Future Dominant Follicle Research Articles

Follicle Selection in Mares as a Model for Illustrating the Many Hormonal and Biochemical Interactions That Drive a Single Physiological Mechanism.

The mechanism for selection of the future dominant or ovulatory follicle in mares involves a relatively abrupt separation in growth rates between the future dominant follicle and several subordinate follicles and is termed diameter deviation. The event is used to illustrate that a coordinated complex of many follicular, hormonal, and biochemical factors interact and interbalance during a single physiological mechanism. For example, a positive effect of follicle stimulating hormone (FSH) on development of all follicles during the growing phase can later involve a positive effect of luteinizing hormone (LH) but apparently only on the future dominant follicle. In turn, the developing and future dominant follicle produces estradiol which at appropriate times and degrees reduces FSH concentrations to accommodate follicle functions at certain levels of FSH. Meanwhile, the estradiol prevents LH from increasing from a useful to an adverse concentration. These interactions enmesh with the production and roles of other factors (e.g., inhibin, insulin-like growth factor) during follicle selection. The wide array of morphological, hormonal, and biochemical activities occur in harmony even when in the same tissue and often at the same time.

Structural and Functional Dynamics of the Ovary and Uterus during the Estrous Cycle in Donkeys in the Eastern Caribbean.

Eight non-bred, non-pregnant, regularly cycling Caribbean jennies were examined daily via transrectal ultrasound to define the ovarian and uterine dynamics during four consecutive estrous cycles. Blood samples were collected every other day for progesterone analysis. The mean (±SD) overall inter-ovulatory interval across all donkeys and cycles was 22.93 ± 1.99 days. The maximum follicular diameter was 34.6 ± 2.9 mm. A two-wave pattern was evident in 97% (30/31) of the cycles. The emergence of the future dominant follicle and the largest subordinate follicle of the major primary wave coincided on Day 5.7 ± 3.6 post-ovulation, whereas the secondary wave emerged on Day 19.8 ± 2.9 during estrus of the previous cycle or early diestrus. The secondary wave was often minor (93%, 28/30 cycles). Follicular deviation occurred 8.2 ± 1.4 days before the subsequent ovulation. Luteal volume increased for the first four days after ovulation and reached a maximum volume of 8.5 ± 2.7 mm3 at Day 5.4 ± 0.4, before gradually regressing after Day 15. Serum progesterone concentration increased from Day 1 after ovulation, peaking at 27.0 ± 9.6 ng/mL between 7 and 10 days after ovulation. Progesterone concentration dropped precipitously around Day 15 after ovulation and was below 2 ng/mL around Day 17 ± 2. A day effect (p < 0.0001) was observed for corpus luteum’s volume, progesterone concentration, and uterine tone, but not for endometrial edema (p > 0.05). This study helps to clarify and define normal estrous characteristics of jennies in the Eastern Caribbean.

FSH Regulates YAP-TEAD Transcriptional Activity in Bovine Granulosa Cells to Allow the Future Dominant Follicle to Exert Its Augmented Estrogenic Capacity.

The molecular mechanisms that drive the granulosa cells' (GC) differentiation into a more estrogenic phenotype during follicular divergence and establishment of follicle dominance have not been completely elucidated. The main Hippo signaling effector, YAP, has, however, emerged as a potential key player to explain such complex processes. Studies using rat and bovine GC demonstrate that, in conditions where the expression of the classic YAP-TEAD target gene tissue growth factor (CTGF) is augmented, CYP19A1 expression and activity and, consequently, estradiol (E2) secretion are reduced. These findings led us to hypothesize that, during ovarian follicular divergence in cattle, FSH downregulates YAP-TEAD-dependent transcriptional activity in GC to allow the future dominant follicle to exert its augmented estrogenic capacity. To address this, we performed a series of experiments employing distinct bovine models. Our in vitro and ex vivo experiments indicated that indeed FSH downregulates, in a concentration-dependent manner, mRNA levels not only for CTGF but also for the other classic YAP-TEAD transcriptional target genes ANKRD1 and CYR61 by a mechanism that involves increased YAP phosphorylation. To better elucidate the functional importance of such FSH-induced YAP activity regulation, we then cultured GC in the presence of verteporfin (VP) or peptide 17 (P17), two pharmacological inhibitors known to interfere with YAP binding to TEADs. The results showed that both VP and P17 increased CYP19A1 basal mRNA levels in a concentration-dependent manner. Most interestingly, by using GC samples obtained in vivo from dominant vs. subordinate follicles, we found that mRNA levels for CTGF, CYR61, and ANKRD1 are higher in subordinate follicles following the follicular divergence. Taken together, our novel results demonstrate that YAP transcriptional activity is regulated in bovine granulosa cells to allow the increased estrogenic capacity of the selected dominant follicle.

Follicular-fluid proteomics during equine follicle development.

The complex composition of the follicular fluid (FF), the intimate proximity to the oocyte, and the continual changes in their composition have a major effect on folliculogenesis and oogenesis. To date, the profiling of FF proteomes during follicle selection, development, and ovulation has not been comprehensively investigated. Therefore, a shotgun proteomics approach and bioinformatics analyses were used to profile the proteomes of equine FF harvested in vivo from follicles at the following development stages: predeviation (18-20 mm), deviation (22-25 mm), postdeviation (26-29 mm), preovulatory (30-35 mm), and impending ovulation. A total of 294 proteins were detected in FF (FDR <1%), corresponding to 65 common proteins and 124, 142, 167, 132, and 142 proteins in the predeviation, deviation, postdeviation, preovulatory, and impending ovulation groups, respectively. The higher expression of properdin and several other proteins belonging to the complement system during the deviation time and ovulation suggested their contribution in the selection of the future dominant follicle and ovulation. Apolipoprotein A-1 and antithrombin-III appeared to be important throughout folliculogenesis. The "complement and coagulation cascades" was the major KEGG pathway across all stages of follicle development. The significant expression of several proteins belonging to the serine-type endopeptidase indicated their likely contribution to follicle and oocyte development. Our data provide an extensive description and functional analyses of the equine FF proteome during follicle selection, development, and ovulation. This information will help improve understanding of the ovarian function and ovulatory dysfunctions and might serve as a reference for future biomarker discovery for oocyte quality assessment.

Accessory corpus luteum induced by human chorionic gonadotropin on day 7 or days 7 and 13 of the estrous cycle affected follicular and luteal dynamics and luteolysis in lactating Holstein cows

Our objective was to determine the effect of inducing an accessory corpus luteum (CL) with human chorionic gonadotropin (hCG; 3,300 IU) on d 7 (hCG7) or 2 accessory CL with hCG on d 7 and 13 (hCG7+13) of the estrous cycle in noninseminated lactating Holstein cows. Cows (n = 86) between 39 and 64 DIM were pretreated with an Ovsynch + CIDR protocol, and only synchronized cows were used (n = 64). The day of the last GnRH of Ovsynch was considered d 0 of the estrous cycle. Follicular and luteal dynamics of cows were evaluated daily during an entire estrous cycle by ovarian ultrasonography. Blood samples were collected daily to measure serum concentration of progesterone (P4). Cows were randomly assigned to CON (n = 22, no treatment), hCG7 (n = 20), or hCG7+13 (n = 22) treatments. Two cows from hCG7+13 failed to ovulate after hCG and were removed from the analyses post-hCG treatment. The first day of luteolysis was considered the day that P4 declined to more than 2 SD of the mean for the 4 consecutive P4 concentrations with the greatest mean in late diestrus for each individual cow. The P4 cut-off for complete luteolysis was <1.0 ng/mL. Mean P4 on d 7 (3.23 ± 0.16 ng/mL) did not differ among treatments. Cows treated with hCG had greater total luteal and original CL volume and serum P4 during diestrus than CON. Cows treated with hCG7+13 had greater serum P4 after d 13 of the cycle than hCG7. Cycles were classified as having atypical cycles if the dominant follicle or future dominant follicle at the time of luteolysis did not ovulate (delayed ovulation; CON, n = 2; hCG7, n = 4; hCG7+13, n = 3), had a short cycle (CON, n = 1), delayed (CON, n = 2) or incomplete luteolysis (CON, n = 1; hCG7, n = 4; hCG7+13, n = 5). The remainder of cycles with normal complete luteolysis followed by ovulation were considered to be typical. Based on blood perfusion, the CON cow with incomplete luteolysis had 2 original CL remaining functional after first onset of luteolysis. The rest of the cows with incomplete luteolysis (9/10) had one or more CL regressing and at least one remaining functional after first onset of luteolysis. No specific pattern for CL side (ipsilateral vs. contralateral to a CL with complete regression) was observed for nonregressed CL. Cows with incomplete luteolysis had a second onset of luteolysis to undergo complete functional luteolysis. The proportion of cows with typical cycle was 73% (16/22) for CON, 60% (12/20) for hCG7, and 55% (11/20) for hCG7+13. Cows with typical cycles treated with hCG (hCG7 and hCG7+13) had a later onset of luteolysis, prolonged time to undergo complete luteolysis, and greater proportion of cows with 3 follicular waves than CON, resulting in a longer interovulatory interval for hCG7 and hCG7+13 than CON. In summary, accessory CL induced by hCG during diestrus not only altered follicular and luteal dynamics but also deferred and prolonged the luteolytic process.

Hormonal mechanisms regulating follicular wave dynamics II: Progesterone decreases diameter at follicle selection regardless of whether circulating FSH or LH are decreased or elevated

Selection of a single dominant follicle is morphologically manifested by diameter deviation between the future dominant follicle (F1) and the future largest subordinate follicle (F2). Conventional deviation is defined as F2≥7 mm when F1 reaches ∼8.5 mm whereas, undersized deviation is if F2<7 mm when F1 reaches ∼8.5 mm. Greater frequency of undersized deviation has been temporally associated with greater circulating progesterone (P4) and greater FSH but reduced LH in observational studies. Experiment 1 was conducted to directly test if elevating P4 increased the likelihood of undersized deviation and altered circulating concentrations of LH and FSH. Experiment 2 was conducted to test if increasing LH action by treatment with exogenous porcine LH or human chorionic gonadotropin (hCG) in the presence of elevated P4, would stimulate growth of F2 and increase the likelihood of conventional deviation. Ovaries were evaluated by ultrasound and blood samples collected every 12 h after development of a new wave following follicle ablation on D6 (D0 = ovulation). Data were normalized to F1≥7.5 mm and compared using SAS software. In experiment 1 (n = 20), the CL was regressed by prostaglandin F2α treatment and heifers were randomized on D6 into control (no P4 treatment) or P4 treatment (75 mg every 12 h for 5.5 d) beginning when F1 reached ∼3 mm (P4-3 mm group) or ∼6 mm (P4-6 mm group). The P4 treatment significantly increased the frequency of undersized deviation from 0% (controls) to 54%, decreased LH by 44%, and increased FSH by 32%. In experiment 2 (n = 27) heifers were randomized on D6 into control (saline) or treatment with the LH analogs - pLH (1.25 mg porcine LH/12 h) or hCG (160 IU initially and subsequently 96 IU/24 h).Treatment with LH analogs significantly increased P4 (control, 4.6 ± 0.3 ng/mL; pLH, 6.6 ± 0.4 ng/mL; and hCG, 8.9 ± 0.4 ng/mL) and decreased FSH (control, 0.46 ± 0.03 ng/mL; combined-pLH/hCG, 0.34 ± 0.02 ng/mL). However, F1 and F2 diameter and frequency of conventional (37%) and undersized (48%) deviations were similar between the control and combined-pLH/hCG groups. In conclusion, elevated P4 was directly linked to undersized deviation but the P4 effect on decreasing F2 diameter occurred independently of the P4 effects on FSH and LH concentrations.

Role of intraovarian mechanisms and side of ovary on characteristics of follicle selection in Bos taurus heifers

Intraovarian effects on diameter of future dominant follicle (DF or F1) and future largest subordinate follicle (F2) during the few days before selection of the future DF and subordinate follicles were studied in 147 bovine interovulatory intervals. Follicle selection involves diameter deviation or the beginning of separation of growth rates between F1 and F2. Diameter deviation is classified as conventional (F2 ≥ 7.0 mm when F1 is 8.5 mm or at expected deviation) and as undersized (F2 < 7.0 mm when F1 is 8.5 mm). Diameter separation of F1 and F2 in conventional and undersized deviations is characteristically abrupt and gradual, respectively. The predeviation diameter of F2 when located in an ovary that later becomes the F1 intraovarian patterns of DF−CL, devoid (ovary without a DF or CL), DF alone, or CL alone and in left or right ovaries (LO, RO) was compared between conventional and undersized deviations. In conventional deviation, ovaries with the future DF (combined DF−CL and DF patterns) were associated with greater (P < 0.02) predeviation growth rate of F2 when the DF was in the right ovary (DF/RO, 1.6 ± 0.1 mm/d) than when in the left ovary (DF/LO, 1.2 ± 0.1 mm/d). The F2 was in DF/RO more frequently (75%, P < 0.002) than in non-DF/RO. When F2 was in the future devoid F1 pattern and F1 was 6 mm, F2 was smaller (P < 0.002) in the undersized class (5.3 ± 0.2 mm) than in the conventional class (6.3 ± 0.1) but not when F2 was in one of the other future F1 patterns. Only the devoid pattern was greater in frequency (P < 0.03) in the undersized class than in the conventional class. The novel hypothesis was supported that location of F2 in ovaries with different future F1 intraovarian patterns and on different sides affects the predeviation diameter and growth rate of F2 and thereby the frequencies of conventional and undersized deviations during follicle selection.

Follicle blood flow and FSH concentration associated with variations in characteristics of follicle selection in heifers

Selection of the dominant follicle during a follicular wave is manifested by diameter deviation. At deviation (day 0), growth rate continues for the future dominant follicle (F1) and begins to decrease for the largest subordinate follicle (F2). The percentage of color-Doppler blood-flow signals in the wall of F1 and F2 and the temporality between FSH concentration and F1 and F2 diameter were determined daily in waves 1 and 2 in 24 Holstein heifers. Diameter and blood flow were compared among classes of deviation: (1) conventional (F2 ≥ 7.0 mm on day 0), (2) F2-undersized (F2 < 7.0 mm on day 0), and (3) F1,F2-switched (F2 larger than F1 on days −1 or 0). A class-by-day interaction for diameter of F2 (P < 0.004) and for blood-flow percentage of F2 (P < 0.02) represented greatest values on days −1 or 0 in the switched class and greater values in the conventional than undersized class. Changes were similar between diameter and blood flow in F1 and F2 before deviation. Blood flow in F2 decreased sooner than diameter after deviation indicating that a decrease in vascular perfusion preceded a decrease in diameter. Relationships between F1 and FSH in conventional deviation were similar between waves 1 and 2 for (1) growth rate of F1 on days −1 to 0, (2) interval from emergence of F1 at 4 mm to deviation, and (3) decrease in FSH on days −2 to 0. Relationships between F2 diameter and FSH were dissimilar between classes and between waves 1 and 2 indicating other hormones or factors are also involved in the complex control of F2. For example, the growth rate of F2 was greater (P < 0.05) for conventional than undersized class during wave 1 but similar between classes during wave 2. The FSH surge 2 was similar in profile and prominence between classes but the interval from the FSH peak of surge 2 to deviation was shorter (P < 0.05) in the undersized class (1.5 ± 0.3 d) than in the conventional class (2.3 ± 0.3 d). This was a novel finding and accounted for some of the dissimilarities in growth rate of follicles between classes in wave 2. Results did not support the hypothesis that the extent of blood flow in the wall of future dominant and largest subordinate follicles before deviation is an earlier indicator of follicle destiny than diameter. Results supported the hypothesis that follicle dynamics and FSH concentrations before deviation are temporally associated within conventional and undersized deviation classes but the temporality differs between classes.

Spontaneous switching of future dominance to a smaller follicle: commonality among monovular species.

Selection of a dominant follicle from a wave of follicles is manifested by diameter deviation between future dominant (F1) and largest subordinate (F2) follicles. On day -1 or 0 (day 0=beginning of deviation), growth rate of F1 continues and growth rate of F2 decreases. Deviation occurs during the decline in the wave-stimulating FSH surge when F1 reaches means of 8.5, 10.5, and 22.5 mm in heifers, women, and mares, respectively. Diameter of F1 at the FSH peak vs at deviation is proportionally similar among these monovular species. In conventional deviation, F1 usually emerges first. In F1,F2-switched deviation, F2 is usually first to emerge and to reach a diameter characteristic of deviation. On day -1 or 0, the larger F2 and the smaller F1 switch so that the formerly larger F2 becomes subordinate and the formerly smaller F1 becomes dominant. In heifers and mares, the profile and prominence of the FSH surge are similar between deviation classes. Surge location relative to deviation differs so that the surge ends earlier in switched deviation. When the larger F2 reaches a diameter characteristic of deviation, FSH concentration is too low for continued growth of F2. The decrease in FSH ceases (heifers) or increases (mares) presumably from a decrease in FSH inhibitors; therefore, F1 continues to grow and becomes dominant. The frequency of switched deviation (e.g., 16 to 37% among species) can be problematic in follicle-selection research. Switching is a natural model for study of the interplay between follicles and FSH.

Gonadotropin concentrations associated with variations in diameter deviation during follicle selection in Holstein heifers

Diameter deviation or selection of the future dominant follicle (F1) from the future largest subordinate follicle (F2) during a follicular wave occurs when F1 is 8.5 mm (expected deviation, day 0). Deviation has been classified as conventional (F2 ≥ 7.0 mm), F2-undersized (F2 < 7.0 mm), and F1,F2-switched (F2 larger than F1 on day −1 or 0). Concentrations of gonadotropins were compared within and among deviation classifications in waves 1 and 2 in 48 heifers. A three-way (wave 1 compared with 2, classification, day) analysis indicated no effect of wave 1 compared with 2 on F2 or FSH. An interaction of classification by day for F2 diameter (P < 0.001) and FSH concentration (P < 0.005) was primarily from differences on day −1. Rankings on day −1 from greatest to least for F2 diameter were switched, conventional, and undersized and for FSH concentration were undersized, conventional, and switched. Lower FSH concentration in conventional compared with undersized deviations during the decline in the FSH surge was presumed to represent greater output of FSH inhibitors by larger follicles. The incline in FSH surge 2 began significantly later for undersized than for conventional deviation. Switched deviation was associated significantly with emergence of F2 before F1, lower FSH concentration during the decline in the FSH surge, and earlier occurrence of the post-surge FSH nadir. Results supported the hypothesis that diameter differences among deviation classifications are temporally associated with differences in FSH concentration within each classification. These novel findings may complicate studies on the mechanisms of follicle selection.

Molecular and endocrine factors involved in future dominant follicle dynamics during the induction of luteolysis in Bos indicus cows

The growth profiles of the future dominant follicle (DF) and subordinate follicle (SF) and the gene expression of the granulosa cells during luteolysis induction in Bos indicus cows were evaluated. Forty cows were synchronized with a progesterone and estradiol based protocol. After synchronization, cows with a corpus luteum (CL) were evaluated by ultrasonography every 12 h, beginning at eight days post ovulation. Cows identified with a follicle of at least 6.0 mm in diameter in the second wave were split into two groups (BD-before follicular deviation and AD-after follicular deviation. In the BD group cows received 500 μg of cloprostenol (a synthetic analogue of prostaglandin F2α) when the DF reached a mean diameter of 7.0 mm (6.5–7.5 mm). In the AD group, cows received 500 μg of cloprostenol when the DF reached a mean diameter of 8.0 mm (7.5–8.5 mm). Cows in both groups were submitted to aspiration of the DF at 96 and 72 h after prostaglandin was given. Follicular aspirations were performed to quantify IGF1R, LHR and PAPPA transcripts in the granulosa cells. The diameter of the DF at the moment of prostaglandin administration (P = 0.001) and the growth rate of the SF (P = 0.05) were greater in the AD group. There was greater abundance of LHR transcripts in BD cows (P = 0.04). The remaining variables tested were similar between the experimental groups (P > 0.05). In conclusion, the induction of luteolysis before follicular deviation does not interfere with dominant follicle dynamics. However, it causes granulosa cell LHR down regulation.

Variations in follicle-diameter deviation and a growth spurt in the dominant follicle at deviation in Bos taurus heifers

Follicle diameter deviation during a follicular wave in cattle is preceded by a common-growth phase and begins with continued growth rate of the future dominant follicle (DF or F1) and reduced growth rate of the future largest subordinate follicle (SF or F2). Follicle diameters were analyzed daily for wave 1 (n=100) and wave 2 (n=91). The beginning of expected deviation was designated day 0 and was defined as the day the largest follicle was closest to 8.5mm. Conventional classification of deviation (F2≥7.0mm at expected deviation) was more frequent (P<0.0001) in wave 1 (59%) than in wave 2 (35%); deviation began abruptly. An F2-undersized classification (F2<7.0mm at expected deviation) was greater (P<0.0001) for wave 2 (40%) than for wave 1 (15%); separation of diameters of F1 and F2 occurred gradually during the last 2days of the common-growth phase. The growth rate of F1 in conventional deviations of wave 1 was greater (P<0.002) on days 0 to 1 (1.8±0.1mm/day) than on days −1 to 0 (1.5±0.1mm/day). Several other novel growth spurts of F1 were observed in association with deviation including during switching of diameter rank between F1 and F2 in each of waves 1 and 2. Hypotheses were supported that (1) conventional deviation is more common during wave 1 whereas F2-undersized deviation is more common during wave 2 and (2) a transient growth spurt occurs in F1 of wave 1 at the beginning of diameter deviation.

Follicles and gonadotropins during waves 2 and 3 in three-wave interovulatory intervals in Bos taurus heifers

Observations were made on follicle dynamics and gonadotropin concentrations in anovulatory wave 2 and ovulatory wave 3 in three-wave interovulatory intervals (n = 15). Hypotheses were not used owing to inadequate availability of rationale. The future dominant follicles for waves 2 and 3 were designated DF2 and DF3 and the largest future subordinate follicles as SF2 and SF3, respectively. The day of expected diameter deviation (day 0) was defined as the day that DF2 or DF3 was closest to 8.5 mm. The first day that DF2 became smaller (P < 0.05) than DF3 was day 2 (10.7 ± 0.2 mm vs 11.8 ± 0.3 mm). The FSH surges 2 and 3 that stimulated waves 2 and 3 were similar at peak concentration, but the postsurge nadir of surge 2 occurred 1 day earlier than for surge 3. An LH increase was not temporally associated with deviation in wave 2, but an increase (P < 0.05) in LH in wave 3 began on day −1. Diameter of SF2 (6.5 ± 0.2 mm) on day 0 was less (P < 0.005) than for SF3 (7.2 ± 0.2 mm). Mean diameter of subordinate follicles in wave 2 did not differ among days. Diameter of subordinate follicles that attained ≥6 mm in wave 3 was greater (interaction, P < 0.02) by day 3 when in the right ovary (RO, 7.4 ± 0.2 mm) than when in the left ovary (LO, 5.6 ± 0.2 mm). The frequency of a conventional classification of deviation (future SF greater than 7.0 mm on day 0) was less (P < 0.001) for wave 2 (1 of 15 waves) than for wave 3 (8 of 15 waves). Novel observations involving DF2 and DF3 were (1) before deviation, diameter of DF2 vs DF3 and an incline in FSH surge 2 vs surge 3 were similar and (2) after deviation, smaller diameter of DF2 vs DF3 by day 2 was associated with an earlier cessation (nadir) in FSH surge 2 vs surge 3 and an absence of an LH increase during deviation. Novel observations involving subordinate follicles ≥6 mm were (1) before deviation, diameters were similar between waves 2 and 3 in association with the similar incline in FSH surges 2 vs 3 and (2) after deviation, a greater diameter increase of subordinates occurred in RO than in LO for wave 3, but an increase did not occur for either ovary in wave 2. The characteristics of diameter deviation were profoundly different between waves 2 and 3 owing to a smaller SF2 than SF3 at deviation but similar diameter of DF2 and DF3.

Blood flow and echotextural differences between the future dominant and subordinate follicles before the beginning of diameter deviation in heifers

Diameter deviation is the beginning of a decrease in growth rate of the largest subordinate follicle (SF) and a continuing growth rate of the dominant follicle (DF). In wave 1 in cattle, deviation begins 2 or 3 days after ovulation when the future DF is about 8.5 mm. Gray scale and power-flow Doppler ultrasound examinations were done in experiment 1 (daily examinations, n = 13) and experiment 2 (examinations every 8 h, n = 15). Blood flow and an anechoic layer in the follicle wall were normalized to the beginning of diameter deviation (day 0 or hour 0). Only waves with conventional diameter deviation (68% of waves) were used as identified by: (1) future SF greater than 7.0 mm when DF was 8.5 mm and (2) future DF and SF did not switch in diameter rank. In experiment 1, deviations in the extent of blood-flow signals and in the extent of anechoic layer began on the same day as deviation in diameter. In experiment 2, deviations in diameter, surface area (πd2), and anechoic layer began in synchrony, and deviation in blood-flow signals began 16 h earlier. Blood-flow deviation before diameter deviation was shown by (1) a first difference (P < 0.02) between follicles at hour −16 and (2) development during the hours −24 to 0 of a greater (P < 0.05) percentage difference between follicles in blood-flow signals (11.1 ± 2.3%) than in surface area (7.4 ± 0.7%) or diameter (4.5 ± 0.4%). Results supported the hypothesis that the extent of blood flow in the future dominant and subordinate follicles deviates before diameter deviates. A similar hypothesis for anechoic layer was not supported; diameter and anechoic layer deviated in synchrony.

An intraovarian mechanism that enhances the effect of an FSH surge on recovery of subordinate follicles in heifers

The effect of the future dominant follicle (DF), corpus luteum (CL), and side (left ovary [LO] and right ovary [RO]) on FSH-induced recovery (increase in diameter) of regressing subordinate follicles was studied in heifers. The DF of wave 2 and the largest subordinate follicle remained intact (controls, n = 14 heifers) or were ablated (n = 14 heifers) on a mean of 13 d postovulation when the DF was ∼10 mm (hour 0). Concentration of FSH (P < 0.0004) and diameter of subordinate follicles (P < 0.0002) decreased between hours −48 to 0 combined for the control and ablation groups. Thereafter, follicle diameter continued to decrease in the controls. Concentration of FSH increased (P < 0.05) and diameter of subordinates began to increase at hour 12 in the ablation group. Follicle-stimulating hormone increased to hour 24 and then returned to the hour 0 concentration by hour 72, completing the induced FSH surge. Concentration of LH began to increase at hour 0 in each group and at a similar rate between groups. Follicle recovery in the ablation group was compared among 8 subgroups as defined by the 2 sides and 4 intraovarian patterns (DF–CL pattern, both structures in same ovary; DF pattern, DF alone; CL pattern, CL alone; and devoid pattern, both structures absent). Follicle diameter increased (P < 0.05) between hours 24 and 48, and diameter at hours 24, 48, 72, and 96 involved a 3-way interaction (P < 0.0001) of pattern, side, and hour. The interaction was similar when diameter of the DF that originated from a recovered subordinate was either included or excluded in the analysis. Diameter of subordinate follicles in the ablation group at hour 96 was greater (P < 0.05) in the DF–CL/RO and DF/RO subgroups than that in the devoid/LO, devoid/RO, and CL/LO subgroups. The DF–CL/LO and CL/RO subgroups were intermediate. For follicles that decreased in diameter before hour 0, a greater (P < 0.05) percentage increased after hour 0 when the ovary contained a DF and was in the RO (DF–CL/RO and DF/RO subgroups) than for the remaining subgroups even after excluding the DF that originated from a subordinate. Results supported the hypotheses that (1) an induced FSH surge can stimulate the recovery of regressing subordinate follicles and (2) recovery of regressing subordinate follicles by FSH involves an intraovarian mechanism. Our interpretation is that the intraovarian mechanism that enhances the stimulatory effect of FSH on recovery of subordinate follicles was effective only in RO and only when it contained a DF.

Characteristics and functions of a minor FSH surge near the end of an interovulatory interval in Bos taurus heifers

The apparent function of a minor FSH surge based on temporality with follicular events was studied in 10 heifers with 2 follicular waves per interovulatory interval. Individual follicles were tracked from their emergence at 2 mm until their outcome was known, and a blood sample was collected for FSH and LH assay every 12 h from day −14 (day 0 = ovulation) to day 4. A minor FSH surge occurred in each heifer (peak, day −4.6 ± 0.2). Concentration of LH increased (P < 0.05) during the FSH increase of the minor surge but did not decrease during the FSH decrease. A minor follicular wave with 8.2 ± 2.0 follicles occurred in 6 of 10 heifers. The maximal diameter (mean, 3.4 ± 0.9 mm) of 77% of the minor-wave follicles occurred in synchrony on day −4.4 ± 0.4. Most (59%) of minor-wave follicles regressed before ovulation and 41% decreased and then increased in diameter (recovered) on day −1.9 ± 0.3 to become part of the subsequent wave 1. A mean of 3.7 ± 0.9 regressing subordinate follicles from wave 2 recovered on the day before or at the peak of the minor FSH surge. The growth rate of the preovulatory follicle decreased (P < 0.02) for 3 d before the peak of the minor FSH surge and then increased (P < 0.03). Concentration of LH increased slightly but significantly temporally with the resurgence in growth rate of the preovulatory follicle. A minor LH surge peaked (P < 0.0002) on day 3 at the expected deviation in growth rates between the future dominant and subordinate follicles. Results indicated on a temporal basis that the recovery of some regressing subordinate follicles of wave 2 was attributable to the minor FSH surge. The hypothesis was supported that some regressing follicles from the minor follicular wave recover to become part of wave 1.

Evidence that fibroblast growth factor 10 plays a role in follicle selection in cattle.

There is evidence that regulation of follicle selection in cattle involves locally produced growth factors. In the present study, we investigated the expression of members of the fibroblast growth factor (FGF) 7 family during follicle deviation. The largest and second largest follicles were recovered during the second day of a synchronised follicle wave and the future dominant and future subordinate follicles were identified based on diameter and cytochrome P450, family 19, subfamily A, polypeptide 1 (CYP19A1) mRNA levels in granulosa cells. Theca cells of the future dominant follicle contained less mRNA encoding FGF7 and FGF10 compared with those from the future subordinate follicle 2.5 days after ovulation, before a significant difference between the diameters of the future dominant and future subordinate follicles could be observed, but FGF22 mRNA levels did not change. Levels of mRNA encoding FGF receptors FGFR1B and FGFR2B in theca and granulosa cells, respectively, were lower in the future dominant follicle compared with the future subordinate follicle. Addition of FGF10 to granulosa cells in vitro significantly decreased oestradiol secretion, as well as CYP19A1, FSH receptor (FSHR) and insulin-like growth factor 1 receptor (IGF1R) mRNA abundance, whereas FGF22 had no effect. We conclude that FGF10 and FGFR2B expression is increased in the future subordinate follicle before morphological deviation, which may contribute to follicle selection.

Differences between follicular waves 1 and 2 in patterns of emergence of 2-mm follicles, associated FSH surges, and ovarian vascular perfusion in heifers

The emergence (first detection) of 2-mm follicles, FSH surges, and ovarian vascular perfusion for follicular wave 1 and surge 1 (n = 26) and wave 2 and surge 2 (n = 25) were studied daily in heifers. The day the future dominant follicle was closest to 5.5 mm was designated Day 0 for each wave. In wave 1, many 2-mm follicles (41%) emerged on Days −5 to −3, whereas FSH surge 1 did not begin until Day −3. Concentration of FSH increased abruptly in 1 day to a peak on the day of maximal number of emerging 2-mm follicles, although the day of maximal number relative to Day 0 differed among individuals. The first emergence of 2-mm follicles in wave 2 occurred concurrently with the first increase in the FSH of surge 2. In wave 1, ovarian resistance to vascular perfusion was negatively correlated (r = −0.48, P < 0.05) with a number of 2-mm follicles on Days −4 to −1 for ovaries that did not contain the preovulatory follicle; vascular perfusion increased with an increase in the number of small follicles. The following hypotheses were supported for wave 1 but not for wave 2: (1) an increase in the number of emerging 2-mm follicles of a follicular wave occurs before the beginning of an increase in FSH, (2) the day of maximal number of emerging 2-mm follicles occurs concurrently with an abrupt FSH increase on different days among individuals, and (3) the association between the number of emerging 2-mm follicles and the extent of ovarian vascular perfusion is positive.

Intraovarian factors associated with switching of a future dominant follicle to a subordinate follicle during induced luteolysis in heifers

The factors involved in the switching of a future dominant follicle (DF) to subordinate status were studied (n = 42) by induction of luteolysis with PGF2α (hour 0) when the largest follicle (F1) in follicular wave 2 was 7.0 or 8.5 mm. Combined for 7.0- and 8.5-mm groups, the frequency of switching was greater (P < 0.01) when F1 and CL were ipsilateral (10 of 28, 36%) than when contralateral (0 of 14). The frequency of switching in the ipsilateral relationship was greater (P < 0.002) when F1 and CL were adjacent (<3 mm apart; 10 of 17) than when separated (0 of 11). The difference in diameter between F1 and F2 was less (P < 0.005) when switching occurred (0.4 ± 0.1 mm) than when switching did not occur (ipsilateral, 1.3 ± 0.2 mm; contralateral, 1.1 ± 0.2 mm). Treatment at hour 0 when F1 was 7.0 mm and ipsilateral to the CL resulted in smaller diameter (P < 0.001) of F1 at hour 12 (7.6 ± 0.2 mm) than when treatment was not given (8.3 ± 0.1 mm). The hypotheses were supported that (1) switching from a future DF to a future subordinate is functionally related to luteolysis, and (2) factors involved in switching include an ipsilateral relationship between the largest follicle and CL and close intraovarian proximity of the follicle to the regressing CL. It is proposed that switching of the future DF to subordinate status during spontaneous luteolysis accounts for the reported greater frequency of the contralateral than ipsilateral relationships between the preovulatory follicle and CL in three-wave interovulatory intervals.

Functional status of STAT3 and MAPK3/1 signaling pathways in granulosa cells during bovine follicular deviation

Follicle development is coordinated by gonadotropins, steroids, and growth factors, which activate multiple signaling pathways. Phosphorylated-MAPK (pMAPK) level was indicated as an early marker of follicle dominance, whereas phosphorylated STAT3 (pSTAT3) was increased in granulosa cells of hypophysectomized rats. We hypothesized that MAPK3/1 and STAT3 pathways are regulated in granulosa cells during follicle deviation in cattle. Cyclic beef cows were synchronized and ovariectomized to recover the two largest follicles. Follicular diameter did not differ on Day 2 but was significantly greater in dominant follicles (DFs) than that in subordinate follicles (SFs) on Days 3 and 4 of the follicular wave. The elevated abundance of CYP19A1 mRNA expression in granulosa cells of DFs and cleaved caspase 3 in Day-4 SFs further validated our in vivo model. Before deviation, pMAPK3/1 levels were significantly higher in granulosa cells of the future DF. STAT3 mRNA and total protein (tSTAT3) were higher in granulosa cells of SFs collected on Day 4. Furthermore, levels of pSTAT3 were dramatically increased in granulosa cells of Day-4 SFs. In conclusion, pMAPK3/1 was increased in the future DF, but such differential abundance between the DF and SF was not evident after deviation. The higher abundance of pSTAT3 in granulosa cells of SFs after deviation suggests that this pathway may be involved in granulosa cell death and follicular atresia.