action sur l'adenohypophyse subissent cependant des ... hormone (LH) during the bovine estrous cycle. ..... Infusion of LH or human chorionic gonado-.
Regulation of the Estrous Cycle in Domestic Animals A Review
Coincident with the advent of were the chemical identification and eventual laboratory synthesis of a number of
improved assay techniques
compounds playing important
roles in female reproductive processes. The availability of such compounds (prostaglandins and hypothalamic releasing hormones are two outstanding examples) in quantities sufficient for experimentation in the larger animals revolutionized research in animal reproductive physiology and endocrinology. Although important in themselves, many of these compounds have become valuable "tools" in the investigation of reproductive mechanisms.
breuses publications qui se rapportent Neuroendocrine and endocrine factors a ce domaine. involved in the regulation of reproducLa secretion et la synthese de l'hortive cycles in domestic animals are dis- mone de rel'achement des gonadocussed. Although research data from stimulines dependent de divers facmany species are considered, emphasis teurs exogenes; son rel'achement et son is placed on their relevance for the action sur l'adenohypophyse subissent cow, sow, ewe and, to a lesser extent, cependant des modifications par the mare. Literature cited is not retroaction des hormones gonadiques. designed to be complete, but rather to Le r6le quejoueraient dans ces modifibe representative of the large volume cations des regulateurs non steroides, of material which has been written on tels que l'inhibine, reste encore ia the subject. prouver. Les prostaglandines exercent Gonadotropin-releasing hormone is une action importante sur la regressynthesized and secreted in response to sion du corps jaune et sur l'ovulation. various exteroceptive stimuli, but both L'auteur examine aussi le r6le its release and its effects on the ante- respectif des gonadotrophines et des rior pituitary are modified by feedback autres substances mentionnees plus of target gland hormones. A modulat- haut, quant ia leur action sur les foncing role for nonsteroidal regulators tions hypothalamiques, adenohyposuch as inhibin has yet to be proven. physaires et ovariennes. I1 cite brieveProstaglandins are important for cor- ment les mecanismes d'action qui pus luteum regression and ovulation. permettent aux hormones de produire The relative roles of the gonado- leurs effets. tropins and the above substances in hypothalamic, adenohypophyseal and I N T R O D U C T I O N ovarian function are considered. Gen- With the development of sensitive and eral mention is made of mechanisms of relatively simple assay techniques such hormonal action. as protein binding and radioimmunoassays, a large volume of data has RtSU M t been accumulated since 1970 on horUne revue de la regulation du cycle mone concentrations in the blood and oestral, chez les femelles domestiques glandular organs of many domestic L'auteur commente les facteurs neuro- animals. Recently, related procedures endocriniens et endocriniens impli- have enabled researchers to demonques dans la regulation du cycle repro- strate the presence of individual horducteur, chez les femelles domestiques. mone receptors (sites at which a subMeme s'il presente les resultats des stance binds to a cell) in various target recherches effectuees chez plusieurs organs or specific target cells within especes, il fait ressortir davantage ceux these organs. As a consequence, the qui se rapportent 'a la vache, 'a la bre- respective reproductive cycles in bis, 'a la truie et, 'a un degre moindre, 'a animals have become better defined lajument. Sans etre complete, la bibli- although, as in all research, many new ographie se veut quand meme re- questions are asked as old ones are presentative de l'ensemble des nom- answered.
THE ESTROUS CYCLE
The duration of the estrous cycle in the cow, the sow and the mare averages 21 days in length as compared to 17 days in the ewe. Estrus, the time during which mating behaviour is exhibited, is variable in length within species with ranges for cow, sow, ewe and mare of 12-24 hr, 48-72 hr, 24-36 hr and 4-8 days, respectively. The cow and sow are polyestrous, whereas the ewe and the mare are seasonally polyestrous with photoperiod playing an important role in the initiation of onset and cessation of ovarian activity. Peripheral plasma concentrations of luteinizing hormone (LH), follicle stimulating hormone (FSH), estrogens and progesterone are schematically illustrated in Figures 1 and 2 using the estrous cycle of the cow as an example. After estrus (Day 0), progesterone concentrations rise beginning on day 3 or 4 and remain elevated for the duration of the corpus luteum life span in all species (27,49). As a general 12
,I"I O'\ I J
10 12 DAYS
FIGURE 1. Schematic diagram of changes in
peripheral plasma concentrations of follicle stimulating hormone (FSH) and luteinizing hormone (LH) during the bovine estrous cycle.
*Department of Animal Science, Macdonald Campus of McGill University, Ste. Anne de Bellevue, Quebec H9X ICO.
Can. vet. J. 21: 301-306 (November 1980)
THE ROLE OF HYPOTHALAMUS
lamus, interruption of the blood vessels between the hypothalamus and anterior pituitary by sectioning the hypophyseal stalk, and the transplantion of the ovary to various sites. Sawyer et al (50) reported that the release of LH and consequently ovulation could be inhibited in the rabbit by the administration of centrally-acting blocking agents such as atropine. Hansel and Trimberger (29) were able to block ovulation in the cow by atropine injections given at the very beginning of estrus and found that the blockade was overcome when LH was administered along with the atropine. The question of how information perceived in the CNS was communicated to the anterior pituitary remained unresolved. Since there were no direct neural connections, it appeared that blood-borne factors carried by hypothalamo-adenohypophysis portal vessels, present in all domestic animals, must be responsible for hypothalamic influence over the anterior pituitary. In 1961, Harris (30) showed that return of ovarian functions after pituitary stalk sectioning is closely correlated with regeneration of the portal vascular system. In 1964 (55, 56), it was demonstrated that materials with the properties of peptides purified from bovine and porcine hypothalami stimulated LH release not only in vivo but also in vitro. Moreover, it was shown by in vitro study that hypothalamic materials release LH by direct action on the pituitary. The decapeptide sequence
It has been recognized for years that environmental factors such as nutrition, light and temperature exert a profound effect on mammalian reproductive processes, and of these, light is probably the most significant. Such effects can best be explained by the nervous system playing an intermediary role between the environment and the endocrine system. In certain mammalian species, i.e. rabbit, cat and mink, ovulation occurs only after coitus, and this reflex ovulation occurs in response to afferent stimuli that converge on the hypothalamus from genitalia, eyes, nose and other organs (14). In 1947, Green and Harris (24) proposed the hypothesis of neurohormonal regulation of gonadotropin secretion, based on experiments such as electrical stimulation of the hypotha-
for luteinizing hormone-releasing hormone/ follicle stimulating hormonereleasing hormone (LH-RH/FSHRH or GnRH) was proposed and confirmed by Schally et al (54). Further confirmation came with synthesis of the material and the demonstration that synthetic LH-RH/FSH-RH possessed the same properties as the natural material (57). Subsequently, Schally and Arimura (52) and others demonstrated that this material enhances the release of LH or LH and FSH in a wide variety of mammalian species including horses, (22), pigs, (9), sheep (47) and cattle (37). The concept that one hypothalamic hormone is responsible for both LH and FSH effects is supported by many physiological as well as immunological data (51).
Z0 4 g
0 0 z *00 _
10 12 DAYS
FIGURE 2. Schematic diagram of changes in peripheral plasma concentrations of estrogens and progesterone during the bovine estrous cycle.
rule, estrogen levels rise following luteolysis and peak prior to or coincident with the onset of estrus. An estrogen peak occurs prior to the rise in progesterone in the sow, ewe and cow, but a mid-cycle peak has only been described for the ewe and cow (28). Recent work in the cow has shown that an FSH peak coincides with the LH rise in early estrus although additional wave-like patterns of FSH occur throughout the luteal phase as well (58). In the ewe (21) and the sow (46), the principal peaks of FSH in plasma have also been shown to coincide with the preovulatory LH rise. Only in the mare, where prolongation of luteal activity occurs with some regularity, do these general patterns vary. Plasma levels of LH do not peak before ovulation but rather have a protracted rise commencing prior to estrus and often declining to basal values some days after ovulation (62).
Numerous experiments involving the production of lesions and stimulation of various hypothalamic and CNS areas have led to the concept of dual hypothalamic control of pituitary gonadotropin secretion. In other words, one region in the hypothalamus is responsible for maintenance of a tonic secretion of gonadotropins sufficient to cause estrogen production at a low level. Another region probably controls the preovulatory cyclic LH surge that is responsible for the very high plasma LH levels seen at the onset of estrus in many species (28). Control of GnRH synthesis and release must depend on numerous external and internal stimuli. Results of numerous experiments attest to the fact that alterations of the light-dark ratio alter the estrous cycles of most animals (28). For example, the majority of sheep in the northern hemisphere are anestrous during the late spring and early summer. Reducing amounts of light, whether artificial or natural, will initiate estrous cycles. On the other hand, mares cycle in response to increasing amounts of light, and for the most part, remain anestrous from late summer through early winter. The effects of light on reproduction in cattle are not so clearly established although improved fertility has been reported in cattle subjected to additional hours of light under conditions of limited natural daylight. Olfactory stimuli, probably acting through the hypothalamus, appear to play an important role in gonadotropin release in at least some animals (28). Internal regulation of GnRH appears to be primarily through circulating levels of the sex steroids, i.e. via feedback from the ovaries in the case of females. Estrogens and progesterone have been shown to have effects, inhibitory or stimulatory, at both the anterior pituitary and the hypothalamic levels (53), and they do significantly alter the magnitude of the GnRH effect (2). To correlate these results with the estrous cycle of animals, an increase in estrogen concentration in plasma preceding the preovulatory surge of LH appears to augment the pituitary responsiveness to GnRH. Conversely, the large amounts of progesterone which are secreted after ovulation may lower this responsiveness
ROLE OF THE ANTERIOR PITUITARY
in follicular fluid (15). The striking elevation of plasma gonadotropin concentration following ovariectomy is well established, and it is well known that injection of sex hormones will depress pituitary gonadotropin output. As noted previously, GnRH from the hypothalamus no doubt plays a role in the induction of the LH surge. Elevated levels of GnRH coinciding with the LH surge have been demonstrated in hypophyseal portal blood in the rat (48).
If the adenohypophysis is removed before puberty, accessory reproductive organs remain infantile, interstitial tissue in the ovaries remains sparse and ovarian follicles fail to ovulate or undergo atresia. If hypophysectomy is performed in an adult female, the uterus and vagina involute, and ovulation and estrous cycles cease. All these changes can be reversed by injections of a crude extract of anterior pituitary tissue which contains the three protein hormones involved in the THE OVARY direct regulation of ovarian function Sex steroid hormone concentrations (28). The first hormone, FSH, is in the peripheral plasma tend to reflect involved in the growth and maturation changes taking place within the ovary. of follicles and stimulates the produc- The mammalian follicle, under gonation of estrogens within these follicles. dotropin stimulation, synthesizes The second, LH, plays the primary estrogen until just prior to ovulation. role in follicular rupture and ovulation As the corpus luteum becomes funcand initiates luteinization of follicular tional, increasing amounts of progescells. It is probably the principal luteo- terone are secreted and high circulattropin responsible for the maintenance ing levels are maintained until the time of corpora lutea. It should be noted of luteolysis. that both FSH and LH are required for most of the above events and usu- Follicular Changes and Ovulation Two cell types, theca interna and ally act synergistically. This synergism between FSH and LH has been dem- granulosa, are responsible for steroid onstrated in several experiments using production within the follicle and, rats and other laboratory animals until recently, the prevailing view was where the relatively poor activity of that estrogens secreted by the follicle highly purified forms of one of the come from theca cells (13, 40, 65). A gonadotropins can be greatly en- two-cell theory is now gaining accephanced by the addition of very small tance in which theca cells produce amounts of the other. Follicle stimu- androgen under LH stimulation and lating hormone and LH appear to act the resultant testosterone is then similarly in the large domestic anim- aromatized to estradiol-17,8 by the als. The third hormone, prolactin, granulosa cells under FSH influence maintains the corpora lutea in hypo- (5). A number of studies support a physectomized rats (6), but attempts two-cell theory including evidence in to show that it is luteotropic in other cow follicles from which granulosa animals has been relatively unsuccess- cells showed a much greater capacity ful (17) with the possible exception of to transform androstenedione to estrogens than did thecal tissues (35). the ewe (16). A similar pattern occurs in isolated Follicle stimulating hormone and theca and granulosa cells from rat folLH are produced in the basophil cells licles (20), in granulosa cells from porin the medullary area of the adenohy- cine follicles and in cultured pophysis. Both FSH- and LH- ovine Graafian(10), follicles As the secreting basophils have been des- follicle matures and (41). ovulation cribed in rats and dogs (43, 44) approaches, granulosa cells begin to whereas only one cell type has been luteinize. In bovine granulosa cells, identified as a gonadotropin producer this luteinization and subsequent corin cattle pituitaries (33). pus luteum formation is associated Plasma gonadotropins appear to be with a loss in androgen-aromatizing kept within a relatively low range by ability (3 1). the negative feedback of gonadal steA number of nonsteroidal follicular roids and possibly by inhibin, an anti- "regulators" have been described FSH proteinaceous material present which are protein in nature and found
in the follicular fluid of most species (12). Perhaps the most important of these substances is inhibin (folliculostatin) which was originally isolated from bovine follicular fluid by DeJong and Sharpe ( 15). This substance inhibits the secretion of FSH, and could conceivably play a role in the regulation of the estrous cycle. Charcoal extracted bovine follicular fluid inhibited follicular development and delayed return to estrus when administered to heifers or ewes (39). Inhibin has been shown to be produced by rat granulosa cells in vitro and this same material suppressed FSH production by cultured anterior pituitary cells
Elevated follicular fluid concentrations of prostaglandins (PG) have been measured in pre-ovulatory porcine follicles (64) and in the follicles of laboratory animals. As well, the administration of PG synthesis inhibitors will block ovulation (1), and this blockade can be reversed by injections of prostaglandin F2a (PGF2a) (18). The mechanism of PGF2a action in ovulation has not yet been determined but it is probably a local effect since this substance has a very short half-life in the circulation. From this point of view, it is unlikely that PG synthesized in the follicle plays a major role in overall estrous cycle regulation.
Corpus Luteum Function and Luteolysis After ovulation, granulosa and theca cells within the ruptured follicle undergo luteinization and form a corpus luteum (CL). In large domestic animals, this CL secretes progesterone throughout the middle two-thirds of the ensuing estrous cycle and probably requires LH as a luteotropin. Infusion of LH or human chorionic gonadotropin (hCG) via the ovarian artery has stimulated an increase in progesterone secretion by the sheep ovary although the latter became refractory to subsequent stimuli (7). Moudgal et al (42) have demonstrated a reduction in progesterone level and a shortening of the luteal phase in primates after administration of antibody to hCG. It would appear then that the pituitary controls the length of the luteal phase. However, in the pig, sheep and cow at least, the uterus plays an important role in terminating the luteal 303
phase since hysterectomy extends the length of the cycle and circulating progesterone is maintained at a high level for an extended period of time (4). The luteolytic action of PGF2a has been demonstrated in a variety of animals (26), and arachidonic acid, a precursor of PGF2a has been identified as a luteolytic agent in extracts of bovine endometrial tissue. Furthermore, injections of arachidonic acid into the bovine CL caused an immediate increase in PG concentration in ovarian venous blood, followed by a sharp decline in plasma progesterone (61). What initiates the signal for uterine PGF2a production is not known, although there is evidence that its secretion is under the influence of estrogen (38) and that estrogen is necessary for the manifestation of luteolysis by PGF2a (26). The mechanism involved in luteolytic action is not well-defined although it has been studied in rats (25). These workers found a marked fall in serum progesterone and in the capacity of luteal tissue to bind hCG which would suggest that luteolysis is due to a reduction in gonadotropin receptors. However, it has been shown in recent work by Rao et al (45) that PGF2a binding in bovine corpora lutea progressively increases throughout the estrous cycle, whereas hCG receptor number and affinity are the same on days 13 and 20 even though plasma progesterone levels drop substantially by day 20. DISCUSSION
The interrelationships among the hypothalamus, anterior pituitary and ovary are outlined in schematic fashion in Figure 3. It is probable that control of gonadotropin release is mediated through a neurohumoral substance produced in hypothalamic nuclei. This substance, called GnRH, is synthesized and secreted in response to various exteroceptive stimuli, but both its release and its effects on the anterior pituitary are modified by feedback of target gland and adenohypophyseal hormones. A modulating role for nonsteroidal regulators such as inhibin has yet to be proven. The sex steroids may exert either a stimulatory or inhibitory influence on gonadotropin release depending on a number of factors such as concentration, duration of exposure to that concentration
] Hypothlamus _ J 1 Anterior
Ovary I u.l,uL J 3. anterior FIGURE pituitary, Hypothalamus, ovary and uterus interrelationships.
and stage of the estrous cycle. The latter factor no doubt relates to relative concentrations of other hormo nes and receptor population on the tars Jet cells. For example, rising estrogei n levels appear to initiate the preovulatory LH surge although exogenous estrogens can be efficient blockers of gonadotropin release, e.g. in birth control pills. The less effective inhibition of gonadotropin release by progesterone is probably beneficial in that it allows some "leakage" of FSH and LH during the luteal phase and thereby facilitates follicular development and CL maintenance. Recent studies in the ewe conclude that progesterone withdrawal at the time of normal luteolysis initiates preovulatory hormonal events (34). Gonadotropin releasing hormone is transported to the pars distalis or anterior pituitary via the portal vessels of the pituitary stalk where it attaches to basophil cell receptors and stimulates the synthesis and/or release of FSH and LH. Circulating FSH and LH stimulate the growth and development of follicles and steroidogenesis within those follicles to the point where high levels of estrogen are produced, the estrogen stimulates an LH surge (and probably induces LH receptors in granulosa cells), and luteinization of fol-
licular cells and ovulation ensue. Estrogen production then declines and, while the luteal cells are producing large amounts of progesterone, it remains low and sporadic until luteolysis. The luteolytic factor appears to be PGF2a (and perhaps other PG's) in the large domestic animals, and its principal source is the uterus. What initiates PG synthesis at this specific time is unknown. Although estrogen may be necessary for PGF2a luteolytic activity, there is no obvious change in the level of circulating estrogen, at least, which theoretically might act as a stimulus. Although some PG's have been shown to be active at the level of the brain, most evidence strongly supports the view that PGF2a has a direct effect on the corpus luteum. From a practical standpoint, this knowledge of estrous cycle regulation has resulted in a number of applications in animal reproduction. For example, the continual administration of progesterone (and numerous synthetic analogues) will effectively inhibit gonadotropin release from the anterior pituitary until withdrawal, at which time follicular development and ovulation will ensue if sufficient time has elapsed for normal luteolysis to occur. This approach has been applied successfully in many countries for the synchronized breeding of ewes (23). Luteolytic agents, such as PGF2a, have provided a second approach to estrous cycle control in which their administration to cows (36) or mares (3) having a functional CL will result in its regression and the initiation of a new cycle. Several of these new compounds are proving useful in conditions other than simple manipulation of the estrous cycle. Resolution of certain pathological states, such as pyometritis in the cow in which the CL is retained, may be facilitated by PGF2a, (or synthetic analogue) administration. The latter will also induce abortion and parturition, particularly in those species requiring a CL for maintenance of pregnancy. Gonadotropin releasing hormone is effective in luteinizing ovarian cysts in cows (1 1). The technique of bovine embryo transfer depends on superovulation for the production of several embryos from a donor mother. This entails the admin-
istration of gonadotropins during the mid-luteal phase of the estrous cycle to stimulate multiple follicle growth, followed by PGF2Ct injection to regress the CL. As a consequence, follicular maturation and synchronized ovulations ensure (60). Injection of the pregnant mare's serum gonadotropin (PMSG) into prepuberal heifers will stimulate follicles to mature and ovulate (63). The PMSG, in combination with hCG administration, will induce follicular development and ovulation in prepuberal gilts (8) and in anestrous sows (59) and given at the time of progesterone withdrawal in ewes, results in better synchronization of heats and a slightly increased lambing rate (23). Although our knowledge of the functioning of the hypothalamicpituitary-ovarian axis has increased greatly in recent years, much remains to be discovered before the estrous cycle will be completely understood. In farm animals particularly, the "time-clock-like cyclicity" of the female reproductive process is remarkable as is the seasonality of the cycle in some species. In all species, there has been much recent work in the area of hormone receptors and factors which influence the number present at any given time. The regulation of receptors in vivo, when fully understood, may be an important key to resolving many of the unanswered questions about the female reproductive cycle. REFERENCES
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