oviposition and ovulation. Reading, RG6 2AJ Injections of oestradiol benzoate and testosterone propionate did not alter plasma (Received 6 June 1979)

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1 CONCENTRATIONS OF CORTICOSTERONE AND LUTEINIZING HORMONE IN PLASMA DURING THE OVULATORY CYCLE OF THE DOMESTIC HEN AND AFTER THE ADMINISTRATION OF GONADAL STEROIDS SUSAN C. WILSON AND F. J. CUNNINGHAM Department of Physiology & Biochemistry, University of Reading, Whiteknights, Reading, RG6 2AJ (Received 6 June 1979) SUMMARY Plasma concentrations of corticosterone and LH were measured in hens in relation to the ovulation of an egg either early or late in a sequence and also in hens injected with gonadal steroids. In hens about to ovulate an egg early in the sequence, the plasma concentration of LH rose at the onset of darkness while levels of corticosterone remained low. As levels of LH fell during the 4 h preceding ovulation, those of corticosterone increased steeply to reach a maximum at about the time of ovulation, which is shortly after dawn. In contrast, in hens about to ovulate an egg late in a sequence, the concentration of LH in plasma fell and that of corticosterone tended to increase during the hours of darkness. During the 7 h preceding ovulation the temporal relationship of LH to corticosterone was similar to that observed before an early ovulation such that levels of corticosterone increased rapidly on the descending slope of the preovulatory LH surge and reached maximum values at the time of oviposition and ovulation. Injections of oestradiol benzoate and testosterone propionate did not alter plasma concentrations of either corticosterone or LH. On the other hand, the injection of progesterone was associated with a rapid fall in levels of corticosterone. It is suggested that progesterone may suppress the pituitary\p=n-\adrenalsystem and thus modulate the pattern of corticosterone secretion during the ovulatory cycle. INTRODUCTION The occurrence of ovulation in the domestic hen is restricted to a period of 8-10 h of the 24 h day. The timing of the first ovulation of a sequence is influenced by sunset (Wilson & Sharp, 1973) and, to a lesser extent, by sunrise (Bhatti & Morris, 1978), and under a photoschedule of 16 h light : 8 h darkness it occurs between 7 and 8 h after the onset of darkness. Subsequent ovulations take place at a slightly later time each day until there is a single day on which no ovulation occurs before the start of a new sequence (Fraps, 1955). In the rat there is some evidence to suggest adrenal involvement in the timing of the preovulatory surge of luteinizing hormone (LH) (Resko, 1969; Feder, Brown-Grant & Corker, 1971; Mann, Korowitz, MacFarland & Cost, 1976). In the hen also, the adrenal may be involved in the control of ovulation since in hens injected with metopirone, a drug which reduces the secretion of corticosterone in the domestic fowl (Nagra, Sauers & Wittmaier, 1965; Frankel, Graber & Nalbandov, 1967; Culbert & Wells, 1975), preovulatory surges of LH can occur at any time during the 24 h day instead of being restricted to a period of 8-10 h of the day (Wilson & Cunningham, 1980). Also, intramuscular injections of dexamethasone

2 acetate (1-7 mg/hen) which depress levels of corticosterone in the circulation by 50% within 3 h (S. C. Wilson, unpublished observations) inhibit both the preovulatory LH surge (Wilson & Lacassagne, 1978) and ovulation (Soliman & Huston, 1974). These events can be restored in the dexamethasone-treated hen by an injection of adrenocorticotrophic hormone (ACTH) (Wilson & Lacassagne, 1978). However, a precise role for the adrenal gland in the control of ovulation has not been established. Progesterone and deoxycorticosterone can stimulate release of LH (Wilson & Sharp, 1975,1976) and ovulation (Etches & Cunningham, 1976a) although it is not known whether either hormone is secreted by the adrenal in sufficient quantities to initiate the spontaneous preovulatory discharge of LH. The injection of corticosterone, a major adrenal corticosteroid in birds (de Roos, 1961; Sandor, Lamoureux & Lanthier, 1963), leads to a rise in the level of LH in plasma of those hens in which the ovary contains a follicle capable of ovulating at the time of injection (Sharp & Beuving, 1978; Wilson & Lacassagne, 1978). The dose of corticosterone required to release LH, however, exceeds normal levels in the circulation (Sharp & Beuving, 1978). The present study was designed to investigate the temporal relationship between corticosterone and LH during the ovulatory cycle of the hen and to determine whether this is modified during the course of a sequence of ovulations. The effects of injections of gonadal steroids on the concentrations of LH and corticosterone in plasma were also studied since the preovulatory increase in LH concentration in plasma is accompanied by increases in the secretion of gonadal steroids. MATERIALS AND METHODS One- to two-year-old Ross tint hens, caged singly and laying regular sequences of eggs, were maintained under a photoschedule of 16 h light : 8 h darkness (lights on h) with food and water available at all times. Ovipositions were recorded to the nearest minute by means of an automatic recording device. Ovulation was estimated to occur about 20 min after oviposition (Warren & Scott, 1935) except at the end of a sequence where the terminal oviposition of one sequence and the first ovulation of the next are separated by about 12 h. The first ovulation of a sequence was estimated to occur at h since the interval between the peak of the preovulatory surge of LH and ovulation is about 4 h (Furr, Bonney, England & Cunningham, 1973). In each study, blood (1-5 ml) was taken by brachial venepuncture into heparinized syringes within 45 s of the removal of each hen from its cage. The blood was centrifuged, and the plasma separated and stored at 20 C until assayed. Levels of corticosterone and LH during the ovulatory cycle Blood was taken at intervals of 2 h during 24 h from 12 hens expected to ovulate either an early (first or second) or late (terminal) egg of a sequence during the period of sampling. To investigate changes in the secretion of corticosterone around the time of ovulation (unaccompanied by an oviposition) and around the time of oviposition (unaccompanied by an ovulation), blood was taken from other hens at intervals of 2 h for a period of 10 h commencing either 30 min before the onset of darkness or about 4-6 h before the terminal oviposition of a sequence. Levels of corticosterone and LH after the injection of gonadal steroids Progesterone, oestradiol benzoate and testosterone propionate were obtained from Sigma, London and doses of 0-7, 015 and 0-5 mg respectively dissolved in 0-3 ml arachis oil were injected i.m. into hens either 7 h after the second ovulation of a sequence or 26 h after the last ovulation of a sequence (i.e. on the day of terminal oviposition of a sequence when the ovary contained a follicle capable of ovulating but when ovulation was not expected to occur). Blood was taken immediately preceding the injection and at intervals of 90 min thereafter for a period of 10-5 h.

3 Radioimmunoassays Luteinizing hormone was measured using the homologous radioimmunoassay for avian LH described by Follett, Scanes & Cunningham (1972). Corticosterone was measured using both the radioimmunoassay method and the antibody to corticosterone developed by Etches (1976). In this laboratory the antibody cross-reacted 17-6% with progesterone and 411% with 11-deoxycorticosterone. Progesterone was removed from plasma by extraction with spectroanalar grade iso-octane (Fisons) and a second extraction with spectroanalar grade dichloromethane (Fisons) recovered 94% [3H]corticosterone but only 5-5% [3H]progesterone that had been added to a sample of pooled plasma. Statistical comparisons were made using Student's paired and unpaired /-tests. RESULTS Levels of corticosterone and LH during the ovulatory cycle In the 12 hens in which levels of corticosterone and LH were measured during 24 h, the mean concentration of corticosterone in plasma increased significantly (P<0-01) from (s.e.m.) ng/ml at 30 min before the onset of darkness (17.30 h) to ng/ml at 1-5 h after the onset of light (03.30 h). Levels of corticosterone then tended to fall and reached ng/ml at h and remained at about that level until the end of sampling at h. In six of the above hens ovulating the first or second eggs of a sequence during the experimental period, the concentrations of LH and corticosterone at h (i.e. 30 min before the onset of darkness) were and 1-49 ±0-37 ng/ml respectively. The concentration of LH then rose steeply ( < 0-02) to a maximum of ng/ml at h, while the concentration ofcorticosterone remained low (Fig. la). Between and h there was a simultaneous fall and rise in the mean concentrations of LH and corticosterone respectively. The concentration of corticosterone rose (P<0-01) to a maximum of ng/ml and then fell (P<002) to ng/ml at h. No further significant changes were observed in the concentrations of either LH or cor ticosterone although there was a tendency for the levels of both hormones to decline slightly between h and the end of sampling at h. In the remaining six hens ovulating their final egg of a sequence during the experimental period, the concentrations of LH and corticosterone in the plasma at h were and ng/ml respectively (Fig. lb). During the following 6 h, levels of LH fell continuously so that the concentration at h (1-57 ±0-13 ng/ml) was signific antly (.P< 0-001) lower than that at h. The concentration of LH then rose steeply CP<0-001) to ng/ml at h, 4 h before ovulation. Plasma levels of cor ticosterone tended to increase during the hours of darkness and to fall between 2 and 4 h after the onset of light. Between and h the concentration of LH in plasma fell (P< 0-05) to ng/ml and the concentration of corticosterone rose from 209 ± 0-35 to 4-53 ± 1-04 ng/ml. Thereafter, levels of LH remained stable while those of corticosterone fell reaching a value at h similar to that observed 24 h earlier. The general pattern of changes in concentrations of corticosterone and LH was similar in hens ovulating their first (i.e. no accompanying oviposition) and last (i.e. accompanying oviposition) eggs of a sequence (Fig. 2a and b) to that seen in hens ovulating an early (first second) or or the terminal egg of a sequence respectively (cf. Fig. 2a and b with Fig. la and b). Maximum levels ofcorticosterone in hens during their first ovulation, however, were reached at h whereas in hens during an early (first or second) ovulation, they were reached at h. The concentration of corticosterone in six hens which laid the final egg of a sequence (i.e. no accompanying ovulation) rose significantly ( <0 001) during the 5 h preceding oviposition from to ng/ml and then fell (P<0-001) to

4 4- (a) 3- ~SS 2 c Predicted ovulation 4 ll _i_ _l_ Time of day (h) Fig. 1. Plasma concentrations (means ± s.e.m.) of corticosterone (O) and LH ( ) in (a) six hens ovulating an early (first or second) egg of a sequence or (b) six hens ovulating the terminal egg of a sequence. Black bars indicate hours of darkness. Arrows indicate in (a) predicted time of ovulation unaccompanied by an oviposition, and in (b) predicted time of ovulation accompanied by an oviposition ng/ml at h (Fig. 2c). Concentrations of LH in plasma did not change significantly around the time of oviposition. Levels of corticosterone and LH after the injection of gonadal steroids No significant changes in the concentration of either corticosterone or LH were observed after the injection, 7 h after ovulation, of 015 mg oestradiol benzoate, 0-5 mg testosterone propionate or oil (Fig. 3a, b and d). In contrast, the injection of progesterone 7 h after

5 (b) 3-3- a g ' 1 î Predicted ovulation I-1-1_1_I_I o 3r M oviposition Time of day (h) Fig. 2. Plasma concentrations (means ± s.e.m.) ofcorticosterone (O) and LH ( ) in ( ) nine hens ovulating a first egg (i.e. no accompanying oviposition), (b) nine hens ovulating the-terminal egg (i.e. accompanying oviposition) of a sequence and (c) six hens around the time of the final oviposition (i.e. no accompanying ovulation) of a sequence. Black bars indicate hours of darkness. ovulation was associated with a significant (P<005) fall in the concentration of corticosterone from to ng/ml (n 5) 90 min later (Fig. 3c). Levels = remained low until 7-5 h after injection when they rose to reach values observed before injection. The concentration of LH tended to rise slightly, though not significantly, during the 4-5 h after the injection of progesterone. However, when progesterone was injected 26 h after the final ovulation of a sequence the concentration of LH rose significantly ( <0 001) from to ng/ml (n 6). This rise occurred in conjunction with = a steep fall in the concentration of corticosterone from to ng/ml (Fig. 3e). Secretion ofcorticosterone then remained low until 6 h after injection (17.30 h) at which time the concentration rose significantly (P<005) from to ng/ml at

6 - 4- (b) -J_I_I 3 3r («O S _i Oviposition Oviposition Time of day (h) _ Fig. 3. Plasma concentrations (means±s.e.m.) of corticosterone (O) and LH ( ) in hens injected at h, 7 h after the second ovulation of a sequence, with (a) 015 mg oestradiol benzoate (n = 4), (i>) 0-5 mg testosterone propionate (n = 4), (c) 0-7 mg progesterone (n = 5), (d) arachis oil (n = 5), or 26 h after the final ovulation of a sequence with (e) 0-7 mg progesterone (n = 6) or (/) arachis oil (n = 4). Black bars indicate hours of darkness.

7 20.30 h (Fig. 3e). In hens injected with oil alone, and in contrast with hens injected with progesterone, the concentration of corticosterone in plasma rose to a maximum level at the time of oviposition (cf. Figs 2c and 3/) and then fell to a nadir at h after which no significant changes were observed. Concentrations of LH increased between and h, as would be expected in hens due to ovulate the first egg of a sequence (cf. Figs 2a and 3f). DISCUSSION A circadian rhythm in secretion of corticosterone has been described in several avian species using the following methods: fluorometry (quail, Boissin & Assenmacher, 1968; whitethroated sparrow, Dusseau & Meier, 1971; pigeon, Sato & George, 1973); competitive protein-binding (pigeon, Joseph & Meier, 1973; domestic hen, Beuving & Vonder, 1977); radioimmunoassay (domestic hen, Etches, 1979). In the present study, if the position of an ovulation in a sequence is disregarded, the mean changes in the concentrations of corticosterone in plasma during the ovulatory cycle are similar to those observed previously (Beuving & Vonder, 1977; Etches, 1979) with a gradual increase in concentration during the hours of darkness followed by a fall around dawn. However, the pattern of secretion of corticosterone during the ovulatory cycle varied according to the position of an ovulation in the sequence. Thus, in hens ovulating the first egg of a sequence during the experimental period the level of corticosterone in plasma rose only towards the end of darkness, whereas in those ovulating the terminal egg of a sequence there was a tendency for secretion of corticosterone to increase at the beginning of darkness and to remain raised until dawn when the levels fell in all the hens. Examination of the temporal relationship of corticosterone to LH shows that corticosterone levels were depressed 7-4 h before ovulation, when the plasma concentration of LH was high but then rose simultaneously with the fall in LH. It is possible that early in the ovulation sequence the tendency for corticosterone to rise at the onset of darkness is suppressed in some way by the preovulatory rise of one or more gonadal steroids. The preovulatory surge of LH is accompanied, or shortly preceded, by rises in the blood level of progesterone (Furr et al. 1973; Lague, van Tienhoven & Cunningham, 1975), testosterone (Etches & Cunningham, 1977) and oestradiol (Senior & Cunningham, 1974; Shodono, Nakamura, Tanabe & Wakabayashi, 1975). While there is some evidence for an inhibitory effect of oestradiol on secretion of corticosterone in the female rat (McKerns & Bell, 1960; Kitay, 1963) neither oestradiol nor testosterone altered levels of corticosterone in plasma within 10-5 h of injection. The injection of progesterone, on the other hand, induced an immediate fall in the secretion of corticosterone both when administered 26 h after ovulation, when it would stimulate both the release of LH and steroidogenesis (Shahabi, Bahr & Nalbandov, 1975; Etches & Cunningham, 19760) and 7 h after ovulation when, although release of LH would be stimulated, there is no accompanying release of endogenous gonadal steroids (Etches & Cunningham, 19766). Nagra et al. (1965) have also shown that progestagens depress the secretion of corticosterone in cockerels. Luteinizing hormone itself is unlikely to have depressed secretion of corticosterone since in hens injected with progesterone 7 h after ovulation a significant fall in secretion of corticosterone was observed within 1-5 h, during which time the plasma concentration of LH did not change. It is probable, therefore, that the normal preovulatory increase in the plasma concentration of progesterone exerts a depressive effect on the secretion of corticosterone and would account for the difference in the corticosterone pattern during the hours of darkness between hens due to ovulate their first and last eggs of a sequence. This hypothesis is supported by the observation that during a mid-sequence ovulatory cycle the preovulatory increase in secretion of progesterone is associated with a fall in the concentration of corticosterone in plasma (Etches, 1979). However, Etches (1979) suggests that the pattern of

8 corticosterone secretion reflects solely a diurnal rhythm and discounts any influence of changes in secretion of gonadal steroids. In all spontaneously ovulating hens, levels of corticosterone began to increase as those of LH started to fall. It has been shown that corticosterone depresses LH release in male tree sparrows (Wilson & Follett, 1976) and when ovariectomized hens are injected with ACTH, concentrations of LH fall within 3 h from a mean of 51-3 to a mean of 36-7 ng/ml plasma (S. C. Wilson, unpublished observation). This raises the possibility that endogenous corticosterone is normally involved in bringing about the decline in plasma LH concen tration following the preovulatory peak. The cause of the rise in secretion of corticosterone is not clear. In the present study levels of corticosterone rose around the time of the last oviposition of a sequence when there was no accompanying ovulation; a rise which Beuving & Vonder (1977) attribute to the stress of egg-laying. However, the possibility cannot be discounted that events associated with ovulation are somehow involved in an increase in secretion of corticosterone since in hens injected with progesterone 26 h after ovulation, levels of corticosterone rose between 6 and 9 h after the injection. This is the time at which ovulation induced by the injection of progesterone would be expected to occur (Fraps, 1955; Etches, 1977) and when there would be no accompanying oviposition. On the other hand in hens injected with progesterone 7 h after ovulation, i.e. when ovulation cannot be induced, a comparatively small increase in the plasma concentration of corticosterone was observed 6-9 h after injection. Since the concentrations of exogenous progesterone in plasma would be falling at this time (Etches & Cunningham, 19766) it is possible that the removal of a suppressive effect exerted by high levels of progesterone in the blood contributes to the observed rises in corticosterone. The significance of the changes in secretion of corticosterone during the ovulatory cycle of the hen remains unclear. It has been suggested that an increase in adrenal activity at the onset of darkness may initiate the hormonal events normally preceding the first ovulation of a sequence (Wilson & Lacassagne, 1978). However, in hens ovulating the first egg of a sequence, plasma concentrations of LH increased at the onset of darkness while levels of" corticosterone remained low. Similarly, Sharp & Beuving (1978) reported no increase in corticosterone levels immediately preceding the preovulatory LH surge. However, the increase in plasma concentration of LH preceding the first ovulation of a sequence was itself preceded about 6 h earlier by a rise in the concentration of corticosterone associated with the terminal oviposition of a sequence. Whether this was of physiological significance in the timing of the first ovulation of a sequence remains to be shown. In conclusion, the changes in secretion of corticosterone during the ovulatory cycle of the hen appear to reflect not only an external 24 h rhythm of light : darkness activity but also the endogenous 26 h cycle of ovulation and oviposition. The authors are grateful for the facilities provided by the staff of Lane End Farm, Shinfield, Reading, and to Dr R. J. Etches, University of Guelph, Canada for his gift of corticosterone antiserum. S. C. W. acknowledges the receipt of a postdoctoral fellowship from the British Egg Marketing Board Research and Education Trust. This work was supported by grant no. AG 45/157 from the Agriculture Research Council. REFERENCES Beuving, G. & Vonder, G. M. A. (1977). Daily rhythm of corticosterone in laying hens and the influence of egg laying. Journal of Reproduction and Fertility 51, Bhatti, B. M. & Morris, T. R. (1978). The relative importance of sunrise and sunset for entrainment of oviposition in the fowl. British Poultry Science 19, Boissin, J. & Assenmacher, I. (1968). Rythmes circadiens des taux sanguin et surrénalien de la corticosterone chez le caille. Comptes Rendus Hebdomadaires des Séances de l'académie des Sciences 265, Culbert, J. & Wells, J. W. (1975). Aspects of adrenal function in the domestic fowl. Journal of Endocrinology 65,

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