Development of codominant follicles in cattle is associated with a follicle-stimulating hormone-dependent insulin-like growth factor binding protein-4 protease.

Low molecular weight insulin-like growth factor binding proteins (IGFBPs), particularly IGFBP-4, are believed to inhibit the actions of insulin-like growth factors (IGFs). We showed previously that ovarian follicular dominance in cattle is associated with the presence of a protease that degrades IGFBP-4. To test the hypothesis that specific IGFBP-4 proteolysis is associated with selection of the dominant follicle, we induced codominant follicles (co-DFs) during the first follicular wave of the estrous cycle. The ovaries of Holstein heifers were examined twice daily by ultrasonography; when the largest follicle reached 6 mm in diameter, saline (control, n = 5) or 2 mg of recombinant bovine (rb) FSH (FSH, n = 5) was injected i.m. every 12 h for 48 h. Follicular fluid was collected by aspiration from the two largest follicles/heifer 12 h after the last injection. IGFBPs in follicular fluid were quantified by Western ligand blotting/phosphorimaging. IGFBP-4 protease activity was measured by incubating follicular fluid with recombinant human (rh) IGFBP-4 substrate, followed by ligand blotting/phosphorimaging to quantify the percent of substrate loss and Western immunoblotting to detect specific proteolytic fragments. Co-DFs of FSH heifers did not differ (P > 0.05) from the single dominant follicle of controls in size, or in concentration of progesterone or level of IGFBP-4 in follicular fluid. In contrast, the largest subordinate follicle of control heifers was smaller, with lower progesterone and higher IGFBP-4 in the follicular fluid (P < 0.05). Concentrations of estradiol in follicular fluid were high in dominant follicles, intermediate in co-DFs, and low in subordinate follicles (P < 0.05). IGFBP-4 protease activity in co-DFs was similar (P > 0.05) to that of dominant follicles, but fourfold higher (P < 0.05) than that of subordinate follicles. The results strongly suggest that an FSH-dependent IGFBP-4 protease is associated with selection of the dominant follicle in cattle.     

Alterations in follicular IGFBP mRNA expression and follicular fluid IGFBP concentrations during the first follicle wave in beef heifers

The objective was to determine the pattern of IGFBP-2, -3 and -4 gene expression and follicular fluid concentrations of IGFBP-2, -3, -4 and -5 during emergence, selection and dominance of the first follicle wave of the estrous cycle in cattle and during exogenous steroid treatment. Heifers (n = 35) were ovariectomized at 36 (n = 7), 66 (n = 8), 84 (n = 12) and 108 (n = 8) h after the onset of estrus. Heifers in the 84 h ovariectomy group were sub-divided to receive either no treatment (n = 6) or were treated with a progesterone-releasing intravaginal device (n = 6, PRID) and 0.75 mg estradiol benzoate i.m. at the approximate time of ovulation, 30 h post estrus until ovariectomy. Within heifers the four largest follicles recovered following ovariectomy were ranked on size (F1, F2, F3 and F4). At 36 h IGFBP gene expression and follicular fluid IGFBP concentrations were similar in all follicles (F1-F4). Mean diameter of the F1 follicle increased (P < 0.05) between 36 and 84 h with no difference between 84 and 108 h. The F1 follicle had the highest (P < 0.05) concentration of estradiol compared with the F2, F3 and F4 at 84 and 108 h. There was no granulosa cell IGFBP-2 mRNA in F1 follicles at 84 or 108 h. Intrafolliclar IGFBP-2 concentrations were lower (P < 0.05) in the F1 compared with F3 and F4 follicles at 108 h. There was no difference in theca cell IGFBP-4 mRNA expression at 108h, but amounts of follicular fluid IGFBP-4 were lower (P < 0.05) in F1 follicles compared with F3 and F4 follicles at 108 h. IGFBP-3 mRNA was localized in the theca layer of all follicles examined with no difference in expression or follicular fluid concentrations during emergence, selection and dominance of the first follicle wave. IGFBP-5 concentrations were higher (P < 0.05) in follicular fluid of F3 follicles at 108 h compared with the F3 at 36 h. In conclusion follicular dominance was associated with low or decreased follicular fluid concentrations of IGFBP-4 and -5, increased estradiol and differential regulation of IGFBP production.     

Identification of potential intrafollicular factors involved in selection of dominant follicles in heifers

A surgical procedure to aspirate follicular fluid concurrently from individual follicles from the same heifer was validated and used to determine if intrafollicular amounts of estradiol, progesterone, inhibins, activin-A, follistatins, and insulin-like growth factor binding proteins (IGFBP) differed for the future dominant compared with subordinate follicles during selection of the first wave dominant follicle. Heifers were subjected to surgery and aspiration of follicular fluid from the two or three largest follicles on Day 3 of the estrous cycle (approximately 1.5 days after emergence). Ultrasound was used to determine the fate of each aspirated follicle after surgery. At aspiration, diameter of the future dominant and largest subordinate follicle was similar in heifers. However, estradiol was higher, whereas IGFBP-4 was lower in the future dominant compared with the largest or next largest subordinate follicles. Also, the future dominant follicle in most cohorts had the highest estradiol and lowest IGFBP-4 compared with future subordinate follicles. We concluded that: IGFBP-4 and estradiol may have key roles in determining the physiological fate of follicles during selection of the first wave dominant follicle in heifers, and that both are reliable markers to predict which follicle in a growing cohort of 5- to 8.5-mm follicles becomes dominant.     

Activin A,estradiol, and free insulin-like growth factor I in follicular fluid preceding the experimental assumption of follicle dominance in cattle

In cattle, the two largest follicles of a wave (F1, F2) begin to deviate into a dominant follicle and a subordinate follicle when F1 is a mean of 8.5 mm in diameter. After the beginning of deviation, F1 and F2 are diameter-defined dominant and subordinate follicles. Changes associated with the conversion of F2 into a future dominant follicle were studied by ablating F1 at the expected beginning of deviation (F1, 8.5 mm; Hour 0) and assessing the follicular-fluid factors in F2. Follicles were designated F1C and F2C in controls and F2A in F1-ablated heifers. Follicular-fluid collections were made at Hours 0, 4, 8, or 12 (n = 7 heifers per hour; fluid from F1C, F2C, and F2A; experiment 1) or at Hours 4, 6, 8, 10, or 12 (n = 9 heifers per hour; fluid from F2A; experiment 2). Postablation concentrations of circulating FSH increased (P < 0.05) between Hours 2 and 6. Diameter of F2A increased (P < 0.05) after Hour 8 in both experiments so that the diameter of F2A at Hours 10 or 12 was not different (P > 0.1) from the diameter of F1 at Hour 0. A transient elevation (P < 0.05) in follicular-fluid activin A occurred in F2A at Hour 8 in both experiments. Concentrations of estradiol (P < 0.05) and insulin-like growth factor I (IGF-I; P < 0.1) decreased in F2C by Hour 8. In F2A, the concentrations of both factors began to increase (P < 0.05) after Hours 4 or 8 so that there was no difference (P > 0.1) between F1C and F2A at Hour 12. Concentrations of IGF-I and IGF binding protein 2 (IGFBP-2) in F2A changed in opposite directions at the same hours. No differences between follicles were found for concentrations of progesterone, androstenedione, inhibin A, and inhibin B. The order of events in the conversion of a future subordinate follicle to a future dominant follicle was an increase in systemic FSH, a transient elevation in follicular-fluid activin A, and a simultaneous increase in follicular-fluid estradiol and restoration of an apparent growth-compatible balance of free IGF-I and IGFBP-2.     

Temporal aspects of ovarian follicular growth and steroidogenesis following exogenous follicle-stimulating hormone in Angus heifers

Ultrasonography and endocrine assay techniques were used to monitor structural and hormonal alterations made by the ovary in response to the biological actions of pituitary-derived follicle-stimulating hormone (FSH-P). Angus heifers (n = 36) were allotted to receive injections (twice per day) of either FSH-P (up to a total of 28 mg over a maximum of 4 days beginning on Day 10 of a synchronized estrous cycle) or saline in order to quantify temporal relationships among follicle growth and steroid hormone profiles. Transrectal ultrasonography was utilized at 12-h intervals to monitor and record follicle growth. Plasma was collected every 12 h for the first 48 h of the experiment and then every 6 h for the remainder of the experiment. At 48 and 60 h after the onset of treatments, prostaglandin F2α (PGF2α; 25 mg) was administered (i.m.). FSH-treated heifers (n = 6 at each time) were terminated at 24, 48, 72 and 96 h following the onset of treatment. Saline-treated heifers were terminated at 24 and 96 h (n = 6 at each time). After ovaries were obtained, follicular number and size were recorded and follicular fluid (FF) was collected. Plasma concentration of progesterone (P) and estradiol (E2) and FF concentration of P, E2, estrone, testosterone and androstenedione were determined by radioimmunoassays. Plasma concentration of E2 increased (P < 0.05) within 36 h of initiation of FSH treatment. Plasma P decreased (P < 0.0001) by 12 h post-PGF2α. Ultrasonographic examination revealed a significant decrease in the number of small follicles by 48 h, whereas the number of medium follicles increased (P < 0.05) by 60 h after the initiation of FSH treatment. The number of large follicles (LF ≥ 10 mm diameter) increased (P < 0.01) over the course of the experiment. The total number of ovarian follicles (TF) 24 h after the start of FSH treatment was correlated (r = 0.99; P < 0.0001) with the number of small follicles (SF ≤ 5 mm). At 72 h after the onset of FSH treatment, the number of medium follicles (i.e. 6–9 mm) was correlated with TF (r = 0.97; P < 0.0001). Estradiol was the predominant FF steroid. Follicular fluid E2 was greatest in follicles at 72 h after FSH treatment. Follicular fluid E2 and plasma E2 were positively correlated (r = 0.66; P < 0.001). Follicular aromatase activity was estimated by evaluating the ratio of FF estrogens (E) to androgens (A). Elevated aromatase activity (E:A ratio > 1.0) was detected in 196 of 206 follicles. The estrogen to progesterone ratio was used as an estimate of follicle viability. Eighty-five percent of the follicles were estimated to be viable (E:P ratio > 1.0). The peak E:A ratio in LF preceded by 24 h the peak concentration in FF E2 and plasma E2. In MF and SF the E:A ratio increased by 72 h. Enhancement of ovarian follicular growth (i.e. increased number and size of follicles; increased steroidogenesis) by exogenous, pituitary-derived FSH is characterized by (1) increased activity of aromatase, and (2) accumulation of FF E2, events which temporally preceded the increase in plasma concentration of E2. These observations will aid efforts to incorporate recombinant bovine FSH and somatotropin in an effort to develop more predictable superstimulation and ovulation induction protocols.     

Selection of the dominant follicle in cattle: role of estradiol

Involvement of estradiol in the deviation in growth rates between the two largest follicles of a wave was studied in 39 heifers. In experiment 1, the largest follicle remained intact in a control group and was ablated in five estradiol-treated groups when the largest follicle reached 8.5 mm or larger (expected beginning of deviation; Hour 0). The ablation groups were given a single injection of 0, 0.004, 0.02, 0.1, or 0.5 mg of estradiol. Blood samples were taken from a jugular vein every hour at Hours 0 to 16. By Hour 8, FSH concentrations were greater (P < 0.05) in the ablation group that received 0 mg of estradiol than in the controls. Among the estradiol groups, that receiving 0.02 mg had the lowest detectable increase in estradiol. In this group, FSH concentrations were not suppressed below the control concentrations, but the increase in FSH concentrations following ablation of the largest follicle was delayed for 2 or 3 h. This delay in the increase of FSH concentrations corresponded to the hours that estradiol was maximal. In experiment 2, blood samples were taken every 4 h from the caudal vena cava cranial to the junction with the ovarian veins in heifers with the largest follicle intact (controls) or ablated at 8.5 mm or larger (Hour 0). Averaged over Hours 4 to 48, estradiol concentrations were higher (P < 0.04) in the controls than in the ablation group. During Hours 0 to 12, estradiol concentrations increased (P < 0.05) in the controls, whereas FSH concentrations decreased (P < 0.05). In the ablation group, estradiol concentrations were lower than in the controls by Hour 4, and FSH concentrations increased (P < 0.05) between Hours 4 and 12. These results support the hypothesis that the largest follicle releases increased estradiol into the blood at the beginning of follicular deviation, and that the released estradiol is involved in the continuing depression of FSH concentrations to below the requirement of the smaller follicles.     

Follicular dynamics around the recruitment of the first follicular wave in the cow

The present study aimed to test the generally accepted view that a follicular wave starts with follicles newly recruited from the population smaller than 3 mm, which later compete for dominance. According to this view, subordinate follicles are expected to be too atretic to join the next follicular wave. Ten cows were ovariectomized shortly prior to the LH surge, thus around the start of the first follicular wave of the cycle. Per cow, on average, 14.4 follicles of >/=3 mm were dissected. Follicular health was determined on the basis of four parameters: 1) judgment of the degree of atresia by stereomicroscope, 2) incidence of apoptotic nuclei among the granulosa cells, 3) estradiol and progesterone concentrations, and 4) insulin-like growth factor-I (IGF-I) binding proteins (IGFBPs)-2, -4, and -5 concentrations in the follicular fluid. In addition to the preovulatory follicle, 3.1 other follicles, mainly sized 3-4.5 mm, were found to be healthy based on the proportion of apoptotic nuclei, and concentrations of estradiol/progesterone, and IGFBPs. The ability of these follicles to respond with growth on the preovulatory and periovulatory FSH surges was supported by a comparison to the follicular population of four cows 31-68 h after the LH surge. The present results point to an alteration of the view on the follicular wave. The larger follicles during the first days of the follicular wave are, in general, derived from follicles that also joined the previous wave. A portion of these growing follicles are estradiol active and compete for dominance. Other growing follicles lack estradiol production and are probably derived from rather atretic follicles. The first newly recruited follicles do not reach the size of 3 mm before 31 h after the preovulatory FSH surge. At that time, the larger follicles are already competing for dominance.     

Association between surges of follicle-stimulating hormone and the emergence of follicular waves in heifers.

The effects of ablation of a dominant follicle and treatment with follicular fluid on circulating concentrations of follicle-stimulating hormone (FSH) were studied and the temporal relationships between surges of FSH and follicular waves were studied in heifers with two or three follicular waves/interovulatory interval. Cauterization of the dominant follicle on Day 3 or Day 5 (ovulation on Day 0) (six control and six treated heifers/day) resulted in a surge (P less than 0.05) in FSH beginning the day after cautery. The FSH surge prior to wave 2 (first post-treatment follicular wave) occurred 4 days (Day 3 cautery) and 2 days (Day 5 cautery) before the surge in control groups, corresponding to a 4-day and a 2-day advance in emergence of wave 2 compared with controls. It was concluded that the dominant follicle on Day 3 and Day 5 was associated with the suppression of circulating FSH concentrations. Heifers (n = 4/group) were untreated or treated intravenously with a proteinaceous fraction of bovine follicular fluid on Days 0-3, 3-6, or 6-11. Concentrations of FSH were suppressed (P less than 0.05) for the duration of treatment, regardless of the days of treatment. Cessation of treatment was followed within 1 day by the start of a surge in FSH. The FSH surge prior to wave 2 occurred 2 days earlier (treatment on Days 0-3), 1 day later (treatment on Days 3-6), and 6 days later (treatment on Days 6-11) than in controls, corresponding to an equivalent advance or delay, respectively, in the emergence of wave 2 compared with controls. The results suggest that the effects of exogenous follicular fluid on follicular development were mediated, in whole or in part, by altering plasma FSH concentrations. Control heifers combined for the two experiments were separated into those with 2-wave (n = 11) or 3-wave (n = 5) interovulatory intervals. Two-wave heifers had two FSH surges and 3-wave heifers had three apparent FSH surges during the interovulatory interval. Results of the cautery and follicular fluid experiments indicated that a surge in FSH necessarily preceded the emergence of a wave. The FSH surges in treated and control heifers began 2-4 days before the detectable (ultrasound) emergence of a follicular wave (follicles of 4 and 5 mm), peaked 1 or 2 days before emergence and began to decrease approximately when the follicles of a wave begin to diverge into a dominant follicle and subordinate follicles (follicles 6-7 mm).     

Effect of follicle size on in vitro production of steroids and insulin-like growth factor (IGF)-I,IGF-II,and the IGF-binding proteins by equine ovarian granulosa cells

Little is known regarding the hormonal regulation of granulosa cell steroidogenesis and the ovarian insulin-like growth factor (IGF) system in the mare. The objectives of this study were to determine, first, if estradiol, insulin, and/or FSH affect steroid production by equine granulosa cells (experiment 1) and, second, if the components of the IGF system are produced by equine granulosa cells in culture as well as whether estradiol, insulin, and/or FSH affects IGF and/or IGF-binding protein (IGFBP) production by equine granulosa cells (experiment 2). Granulosa cells from small (6-15 mm), medium (16-25 mm), and large (25-48 mm) follicles were collected from cyclic mares (n = 14), cultured for 2 days in medium containing 10% fetal calf serum, washed, and then treated for an additional 2 days in serum-free medium with or without added hormones. In experiment 1, large-follicle granulosa cells produced less progesterone and more estradiol than did medium- and/or small-follicle granulosa cells (P < 0.05). Progesterone production was inhibited (P < 0.05) by FSH and insulin in small- and medium- but not in large-follicle granulosa cells; estradiol was without effect. Insulin increased (P < 0.05) estradiol production in small- and medium-follicle granulosa cells but had no effect in large-follicle granulosa cells. In experiment 2, IGF-I production was inhibited (P < 0.05) by insulin across all follicle sizes but was not affected by estradiol or FSH. Granulosa cells of medium and large follicles produced more IGF-II than did granulosa cells of small follicles (P < 0.05). Insulin and FSH inhibited (P < 0.05) IGF-II production by granulosa cells of large and medium but not of small follicles; estradiol was without effect. Only IGFBP-2 and -5 were produced by equine granulosa cells. Production of IGFBP-2 was less (P < 0.10) in granulosa cells of large versus those of small and medium follicles, whereas medium-follicle granulosa cells produced more (P < 0.05) IGFBP-5 than did small- or large-follicle granulosa cells. Averaged across follicle sizes, estradiol increased (P < 0.05) IGFBP-2 production, FSH increased (P < 0.10) IGFBP-2 and -5 production, and insulin was without effect. These results indicate that IGF-I, IGF-II, IGFBP-2, and IGFBP-5 are produced by equine granulosa cells and that insulin, FSH, and estradiol play a role in the regulation of steroidogenesis and the IGF system of equine granulosa cells.     

Alterations in follicular estradiol and gonadotropin receptors during development of bovine antral follicles

It was hypothesized that growth divergence of dominant and subordinate follicles during Wave 1 and growth termination of the dominant follicle would be associated with changes in the number of gonadotropin receptors on granulosa cells and estradiol in follicular fluid. To test this hypothesis, follicular development of 16 Holstein heifers was monitored by ultrasound, and follicles were collected on Days 2,4,6 and 10 (Day 0 = ovulation). Dominant follicles were compared across days, whereas dominant and largest subordinate follicles were compared on Days 2 and 4 only. The numbers of LH and FSH receptors on the granulosa cells of dominant follicles did not differ significantly over Days 2, 4, 6 and 10. In contrast, concentrations of estradiol in follicular fluid decreased (P < 0.05) from Days 2 to 10 (373 +/- 150 to 42 +/- 12 ng/ml) and concentrations of progesterone in follicular fluid increased (P < 0.05) from Days 2 to 10 (12.2 +/- 2.3 to 24.4 +/- 4.8 ng/ml). Correspondingly, the ratio of estradiol:progesterone in the dominant follicles decreased (P < 0.05) from Days 2 to 10. Comparisons between dominant and subordinate follicles indicated greater (P < 0.05) estradiol concentrations in the dominant follicle on Day 2, but the number of gonadotropin receptors was not different until Day 4. Thus, differences in concentrations of follicular fluid estradiol, but not numbers of granulosa cell gonadotropin receptors, were associated with the early growth divergence of dominant and subordinate follicles (Day 2) and the eventual growth termination of the dominant follicle (Day 10). Late divergence (Day 4) was associated with higher gonadotropin receptor numbers and follicular estradiol concentrations in the dominant than in the subordinate follicles. These results indicate that an increase in estradiol productivity of the selected dominant follicle occurred before an increase in the number of gonadotropin receptors.     

Follicular and hormonal response to experimental suppression of FSH during follicle deviation in cattle

A near steroid-free fraction of bovine follicular fluid was used to suppress FSH concentrations at the expected time of follicle deviation or when the largest follicle of Wave 1 reached > or = 8.0 mm (actual mean diameter, 8.4 mm; Hour 0). It was hypothesized that the low concentrations of FSH associated with deviation are inadequate for the smaller follicles but are needed for continued growth of the largest follicle. Control heifers (n=8) received 10 mL of saline, and treated heifers (n=16) received either 8.8 mL or 13.3 mL of the follicular-fluid fraction at Hours 0, 12, and 24. Between Hours -48 and 0, FSH concentrations decreased (P<0.05) and diameters of the 4 largest follicles increased (Hour effect, P<0.0001) similarly between groups. Concentrations of LH in the controls increased (P<0.05) between Hours -24 and -12 and decreased (P<0.05) between Hours 8 and 36, demonstrating a transient LH surge encompassing the expected beginning of deviation. In the treated group, a comparable increase in LH occurred before deviation but a decrease did not occur until after Hour 48. By Hour 4.5, the FSH concentrations in the treated group decreased (P<0.05) to below the concentrations in the controls. Suppressed diameter (P<0.001) of the largest follicle was detected at the first post-treatment examination (Hour 12; 7.5 h after FSH suppression) and was accompanied by reduced (P<0.04) systemic estradiol concentrations. The mean growth rates of the 3 smaller follicles in both the treated and control groups began to decrease at Hours -12 to 24 and were not different between groups during Hours 0 to 36. Concentrations of FSH in the treated group returned to control concentrations by Hour 24 (hour of last treatment). A rebound (P<0.05) in concentrations of FSH to >100% above control concentrations occurred by Hour 48 and was accompanied by resumed growth of the largest follicle in 75% of the heifers between Hours 48 and 72. The results demonstrated that the low concentrations of FSH associated with deviation can be further reduced by treatment with a nonsteroidal factor of follicular origin. Transient reduction of FSH concentrations to below the already low control concentrations inhibited the largest follicle but did not further inhibit the smaller follicles. These results support the hypothesis that the low FSH concentrations associated with follicle deviation are below the minimal requirements of the smaller or subordinate follicles but are needed for continued growth of the largest or dominant follicle in cattle.     

Steroidogenic changes and steady state amount of messenger RNA encoding steroidogenic enzymes, gonadotropin receptors and cell-death signalling in the dominant ovarian follicle during estradiol-induced atresia in cattle

Changes in steroidogenic function and associated gene expression were characterized in dominant ovarian follicles (DF) of cattle where follicles were induced to become atretic by systemic administration of estradiol benzoate (EB). In experiment 1, follicular fluid (FF) steroid concentrations in the DF were measured at 12-hourly time points for 48 h in heifers treated with 1 mg EB i.m./500 kg body weight (EB; n=20) as compared with untreated controls (C; n=19). Treatment with EB promoted a transient reduction in circulating FSH, a rapid (12 h) and sustained reduction in FF estradiol, a rapid (12 h) but transient reduction in FF progesterone and a delayed (36 h) increase in FF testosterone concentrations. In experiment 2, whole follicular wall tissue was collected from DF of mature non-lactating cows allocated to a 0 h control group (0 HC: n=7), a 24h control group (24 HC; n=7) or an EB-treated group where tissue was collected 24 h after administration of 1 mg EB i.m./500 kg body weight (EB; n=8). As for experiment 1, EB promoted a transient reduction in circulating FSH, a pronounced reduction in FF estradiol and a smaller but significant reduction in FF progesterone concentrations. Semi-quantitative RT-PCR on follicular wall tissue revealed that the loss in estrogen activity at 24 h after EB was associated with two-fold reduction in aromatase mRNA, with an apparent acceleration in loss of 17alpha-hydroxylase mRNA. Expression of genes for gonadotropin receptors (LHR and FSHR) and a cell-death signalling pathway (Fas antigen and Fas ligand) were unchanged during the initial 24h of EB-induced atresia. These results suggest that EB initiates atresia in dominant ovarian follicles through a rapid suppression of follicular estradiol synthesis, an effect associated with down-regulation of the aromatase gene. A transient suppression in circulating FSH following administration of EB appears to have initiated these events, and it is suggested that subsequent processes involved in atresia follow this loss in estrogenic function.     

Follicular and hormonal dynamics during the first follicular wave in heifers

A few days after the first follicular wave emerges as 4-mm follicles, follicular deviation occurs wherein 1 follicle of the wave continues to grow (dominant follicle) while the others regress. The objectives of this study were to characterize follicle growth and associated changes in systemic concentrations of gonadotropins and estradiol at 8-h intervals encompassing the time of follicle deviation. Blood samples from heifers (n = 11) were collected and the ovaries scanned by ultrasound every 8 h from 48 h before to 112 h after the maximal value for the preovulatory LH surge. The follicular wave emerged at 5.8 +/- 5.5 h (mean +/- SEM) after the LH surge, and at this time the future dominant follicle (4.2 +/- 0.8 mm) was larger (P < 0.001) than the future largest subordinate follicle (3.6 +/- 0.1 mm). There was no difference in growth rates between the 2 follicles from emergence to the beginning of the deviation (0.5 mm/8 h for each follicle), indicating that, on average, the future dominant follicle maintained a size advantage over the future subordinate follicle. Deviation occurred when the 2 largest follicles were 8.3 +/- 0.2 and 7.8 +/- 0.2 mm in diameter, at 61.0 +/- 3.7 h after wave emergence. Diameter deviation was manifested between 2 adjacent examinations at 8-h intervals. Mean concentrations of FSH decreased, while mean concentrations of LH increased 24 and 32 h before deviation, respectively, and remained constant (no significant differences) for several 8-h intervals encompassing deviation. In addition to the increase and decrease in circulating estradiol concentrations associated with the preovulatory LH surge, an increase (P < 0.05) occurred between the beginning of deviation and 32 h after deviation. The results supported the hypotheses that deviation occurs rapidly (within 8 h), that elevated systemic LH concentrations are present during deviation, and that deviation is not preceded by an increase in systemic estradiol.     

Oxytocin,prostaglandins E and F,estradiol, progesterone,sodium, and potassium in preovulatory bovine follicles either developed normally or stimulated by follicle stimulating hormone

To determine the differences between stimulation by follicle stimulating hormone (FSH) and normal development of follicles in heifers and the endocrinologic events associated with ovulatory development of follicles in heifers, follicular fluid was aspirated from follicles (n = 627) at 24, 48, and 72 h after estrus synchronization (via prostaglandin F_2α (PGF_2α)) from control animals (n = 10/time period) and a treatment group (n = 10/time period) that received FSH. No endocrinologic or ionic differences were noted between follicles harvested from control or FSH-stimulated animals within follicular size or time (24, 48, or 72 h). No interaction of treatment (FSH stimulation vs controls) by time was detected; thus the effect and timing of the LH surge was similar between treatments.At 48 h after PGF_2α administration, sodium from follicular fluid decreased and potassium increased, signifying a considerable physiologic change in the follicle. Follicular prostaglandins E and F increased ten-fold by 72 h, and these changes were primarily found in the estrogen-inactive follicles at 72 h. Though progesterone concentrations within follicular fluid tended to increase throughout the three periods and increased with enlargement of follicular size, a major increase was detected 72 h after PGF_2α injection. Estradiol concentrations tended to increase with enlargement of follicular size within a time period, but estradiol concentrations in the follicles greater than 10 mm in diameter decreased with time (24, 48, and 72 h) after PGF_2α. Concentrations of oxytocin increased with increases in follicular diameter, and the time trends were similar to changes in follicular progesterone. By 72 h after PGF_2α administration, follicular estradiol) was decreasing in concentration and progesterone and oxytocin were increasing, thus signifying a change in the secretory role of the granulosa cells.     

Follicle selection in cattle: follicle deviation and codominance within sequential waves

Follicle deviation during bovine follicular waves is characterized by continued growth of a developing dominant follicle and reduction or cessation of growth of subordinate follicles. Characteristics of follicle deviation for waves with a single dominant follicle were compared between wave 1 (begins near ovulation; n = 15) and wave 2 (n = 15). Follicles were defined as F1 (largest), F2, and F3, according to maximum diameter. No mean differences were found between waves for follicle diameters at expected deviation (F1, > or =8.5 mm; Hour 0) or observed deviation or in the interval from follicle emergence at 4.0 mm to deviation. For both waves, circulating FSH continued to decrease (P < 0.05) after Hour 0, estradiol began to increase (P < 0.05) at Hour 0, and immunoreactive inhibin began to decrease (P < 0.05) before Hour 0. A transient elevation in circulating LH reached maximum concentration at Hour 0 (P < 0.01) in both waves and was more prominent (P < 0.0001) for wave 1. Waves with codominant follicles (both follicles >10 mm) were more common (P < 0.02) for wave 1 (35%) than for wave 2 (4%). Codominants (n = 6) were associated with more (P < 0.05) follicles > or=4 mm and a greater concentration (P < 0.04) of circulating estradiol at Hours -48 to -8 than were single dominant follicles (n = 15). A mean transient increase in FSH and LH occurred in the codominant group at Hour -24 and may have interfered with deviation of F2. In codominant waves, deviation of F3 occurred near Hour 0 (F1, approximately 8.5 mm). A second deviation involving F2 occurred in four of six waves a mean of 50 h after the F3 deviation and may have resulted from a greater suppression (P < 0.05) of FSH in the codominant group after Hour 0. In conclusion, follicle or hormone differences were similar for waves 1 and 2, indicating that the deviation mechanisms were the same for both waves. Waves that developed codominant follicles differed in hormone as well as follicle dynamics.     

Long-term follicular dynamics and biochemical characteristics of dominant follicles in dairy cows subjected to acute heat stress

The objective of this study was to examine the quality of successive dominant follicles (DFs) after induced heat stress. Non-lactating dairy cows expressing estrus at normal intervals were allocated randomly to heat stress (HS; n=8) and control (C; n=8) groups. Cows received GnRH (100 microg, i.m.) on Day 0, a progesterone CIDR-B device on Day 4 and prostaglandin (PGF(2alpha); 25mg, i.m.) on Day 7 upon removal of the CIDR device. The DF and follicles >5mm were aspirated on Day 8, and GnRH (100 microg) injected following aspiration, to initiate a new follicular wave. In this manner, a DF was aspirated every 8 days (one "follicular cycle") for 10 cycles. After the first follicular cycle, HS cows were placed in environmental chambers for 7 days during the second follicular cycle (8h per day at 43.3 degrees C set point and 16h per day at 24 degrees C for 4 days, and 8h per day at 43.3 degrees C set point and 16h per day at 32.2 degrees C set point for 3 days; relative humidity, 40%) and thereafter maintained outdoors with control cows at a mean ambient temperature (18.5 degrees C; range 12.7-26 degrees C). Rectal temperature increased (P<0.001) in HS as compared with C cows (39.28+/-0.01 degrees C versus 38.78+/-0.01 degrees C). Concentrations of estradiol (E(2); 1662+/-189 versus 1493+/-188ng/ml) and progesterone (P(4); 44.7+/-5 versus 54.1+/-5.1ng/ml) in follicular fluid (FF) of DF did not differ between C and HS treatments, respectively. Total FF protein concentration was greater (P<0.05) in HS (99.7+/-2.3mg/ml) than in C (92.7+/-2.3mg/ml). Heat shock protein 90 (Hsp 90) in FF was not altered by heat stress. IGF-II ligand blots were conducted with FF samples (n=79) from four HS and four C cows. There was a predominance of IGFBP-3 in 76 of 79 FF samples, indicating healthy follicular status, and only three FF samples had the lower molecular weight IGFBP-2 indicative of a poor quality follicle. Plasma P(4) and E(2) concentrations did not differ between C and HS groups. The number of class 1 and 3 follicles increased during and just after heat stress, but the number of class 2 follicles did not differ between C and HS cows. Heat stress appeared to induce a decrease in follicular dominance, but GnRH-induced follicular cycles resulted in development of healthy preovulatory follicles in both groups.     

Differentiation of dominant versus subordinate follicles in cattle

Selection of a dominant follicle, capable of ovulating, from among a cohort of similarly sized follicles is a critical transition in follicular development. The mechanisms that regulate the selection of a species-specific number of dominant follicles for ovulation are not well understood. Cattle provide a very useful animal model for studies on follicular selection and dominance. During the bovine estrous cycle, two or three sequential waves of follicular development occur, each producing a dominant follicle capable of ovulating if luteal regression occurs. Follicles are large enough to allow analysis of multiple endpoints within a single follicle, and follicular development and regression can be followed via ultrasonographic imaging. Characteristics of recruited and selected follicles, obtained at various times during the first follicular wave, have been determined in some studies, whereas dominant and subordinate follicles have been compared around the time of selection in others. As follicular recruitment proceeds, mRNA for P450 aromatase increases. By the time of morphological selection, the dominant follicle has much higher concentrations of estradiol in follicular fluid, and its granulosa cells produce more estradiol in vitro than cells from subordinate follicles. Shortly after selection, dominant follicles have higher levels of mRNAs for gonadotropin receptors and steroidogenic enzymes. It has been hypothesized that granulosa cells of the selected follicle acquire LH receptors (LHr) to allow them to increase aromatization in response to LH, as well as FSH. However, LH does not appear to stimulate estradiol production by bovine granulosa cells, and the role of LHr acquisition remains to be determined. Recent evidence suggests a key role for changes in the intrafollicular insulin-like growth factor (IGF) system in selection of the dominant follicle. When follicular fluid was sampled in vivo before morphological selection, the lowest concentration of IGF binding protein-4 (IGFBP-4) was more predictive of future dominance than size or estradiol concentration. Consistent with this finding, dominant follicles acquire an FSH-induced IGFBP-4 protease activity. Thus, a decrease in IGFBP-4, which would make more IGF available to interact with its receptors and synergize with FSH to promote follicular growth and aromatization, appears to be a critical determinant of follicular selection for dominance.     

Follicle selection in monovular species

Follicle deviation is proposed to be the eminent event in follicle selection in monovular species. At deviation, the largest follicle establishes dominance apparently before the second-largest follicle can reach a similar diameter. In cattle, based on diameters of the two follicles at the beginning of deviation, the mechanism becomes established in <8 h. An FSH:follicle-coupling hypothesis has been supported as the essence of follicle selection. According to the hypothesis, the growing follicles cause the FSH decline from the peak of the wave-stimulating FSH surge until deviation, even though the follicles continue to require FSH (two-way functional coupling involving multiple follicles). During multiple-follicle coupling, inhibin is the primary FSH suppressant. Near the beginning of deviation, the largest follicle secretes increased estradiol, and apparently both estradiol and inhibin contribute to the continuing FSH decline; only the more-developed largest follicle is able to utilize the low FSH concentrations (single-follicle coupling). Deviation is encompassed by a transient elevation in LH in heifers and by a component, often distinct, of the long ovulatory LH surge in mares. In heifers, receptors for LH appear in the granulosa cells of the future dominant follicle about 8 h before the beginning of deviation. The LH stimulates the production of estradiol and insulin-like growth factor-1. These intrafollicular factors and perhaps others account for the responsiveness of the largest follicle to the low concentrations of FSH. The smaller follicles have not reached a similar developmental stage and because of their continued and close dependency on FSH become susceptible to the low concentrations. Thereby, follicle selection is established.     

Effects of follicle stimulating hormone, cholera toxin, pertussis toxin and forskolin on adenosine cyclic 3',5'-monophosphate output by granulosa cells from Booroola ewes with or without the F gene

The cAMP outputs by granulosa cells from 3-4.5 mm diameter (medium) follicles of Booroola FF ewes were similar to those by cells from greater than or equal to 5 mm diameter (large) follicles of ++ ewes with respect to time or dose of FSH, cholera toxin or forskolin. Likewise, the cAMP outputs by cells from 1-2.5 mm diameter (small) FF follicles were similar to those by cells from small and medium ++ follicles with respect to time or dose of FSH, cholera toxin or forskolin. At FSH, cholera toxin or forskolin doses of 1 microgram/ml, 0.5 microgram/ml and 10(-4) M respectively, the granulosa cell cAMP outputs of medium FF or large ++ follicles were approximately 2-fold (P less than 0.05) higher than in the respective small FF and medium ++ follicles. The effects of cholera toxin plus forskolin or FSH plus forskolin were additive irrespective of genotype or follicle size, with significant differences (P less than 0.05) observed between follicle sizes but not genotype. No differences were noted between cholera toxin plus forskolin or FSH plus forskolin on granulosa cell cAMP output. For the FSH and forskolin treatments, increased mean cAMP outputs were evident after 10 min, whereas after cholera toxin treatment they were not evident until after 20 min incubation. For all treatments the rate of cAMP production tended to slow down after 40-60 min. Pre-incubation of granulosa cells with pertussis toxin subsequently resulted in a significantly greater (P less than 0.05) FSH-induced output of cAMP relative to the untreated controls irrespective of follicle size. However, no gene-specific differences were noted when the cAMP outputs of cells from medium or small FF follicles were compared with cells from large or small-medium ++ follicles respectively. These results indicate that the activity (or composition) of the regulatory and catalytic components of adenylate cyclase in the FF granulosa cells change in a manner similar to those observed in ++ cells with the only difference being that the increases in cyclase in FF ewes occurs as follicles enlarge from 1-2.5 to 3-4.5 mm in diameter, whereas in ++ ewes they occur as follicles enlarge from 3-4.5 to greater than or equal to 5 mm in diameter. No evidence was found to link the F gene to the granulosa cell cAMP response independently of follicle size. It is suggested that the association between the F gene and the size-specific difference in follicle maturation may be unrelated to the FSH receptor/cAMP generating system.     

Effect of estradiol valerate on ovarian follicles, emergence of follicular waves and circulating gonadotropins in heifers

An experiment was designed to examine the effect of estradiol valerate (EV) on the growth and regression of follicles of a wave and on the emergence of the next follicular wave. Twenty-six beef heifers were xamined daily by ultrasonography and randomly allocated to 1 of 4 treatment groups at the time of ovulation (Day 0): unterated control heifers and those that received 5 mg EV intramuscularly on Day 1, Day 3 or Day 6. Maximum diameter of the dominant follicle was greater (P<0.05) in control heifers than in heifers treated on Day 1 or Day 3. Mean day of onset of regression of the dominant follicle was later (P<0.05) in control heifers than in heifers treated on Day 1 but was not different from heifers treated on Day 3. In heifers treated on Day 6, cessation of growth, maximum diameter and onset of regression were not different from that of control heifers. The emergence of the next follicular wave was earlier (P<0.05) in heifers treated on Day 1 than in control heifers, whereas wave emergence was delayed (P<0.05) in heifers treated on Day 3 or Day 6. The mean day of maximum concentration of FSH prior to the emergence of the next wave was earlier in heifers treated with EV on Day 1 and later in heifers treated on Day 3 or Day 6 compared with that of the controls (P<0.05). Treatment on Day 1 or Day 3 resulted in a significant LH surge in 8 13 heifers, whereas no LH surges were detected in control heifers or in heifers treated on Day 6. The hypothesis that EV suppresses the growth of the dominant follicle, was supported. Estradiol valerate treatment resulted in early emergence of the next follicular wave in heifers treated on Day 1, but treatment on Day 3 or Day 6 resulted in delayed emergence of the next follicular wave.