Neuroendocrine Regulation of the Estrous Cycle and Seasonal Breeding in the Ewe

Transcription

1 BIOLOGY OF REPRODUCTION 20, (1979) Neuroendocrine Regulation of the Estrous Cycle and Seasonal Breeding in the Ewe SANDRA J. LEGAN and FRED J. KARSCH INTRODUCTION One of the most intriguing aspects of the reproductive process is the reversible fertility resulting from seasonal breeding. From the earliest of times, it has been recognized that "for everything there is a season...a time to be born and a time to die...." (Ecclesiastes, Ch. 3, Vs. 1 2). In recent years, we have gained important new insight into the neuroendocrine mechanisms governing seasonal breeding in female mammals. To a large degree, this has evolved directly from elucidation of the intricate details of the feedback interplay between the gonadal and gonadotropic hormones during the estrous cycle. This report reviews our present understanding of the endocrine basis of the estrous cycle and seasonal breeding in female sheep. Although theories will be developed based on data obtained solely in sheep, it is likely that the fundamental characteristics of the control systems in sheep are similar, at least conceptually, to those in other seasonally breeding females. Temporal Organization of Reproductive Cycles in the Ewe The annual reproductive cycle of the ewe consists of a breeding and a nonbreeding (or anestrous) season. In most breeds of sheep, the breeding season begins in late summer and is characterized by successive 16 day estrous cycles. The anestrous season begins in late winter and is characterized by the absence of regular ovarian cycles (Yeates, 1949; Hafez, 1952). The organization of a single 16 day estrous cycle is illustrated schematically in Fig. 1. The cycle may be divided into a day luteal phase and a 3 4 periovuhtory period. The pattern of circulating progesterone reflects the waxing and waning of the secretory activity of the corpus luteum and is governed by an Reproductive Endocrinology Program, Departments of Pathology and Physiology, The University of Michigan, Ann Arbor, Michigan interplay between stimulatory factors from the pituitary and inhibitory ones from the uterus (Nalbondov, 1973; Hansel et al, 1973; Goding, 1974). The fluctuating profile of circulating estradiol is derived almost exclusively from ovarian follicles, several of which develop and undergo atresia during the course of each cycle (Smeaton and Robertson, 1971; Baird and Scaramuzzi, 1976a). The pattern of circulating luteinizing hormone (LH) reflects the operation of 2 separate regulatory systems, a tonic system which produces relatively low pulsatile discharges of gonadotropins during most of the cycle and a surge system which generates the massive preovulatory LH discharge (Goding et I I PERIOVULATORY PERIOD PROGESTERONE [ ( * \» \ t \ I I DAY OF ESTROUS CYCLE ESTRUS FIG. 1. Schematic representation of the 16 day estrous cycle of the ewe. Serum hormone patterns are from Hauger et al., (1977). Follicle size is from Smeaton and Robertson (1971). 74

2 CONTROL OF OVARIAN AND SEASONAL CYCLICITY IN THE EWE 75 al., 1970; Scaramuzzi et al., 1971; Foster et al., 1975; Hauger et al., 1977). The LH surge is accompanied by behavioral estrus which precedes ovulation by approximately 24 h (McKenzie and Terrill, 1937; Robertson, 1967; Hauger et al., 1977). The role of follicle stimulating hormone (FSH) will not be considered here because of methodological problems in measurement of serum FSH and a paucity of definitive information concerning its physiological control by gonadal steroids. The regulatory systems described in the following sections, therefore, must be considered incomplete until FSH can be incorporated into the scheme. Conceptual Approach A conceptual framework for elucidating the endocrine control of the estrous cycle and the seasonal reproductive cycle is provided in Fig. 2. In its simplest sense, the estrous cycle may be envisioned as a sequence of component events, each of which must occur for successful completion of one cycle as well as for progression of one cycle to the next. Interruption of any single event in the sequence would terminate the cycle. Restoration of this event would reinstate the cycle thereby endowing the system with the capacity for reversible fertility. Implicit in this scheme for seasonal breeding is that the remaining events need not be disrupted for cycles to cease. The sections which follow will: 1) identify key regulatory steps in the sequence of hormonal events leading to ovulation; 2) examine the evidence for each step separately; 3) identify which step in the sequence is broken to cause anestrus and 4) describe how this step is broken as a result of exposure to inhibitory environmental photoperiods. BREEDING SEASON ANESTROUSSEASON FIG. 2. Scheme for control of estrous cycle and seasonal breeding. The large arrow represents a critical step which is interrupted to cause anestrus. Hormonal Events Leading to Ovulation The temporal relationships between LH, estradiol and progesterone in peripheral serum during the preovulatory period underlie the preceding conceptual framework and are illustrated in Fig. 3. Approximately h prior to onset of the LH surge, circulating progesterone begins its precipitous descent as a consequence of the rapid demise of the corpus luteum. Associated with this decrease in progesterone, mean serum LH concentrations rise progressively reaching levels at least 5-fold greater than baseline by the time of onset of the preovulatory LH surge. This rise reflects an increase in the frequency of pulsatile LH discharges (Foster et al., 1975; Baird, 1978) and it constitutes an increase in tonic LH secretion separate from the LH surge (Karsch, unpublished observations). The sustained increase in basal serum LH concentrations, which spans a period of about 48 h, is accom- 100: 50 h P I 0 +1 DAYS FROM LH PEAK 10 _ E 5 CL < a. FIG. 3. Mean + SEM (shaded area) concentrations of LH, estradiol (E) and progesterone (P) in peripheral serum during the preovulatory period in 6 ewes. Samples were taken every 4 h and data are normalized to the preovulatory LH peak. The circle in the upper portion represents a hypothetical sequence of events leading to the LH surge. Note serum hormone concentrations are plotted on a logarithmic scale (from Karsch et al., 1977c).

3 76 LEGAN AND KARSCH panied by a 5-fold increase in circulating estradiol secreted by the rapidly enlarging follicle(s). This increase in estradiol leads to the preovulatory LH surge (Goding et al., 1969; Scaramuzzi et al., 1971, Karsch and Foster, 1975). A hypothesis for the feedback control of the events leading to ovulation has been proposed (Baird and Scaramuzzi, 1976b; Karsch et al., 1977a) based on the following hormonal interrelationships observed during the preovulatory period: 1) the inverse relationship between LH and progesterone and 2) the parallel increases in LH and estradiol. In this hypothesis, which is depicted schematically in the upper portion of Fig. 3, it is proposed that progesterone secreted by the corpus luteum exerts a dominant inhibition of tonic LH secretion. Thus, the progesterone fall resulting from luteolysis permits an increase in tonic LH secretion. In turn, the resultant rise in basal serum LH concentrations stimulates an increase in estradiol secretion. The sustained nature of the LH increment (48 h) provides the impetus necessary to drive circulating estradiol to reach the threshold necessary to trigger the LH surge. This leads to a new cycle, beginning with ovulation and formation of the corpus luteum. During the time between formation of the corpus luteum and its regression, the elevated progesterone concentrations maintain low serum LH and estradiol concentrations. At the end of the cycle, luteolysis occurs, progesterone falls and the entire sequence is repeated. It is important to note that the parallel increase in LH and estradiol precludes the possibility that estradiol is a potent inhibitor of tonic LH secretion during the preovulatory period. This becomes critical in considering the basis of seasonal breeding as will be described below. Evidence for Proposed Sequence of Preovulatory Events What evidence supports the first step in the sequence, that withdrawal of progesterone leads to the sustained increase in tonic LH secretion? First, physiological serum levels of progesterone can exert a potent inhibition of tonic LH secretion in the ewe, an effect which is enhanced by basal levels of estradiol (Foster and Karsch, 1976; Karsch et al, 1977b; Goodman and Karsch, 1978; Karsch and Legan, 1978). Further, the effect of progesterone is dose dependent (Goodman and Karsch, 1978), thus accounting for the inverse relationship between progesterone and basal serum LH concentrations throughout the cycle (see Fig. 1). The best evidence for this step, however, was obtained in experiments in which the sustained, preovulatory increase in tonic LH secretion was induced during the mid-luteal phase by experimental procedures which effected a premature withdrawal of progesterone (Baird and Scaramuzzi, 1976b; Karsch et al., 1977c). In one such experiment, surgical removal of the corpus luteum on Day 8 after the LH surge caused a rapid, premature decrease in circulating progesterone and an immediate 4- to 5-fold increase in circulating LH (Fig. 4, left panel). The LH increase was sustained for approximately 48 h, FIG. 4. Induction of preovulatory events by premature withdrawal of progesterone on Day 8 of the cycle. Left panel depicts mean + SEM serum concentrations of LH, estradiol and progesterone before and after surgical removal of the corpus luteum (CL). Right panel illustrates hormone concentrations resulting from removal of CL and insertion of Silastic implants containing progesterone (P). The number of experimental animals is indicated by n. The shaded area depicts mean serum hormone concentrations in a group of 6 sham operated controls implanted with empty capsules. Data are normalized to the time of CL removal (vertical broken line). The circle in the upper right panel indicates the step in the hypothetical preovulatory sequence tested in this experiment (from Karsch et al., 1977c).

4 CONTROL OF OVARIAN AND SEASONAL CYCLICITY IN THE EWE 77 a duration remarkably similar to that of the increase in tonic LH secretion occurring at the time of spontaneous luteolysis. Further, the LH increase was accompanied by a parallel sustained increment in circulating estradiol sufficient to initiate an LH surge and estrus, followed by ovulation. The premature occurrence of the increase in tonic LH secretion which followed corpus luteum removal was prevented when circulating progesterone was maintained at mid-luteal phase levels. This was accomplished by insertion of progesterone implants at the time of surgery on Day 8 (Fig. 4, right panel). Further, there was no sustained increase in circulating estradiol, no premature estrus or LH surge and thus, no ovulation. Serum concentrations of LH and estradiol remained low until the progesterone implants were removed on Day 14, when there were sustained increases in LH and estradiol (not illustrated). These increases were indistinguishable from those following corpus luteum removal and they culminated in an LH surge, estrus and ovulation. The preceding observations provide compelling support for the postulate that the fall in progesterone associated with luteolysis leads to the sustained increase in tonic LH secretion. The evidence further suggests that this automatically leads to the subsequent steps in the preovulatory sequence. This brings us to the second step in the sequence and an examination of the evidence that the sustained increase in tonic LH secretion stimulates the preovulatory estradiol rise. Supportive evidence was obtained in the experiment just described in that the increase in circulating LH was always associated with an estradiol rise. Additional evidence has been provided by Baird and his colleagues who observed a marked increase in ovarian secretion of estradiol within minutes following the discrete, pulsatile discharges of LH which occur throughout the cycle (Baird et al., 1976; Baird, 1978). Results from one such study performed in the late luteal phase are illustrated in Fig. 5. Their findings have led to the attractive hypothesis that the increased frequency of LH pulses between luteolysis and the LH surge generates the preovulatory estradiol rise (Baird, 1978). The tight coupling between LH and estradiol provides strong circumstantial evidence for the second step in the proposed sequence. Nevertheless, there is no direct definitive evidence that the physiological sustained increase in I 5 c 0.1 I 1 BREEDING SEASON Late Luteal -LH n = 4-13 Ewes From Baird MINUTES FROM LH PULSE FIG. 5. Mean ± SEM concentrations of LH and ovarian secretion rates of estradiol (E) during the late luteal phase of the estrous cycle in ewes with uteroovarian transplants. Observations were made every 10 min and data are normalized to the peak of the pulsatile LH discharge. Redrawn from Baird (1978). tonic LH secretion is either a necessary or a sufficient stimulus for the preovulatory estradiol rise. It has been demonstrated, however, that ovarian secretion of estradiol in the ewe can be enhanced by the administration of LH (Mc- Cracken et al., 1969). Further, administration of human chorionic gonadotropin (hcg), an exogenous LH-like stimulus, elicited an increase in circulating estradiol in the anestrous ewe (Fig. 6). Although this estradiol rise was somewhat smaller than that which normally occurs during the preovulatory period in the breeding season, it was of sufficient magnitude and duration to trigger an LH surge followed by ovulation. The foregoing evidence provides strong support for the hypothesis that the sustained increase in tonic LH secretion leads to the preovulatory increase in estradiol secretion and that this automatically leads to the subsequent preovulatory events. This brings us to the next step the LH surge which causes ovulation. A voluminous body of data, which has been obtained in a wide variety of mammals including sheep, provides compel-

5 78 LEGAN AND KARSCH DAYS FIG. 6. Mean + SEM concentrations of LH and estradiol in 4 ewes before and after injection of hcg (100 IU, i.m. twice daily) during the mid-anestrous season (June). Samples were obtained every 4 h. LH was assayed in each sample; estradiol was measured in samples pooled over the time period indicated by the width of each bar. Data are normalized to time of first hcg injection (vertical broken line). Dark arrow in circle in upper portion of figure indicates step in hypothetical sequence tested by this experiment (from Karsch et al., 1977c). ling evidence that the sustained rise in circulating estradiol is the ovarian signal for the preovulatory LH surge (Ferin et al., 1969; Goding et al., 1969; Scaramuzzi et al., 1971; Legan and Karsch, 1974; Knobil, 1974). This evidence will not be reviewed here other than to indicate that the preovulatory increase in estradiol is, in itself, of sufficient magnitude and duration to initiate the LH surge following progesterone withdrawal (Goodman, 1978). The occurrence of the LH surge completes the sequence of preovulatory endocrine events. Available evidence strongly supports the concept that each step in the sequence leads to the next and that all are required for the successful completion of the estrous cycle. Although this scheme can account for the succession of cycles throughout the breeding season, it does not explain the mechanism whereby the estrous cycle is interrupted to cause anestrus. The remaining sections will focus on the control of seasonal breeding in the context of our understanding of the estrous cycle. Endocrine Basis of Seasonal Breeding Where is the Preovulatory Sequence of Events Interrupted? Most of the essential components of the hypothalamo-hypophyseal-ovarian axis remains functional in ewes during the anestrous season, Ovarian follicles develop, produce steroids and are capable of ovulating; gonadotropic hormones are secreted; and both positive and negative feedback effects of ovarian steroids on gonadotropin secretion are readily demonstrable (Cole and Miller, 1935; Hutchinson and Robertson, 1966; Goding et al., 1969; Roche et al., 1970; Symons et al., 1973; Yuthasastrakosol et al., 1975; Karsch and Foster, 1975; Martensz et al., 1976; Scaramuzzi and Baird, 1977). Nonetheless, estrous cycles cease. As was proposed, interruption of any one step in the preovulatory sequence would be sufficient to terminate cyclicity. That step will now be identified by considering each of the regulatory steps, beginning with the LH surge and working back. Spontaneous, preovulatory LH surges Have not been observed during anestrus. Does anestrus therefore result from a change in the LH surge system such that it fails to respond to a preovulatory increase in estradiol? Some support for this possibility has been obtained from results which suggest that estrogens may become less effective in inducing the LH surge in ewes during anestrus (Land et al., 1976). Interpretation of that finding, however, is difficult because a single large dose of estradiol benzoate was injected and serum levels of estradiol were not measured. Further, long term ovariectomized ewes were employed and it is well documented that feedback responses to steroids can change markedly in the long term absence of the gonads (Brown et al., 1972; Karsch et al., 1973; Legan and Karsch, 1975; Karsch et al., 1977b). The real issue regarding

6 T CONTROL OF OVARIAN AND SEASONAL CYCLICITY IN THE EWE 79 the cause of anestrus is whether the sustained discharges of LH during the anestrous season is preovulatory estradiol rise from 2 to 10 pg/ml as great, or greater, than that which occurs is sufficient to induce an LH surge during the following LH pulses during the estrous cycle anestrous season in intact ewes. The results (Fig. 7). It would appear, therefore, that the illustrated in Fig. 6, in which an estradiol rise follicle does not become less responsive to LH from 3 to 7 pg/ml induced an LH surge and in anestrus. ovulation during anestrus, suggest that it is The foregoing considerations suggest that the sufficient. Further, results from a detailed dose primary cause for seasonal anestrus is neither response study conducted by one of our failure of the LH surge, behavioral estrus colleagues, Dr. Robert L. Goodman, suggest nor absence of the estradiol rise. This leaves the LH surge mechanism in the ewe does not only one step in the preovulatory sequence of become less sensitive to estradiol during anestrus. In light of these considerations, a primary secretion following the decrease in progesterone. events the sustained increase in tonic LH deficiency in the LH surge mechanism becomes Does anestrus result from absence of the a rather unattractive explanation for seasonal sustained increase in tonic LH secretion? A anestrus. number of observations indicate this is the step at which the cycle is interrupted. Although pulsatile LH secretion persists during anestrus, producing brief episodes of elevated serum LH concentrations, these increases are not sustained for the 48 h period characteristic of the preovulatory increase in tonic LH secretion (Scaramuzzi and Baird, 1977; Legan, unpublished observations). Further, following regression of an experimentally induced corpus luteum during anestrus, there is not a sustained increase in tonic LH secretion nor do any of the other preovulatory events occur (Ryan and Foster, 1978). Finally, if this hypothesis is correct, there should be no sustained 48 h increase in tonic LH secretion following regression of the last corpus luteum of the breeding season. Does anestrus result from a change in the centers which control estrous behavior such that the preovulatory rise in estradiol is no longer sufficient to induce estrus? From this point on, the breeding season would be effectively terminated even if ovulatory cycles continued for a short time. Seasonal changes in sensitivity to the behavioral effects of estradiol have been reported (Reardon and Robinson, 1961; Gibson and Robinson, 1971; Land et al., 1976). However, the real issue (as with the LH surge system) is whether the preovulatory estradiol rise is sufficient to induce behavioral estrus during the anestrous season. Preliminary results of a detailed dose response study suggest that it is; therefore, anestrus is probably not the result of a change in the behavioral estrus response (Goodman, unpublished observations). Does anestrus result from a decreased capacity of the follicle to respond to the sustained increase in tonic LH secretion, such that there is no preovulatory estradiol rise? In our studies, a spontaneous, sustained increase in circulating estradiol has never been observed in anestrus. It is unlikely, however, that the ovarian follicle becomes totally unresponsive to LH as indicated by the estradiol rise following injection of hcg into anestrous ewes (Fig. 6). Nonetheless, this observation does not exclude the possibility that there is a subtle shift in capacity of the follicle to respond to LH such that a sustained increase in LH secretion, such as that following luteal regression, is no longer a sufficient stimulus. Although a definitive test of this possibility remains to be conducted, the findings of Scaramuzzi and Baird (1977) suggest that it is unlikely. Specifically, the increased rate of ovarian estradiol secretion which follows the discrete, pulsatile Figure 8 illustrates the preliminary results from such a study in which blood samples were obtained from intact ewes every 4 h from mid-january to mid-march under natural environmental conditions of temperature and MINUTES FROM LH PULSE FIG. 7. Seasonal comparison of secretion rate of estradiol (E) following pulsatile discharges of LH. Data in left panel (breeding season) redrawn from Baird (1978); data in right panel (anestrus) redrawn from Scaramuzzi and Baird (1977). Further details in legend to Fig. 5.

7 80 LEGAN AND KARSCH DAYS FROM LH PEAK DAYS FROM PROGESTERONE FALL FIG. 8. Comparison of mean ± SEM concentrations of LH, estradiol and progesterone around the time of luteolysis during late breeding season (Jan-Feb) (left panel) and transition into anestrus (right panel). Data in left panel normalized to LH peak; data in right panel normalized to first decrease in progesterone. The horizontal bar in each top panel depicts the approximate duration of the preovulatory rise in tonic LH secretion. Samples were obtained every 4 h. No artificial light was used for night samples. photoperiod. The patterns of circulating LH, estradiol and progesterone at the time of the first missed ovulation following regression of the last corpus luteum of the breeding season are illustrated in the right panel. For reference, the hormonal profiles for the same 3 ewes during a preovulatory period in the late breeding season are shown in the left panel. The patterns of progesterone associated with luteolysis at both times were virtually identical. Regression of the last corpus luteum of the breeding season, however, was not followed by a typical sustained increase in tonic LH secretion. Although basal LH levels did increase at this time, this increase was abbreviated, lasting only half as long as the 48 h LH increase typical of the preovulatory period. In the absence of the usual sustained increase in tonic LH secretion, there was no sustained estradiol rise, therefore no LH surge and anestrus began. Thus, seasonal anestrus results from absence of the sustained increase in tonic LH secretion. The cause for this disruption of the preovulatory sequence will now be considered. Mechanism for Photoperiodic Control of Seasonal Breeding What prevents the sustained preovulatory increase in tonic LH secretion during anestrus? A clue for answering this question is provided by the contrast between the patterns of circulating LH during a preovulatory estradiol rise of the breeding season and during an induced estradiol rise in anestrus (compare Figs. 3 and 6). Each preovulatory estradiol rise of the breeding season is accompanied by a sustained parallel increase in LH, a relationship which precludes the possibility that estradiol is a potent inhibitor of tonic LH secretion during the breeding season (Fig. 3). In marked contrast, an induced estradiol rise during anestrus is accompanied by a pronounced decrease in circulating LH (Fig. 6). This inverse relationship suggests that estradiol may be a potent negative feedback steroid during the anestrous season. In 1972, Hoffmann proposed that seasonal breeding results from photoperiod-induced changes in sensitivity of the hypothalamohypophyseal axis to the negative feedback action of gonadal steroids (Hoffmann, 1973). This hypothesis, however, did not describe how such a change in sensitivity would terminate an estrous cycle. Based on the foregoing explanation for control of the cycle, it can easily be seen how a heightened response to the negative feedback action of estradiol causes estrous cycles to cease by preventing the sustained preovulatory increase in tonic LH secretion. This scheme for the onset of anestrus is illustrated in Fig. 9. Once the last corpus luteum of the breeding season begins to regress, tonic LH secretion would increase, thus stimulating an increase in estradiol secretion. Unlike in the breeding season, however, the rising titers of estradiol would feed back and inhibit LH, thereby preventing the occurrence of a sustained, 48 h rise in tonic LH secretion. As a consequence, estradiol secretion would decrease before attaining

8 CONTROL OF OVARIAN AND SEASONAL CYCLICITY IN THE EWE 81 LH zy Surged / (Estrus) \ I \ ^ * L H ^ * ^ t ^ HIGH RESPONSE TO ESTRADIOL ANESTRUS FIG. 9. Schematic diagram for hypothetical sequence of events for transition from breeding season to anestrus. Due to increased response to negative feedback action of estradiol (E) on LH, which establishes the negative feedback loop shown on right, the decrease in progesterone (P) no longer leads to a sustained increase in tonic LH secretion. threshold for triggering the LH surge; estrous cycles would cease and a classical negative feedback loop between LH and estradiol would be established. It should be noted that the hormonal interrelationships which actually follow regression of the last corpus luteum of the breeding season are fully compatible with this scheme (Fig. 8). A necessary corollary to this proposal is that response to the negative feedback action of estradiol would decrease at the end of the anestrous season. This would permit sustained, parallel increases in LH and estradiol secretion and a consequent resumption of estrous cycles. The scheme illustrated in Fig. 9 is plausible only if the following criteria can be satisfied: 1) there is a seasonal change in response to the negative feedback action of estradiol on tonic LH secretion in the ewe; 2) these changes coincide with transitions between breeding and anestrous seasons and 3) the changes in response to estradiol are dictated by photoperiod, the major environmental factor governing seasonal breeding in the ewe (Yeates, 1949; Hafez, 1952;Mauleon and Rougeot, 1962). The first criterion, existence of a seasonal change in response to estradiol, is satisfied by the results of the following experiment. Ewes were ovariectomized and immediately treated with s.c. Silastic implants of estradiol which maintained luteal phase levels of serum estradiol. During the ensuing 2 years, there was a striking seasonal change in circulating LH (Legan et al., 1977). Results for 1 year are illustrated in Fig. 10. Serum LH was elevated to levels similar to those in untreated ovariectomized ewes during the autumn and early winter. In February, LH declined precipitously to undetectable levels and remained low until August, when they returned to elevated levels. In individual ewes, the seasonal shifts in LH concentrations, which exceeded 30-fold, occurred within the 16 day period of an estrous cycle (Legan etal., 1977). These dramatic changes in circulating LH occurred despite relatively constant serum estradiol levels generated by the implants, thus indicating the LH pattern was not due to changes in metabolic clearance of estradiol. However, a seasonal change in LH does not necessarily reflect a change in response to steroid, since there may be a steroid independent change in the LH release mechanism as has been shown in rams and in other seasonal breeders (Davis and Meyer, 1973; Turek et al., 1975; Pelletier and Ortavant, 1975; Garcia and Ginther, 1976). Nevertheless, examination of LH levels throughout the year in a control group of ovariectomized ewes not treated with estradiol implants revealed that the seasonal fluctuations in LH require the presence of estradiol (Legan et al., 1977). These findings demonstrate that in the ewe, there is a marked seasonal change in response to the negative feedback action of estradiol on tonic LH secretion. The second criterion, coincidence of the changes in response to estradiol with transitions between breeding and anestrous seasons, has also been satisfied. The upper panel of Fig. 11 illustrates the time of onset of the breeding and anestrous seasons in intact ewes; the lower panel depicts the seasonal pattern of circulating S3 ' NOV DEC JAN FEB MAR APR MAY JUN JUL AUG SEP OCT FIG. 10. Seasonal change in LH (mean + SEM, shaded area, upper panel) and mean ± SEM concentrations of estradiol (lower panel) in 6 ovariectomized (OVX) ewes treated s.c. with Silastic implants containing estradiol (E). Redrawn from Legan et al., (1977).

9 82 LEGAN AND KARSCH 5- DEC JAN FEB MAR APH MAI JUN JUL AUG SEP OCT 1976 FIG. 11. Comparison of time of transitions between breeding and anestrous seasons in 14 intact ewes (upper panel) with response to estradiol (E) in ovariectomized ewes (lower panel). Onset of anestrus in individual intact ewes defined as first missed ovulation; onset of breeding season defined as first ovulation based on serum progesterone concentrations. Further details in legend to Fig. 10. Redrawn from Legan et al., (1977). LH in estradiol-treated ovariectomized ewes. It is evident that onset of the anestrous season occurred at the same time as the increase in response to estradiol, reflected by the decrease in LH in ovariectomized ewes. Likewise, onset of the breeding season coincided with the decrease in response to estradiol. The final criterion, control of response to estradiol by photoperiod, has also been satisfied. In sheep, the decreasing photoperiods of late summer are stimulatory and induce onset of the breeding season; the increasing photoperiods of late winter are inhibitory and bring about onset of anestrus (Yeates, 1949; Hafez, 1952). Figure 12 illustrates that both seasonal breeding and response to estradiol can be manipulated by photoperiod in much the same manner. In this study, both intact and estradioltreated ovariectomized ewes were subjected to an artificial photoperiod of alternating long (16L:8D) and short days (8L:16D) every 90 days. The artificial photoperiod accelerated the natural rhythms by inducing alternating breeding and anestrous seasons every 90 days in intact ewes accompanied by coincident shifts in serum LH concentrations in estradiol-treated ovariectomized ewes (middle and lower panels, respectively). Short days were associated with breeding season and high LH levels, long days with anestrus and low LH levels. This was the case even when the seasonal transitions were completely out of phase with those occurring under the natural environmental photoperiod. These observations leave little doubt that response of the hypothalamo-hypophyseal axis to the negative feedback effect of estradiol is governed by environmental photoperiod. Finally, they lend strong support to the hypothesis that photoperiod controls seasonal breeding by inducing a change in response to the feedback action of estradiol. This completes our consideration of the control of seasonal breeding. The evidence presented here substantiates the view that the sustained preovulatory increase in tonic LH secretion is the pivotal step, the interruption or completion of which dictates the seasonal reproductive state. The absence of estrous cycles during anestrus is a consequence of a negative feedback loop between LH and estradiol which prevents the sustained LH rise necessary to drive the estradiol signal for the LH surge. The occurrence of estrous cycles during the breeding season requires the opening of this negative feedback loop, thus permitting the preovulatory sequence to occur. gee ju fei >AI m m JUL AUG SEP OCT NOV DEC JM FEI FIG. 12. Manipulation of breeding season in 6 intact ewes (middle panel) and response to estradiol in 5 ovariectomized (OVX) ewes treated s.c. with estradiol (E) implants (lower panel). Photoperiod is indicated by horizontal bar below each panel, open i closed portions depicting hours of light and dark per day, respectively. Artificial photoperiods of long days (16L:8D) and short days (8L:16D) were alternated every 90 days. Breeding season in natural environmental photoperiod shown in upper panel. Vertical line in natural photoperiod bar indicates summer solstice. Further details in legends to Figs. 10 and 11. From Legan, S. J. and Karsch, F. J. (1977). Mechanism for photoperiodic control of seasonal breeding in the ewe. Fed. Proc. 3 7, 297.

10 CONTROL OF OVARIAN AND SEASONAL CYCLICITY IN THE EWE 83 SUMMARY A generalized scheme for neuroendocrine regulation of the estrous cycle and seasonal breeding is illustrated in Fig. 13. During the breeding season, occurrence of each estrous cycle requires a cascade of causally related events culminating in ovulation (IP, tlh, te, LH surge and estrus). Interruption of a single step in the sequence (ip-^-tlh) breaks the entire cycle and results in anestrus. Much emphasis has been placed on control of the tonic mode of LH secretion because this system appears to occupy a central position in regulating the estrous cycle and in determining the seasonal reproductive state. During the breeding season progesterone, with the help of estradiol and possibly other ovarian steroids, plays the dominant role in governing tonic LH secretion; its withdrawal initiates the sequence of events which leads to ovulation. In this sense, progesterone may be viewed as an organizer of the preovulatory events of the estrous cycle of the ewe. Estradiol, on the other hand, may be considered an organizer of the seasonal reproductive cycle. By virtue of seasonal changes in BREEDING SEASON < P7 "\ /? SHORT DAYS NEGATIVE FEEDBACK OF ESTRADIOL /I PHOTOPERIOD >ANESTRUS LONG DAYS FIG. 13. Scheme for neuroendocrine regulation of the estrous cycle and seasonal breeding. See text for details. its effects on the system which governs tonic LH secretion, estradiol determines whether or not the normal progression of preovulatory events can occur. Thus, the sequence is permitted when the response to estradiol is low; it is actively inhibited when the response to estradiol is high. The actions of estradiol on the system governing tonic LH secretion, in turn, subserve the influences of environmental photoperiod on the ovarian cycle. Thus, by means of seasonal alternations between stimulatory (short day) and inhibitory (long day) photoperiods and the attendant changes in response to estradiol, the natural process of reversible fertility is achieved. ACKNOWLEDGMENTS We are indebted to our colleagues, Drs. Douglas L. Foster, Robert L. Goodman and Kathleen D. Ryan, who helped conceive these studies, critically evaluated their design and gave their time generously to help conduct the experiments. We also thank Mr. Douglas Doop for most valuable assistance in all aspects of the animal experimentation, Ms. Barbara Glover and Ms. Marjorie Hepburn for conducting radioimmunoassays and Drs. Gordon D. Niswenderand Leo E. Reichert, Jr., for providing reagents used in radioimmunoassays. This work was supported by grants from NIH (HD ) and the Ford Foundation. REFERENCES Baird, D. T. (1978). Pulsatile secretion of LH and ovarian estradiol during the follicular phase of the sheep estrous cycle. Biol. Reprod. 18, Baird, D. T. and Scaramuzzi, R. J. (1976a). The source of ovarian estradiol and androstenedione in the sheep during the luteal phase. Acta Endocrinol. 83, Baird, D. T. and Scaramuzzi, R. J. (1976b). Changes in the secretion of ovarian steroids and pituitary luteinizing hormone in the peri-ovulatory period in the ewe: The effect of progesterone. J. Endocrinol. 70, Baird, D. T., Swanston, I. and Scaramuzzi, R. J. (1976). Pulsatile release of LH and secretion of ovarian steroids in sheep during the luteal phase of the estrous cycle. Endocrinology 98, Brown, J. M., Cumming, I. A., Goding, J. R. and Hearnshaw, H. (1972). Control of baseline levels of luteinizing hormone in the ewe. J. Reprod. Fert. 28, Cole, H. H. and Miller, R. F. (1935). The changes in the reproductive organs of the ewe with some data bearing on their control. Am. J. Anat. 57, Davis, G. J. and Meyer, R. K. (1973). Seasonal variation in LH and FSH in bilaterally castrated snowshoe hares. Gen. Comp. Endocrinol. 20, Ferin, M., Tempone, A., Zimmering, P. E. and Vande Wiele, R. L. (1969). Effect of antibodies to 17 3-estradiol and progesterone on the estrous

11 84 LEGAN AND KARSCH cycle of the rat. Endocrinology 85, Foster, D. L. and Karsch, F. J. (1976). Inhibition of tonic secretion of luteinizing hormone by progesterone in immature female sheep. Endocrinology 99, 1-6. Foster, D. L., Lemons, J. A., Jaffe, R. B. and Niswender, G. D. (1975). Sequential patterns of circulating luteinizing hormone and follicle-stimulating hormone in female sheep from early postnatal life through the first estrous cycles. Endocrinology 97, Garcia, M. C. and Ginther, O. J. (1976). Effects of ovariectomy and season on plasma luteinizing hormone in mares. Endocrinology 98, Gibson, W. R. and Robinson, T. J. (1971). The seasonal nature of reproductive phenomena in sheep. I. Variation in sensitivity to oestrogen. J. Reprod. Fert. 24, Goding, J. R. (1974). The demonstration thatpgf 2a is the uterine luteolysin in the ewe. J. Reprod. Fert. 38, Goding, J. R., Blockey, M. A. deb., Brown, J. M., Catt, K. J. and Cumming, I. A. (1970). The role of oestrogen in the control of the oestrous cycle in the ewe. J. Reprod. Fert. 21, Goding, J. R., Catt, K. J., Brown, J. M., Kaltenbach, C. C, Cumming, I. A. and Mole, B. J. (1969). Radioimmunoassay for ovine luteinizing hormone. Secretion of luteinizing hormone during estrus and following estrogen administration in the sheep. Endocrinology 85, Goodman, R. L. (1978). Role of ovarian steroids in the initiation and synchronization of behavioral estrus and the LH surge in the ewe. Biol. Reprod. 18(Suppl. 1),46A. Goodman, R. L. and Karsch, F. J. (1978). Actions of estradiol in the control of tonic LH secretion during the breeding season in the ewe. Program 60th Annual Meeting, The Endocrine Society, Abstr Hafez, E.S.E. (1952). Studies on the breeding season and reproduction of the ewe. J. Agric. Sci. 42, Hansel, W., Concannon, P. and Lukaszewska, J. H. (1973). Corpora lutea of the large domestic animals. Biol. Reprod. 8, Hauger, R. L., Karsch, F. J. and Foster, D. L. (1977). A new concept for control of the estrous cycle of the ewe based on the temporal relationships between luteinizing hormone, estradiol and progesterone in peripheral serum and evidence that progesterone inhibits tonic LH secretion. Endocrinology 101, Hoffmann, J. C. (1973). Light and feedback control of gonadotropin secretion. In: Endocrinology, Proceedings of the IV International Congress of Endocrinology. Washington, D. C, June 18 24, (R. O. Scow, ed.). Excerpta Medica, Amsterdam and American Elsevier Publishing Co., New York. pp Hutchinson, J.S.M. and Robertson, H. A. (1966). The growth of the follicle and corpus luteum in the ovary of the sheep. Res. Vet. Sci. 7, Karsch, F. J. and Foster, D. L. (1975). Sexual differentiation of the mechanism controlling the preovulatory discharge of luteinizing hormone in sheep. Endocrinology 97, Karsch, F. J., Foster, D. L., Legan, S. J. and Hauger, R. L. (1977a). On the control of tonic LH secretion in sheep: A new concept for regulation of the estrous cycle and breeding season. In: Endocrinology, Vol. 1. Proceedings of V International Congress of Endocrinology. Hamburg, July 18-24, (V.H.T. James, ed.). Excerpta Medica, Amsterdam-Oxford, pp Karsch, F. J., Foster, D. L., Legan, S. J., Peter, G. K. and Ryan, K. D. (1977c). Hormonal interrelationships during the periovulatory period in the ewe. Program 59th Annual Meeting, The Endocrine Society, Abstr Karsch, F. J. and Legan, S. J. (1978). Roles of progesterone and estradiol in controlling LH secretion during the estrous cycle of the ewe. Biol. Reprod. 18(Suppl. 1), 50A. Karsch, F. J., Legan, S. J., Hauger, R. L. and Foster, D. L. (1977b). Negative feedback action of progesterone on tonic luteinizing hormone secretion in the ewe: Dependence on the ovaries. Endocrinology 101, Karsch, F. J., Weick, R. F., Hotchkiss, J., Dierschke, D. J. and Knobil, E. (1973). An analysis of the negative feedback control of gonadotropin secretion utilizing chronic implantation of ovarian steroids in ovariectomized rhesus monkeys. Endocrinology 93, Knobil, E. (1974). On the control of gonadotropin secretion in the rhesus monkey. Rec. Progr. Horm. Res. 30, Land, R. B., Wheeler, A. G. and Carr, W. R. (1976). Seasonal variation in the oestrogen induced LH discharge of ovariectomized Finnish Landrace and Scottish Blackface ewes. Ann. Biol. Anim. Bioch. Biophys. 16, Legan, S. J. and Karsch, F. J. (1974). An analysis of the positive feedback action of estradiol on LH secretion in the rat. In: Recent Studies of Hypothalamic Function, Int. Symp. Calgary (K. Lederis and K. E. Cooper, eds.). Karger, Basel, pp Legan, S. J. and Karsch, F. J. (1975). A daily signal for the LH surge in the rat. Endocrinology 96, Legan, S. J., Karsch, F. J. and Foster, D. L. (1977). The endocrine control of seasonal reproductive function in the ewe: A marked change in the response to the negative feedback action of estradiol on luteinizing hormone secretion, Endocrinology 101, Martensz, N. D., Baird, D. T., Scaramuzzi, R. J. and Van Look, P.F.A. (1976). Androstenedione and the control of luteinizing hormone in the ewe during anestrus. J. Endocrinol. 69, Mauleon, P. and Rougeot, J. (1962). Regulation des saisons sexuelles chez des brebis de races differentes au moyen de divers rhythmes lumineux. Ann. Biol. Anim. Bioch. Biophys. 2, McCracken, J. A., Uno, A., Goding, J. R., Ichikawa, Y. and Baird, D. T. (1969). The in-vivo effect of sheep pituitary gonadotrophins on the secretion of steroids by the autotransplanted ovary of the ewe. J. Endocrinol. 45, McKenzie, F. F. and Terrill, C. E. (1937). Estrus, ovulation, and related phenomena in the ewe. Missouri Agric. Exp. Sta. Res. Bull. 264, 5-88.

12 CONTROL OF OVARIAN AND SEASONAL CYCLICITY IN THE EWE 85 Nalbandov, A. V. (1973). Control of luteal function in mammals. In: Handbook of Physiology. Sect. 7, Endocrinology, Vol. 2, Pt. 1. (R. 0. Greep, ed.). American Physiological Society, Washington, D.C. pp Pelletier, J. and Ortavant, R. (1975). Photoperiodic control of LH release in the ram. I. Influence of increasing and decreasing light photoperiods. Acta Endocrinol. 78, Reardon, T. F. and Robinson, T. J. (1961). Seasonal variation in the reactivity to oestrogen of the ovariectomized ewe. Austr. J. Agric. Res. 12, Robertson, H. A. (1967). Gonadotrophin secretion in relation to oestrus and to ovulation. In: Reproduction in the Female Mammal. Proceedings of Thirteenth Easter School in Agricultural Science. University of Nottingham, (G. E. Lamming and E. C. Amoroso, eds.). Plenum Press, New York and Butterworths, London, pp Roche, J. F., Foster, D. L., Karsch, F. J., Cook, B. and Dziuk, P. J. (1970). Levels of luteinizing hormone in sera and pituitaries of ewes during the estrus cycle and anestrus. Endocrinology 86, Ryan, K. D. and Foster, D. L. (1978). Necessity for a decrease in negative feedback of ovarian steroids on LH secretion at puberty in the lamb. Program 60th Annual Meeting, The Endocrine Society, Abstr Scaramuzzi, R. J. and Baird, D. T. (1977). Pulsatile release of luteinizing hormone and the secretion of ovarian steroids in sheep during anestrus. Endocrinology 101, Scaramuzzi, R. J., Tillson, S. A., Thorneycroft, I. H. and Caldwell, B. V. (1971). Action of exogenous progesterone and estrogen on behavioral estrus and luteinizing hormone levels in the ovariectomized ewe. Endocrinology 88, Smeaton, T. C. and Robertson, H. A. (1971). Studies on the growth and atresia of Graafian follicles in the ovary of the sheep. J. Reprod. Fert. 25, Symons, A. M., Cunningham, N. F. and Saba, N. (1973). Oestrogen-induced LH surges in the anoestrous and cyclic ewe. J. Reprod. Fert. 3 5, Turek, F. W., Elliot, J. A., Alvis, J. D. and Menaker, M. (1975). The interaction of castration and photoperiod in the regulation of hypophyseal and serum gonadotropin levels in male golden hamsters. Endocrinology 96, Yeates, N.T.M. (1949). The breeding season of the sheep with particular reference to its modification by artificial means using light. J. Agric. Sci. 39, Yuthasastrakosol, P., Palmer, W. M. and Howland, B. E. (1975). Luteinizing hormone, oestrogen, and progesterone levels in peripheral serum of anoestrous and cyclic ewes as determined by radioimmunoassay. J. Reprod. Fert. 43,