913 Braz J Med Biol Res 37(6) 2004 Puberty in female ponyBrazilian Journal of Medical and Biological Research (2004) 37: 913-922 ISSN 0100-879X Follicle profile and plasma gonadotropin concentration in pubertal female ponies Departamento de Apoio, Produção e Saúde Animal, Faculdade de Medicina Veterinária, Universidade Estadual Paulista, Araçatuba, SP, Brasil G.P. Nogueira Abstract Twelve female ponies were examined daily for 30 days and classified as ovulating (OV; N = 6; 197 ± 6 kg) or prepubertal (PP; N = 6; 196 ± 9 kg). Follicles were detected by ultrasound and gonadotropins quan- tified by radioimmunoassay. The mean diameter of the largest follicles was significantly larger in OV (38 ± 1 mm) than in PP (26 ± 2 mm) but there was no difference between groups in the size of the second largest follicle. There were more small follicles (<24 mm) in the PP than in the OV group, but PP fillies had a smaller number of follicles >29 mm than the OV fillies. Follicle-stimulating hormone (FSH) levels did not differ between groups but PP fillies had lower luteiniz- ing hormone (LH) peak (8 ± 1 ng/ml) and basal (4 ± 0.5 ng/ml) levels, lower peak magnitude (2 ± 0.2 ng/ml) and period average (5 ± 0.6 ng/ ml) than OV fillies (32 ± 4.5, 8 ± 1.2, 17.1 ± 6, and 15 ± 2.3 ng/ml, respectively). The PP group, in contrast to the OV group, showed no relationship between FSH surge and follicle wave emergence. We conclude that an LH concentration higher than 8 ng/ml is needed for follicle growth to a preovulatory size. Wave emergence and FSH secretion seem to be independent events, probably due to an inhibitory neural system in these PP animals. PP fillies may provide a physiologi- cal model for the study of follicle wave emergence which apparently does not depend on gonadotropin levels. Correspondence G.P. Nogueira Departamento de Apoio, Produção e Saúde Animal, UNESP 16050-680 Araçatuba, SP Brasil Fax: +55-18-3622-6487 E-mail: gpn@fmva.unesp.br Presented at the Annual Conference of the International Embryo Transfer Society in Foz do Iguaçu, PR, Brazil in 2002, and reported in abstract form (Theriogenology, 57: 617, 2002). Research supported by Equiservice Publishing and by EquiCulture, Inc., Cross Plains, WI, USA. G.P. Nogueira was the recipient of a FAPESP fellowship (No. 98-00287-6) and UNESP. Received June 9, 2003 Accepted January 27, 2004 Key words • Follicles • FSH • LH and prepubertal fillies Introduction Puberty is the transitional period between sexual immaturity and maturity, after which animals reach the ability to reproduce for the preservation of the species. Each species has its own time course of puberty according to its life span (1). Puberty in fillies is generally considered to be at approximately 12 to 15 months if the animals were born early in the breeding season. Fillies born late may reach sexual maturation during the second spring of life (2). Ponies born during the previous summer and early autumn tend to have fewer ovulations and shorter breeding seasons than those born during the spring, whereas those born late in August and September do not ovulate during their first breeding season (3). Follicular development occurs in waves in adult ponies, with the emergence of the 914 Braz J Med Biol Res 37(6) 2004 G.P. Nogueira wave coinciding with a surge in follicle- stimulating hormone (FSH) (4). A similar association between wave emergence and FSH surge (2) also occurs during the transi- tional period that precedes the onset of the ovulatory season in adults. In fillies, the association between gonadotropin changes and puberty has been described (3), but the association with follicular waves has not been studied. In adults, major waves cause the differ- ential development of a dominant follicle to 30 mm (5) in parallel to regression of the smaller subordinate follicles (4). Minor waves also develop in which dominant fol- licles do not differentiate (5) and the largest follicle reaches only 19 to 28 mm during the wave. The emergence of minor as well as major waves is associated with a surge in FSH concentrations (6). Surges in circulat- ing FSH and luteinizing hormone (LH) con- centrations are more synchronous during the luteal phase than during the follicular phase in mares (7,8). In a previous study with prepubertal (PP) fillies aged 2-10 months, follicular waves and FSH surges were not temporally associ- ated and the waves did not partition into dominant and subordinate follicles (9). The objective of the present study was to com- pare, during the peripubertal period, the fol- licular and gonadotropin characteristics of yearling fillies which did and did not ovulate by the end of spring of the year following their birth. Special consideration was given to temporal associations between changing FSH concentrations, emergence of waves and follicle diameter in fillies around the peripubertal period. Material and Methods Animals and ultrasound scanning Twelve pony fillies born in April-July of the previous year (month unknown for each individual) and weighing 170-222 kg at the end of the experiment were used. The fillies were kept in an outdoor paddock and were maintained on alfalfa/grass hay with free access to water and trace-mineralized salt. The ultrasound scanner was equipped with a 5-MHz linear-array transrectal transducer (Tokyo Keiki LS 300; Products Group Inter- national, Lyons, CO, USA) for the examina- tion of ovaries and uterus, performed as described previously (10). On June 22, the fillies were divided into two groups: those that had ovulated or appeared to be approach- ing ovulation (ovulatory or post-pubertal group, OV; N = 6) and those that had not (non-ovulatory or PP group; N = 6). The separation into groups was based on the presence or absence of a corpus luteum or a preovulatory-sized follicle (>30 mm). The experimental period of 30 days extended from June 23 to July 22 and the research was conducted in Cross Plains, WI, USA. Follicles On each day of the 30-day period, the three largest follicles per ovary were meas- ured with the electronic calipers of the ultra- sound. The others (>10 mm) were counted and their diameter was estimated using the centimeter scale displayed on the side of the ultrasound image. Follicles were grouped into diameter categories of 10-14, 15-19, 20- 24, 25-29, and >30 mm. No attempt was made to maintain day-to-day identity of fol- licles except for follicles that attained 25 mm. Follicular waves were detected and char- acterized according to a mathematical model described elsewhere (11). Briefly, each year- ling was evaluated according to the follow- ing procedures: 1) constructing profiles of the diameters of each of the 3 largest fol- licles per ovary without considering day-to- day identification of follicles, but with main- tenance of the distinction between left and right ovaries; 2) dividing the follicles into large and small categories using the largest 915 Braz J Med Biol Res 37(6) 2004 Puberty in female pony diameter reached by the second largest fol- licle (large-category follicle) and the remain- ing subordinate follicles (small-category fol- licles); 3) using the large category to profile the diameters of individual large follicles, these values were plotted graphically in the figures; 4) using the 6 largest follicles per yearling in the small-follicle category to de- tect significant waves of follicular activity. A significant (P < 0.05) increase fol- lowed by a significant decrease in mean diameter of the 6 largest small-category fol- licles per yearling, based on the Tukey mul- tiple range test, was used to identify a folli- cular wave. If the placement of the nadir was not clear from the means because of fluctua- tions, the decision was supported by inspec- tion of the diameter profile of the 3 largest follicles per ovary and by paired t-tests. The maximal diameter of the largest follicle and the second largest follicle and the difference in maximal diameter were also determined for each animal. Blood sampling and hormone assay Blood was collected daily by jugular veni- puncture into heparinized tubes and refriger- ated before being centrifuged for plasma separation. Plasma was then placed in poly- ethylene vials for cold storage (-20ºC) until the time for assay. Circulating concentra- tions of FSH (12) and LH (13) were deter- mined using radioimmunoassays previously validated for this species. The intra-assay and inter-assay coefficients of variation and the sensitivity were 10.9%, 13.2% and 0.1 ng/ml for FSH and 5.9%, 9.4% and 0.8 ng/ml for LH, respectively, as determined in two assays each for FSH and LH. A technique developed to study episodic fluctuations in circulating hormones (14) was used to detect peak concentrations of FSH and LH in indi- vidual yearlings. This procedure had been used to detect FSH and LH surges in blood samples collected daily from monkeys dur- ing the menstrual cycle (15). Briefly, the program determines threshold concentrations of FSH or LH based on the within-day vari- ability of the assay results between duplicate samples for each yearling. Concentrations higher than the threshold values were de- tected and identified as peak hormone con- centrations. The FSH or LH surge in an individual yearling was defined by a pro- gressive increase and decrease in concentra- tions higher than 1 ng/ml that encompassed a peak concentration (nadir-to-peak-to-nadir). The frequency of occurrence of individual FSH and LH surges, the number of FSH and LH surges per yearling and the magnitude of the surge were analyzed. Statistical analysis Analysis of variance (ANOVA) was used for sequential data to identify follicular waves in the mean values of small follicles, fol- lowed by the Tukey multiple range test. Main effects of group and day and group-by-day interaction were determined and when a sig- nificant main effect or interaction was de- tected, the Tukey multiple comparison test was used to determine the mean difference between groups within days and the paired t- test was used to determine the main differ- ence between days within groups. Daily changes in hormone concentration and fol- licle profile centered on the largest follicle diameter for the PP fillies were compared among groups of yearlings until the last day on which the data from all fillies in each group could be included. ANOVA was used to determine a group effect for single-point measurements. To determine whether simul- taneous peaks of both LH and FSH were occurring at random or not, the observed number of times that both surges occurred together was compared to the expected num- ber if each surge occurred independently of the other. The expected frequencies of both hormone surges occurring on the same day were calculated from the observed occur- rence of surges of each hormone during the 916 Braz J Med Biol Res 37(6) 2004 G.P. Nogueira 30-day period. For example, if the observed frequency of surges was 5/30 (17%) for LH and 8/30 (27%) for FSH the expected fre- quency of both surges occurring together would be 4.6% (17 x 27%). This expectancy calculation assumes that a surge occurs inde- pendently of the other (16). The observed values were compared to the expected val- ues by chi-square analysis. Proportional data were examined by chi-square analysis to determine group effects. Data are reported as means ± SEM unless otherwise indicated. Significance was indicated by a probability of P < 0.05. Results From the first ultrasound scan it was possible to divide the fillies into two catego- ries, i.e., six fillies with larger follicles (≥30 mm) or a corpus luteum, called OV, and 6 fillies with smaller follicles and without a corpus luteum, called PP. This first observa- tion was consistent throughout the 30-day period when 6 fillies ovulated and 6 did not. The weight at the end of the experiment did not differ between groups (197 ± 6, 188- 225, and 196 ± 9, 170-225 kg, respectively). The diameter of the largest follicle was wider for OV than for PP fillies (P = 0.001), but there was no significant difference in the diameter of the second largest follicle. There were more small follicles in PP than OV fillies. The mean ± SEM values for the fol- licle profile of OV and PP fillies are shown in Table 1. Most of the follicular waves did not partition into dominant and subordinate follicles (Figure 1). Ovulating fillies had more circulating LH (highest value, lowest value, basal levels, average throughout the 30-day period) than PP fillies. Circulating FSH did not differ between groups. LH and FSH pulses oc- curred together more times than expected by chance in PP fillies. The gonadotropin end points during the 30-day period are shown in Table 2. Individual follicle profile, the aver- age for the mathematical model and daily gonadotropin levels for the PP group are shown in Figure 1. Gonadotropin concentra- tion was normalized according to the largest follicle diameter in the PP group and to the corresponding diameter in the OV fillies (Figure 2) to determine the interference of endocrine profile with follicle growth. At the time when the largest follicle stopped growing in PP fillies, LH level was lower than in OV fillies (P > 0.05) but there was no difference in FSH concentration. The diam- eters of the largest and second largest follicle were centered on the day of the main FSH surge during the 30-day period to determine the relationship between FSH secretion and wave emergence (Figure 3). PP fillies showed no variation in follicle diameter (average of the 6 largest ones) after the FSH peak, whereas OV fillies showed a significant in- crease in follicle diameter. There was no apparent relationship between gonadotropin secretion and follicle wave emergence in PP fillies. Table 1. Characteristics of follicle profile during a 30-day period in ovulating fillies and non-ovulating (prepubertal) fillies . Fillies Ovulating Prepubertal Largest follicle Maximum diameter (mm) 38.3 ± 0.7* 25.9 ± 1.6 Second largest follicle Maximum diameter (mm) 18.8 ± 0.9 19.9 ± 0.7 Diameter difference between the two largest follicles (mm)a 8.1 ± 0.6* 2.7 ± 0.5 Difference in maximum diameter between the largest and second largest follicles (mm)b 27.2 ± 0.8* 8.33 ± 1.4 Number of follicles of different diametersc 10-14 mm 2.6 ± 0.4* 7.3 ± 1.5 15-19 mm 0.9 ± 0.2* 2.7 ± 0.4 20-24 mm 0.2 ± 0.05* 0.5 ± 0.1 25-29 mm 0.1 ± 0.02 0.04 ± 0.02 30 mm or more 0.17 ± 0.03* 0.01 ± 0.01 Data are reported as mean ± SEM for 6 animals in each group. aMonthly average of daily differences between the largest and the second largest follicle. bDifference between the two largest diameters attained by the largest and second largest follicle during the 30-day period. cMean daily number of follicles during a 30-day period. *P < 0.05 compared to prepubertal fillies (ANOVA). 917 Braz J Med Biol Res 37(6) 2004 Puberty in female pony Figure 1. Follicular data for each of the 6 prepubertal fillies that did not ovulate during the 30- day observation period. Panels A, B, E, F: diameter profiles of the 3 largest follicles in the right and left ovary without consider- ing day-to-day identity. Panels C, G: day-to-day diameter profile of follicles in the large category and mean profile of the 6 largest follicles per filly after excluding the follicles in the large cat- egory. Panels D, H: LH and FSH profile during the 30-day period. Asterisks indicate differences between days. The numbers at the top of each panel indicate the number of the animal. FSH = follicle-stimulating hormone; LH = luteinizing hormone. P < 0.05 compared to the other days (paired t-test). D ia m et er ( m m ) 30 25 2 - Right ovary 58 - Right ovary 67 - Right ovary 2 - Left ovary 58 - Left ovary 67 - Left ovary 77 - Right ovary 310 - Right ovary 506 - Right ovary 20 15 10 30 25 20 15 10 30 25 20 15 10 30 25 20 15 10 20 18 16 14 12 10 30 25 20 15 10 30 25 20 15 10 20 18 16 14 12 10 20 18 16 14 12 10 10 8 6 4 2 10 8 6 4 2 10 8 6 4 2 0 30 25 20 15 10 30 25 20 15 10 30 25 20 15 10 30 25 20 15 10 30 25 20 15 10 30 25 20 15 10 20 18 16 14 10 12 20 18 16 14 12 20 18 16 14 12 10 10 8 6 4 2 10 10 8 6 4 10 8 6 4 2 77- Left ovary 310 - Left ovary 506 - Left ovary LH D ia m et er ( m m ) D ia m et er ( m m ) ng /m l D ia m et er ( m m ) D ia m et er ( m m ) D ia m et er ( m m ) ng /m l 2 A B C D E F G H FSH * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 0 4 8 12 16 20 24 28 0 4 8 12 16 20 24 28 0 4 8 12 16 20 24 28 0 4 8 12 16 20 24 28 0 4 8 12 16 20 24 28 0 4 8 12 16 20 24 28 Days of observation LH FSH 918 Braz J Med Biol Res 37(6) 2004 G.P. Nogueira Figure 2. Follicle and gonadotropin profile normalized to the day of largest diameter for the prepubertal fillies (26 mm = day zero) and to the day of the corresponding follicle diameter for the ovulating fillies. Data are reported as means ± SEM for 6 animals in each group. FSH = follicle-stimulating hormone; LH = luteinizing hormone; OV = ovulating fillies; PP = prepubertal fillies. LH level differed (P < 0.01) between OV and PP only on day 0 (ANOVA repeated measures and Tukey test). Figure 3. Largest (filled circles) and 2nd largest (open circles) follicle diameter centered on the higher value of FSH (filled lozenges). PP = prepubertal (left graph) and OV = ovulating (right graph) fillies. Data are reported as means ± SEM for 6 animals in each group. Differences between days are indicated by asterisks (ANOVA repeated measures and Tukey test). D ia m et er ( m m ) 26 F S H ( ng /m l) 15 12 9 6 3 0 24 22 20 18 16 14 12 10 8 D ia m et er ( m m ) 26 24 22 20 18 16 14 12 10 8 F S H ( ng /m l) 15 12 9 6 3 0 FSH/centered/PP FSH/centered/OV -5 -4 -3 -2 -1 0 1 2 3 4 5 Days from FSH surge -5 -4 -3 -2 -1 0 1 2 3 4 5 Days from FSH surge Fo lli cl e di am et er ( m m ) 40 ng /m l 15 30 20 10 10 5 0 Fo lli cl e di am et er ( m m ) 40 30 20 10 ng /m l 25 15 5 0 OV PP OV PP OV PP OV PP 35 25 15 20 10 Largest follicle FSH LH Second largest follicle -9 -8 -7 -6 -5 -4 -3 -2 -1 0 1 2 3 4 5 6 Days after the largest diameter (PP) -9 -8 -7 -6 -5 -4 -3 -2 -1 0 1 2 3 4 5 6 * * * 919 Braz J Med Biol Res 37(6) 2004 Puberty in female pony Table 2. Characteristics of individual FSH and LH concentrations during a 30-day period for ovulat- ing fillies and non-ovulating (prepubertal) fillies. Fillies Ovulating Prepubertal Highest value (ng/ml) FSH 12.5 ± 4.1 6.6 ± 0.8 LH 31.9 ± 4.5* 7.7 ± 0.9 Lowest value (ng/ml) FSH 1.7 ± 0.3 2.3 ± 0.4 LH 5.7 ± 0.9* 3.2 ± 0.4 Basal levels (ng/ml) FSH 2.5 ± 0.4 2.8 ± 0.3 LH 7.8 ± 1.2* 3.9 ± 0.5 Average through the 30-day period (ng/ml) FSH 5.2 ± 1.4 3.7 ± 0.4 LH 15.0 ± 2.3* 4.7 ± 0.6 Number of peaks FSH 4.0 ± 0.7 5.8 ± 0.9 LH 4.5 ± 0.6 5.0 ± 0.6 Magnitude of the peaks above basal levels (ng/ml) FSH 7.6 ± 2.8 2.2 ± 0.3 LH 17.1 ± 6.2* 2.3 ± 0.2 Number of days between peaks FSH 5.2 ± 1.1 4.2 ± 0.5 LH 5.3 ± 0.6 4.9 ± 0.6 Frequency of occurrence of both surges together (%) Observed 7.2 9.9* Expected 2.2 3.6 Data are reported as means ± SEM for 6 animals in each group. FSH = follicle-stimulating hormone; LH = luteinizing hormone. *P < 0.05 compared to prepubertal fillies (ANOVA). Discussion In PP fillies, follicle diameter increased to a maximum of 26 mm, after which it regressed without reaching a preovulatory size (>30 mm). The main characteristic of the endocrine profile at the time of follicle regression in PP fillies was lower circulating LH concentrations. Although low, LH levels did not interfere with the growth of the sec- ond largest follicle. In PP fillies there was no relationship between FSH secretion and wave emergence and the partition of dominant and subordinate follicles during a follicular wave was not evident. In cattle and sheep, the gonadal block is governed by the gonado- static mechanism but in horses (3,9), as in primates (1), the PP hiatus is of central ori- gin. In the present experiment six fillies did not cycle over the 30-day experimental pe- riod (June-July). Fillies born during the first half of the year and well-nourished can be expected to reach puberty by spring of the next year (2,17). The phenomenon of pu- berty retardation when fillies are exposed prematurely to long photoperiods may ac- count for the failure of fillies born in late summer to ovulate during the following sum- mer. Such fillies would be exposed to in- creasing day length earlier and this may pre- clude ovulation at 12 months of age, delay- ing puberty until the following spring when they would be about 18 months old. Thus, a light-controlled puberty retardation phenom- enon may have evolved in late born fillies so that puberty does not occur during the fol- lowing year (17). The largest follicle diameter during the 30-day period was larger in the OV group than in the PP group, but the diameter of the second largest follicle did not differ between groups. The daily difference between the largest and second largest follicles was higher for OV than PP, as also was the difference between the day of the largest follicle diam- eter minus the diameter of the second largest follicle (among the 30 days; Table 1). Fol- licles grew to a certain diameter throughout the PP period, after which they regressed as a consequence of the hormonal milieu dur- ing the PP period. The PP fillies had more follicles smaller than 24 mm but fewer follicles larger than 24 mm. The higher number of small follicles for the PP group may be a consequence of the lack of interaction between follicles and hy- pothalamus-pituitary axis. Since the largest follicle grew beyond dominance diameter it should have been able to exert dominance 920 Braz J Med Biol Res 37(6) 2004 G.P. Nogueira over other follicles (18), but this was not the case. A distinct follicle could be observed in fillies 2 and 506 that apparently exerted dominance since the diameter of other fol- licles diminished during this period (Figure 1). The reduction in diameter of the subordi- nate follicles appeared as a significant de- crease in the average for the follicles. This event was not observed in the other fillies during the 30-day period, a fact possibly reflecting the sexual maturation of these fil- lies. There was a broad variation in the ap- parent follicular waves of the PP group, and in addition daily FSH concentrations fluctu- ated widely, as indicated by irregular levels (Figure 1). This seems to be similar to the minor follicle waves observed in adults, in which the largest follicle reaches only 19 to 28 mm and does not differentiate into the dominant follicle during the wave (5) but in which, in contrast to these fillies, an FSH surge occurs prior to the wave emergence. FSH varied throughout the experimental period but its levels were similar in the two groups, whereas LH levels were apparently suppressed in PP fillies. The central inhibi- tion occurring in PP fillies seems to be simi- lar to that observed in primates, in which the activity of the hypothalamic-pituitary axis is arrested in late infancy, gonadotropin secre- tion declines to low levels characteristic of PP primates, and the juvenile period of go- nadal quiescence is initiated. The gonado- static hypothesis, whereby an enhanced sen- sitivity of the gonadostat must be the major determinant underlying the diminished se- cretion of gonadotropin, is not applicable to higher primates (19) or horses (3). Centralizing the hormone and gonado- tropin profile to the largest follicle diameter in the PP group proved to be illustrative about the mechanism involved in the inhibi- tion of follicle increase up to a certain diam- eter (Figure 2). The largest follicle grew to 26 ± 2 mm in the PP group and then re- gressed, while in the OV fillies it grew until ovulation (38.3 ± 1 mm). At the time when the largest follicle started to regress in the PP group, FSH showed the same concentration in both groups (OV fillies: 3.34 ± 0.5 ng/ml, PP: 2.67 ± 0.5 ng/ml), whereas LH levels were higher in OV fillies (9.56 ± 1.7 ng/ml) compared to PP (5.01 ± 0.9 ng/ml). As shown for adults (20), the most important gonado- tropin that governs follicle growth after de- viation, around 22 mm, is LH. A decline in FSH concentration occurred in OV fillies from day -7 to day 0 as the largest follicle was growing (Figure 2). Low FSH has been postulated to play a role in follicle deviation (7); however, this decline in FSH was not observed in the PP fillies (Figure 2), prob- ably due to lack of LH (20). The lack of circulating LH interfered with the develop- ment of the largest follicle but not of the second largest follicle. The reduced LH con- centration in the PP fillies was observed throughout the 30-day period. The maxi- mum diameter of the second largest follicle in PP fillies was not different from this end- point in OV fillies. A low LH concentration did not reduce the growth rate of the second largest follicle during the study period be- cause the size attained by the 2nd largest follicle (19 ± 1 mm) was not enough for the LH requirement that occurs after the devia- tion diameter. A deviation mechanism was not appar- ently activated in most PP fillies either be- cause of low LH concentration or because of the central origin of the puberal hiatus. These results indicate that LH was needed for con- tinued growth of the largest follicle after 25 mm. A temporal indication for a role of LH in the continued growth of the largest follicle after the beginning of deviation is that a transient elevation in LH concentration usu- ally encompasses the day of deviation in mares (4,20). FSH levels were similar in the OV and PP groups but PP fillies had lower LH values for most of the end points, except for the number of surges and number of days between surges (Table 2). 921 Braz J Med Biol Res 37(6) 2004 Puberty in female pony There was a greater association between the LH and FSH surges, and the number of surges occurring in association was larger than that expected to occur by chance in the PP but not in the OV fillies (Table 2). In pregnant mares there seems to be a relation- ship between periodic FSH fluctuations (fre- quency and magnitude) and the pattern and type of follicular wave. Wave characteristics ranged from rhythmical occurrence with a distinct dominant follicle to sporadic occur- rence of non-prominent waves without domi- nance (6). Without central interference from the follicles during central gonadotropin se- cretion suppression, either before puberty (3,9) or during the luteal phase (21) the fluctuations of LH and FSH seem to occur in a synchronous manner, with a higher degree of coupling between surges. Normalizing FSH surge with follicle di- ameter average allowed us to reach the con- clusion that there is no relationship between FSH surge and follicle wave emergence in non-ovulating fillies (Figure 3). Prior to pu- berty, the follicles grew and regressed in an apparent wave fashion but never attained the preovulatory size. This behavior is similar to the minor waves that occur in mares, without the development of a dominant follicle (5,6). Gonadotropin release in the adult is regu- lated by negative feedback of the gonadal hormones on LH and FSH secretion that may be exerted either directly on the gonado- tropes to suppress their responsiveness to GnRH stimulation or indirectly at a suprapi- tuitary level to modulate the frequency and/ or amplitude of the GnRH pulse generator (22). In the immature filly, low LH levels do not provide the necessary stimulus for com- plete follicular development after the devia- tion diameter, and therefore the sustained rise in estradiol cannot be produced to acti- vate the preovulatory surge mechanism. Thus, it is possible to conclude that PP fillies can be used as a model for the study of intraovarian factors involved in follicle growth since they have a milieu with low LH levels and no apparent relationship between FSH level and wave emergence. Acknowledgments We are grateful to the National Hormone and Pituitary Program (NIDDK, Dr. A.F. Parlow) for providing equine FSH and LH (eFSH and eLH) RIA materials, and to O.J. Ginther for providing the animals and hor- mone measurements. References 1. Therasawa E & Fernandez DL (2001). Neurobiological mechanisms of the onset of puberty in primates. Endocrine Reviews, 22: 111- 151. 2. Ginther OJ (1992). Reproductive Biology of the Mare, Basic and Applied Aspects. 2nd edn. Equiservices Publishing, Cross Plains, WI, USA. 3. Wesson JA & Ginther OJ (1981). Puberty in the female pony: reproductive behavior, ovulation, and plasma gonadotropin concen- tration. Biology of Reproduction, 24: 977-986. 4. Gastal EL, Gastal MO, Bergfelt DR & Ginther OJ (1997). Role of diameter difference among follicles in selection of a future domi- nant follicle in mares. Biology of Reproduction, 57: 1320-1327. 5. Ginther OJ (1993). Major and minor waves during the equine es- trous cycle. Journal of Equine Veterinary Science, 13: 18-25. 6. Ginther OJ & Bergfelt DR (1992). Association between FSH concen- trations and major and minor follicular waves in pregnant mares. Theriogenology, 38: 817-821. 7. Bergfelt DR & Ginther OJ (1993). Synchronous fluctuations of LH and FSH in plasma samples collected daily during the estrous cycle in mares. Theriogenology, 40: 1137-1145. 8. Irvine CHG, Turner JE, Alexander SL, Shand N & van Noordt S (1998). Gonadotrophin profiles and dioestrous pulsatile release pat- terns in mares as determined by collection of jugular blood at 4 h intervals throughout an oestrous cycle. Journal of Reproduction and Fertility, 113: 315-322. 9. Nogueira GP & Ginther OJ (2000). Dynamics of follicle populations and gonadotropin concentrations in fillies age two to ten months. Equine Veterinary Journal, 32: 482-488. 10. Ginther OJ (1995). Ultrasonic Imaging and Animal Reproduction: Horses. Book 2. Equiservices Publishing, Cross Plains, WI, USA. 11. Ginther OJ & Bergfelt DR (1992). 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