Reproductive endocrinology of free-living nestling and juvenile starlings, Sturnus vulgaris; an altricial species

Plasma concentrations of luteinizing hormone (LH), prolactin and testosterone, and pituitary content of LH and prolactin, were measured in free-living starlings, Sturnus vulgaris , from hatching until 12 weeks of age.
Plasma LH concentrations were elevated in both sexes until four days after hatching, then they decreased. Throughout the period, plasma LH levels were low compared to those in breeding adults but were comparable to levels in post-breeding photorefractory adults. Pituitary LH content increased until 12 days after hatching, but this increase was due to physical growth during this period. Plasma prolactin concentration and pituitary prolactin content increased dramatically during the nestling period. The increase in pituitary prolactin content was in excess of that accounted for by increasing size. Plasma prolactin remained high during the immediate post-fledging period, but had started to decrease by 12 weeks after hatching. Plasma testosterone concentrations were lower than those in breeding adults, but generally higher than in post-breeding photorefractory adults. The gonads of both sexes remained regressed.
These results suggest that the reproductive system of nestling and juvenile starlings is in a similar state to that of post-breeding photorefractory adult starlings. The comparatively high levels of testosterone may reflect involvement in sexual differentiation.     

Lack of gonadotrophin-releasing hormone (GnRH) neuron response to decreasing photoperiod in thyroidectomized male starlings (Sturnus vulgaris)

Exposure of starlings to long days initially causes reproductive maturation, but eventually leads to photorefractoriness. During photorefractoriness, gonadotrophin-releasing hormone (GnRH) decreases in the GnRH cell bodies and fibers emanating from these to the median eminence, circulating gonadotrophin concentrations decrease to a minimum, and the gonads regress. Thyroidectomy profoundly affects these photoperiodic responses. In chronically thyroidectomized starlings, gonadal responses to changes in day length are attenuated. This investigation was conducted to determine whether, in the absence of gonadal responsiveness, the GnRH system of chronically thyroidectomized starlings responds to changes in day length. Two groups of thyroidectomized male starlings were transferred from short days (8L:16D) to long days (18L:6D) for four weeks, and testicular volume increased. One group was kept on long days (TxLD) and the other was returned to short days (TxSD). Testicular volume did not decrease in the TxSD group. The GnRH neurons of the two thyroidectomized groups were compared to those of two groups of intact starlings, one of them on long days and photorefractory (ILD), the other on short days and photosensitive (ISD). Group ILD had lower numbers of GnRH-stained cells than groups TxLD, TxSD and ISD, which did not differ in this respect. Similar differences were observed for GnRH cell size in the pre-optic area (POA) and for density of staining of GnRH fibers in the median eminence. The results confirm that thyroidectomy attenuates gonadal responses to change in day length and suggest that this results from an effect upon the central nervous system rather than a peripheral effect.     

Photorefractoriness in European starlings: associated hypothalamic changes and the involvement of thyroid hormones and prolactin

Responsivity to photostimulation in previously photorefractory European starlings is caused by subjection to short daylengths and is characterized by a marked activation of the hypothalamus in terms of synthesis of gonadotropin releasing hormone. This active hypothalamic state is amplified for a time by a subsequent exposure to long days but is soon completely reversed as the birds become photorefractory again. This latter effect of long photoperiods and the concurrent secretion of prolactin are dependent on the presence of thyroid hormones. Conceivably, prolactin causes photorefractoriness by inhibition at a hypothalamic level.     

Short days induce premature reproductive maturation in juvenile starlings, Sturnus vulgaris

Castration of juvenile and photorefractory adult starlings caused no immediate increase in circulating concentrations of LH. In castrated juveniles and adults exposed to natural changes in daylength, plasma LH increased between mid-October and mid-November, although the increase was more rapid in adults. In castrated photorefractory adults, plasma LH increased 3-5 weeks after transfer to artificial short days (8L:16D). In castrated juvenile starlings plasma LH increased 4-6 weeks after transfer to 8L:16D, irrespective of the age of the birds. Birds as young as 17 weeks had high LH concentrations. These results suggest that the reproductive system of juvenile starlings is in the same state as that of photorefractory adults, and therefore the activation of the hypothalamo-pituitary axis for the first time in juveniles is analagous to the termination of photorefractoriness in adults.     

The LH-RH system of the male European starling: photoperiod induces changes to a possible multifunctional peptide system

In many birds reproduction is triggered by long daylengths but, paradoxically, continued exposure to long days leads to photorefractoriness and a complete shut down of the reproductive system. As these effects are thought to be mediated through the secretion of LH-RH, immunocytochemical techniques were used to investigate changes in the LH-RH system when European starlings were exposed to different photoperiods. Starlings exposed to 11L:13D and with mature testes show strong immunostaining both of LH-RH perikarya and fibers. Photosensitive short-day (8L:16D) starlings with undeveloped testes show an almost identical distribution of strongly immunoreactive perikarya but with less dense fibre staining. However, long-day (18L:6D) photorefractory starlings with fully regressed testes, show a profound reduction in LH-RH immunostaining. Perikarya have the same distribution but show a much reduced intensity of staining and fibers had almost entirely disappeared from all regions of the brain. Preliminary observations on the ultrastructure of immunocytochemically identified LH-RH neurones are also reported.     

Effect of daylength on the rate of recovery of photosensitivity in male starlings (Sturnus vulgaris).

Four groups of castrated photorefractory starlings were transferred from a photoperiod of 18 h light/day (18 L) to photoperiods of 6 L, 8 L, 11 L or 12 L. A control group was kept on 18 L. Plasma concentrations of luteinizing hormone (LH) were low in all groups initially. The first significant increase in LH, which signals the recovery of photosensitivity, occurred after 4 weeks in the groups on 6 L and 8 L, after 8 weeks in the 11 L group and after 13 weeks in the 12 L group. There was no increase in the group on 18 L. The rate of recovery of photosensitivity is therefore inversely proportional to daylength, for daylengths between 8 L and 12 L.     

Modulation of gonadotrophin and prolacrin secretion by daylength and breeding behaviour in free-living starlings, Sturnus vulgaris

Plasma levels of luteinizing hormone (LH), follicle-stimulating hormone (FSH) and prolactin were measured by radioimmunoassay in plasma samples collected from free-living starlings, Sturnus vulgaris , trapped in nest-boxes. By leaving some nest-boxes undisturbed, and repeatedly destroying nests in others, birds from a single-brooded population were trapped whilst nest-building, incubating or feeding nestlings, at different times throughout the normal breeding season. In both males and females trapped whilst nest-building, plasma LH and prolactin levels increased progressively from mid March until late May. In females sampled during incubation, LH and FSH levels were high throughout May but decreased in early June. Prolactin levels were highest in late May. In both males and females trapped during mid May, LH levels were highest in these birds which were nest-building at this time and lowest in those feeding nestlings, FSH did not change significantly, and prolactin was low in those birds which were nest-building and high in those incubating or feeding nestlings. In female starlings from a double-brooded population, levels of LH and FSH were much lower whilst feeding the second brood than when feeding the first brood, whereas prolactin levels were similar. The results suggest that incubation and feeding behaviour inhibit the increase in LH secretion caused by increasing daylength, but stimulate prolactin secretion in excess of that caused by increasing daylength.     

Effect of thyroidectomy on rhythmic gonadotropin release.

Nonstress blood samples were obtained from intact and thyroidectomized (TE) male rats at 3-hr intervals over a 24-hr period via rapid decapitation. The animals were thyroidectomized when 40 days old and used 6 weeks later. Intact animals showed periodicity in serum LH (P less than 0.01) and prolactin (P less than 0.01). Both gonadotropins began increasing after 8 PM and peak levels occurred at 11 PM. In contrast, 24-hr periodicity was not observed in serum FSH. Corticosterone levels in these same serum samples showed the expected circadian periodicity. After TE, the 24-hr pattern in all gonadotropins was altered significantly. Serum LH increased (P less than 0.01) and circadian periodicity appeared to be absent. FSH and prolactin levels were increased and decreased, respectively (P less than 0.01), with serum prolactin showing a 9-hr phase shift. Prolactin began increasing at 2 AM and reached a peak at 8 AM. Corticosterone in TE animals showed a 24-hr rhythm similar to that of intact rats. These findings confirm our previous observations that nonstress serum LH and prolactin levels fluctuate with a 24-hr periodicity and suggest that the level of, and the phase angle betweeen, these rhythms is markedly influenced by pituitary-thyroid activity.     

Differential effects of photoperiodic history on the responses of gonadotrophins and prolactin to intermediate daylengths in the male Syrian hamster

The effect of photoperiodic history on the neuroendocrine response to intermediate daylengths (11-13.5 hr of light) was investigated in the male Syrian hamster. The duration of the nocturnal peak of pineal melatonin content was inversely proportional to photoperiod and independent of photoperiodic history. Serum levels of prolactin were lower in animals exposed to shorter photoperiods. Photoperiodic history had little effect on the response of serum prolactin to intermediate daylengths. Serum luteinizing hormone (LH) concentrations were also lower in shorter photoperiods, but in addition were sensitive to the direction of photoperiodic change, so that a single photoperiod could be interpreted as either stimulatory or inhibitory to LH secretion. This effect of photoperiodic history was expressed at intermediate photoperiods with 12-13.5 hr of light. The sensitivity of serum follicle-stimulating hormone (FSH) levels to photoperiodic history was masked by an early onset of photorefractoriness. Testicular size and serum testosterone levels revealed weaker effects of photoperiodic history; these were attributed to the dissociation between gonadotrophin and prolactin secretion induced by intermediate daylengths. The contrasting effects of photoperiodic history on the secretion of LH and prolactin may represent the expression of multiple photoperiodic time-measuring systems.     

Thyroxine can mimic photoperiodically induced gonadal growth in Japanese quail

Summary Pharmacological doses of thyroxine are able to mimic the effects of long photoperiods in Japanese quail. In birds maintained on short daylengths thrice-weekly injections of 100 g thyroxine cause full testicular maturation at rates not greatly different from those seen if quail are exposed to long days. Thyroxine stimulates increases in the secretion of FSH and LH, in pituitary prolactin content and in the hypothalamic content of Gn-RH. The effects are dose-dependent. If female quail kept on short daylengths are given thyroxine their ovaries develop and they lay eggs. In castrated male quail on short days thyroxine causes a ten-fold increase in circulating LH within a week. Thyroxine injections are also capable of maintaining quail in a photorefractory state even when they are transferred to short daylengths. The results suggest that thyroxine mimics long days by acting high in the photoneuroendocrine system and does not simply act to facilitate hormone secretion per se. This is in line with growing evidence in mammals and birds that parts of the photoperiodic machinery are sensitive to thyroid hormones.Abbreviations Gn-RH gonadotropin releasing hormone - FSH follicle stimulating hormone - LH luteinizing hormone - T ₄ thyroxine     

Gonadotrophin secretion and pituitary responsiveness to LHRH in castrated and intact male rabbits exposed to different photoperiods

Adult male wild rabbits were exposed to at least 16 weeks of 16L:8 D before experiments began. Plasma LH and FSH concentrations increased significantly (P less than 0.001) when rabbits were castrated in 16L:8D but declined when rabbits were transferred to 8L:16D. Concentrations had returned to normal for castrated rabbits in 16L:8D by 74 days after the start of the 8L:16D treatment. Treatment of intact male rabbits with an injection of LHRH before and after transfer to short daylengths caused a transient increase in plasma LH which lasted 50-80 min and this produced a concomitant rise in plasma testosterone. The daylength change had no effect on this response even though testicular size declined after the transfer to short daylengths. Rabbits moulted in response to exposure to 8L:16D. This suggests that hypothalamic activity responds to photoperiod and that changes in pituitary responsiveness to LHRH and steroid negative feedback are unimportant.     

Effect of triiodothyronine on the expression of T cell markers and immune function in thyroidectomized White Leghorn chickens.

Hypothyroid K-strain chickens were produced by neonatal thyroidectomy and treatment with 6-propyl-2-thiouracil. Thyroidectomized birds were given 0, 1.5, 4.5, 15, or 45 micrograms/kg body wt of triiodothyronine (T3) by daily injection. At 5 weeks of age, thymocytes were prepared for flow cytometric analysis of CT-1a, CD3, CD4, and CD8 expression. Sham-operated birds had the smallest proportion of CT-1a+ cells and the brightest CT-1a+ cells. Unsupplemented thyroidectomized birds presented an inverse picture, while T3-treated thyroidectomized birds were intermediate. Fewer and less brightly labeled CD3+, CD4+, and CD8+ cells were associated with sham-operated birds or with higher levels of T3 replacement. Low levels (1.5 microgram/kg body wt) or no T3 treatment produced a greater proportion of positive, highly fluorescent cells. The ratios of CD4+ to CD8+ thymocytes were increased (P less than or equal to 0.05) by T3 supplementation. Functionally, thyroidectomy produced a decrease in mitogen-stimulated proliferation of peripheral blood lymphocytes. This effect was ameliorated by T3 supplementation. Further, thyroidectomy produced an elevation of plasma growth hormone concentrations. These results suggest that thyroid factors and alterations of thymic status significantly affect the generation of specific thymus-derived lymphocyte populations and their functional capabilities, perhaps due to changes in the thymic microenvironment. These alterations may have important consequences for the development of immunocompetence and disease resistance in chickens.     

Thyroxine treatment prevents the development of photosensitivity in european starlings (Sturnus vulgaris)

Summary In starlings, the breeding season is terminated by a state of photorefractoriness. Birds remain completely reproductively inactive as long as long days are maintained, and only exposure to short days restores photosensitivity. Two experiments investigated the role of different doses of thyroxine in the development of photosensitivity in castrated starlings. First, photorefractory castrated male starlings were moved from long (18L:6D) to short (8L:16D) days, and received in the drinking water either 1 or 10 mg · 1^-1 thyroxine for the first 7 weeks of a 14-week observation period. Control birds regained photosensitivity after 5 weeks of short days, as signaled by a spontaneous increase in plasma LH, whereas the return to photosensitivity was delayed until weeks 7 and 9 in the 1- and 10-mg · 1^-1 thyroxine-treated birds, respectively. In the second experiment, the effect of different doses of thyroxine was explored at the level of the hypothalamic Gn-RH neurosecretory neurones. The acquisition of photosensitivity in control birds transferred from long to short days was characterized by a marked increase in hypothalamic Gn-RH content (while long-day controls maintained low Gn-RH content). Doses of 10 and 20 mg · 1^-1 of thyroxine completely prevented the return to photosensitivity, as seen through changes in either plasma LH concentrations or hypothalamic Gn-RH content, while a dose of 1 mg · 1^-1 allowed a partial recovery of photosensitivity, as hypothalamic Gn-RH content increased to an intermediate level and the spontaneous rise in plasma LH occurred slowly but steadily.Abbreviations Gn-RH gonadotrophin-releasing hormone - LH luteinizing hormone - LHRH-I luteinizing hormone releasing hormone     

Plasma prolactin levels in maturing intact and cryptorchid male rats: development of stress response.

In order to determine the possible role of the seminiferous tubules in the regulation of prolactin secretion during sexual development, male rats were rendered cryptorchid at 22 days of age, and thereafter different groups of animals were decapitated at 8-10 day intervals between Day 32 and Day 70. Cryptorchid rats showed destruction of the germinal epithelium accompanied by increased plasma FSH and, to a much lesser extent, increased plasma LH titers. Nevertheless, plasma prolactin levels were similar to those of intact controls throughout the entire period studied. Plasma prolactin titers in intact controls remained uniformly low from Day 20 to Day 70, contrasting with previous reports in which increasing prolactin levels have been observed during sexual development. To determine the reason for this apparent discrepancy, a longitudinal experiment was conducted in which intact and cryptorchid male rats were bled every 10 days from Day 30 to Day 70, following a 3-min period of exposure to ether fumes. The prolactin response to this stress increased markedly with age. A similar pattern of prolactin was observed in a cross-sectional study in which different groups of intact animals were bled following a 3-min period of ether exposure, at ages ranging from 20 to 70 daysmthe results indicate that unlike FSH secretion, prolactin secretion is not controlled by the seminiferous tubules. In addition, they suggest that the pattern of increasing plasma prolactin previously described in the developing male rat is at least in part caused by an age-dependent increase in responsiveness of prolactin to stress.     

Prior experience with photostimulation enhances photo-induced reproductive development in female European starlings: a possible basis for the age-related increase in avian reproductive performance

Reproductive performance in female birds improves with age, and this is generally attributed to experiences obtained during breeding. In temperate-zone species, experience with photostimulation during the first breeding year may prime the hypothalamo-pituitary-gonadal axis to respond to photic cues more rapidly or robustly in subsequent years. To test this idea, we captured 32 photorefractory juvenile (hence naive to photostimulation) female European starlings (Sturnus vulgaris) and held half of them (naive group) on a photoperiod of 8L:16D for 32 wk and the other half (experienced group) on 8L:16D for 12 wk, 16L:8D for 12 wk, and then 8L:16D for 8 wk. When we subsequently transferred all birds to 16L:8D, the increase in body mass, which may presage egg laying in the wild, was more robust in experienced than in naive females. Experienced females also showed a more robust elevation in plasma concentrations of the yolk-precursor protein vitellogenin, although naive females showed an initial rapid but transient rise in vitellogenin that we attribute to their extended exposure to short-day photoperiods prior to photostimulation. Finally, the photo-induced increase in diameter of the largest ovarian follicle, in plasma concentrations of luteinizing hormone, and in the number of septo-preoptic fibers relative to the number of cell bodies immunoreactive to GnRH was greater in experienced than in naive females. Thus, prior experience with photostimulation enhances some initial phases of photo-induced reproductive development and may explain, in part, why reproductive performance improves with age in temperate-zone birds.     

Timing of photorefractoriness in the European starling: significance of photoperiod early and late in the reproductive cycle

In European starlings, as in many other birds inhabiting higher latitudes, gonads develop in response to the increasing daylengths in early spring. Later in the year, however, the hypothalamo-pituitary-gonadal axis becomes refractory to the previously stimulatory long photoperiods and the gonads regress in summer. The present study addresses the question of when during the gonadal growth phase photorefractoriness is determined. A 13-h photoperiod induces testicular development and subsequent testicular regression associated with refractoriness in male starlings. An 11-h photoperiod, in contrast, induces only testicular development, and photorefractoriness never develops. When starlings were transferred to an 11-h photoperiod, either 12 or 25 days following exposure to a 13-h photoperiod, their testes developed to full size, but remained large to the end of the experiment, i.e. refractoriness did not develop. The same was even true of most birds in a third group that were transferred to an 11-h photoperiod after 46 days of the 13-h photoperiod, when gonads had developed to near maximal size. These data show that, in contrast to some other species of passerine birds, the onset of photorefractoriness does not become fixed before the testes have undergone considerable development, and that the photoperiodic conditions experienced at the end of the testicular growth phase are still effective in determining the precise time of onset of photorefractoriness. It is suggested that this peculiarity of the starling is related to the fact that its gonadal development begins rather early in spring and, hence, under much shorter photoperiods than the other species studied.     

Termination of photorefractoriness in the brahminy myna, Sturnus pagodarum: role of photoperiods and gonadal hormones.

In brahminy myna a photosensitive species, long days caused full gonadal development followed by rapid regression, whereas short days inhibited these responses. Experiments were performed to investigate the effects of duration of photoperiod and gonadal hormones on the recovery of photosensitivity to long photoperiods in male birds. Groups of photorefractory birds were subjected to 8-, 9- or 11-hr daily photoperiods for 45 (6.5 weeks) or 63 (9 weeks) days and then transferred to 15 h daily photoperiods for 60 days to check for the regaining of photoresponsivity. A control group was held under 15L:9D throughout the period of study. Another experiment included three groups of photorefractory males, which were maintained on 9L:15D for 9 weeks and administered with, birth-1day-1 alternately for first 30 days olive oil or different doses (10 or 100 micrograms) of testosterone propionate (TP)/bird/day alternately for first 30 days, and then transferred to 15L:9D for another 30 days to test the recovery of photosensitivity. The results indicated that (i) a period of exposure to short daylengths is required to dissipate photorefractoriness, (ii) termination of photorefractoriness is dependent on the length and duration of photoperiods and (iii) TP inhibits the recovery of photosensitivity in a dose dependent manner.     

Effect of breast-feeding on pituitary-ovarian function after childbirth

Pituitary and ovarian function at the end of pregnancy and during the first six weeks after delivery was investigated serially in women who fully breast-fed their infants and in women who did not. In the women who did not breast-feed the plasma prolactin level decreased rapidly and from the third day after delivery was significantly lower than in the breast-feeding mothers, reaching the normal range of the menstrual cycle by the third week of the puerperium. In the breast-feeding mothers the plasma prolactin was still raised six weeks after delivery. The levels of FSH in both groups were identical and increased over the third week of the puerperium. Plasma oestrogen fell steeply in both groups during the first two weeks after delivery. In the breast-feeding mothers plasma oestrogen remained depressed but increased in the non-lactating women, reflecting follicular development in the ovary in response to FSH; the plasma oestrogen levels were significantly higher in the non-lactating women from the 17th day of the puerperium onwards. These findings support the concept that in breast-feeding women prolactin delays the return of ovulation by inhibiting the ovarian response to FSH stimulation.     

Melatonin,immunity and cost of reproductive state in male European starlings.

The effects of reproductive condition and exogenous melatonin on immune function were investigated in castrated European starlings, Sturnus vulgaris. Photorefractory and photostimulated starlings exposed to long days were implanted with melatonin or with blank capsules. Photostimulated starlings with blank capsules exhibited reduced splenocyte proliferation in response to the T-cell mitogen, concanavalin A, compared with the other long-day birds. Exogenous melatonin prevented the suppression of immune function by photostimulation. Photorefractory starlings, with or without melatonin implants, exhibited enhanced immune function compared with photostimulated starlings implanted with blanks. This enhancement was not mediated by endogenous melatonin, but appeared to be related to changes in reproductive state. In addition to the traditional costs of reproduction in birds (e.g. raising of young), there may be a cost of the reproductive state of starlings (i.e. whether they are photorefractory or photostimulated). These data are, we believe, the first to indicate a direct effect of reproductive state on immune function that is independent of both photoperiod (i.e., changes in the duration of melatonin secretion) and gonadal steroids.     

Disjunction between the daily duration of light and the plasma level of prolactin under hyperlong days in rams

Adult rams were submitted to an artificial light regime with increasing and decreasing daylengths between 6 and 20 hrs. over a period of 8 months. Blood samples occurred at different durations of daylengths for measuring plasma levels of prolactin. Levels were high for a daily duration of light of 14.20, 17 and 18.30 hrs. along the increasing light-phase and of 18.30 hrs. during the decreasing light-phase. On the other hand prolactin levels were low for short daylengths, but also for a daylength of 20 hrs. It clearly appears that hyperlong days (greater than 18 hrs.) do not stimulate (or do actively inhibit) the secretion of prolactin in ovines.