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LH responses to chicken luteinizing hormone-releasing hormone I and II in laying, incubating, and out of lay turkey hens
Domestic Animal Endocrinology 17 (1999) 1–15
LH responses to chicken luteinizing hormone-releasing hormone I and II in laying, incubating, and out of lay turkey hens D. Gue´mene´*, J.B. Williams I.N.R.A., Centre de Tours-Nouzilly, Station de Recherches Avicoles, 37380 Nouzilly, France Received 26 August 1998; accepted 6 April 1999
Abstract An experiment was conducted to assess the relative in vivo and in vitro activities of chicken LH-RH-I and -II in laying, incubating and out-of-lay turkey hens. The highest plasma concentrations of LH were measured in laying turkey hens, whereas hypophyseal concentrations were highest in incubating hens (I) and lowest in the laying hens at the end of the laying period (EL). Hypophyseal and plasma concentrations of LH decreased with aging in laying hens (L) and the greater decrease occurred in the hypophyses. An in vitro hypophyseal acute challenge with 2-min pulses of cLHRH I or II (10⫺7 M) using a perifusion technique resulted in an increase in the release of LH in out-of-lay (OL) and incubating (I) hens, but not in laying (L) hens. Although both peptides elicited comparable responses in I hens, cLHRH II was more effective in OL hens. This difference was attributable to a greater amplitude of the response, whose duration was unchanged. Hypophyseal desensitization to a subsequent stimulation was observed in OL hens when the interval between stimulations was 30 min, but this did not occur at 60- or 120-min intervals. In vivo, the injection of cLHRH I or II, at doses of 10⫺8 and 10⫺10 M/kg B.W. stimulated increases in the plasma concentrations of LH, which were initiated within 1 min of injection in OL and I hens but from 5 to 20 min postinjection in L hens. The responses were dose-related and greater immediate responses were measured with cLHRH I than with cLHRH II. Also, after the injection of cLHRH II at the 10⫺8 M/kg B.W. dose, the shape of the LH response consisted of an initial increase, followed by a more sustained phase during which LH concentrations were either stable (I hens) or continued to increase (L and OL hens) from 20 to 60 min after injection. In contrast, the injection of cLHRH I at doses of 10⫺8 or 10⫺10 M/kg or cLHRH II
* Corresponding author. Tel.: ⫹02-47-42-76-43; fax: ⫹02-47-42-77-78. E-mail address: [email protected] (D. Guemene) 0739-7240/99/$ – see front matter © 1999 Elsevier Science Inc. All rights reserved. PII: S 0 7 3 9 - 7 2 4 0 ( 9 9 ) 0 0 0 2 0 - X
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at a dose of 10⫺10 M/kg in I and OL hens, produced a peak of LH concentrations in plasma within 5 min and thereafter declined gradually. The difference in the in vivo responses to LHRH I and II could not be attributed to a greater potency of cLHRH II, but to a more prolonged action. In summary, the responses to both forms of chicken LH-RH varies markedly with the stage of the reproductive cycle (L, I, and OL) and differs between the in vivo and in vitro situations. Although cLHRH II may be more active than cLHRH I, controversy still surrounds its precise physiological role. © 1999 Elsevier Science Inc. All rights reserved. Keywords: Turkey hens; LHRH; LH; Pituitary gland; Physiological stages
1. Introduction Brain tissues from several nonmammalian species contain at least two distinct luteinizing hormone-releasing hormones (LHRH). Thus, two forms of LHRH have been isolated in the chicken: cLHRH I, which is (Gln8)-LHRH [1,2] and cLHRH II, which is (His5-Trp7-Tyr8)-LHRH [3]. These two forms of LHRH were later identified in the ostrich [4], starling [5] and in female turkeys [6]. Results of studies on their relative efficiencies in stimulating LH release in birds are conflicting [7–14]. At the present time, cLHRH II is reported to be more potent in the male turkey both in vitro and in vivo and to have long–acting effects after in vivo injections [14]. However, there are no data available on the effects of these two peptides in turkey hens which are known to respond to the administration of synthetic mammalian LHRH in vivo with the release of LH [15–17]. There is controversy surrounding the physiological role of cLHRH II. Sharp et al. [13] have demonstrated by RIA on chicken brain sections that it is not a predominantly hypothalamic peptide but that it occurs in low amounts throughout the brain. Its concentration in the median eminence and the pre-optic area does not differ significantly in laying (L) and out-of-lay (OL) hens and active immunization against the peptide does not affect egg production. This argues against a role as a releasing hormone. However, an in vitro study of pituitary release of LH in chickens that had previously been passively immunized against cLHRH I or cLHRH II argue in favor of a physiological role of cLHRH II [18]. Moreover, it has been shown that both forms are present in the turkey hen hypothalamus [6] and that hypothalamic cLHRH II concentrations are only slightly lower than those of cLHRH I [19]. Furthermore, measurement of the hypothalamic concentrations of the two peptides in various reproductive states reveals a pattern of change of cLHRH-II that parallels that of cLHRH-I [19]. It has also been observed that both are present in the pituitary gland [20] and that treatment with ovine prolactin causes a rapid decrease in hypothalamic cLHRH I content and a more delayed decline in that of cLHRH II [19]. This study was designed to investigate the biologic properties of both forms of chicken LHRH by comparing their ability to induce the release of LH from turkey hen hypophyses
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in vitro using a perifusion technique, and the in vivo LH responses in L, OL or incubating (I) turkey hens.
2. Materials and methods 2.1. Animals Medium white turkey hens obtained before sexual maturity (BETINA, 56250, St. Nolff, Elven, France), were used in their first (experiments 2 and 3) or second productive period (Experiment 1). They were reared in individual pens and maintained under a lighting schedule of 14-hr light and 10-hr darkness during the productive period. They were fed on commercial diets available ad libitum and had free access to water. In Experiment 1, hens (5 to 6 per group) were assigned to the following physiological groups: new (NL) or old laying (end of productive period, EL) hens that had laid for 4 to 6 wk or for over 20 wk respectively, I and OL hens. The ages of the hens in these stages were 94 (OL), 95 (NL), 97 (EL), and 98 (I) wk of age, respectively. In Experiments 2 and 3, hens were used at three different physiological stages (L, I, and OL) between 35 and 42 wk of age. Hens were considered to be I or OL according to previously described criteria [17,22]. Briefly, a hen had to express broody behavior for at least 7 d or not to have laid an egg for at least 14 d to be classified as I or OL, respectively. The L hens used in this study had laid an egg in the 24-hr before the experiment. 2.2. Experimental approach 2.2.1 Experiment 1: Plasma and hypophyseal levels of LH in turkey hens New (NL) and EL, incubating (IL), and OL hens were killed by cervical dislocation. Just before death, a blood sample was withdrawn from a wing vein into a heparinized syringe. Thereafter, plasma was separated from blood by centrifugation at 2000 g for 10 min at 4°C and kept at ⫺20°C until assayed for LH. Hypophyses were removed, snap-frozen in liquid nitrogen and stored until assayed for LH. 2.2.2. Experiment 2: In vitro effects of cLHRH I and II on LH release in turkey hens As in Experiment 1, the hens were killed by cervical dislocation and a blood sample withdrawn from a wing vein into a heparinized syringe, just before death. Plasma was separated as above and samples assayed for LH, prolactin, progesterone, and estradiol. The ovary and the oviduct were removed and weighed. Hypophyses were removed, weighed, then placed in ice-cold sterile culture medium 199 [M199; Sigma-Chimie S.A.R.L., 38297, La Verpilliere Cedex France] supplemented with glucose (2.5 g/l), amphotericin B (2.5 mg/l), gentamicin (0.1%, w/v), BSA (0.1%, w/v), sodium bicarbonate (0.35 g/l), and (1 mM)] within 2 min of death. The gland were rinsed with M199 and diced with a scalpel blade. The equivalent of one-half of an hypophysis was placed in a 0.5-ml perifusion chamber of a specialized apparatus (Acusyst S, Endotronics, Coon Rapids, MN. 55433, USA).
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A preliminary experiment was conducted to establish the optimum flow rate using six preparations of OL hens subjected to three successive 100-min perifusion periods at 0.125, 0.25, and 0.5 mL/min. Chambers were perifused with M199 (pH ⫽ 7.2, temperature ⫽ 39°C) continuously gassed with 95% O2 and 5% CO2. After a 60-min equilibration period, the preparations were challenged with 10⫺7 M cLHRH II (Peninsula Laboratories Europe LTD., St. Helens, Merseyside, WA9 3AJ, England) using pulse lengths of 8 (n ⫽ 3) or 2 min (n ⫽ 3), then 4 or 2 min and finally 2 min. A three-way tap was used to switch between reservoirs of M199 and the test substance. In the subsequent experiments, six preparations were generally run simultaneously (n ⫽ 4 to 7 repetitions per physiological stage and treatment) at a flow rate of 0.5 ml/min. After 1 hr of equilibration with M199, the hypophysis fragments were exposed to 2-min pulses of test substances dissolved in M199. The preparations were challenged successively at intervals of 120, 60, and 30 min with cLHRH I (Sigma-Chimie S.A.R.L., France) or II at a dose of 10⫺7 M in M199. Between stimulations, the preparations were perifused with M199 alone. In all cases, the effluent was collected as 3-min fractions and stored at⫺20°C until used for radioimmunoassay of LH. The profiles of LH release were measured individually and the release of LH due to the stimulation was defined as the amount of LH secreted per chamber above basal level during the 21 min after stimulation [14]. Concentrations measured during the 30 min before the initial stimulation and during the 10 min before the subsequent stimulations were used to define each chamber’s basal level. 2.2.3. Experiment 3: In vivo effects of cLHRH I and II on LH release in turkey hens At each physiological stage (L, I, and OL), a group of 30 turkey hens (n ⫽ 30 ⫻ 3) were randomly allocated to one of the five following treatments (control, 0.5 ml of 0.9% (w/v) saline solution, n ⫽ 6; cLHRH I 10⫺8 M/kg body weight, n ⫽ 6; cLHRH I 10⫺10 M/kg B.W., n ⫽ 6; cLHRH II 10⫺8 M/kg B.W., n ⫽ 6; cLHRH II 10⫺10 M/kg B.W., n ⫽ 6). Treatments were dissolved in 0.9% (w/v) saline solution and administered (0.5 ml per) i.v. Blood samples (5 ml) were withdrawn just before injection (time 0), and at 1, 2, 5, 10, 20, and 60 min after injection. All samples were assayed for LH. 2.3. Radioimmunoassays 2.3.1. LH Plasma (Experiments 1, 2, and 3), hypophyseal extracts (experiments 1 & 2) and aliquots of perifusate fractions (Experiment 2) were assayed in triplicate according to Sharp et al. [10] using fraction PRC-AE1-s-1 [10] as labeled hormone and standard, and LH 3-3 antiserum [10]. This assay has been previously validated for the measurement of LH in turkey [17]. Mean within and between assay coefficients of variation were 5.7% and 13.2%, respectively. 2.3.2. Progesterone Plasma samples collected from all hens used in Experiment 2 were assayed using the method described by Duplaix et al., [23]. The mean within assay coefficient of variation was 2.9%.
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2.3.3. Estradiol Plasma samples collected from all hens used in Experiment 2 were assayed according to Etches et al. [24]. The mean within assay coefficient of variation was 4.6%. 2.3.4. Prolactin Plasma (Experiment 2) were assayed in triplicate 100 l samples following the method of Etches and Cheng [25], but using tPRL provided by Proudman and Opel [26] as labeled hormone and standard and the anti-tPRL-2-B3 provided by Etches and Cheng [25] as antiserum. The mean within assay coefficient of variation was 7.6%. 2.4. Calculations and statistical analysis Anatomic and physiological data from the hens included in experiments 1 and 2 were compared using a one-way ANOVA and Fisher’s PLSD (Protected Least Significant Difference) post-hoc test if appropriate (P ⬍ 0.05, ANOVA). The release of LH due to the stimulation itself was estimated as described previously [17] by linear regression of LH concentrations on time, using the initial basal values plus the concentrations measured in the three samples preceding each stimulation. The amount of LH released from each chamber during the 21 min after stimulation was calculated by estimating the difference between the LH concentration in each of the seven samples collected after stimulation and the expected basal release estimated by linear regression at the appropriate given times. The amounts of LH were thereafter compared using a two-factor analysis of variance (ANOVA. Dependent variables: length of the interval between stimulations; treatment). All data from Experiment 3 were analyzed using a repeated measures ANOVA. When significant treatment effects were observed (P ⬍ 0.05, ANOVA), data were further analyzed using Fisher’s PLSD post hoc test. All statistical analyses were performed using the StatviewTM II program (Abacus Concepts Inc., Berkeley, CA, USA) for the Apple Macintosh computer.
3. Results 3.1. Experiment 1: Plasma and hypophyseal levels of LH in turkey hens (Fig. 1) Plasma levels of LH were the highest in both groups of laying hens (NL and EL) and the lowest in I and OL hens. Differences reached significance (P ⬍ 0.05) between NL and I or OL hens. Hypophyseal concentrations of LH were the highest in I hens and the lowest in EL hens, whereas intermediate concentrations were measured in NL and OL hens and the physiological stage effect was close from statistical significance (P ⫽ 0.06). 3.2. Experiment 2: Anatomic and hormonal characteristics of the turkey hens (Tables 1 and 2) Regressed reproductive tracts were observed in all OL and I turkey hens whereas they were fully developed and functional in L hens. The pituitary gland wet weights did not differ
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Fig. 1. Plasma (ng/ml) and hypophyseal (g/hypophysis) concentrations of LH in laying turkey hens at the beginning or at the end of a productive period, and in I and OL turkey hens, respectively (means ⫾ SEM). * P ⬍ 0.05, Fisher’s PLSD test)
between physiological stages. LH plasma concentrations differed between physiological stages as in experiment one. Similarly, prolactin plasma concentrations differed significantly between physiological stages (P ⬍ 0.05). They were at the lowest in OL hens while intermediate and high concentrations were measured in L and I hens, respectively. Both progesterone and estradiol plasma concentrations measured in L hens were significantly (P ⬍ 0.05) higher than those measured in I and OL hens. 3.3. Experiment 2: Effects of cLHRH I and II on the in vitro LH release (Figs. 2– 4 ) There was no significant effect (P ⬎ 0.05) of flow rate on basal LH release by pituitary gland nor the response to 2 min challenge with cLHRH II using preparations from OL Table 1 Pituitary, ovary and oviduct weights of laying, incubating and out-of-lay turkey hens (means ⫾ S.E.M.)
Hypophysis (mg) Oviduct (g) Ovary (g) a,b
Laying (n ⫽ 9)
Incubating (n ⫽ 7)
Out-of-lay (n ⫽ 6)
ANOVA
23.83 ⫾ 1.73a 105.43 ⫾ 5.77a 137.69 ⫾ 4.50a
24.74 ⫾ 1.33a 18.57 ⫾ 4.25b 13.12 ⫾ 3.47b
21.11 ⫾ 1.14a 15.92 ⫾ 4.13b 9.00 ⫾ 1.33b
p ⫽ 0.29 p ⬍ 0.001 p ⬍ 0.001
Means within a row followed by different superscripts differ at the 5% probability level.
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Table 2 Plasma concentrations of LH, prolactin, progesterone and estradiol (ng/ml plasma) of laying, incubating and out-of-lay turkey hens (means ⫾ S.E.M.)
LH Prolactin Progesterone Estradiol a,b,c
Laying (n ⫽ 7 to 9)
Incubating (n ⫽ 5 to 7)
Out-of-lay (n ⫽ 4 to 6)
ANOVA
1.45 ⫾ 0.22a 51.1 ⫾ 7.6a 1.57 ⫾ 0.10a 0.29 ⫾ 0.09a
0.97 ⫾ 0.12b 271.7 ⫾ 16.8b 0.70 ⫾ 0.10b 0.12 ⫾ 0.08b
0.51 ⫾ 0.09c 4.1 ⫾ 0.4c 0.45 ⫾ 0.04b 0.14 ⫾ 0.04b
P P P P
⬍ ⬍ ⬍ ⬍
0.01 0.001 0.01 0.01
Means within a row followed by different superscripts differ at the 5% probability level.
hens (Fig. 2A and 2B. Consequently, the highest flow rate of 0.5 ml/min was chosen for the subsequent experiments. Pulse lengths of cLHRH II greater than 4 min significantly (P ⬍ 0.05) depressed the cumulative LH release (Fig. 2B). Using a 10⫺7 M dose, neither of the two forms of LHRH had any effect on LH release on preparations from L hens (data not shown). On the other hand, the same dose of cLHRH I or cLHRH II, induced a rapid response in LH secretion from the hypophysis of I and OL hens (Fig. 3). The time course of LH release after stimulations at intervals of 120, 60, and 30 min are shown in Fig. 3. Responses of comparable duration were observed when cLHRH I or II were used in both I and OL hens (Fig. 3). On the other hand, cLHRH II was significantly (P ⬍ 0.01) more potent than cLHRH I in OL hens and this difference in potency resulted in a difference in response ranging from 1.5- to threefold (Figs. 3 and 4). On the other hand, both compounds were approximately equipotent in I hens (Figs. 3 and 4). The responses were reduced for preparations from OL hens when the pituitary was stimulated at 30-min intervals (Figs. 3 and 4). In OL hens, the initial stimulation also resulted in lower responses than the subsequent challenges. 3.4. Experiment 3: Effects of cLHRH I and II on LH release in vivo in turkey hens (Fig. 5) Treatment of turkey hens with a single i.v. injection of either cLHRH I or II stimulated increases in the plasma concentration of LH. Plasma LH increased within 1 min of injection of either compound at both doses in I and OL turkey hens, but the increases in L hens were of much smaller magnitude and occurred later, at approximately 20 min post-injection. Dose-response relationships were found between the doses of LHRH I or II administered and the resulting responses in LH for both compounds and at all physiological stages tested. However, the time-course of the LH response was dependent upon the nature and/or dose of the LHRH injected. Thus, in I and OL hens, the maximum increase was observed within 2 min of injection of either dose of cLHRH I and of the dose of 10⫺10 M/kg B.W. of cLHRH II. On the other hand, maximal LH responses were observed 60 min post injection of 10⫺8 M/kg B.W. cLHRH II when the last sample was taken at all physiological stages. In spite of these discrepancies in duration, the maximum increase observed with either cLHRH I or II was similar in amplitude and ranged from a three- to fivefold increase in I and OL hens to a 1.5-fold increase for the L hens.
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Fig. 2. (A) Time course of LH response (means ⫾ SD; n ⫽ 3) of diced pituitary glands form OL turkey hens perifused at successive flow rates of 0.125, 0.25, and 0.5 ml/min and challenged (indicated by arrows) with 10 –7 M cLHRH II for 8, 4, or 2 min; Controls (n ⫽ 3) received 2-min pulses. (B) Cumulative LH release over basal LH level) of diced pituitary glands form OL turkey hens perifused at successive flow rates of 0.125, 0.25, and 0.5 ml/min and challenged (indicated by arrows) with 10 –7 M cLHRH II for 8, 4, or 2 min; Controls (n ⫽ 3) received 2-min pulses. Asterisks indicate significant differences (P ⬍ 0.05; Fisher’s PLSD test).
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Fig. 3. Mean LH responses (ng/min per mg hypophysis, n ⫽ 6) of I (upper panel) and OL (lower panel) turkey hens diced hypophyses perifused and challenged successively at intervals of 120, 60, and 30 min for 2 min with 10⫺7 M chicken LHRH-I (solid symbols) or chicken LHRH-II (open symbols). Arrows indicate time of LHRH pulses. Error bars ommitted for the sake of clarity.
4. Discussion The present results indicates that both forms of cLHRH, cLHRH I and II, which have been shown to be present in the turkey hen [6,19], can stimulate LH release both in vivo or in vitro in I and OL hens and in vivo in L hens for the dose range examined. Furthermore, these
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Fig. 4. Summed LH release (means ⫾ SD; ng/mg hypophysis) above basal levels from perifused of I (upper panel) and OL (lower panel) turkey hens diced hypophyses during the 21 min after successive challenges at intervals of 120, 60 and 30 min between successive stimulations for 2 min with 10⫺7 M of chicken LHRH-I (solid bars), or chicken LHRH-II (hatched bars). * P ⬍ 0.05, Fisher’s PLSD test.
results and those published previously concerning the effects of LHRH in the male turkey [14] clearly show that although sex-related differences in the response amplitude exist in this species, they are much smaller than the very marked sex-related response reported in chickens [10]. On the other hand, the profile of the LH response clearly differs with the stage of the reproductive cycle, which is in overall agreement with previous findings [16]. Using an LHRH of mammalian origin, these authors found greater LH in vivo release in I than in OL hens, and in OL than in L hens. Likewise, in the present study, cLHRH I, which has been
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Fig. 5. Changes in LH plasma concentrations (ng/ml) after a single i.v. injection of cLH-RH I or II at doses of 10⫺8 or 10⫺10 M/kg body weight or 9% saline solution, in laying (A), incubating (B) or out of lay turkey hens (C). (Means ⫾ SEM; n ⫽ 6 per group). Treatments were applied at time 0. **: P ⬍ 0.01, *: P ⬍ 0.05 significant difference with control at a specific time.
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shown to be equipotent to pLHRH in bantam hens [27], was inactive in vivo at a dose of 10⫺10 M/kg B.W. in OL hens yet active in I hens. However, basal release or the response to stimulation in vitro using either cLHRH, and in vivo using cLHRH II or a higher dose of cLHRH I (10⫺8 M/kg B.W.), was of similar or higher amplitude in OL than in I hens. This differs from previous published data [16] and may arise from differences in the experimental procedures: the route of administration (i.v. or i.m.); LHRH origin (mammalian or chicken); sampling frequency; strain of animal used. Thus, a variation in the wet weight of the pituitary gland associated with changes in the reproductive state has been reported [28] that may influence the overall response. Conversely, for the strain of turkey hens used in the present study, the pituitary gland wet weight did not vary significantly with the physiological stages (17; Table 1). The present results also indicate that both peptides were equipotent in eliciting LH release in vitro in I hens, whereas cLHRH II was more potent than cLHRH I in OL hens, although the time-course of the in vitro LH responses were comparable in I and OL hens for both peptides. One cannot exclude that the high circulatory concentrations of prolactin in I hens may have an effect on LHRH-stimulated LH release. An inhibitory effect of this hormone upon LH secretion has been suggested [29], but this contradicts previous finding from the same laboratory [16] that reported higher LH responses in I than in OL hens. In our study, I and OL hens showed comparable and low plasma concentrations of estradiol and progesterone (Table 2) and although we cannot exclude a difference in sensitivity to low concentrations of circulating steroids, the differences in response reported here may be independent of these hormones. In vitro, the LH release resulting from a second stimulation given 30 min after the initial challenge was of a somewhat lower amplitude. However, no significant effect of the interval between stimulations was observed in I hens. On the other hand, in OL hens, the LH release resulting from a second stimulation given 30 min later was significantly lower than that resulting from stimulations applied at intervals of 60 or 120 min. Thus, in OL hens, each form of LHRH induces a specific partial desensitization of the adenohypophysis to a subsequent stimulation given 30 min later, as previously reported in male turkeys [14]. The two forms of cLHRH induce heterologous desensitization of dispersed pituitary cells from immature cockerels [11], which argues for a single type of LHRH receptor. However, preliminary data suggest that the two forms of cLHRH do not induce reciprocal desensitization in preparations from OL hens, although their effects are not additive (Gue´mene´ et al., unpublished observations). This could mean that the functional receptors in immature birds are different from those present in adults of other avian species. This study also confirms that there is no correlation between the plasma concentration of LH and the pituitary basal release in vitro (cf. Table 2 and Fig. 2) as reported previously [17]. The pituitary gland of L turkey hens releases less LH in vivo in response to LHRH stimulation than that of I or OL hens and their response profiles also differ. However, the smaller amplitude of the response, or the absence of a response to LHRH in domestic laying hens is well documented [16,30 –32]. A diminished response is probably related to the presence of elevated concentrations of circulating gonadal steroids that depress LH release through negative feedback. The complete absence of such an effect in vitro using preparations from L hens indicates a lack of sensitivity which is puzzling since data from a previous experiment [17] indicated that mammalian LHRH (pLHRH) could elicit LH release at doses
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ranging from 10⫺10 M to 10⫺5 M. Although these hens were from different flocks, they were of the same strain. However, hypophyseal LH content is lower at the end than at the beginning of the egg productive period in turkey hens and than in I and OL hens, as reported in the domestic hens [33]. The higher rate of decrease in hypophyseal than in plasma LH levels throughout the productive period observed in the present study may be, at least in part, an explanation for the previously observed decrease in the response to LHRH associated with aging in chickens [12]. A possible explanation is that these decreases in LH are a major factor controlling the persistence in ovarian activity and consequently egg laying. Indeed, as the rate of decrease in LH levels is lower in the plasma than in the hypophysis, the rate of synthesis is likely to be insufficient to sustain high plasma LH levels and the hen must progressively rely on stored LH, which will ultimately lead to a decrease in LH secretion. Therefore, this finding may indicate that a reduction in the capacity to store LH by the gonadotrophs is one of the major factors underlying reproductive aging in turkey hens, as in chickens [33]. A clear difference in characteristics of the response was apparent after injection of 10⫺8 M/Kg body weight of cLHRH I and II in vivo. This has also been demonstrated in the male turkey [14]. Similarly, comparable doses of cLHRH II induced a sustained release of LH in both incubating bantam hens [34] and domestic hens [12]. This biphasic action of LHRH has been well studied in mammals and is known to be due to the stimulatory effect of LHRH on gonadotrophin synthesis. The results from these in vivo studies also indicate a dose-response relationship between LHRH and LH release in the female turkey. This result emphasizes that the existence of a dose-response relationship in LH responses to LHRH in the adult bird is not specific to the male. In summary, this study demonstrates that aging is associated with a decrease in the pituitary concentration of LH, which may lead to cessation of reproductive activity. Also, the relative potency of cLHRH I and II in turkey hens depends on the physiological status of the birds. In turkey hens, cLHRH II is either equipotent or more potent than cLHRH I as in male turkeys [14] or other species of birds [7–13]. As, the physiological significance of cLHRH II has been questioned, further investigations are needed to clarify the physiological role of both cLHRH forms, especially that of cLHRH II, which can induce long-term release of LH. Small and possibly undetectable changes in its concentration in the hypothalamus might therefore be sufficient to directly or indirectly induce an LH release of physiological significance. It therefore remains to be established how each form of cLHRH may influence reproductive activity, directly or indirectly.
Acknowledgments The co-operation of BETINA, in providing the birds, was greatly appreciated. Our thanks are extended to Drs Etches, Proudman and Sharp for supplying the materials used in the prolactin and LH assays respectively. We are indebted to Mrs M.T. Durand, Mrs M. Garreau-Mills, Mr B. Gaultier and Mr S. Nevoit for expert technical assistance.
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References [1] King JA, Millar RP. Structure of chicken hypothalamic luteinizing hormone releasing hormone. I. Structural determination on partially purified material. J Biol Chem 1982;257:10722– 8. [2] Miyamoto K, Hasegawa Y, Minegishi T, Nomura M, Takahashi Y, Igarashi M, Kangawa K, Matsuo H. Isolation and characterization of chicken hypothalamic luteinizing hormone releasing hormone. Biochem Biophys Res Commun 1982;107:820 –7. [3] Miyamoto K, Hasegawa Y, Nomura M, Igarashi M, Kangawa K, Matsuo H. Identification of the second gonadotropin-releasing hormone in chicken hypothalamus:evidence that gonadotropin secretion is probably controlled by two distinct gonadotropin-releasing hormones in avian species. Proc Natl Acad Sci USA 1984;81:3874 – 8. [4] Powell RC, Jach H, Millar RP, King JA. Identification of Gln8-GnRH and His5,Trp7,Tyr8-GnRH in the hypothalamus and extrahypothalamic brain of the ostrich (Struthio camelus). Peptides 1987;8:185–90. [5] Sherwood N, Wingfield JC, Ball GF, Dufty AM. Identity of gonadotropin-releasing hormone in passerine birds:comparison of GnRH in song sparrow (Melospiza melodia) and starling (Sturnus vulgaris) with five vertebrate GnRHs. Gen Comp Endocrinol 1988;69:341–51. [6] Millam JR, Craig-Veit CB, Adams TE, Adams BM. Avian gonadotropin-releasing hormones I and II in brain and other tissues in turkey hens. Comp Biochem Physiol 1989;94A:771– 6. [7] Chou HF, Johnson AL, Williams JB. Luteinizing hormone releasing activity of (Gln8)-LHRH and (His5,Trp7,Tyr8)-LHRH in the cockerel, in vivo and in vitro. Life Sci 1985;37:2459 – 65. [8] Hattori A, Ishii S, Wada M. Effects of two kinds of chicken luteinizing hormone releasing hormone (LHRH), mammalian LHRH and its analogs on the release of LH and FSH in Japanese quail and chicken. Gen Comp Endocrinol 1986;64:446 –55. [9] Millar RP, Milton RC de L, Follett BK, King JA. Receptor binding and gonadotropin-releasing activity of a novel chicken gonadotropin-releasing hormone ([His5,Trp7,Tyr8] GnRH) and a D-Arg6 analog. Endocrinology 1986;119:224 –31. [10] Sharp PJ, Dunn IC, Talbot RT. Sex differences in the LH responses to chicken LHRH-I and -II in the domestic fowl. J Endocrinol 1987;115:323–31. [11] King JA, Davidson JS, Millar RP. Interaction of endogenous chicken gonadotrophin-releasing hormone-I and -II on chicken pituitary cells. J Endocrinol 1988;117:43–9. [12] Wilson SC, Cunningham FJ, Chairil RA, Gladwell RT. Maturational changes in the LH response of domestic fowl to synthetic chicken LHRH-I and -II. J Endocrinol 1989;123:311– 8. [13] Sharp PJ, Talbot RT, Main GM, Dunn IC, Fraser HM, Huskisson NS. Physiological roles of chicken LHRH-I and -II in the control of gonadotrophin release in the domestic chicken. J Endocrinol 1990;124: 291–9. [14] Gue´mene´ D, Williams JB. In vitro and In vivo responses to chicken LHRH-I and chicken LHRH-II in male turkeys (Meleagris gallopavo). J Endocrinol 1992;132:387–93. [15] Burke W, Cogger EA. The effect of luteinizing hormone releasing hormone (LHRH) on serum LH and ovarian growth in turkeys. Poult Sci 1977;56:234 – 42. [16] El Halawani ME, Fehrer SC, Silsby JL. Enhanced luteinizing hormone release by luteinizing hormone releasing hormone in incubating female turkeys (Meleagris gallopavo). Biol Reprod 1987;36:884 –9. [17] Gue´mene´ D, Williams JB. Comparison of the basal and luteinizing hormone-releasing hormone (LH-RH) induced luteinizing hormone (LH) release by perifused hypophyses from turkey hens (Meleagris gallopavo) at different physiological stages. Br Poult Sci 1992;33:153– 63. [18] Chairil RA, Wilson SC, Gladwell RT, Cunningham FJ. Immunoneutralization in vivo and in vitro of cLHRH-I and -II releasing activities suggest a possible modulatory role for cLHRH-II in the hypothalamic mechanisms regulating LH secretion. J Reprod Fertil 1993;89;11–50. [19] Rozenboim I, Silsby JL, Tabibzadeh C, Pitts GR, Youngren OM, El Halawani M. Hypothalamic and posterior pituitary content of vasoactive intestinal peptide and gonadotropin-releasing hormones in the turkey hen. Biol Reprod 1993;49:622– 6.
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[20] Pitts GR, Youngren OM, Silsby JL, Rozenboim I, Chaiseha Y, Phillips RE, Foster DN, El Halawani M. Role of vasoactive intestinal peptide in the control of prolactin-induced turkey incubation behavior. II. Chronic infusion of vasoactive intestinal peptide. Biol Reprod 1994;50:1350 – 6. [21] Rozenboim I, Tabibzadeh C, Silsby JL, El Halawani M. Effect of ovine prolactin administration on hypothalamic vasoactive intestinal peptide (VIP), gonadotropin releasing hormone I and II content, and anterior pituitary VIP receptors in laying turkey hens. Biol Reprod 1993;48:1246 –50. [22] Gue´mene´ D, Etches RJ. Endocrinological and behavioural effects of p– chlorophenyl-alanine (PCPA) oral administration to broody turkey hens (Meleagris gallopavo). Reprod Nutr Dev 1989;29:469 –76. [23] Duplaix M, Williams JB, Mongin P. Effects of an intermittent lighting schedule on the time of egg-laying and the levels of luteinizing hormone, progesterone and corticosterone in the plasma of the domestic hen. J Endocrinol 1981;91:375– 83. [24] Etches RJ, Williams JB, Rzasa J. Effects of corticosterone and dietary changes in the hen on ovarian function, plasma LH and steroids and the responses to exogenous LH-RH. J Reprod Fert 1984;70:121–30. [25] Etches RJ, Cheng KW. A homologous radioimmunoassay for turkey prolactin:changes during the reproductive and ovulatory cycle. Poult Sci 1982;61:1354 – 62. [26] Proudman JA, Opel H. Turkey prolactin: validation of a radioimmunoassay and measurement of changes associated with broodiness. Biol Reprod 1981;25:573– 80. [27] Sharp PJ, Sterling RJ, Milton RC de L, Millar RP. Effect of luteinizing hormone-releasing hormone and its analogues on plasma luteinizing hormone concentrations in incubating bantam hens. Br Poult Sci 1986;27: 129 –35. [28] El Halawani ME, Silsby JL, Youngren OM, Phillips RE. Exogenous prolactin delays photo-induced sexual maturity and suppresses ovariectomy-induced luteinizing hormone secretion in the turkey (Meleagris gallopavo). Biol Reprod 1991;44:420 – 4. [29] El Halawani ME, Silsby JL, Fehrer SC. Basal and hypothalamic extract-induced luteinizing hormone and prolactin secretion by cultured anterior pituitary cells from female turkeys in various stages of the reproductive cycle. Gen Comp Endocrinol 1988;71:45–54. [30] Bonney RC, Cunningham FJ, Furr BJA. Effect of synthetic luteinizing hormone releasing hormone on plasma luteinizing hormone in the female domestic fowl. J Endocrinol 1974;63:539 – 47. [31] Gue´mene´ D, Williams JB. Comparison of mammalian luteinizing hormone releasing hormone (LHRH), and of an analog (ICI 118630), on luteinizing hormone and ovarian steroid (progesterone, œstradiol) secretions in laying hens. (Gallus domesticus). Life Sci 1986;39:541–7. [32] Wilson SC, Gladwell RT, Cunningham FJ. Differential responses of hypothalamic LHRH-I and -II to castration and gonadal steroid or tamoxifen treatment in cockerels. J Endocrinol 1990;125:139 – 46. [33] Sharp PJ, Dunn IC, Cerolini S. Neuroendocrine control of reduced persistence of egg-laying in domestic hens: evidence for the development of photorefractoriness. J Reprod Fertil 1992;94:221–35.
LH responses to chicken luteinizing hormone-releasing hormone I and II in laying, incubating, and out of lay turkey hens D. Gue´mene´*, J.B. Williams I.N.R.A., Centre de Tours-Nouzilly, Station de Recherches Avicoles, 37380 Nouzilly, France Received 26 August 1998; accepted 6 April 1999
Abstract An experiment was conducted to assess the relative in vivo and in vitro activities of chicken LH-RH-I and -II in laying, incubating and out-of-lay turkey hens. The highest plasma concentrations of LH were measured in laying turkey hens, whereas hypophyseal concentrations were highest in incubating hens (I) and lowest in the laying hens at the end of the laying period (EL). Hypophyseal and plasma concentrations of LH decreased with aging in laying hens (L) and the greater decrease occurred in the hypophyses. An in vitro hypophyseal acute challenge with 2-min pulses of cLHRH I or II (10⫺7 M) using a perifusion technique resulted in an increase in the release of LH in out-of-lay (OL) and incubating (I) hens, but not in laying (L) hens. Although both peptides elicited comparable responses in I hens, cLHRH II was more effective in OL hens. This difference was attributable to a greater amplitude of the response, whose duration was unchanged. Hypophyseal desensitization to a subsequent stimulation was observed in OL hens when the interval between stimulations was 30 min, but this did not occur at 60- or 120-min intervals. In vivo, the injection of cLHRH I or II, at doses of 10⫺8 and 10⫺10 M/kg B.W. stimulated increases in the plasma concentrations of LH, which were initiated within 1 min of injection in OL and I hens but from 5 to 20 min postinjection in L hens. The responses were dose-related and greater immediate responses were measured with cLHRH I than with cLHRH II. Also, after the injection of cLHRH II at the 10⫺8 M/kg B.W. dose, the shape of the LH response consisted of an initial increase, followed by a more sustained phase during which LH concentrations were either stable (I hens) or continued to increase (L and OL hens) from 20 to 60 min after injection. In contrast, the injection of cLHRH I at doses of 10⫺8 or 10⫺10 M/kg or cLHRH II
* Corresponding author. Tel.: ⫹02-47-42-76-43; fax: ⫹02-47-42-77-78. E-mail address: [email protected] (D. Guemene) 0739-7240/99/$ – see front matter © 1999 Elsevier Science Inc. All rights reserved. PII: S 0 7 3 9 - 7 2 4 0 ( 9 9 ) 0 0 0 2 0 - X
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at a dose of 10⫺10 M/kg in I and OL hens, produced a peak of LH concentrations in plasma within 5 min and thereafter declined gradually. The difference in the in vivo responses to LHRH I and II could not be attributed to a greater potency of cLHRH II, but to a more prolonged action. In summary, the responses to both forms of chicken LH-RH varies markedly with the stage of the reproductive cycle (L, I, and OL) and differs between the in vivo and in vitro situations. Although cLHRH II may be more active than cLHRH I, controversy still surrounds its precise physiological role. © 1999 Elsevier Science Inc. All rights reserved. Keywords: Turkey hens; LHRH; LH; Pituitary gland; Physiological stages
1. Introduction Brain tissues from several nonmammalian species contain at least two distinct luteinizing hormone-releasing hormones (LHRH). Thus, two forms of LHRH have been isolated in the chicken: cLHRH I, which is (Gln8)-LHRH [1,2] and cLHRH II, which is (His5-Trp7-Tyr8)-LHRH [3]. These two forms of LHRH were later identified in the ostrich [4], starling [5] and in female turkeys [6]. Results of studies on their relative efficiencies in stimulating LH release in birds are conflicting [7–14]. At the present time, cLHRH II is reported to be more potent in the male turkey both in vitro and in vivo and to have long–acting effects after in vivo injections [14]. However, there are no data available on the effects of these two peptides in turkey hens which are known to respond to the administration of synthetic mammalian LHRH in vivo with the release of LH [15–17]. There is controversy surrounding the physiological role of cLHRH II. Sharp et al. [13] have demonstrated by RIA on chicken brain sections that it is not a predominantly hypothalamic peptide but that it occurs in low amounts throughout the brain. Its concentration in the median eminence and the pre-optic area does not differ significantly in laying (L) and out-of-lay (OL) hens and active immunization against the peptide does not affect egg production. This argues against a role as a releasing hormone. However, an in vitro study of pituitary release of LH in chickens that had previously been passively immunized against cLHRH I or cLHRH II argue in favor of a physiological role of cLHRH II [18]. Moreover, it has been shown that both forms are present in the turkey hen hypothalamus [6] and that hypothalamic cLHRH II concentrations are only slightly lower than those of cLHRH I [19]. Furthermore, measurement of the hypothalamic concentrations of the two peptides in various reproductive states reveals a pattern of change of cLHRH-II that parallels that of cLHRH-I [19]. It has also been observed that both are present in the pituitary gland [20] and that treatment with ovine prolactin causes a rapid decrease in hypothalamic cLHRH I content and a more delayed decline in that of cLHRH II [19]. This study was designed to investigate the biologic properties of both forms of chicken LHRH by comparing their ability to induce the release of LH from turkey hen hypophyses
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in vitro using a perifusion technique, and the in vivo LH responses in L, OL or incubating (I) turkey hens.
2. Materials and methods 2.1. Animals Medium white turkey hens obtained before sexual maturity (BETINA, 56250, St. Nolff, Elven, France), were used in their first (experiments 2 and 3) or second productive period (Experiment 1). They were reared in individual pens and maintained under a lighting schedule of 14-hr light and 10-hr darkness during the productive period. They were fed on commercial diets available ad libitum and had free access to water. In Experiment 1, hens (5 to 6 per group) were assigned to the following physiological groups: new (NL) or old laying (end of productive period, EL) hens that had laid for 4 to 6 wk or for over 20 wk respectively, I and OL hens. The ages of the hens in these stages were 94 (OL), 95 (NL), 97 (EL), and 98 (I) wk of age, respectively. In Experiments 2 and 3, hens were used at three different physiological stages (L, I, and OL) between 35 and 42 wk of age. Hens were considered to be I or OL according to previously described criteria [17,22]. Briefly, a hen had to express broody behavior for at least 7 d or not to have laid an egg for at least 14 d to be classified as I or OL, respectively. The L hens used in this study had laid an egg in the 24-hr before the experiment. 2.2. Experimental approach 2.2.1 Experiment 1: Plasma and hypophyseal levels of LH in turkey hens New (NL) and EL, incubating (IL), and OL hens were killed by cervical dislocation. Just before death, a blood sample was withdrawn from a wing vein into a heparinized syringe. Thereafter, plasma was separated from blood by centrifugation at 2000 g for 10 min at 4°C and kept at ⫺20°C until assayed for LH. Hypophyses were removed, snap-frozen in liquid nitrogen and stored until assayed for LH. 2.2.2. Experiment 2: In vitro effects of cLHRH I and II on LH release in turkey hens As in Experiment 1, the hens were killed by cervical dislocation and a blood sample withdrawn from a wing vein into a heparinized syringe, just before death. Plasma was separated as above and samples assayed for LH, prolactin, progesterone, and estradiol. The ovary and the oviduct were removed and weighed. Hypophyses were removed, weighed, then placed in ice-cold sterile culture medium 199 [M199; Sigma-Chimie S.A.R.L., 38297, La Verpilliere Cedex France] supplemented with glucose (2.5 g/l), amphotericin B (2.5 mg/l), gentamicin (0.1%, w/v), BSA (0.1%, w/v), sodium bicarbonate (0.35 g/l), and (1 mM)] within 2 min of death. The gland were rinsed with M199 and diced with a scalpel blade. The equivalent of one-half of an hypophysis was placed in a 0.5-ml perifusion chamber of a specialized apparatus (Acusyst S, Endotronics, Coon Rapids, MN. 55433, USA).
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A preliminary experiment was conducted to establish the optimum flow rate using six preparations of OL hens subjected to three successive 100-min perifusion periods at 0.125, 0.25, and 0.5 mL/min. Chambers were perifused with M199 (pH ⫽ 7.2, temperature ⫽ 39°C) continuously gassed with 95% O2 and 5% CO2. After a 60-min equilibration period, the preparations were challenged with 10⫺7 M cLHRH II (Peninsula Laboratories Europe LTD., St. Helens, Merseyside, WA9 3AJ, England) using pulse lengths of 8 (n ⫽ 3) or 2 min (n ⫽ 3), then 4 or 2 min and finally 2 min. A three-way tap was used to switch between reservoirs of M199 and the test substance. In the subsequent experiments, six preparations were generally run simultaneously (n ⫽ 4 to 7 repetitions per physiological stage and treatment) at a flow rate of 0.5 ml/min. After 1 hr of equilibration with M199, the hypophysis fragments were exposed to 2-min pulses of test substances dissolved in M199. The preparations were challenged successively at intervals of 120, 60, and 30 min with cLHRH I (Sigma-Chimie S.A.R.L., France) or II at a dose of 10⫺7 M in M199. Between stimulations, the preparations were perifused with M199 alone. In all cases, the effluent was collected as 3-min fractions and stored at⫺20°C until used for radioimmunoassay of LH. The profiles of LH release were measured individually and the release of LH due to the stimulation was defined as the amount of LH secreted per chamber above basal level during the 21 min after stimulation [14]. Concentrations measured during the 30 min before the initial stimulation and during the 10 min before the subsequent stimulations were used to define each chamber’s basal level. 2.2.3. Experiment 3: In vivo effects of cLHRH I and II on LH release in turkey hens At each physiological stage (L, I, and OL), a group of 30 turkey hens (n ⫽ 30 ⫻ 3) were randomly allocated to one of the five following treatments (control, 0.5 ml of 0.9% (w/v) saline solution, n ⫽ 6; cLHRH I 10⫺8 M/kg body weight, n ⫽ 6; cLHRH I 10⫺10 M/kg B.W., n ⫽ 6; cLHRH II 10⫺8 M/kg B.W., n ⫽ 6; cLHRH II 10⫺10 M/kg B.W., n ⫽ 6). Treatments were dissolved in 0.9% (w/v) saline solution and administered (0.5 ml per) i.v. Blood samples (5 ml) were withdrawn just before injection (time 0), and at 1, 2, 5, 10, 20, and 60 min after injection. All samples were assayed for LH. 2.3. Radioimmunoassays 2.3.1. LH Plasma (Experiments 1, 2, and 3), hypophyseal extracts (experiments 1 & 2) and aliquots of perifusate fractions (Experiment 2) were assayed in triplicate according to Sharp et al. [10] using fraction PRC-AE1-s-1 [10] as labeled hormone and standard, and LH 3-3 antiserum [10]. This assay has been previously validated for the measurement of LH in turkey [17]. Mean within and between assay coefficients of variation were 5.7% and 13.2%, respectively. 2.3.2. Progesterone Plasma samples collected from all hens used in Experiment 2 were assayed using the method described by Duplaix et al., [23]. The mean within assay coefficient of variation was 2.9%.
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2.3.3. Estradiol Plasma samples collected from all hens used in Experiment 2 were assayed according to Etches et al. [24]. The mean within assay coefficient of variation was 4.6%. 2.3.4. Prolactin Plasma (Experiment 2) were assayed in triplicate 100 l samples following the method of Etches and Cheng [25], but using tPRL provided by Proudman and Opel [26] as labeled hormone and standard and the anti-tPRL-2-B3 provided by Etches and Cheng [25] as antiserum. The mean within assay coefficient of variation was 7.6%. 2.4. Calculations and statistical analysis Anatomic and physiological data from the hens included in experiments 1 and 2 were compared using a one-way ANOVA and Fisher’s PLSD (Protected Least Significant Difference) post-hoc test if appropriate (P ⬍ 0.05, ANOVA). The release of LH due to the stimulation itself was estimated as described previously [17] by linear regression of LH concentrations on time, using the initial basal values plus the concentrations measured in the three samples preceding each stimulation. The amount of LH released from each chamber during the 21 min after stimulation was calculated by estimating the difference between the LH concentration in each of the seven samples collected after stimulation and the expected basal release estimated by linear regression at the appropriate given times. The amounts of LH were thereafter compared using a two-factor analysis of variance (ANOVA. Dependent variables: length of the interval between stimulations; treatment). All data from Experiment 3 were analyzed using a repeated measures ANOVA. When significant treatment effects were observed (P ⬍ 0.05, ANOVA), data were further analyzed using Fisher’s PLSD post hoc test. All statistical analyses were performed using the StatviewTM II program (Abacus Concepts Inc., Berkeley, CA, USA) for the Apple Macintosh computer.
3. Results 3.1. Experiment 1: Plasma and hypophyseal levels of LH in turkey hens (Fig. 1) Plasma levels of LH were the highest in both groups of laying hens (NL and EL) and the lowest in I and OL hens. Differences reached significance (P ⬍ 0.05) between NL and I or OL hens. Hypophyseal concentrations of LH were the highest in I hens and the lowest in EL hens, whereas intermediate concentrations were measured in NL and OL hens and the physiological stage effect was close from statistical significance (P ⫽ 0.06). 3.2. Experiment 2: Anatomic and hormonal characteristics of the turkey hens (Tables 1 and 2) Regressed reproductive tracts were observed in all OL and I turkey hens whereas they were fully developed and functional in L hens. The pituitary gland wet weights did not differ
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Fig. 1. Plasma (ng/ml) and hypophyseal (g/hypophysis) concentrations of LH in laying turkey hens at the beginning or at the end of a productive period, and in I and OL turkey hens, respectively (means ⫾ SEM). * P ⬍ 0.05, Fisher’s PLSD test)
between physiological stages. LH plasma concentrations differed between physiological stages as in experiment one. Similarly, prolactin plasma concentrations differed significantly between physiological stages (P ⬍ 0.05). They were at the lowest in OL hens while intermediate and high concentrations were measured in L and I hens, respectively. Both progesterone and estradiol plasma concentrations measured in L hens were significantly (P ⬍ 0.05) higher than those measured in I and OL hens. 3.3. Experiment 2: Effects of cLHRH I and II on the in vitro LH release (Figs. 2– 4 ) There was no significant effect (P ⬎ 0.05) of flow rate on basal LH release by pituitary gland nor the response to 2 min challenge with cLHRH II using preparations from OL Table 1 Pituitary, ovary and oviduct weights of laying, incubating and out-of-lay turkey hens (means ⫾ S.E.M.)
Hypophysis (mg) Oviduct (g) Ovary (g) a,b
Laying (n ⫽ 9)
Incubating (n ⫽ 7)
Out-of-lay (n ⫽ 6)
ANOVA
23.83 ⫾ 1.73a 105.43 ⫾ 5.77a 137.69 ⫾ 4.50a
24.74 ⫾ 1.33a 18.57 ⫾ 4.25b 13.12 ⫾ 3.47b
21.11 ⫾ 1.14a 15.92 ⫾ 4.13b 9.00 ⫾ 1.33b
p ⫽ 0.29 p ⬍ 0.001 p ⬍ 0.001
Means within a row followed by different superscripts differ at the 5% probability level.
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Table 2 Plasma concentrations of LH, prolactin, progesterone and estradiol (ng/ml plasma) of laying, incubating and out-of-lay turkey hens (means ⫾ S.E.M.)
LH Prolactin Progesterone Estradiol a,b,c
Laying (n ⫽ 7 to 9)
Incubating (n ⫽ 5 to 7)
Out-of-lay (n ⫽ 4 to 6)
ANOVA
1.45 ⫾ 0.22a 51.1 ⫾ 7.6a 1.57 ⫾ 0.10a 0.29 ⫾ 0.09a
0.97 ⫾ 0.12b 271.7 ⫾ 16.8b 0.70 ⫾ 0.10b 0.12 ⫾ 0.08b
0.51 ⫾ 0.09c 4.1 ⫾ 0.4c 0.45 ⫾ 0.04b 0.14 ⫾ 0.04b
P P P P
⬍ ⬍ ⬍ ⬍
0.01 0.001 0.01 0.01
Means within a row followed by different superscripts differ at the 5% probability level.
hens (Fig. 2A and 2B. Consequently, the highest flow rate of 0.5 ml/min was chosen for the subsequent experiments. Pulse lengths of cLHRH II greater than 4 min significantly (P ⬍ 0.05) depressed the cumulative LH release (Fig. 2B). Using a 10⫺7 M dose, neither of the two forms of LHRH had any effect on LH release on preparations from L hens (data not shown). On the other hand, the same dose of cLHRH I or cLHRH II, induced a rapid response in LH secretion from the hypophysis of I and OL hens (Fig. 3). The time course of LH release after stimulations at intervals of 120, 60, and 30 min are shown in Fig. 3. Responses of comparable duration were observed when cLHRH I or II were used in both I and OL hens (Fig. 3). On the other hand, cLHRH II was significantly (P ⬍ 0.01) more potent than cLHRH I in OL hens and this difference in potency resulted in a difference in response ranging from 1.5- to threefold (Figs. 3 and 4). On the other hand, both compounds were approximately equipotent in I hens (Figs. 3 and 4). The responses were reduced for preparations from OL hens when the pituitary was stimulated at 30-min intervals (Figs. 3 and 4). In OL hens, the initial stimulation also resulted in lower responses than the subsequent challenges. 3.4. Experiment 3: Effects of cLHRH I and II on LH release in vivo in turkey hens (Fig. 5) Treatment of turkey hens with a single i.v. injection of either cLHRH I or II stimulated increases in the plasma concentration of LH. Plasma LH increased within 1 min of injection of either compound at both doses in I and OL turkey hens, but the increases in L hens were of much smaller magnitude and occurred later, at approximately 20 min post-injection. Dose-response relationships were found between the doses of LHRH I or II administered and the resulting responses in LH for both compounds and at all physiological stages tested. However, the time-course of the LH response was dependent upon the nature and/or dose of the LHRH injected. Thus, in I and OL hens, the maximum increase was observed within 2 min of injection of either dose of cLHRH I and of the dose of 10⫺10 M/kg B.W. of cLHRH II. On the other hand, maximal LH responses were observed 60 min post injection of 10⫺8 M/kg B.W. cLHRH II when the last sample was taken at all physiological stages. In spite of these discrepancies in duration, the maximum increase observed with either cLHRH I or II was similar in amplitude and ranged from a three- to fivefold increase in I and OL hens to a 1.5-fold increase for the L hens.
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Fig. 2. (A) Time course of LH response (means ⫾ SD; n ⫽ 3) of diced pituitary glands form OL turkey hens perifused at successive flow rates of 0.125, 0.25, and 0.5 ml/min and challenged (indicated by arrows) with 10 –7 M cLHRH II for 8, 4, or 2 min; Controls (n ⫽ 3) received 2-min pulses. (B) Cumulative LH release over basal LH level) of diced pituitary glands form OL turkey hens perifused at successive flow rates of 0.125, 0.25, and 0.5 ml/min and challenged (indicated by arrows) with 10 –7 M cLHRH II for 8, 4, or 2 min; Controls (n ⫽ 3) received 2-min pulses. Asterisks indicate significant differences (P ⬍ 0.05; Fisher’s PLSD test).
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Fig. 3. Mean LH responses (ng/min per mg hypophysis, n ⫽ 6) of I (upper panel) and OL (lower panel) turkey hens diced hypophyses perifused and challenged successively at intervals of 120, 60, and 30 min for 2 min with 10⫺7 M chicken LHRH-I (solid symbols) or chicken LHRH-II (open symbols). Arrows indicate time of LHRH pulses. Error bars ommitted for the sake of clarity.
4. Discussion The present results indicates that both forms of cLHRH, cLHRH I and II, which have been shown to be present in the turkey hen [6,19], can stimulate LH release both in vivo or in vitro in I and OL hens and in vivo in L hens for the dose range examined. Furthermore, these
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Fig. 4. Summed LH release (means ⫾ SD; ng/mg hypophysis) above basal levels from perifused of I (upper panel) and OL (lower panel) turkey hens diced hypophyses during the 21 min after successive challenges at intervals of 120, 60 and 30 min between successive stimulations for 2 min with 10⫺7 M of chicken LHRH-I (solid bars), or chicken LHRH-II (hatched bars). * P ⬍ 0.05, Fisher’s PLSD test.
results and those published previously concerning the effects of LHRH in the male turkey [14] clearly show that although sex-related differences in the response amplitude exist in this species, they are much smaller than the very marked sex-related response reported in chickens [10]. On the other hand, the profile of the LH response clearly differs with the stage of the reproductive cycle, which is in overall agreement with previous findings [16]. Using an LHRH of mammalian origin, these authors found greater LH in vivo release in I than in OL hens, and in OL than in L hens. Likewise, in the present study, cLHRH I, which has been
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Fig. 5. Changes in LH plasma concentrations (ng/ml) after a single i.v. injection of cLH-RH I or II at doses of 10⫺8 or 10⫺10 M/kg body weight or 9% saline solution, in laying (A), incubating (B) or out of lay turkey hens (C). (Means ⫾ SEM; n ⫽ 6 per group). Treatments were applied at time 0. **: P ⬍ 0.01, *: P ⬍ 0.05 significant difference with control at a specific time.
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shown to be equipotent to pLHRH in bantam hens [27], was inactive in vivo at a dose of 10⫺10 M/kg B.W. in OL hens yet active in I hens. However, basal release or the response to stimulation in vitro using either cLHRH, and in vivo using cLHRH II or a higher dose of cLHRH I (10⫺8 M/kg B.W.), was of similar or higher amplitude in OL than in I hens. This differs from previous published data [16] and may arise from differences in the experimental procedures: the route of administration (i.v. or i.m.); LHRH origin (mammalian or chicken); sampling frequency; strain of animal used. Thus, a variation in the wet weight of the pituitary gland associated with changes in the reproductive state has been reported [28] that may influence the overall response. Conversely, for the strain of turkey hens used in the present study, the pituitary gland wet weight did not vary significantly with the physiological stages (17; Table 1). The present results also indicate that both peptides were equipotent in eliciting LH release in vitro in I hens, whereas cLHRH II was more potent than cLHRH I in OL hens, although the time-course of the in vitro LH responses were comparable in I and OL hens for both peptides. One cannot exclude that the high circulatory concentrations of prolactin in I hens may have an effect on LHRH-stimulated LH release. An inhibitory effect of this hormone upon LH secretion has been suggested [29], but this contradicts previous finding from the same laboratory [16] that reported higher LH responses in I than in OL hens. In our study, I and OL hens showed comparable and low plasma concentrations of estradiol and progesterone (Table 2) and although we cannot exclude a difference in sensitivity to low concentrations of circulating steroids, the differences in response reported here may be independent of these hormones. In vitro, the LH release resulting from a second stimulation given 30 min after the initial challenge was of a somewhat lower amplitude. However, no significant effect of the interval between stimulations was observed in I hens. On the other hand, in OL hens, the LH release resulting from a second stimulation given 30 min later was significantly lower than that resulting from stimulations applied at intervals of 60 or 120 min. Thus, in OL hens, each form of LHRH induces a specific partial desensitization of the adenohypophysis to a subsequent stimulation given 30 min later, as previously reported in male turkeys [14]. The two forms of cLHRH induce heterologous desensitization of dispersed pituitary cells from immature cockerels [11], which argues for a single type of LHRH receptor. However, preliminary data suggest that the two forms of cLHRH do not induce reciprocal desensitization in preparations from OL hens, although their effects are not additive (Gue´mene´ et al., unpublished observations). This could mean that the functional receptors in immature birds are different from those present in adults of other avian species. This study also confirms that there is no correlation between the plasma concentration of LH and the pituitary basal release in vitro (cf. Table 2 and Fig. 2) as reported previously [17]. The pituitary gland of L turkey hens releases less LH in vivo in response to LHRH stimulation than that of I or OL hens and their response profiles also differ. However, the smaller amplitude of the response, or the absence of a response to LHRH in domestic laying hens is well documented [16,30 –32]. A diminished response is probably related to the presence of elevated concentrations of circulating gonadal steroids that depress LH release through negative feedback. The complete absence of such an effect in vitro using preparations from L hens indicates a lack of sensitivity which is puzzling since data from a previous experiment [17] indicated that mammalian LHRH (pLHRH) could elicit LH release at doses
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ranging from 10⫺10 M to 10⫺5 M. Although these hens were from different flocks, they were of the same strain. However, hypophyseal LH content is lower at the end than at the beginning of the egg productive period in turkey hens and than in I and OL hens, as reported in the domestic hens [33]. The higher rate of decrease in hypophyseal than in plasma LH levels throughout the productive period observed in the present study may be, at least in part, an explanation for the previously observed decrease in the response to LHRH associated with aging in chickens [12]. A possible explanation is that these decreases in LH are a major factor controlling the persistence in ovarian activity and consequently egg laying. Indeed, as the rate of decrease in LH levels is lower in the plasma than in the hypophysis, the rate of synthesis is likely to be insufficient to sustain high plasma LH levels and the hen must progressively rely on stored LH, which will ultimately lead to a decrease in LH secretion. Therefore, this finding may indicate that a reduction in the capacity to store LH by the gonadotrophs is one of the major factors underlying reproductive aging in turkey hens, as in chickens [33]. A clear difference in characteristics of the response was apparent after injection of 10⫺8 M/Kg body weight of cLHRH I and II in vivo. This has also been demonstrated in the male turkey [14]. Similarly, comparable doses of cLHRH II induced a sustained release of LH in both incubating bantam hens [34] and domestic hens [12]. This biphasic action of LHRH has been well studied in mammals and is known to be due to the stimulatory effect of LHRH on gonadotrophin synthesis. The results from these in vivo studies also indicate a dose-response relationship between LHRH and LH release in the female turkey. This result emphasizes that the existence of a dose-response relationship in LH responses to LHRH in the adult bird is not specific to the male. In summary, this study demonstrates that aging is associated with a decrease in the pituitary concentration of LH, which may lead to cessation of reproductive activity. Also, the relative potency of cLHRH I and II in turkey hens depends on the physiological status of the birds. In turkey hens, cLHRH II is either equipotent or more potent than cLHRH I as in male turkeys [14] or other species of birds [7–13]. As, the physiological significance of cLHRH II has been questioned, further investigations are needed to clarify the physiological role of both cLHRH forms, especially that of cLHRH II, which can induce long-term release of LH. Small and possibly undetectable changes in its concentration in the hypothalamus might therefore be sufficient to directly or indirectly induce an LH release of physiological significance. It therefore remains to be established how each form of cLHRH may influence reproductive activity, directly or indirectly.
Acknowledgments The co-operation of BETINA, in providing the birds, was greatly appreciated. Our thanks are extended to Drs Etches, Proudman and Sharp for supplying the materials used in the prolactin and LH assays respectively. We are indebted to Mrs M.T. Durand, Mrs M. Garreau-Mills, Mr B. Gaultier and Mr S. Nevoit for expert technical assistance.
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