmillard drake which WBS incompletely castrated. {a) Tissue at the time of castration un Aug. 4th. Narro% tubules and PASposit&e inclusions in the lining epithetium. (b) Tissue from the same bird on Sept. i&h after 6 weeks of incomplete castration. Tubules are widened, cleared from degenerating mateM. and ~perrn~~t~g~~et~c recrudescence has occurred. (a) and (b) at same magnification. PAS Ehrlich‘s hemtltoxylin.
Annual Table 2. Androgen Hypoth.
Date Apr. 18
61 f
June 25
uptake
by wild mallard
Ant. pit.
11*
reproductive
tissues 30 min after i.v. injection
Testis
Pect. m.
Cerebel.
114 + 7 (5)
12 + 11 (4) 65 + 3 (5)
135 F 9*
215 2 15
995 4 236
(5)
(5)
(5)
(5)
80 + 4*
331 f 29*
128 * 8*
179 + 38
678 & 124
117 * 14
(5)
(5)
(5)
(3)
(5)
significance
(r-test, P < 0.05) between
of the drakes during the course of the experiment but resulted from the ablation of testicular tissue. tivity
ANDROCEN
UPTAKE
The evidence so far presented for an increased sensitivity of the hypothalamo-hypophyseal system to the negative testicular feedback during photorefractoriness provides no information as to the chemical nature of the feedback substance(s), However, data from other laboratories point to testosterone and/or its metabolites as promising candidates. As mentioned above testosterone propionate injections can prevent the post-castrational rise of plasma gonadotropins in domestic drakes [3 1,321 and can induce testicular regression in drakes with increasing sensitivity of the system at the time of seasonal testicular regression [38 J. Microimplants of testosterone crystals in the preoptic region or the ventromedial nucleus of the hypothalamus of drakes inhibited testicular growth or induced testicular regression [39]. Moreover, the steroid seems to act at the level of the anterior pituitary by changing its responsiveness to GnRH [40,41]. I have tried to find out whether during different phases of the reproductive cycle the hypothalamohypophyseal system and other organs of drakes varied in the uptake of labelled testosterone. On April 18th and June 29th wild mallard drakes were i.v. injected with [ 1,2,6,7-3H]-testosterone (NEN, SA 98.8 Cijmmol; 80 &i/kg body weight) and sacrified after 30min. Different organs were quickly removed, weighed and dissolved in Soluene (Packard). Uptake of testosterone and its met~~bolites was expressed in d.p.m./mg tissue. The results are shown in Table 2. Interestingly the structures of the hypothalamo-pituitary-testicular axis but not the other organs significantly differed in the uptake of radioactivity between the two phases of the breeding cycle. During photorefractoriness, when the hypothalamohypophyseal system was shown to be more sensitive to the testicular feedback it took up more labelled androgen than during the reproductive phase. Thus, the increased sensitivity to the testicular feedback seems to be correlated to an increased ability to take up androgens. The increased androgen uptake could be due to an augmented number of androgen recepUnfortunately,
Liver
229 + 23*
x & SEM (n); *statistical
scarcely
Plasma
of [3H]-testosterone
(4) (5)
tors.
735
cycle in mallards
androgen
receptors
in birds
have
been studied. They occur in the pituitary
the 2 dates.
gland of chickens [42] but data on the hypothalamus are lacking. In this situation it means entering upon soft ground to propose a model on the endocrine mechanism controlling photorefractoriness in wild mallards. The model is consistent with the findings reported here and may serve as a working hypothesis: During the annual gonadal cycle of mallard drakes the termination of the reproductive phase is caused by an increased sensitivity of the hypothalamo-hypophyseal system to the negative androgenic feedback of the testes. This results in a reduced gonadotropin secretion and testicular regression. The increased sensitivity of the hypothalamo-hypophyseal system is due to an augmented number of androgen receptors in these structures. A possible reason for the augmented number of androgen receptors could lie in the increased plasma prolactin concentrations at the end of the reproductive phase and during refractoriness. In mammals prolactin can stimulate the uptake of labelled androgens into male accessory sex glands [43] and it seems to increase the amount of androgen receptor complex in androgen responsive tissue [44]. Acknowledl/mmrrrts-This work was supported by the Deutsche Forschungsgemeinschaft SPP “Mechanismen biologischer Uhren” Ha 82017. I wish to thank Mr R. Talbot, W. Kochling, and R. Schmedemann for RIA work. Dr E. Paulke performed the studies on [‘Hltestosterone uptake. Professor Nieschlag kindly provided anti-serum for the testosterone assay. REFERENCES
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(19X0)113.-1’3. M.. Inagami M. and Kawash52. Tanaka K.. Kamiyoshi ima M.: Presence of a testosterone receptor in the pituitary of the male chicken. Proc. XVI Wnfftl’.s Poltll. Ci>il(/“.. Vol. 7 ( 1978) 1I77 I I x3. J. A.. Mawhinney M. G. and 43. Zepp E,. A.. Thomas Lloyd J. W.: Differential response to either bovine or ovine prolnctin on testosterone-‘H (T-Ha) metabolism of various lobes of the rat prostate gland. Plz~rrr~tcccolo(/iSl 1s (I 973) 256. 44. Bardin C. W.: Pituitary--testicular axis. In Rrprdwtiro El~dot,~r,~cl/o~~~ (Edited by S. S. <‘. Yen and R. R. Jaffe). Saunders. Philadelphia f 197X) p_ 123, pp. I lo- 175.
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