Magnetic resonance image attributes of the ovarian follicle wall during development and regression

We analyzed image characteristics in T(1)-, T(2)-, and diffusion-weighted in vitro magnetic resonance (MR) images acquired at predefined stages of the ovarian cycle in 36 heifers to test the hypothesis that MR image attributes of the follicle wall reflect the physiologic status of ovarian follicles (viable, atretic, dominant, subordinate). Numerical pixel values (NPV), standard deviation of pixel values (heterogeneity), and area under the curve were used to assess images of follicle walls. Pixel values of the wall were used to calculate a regression line from which intercept, slope, and coefficient of determination were calculated. In T(1) images, NPV of dominant follicles were less likely to fit a regression line at the preovulatory phase than at any other phase (P < 0.1). Preovulatory dominant follicles had lower area under the curve in diffusion-weighted images than early and late static dominant follicles of the anovulatory wave (P < 0.02). Subordinate follicles in the presence of a preovulatory dominant follicle had lower mean NPV in T(1)- and T(2)-weighted images and lower intercepts in T(1)-weighted images than subordinate follicles of the anovulatory wave (P < 0.02). Early atresia of dominant follicles was identified at the late static phase by greater area, mean NPV, and slope in T(2)-weighted images (P < 0.02). Preovulatory dominant follicles had poor fit of NPV to a regression line in T(1)-weighted images and lower area under the curve in diffusion images. Atretic follicles had brighter walls with more acute transitions from follicular fluid to stroma in T(2)-weighted images and more heterogeneous walls in diffusion images. The MR image attributes of the follicle wall reflected the physiologic status of dominant and largest subordinate follicles.     

Quantitative echotexture analysis of bovine ovarian follicles

Computer-assisted image analysis was used to evaluate ultrasound images of bovine ovarian follicles. The ovaries of 8 sexually mature heifers were examined daily by transrectal ultrasonography for 2 estrous cycles. Ultrasonographic examinations of the ovaries were then videotaped, and the dominant and subordinate follicles of successive waves were individually identified and monitored. Recorded images of the dominant anovulatory follicle of the first wave (n = 15) and the ovulatory follicle of the last wave (n = 15) of the estrous cycle were subsequently digitized for computer analysis of echotexture (mean pixel value and pixel heterogeneity). Regions of the image spanning the breadth of the follicle wall were selected, and image analysis revealed that mean pixel value of the dominant anovulatory follicle changed over time (P = 0.0005). Mean pixel value decreased (P = 0.0005) dramatically during the early static phase (Days 6 to 8, Day 0 = day of ovulation), increased (P = 0.0005) at the onset of the regressing phase (Day 12), and reached maximal levels (P = 0.0005) on Day 14. Similarly, image echotexture of the ovulatory follicle revealed a time-dependent effect (P = 0.0001) due to a rapid decrease in mean pixel values between 7 and 4 d before ovulation, followed by an increase until the day before ovulation. The echotexture of images of the follicular antrum were also evaluated and with regard to the dominant anovulatory follicle, a time-dependent effect was not detected for mean pixel value (P = 0.62) but was observed for pixel heterogeneity (P = 0.02). In addition, there was a positive correlation between mean pixel value and heterogeneity (r = 0.61, P = 0.0001). Heterogeneity initially decreased (P = 0.02) and remained low until the emergence of the second follicular wave (mean Day 9). Values subsequently increased and became variable during the late static and regressing phases (> Day 9). Mean pixel value of the antrum of the dominant ovulatory follicle increased (P = 0.0001) as the day of ovulation approached. Heterogeneity did not change (P = 0.14), nor was there any correlation between mean pixel value and heterogeneity for the antrum of the ovulatory follicle (r = 0.06, P = 0.49). We concluded that changes in echotexture (mean pixel value and heterogeneity) of bovine ovarian follicles assessed by computer analysis of ultrasound images were temporally related to functional status (i.e., anovulatory versus ovulatory; growing, static or regressing). The results were strongly supportive of the concept that ultrasonographically detected image attributes are a reflection of physiologic status.     

A potential role for insulin-like growth factor binding protein-4 proteolysis in the establishment of ovarian follicular dominance in cattle.

A critical transition in ovarian follicular development is the selection of a dominant follicle, capable of ovulating, from a cohort of synchronously growing antral follicles. However, little is known about mechanisms and factors that regulate the selection and growth of dominant ovarian follicles. We have investigated whether a component of the insulin-like growth factor (IGF) system, namely IGFBP-4 protease, is associated with the establishment of follicular dominance in cattle. IGFBP proteases degrade IGFBPs, freeing IGFs to interact with their receptors. In experiment 1, follicular fluid from preovulatory follicles (n = 4) degraded about 80% of the added recombinant human (rh) IGFBP-4 within 18 h of incubation. The IGFBP-4 protease exhibited optimal activity at neutral/basic pH and its sensitivity to various protease inhibitors suggested a metalloprotease. The decline in the intensity of the band corresponding to intact rhIGFBP-4 was accompanied by the appearance of immunoreactive fragments of molecular weights approximately 18 and 14 kDa, which were not detectable by ligand blot analysis. In experiment 2, follicular fluid samples were collected from dominant and subordinate follicles on Day 2 or 3 of the first follicular wave, after ovariectomy (experiment 2a, n = 3/day) or by ultrasound-guided follicular aspiration (experiment 2b, n = 4-5/day). Estradiol concentrations in follicular fluid from dominant vs. subordinate follicles confirmed their identities and indicated that the dominant follicle had been selected by Day 2 of the follicular wave. In both experiments 2a and 2b, IGFBP-4 proteolytic activity was 2- to 3.5-fold (P < 0.05) and 5-fold (P < 0.01) higher in follicular fluid from dominant than subordinate follicles on Days 2 and 3 of the follicular wave, respectively. The finding that IGFBP-4 proteolytic activity is higher in dominant, estrogen-active follicles than in subordinate follicles of the same cohort, as early as Day 2 of the follicular wave, strongly suggests a role for IGFBP-4 protease in the establishment of ovarian follicular dominance.     

Ultrasound image characteristics of ovarian follicles in relation to oocyte competence and follicular status in cattle

Assessment of the quality of the female gamete has become paramount for in vitro procedures. There is a need to identify reliable indicators of oocyte competence and develop a simple, non-invasive method to assess competence. The aim of this study was to investigate the relationships among ultrasonographic attributes of a follicle, its stage of development and the competence of the oocyte that it contains. We tested the hypotheses that follicular echotexture characteristics are related to: (1) the phase of development of the follicle, (2) the presence of the corpus luteum (CL) and/or the dominant follicle in the ovary, and (3) developmental competence of cumulus oocyte complexes (COC) from the same ovary. Crossbred beef cows (n=143), age 4-14 years, were given a luteolytic dose of dinoprost to cause ovulation. Ultrasound-guided ablation of all follicles > or = 4mm was done 8 days later to induce new follicular wave emergence during a luteal phase. Ultrasonographic images of dominant follicles and the three largest subordinate follicles (n=402 follicles; 84 cows) were acquired on Days 2, 3, 5 or 7 of the follicular wave (Day 0: wave emergence), i.e. growing, early-static, late static, and regressing phases of subordinate follicle development, respectively. From a subset of these animals (n=33), ovaries were collected within 30 min of slaughter and COC from subordinate follicles > or = 3mm underwent in vitro maturation, fertilization and culture to the blastocyst stage.Image analysis revealed differences in echotexture between dominant and subordinate follicles among Days 2-7 of the follicular wave. Images of dominant and subordinate follicles at Day 7 of the wave displayed consistently lower grey-scale values (P<0.05) in the peripheral antrum, follicular wall and perifollicular stroma than all other days. Follicle images displayed a consistent pattern of variation in echotexture among follicular phases. Data did not support the hypothesis of a local effect of the CL or dominant follicle on follicular echotexture. Echotexture values of the perifollicular stroma were lower in ovaries that did not produce embryos compared to ovaries that produced embryos. Our results showed that the changes in follicular image attributes are consistent with changes in follicular status. The sensitivity of the technique is not yet sufficient for use in a diagnostic setting, but results provide rationale for further development of image analysis as a tool for evaluating oocyte competence in situ.     

Evaluation of the ultrasound image attributes of developing ovarian follicles in the four follicular waves of the interovulatory interval in ewes

Computer-assisted quantitative echotextural analysis was applied to ultrasound images of antral follicles in the follicular waves of an interovulatory interval in sheep. The ewe has three or four waves per cycle. Seven healthy, cyclic Western White Face ewes (Ovis aris) underwent daily, transrectal, ovarian ultrasonography for an interovulatory interval. Follicles in the third wave of the ovulatory interval had a longer static phase than that of those in Waves 1 and 2 (P < 0.05). The numeric pixel value for the wall of anovulatory follicles emerging in the third wave of the cycle was significantly higher than that for Waves 1 and 2 at the time of emergence (156.7 ± 8.09, 101.6 ± 3.72, and 116.5 ± 13.93, respectively), and it decreased as follicles in Wave 3 reached maximum follicular diameter (P < 0.05). The numeric pixel value of the antrum in the ovulatory follicles decreased as follicular diameter increased to ≥5 mm in diameter (P < 0.05). The pixel heterogeneity of the follicular antrum in Wave 1 increased from the end of the growth phase to the end of the regression phase for follicles in that wave (P < 0.05). The total area for the wall and antrum of the follicles studied were correlated with follicular diameter in all follicular waves (r = 0.938, P < 0.01 and r = 0.941, P < 0.01 for the wall and antrum, respectively). Changes in image attributes of the follicular wall and antrum indicate potential morphologic and functional differences among antral follicles emerging at different stages of the interovulatory interval in cyclic ewes.     

Changes in the cumulus-oocyte complex of subordinate follicles relative to follicular wave status in cattle

We investigated factors that affect cumulus-oocyte complex (COC) morphology and oocyte developmental competence in subordinate follicles on different days after follicular wave emergence in beef heifers. In Experiment 1, heifers (n = 13) were assigned at random to COC aspiration during the growing/static (Days 1 to 3) or regressing (Day 5) phase of subordinate follicle development (follicular wave emergence = Day 0). Follicular wave emergence was induced by transvaginal ultrasound-guided follicular ablation, ovaries were collected at slaughter, all follicles > or = 2 mm except the dominant follicle were aspirated, and COC were microscopically evaluated for morphology. There was a greater percentage of COC with expanded cumulus layers on Day 5 (42.4%) than on Days 1 to 3 (2.2%). In Experiment 2, heifers (n = 64) at random stages of the estrous cycle had all follicles > or = 5 mm ablated and 4 d later, 2 doses of PGF were injected 12 h apart; heifers were monitored daily by ultrasonography for ovulation (Day 0 = follicular wave emergence). Heifers were assigned to the following time periods for oocyte collection from subordinate follicles: Days 0 and 1 (growing phase), Days 2, 3 and 4 (static phase), and Days 5 and 6 (regressing phase). Ovaries were individually collected at slaughter, and all follicles > or 2 mm except for the dominant follicle were aspirated. The COC were morphologically evaluated and then matured, fertilized and cultured in vitro. Expanded COC were more frequent during the regressing phase (53.4%) than the growing or static phase (14.4 and 17.8%, respectively; P < 0.05). While the proportions of COC with > or = 4 layers of cumulus cells and denuded oocytes were higher (P < 0.05) in the growing and static phases, the production of morulae was highest (P < 0.05) with COC collected from subordinate follicles during the regressing phase. In Experiment 3, heifers (n = 18) were assigned at random to oocyte collection from subordinate follicles 3 and 4 d (static phase) or 5 and 6 d (regressing phase) after follicular wave emergence. The heifers were monitored ultrasonically for ovulation (Day 0 = follicular wave emergence); COC were collected from all follicles (> or = 5 mm) except for the dominant follicle by transvaginal ultrasound-guided follicle aspiration 3 to 6 d later. Recovered oocytes were stained and examined microscopically to evaluate nuclear maturation. A higher proportion of oocytes collected on Days 5 and 6 showed evidence of nuclear maturation (50%) than on Days 3 and 4 (8.3%; P < 0.05). Results support the hypothesis that COC morphology and oocyte developmental competence change during the growing, static and regressing phases of subordinate follicle development.     

Effect of environmental heat stress on follicular development and steroidogenesis in lactating Holstein cows

Lactating Holstein cows were utilized over two replicate periods (July and September, 1990) to examine the effect of summer heat stress on follicular growth and steroidogenesis. On day of synchronized ovulations, cows were assigned to shade (n=11) or no shade (n=12) management systems. Follicular development was monitored daily by ultrasonography until ovariectomy on Day 8 post estrus. At time of ovariectomy, dominant and second largest follicles were dissected from the ovary. Aromatase activity and steroid concentrations in dominant and subordinate follicles were measured. Acute heat stress had no effects on patterns of growth of first wave dominant and subordinate follicles between Days 1 and 7 of the cycle. Compared with shaded cows, the heat stressed cows did not have suppression of medium size (6 to 9 mm) follicles between Days 5 and 7. A treatment x follicle interaction was detected (P<0.01) for follicular diameter and fluid volume at Day 8. Dominant follicles in shade were bigger (16.4>14.5 mm) and contained more fluid (1.9>1.1 ml) than dominant follicles in no shade. Conversely, subordinate follicles in no shade were bigger (10.1>7.9 mm) and contained more fluid (0.4>0.2 ml) than subordinate follicles in shade. Concentrations of estradiol in plasma and follicular fluid were higher (P<0.01) in July than in September. Heat stress appears to alter the efficiency of follicular selection and dominance, and to have adverse effects on the quality of ovarian follicles.     

Ultrasonographic image attributes of non-ovulatory follicles and follicles with different luteal outcomes in gonadotropin-releasing hormone (GnRH)-treated anestrous ewes

Ultrasonographic images are composed of multiple square picture elements called pixels. Quantitative changes in numerical pixel values (echotexture) determined by computer-assisted analysis of digital images reflect discrete changes in the microscopic structure and physiological status of ovarian antral follicles. The objective of the present study was to determine and compare the ultrasonographic attributes of non-ovulatory antral follicles that grew to an ostensibly ovulatory diameter (> or =5mm) and follicles with different luteal outcomes in response to gonadotropin-releasing hormone (GnRH) in anestrous Western White Face ewes (n=34). All animals received GnRH injections (250ng i.v. every 2h for 24h) followed by a bolus injection of 125microg of GnRH i.v. Ovarian images obtained by repeated transrectal ultrasonography were digitized and subjected to computerized analyses to determine the changes in follicular size and echotexture of the follicular antrum and wall. At the beginning of GnRH treatment, follicles that formed inadequate corpora lutea following ovulation (ICL; n=22) had higher (P<0.001) pixel intensity of the central and peripheral antrum compared with non-ovulatory follicles (n=40). Pixel intensity of the central follicular antrum was greater (P<0.01) in follicles that formed ICL compared with follicles that formed normal (full-lifespan) CL post-treatment (NCL; n=20) and mean pixel heterogeneity of the follicular wall was greater (P<0.05) in non-ovulatory follicles compared with follicles that gave rise to NCL. At the time of GnRH bolus injection (i.e., induction of a synchronous LH surge), the mean diameter of non-ovulatory follicles was greater (P<0.01) than that of all ovulating follicles, and pixel heterogeneity of the central follicular antrum was lowest (P<0.05) in non-ovulatory follicles. The mean diameter of luteinized unovulated follicles (n=9) tended to be greater (P<0.10) at 2.5 and 3 days after emergence, and pixel intensity of the follicular wall was lower (P<0.05) compared with non-luteinized follicles (n=8) at 1.5 and 2.5 days after emergence (beginning of the growth from approximately 3mm onwards). In conclusion, ovarian antral follicles with different outcomes after GnRH treatment (in seasonally anestrous ewes) had distinctive ultrasonographic characteristics.     

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.     

The ability of subordinate follicles of the second follicular wave to become dominant is lost by day 15 of the estrous cycle in cattle

Generally, unilateral ovariectomy before a critical period in the latter part of the estrous cycle induces a transitory increase in plasma FSH, which causes subordinate follicles to develop and maintain ovulation rates characteristic of the species. A limiting period for subordinate follicles to assume dominance and from which ovulation occurs has not been shown for cattle. Growth and/or regression of subordinate follicles were characterized following removal of the dominant follicle at different days of the luteal phase of the estrous cycle in cattle in this study. In the mid-luteal phase (Day 13 or 15), the ovary with the dominant follicle of the second wave was ablated via unilateral ovariectomy; the corpus luteum also was removed. In the late luteal phase (Day 17 or 19), the dominant follicle was ablated with an ultrasonically guided 20 gauge needle. When the dominant follicle was removed on Day 13, the largest subordinate follicle of the second wave of follicular development became dominant and ovulation occurred from this follicle in 4 of 4 animals. However, when the dominant follicle was removed on Day 15, 17 or 19, a new wave of follicular development was induced in 14 of 15 animals. Moreover, the recovered subordinate follicle of the second wave of follicular development had similar growth characteristics to naturally occurring dominant follicles. In conclusion, the subordinate follicle in the second follicular wave in cattle retained the ability to become dominant, but this ability was lost by Day 15 of the estrous cycle. However, cattle then were able to maintain ovulation by developing a new wave of follicular growth.     

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.     

A method for characterizing ultrasonically-derived follicular data in heifers

A method was developed for ultrasonically characterizing follicular waves in heifers without the necessity of maintaining day-to-day identities of individual follicles (nonidentity method). Results were compared to a method in which the identities of individual follicles were maintained from day to day (identity method). Data collected daily during 5 estrous cycles were processed by each method, independently, by different operators. The nonidentity method involved grouping and then profiling follicles in order of decreasing diameters without regard to day-to-day identities. The profiling scheme distinguished between follicles of the left versus the right ovary. The dominant and subordinate follicles were readily distinguishable in the nonidentity profiles. When successive dominant follicles developed in the opposite ovary, the follicles were profiled directly. When two successive dominant follicles developed on the same ovary, size information was obscured for a few days where the profiles for each follicle crossed, but continuity of the profiles on each side of the area of ambiguity was maintained. The nonidentity method seemed equivalent to the identity method in determining characteristics of the dominant follicle (e.g., maximal diameter, growth rate, regression rate). Day of emergence of a wave and day of divergence in diameters between dominant and subordinate follicles were readily determined by inspection of the nonidentity profiles. A greater number of subordinate follicles per wave was detected by the nonidentity method due to the inability to individually identify all detected follicles by the identity method. Regression of follicles from a previous wave into the subordinate follicles of a succeeding wave was apparent by either method. The nonidentity method seemed suitable for most needs, was less tedious, and required less skill than the identity method.     

Intraovarian relationships among dominant and subordinate follicles and the corpus luteum in heifers

Previous studies demonstrated that waves of follicular activity develop approximately every 9 d in cattle during the estrous cycle and early pregnancy. A dominant follicle develops from each wave and the remaining follicles (subordinates) begin to regress after a few days. In this study, intraovarian luteal and follicular interrelationships were examined during the follicular waves of the estrous cycle and pregnancy using data obtained by ultrasonography. During the estrous cycle, no intraovarian relationships were found between the ovary containing the corpus luteum and the ovary containing the dominant follicle (n = 165), or between the location of the corpus luteum and the characteristics of the dominant follicle. During pregnancy, however, the frequency distribution for the number of follicular waves with the dominant follicle and corpus luteum on the same or opposite ovaries differed (P<0.05) among Waves 1 to 10. The two structures (dominant follicle and corpus luteum) were more often in opposite ovaries during Waves 3 to 10 (combined frequency, 75%) than during Waves 1 and 2. During pregnancy, dominant follicles of consecutive waves differed (P<0.05) among Waves 1 to 8 in the frequency with which they appeared in the same versus the opposite ovary. The difference seemed primarily due to an increased frequency of consecutive follicles on the same ovary for Waves 4 to 8 (combined frequency, 80%). During both the estrous cycle and pregnancy, there was no significant intraovarian effect of the dominant follicle on the day of detection of the next dominant follicle, on the growth rate of the largest subordinate follicle, or on the length of the interval from wave origin to cessation of growth of the largest subordinate; these results indicate that previously postulated suppressive effects between follicles are exerted through systemic channels.     

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).     

Identification of genes involved in apoptosis and dominant follicle development during follicular waves in cattle

We hypothesize that granulosa and theca cells from growing dominant follicles, with relatively high intrafollicular concentrations of estradiol, have a greater expression of genes involved in inhibiting apoptosis pathways and lower expression of genes involved in apoptosis pathways than growing subordinate follicles with lower estradiol concentrations. Using the well-characterized bovine dominant follicle model, we collected granulosa and theca cells from individual dominant and the largest subordinate follicle 3 days after initiation of a follicular wave in four animals. Based on ultrasound analysis, both follicle types were in the growth phase at the time of ovariectomy. However, dominant follicles were larger (9.8 +/- 1.0 versus 7.6 +/- 0.6 mm in diameter, P < 0.05) and had greater intrafollicular concentrations of estradiol (132.2 +/-3 8.5 versus 24.1 +/- 12.1 ng/ml, P < 0.05), compared with the largest subordinate follicles. We used bovine cDNA microarrays, which contained a total of 1400 genes, including a subset of 53 genes known to be involved in apoptosis pathways, to determine which apoptosis and marker genes from each of the four dominant versus subordinate follicles were potentially differentially expressed. Using a low stringency-screening criterion, 22 genes were identified. Quantitative real-time polymerase chain reaction confirmed that 16 of these genes were differentially expressed. Our novel results demonstrate that the high intrafollicular concentrations of estradiol in growing dominant follicles were positively associated with enhanced expression of mRNAs in granulosa cells for aromatase, LH receptor, estradiol receptor beta, DICE-1, and MCL-1, compared with granulosa cells from subordinate follicles (all survival-associated genes). In contrast, the relatively low intrafollicular concentrations of estradiol in growing subordinate follicles were positively associated with enhanced expression of mRNAs in granulosa cells for beta glycan, cyclo-oxygenase-1, tumor necrosis factor alpha, caspase-activated DNase, and DRAK-2, and in theca cells for beta glycan, caspase 13, P58(IPK), Apaf-1, BTG-3, and TS-BCLL, compared with granulosa or theca cells from dominant follicles (genes that are all associated with cell death and/or apoptosis). We suggest that that these genes may be candidate estradiol target genes and that they may be early markers for the final stages of follicle differentiation or initiation of apoptosis and thus selection of dominant follicles during follicular waves.     

Relationships of changes in ultrasonographic image attributes to ovulatory and steroidogenic capacity of large antral follicles in sheep

Ovarian steroidogenesis and antral follicular development in ewes, following the treatment with medroxyprogesterone acetate (MAP) and equine chorionic gonadotrophin (eCG), are affected by the reproductive season. The objective of this study was to compare the ultrasonographic attributes of large antral follicles between cyclic (December) and seasonally anovular (June–July) ewes, after a 12-day treatment with MAP-soaked intravaginal sponges, with or without the administration of 500 IU of eCG at sponge removal, and to determine whether there is a correlation between the ultrasonographic attributes of the follicular wall and serum concentrations of oestradiol. Digital images of ovulatory follicles from cyclic ewes and eCG-treated anoestrous ewes (n = 34 follicles), and of anovulatory follicles attaining ≥5 mm in control anoestrous ewes (n = 8 follicles), were analysed using the spot and line techniques designed to determine the echotextural characteristics of the follicular antrum (central and peripheral), follicular wall and perifollicular ovarian stroma. The mean diameter of ovulatory follicles was greater (P < 0.001) in cyclic than anoestrous ewes, with or without the eCG treatment. The mean pixel heterogeneity (SD of numerical pixel values) of the follicular antrum (P < 0.05), as well as mean pixel intensity and heterogeneity of the peripheral antrum, follicular wall proper and perifollicular ovarian stroma (P < 0.05), were consistently greater in anoestrous than cyclic ewes at the time of sponge removal and 24 h after the treatment with MAP sponges or MAP/eCG. Mean oestradiol concentrations were greater (P < 0.05) in cyclic compared to anoestrous ewes in both MAP- and MAP/eCG-treated animals, from 1 to 2 days after sponge withdrawal. There was a moderate negative correlation (r² = 0.12, P < 0.05; Pearson's Product Moment and r² = 0.23, P < 0.05; ANCOVA) between mean pixel heterogeneity (standard deviation of mean pixel values) of the follicular wall proper (all follicles ≥5 mm in diameter) and serum concentrations of oestradiol after sponge withdrawal. Our results indicate that large antral follicles from cyclic and seasonally anovular ewes exhibit distinctive ultrasonographic characteristics. The differences in follicular echotexture appear to be related mainly to seasonal variations in ovarian follicular morphology and oestradiol production.     

Complex interrelationships among CL,preovulatory follicle,number of follicular waves,and right or left ovaries in heifers

The diameter of the dominant follicle (DF) of wave 1 was studied on Days 9 to 17 (Day 0 = ovulation) in a survey of the ipsilateral and contralateral relationships between the location of the DF and CL, and number of follicular waves per interovulatory interval (IOI). For contralateral relationships, regardless of number of waves the diameter of the DF of wave 1 decreased (P < 0.03) between Days 11 and 13 when referenced to the follicle–CL relationship of wave 1 and decreased (P < 0.008) between Days 9 and 11 when referenced to the preovulatory follicle (PF)–CL relationship. For wave 2 in two-wave IOIs, the CL ovary of ipsilateral relationships had more (P < 0.05) follicles that reached at least 6 mm than the non-CL ovary. In three-wave IOIs, frequency of IOIs with the DF in the CL ovary was greater (P < 0.02) for wave 2 than for wave 3. In wave 3, the preovulatory and the largest subordinate follicles were located more frequently (P < 0.005) in the contralateral ovary. Ovulation in two-wave IOIs occurred more frequently (P < 0.0009) from the right ovary. In three-wave IOIs with a contralateral relationship ovulation occurred more frequently (P < 0.003) from the left ovary; a negative intraovarian effect of the CL on location of the PF may account for more ovulations from the left ovary and a reported greater frequency of the contralateral relationship. The hypothesis was supported that the ipsilateral versus contralateral relationship between the PF and CL is affected by the DF–CL relationship during the previous follicular waves and by the number and identity of waves per IOI.     

Hormonal dynamics and follicular turnover in prepuberal Mediterranean Italian buffaloes (Bubalus bubalis)

The aim of this study was the investigation of hormonal and ovarian follicular dynamics in prepuberal buffaloes (Bubalus bubalis) bred in Italy. Eleven 5-9-month old buffalo calves ranging in weight from 122 to 270kg, maintained under controlled nutritional and environmental conditions, underwent 50 days of ultrasonographic ovarian follicular monitoring in the months of October-December. Blood sampling for E(2) and FSH determination and ultrasonographic monitoring using a 7.5MHz linear probe and an ALOKA SSD-500 monitor were performed daily. No differences in any of the parameters under study were highlighted when calves were divided into two weight categories (<200 and >200kg) and thus data were pooled. In this study, values are reported as mean+/-S.D. A range of two-six regular follicular waves was reported among calves with an average of 4+/-1.1. Overall interval (days) between wave emergence was 9.9+/-2.8 and largest diameters (mm) of dominant and first subordinate follicles were 8.4+/-1.2 and 4.8+/-0.6, respectively (P<0.05). With the exception of one calf, some minor follicular waves (short waves or SWs; 1.6+/-1), lasting <10 days (6.1+/-1.2) were reported. They were monitored contemporaneously on the ovary contralateral (n=7) or ipsilateral (n=3) to the main follicular wave. Growth rate (mm per day) of dominant follicles (DF) was significantly faster than for corresponding subordinate follicles (SF) and follicles of SWs (1.08+/-0.2 versus 0.79+/-0.1 and 0.83+/-0.1, respectively, P<0.05). The static phase (days) lasted longer in DF compared to SF and SW (5.4+/-1.8 versus 2.4+/-1.2 and 2.6+/-1, respectively, P<0.05). The regressing phase (mm per day) was similar among DF, SF and SW (0.86+/-0.2, 0.94+/-0.2 and 0.84+/-0.1, respectively, P=0.09). Episodic spikes of E(2) and FSH were reported, corresponding to wave development throughout the course of investigation. In conclusion, the majority of buffalo calves displayed a typical pattern of regular follicular development in conjunction with a dynamic trend of ovarian and hypophyseal hormones. Some minor follicle turnover was reported with parallel main follicular waves.     

The mare: a 1000-pound guinea pig for study of the ovulatory follicular wave in women

The mare is a good comparative model for study of ovarian follicles in women, owing to striking similarities in follicular waves and the mechanism for selection of a dominant follicle. Commonality in follicle dynamics between mares and women include: (1) a ratio of 2.2:1 (mare:woman) in diameter of the largest follicle at wave emergence when the wave-stimulating FSH surge reaches maximum, in diameter increase of the two largest follicles between emergence and the beginning of deviation between the future dominant and subordinate follicles, in diameter of each of the two largest follicles at the beginning of deviation, and in maximum diameter of the preovulatory follicle; (2) emergence of the future ovulatory follicle before the largest subordinate follicle; (3) a mean interval of 1 day between emergence of individual follicles of the wave; (4) percentage increase in diameter of follicles for the 3 days before deviation; (5) deviation 3 or 4 days after emergence; (6) 25% incidence of a major anovulatory follicular wave emerging before the ovulatory wave; (7) 40% incidence of a predeviation follicle preceding the ovulatory wave; (8) small but significant increase in estradiol and LH before deviation; (9) cooperative roles of FSH and insulin-like growth factor 1 and its proteases in the deviation process; (10) age-related effects on the follicles and oocytes; (11) approximate 37-hour interval between administration of hCG and ovulation; and (12) similar gray-scale and color-Doppler ultrasound changes in the preovulatory follicle. In conclusion, the mare may be the premier nonprimate model for study of follicle dynamics in women.     

Follicular dynamics and plasma FSH and progesterone concentrations during follicular deviation in the first post-ovulatory wave in Nelore (Bos indicus) heifers

The objective of the present study was to characterize ovarian follicular dynamics and hormone concentrations during follicular deviation in the first wave after ovulation in Nelore (Bos indicus) heifers. Ultrasonographic exams were performed and blood samples were collected every 12h from the day of estrus until 120-144 h after ovulation in seven females. Deviation was defined as the point at which the growth rate of the dominant follicle became greater than the growth rate of the largest subordinate follicle. Deviation occurred approximately 65 h after ovulation. Growth rate of the dominant follicle increased (P<0.05) after deviation, while growth rate of the subordinate follicle decreased (P<0.05). Diameter of the dominant follicle did not differ from the subordinate follicle at deviation (approximately 5.4mm). The dominant follicle (7.6mm) was larger (P<0.05) than the subordinate follicle (5.3mm) 96 h after ovulation or 24h after deviation. Plasma FSH concentrations did not change significantly during the post-ovulatory period. The first significant increase in mean plasma progesterone concentration occurred on the day of follicular deviation. In conclusion, the interval from ovulation to follicular deviation (2.7 days) was similar to that previously reported in B. taurus females, but follicles were smaller. Diameters of the dominant follicle and subordinate follicle did not differ before deviation and deviation was characterized by an increase in dominant follicle and decrease in subordinate follicle growth rate. Variations in FSH concentrations within 12-h intervals were not involved in follicular deviation in Nelore heifers.