Plasma Concentrations of Progesterone and Corticosterone During the Ovulation Cycle of the Hen (Gallus Domesticus)

Transcription

1 Plasma Concentrations of Progesterone and Corticosterone During the Ovulation Cycle of the Hen (Gallus Domesticus) R.J. ETCHES Department of Animal and Poultry Science, The University of Guelph, Guelph, Ontario, Canada NIG 2W1 (Received for publication December 8, 1977) ABSTRACT The observation that injections of either ACTH or corticosterone induced ovulation in the hen has prompted speculation which implicates the adrenal gland in the mechanisms controlling ovulation. In this study, the role of the major adrenal steroid in birds was further examined by measuring the plasma concentration of corticosterone during the 24 hr period preceding midsequence ovipositions and the 28 hr period preceding terminal ovipositions. Progesterone, which is an accurate index of preovulatory ovarian activity, was also measured in the same samples. An increase in plasma progesterone preceded mid-sequence ovipositions which are accompanied by an ovulation. This preovulatory surge in plasma progesterone began approximately 7 hr before ovulation, reached a maximum of 6.9 ng/ml 3 to 2 hr before ovulation, and returned to baseline concentrations at the time of ovulation. A major preovulatory peak of progesterone was not observed during the interval between sequences, although a small but statistically significant rise in progesterone was observed between midnight and 0600 hr on the day of the last oviposition of the sequence. The concentration of corticosterone increased approximately two-fold during the dark portion of the photoperiod regardless of the position of the oviposition in the sequence. Baseline concentrations during the illuminated portion of the photoperiod ranged between 1 and 4 ng of corticosterone per ml of plasma whereas during the period of darkness, they ranged between 2 and 6 ng of corticosterone per ml of plasma. It was concluded, therefore, that the circulating concentration of corticosterone was regulated by a circadian rhythm which operates independently of follicular maturation. INTRODUCTION Under standard light-dark conditions (i.e. 14L:10D, where the lighted period extends from 0600 to 2000 hr), the release of luteinizing hormone (LH) for ovulation in the hen is initiated spontaneously between approximately midnight and 0800 hr. This interval has been termed the open period and ovulation occurs approximately 6 hr after the initiation of the release of LH. Ovulations occur in sequences and consecutive ovulations within a sequence are separated by intervals of approximately 25 to 27 hr (Fraps, 1955). If the first egg of a sequence is ovulated at approximately 0800 hr, then subsequent ovulations occur at later times on successive days until the sequence is ended by ovulation of the last egg at approximately 1600 hr. The next egg, which is the first egg of the next sequence, is ovulated approximately 40 hr after the last ovulation of the previous sequence (Fraps, 1955). The restriction of LH release to 8 hr of the day can be theoretically explained by a model in which two systems interact. The first system is the follicular maturation cycle which presumably is initiated when the preceding egg is ovulated and ends approximately 27 hr later (Bastian and Zarrow, 1955; Fraps, 1970). The second system is the circadian system ( the open period) which restricts the release of LH to an 8 hr period of the day. If the follicle matures during the open period for LH release, ovulation will occur. If maturation occurs after the open period, then the mature follicle must wait until the next open period before it can be ovulated. Although there is considerable evidence that the open period possesses circadian periodicity (Fraps, 1955; Morris, 1973), its exact nature is unknown. In recent years, there have been several attemps to associate changes in the plasma concentrations of LH and the gonadal steroids to either the follicular maturation process or the open period for LH release. Bastian and Zarrow (1955) proposed that the open period was the consequence of a circadian rhythm in LH release while Fraps (1955) proposed that progesterone was secreted in increasing quantities by the developing follicle. By measuring the concentrations of both of these hormones in plasma during the 27 hr period between midsequence ovulations and the 40 hr period between the last ovulation of one sequence and the first ovulation of the next, it has been 1979 Poultry Sci 58:

2 212 ETCHES shown that the changes in the plasma concentrations of progesterone and LH are associated only with the imminent ovulation (Furr et al., 1973). Similarly, the changes in estradiol (Senior, 1974) and testosterone (Etches and Cunningham, 1977) are associated only with the impending ovulation. Presumably, the potential to secrete at least some of these steroids is involved in the acquisition of follicular maturity (Etches and Cunningham, 1976a), but none of these hormones has been associated with the open period for LH release. The involvement of the adrenal gland in the endocrine mechanisms controlling ovulation has been suggested recently by Etches and Cunningham (1976b). Corticosterone, the major adrenal steroid in birds (Assenmacher, 1973), will induce ovulation when injected 14 hr before the expected time of the first ovulation of a sequence, and similar results can be obtained using adrenocorticotrophic hormone (ACTH) of either avian (Fraps et al, 1947) or mammalian (van Tienhoven, 1961) origin. However, the changes in the plasma concentration of corticosterone during the ovulation cycle of the hen have not been monitored nor have attempts been made to associate the ovulation-inducing properties of the adrenal steroids with the open period or with the process of follicular maturation. The objective of this investigation was to measure the changes in the concentration of corticosterone and to relate these changes to the endocrine control of ovulation. MATERIALS AND METHODS White Leghorn hens of a commercial strain were used in their first year of lay. They were caged singly, had free access to food and water, and were maintained on a lighting regime of 14L:10D. Individual birds were selected on the basis of regular sequence lengths, inter-sequence intervals of 40 to 46 hr, and normal oviposition times for each egg within the sequence. The times of oviposition were recorded by the method of Wilson and Zarrow (1961). Blood samples were collected during the 24 hr period separating mid-sequence ovipositions (which are coupled with an ovulation) and during the 28 hr period preceding the terminal oviposition of the sequence (which is unaccompanied by ovulation). Although ovulation was associated only with the mid-sequence ovipositions, both of these periods scanned the open period for LH release. Blood samples were collected at hourly intervals for 12 hr from each bird. Each day was arbitrarily divided into four 12 hr portions, each of which overlapped by 6 hr. Blood samples were removed from 6 to 8 hens in each of the 12 hr periods. The photoperiod in each of the four 12 hr periods was 14L:10D, but the times at which the lights were turned off and on was adjusted so that the 12 hr period of the day during which blood samples were collected fell within the solar daylight hours. For the purpose of analysis, the data were pooled to represent a 14L:10D photoperiod where the lights were turned on at 0600 hr and turned off at 2000 hr. The blood samples were collected by brachial venipuncture and the blood was immediately cooled and the red cells removed by centrifugation. The plasma was frozen at 20 C until it was assayed for progesterone and corticosterone. During the dark portion of the photoperiod, the hens were removed to an illuminated room for approximately 1 min while the blood sample was obtained. The effects of this procedure were assessed by examining the times of oviposition after the samples were removed. If subsequent ovipositions deviated by more than 20 min from the time which was predicted from previous records, the oviposition was considered abnormal. Of the 63 hens from which samples were removed, only 6 hens showed any subsequent alteration in the times of oviposition. These hens were discarded from the series. Corticosterone was assayed by the method of Etches (1976). Briefly, each sample was extracted with iso-octane and progesterone was assayed in this extract using a slightly modified (Etches and Cunningham, 1976a) version of the method reported by Furr (1973). The plasma was subsequently extracted with dichloromethane and corticosterone was quantified in this fraction. Therefore, both corticosterone and progesterone were measured simultaneously in all plasma samples. RESULTS The changes in plasma concentrations of progesterone and corticosterone between midsequence ovulations are illustrated in Figure 1. The preovulatory surge in plasma progesterone began approximately 7 hr before ovulation, reached a maximum of 6.9 ng/ml 3 to 2 hr before ovulation, and returned to baseline concentrations at the time of ovulation. Plasma concentrations of corticosterone were lowest during the illuminated portion of the photoperiod

3 PLASMA CONCENTRATIONS DURING OVULATION CYCLE OF THE HEN TIME OF DAY FIG. 1. Plasma concentrations (means ± SEM) of progesterone and corticosterone between midsequence ovulations in the hen. Twelve observations are included in each of these means. The shaded rectangles represent the dark portion of the photoperiod and the open rectangles represent the illuminated portion of the photoperiod. The time of ovulation which is indicated by the arrow, was ±.17 hr (mean ± SEM). This value was calculated by adding.5 hr to the time of oviposition. and highest during darkness. In each of the hourly samples which were collected during the illuminated portion of the photoperiod, the mean plasma concentration of corticosterone was usually between 1 and 2 ng/ml, whereas the mean concentration of corticosterone was usually 2 to 3 ng/ml in each of the hourly blood samples which were removed during the period of darkness. The maximum concentration of 3.3 ng of corticosterone per ml of plasma was observed at 0200 hr. The mean of all samples removed during the illuminated portion of the photoperiod (x = 1.6 ng corticosterone/ml plasma) was significantly (t = 5.67, P<.01) lower than the mean of all values removed during the dark portion of the photoperiod (x = 2.5 ng of corticosterone per ml of plasma). The changes in the plasma concentrations of progesterone and corticosterone during the inter-sequence interval are illustrated in Figure 2. In contrast to the large preovulatory surge of progesterone which was observed prior to the mid-sequence oviposition which was coupled with ovulation, the terminal oviposition was not preceded by a large increase in plasma progesterone. However, there was a slight increase in both the mean and the variance of the mean plasma concentrations of progesterone between 2400 and 0600 hr (Table 1). The mean concentration of progesterone during the illuminated FIG. 2. Plasma concentrations (means ± SEM) of progesterone and corticosterone before the terminal oviposition of a sequence. Six to twelve observations are included in each of these means. The shaded rectangles represent the dark portion of the photoperiod and the open rectangles represent the illuminated portion of the photoperiod. The arrow on the left indicates the time of the penultimate oviposition and the arrow on the right indicates the time of the terminal oviposition. These values (means ± SEM) are ±.19 hr and ±.22 hr, respectively. portion of the photoperiod (x =.74 ng/ml) was significantly lower (t = 3.42, P<.01) than the mean concentration of progesterone during the dark portion of the photoperiod (x = 1.12 ng/ml). In general, the changes in plasma concentrations of corticosterone were similar to those observed between mid-sequence ovulations, although both the baseline and the maximum concentrations were higher during this interval. During the illuminated portion of the photoperiod, plasma corticosterone values ranged between 2 and 4 ng/ml whereas they ranged between 4 and 6 ng/ml during the 10 hr dark period. The maximum concentration of 6.1 ng of corticosterone per ml of plasma was observed at 2400 hr. The mean concentration of corticosterone during the lighted period (3.6 ng/ml) was significantly (t = 4.56, P<.01) lower than the mean concentration of corticosterone during the dark period (4.8 ng/ml). DISCUSSION The changes in the plasma concentration of progesterone before ovulation (Fig. 1) were similar to those observed by several other investigators (Peterson and Common, 1971; Furr et al, 1973; Lague et al, 1975; Shahabi et al., 1975). Both the magnitude and the duration of

4 214 ETCHES TABLE 1. Plasma concentrations (mean ± SEM) of progesterone prior to the last oviposition of a sequence Time of day Dark period Mean Concentration (iig/iiii; SEM the preovulatory progesterone peak were similar to those reported previously. Similarly, the absence of a large increase in plasma progesterone before the terminal oviposition (Fig. 2) is in agreement v/ith previous reports (Peterson and Common, 1971; Furr et al, 1973). The most significant nhspa/atirvn jn fkk study is the increase in plasma corticosretone during the dark period. This increase is independent of ovulation since it occurs before a midsequence ovulation (Fig. 1) and before a terminal oviposition which is unaccompanied by ovulation (Fig. 2). It is therefore reasonable to conclude that the plasma concentration of corticosterone is regulated by a circadian rhythm in the laying hen. Similar adrenal rhythms_of corticosterone with an acrophase during the dark period have_been reported in the laying hen (Beuving and Vonder, 1977), in the quail (Boissin and Assenmacher, 1970, 1971), and in the pigeon (Joseph and Meier, 1973). A circadian rhythm in plasma corticosterone has also been reported in immature cockerels (Seigel et al., 1976), although the acrophase of this rhythm occurred towards the end of the illuminated portion of the day. The reasons for this difference in the rhythm is unknown, although it is possible that a phase shift in adrenal activity is involved in sexual maturation (see Meier and MacGregor, 1972). Although the relative changes in plasma corticosterone preceding mid-sequence and terrninal ovipositions clearly demonstrated that the concentration of corticosterone was controlled by a circadian rhythm rather than an ovarian rhythm, the absolute values were higher before a t nrnnal oviposition. These higher concentrations might be attributed to differences between groups of hens, since those bled before a mid-sequence ovulation were received before those which were bled before a terminal oviposition. The differences in basal concentrations between mid-sequence ovulations and before the final oviposition of a sequence may, however, be related to the absence of a large preovulatory peak of progesterone and/or testosterone. In some species both progestins and androgens have negative feedback effects on adrenal hormone secretion (see Nagra et al., 1965) and the elevated concentrations of corticosterone which were associated with the terminal oviposition may be related to the lack of a negative feedback stimulus during this portion of the ovulation sequence. Since corticosterone is regulated by a circadian rhythm and injections of corticosterone will induce ovulation, it is tempting to speculate that these two events are related. One possible relationship may involve the regulation of the "open period for LH release" by the circadian rhythm in corticosterone. Indeed, there is circumstantial evidence to support this concept. The open period is presumed to possess circadian periodicity (Fraps, 1955; Morris, 1973) and the evidence presented in this paper indicates that a rhythm of corticosterone in plasma has a similar periodicity. Furthermore, the time of ovulation can be altered by injections of moderate doses of corticosterone (Etches and Cunningham, 1976b) and compounds which block adrenal function also block ovulation. Soliman and Huston (1974) have shown that injections of dexamethasone will in-

5 PLASMA CONCENTRATIONS DURING OVULATION CYCLE OF THE HEN 215 hibit ovulation if given 14 hr before ovulation whereas injections given at shorter intervals before ovulation fail to prevent its occurrence. This may indicate that the adrenal involvement in ovulation occurs when corticosterone concentrations are rising, i.e., during the first half of the dark period. In the hen, an increase in the plasma concentration of testosterone is the first endocrine signal that ovulation is imminent (Etches and Cunningham, 1977). It may be significant that this increase in testosterone usually occurs when plasma concentrations of corticosterone are rising. However, the interrelationships between testosterone and corticosterone during the ovulation cycle have not been investigated. In the rat, LH release for ovulation is also restricted to a critical portion of the day by a circadian rhythm. From a variety of investigations which have altered the adrenal rhythm by surgical manipulation, it has generally been concluded that the timing of LH release in the rat is influenced by a circadian rhythm in adrenal function (Mann et al, 1975, 1976). Furthermore, Ramaley (1975) reported that rats without an apparent adrenal cycle fail to exhibit regular estrus cycles. It is possible, therefore, that the circadian adrenal cycle could act as a pacemaker for the timing of LH release in both the chicken and the rat. An alternative hypothesis for the involvement of the adrenal gland in the mechanisms of ovulation revolves around the contribution of the adrenal gland to peripheral concentrations of plasma progesterone. Mann and Barraclough (1973) showed that in the ovariectomized rat there was a circadian rhythm in plasma progesterone which was 180 out of phase with the corticosterone rhythm. These investigators suggested that the adrenal involvement in the timing of LH release was mediated through adrenal progesterone. Although the adrenal contribution to peripheral progesterone is apparently insignificant in the domestic fowl (Furr, 1973), there is a slight increase in plasma progesterone during the second half of the dark period (Fig. 2, Table 1), and it is possible that this slight increase in progesterone is capable of initiating the preovulatory endocrine events. Although this increase in progesterone has not been observed previously, the sampling intervals in previous investigations (Peterson and Common, 1971; Furr et al, 1973) may have precluded the possibility of a similar observation. It is possible, therefore, that the timing of LH release in the hen is regulated by a circadian rhythm in progesterone which is produced by the adrenal gland. ACKNOWLEDGMENTS This work was supported by the Banting Research Foundation, the National Research Council, and the Ontario Ministry of Agriculture and Food. The assistance of C. Duke and L. Spilsbury was greatly appreciated. REFERENCES Assenmacher, I., The peripheral endocrine glands. In Avian biology. D. S. Farner and J. R. King, eds. Academic Press, New York and London. Bastian, J. W., and M. X. Zarrow, A new hypothesis for the asynchronous ovulatory cycle of the domestic hen Gallus domesticus. Poultry Sci. 34: Beuving, G., and G. M. A. Vonder, Daily rhythm of corticosterone in laying hens and the influence of egg laying. J. Reprod. Fert. 51: Boissin, J., and I. Assenmacher, Circadian rhythms in adrenal cortical activity in the quail. J. Interdisc. Cycle Res. 1: Boissin, J., and I. Assenmacher, Entrainment of the adrenal cortical rhythm and of locomotor activity rhythm by ahemeral photoperiods in the quail. J. Interdisc. Cycle Res. 2: Etches, R. 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