REPRODUCTION IN MALE BIRDS BY MEANS OF EXOGENOUS PROLACTIN AND FOLLICLE STIMULATING HORMONE

Transcription

1 AN INVESTIGATION OF THE REFRACTORY PERIOD OF REPRODUCTION IN MALE BIRDS BY MEANS OF EXOGENOUS PROLACTIN AND FOLLICLE STIMULATING HORMONE By B. LOFTS and A. J. MARSHALL From the Department of Zoology and Comparative Anatomy, St Bartholomew's Medical College, University of London (Received 5 December 1957) SUMMARY At the end of the breeding season wild birds become refractory to photostimulation, and at the same time there is cessation of spermatogenesis, production of cholesterol-positive tubular lipids, tubular collapse and an overall reduction in size of the testis. These natural events can be duplicated by hypophysectomy or prolactin injection. Just before the onset of such changes the males of at least some wild species produce prolactin. It is shown in pigeons that exogenous prolactin is probably without direct effect on the avian testis, operating merely by inhibiting adenohypophysial activity. However, since in pluralbrooded wild species such collapse is delayed until the last clutch of the season has been fertilized and the males of some of these also brood the eggs, it seems likely that, in nature, prolactin can only temporarily depress, and not inhibit, the output of gonadotrophin. The seasonal inhibition of adenohypophysial function, and the resultant metamorphosis of the testis, is probably essentially under neural control with external stimuli ultimately involved. As regards the termination of the refractory period, it is demonstrated that very small quantities (0\m=.\6i.u.) of exogenous follicle stimulating hormone will clear the tubules of experimentally produced (by hypophysectomy) 'post-nuptial' lipids and cholesterol almost immediately after their appearance. This is interpreted as confirmatory evidence that it is the anterior pituitary, and not the testis, that becomes seasonally refractory. The time taken for the rhythmical annual recovery of adenohypophysial function is probably one of the most important events in the regulation of avian breeding seasons and migration. At the end of the breeding season of wild birds the interstitial cells become exhausted, the seminiferous tubules collapse, and their contents undergo a massive steatogenesis involving the production of cholesterol [Marshall, 1949]. It has long been known that both hypophysectomy [Hill & Parkes, 1934] and injections of prolactin [Riddle & Bates, 1933] will stop spermatogenesis and lead to collapse of the testis. Further, it has been demonstrated by Coombs & Marshall [1956] and Lofts & Marshall [1956] that such changes are accompanied by the cholesterol-producing metamor phosis described above. At the period of collapse and steatogenesis the bird becomes unresponsive to experimental photostimulation and enters a refractory period during which sexual behaviour is extinguished. For several months, the duration depending on the species, reproduction is impossible. It is probable that the essential cause of the metamor phosis of the testis is a seasonal, post-nuptial reduction in the output of gonado trophins [Marshall, 1952], but it has been a matter of doubt whether the testis, in addition to the adenohypophysis, becomes refractory.

2 92. LOFTS AND A. J. MARSHALL At the period of incubation, about the time when the refractory period starts in single-brooded species, increased amounts of prolactin are liberated by both sexes [Riddle & Bates, 1933; Bailey, 1952]. Whether the anti-gonadal effect of prolactin is mediated by the suppression of gonadotrophic influences [Bates, Riddle & Lahr, 1937] or whether there is a direct effect exerted on the testes has not been proved. The aim of the present work was twofold: (1) to determine whether prolactin directly influences the seasonal metamorphosis of the testis, and (2) to observe the effect of small doses of follicle stimulating hormone (FSH) on the clearance of the products of metamorphosis (tubular lipids and cholesterol) which are not normally dispersed for several months and sometimes not until the beginning of the next sexual season. MATERIALS AND METHODS (1) Effect of prolactin Sixteen adult male domestic pigeons were unilaterally laparotomized during the breeding season and the size of the left testis of each one measured (Table 1) in situ under open ether anaesthesia. The wounds healed uneventfully within a few days. The anterior pituitaries were then removed from fourteen such birds by the parapharyngeal technique described by Schooley [1939]. The birds were anaesthetized an by in jection of veterinary 'Nembutal' into the radial vein. After a recovery period of 2 days, the birds were divided into three groups. The two non-hypophysectomized birds constituted group I. Those of group II (five) were given ten daily injections of 1 ml. distilled water. The birds of group III (nine) were given ten daily injections of various dosages of prolactin in 1 ml. distilled water (Table 1). All injections were given i.m. into the pectoral muscles. It was believed that if prolactin is capable of producing a direct effect on the testes (apart from merely inhibiting gonadotrophin output), differences should appear in the degree of the metamorphosis of the testis exhibited by birds of groups II and III. Twenty-four hr after the last injections all specimens were killed and the testes dissected out for histological examination. (2) Effect of FSH Twenty-eight male pigeons were subjected to 4 weeks of intensive photostimula tion to ensure that their gonads enlarged to approximately maximum size (as sub sequently confirmed by laparotomy). Twenty-six birds were then hypophysecto mized and left for a period of 25 days during which time metamorphosis occurred, as proved by unilateral gonadectomy and histological examination. The two intact control birds were also killed and their gonads examined. After 3 days (which allowed castration wounds to heal) the twenty-six hypophys ectomized, unilaterally castrated birds were injected with various amounts of FSH (Table 2) in order to discover whether a differential clearance of cholesterol-positive 'post-nuptial' lipids would take place. All injections were made up in 0-5 ml. distilled water in ten consecutive daily doses. Each bird was then killed and the remaining (right) testis examined. The histological material from both studies was treated in the following uniform manner : after measurement of the whole organ a portion was fixed in formal-calcium solution and another part in Bouin's fluid. The formal-calcium fixed material was

3 REPRODUCTION IN MALE BIRDS 93 Sections were embedded in gelatine and sectioned at 8µ on a freezing microtome. coloured with Sudan black to reveal lipids and counterstained with haemalum. The Bouin-fixed material was embedded in wax, cut at 6µ and stained with iron haemat oxylin and orange G to reveal spermatogenetic stages. Gelatine sections were set aside for the Schultz test for cholesterol. Table 1 RESULTS (1) Effect of prolactin shows that the testes of all hypohysectomized birds were reduced from an average of mm to an average of mm. Those of the two nonhypophysectomized control birds remained large and one showed a slight increase in size. The testes of the birds injected with prolactin averaged mm and were no smaller than those of the ones injected with water. Table 1. Effect of various doses of prolactin on size of the testis in hypophysectomized pigeons after 12 days Size of left Size of left testis at testis at Specimen no. Daily injection laparotomy (mm) death (mm) 1* None x10 2* None ml. water x8 4 1 ml. water ml. water ml. water x5 7 1 ml. water i.u. prolactin x i.u. prolactin i.u. prolactin x i.u. prolactin 24-5x i.u. prolactin X i.u. prolactin x i.u. prolactin i.u. prolactin x i.u. prolactin x5 * Non-hypophysectomized controls. Histological appearance (a) Controls (two birds). Spermatogenesis was active in both birds. The tunica of the testis was 25 µ thick and enclosed expanded seminiferous tubules 350 µ wide containing bunches of spermatozoa. The lipid material had arisen within the tubules. The Leydig cells contained normal cholesterol-positive lipid droplets in their cytoplasm (Plate, fig. 1). (b) Hypophysectomized birds injected with water (five birds). These showed striking changes. Most tubules were in the first stages of 'post-nuptial' steatogenesis and contained a large number of small lipid droplets which gave a faint positive reaction with the Schultz test (Plate, fig. 2). In all birds spermatogenesis was suppressed. In most tubules only spermatogonia and occasional primary spermatocytes were present, although odd tubules still retained a few secondary spermatocytes and spermatids. The tunica of the testis had thickened to an average width of 80 µ and

4 94. LOFTS AND A. J. MARSHALL the tubules had shrunk to an average diameter of only 135 µ. The cytoplasm of the interstitial cells contained dense cholesterol-positive lipids. (c) Hypophysectomized birds injected with prolactin (nine birds). These pigeons, like those injected with water, showed the first stages of tubular steatogenesis. The average thickness of the tunica was 65/x. The tubules averaged 134µ, in diameter and were similar to those of the preceding group in their histological appearance and lipid content. Like the above, none had yet produced the dense amorphous mass of strongly cholesterol-positi ve lipids that occludes the narrow lumen of a fully regressed tubule (Plate, fig. 3). The Leydig cells were lipoidal and resembled those of the specimens injected with water. The organs were neither more nor less metamor phosed than those of birds injected with water. (2) Effect of FSH Histological appearance (a) Intact birds (two birds). These displayed the same morphological and histo logical appearance as the unhypophysectomized birds of the preceding experiment. The enlarged tubules contained spermatozoa but no hpid or cholesterol. The interstitium was densely sudanophilic and cholesterol-positive, and was restricted by the expanded tubules to tight wedges of tissue (Plate, fig. 1). ( ) Hypophysectomized birds injected with water (four birds). Left testis (removed before injections) : as in the prolactin experiment, removal ofthe anterior pituitary was followed by collapse of the testis and tubular steatogenesis (Plate, fig. 3). In the present cases the metamorphosis was greater due, no doubt, to the longer post operative period allowed in the present experiment before removal of the gonad. All four birds had greatly reduced gonads (average size : 8 3 mm). The tunica measured between 85 and 110 µ. in thickness and consisted of the old sheath and a newly formed layer of fibroblasts beneath. The tubules were reduced to a width of only 65 µ and contained only spermatogonia and a few necrotic nuclei (Plate, fig. 4). A dense mass of cholesterol-positive lipid completely blocked the lumina. The Leydig cells con tained numerous lipid droplets and cholesterol. Right testis (removed after injections): in each bird it was identical in size and histological appearance with the companion (left) organ removed 13 days earlier. (c) Hypophysectomized birds injected with FSH (twenty-two birds). Left testis (removed before injections) : these measured between 8x2 mm and 11x4-5 mm and had a tunica varying in thickness between 55 and 85µ. The histological appearance of each was that of a typical post-nuptial metamorphosed gonad and identical with that described in the preceding group. Right testis (removed after injections) : in all birds except two (which died after 3 and 4 days' injections respectively) FSH (even in doses as small as 0-6 i.u.) caused the total disappearance of the dense mass of 'post-nuptial' cholesterol and lipid material from the metamorphosed seminiferous tubules (Plate, fig. 5). It is im possible accurately to gauge what would approach a normal ' physiological ' amount of FSH but the observed effect of 0-6 i.u. on the tubular lipids seems to indicate that the products of tubular metamorphosis exert no barrier to FSH once the adeno hypophysis has again begun to produce it. Even the smallest amount (0-6 i.u.) of FSH led also to a resurgence of spermatogenetic activity and a correspondingly

5 - REPRODUCTION IN MALE BIRDS 95 increased size of the testis. The degree of recrudescence varied with the amount of FSH injected (Table 2). Thus, birds 7 and 8 (maximum dose) possessed a surviving testis that had enlarged to mm (stretched tunica reduced to a thickness of 15/t), with seminiferous tubules between 150 and 200µ, in diameter. Birds that had received from 2-5 to 10 i.u. showed an expansion to an average of mm (tunica 25-50jU. thick) with tubules 130µ, wide. Below this dosage (specimens 23-28) Table 2. Specimen no. Pituitary * 15 lot Effect of various doses of FSH on the intra-tubular lipids of hypophysectomized pigeons after 25 days Injection None None 0-5 ml. water 0-5 ml. water 0-5 ml. water 0-5 ml. water i.u. FSH 10 i.u. FSH 10 i.u. FSH 10 i.u. FSH i.u. FSH 1-2 i.u. FSH 1-2 i.u. FSH 0-6 i.u. FSH 0-6 i.u. FSH 0-6 i.u. FSH Intra-tubular lipids,-'-> Left testis Right testis Degree of spermatogenesis Spermatozoa Spermatozoa Spermatozoa Spermatozoa, Present;, absent; * died after three injections; f died after four injections. the remaining testis showed only a slight increase to an average of 12-5x4 mm (tunica 75µ thick) and a tubular diameter of 95µ. In specimens 7-12 all stages of spermatogenesis up to spermatids were present ; in two birds (8 and 9) spermatozoa were seen. Specimens contained numerous spermatogonia and primary sperma tocytes, but secondary spermatocytes were much less common (Plate, fig. 6), and spermatids were present only in birds 17 and 22. The remainder (23-28) had produced merely spermatogonia and a few primary spermatocytes. Birds 14 and 16, which died prematurely, had the right testis in an earlier stage of lipid clearance than the longer-lived birds. Yet clearance was already well advanced, only comparatively small amounts remaining in the form of small droplets tightly clustered in what appeared to be Sertoli cells. These droplets gave a positive Schultz reaction. The overall testis size was mm in no. 14 and 11x4 mm in no. 16

6 96. LOFTS AND A. J. MARSHALL with a tunica of 40 and 60µ, thickness, respectively. The tubules had expanded in each individual to a diameter of only 80/x, and primary spermatocytes were the most advanced spermatogenetic stage present. The interstitium was lipoidal and cholesterol-positive and appeared to be identical with that observed in the others. DISCUSSION The above results strongly suggest that prolactin exerts no direct influence on the seminiferous tubules. Once a bird is hypophysectomized, tubular steatogenesis (identical with the normal post-nuptial event) is inevitable without further inter ference. This was shown by Coombs & Marshall [1956] and is demonstrated addition ally by birds 3-7 in Table 1. If prolactin had a direct influence (instead of merely depressing pituitary activity), the various dosages of injected hormone and water (Table 1) would be expected to produce differential effects by the end of 11 days. No variation in the degree of steatogenesis was found. Although such comparatively large, non-physiological doses of exogenous pro lactin demonstrably cause metamorphosis of the tubules, it would nevertheless be unjustifiable to conclude that the endogenous hormone does more than partially and temporarily depress adenohypophysial function. Proof that the seasonal post nuptial steatogenesis and collapse of the testis is not due solely to the action of pro lactin seems to be inherent in the capacity of many plural-brooded species to produce successive clutches, the eggs of which are fertilized without intervening testicular metamorphosis. The only alternative is to assume that the males of double or treblebrooded passerines (e.g. house sparrow, Passer domesticus) do not produce prolactin even though they indubitably brood and share other parental duties [Summers-Smith, personal communication]. The principal cause in nature of pituitary refractoriness (leading to tubular steato genesis) is probably not hormonal but neural, with external stimuli, including be haviour, directly involved. When field conditions do not satisfy traditional breeding requirements, the testes of certain Arctic and xerophilous species undergo a prema ture metamorphosis [Marshall, 1952; Keast & Marshall, 1954]. At the same time, gross artificial photostimulation, perhaps leading to adenohypophysial exhaustion, will bring about comparable effects [Bissonnette & Wadlund, 1932]. The results of both the prolactin and FSH investigation described above show that the events occurring in the gonads of domestic cockerels after removal of the anterior pituitary [Coombs & Marshall, 1956] are duplicated in hypophysectomized pigeons, and that this artificially-induced metamorphosis is identical with that observed after repro duction in many breeds of wild birds. Miller [1949] had previously produced primary spermatocytes in a migratory passerine species (Zonotrichia coronata) with relatively massive, non-physiological doses (50 i.u.) of pregnant mares' serum (PMS) just before the natural end of its refrac tory period. We have now shown that, in pigeons at least, the administration of doses of as little as 0-6 i.u. of FSH (i.e. about the equivalent of 1 % of Miller's dosage) causes the clearance of dense masses of recently formed cholesterol-positive tubular lipids and produces an overall enlargement of the testis and the production of spermatocytes. Such results support the evidence of Benoit, Assenmacher & Walter

7 REPRODUCTION IN MALE BIRDS 97 [1950] and Greeley & Meyer [1953], obtained by other means, that the pituitary periodically becomes refractory. It has been shown that, even after the removal of the adenohypophysis, the interstitium renews itself and a new testicular tunica is formed as part of an intrinsic testis rhythm [Coombs & Marshall, 1956]. The above results indicate that the linger ing tubular lipids do not exert an antagonizing effect on even small amounts of FSH and suggest that the testis can be refractory for only the briefest period during which the new post-nuptial generation of Leydig cells achieve maturity. That it is principally the seasonal post-nuptial refractoriness of the anterior pitu itary which prevents reproduction for some months after each breeding season now seems undeniable. The seasonal recovery of gonadotrophic function is probably one of the most significant regulators in the avian sexual cycle. Thus, the sooty tern (Sterna fusca) of equatorial Ascension Island breeds about every 9-6 months [Chapín, 1954]. It is probable that each breeding rhythm follows closely upon the recovery of gonado trophic function after the preceding one, the vast flocks being synchronized by social stimulation. Trans-equatorial migrants (and perhaps even many sedentary species), hkewise, probably come spontaneously into seasonal reproductive condition and, after a period of display and resultant sexual synchronization, reach a condition that will send them towards their breeding ground at about the same time each year. Marshall & Serventy [unpublished observations] have shown that captive migratory shearwaters (Pufßnus tenuirostris) came into breeding condition at the same period as the wild birds that had flown across the equator to Japan, 'wintered' in Aleutian seas, and returned south to Tasmania again. This is not to say, of course, that such an endo genous rhythm could remain perpetually in step with the seasons. An external regulator must operate somewhere : but there is now evidence that at least some avian species possess an internal rhythm of remarkable regularity. We express our gratitude to the Endocrinology Study Section of the National Institutes of Health, Bethesda, Maryland, U.S.A., for the prolactin used in this investigation, and to Parke, Davis and Co. for the gift of FSH. REFERENCES Bailey, R. E. [1952]. Condor, 54, 125. Bates, R. W., Riddle, O. & Lahr, E. L. [1937]. Amer. J. Physiol. 119, 610. Benoit, J., Assenmacher, I. & Walter, F. X. [1950]. C.R. Soc. Biol., Paris., 144, 573. Bissonnette, T. H. & Wadlund, A. P. R. [1932]. J. exp. Biol. 9, 339. Chapín, H. P. [1954]. Auk, 71, 1. Coombs, C. J. F. & Marshall, A. J. [1956]. J. Endocrin. 13, 107. Greeley, F. & Meyer, R. K. [1953]. Auk, 70, 350. Hill, R. T. & Parkes, A. S. [1934]. Proc. Roy. Soc. B, 116, 221. Keast, J. A. & Marshall, A. J. [1954]. Proc. zool. Soc, Lond., 124, 493. Lofts, B. & Marshall, A. J. [1956]. J. Endocrin. 13, 101. Marshall, A. J. [1949]. Quart. J. micr. Sci. 90, 265. MarshaU, A. J. [1952]. Ibis, 94, 310. Miller, A. H. [1949]. Science, 109, 546. Riddle, O. & Bates, R. W. [1933]. Endocrinology, 17, 689. Schooley, J. P. [1939]. Endocrinology, 25, 372.

8 98. LOFTS AND A. J. MARSHALL DESCRIPTION OF PLATE Fig. 1. Testis of intact pigeon showing bunched spermatozoa in the large, lipid-free tubules. The inter stitial cells are heavily lipoidal. (Formal-calcium and Sudan black, 8µ; 300.) Fig. 2. Testis of pigeon 12 days after hypophysectomy. The black mottling indicates the beginning of tubular steatogenesis. (Technique and magnification as in Fig. 1.) Fig. 3. Testis of pigeon 25 days after hypophysectomy, showing tubular collapse and massive steato genesis. (Technique as in Fig ) Fig. 4. As Fig. 3, but at magnification of 500. The lipids have been dissolved out by alcohol. (Bouin and iron haematoxylin, 6µ.) Fig. 5. Right testis of hypophysectomized pigeon after ten daily injections of 2-. Tubular lipids have disappeared and the tubules have increased in size (cf. Fig. 3). (Technique and magnifica tion as in Fig. 3.) Fig. 6. As Fig. 5, but at magnification of 550. (Bouin and iron haematoxylin, 6/u.)

9 JOURNAL OF ENDOCRINOLOGY, Vol. 17, No. 1 B. LOFTS and A. J. MARSHALL PLATE (Facing p. 98)