Selection of the dominant follicle in cattle and horses

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1 Ž. Animal Reproduction Science Selection of the dominant follicle in cattle and horses O.J. Ginther ) Animal Health and Biomedical Sciences, 1656 Linden DriÕe, UniÕersity of Wisconsin-Madison, Madison, WI 53706, USA Abstract The nature of selection of the dominant follicle is reviewed by comparing research results between cattle and horses. In both species, emergence of a follicular wave is stimulated by an FSH surge. The surge reaches a peak by the time the follicles attain 4 mm in diameter in cattle and 13 mm in mares. In cattle, all of the growing follicles G5 mm contribute to the decline in FSH concentrations. However, the declining FSH concentrations are still needed by the growing follicles. Several days after the peak of the FSH surge and emergence of the wave, the two largest follicles reach means of 8.5 and 7.7 mm in cattle and 22 and 19 mm in horses. At this approximate time, the follicles begin to undergo deviation in follicle diameters, which is characterized by continued growth of the largest follicle to become the dominant follicle and reduced or terminated growth of the remaining follicles to become subordinate follicles. In both species, on average, the future dominant follicle emerges before the future largest subordinate follicle, and the two follicles grow in parallel until deviation. The difference in diameter between the two largest follicles at the beginning of deviation is equivalent in growth to approximately 8 h in cattle and 24 h in mares. Apparently, this is adequate time for the largest follicle to establish the deviation process before the second-largest follicle reaches a similar diameter. During this time, the largest follicle plays the primary role in further suppressing circulating FSH concentrations to below the requirements of the smaller follicles, which causes their regression. The follicle-produced FSH suppressants appear to be estradiol and inhibin. In addition to enhancing its FSH-suppressing ability, the largest follicle also develops the ability to utilize the reduced concentrations of FSH for its continued growth. It is therefore postulated that the essence of selection of a dominant follicle in these two species is a close two-way functional coupling between changing FSH concentrations and follicle growth and development. Elevated concentrations of circulating LH encompass deviation in both species and may play a role in continued growth of the largest follicle. It is not known if LH begins to be utilized by the largest follicle ) Tel.: q ; fax: q Ž. address: ojg@ahabs.wisc.edu O.J. Ginther r00r$ - see front matterq 2000 Elsevier Science B.V. All rights reserved. Ž. PII: S X

2 62 ( ) O.J. GintherrAnimal Reproduction Science before, at, or after the beginning of diameter deviation. However, results of studies in mares suggested that LH does not influence growth of the dominant follicle until after the beginning of deviation. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Cattle; Dominant follicle; Horses; Follicle selection; Follicular waves; Subordinate follicles 1. Introduction Follicle selection is the mechanism whereby only one of the many available follicles becomes the ovulatory follicle in monovular species and has been a long-time mystery in reproductive biology. Primarily because of the transrectal ultrasound technique, changes in follicle populations have been well characterized in the large farm species, and progress is being made toward resolution of the follicle-selection mystery. The ultrasound technique is being used for characterizing follicle-population changes, tracking individual follicles from examination to examination, monitoring the effects of treatment, and eliminating, treating, or sampling specific follicles at specific times during a follicular wave. Among farm species, cattle and horses have the most effective selection mechanism as indicated by a greater frequency of single ovulations than for swine, sheep, and goats. The purpose of this report is to examine the current scientific status of the follicle-selection phenomenon by comparing research results between cattle and horses. Most of the review considers the morphologic extrafollicular aspects of selection Žchanges in follicle diameters and circulating gonadotropin and estradiol concentrations.; less is known about the intrafollicular biochemical aspects. 2. Follicular waves Cattle and horses have similar follicle-selection characteristics. The two species make good comparative research models because of suitability for different experimental approaches, utilizing, for example, a consistent early diestrous follicular wave in cattle and two- to threefold larger follicles in mares Cattle Based on histologic study, two waves of antral follicular development were proposed initially for the bovine estrous cycle, and each wave resulted in a follicle of preovulatory diameter Ž Rajakoski, Subsequently, results of various experimental approaches were interpreted to agree or disagree with the two-wave proposal Žfor reviews see Matton et al., 1981; Fortune et al., 1991; Ginther et al., 1996b.. Transrectal ultrasonic imaging for the study of bovine ovarian follicles was introduced in Pierson and Ginther Ž Using this technology, it was reported that, on average, two waves of follicular growth for various diameter categories occurred during the estrous cycle ŽPierson and Ginther, 1987b.. It was concluded that each wave involved a selection process, wherein a large follicle grew to a preovulation diameter, and other follicles began to regress

3 ( ) O.J. GintherrAnimal Reproduction Science several days before the largest follicle reached maximum diameter. Functional selection against nonovulatory follicles approximately 5 days before ovulation was confirmed by a reduced response to a superovulatory gonadotropin regimen ŽPierson and Ginther, 1988a.. Ultrasonic tracking of individual follicles from day-to-day ŽFortune et al., 1988; Pierson and Ginther, 1988b; Savio et al., 1988; Sirois and Fortune, 1988; Knopf et al., confirmed these interpretations and provided additional and more detailed characterization. In various herds, a predominance of either two-wave or three-wave estrous cycles was found, accounting for earlier reports of two versus three waves. Each wave is characterized by emergence of a group of follicles at 4 mm, growth of all follicles for a few days, and then dissociation into a large follicle that continues to grow and smaller follicles that regress. The anovulatory wave which begins as 4-mm follicles during the periovulatory period will be featured in this report because it has the most consistent characteristics and has been most extensively studied. In the earlier studies, the follicles were defined as largest Ž F1. and second largest Ž F2., followed by the terminology dominant Ž Goodman and Hodgen, 1983., dominant and nondominant ŽIreland and Roche, 1987; Sirois and Fortune, 1988., or dominant and secondary ŽIreland and Roche, 1987; Savio et al., Thereafter, the terms dominant and subordinate follicles have been used most frequently Mares The types of follicular waves that develop in mares are major waves Žcharacterized by dominant and subordinate follicles. and minor waves Žlargest follicle does not attain the diameter of a dominant follicle.. Based on transrectal palpation, a single major follicular wave was proposed initially for the equine estrous cycle Ž Ginther, The wave of follicles dissociated about 6 days before ovulation into a single growing preovulatory follicle and several regressing follicles. The palpation work was subsequently substantiated by ultrasound, based on grouping of follicles into diameter categories Ž Palmer, 1987; Pierson and Ginther, 1987a. and tracking of individual follicles Ž Sirois et al., 1989; Ginther, There are profound breed differences in wave patterns during the estrous cycle Ž for review see Ginther, In some breeds Ž quarter horses, ponies., usually only one major wave develops in late diestrus and culminates in the estrous ovulation. In other breeds Ž thoroughbreds., a secondary major wave frequently develops in early diestrus, and the dominant follicle may be anovulatory, as in cattle, or ovulatory. The secondary-wave phenomenon accounts for the earlier intriguing discovery Ž Hughes et al., of diestrous ovulations. Minor follicular waves have been demonstrated statistically in mares ŽGinther, 1993; Ginther and Bergfelt, The selection phenomenon of the major ovulatory wave that begins at midcycle for all breeds will be used for this report and will be compared to the selection aspects of the anovulatory wave that begins near ovulation in cattle. 3. Stimulation of waves by FSH surges Surges of FSH stimulate the occurrence of waves during several reproductive statuses in addition to the estrous cycle and postpartum period. In cattle, FSH stimulation of

4 64 ( ) O.J. GintherrAnimal Reproduction Science major waves occurs in calves Ž Evans et al., 1994., during much of pregnancy ŽGinther et al., 1996a., and during prolonged progesterone administration Ž Bergfelt et al., 1991a.. Also, in mares, the major and minor waves develop during various status levels Ž Bergfelt and Ginther, 1992; Ginther, 1993; Ginther and Bergfelt, The occurrence of follicular waves during many diversified hormonal environments attests to the robustness of the follicle-selection phenomenon and is a consideration in the development of hypotheses on controlling mechanisms. Emergence of follicular waves refers to the earliest ultrasonic detection of follicles compatible with retrospective tracking. Emergence of each wave is temporally associated with an FSH surge for the major waves in cattle ŽAdams et al., 1992; Sunderland et al., 1994; Gong et al., 1995; Evans et al., and horses ŽPalmer, 1987; Ginther and Bergfelt, 1992; Bergfelt and Ginther, 1993; Fig. 1. and for the minor waves in horses Ž Ginther, 1993; Ginther and Bergfelt, The FSH surge reaches a peak or plateau when the largest follicle reaches about 4 5 mm in cattle ŽBodensteiner et al., 1996a; Kulick et al., and about 13 mm in mares Ž Gastal et al., The concentrations then decline. In cattle, 3-mm follicles did not suppress FSH, but acquired this capability during their growth to 5 mm Ž Gibbons et al., More than one of the growing follicles contributes to the FSH decline as indicated by the rate of decline when all follicles, all but one follicle, all but two follicles, or no follicles were ablated ŽGibbons et al., 1997.; FSH declined more slowly when fewer follicles were retained. Circulating FSH continues to be needed by the growing follicles in heifers, even when the FSH concentrations are decreasing during the declining portion of the FSH surge Ž Ginther et al., A minimal dose of estradiol was used to decrease FSH concentrations without an associated change in LH concentrations. Estradiol treatment Fig. 1. Mean Ž "SEM. day-to-day diameter of follicles and circulating concentrations of FSH in 14 mares. Normalizing the data to the day of deviation Ž. b produced a sharper dissociation between follicles than when data were normalized to the day of emergence of the future dominant follicle Ž. a. A star indicates the first difference Ž P in the increases or decreases in concentrations. Adapted from Gastal et al. Ž

5 ( ) O.J. GintherrAnimal Reproduction Science when the largest follicle reached G 6.0 mm resulted in depression of both FSH concentrations and diameter of the largest follicle within 8 h. The smaller follicles were also inhibited. Thus, there is a close two-way functional coupling between FSH and the follicles during the declining portion of the FSH surge. The growing follicles cause the FSH decline and, even though decreasing in concentrations, the FSH remains essential for the growing follicles. 4. Follicle selection and deviation Selection is a general term used for monovular species to indicate that usually only one follicle of a follicular wave reaches dominant status, especially as indicated by ovulation. More broadly, the term could be used for multiovular species when the number of follicles of a wave exceeds the number of ovulations. This does not imply that the mechanism of selection is similar in both monovular and multiovular species. However, the occasional occurrence of multiovulations in monovular species and the frequent occurrence in other species could be useful for studying the nature of selection of one follicle Ž Wiltbank et al., There is no consensus on use of the term selection for a specific point during follicle growth, and the term is confusing when used to discuss time of occurrence. In the earlier ultrasound studies in cattle ŽGinther et al., and mares Ž Bergfelt and Ginther, 1993., means for the future dominant and largest subordinate follicles gradually diverged in diameter beginning on the day of follicle emergence. This gave the impression that the dissociation into dominant and subordinate follicles was a gradual event beginning at emergence. Later inspection of individual follicle-diameter profiles indicated that dissociation was often an abrupt event and was termed deviation in cattle Ž Ginther et al., 1996a, 1997a. and mares Ž Gastal et al., The gradual mean divergence was attributable to the occurrence of deviation at different times among waves Ž Fig. 1.. Deviation is characterized by continued growth of the largest follicle to become the dominant follicle and a reduction or cessation of growth by the remaining follicles to become subordinate follicles. Diameter deviation is retrospectively judged to have begun at the ultrasound examination preceding the first examination with an apparent change in diameter difference between the two largest follicles. For some waves, the time of deviation may not be obvious, especially because the subordinate follicles may continue to grow for )1 day, but at a reduced rate. Averaged over several reports, the mean diameters of the two largest follicles in cattle at the beginning of deviation were 8.5 and 7.7 mm with deviation beginning a mean of 2.5 days after emergence of the largest follicle at 4 mm Ž Ginther et al., 1997a, 1998, 1999; Kulick et al., The corresponding values in mares were 22 and 19 mm and 6.2 days after emergence of a 6-mm follicle Ž Gastal et al., 1997, 1999a,c,d.. The future dominant follicle emerges earlier, on average, than the other follicles of the wave. In cattle, the future dominant follicle emerged at 3 mm a mean of 6 h ŽGinther et al., 1997a. or at 4 mm a mean of 7 h Ž Kulick et al., earlier than the future largest subordinate follicle. The follicles grow in parallel, on average ŽGinther et al., 1997a; Kulick et al., 1999., so that the largest follicle maintains about a 0.5-mm-diame-

6 66 ( ) O.J. GintherrAnimal Reproduction Science ter advantage until deviation Ž Kulick et al., Similarly in mares, on average, the future dominant follicle reached 6 mm before the future largest subordinate follicle and maintained a mean diameter advantage of 3 mm until the beginning of deviation ŽGastal et al., Although none of several experiments have statistically detected a mean difference in diameter growth rates between the two largest follicles from emergence to deviation, considerable variation occurs among individual waves, presumably reflecting biologic variation as well as measuring error. Error can be considerable, especially when distortion of the ultrasound image of the follicle occurs because the face of the transducer is not optimally oriented relative to the surface of the follicle or the walls of the follicle are obscured by artifacts Ž Ginther, In heifers, the future dominant follicle was the largest follicle 2 days after wave emergence at 4 mm ŽBodensteiner et al., 1996a. or 4 5 mm Ž Evans and Fortune, in 82% and 79% of follicular waves, respectively. Examples of the occasional occurrence of the largest follicle reaching a diameter equivalent to the expected diameter at deviation and then ceasing to grow have been published for cattle Ž Ginther et al., 1996b. and mares Ž Gastal et al., Because of the diameter advantage of the largest follicle, it could be concluded that selection occurs or begins before ultrasonic detection of emerging follicles. With this reasoning, however, the beginning of selection would not be definable, since other factors may determine which of the underlying follicles becomes sensitive to an FSH surge; the largest follicle in a range of responsive diameters may be designated by chance. A further consideration against use of the term selection before deviation is that the future nondominant follicles retain their capacity to become dominant until after deviation; that is, they have not been selected against. In cattle, many of the growing follicles are capable of becoming dominant; a randomly selected 5-mm follicle can be directed toward dominance by destroying other 5-mm follicles as they appear ŽGibbons et al., In both heifers Ž Ginther et al., and mares Ž Gastal et al., 1999c., the second-largest follicle becomes dominant when the largest follicle is ablated at the expected beginning of deviation. Furthermore, initiation of FSH treatment early in a wave resulted in a delay in the apparent equivalent of deviation in cattle ŽAdams et al., 1993; Mihm et al., and development of dominance by several follicles in horses Ž Squires et al., 1986; Rosas et al., 1998., demonstrating the pre-deviation capabilities of follicles. It appears that the terms deviation and selection can be used synonymously, but to minimize confusion, deviation will be used in the remainder of this report to assure focusing on the relatively narrow time span where the future dominant follicle and future largest subordinate follicle begin to differ in growth rates in individual follicular waves. 5. Control of deviation When two spherical follicles of different diameters increase in diameter at the same rate, the rate of change in surface area over the same time span is greater for the larger follicle Ž Ginther, 1998.; surface area of a sphere is calculated exponentially from radius. This consideration indicates that the surface area of the largest follicle gains an

7 ( ) O.J. GintherrAnimal Reproduction Science increasing advantage over the next-largest follicle, even though the difference in diameter between the two follicles is constant. It is not known if the increasing advantage in surface area for the largest follicle plays a role in deviation. Surface area changes seem more representative of functional changes, because function involves the cells that line the follicle. Nevertheless, this review will use diameters because of tradition. Concentrations of FSH decline for a few days after the peak of the FSH surge in cattle Ž Adams et al., and mares Ž Bergfelt and Ginther, The decline, therefore, encompasses deviation. More specifically, low FSH concentrations are temporally associated with deviation in cattle ŽGinther et al., 1997a, 1998, 1999; Kulick et al., and mares Ž Gastal et al., The concentrations continue to decline for several days after deviation in mares Ž Fig. 1., whereas the decline ends within hours after expected deviation in cattle Ž Fig. 2.. In a recent study in cattle Ž Ginther et al., 1999., attainment of a diameter of G8.5 mm by the largest follicle was used as a reference for the expected beginning of deviation Ž Hour 0.; FSH concentrations were determined for Hours y16, y8, and 0, every hour between Hours 0 and 16, and then every 8 h. Concentrations decreased between Hours y16 and 0, continued to decrease until Hour 10, and then increased after Hour 16 Ž Fig. 2.. The results indicated a close association between the attainment of a mean diameter of G8.5 mm and a continued decrease in FSH concentrations for a short time thereafter. As noted earlier, a role for the low concentrations of FSH in the deviation mechanism in both species is consistent with the findings of a delay or prevention of deviation following administration of FSH. We postulated in cattle Ž Ginther et al., 1996b; 1997a. and mares Ž Gastal et al., that the events underlying the beginning of diameter deviation are abrupt. Even when the intervals between scanning in cattle was 8 h, the beginning of deviation was readily assigned to a specific examination in six of eight profiles of follicular waves ŽKulick et Fig. 2. Mean Ž "SEM. circulating concentrations of FSH at 8-h Ž a. and 1-h Ž b. intervals normalized to the expected time of deviation Ž largest follicle at G8.5 mm; Hour 0.. In the ablation group, the G8.5-mm follicle was ablated. The stars indicate changes Ž P between hours for the indicated groups, and the pound marks indicate a difference Ž P between groups within hours. The data indicate that FSH concentrations continued to decrease for 8 h after expected deviation and that FSH concentrations increased between 5 and 8 h after ablation of the largest follicle. Adapted from Ginther et al. Ž

8 68 ( ) O.J. GintherrAnimal Reproduction Science al., Apparently, when the largest follicle reaches a decisive developmental stage, rapid activation of the deviation mechanism blocks the second largest follicle before it reaches a similar decisive diameter. Thus, the difference in diameter between the two largest follicles indicates that the smaller follicle must be inhibited in - 8 h in cattle Ž equivalent to a diameter difference of 0.5 mm. and in -1 day in mares Žequivalent to a difference of 3 mm.. Diameter deviation is likely preceded by biochemical or functional deviation. In this regard, echotextural changes were detected in the wall of the largest follicle on the day before the beginning of diameter deviation in mares ŽGastal et al., 1999b.. Thus, diameter deviation was preceded by echotextural or structural deviation. The depression of FSH concentrations could be the pivotal event in deviation if the FSH is depressed below the quantities required by the smaller follicles, but not the largest follicle, and the changes in FSH concentrations and follicle development are closely coupled. This last stipulation is to satisfy the postulate that deviation must occur rapidly before the next largest follicle reaches a diameter similar to the diameter of the largest follicle at the beginning of deviation. The closeness of coupling between the two events was investigated in heifers by ablating the largest follicle when it reached G 8.5 mm Ž expected beginning of deviation. and, in another experiment, administering a known FSH depressant Ž Ginther et al., Ablation of the largest follicle when it reached G 8.5 mm Ž Hour 0. resulted in increased circulating FSH concentrations between Hours 5 and 8 Ž Fig. 2.. Growth rate of the retained second-largest follicle between Hours 0 and 8 was greater in the ablation group than in the controls. A single injection of a minimal dose of an FSH depressant Ž4.4 ml of a near steroid-free follicular fluid. at the expected time of deviation resulted in decreased FSH concentrations by Hour 6. Reduced growth rate of the largest follicle occurred within 6 h after the suppression in FSH concentrations. These studies demonstrated that a close temporal coupling between a change in FSH concentrations and the follicle response could establish the deviation mechanism in - 8 h before the second-largest follicle reaches a similar critical diameter. In conclusion, the largest follicle affected FSH concentrations and FSH affected the follicles within the time represented by the difference in diameters between the two largest follicles at deviation. Following administration of a near steroid-free fraction of follicular fluid, the FSH concentrations and diameters of the largest follicle were lower than the corresponding values in controls at the expected time of deviation Ž Ginther et al., These results indicated that the largest follicle in controls required the basal FSH concentrations. The requirement for the basal FSH concentrations for continued growth of the largest follicle has also been demonstrated by administration of a single minimal dose of estradiol Ž Ginther et al., 2000; Fig. 3.. Although regression of the smaller follicles at the time of deviation can be attributed to inadequate FSH, these results indicate that the low concentrations of FSH are required for continued growth of the largest follicle. Thus, deviation involves a change in the largest follicle so that it is sensitive to a concentration of FSH that is inadequate for the smaller follicles. Ablation or retention Ž Hour 0. of the largest follicle was done at G7.5 mm vs. G8.5 mm Ž Ginther et al., The mean FSH concentrations for the 8.5-mm groups were greater for the ablation group than for the control group at Hours 8 and 12, but there was no difference between the 7.5-mm groups at any hour. These results supported the

9 ( ) O.J. GintherrAnimal Reproduction Science Fig. 3. Mean Ž "SEM. circulating concentrations of FSH Ž a. and diameters of the largest follicle Ž b. when estradiol injections were given when the largest follicle reached G8.5 mm Žexpected time of deviation; Hour 0.. There was a significant interaction between estradiol-treated and control groups for both end points. The data indicate that the low concentrations of FSH after deviation were required for continued growth of the dominant follicle. Adapted from Ginther et al. Ž hypothesis that by the time the largest follicle reaches the expected beginning of deviation it has developed a greater capacity for suppressing FSH. In a similar ablation study in mares, a two-follicle model was used Ž Gastal et al., When the larger follicle reached G 20 mm Ž actual diameter, 21.2 mm; expected day of deviation; Day 0. follicle ablation was done so that both follicles, only the larger follicle, or only the smaller follicle was retained. The decline in FSH concentrations of the wave-stimulating FSH surge continued for several days after Day 0 in the groups with both follicles or only the larger follicle retained Ž Fig. 4.. In the group with only the smaller follicle retained, the FSH concentrations and diameter of the smaller follicle increased between Days 0 and 1. The continued decrease in FSH at the expected beginning of deviation Fig. 4. Mean Ž "SEM. circulating concentrations of FSH Ž a. and estradiol Ž b. in mares in which two follicles were studied; the remaining follicles were ablated. When the larger follicle reached G20 mm Žexpected day of deviation., one of the follicles was ablated, resulting in groups with retention of the smaller, larger, or both follicles. In both groups with the larger follicle present, FSH continued to decrease and estradiol increased. None of these effects were attributable to the smaller follicle. Adapted from Gastal et al. Ž 1999c..

10 70 ( ) O.J. GintherrAnimal Reproduction Science was attributable to the larger follicle; there was no indication that the smaller follicle was involved. Thus, the continued FSH suppression at the expected beginning of deviation was a function of the larger follicle. These findings support the hypothesis, in mares as in heifers, that when the largest follicle reaches a critical diameter the FSH depression continues and thereby the next largest follicle is depressed before it reaches a similar critical diameter. Thus, two abilities develop in the future dominant follicle by the beginning of diameter deviation: Ž. 1 ability to suppress circulating FSH to below the concentrations required by other follicles and Ž. 2 ability to utilize the low FSH concentrations in its further growth and development. Fig. 5. Schematic model of the proposed functional coupling between circulating FSH concentrations and diameters of the two largest follicles during development of a follicular wave. The following numbered statements refer to the circled numbers in the figure. Ž. 1 Emergence of a follicular wave is stimulated by an FSH surge that reaches its peak approximately when the largest follicle of the wave is 4.0 mm. The declining FSH concentrations continue to exert a required positive effect on the follicles Ž solid arrows.. Ž 2. By the time the follicles reach 5.0 mm, they develop an FSH-suppressing ability. All of the growing follicles G5.0 mm contribute to a decline in the FSH surge for approximately the next 2 days Ž broken arrows.. Ž 3. By the time the largest follicle reaches a diameter of 8.5 mm, it plays a major role in the continued FSH decline. Follicle deviation begins when the FSH concentrations are suppressed and temporarily maintained below the concentrations required by the smaller follicles. Hence, the smaller follicles become subordinate follicles. Ž. 4 The suppressed FSH concentrations are adequate and required for at least the initial continued growth of the largest follicle Ž dominant follicle. after the beginning of deviation. Ž 5. At an unknown point relative to deviation, LH begins to play a role in the continued growth of the dominant follicle. From Ginther et al. Ž

11 ( ) O.J. GintherrAnimal Reproduction Science It has been proposed that regression of the smaller follicles involves a direct effect of follicle inhibitors secreted by the larger follicle Žfor review see Armstrong and Webb, 1997., but the evidence for such an effect is not convincing Ž Fortune, There have been no reports that suggest that such inhibitors can be produced specifically by the largest follicle at the beginning of deviation and enter the vascular system to affect other follicles. Taken together, the ablation studies in cattle and horses indicate that inhibition of the smaller follicles during follicle deviation is attributable to continued suppression of FSH concentrations below the concentrations required by the smaller follicles, but not the largest follicle. It does not seem necessary to invoke a follicle-to-follicle inhibitory mechanism, unless indicated by further studies. It is postulated, instead, that the essence of the selection of a dominant follicle is a close two-way functional coupling between changing FSH concentrations and follicular growth and development Ž Fig Follicular systemic inhibitors of FSH The identities of the substances produced by follicles and causing the FSH decline after the peak of the surge and at the beginning of follicle deviation are not known. Estradiol, as well as androgens, are secreted by the developing follicles and are candidates for an inhibitory effect on FSH secretion Ž Evans et al., However, it is not known if estradiol enters the circulation in concentrations adequate for a negative feedback effect on FSH, especially during the initial portion of the FSH decline. In cattle, estradiol concentrations increase in the follicular fluid of the largest follicle and in the systemic circulation at about the time deviation would be expected to occur ŽKaneko et al., 1991; Mihm et al., 1997; Evans et al., The increase in plasma estradiol occurred from a single ovary and encompassed 3 to 7 days after the LH surge ŽIreland et al., or occurred from the ovary with the dominant follicle Ž Evans et al., In a study in which follicles were sampled in vivo before, during, and after deviation, the mean increase in estradiol in the largest follicle relative to the second-largest follicle began on the same day as follicle deviation Ž Fig. 6; Ginther et al., 1997b.. In mares, a similar study was done, using a two-follicle model Ž Gastal et al., 1999d.. Fluid from each follicle was sampled on the day the larger follicle reached 15, 20, or 25 mm. An increased difference between the two follicles in estradiol concentrations occurred when the larger follicle was 20 mm Ž expected beginning of deviation; Fig. 7.. In contrast, an increased difference in diameter between follicles did not occur until the largest follicle was 25 mm. Thus, the beginning of a difference in estradiol concentrations preceded the beginning of diameter deviation. In another study using the two-follicle model, ablation of one or none of the follicles was done on the day of the expected beginning of deviation Ž Day 0; Gastal et al., 1999c.. Systemic estradiol increased between Days 0 and 2 in the groups with both follicles or only the larger follicle retained Ž Fig. 4.. No such increase occurred when only the smaller follicle was retained. In conclusion, in mares, estradiol production increases in the largest follicle and estrogen-like echotextural changes occur in the follicular wall on the day before the beginning of diameter deviation. In cattle, follicle deviation and increased estradiol production seem to occur simultaneously.

12 72 ( ) O.J. GintherrAnimal Reproduction Science Fig. 6. Mean Ž "SEM. diameters of the two largest follicles Ž a. and estradiol concentrations in follicular fluid Ž. b before and after the beginning of deviation. Black bars Ž. b are for the largest follicle, and white bars are for the second-largest follicle. The number of waves with sampled follicles is shown in parentheses for each day. Means for the two largest follicles for each end point were significantly different Ž P only on the day after the beginning of deviation as indicated by different letters. Adapted from Ginther et al. Ž 1997b.. A negative feedback effect of inhibin or other proteinaceous products of the follicles also may regulate the declining portion of the FSH surge Ž Mihm et al., 1997., including the low levels during deviation. Inhibin has been measured in growing and atretic Fig. 7. Mean Ž "SEM. diameters of follicles Ž a. and estradiol concentrations in follicular fluid Ž b. for larger Ž black bars. and smaller Ž white bars. follicles in mares with two retained follicles. The follicles were monitored by ultrasound and were sampled when the larger follicle first reached the indicated diameters. The number in parentheses is the number of mares in each diameter group. The letters Ž XYZ. indicate that the difference in diameters between the two follicles was greater Ž P for the 25-mm group than for the other two groups, but the differences between follicles for estradiol concentrations progressively increased over groups. An increased difference in diameter between follicles did not occur until the larger follicle was 25 mm, whereas an increased difference in estradiol concentrations occurred when the larger follicle was 20 mm Ž expected beginning of deviation.. Adapted from Gastal et al. Ž 1999d..

13 ( ) O.J. GintherrAnimal Reproduction Science follicles of different diameters in cattle at different reproductive stages ŽMartin et al., 1991; Ireland et al., 1994; Sunderland et al., 1996; Mihm et al., The results are complex, resulting from the subunit make-up and the wide array of forms with different molecular weights and the manner in which the subunits converge to form structurally similar, but functionally different, compounds. Administration of an inhibin antiserum increases plasma FSH Ž Glencross et al., and increases the number of ) 8mmor G 10 mm follicles during a follicular wave and the number of ovulations in cattle Ž Kaneko et al., 1993; Glencross et al., 1994; Hillard et al., and the number of ovulations in mares Ž McKinnon et al., 1992; McCue et al., 1993; Nambo et al., These results with anti-inhibin may reflect an interference with follicle deviation, indicating that inhibin is needed for the deviation process; however, this has not been studied directly. Immunoreactive inhibin concentrations increase in the circulation at the time FSH concentrations decline in mares ŽBergfelt et al., 1991b; Nagamine et al., This occurs at the reported time of development of the ovulatory follicular wave. These results with inhibin assays and inhibin antisera suggest that inhibin or other proteinaceous follicular factors may be involved in the deviation mechanism through depression of circulating FSH. It has been proposed that the decline in FSH plays a role in stimulating the follicle to synthesize inhibin, as well as estradiol and growth factors Ž Mihm et al., It appears that the declining concentrations of FSH and the developmental stage reached by the largest follicle interact to initiate deviation before other follicles reach the critical diameter. The initiation involves a further release of FSH suppressants, which further depresses FSH below the requirements of the smaller follicles. As noted earlier, a treatment at the time of expected deviation with a minimal dose of either estradiol or a near estrogen-free fraction of follicular fluid reduced the FSH concentrations to a level below the concentrations found in controls. These findings and the temporal relationships, discussed above indicate that both estradiol and inhibin are candidates for the role of FSH suppressants during diameter deviation. 7. Role of LH Several studies have suggested that LH is utilized as a gonadotropin stimulant by the selected dominant follicle Žfor reviews see Goodman and Hodgen, 1983; Gong et al., 1996; Ginther et al., 1996b.. In cattle, a change in emphasis to LH dependency so that continued development of the dominant follicle is driven at least partly by LH is consistent with the following results: Ž. 1 Follicles did not grow beyond 7 9 mm when LH was suppressed Ž Gong et al., 1995, Ž 2. The growth phase of the dominant follicle was associated with higher-frequency LH pulses than for the plateau phase Ž Rhodes et al., Ž 3. The life span of the dominant follicle can be extended by increasing the LH-pulse frequency Ž Fortune et al., 1991; Savio et al., Ž 4. The largest follicle acquires LH receptors or gene expression for LH receptors between 2 and 4 days after wave emergence ŽXu et al., 1995; Bodensteiner et al., 1996b; Evans and Fortune, 1997., which encompasses the reported day of the beginning of deviation. Ž 5. Lactating cows on a low-energy diet had a lower LH-pulse frequency, and the diameter

14 74 ( ) O.J. GintherrAnimal Reproduction Science of the largest follicle was less than in controls Ž Grimard et al., Ž 6. After the 90th day of pregnancy, there was both a transitional decrease in diameter of the largest follicle of successive waves Ž Ginther et al., 1996a. and a decrease in LH-pulse frequency Ž Schallenberger et al., 1985., indicating the two events are temporally related. Ž. 7 LH concentrations increased to a plateau before deviation, remained elevated Žno significant changes. until after deviation, and then decreased ŽGinther et al., 1998, 1999; Kulick et al., 1999., indicating that elevated LH as well as reduced FSH concentrations encompass the time of deviation. However, whether the dominant follicle utilizes LH in cattle in association with diameter deviation is not known. In mares, LH receptors were higher in the theca when the mean diameter of the largest follicle was 29 mm Ž Fay and Douglas, 1987.; this would presumably be after deviation. Recently, values for LH receptors, a-inhibin, and aromatase were reported to be lower in the granulosa of follicles 5 9 mm than in larger follicles ŽGoudet et al., 1999., but this would be well before deviation. The status of LH receptors and other factors in follicles before, during, and after deviation needs study in mares. An increase in circulating LH concentrations occurs before deviation Ž Gastal et al., 1997, 1999c.. In some mares, the increase continued as part of the prolonged ovulatory LH surge in this species, but in others, a plateau encompassed deviation or a transient decrease occurred between deviation and ovulation. Thus, in mares, as well as cattle, elevated circulating LH is available during deviation. In recent studies in ponies Ž Gastal et al., 1999a,e., LH concentrations were manipulated by daily treatment with various doses of progesterone beginning before the emergence of the follicular wave. A dose that did not alter FSH concentrations did affect LH. After an LH increase in both treated and control mares, a decrease occurred in the treated group 1 or 2 days before deviation in the controls; the reduced levels were similar to pretreatment levels. The decrease in the progesterone group was associated to reduced diameter of the largest follicle within 2 days after deviation in the controls. However, the reduced LH did not affect the second-largest follicle. Thus, the onset of deviation, as assessed by the second-largest follicle, was not delayed by the reduced LH, but the post-deviation growth of the largest follicle was reduced. Although not conclusive, these results indicate that LH was not involved in the initiation of follicle deviation Ž inhibition of the other follicles through FSH depression. in mares, but was required for continued growth of the largest follicle after the beginning of deviation. 8. Intrafollicular facilitators of gonadotropins The intrafollicular facilitating substances that favor a lower requirement for FSH and a hypothesized change in gonadotropin dependency from FSH to LH by the largest follicle have not been determined Ž for reviews see Ireland, 1987; Roche, Such intrafollicular factors could account for a requirement for less circulating FSH by the largest follicle than for the smaller follicles, and thus may be fundamental to the initiation of deviation. In addition, such factors may enable the dominant follicle to utilize circulating LH as a growth stimulant. Estradiol is a candidate for such a role, based on temporal considerations as discussed earlier.

15 ( ) O.J. GintherrAnimal Reproduction Science There is considerable evidence implicating a number of growth factors in local regulation of follicular development in cattle ŽFindlay, 1994; Campbell et al., 1995; Erickson and Danforth, 1995; Stewart et al., 1996; Khamsi and Armstrong, 1997; Mihm et al., 1997; Monniaux et al., and have also been associated with an increase in intrafollicular estradiol in mares Ž Gerard and Monget, Reportedly, the intrafollicular growth factors have many paracrinerautocrine roles, including enhancing FSH action, inducing expression of LH receptors, and regulating aromatase activity. The insulin-like growth factor Ž IGF-1. system has received particular attention, especially in its influence on facilitating the utilization of low circulating levels of FSH ŽMihm et al., Activity of IGF-1 increases rapidly in the dominant follicle because of the disappearance of binding proteins, whereas the binding proteins remain active in the subordinate follicles. In this regard, administration of growth hormone in cattle resulted in an increased number of gonadotropin-responsive follicles Ž Campbell et al., 1995., perhaps by increasing liver production and circulatory concentrations of IGF-1. The conclusion that the IGF system acts as a facilitator within the dominant follicle and thereby plays a role in deviation will remain tentative until the temporal and functional relationships between the IGF system and follicle-diameter deviation and growth of the dominant follicle after deviation are verified. The role of other intrafollicular factors in deviation or in subsequent growth of the dominant follicle also have not been clarified. These other factors include aromatase inhibitors Ž Goudet et al., 1999., follicle regulatory protein Ž dizerega et al., and androgens Ž Evans et al., Acknowledgements Original research supported by grants from the United States Department of Agriculture, by the University of Wisconsin-Madison, and by Equiservices Publishing, Cross Plains, Wisconsin. Research from this laboratory is based on reported studies seniorauthored by O.J. Ginther or one of the following Research Assistants and Associates: G.P. Adams, D.R. Bergfelt, K.J. Bodensteiner, E.L. Gastal, J.R. Gibbons, L. Knopf, K. Kot, L.J. Kulick, and R.A. Pierson. References Adams, G.P., Matteri, R.L., Kastelic, J.P., Ko, J.C.H., Ginther, O.J., Association between surges of follicle-stimulating hormone and the emergence of follicular waves in heifers. J. Reprod. Fertil. 94, Adams, G.P., Kot, K., Smith, C.A., Ginther, O.J., Selection of a dominant follicle and suppression of follicular growth in heifers. Anim. Reprod. Sci. 30, Armstrong, D.G., Webb, R., Ovarian follicular dominance: the role of intraovarian growth factors and novel proteins. Rev. Reprod. 2, Bergfelt, D.R., Ginther, O.J., Relationships between circulating concentrations of FSH and follicular waves during early pregnancy in mares. Equine Vet. Sci. 12, Bergfelt, D.R., Ginther, O.J., Relationships between FSH surges and follicular waves during the estrous cycle in mares. Theriogenology 39,

16 76 ( ) O.J. GintherrAnimal Reproduction Science Bergfelt, D.R., Kastelic, J.P., Ginther, O.J., 1991a. Continued periodic emergence of follicular waves in non-bred progesterone-treated heifers. Anim. Reprod. Sci. 24, Bergfelt, D.R., Mann, B.G., Schwartz, N.B., Ginther, O.J., 1991b. Circulating concentrations of immunoreactive inhibin and FSH during the estrous cycle of mares. Equine Vet. Sci. 11, Bodensteiner, K.J., Kot, K., Wiltbank, M.C., Ginther, O.J., 1996a. Synchronization of emergence of follicular waves in cattle. Theriogenology 45, Bodensteiner, K.J., Wiltbank, M.C., Bergfelt, D.R., Ginther, O.J., 1996b. Alterations in follicular estradiol and gonadotropin receptors during development of bovine antral follicles. Theriogenology 45, Campbell, B., Scaramuzzi, R., Webb, R., Control of antral follicle development and selection in sheep and cattle. J. Reprod. Fertil., Suppl. 49, dizerega, G.S., Tonetta, S.A., Westhof, G., A postulated role for naturally occurring aromatase inhibitors in follicle selection. J. Steroid Biochem. 27, Erickson, G.F., Danforth, D.R., Ovarian control of follicle development. Am. J. Obstet. Gynecol. 172, Evans, A.C.O., Fortune, J.E., Selection of the dominant follicle in cattle occurs in the absence of differences in the expression of messenger ribonucleic acid for gonadotropin receptors. Endocrinology 138, Evans, A.C.O., Adams, G.P., Rawlings, N.C., Follicular and hormonal development in prepubertal heifers from 2 to 36 weeks of age. J. Reprod. Fertil. 102, Evans, A.C.O., Komar, C.M., Wandji, S.-A., Fortune, J.E., Changes in androgen secretion and luteinizing hormone pulse amplitude are associated with the recruitment and growth of ovarian follicles during the luteal phase of the bovine estrous cycle. Biol. Reprod. 57, Fay, J.E., Douglas, R.H., Changes in thecal and granulosa cell LH and FSH receptor content associated with follicular fluid and peripheral plasma gonadotrophin and steroid hormone concentrations in preovulatory follicles of mares. J. Reprod. Fertil., Suppl. 35, Findlay, J.K., Peripheral and local regulators of folliculogenesis. Reprod. Fertil. Dev. 6, Fortune, J.E., Ovarian follicular growth and development in mammals. Biol. Reprod. 50, Fortune, J.E., Sirois, J., Quirk, S.M., The growth and differentiation of ovarian follicles during the bovine estrous cycle. Theriogenology 29, Fortune, J.E., Sirois, J., Turzillo, A.M., Lavoir, M., Follicle selection in domestic ruminants. J. Reprod. Fertil., Suppl. 43, Gastal, E.L., Gastal, M.O., Bergfelt, D.R., Ginther, O.J., Role of diameter differences among follicles in selection of a future dominant follicle in mares. Biol. Reprod. 57, Gastal, E.L., Bergfelt, D.R., Nogueira, G.P., Gastal, M.O., Ginther, O.J., 1999a. Role of luteinizing hormone in follicle deviation based on manipulating progesterone concentrations in mares. Biol. Reprod. 61, Gastal, E.L., Donadeu, F.X., Gastal, M.O., Ginther, O.J., 1999b. Echotextural changes in the follicular wall during follicle deviation in mares. Theriogenology 52, Gastal, E.L., Gastal, M.O., Ginther, O.J., 1999c. Experimental assumption of dominance by a smaller follicle and associated hormonal changes in mares. Biol. Reprod. 61, Gastal, E.L., Gastal, M.O., Wiltbank, M.C., Ginther, O.J., 1999d. Follicle deviation and intrafollicular and systemic estradiol concentrations in mares. Biol. Reprod. 61, Gastal, E.L., Gastal, M.O., Norgueira, G.P., Bergfelt, D.R., Ginther, O.J., Temporal interrelationships among luteolysis, FSH, and LH concentrations and follicle deviation in mares. Theriogenology 53, Gerard, N., Monget, P., Intrafollicular insulin-like growth factor-binding protein levels in equine ovarian follicles during preovulatory maturation and regression. Biol. Reprod. 58, Gibbons, J.R., Wiltbank, M.C., Ginther, O.J., Functional interrelationships between follicles greater than 4 mm and the follicle-stimulating hormone surge in heifers. Biol. Reprod. 57, Gibbons, J.R., Wiltbank, M.C., Ginther, O.J., Relationship between follicular development and the decline in the follicle-stimulating hormone surge in heifers. Biol. Reprod. 60, Ginther, O.J., In: Reproductive Biology of the Mare, Basic and Applied Aspects. Equiservices Publishing, Cross Plains, WI, USA, pp