Largest Subordinate Follicle Research Articles

Ovarian follicular dynamics, hormonal profiles and ovulation time in Mithun cows (Bos frontalis).

The study aimed to evaluate follicular dynamics and concentrations of estradiol-17β (E2), progesterone (P4), follicle stimulating hormone (FSH) and luteinizing hormone (LH) during the oestrous cycle and to determine ovulation time in Mithun cows. Ovaries of experimental cows (n=7) were examined daily by transrectal-ultrasonography for three consecutive oestrous cycles (n=21). The characteristics of follicular waves, dominant follicle, largest subordinate follicle and corpus luteum and ovulation time were evaluated. The plasma samples were analysed throughout the interovulatory interval to determine the differences in the hormonal profiles (E2, P4, FSH and LH) between different follicular wave cycles. Out of eighteen oestrous cycles analysed, three-wave follicular cycles were maximum (n=12: 66.66%) followed by two (n=4: 22.22%) and four waves (n=2: 11.11%). The two and three waves were statistically compared, and no significant (p > .05) differences were observed in day of wave emergence, number of follicles (≥3 mm) recruited, maximum diameter of the ovulatory dominant follicle, growth rates of ovulatory and anovulatory dominant follicles and maximum diameter of corpus luteum. The diameter of dominant follicles was significantly (p < .05) greater than subordinate follicles in both ovulatory and anovulatory waves. No significant differences were observed in peak concentrations of estradiol-17β and follicle stimulating hormone between ovulatory and anovulatory waves in all wave cycles. A preovulatory luteinizing hormone surge was observed a day before ovulation in all wave cycles. Progesterone concentrations were lower than 0.5ng/ml during oestrus and increased sharply to the maximum levels of ≥3.8ng/ml in all wave cycles. Ovulation time (mean ± SEM), irrespective of follicular waves was 10.5 ± 0.64 h after the end of oestrus. It was concluded that Mithun cows have a preponderance of three follicular waves with little difference between the two- and three-follicular waves and ovulation occurred 10.5h after the end of oestrus.

The dynamics of trkA expression in the bovine ovary are associated with a luteotrophic effect of ovulation-inducing factor/nerve growth factor (OIF/NGF).

BackgroundOvulation-inducing factor in semen (OIF/NGF) influences ovulation and CL form and function in camelids and, remarkably, in cows. To test the hypothesis that the luteotrophic effect of OIF/NGF is mediated by an increase in trkA receptors in the ovulatory follicle and early CL, a study was designed to characterize the spatial and temporal distribution of trkA in ovarian follicles and CL at known stages of the bovine estrous cycle.MethodsSexually mature cattle (n = 14) were examined daily by transrectal ultrasonography to determine the day of ovulation (Day 0), and assigned randomly to be unilaterally ovariectomized on Day 2, 4, 6 or in the pre-ovulatory period just before or after exogenous LH treatment. After a complete interovulatory interval, the cows were re-assigned to a different day–group on which the remaining ovary was removed. Sections of ovarian tissue representing the dominant follicle, largest subordinate follicle, and the CL were processed for immunofluorescent detection and quantification of trkA receptor.ResultsTrkA immuno-fluorescence in ovarian tissues was restricted to follicles and the CL (no reaction in stroma or vessels), and was restricted to the cytoplasm (no nuclear staining). The trkA staining intensity, area of staining, and proportion of cells stained was greater in both theca and granulosa layers of dominant follicles than in that of subordinate follicles (P ≤ 0.05) in all day-groups except the Pre-LH group. Among dominant follicles, a progressive reduction in the immuno-positive reaction was detected from Day 2 to Day 6. Among subordinate follicles, immuno-reactivity remained low and unchanged except a rise in the Pre-LH group. The number of immuno-positive cells was greater in early developing CL (Days 2 and 4 combined) than in mature or regressing stage CL (Day 6, Pre- and Post-LH combined; P = 0.01). The intracellular distribution of trkA was more diffuse and widespread in dominant than subordinate follicles, particularly on Day 2 and Post-LH (P < 0.05).ConclusionsDistinct differences in trkA expression between dominant and subordinate follicles, particularly when circulating progesterone is minimal (early luteal development and after luteolysis) is consistent with a local role of OIF/NGF in follicle selection and early luteogenesis.

171 EXPRESSION OF THE OVULATION-INDUCING FACTOR/NERVE GROWTH FACTOR HIGH AFFINITY RECEPTOR IN THE OVARIES OF COWS DURING THE PERIOVULATORY PERIOD

Ovulation-inducing factor/nerve growth factor (OIF/NGF) influences ovulation and corpus luteum (CL) size and function in camelids and, remarkably, cows. The ovulation effect in induced ovulators is mediated at the hypothalamo-pituitary-axis, but the site and mechanism of action of the luteotrophic effect is unknown. This mechanism may be an important aspect of OIF/NGF in spontaneous-ovulators. The objective of this experiment was to detect changes in expression of the high affinity OIF/NGF receptor (TrkA) in the ovary during the periovulatory period in cattle. Cows (n = 14) were examined daily by transrectal ultrasonography to determine the day of ovulation (Day 0), and were assigned randomly to be unilaterally ovariectomized on Day 2, 4, 6, or in the preovulatory period before or after the LH surge. Cows assigned to preovulatory groups were given a luteolytic dose of prostaglandin when the dominant follicle of the second follicular wave was ≥10 mm and growing. Cows assigned to the pre-LH group were ovariectomized 24 h after prostaglandin treatment. Cows assigned to the post-LH group were given LH 24 h after prostaglandin treatment and were ovariectomized 18–20 h later. Cows were allowed to rest for one complete interovulatory interval and re-assigned to a different day-group on which the remaining ovary was removed (n = 4 to 5 ovaries/day-group). Ovaries were fixed in paraformaldehyde, and 5-μm sections of ovarian tissue representing the dominant follicle, largest subordinate follicle, and the CL were treated for enzymatic antigen retrieval and blocked in 1% BSA for 1 h. Slides were incubated overnight with the primary antibody (rabbit anti-human TrkA) and for 2 h with the secondary antibody (goat anti-rabbit IgG). Slides were evaluated by fluorescence microscopy. Images of 3 arbitrarily chosen fields (40×) were captured for each structure for each day-group, and the average proportion of immunoreactive cells in the theca layer and luteal tissue was estimated using ImageJ software (National Institutes of Health, Bethesda, MD, USA). The intensity of individual cell immunofluorescence was also scored as no reaction (0), faint (1), weak to moderate (2), or strong (3). Data were compared among groups by two-way ANOVA. The proportion of immunoreactive cells was higher in the dominant follicle than in the subordinate follicle or the CL (P &lt; 0.001); no effect of day-group or interaction was detected. The intensity score increased from faint on Day 2 to strong on Day 6, and remained strong in the pre- and post-LH groups. A discernible pattern of change in intensity score was not evident for the subordinate follicle or the CL. The greater proportion of TrkA positive cells and greater immunoreactive intensity in the maturing dominant follicle support the hypothesis that the luteotrophic effect of OIF/NGF in cattle is a result of a local increase in the expression of TrkA in the theca layer of the dominant follicle. Research was supported by the Natural Sciences and Engineering Research Council of Canada.

Ultrasonographic Characterization of Follicle Deviation in Follicular Waves with Single Dominant and Codominant Follicles in Dromedary Camels (Camelus dromedarius)

Follicular wave emergence was synchronized by treating camels with GnRH when a dominant follicle (DF) was present in the ovaries. Animals were scanned twice a day from day 0 (day of GnRH treatment) to day 10, to characterize emergence and deviation of follicles during the development of the follicular wave. Follicle deviation in individual animals was determined by graphical method. Single DFs were found in 16, double DFs in 9 and triple DFs in two camels. The incidence of codominant (double and triple DFs) follicles was 41%. The interval from GnRH treatment to wave emergence, wave emergence to deviation, diameter and growth rate of F1 follicle before or after deviation did not differ between the animals with single and double DFs. The size difference between future DF(s) and the largest subordinate follicle (SF) was apparent from the day of wave emergence in single and double DFs. Overall, interval from GnRH treatment to wave emergence and wave emergence to the beginning of follicle deviation was 70.6 ± 1.4 and 58.6 ± 2.7 h, respectively. Mean size of the DF and largest SF at the beginning of deviation was 7.4 ± 0.2 and 6.3 ± 0.1 mm, respectively. In conclusion, the characteristics of follicle deviation are similar between the animals that developed single or double DFs.

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.

174 ULTRASOUND BIOMICROSCOPIC IMAGE ATTRIBUTES OF OVARIAN FOLLICLES IN HEIFERS

High-resolution ultrasound biomicroscopy (UBM) with high-frequency transducers (20 to 50 MHz) has enabled imaging of small antral follicles (&lt;1 mm) and acoustic characterisation of the follicular antrum and wall. Quantitative echotextural analysis of the images generated by UBM provides detailed functional information about the granulosa and theca layers of follicle walls. The objectives of our study were to compare biomicroscope image echotextures between dominant and largest subordinate follicles during the first follicular wave and to characterise UBM attributes of the ovulatory follicle. Hereford crossbred heifers (14 to 16 months old, n = 12) were used. Ovarian examinations were performed transvaginally from Day –4 or –3 (Day 0 = ovulation) to Day 10 using a UBM imaging instrument (Vevo 660; Visual Sonics Inc., Toronto, Canada) equipped with a 25-MHz end-fire transducer. Echotextural assessment was performed by spot metrics of the follicle antrum and follicle wall by using a series of custom-developed computer algorithms optimized for ultrasonography (Synergyne©, Version 2.8, WHIRL, Saskatoon, SK, Canada). Although subordinate and dominant follicles development were tracked from Day –2 (around 1 mm in diameter) to Day 10, the ovulatory follicle was tracked only from Day –4 to ovulation. All data were analysed by analysis of variance for repeated measures using the Mixed procedure (Littell et al. 1998) in the Statistical Analysis System software package (SAS version 8.2 for MS Windows; SAS Institute Inc., Cary, NC, USA). No differences were detected between dominant and largest subordinate follicle in the mean pixel value (MPV) or pixel heterogeneity (PH) of the antrum. However, the MPV of the wall of dominant follicles was greater and PH tended to be greater than that of the largest subordinate follicles (P = 0.05 and P = 0.1, respectively). This result could be evidence that the dominant follicle would be healthier. The PH of the wall the ovulatory follicle decreased (P = 0.03) from Day –4 to Day –1; however, no changes in MPV were observed. Similarly, no changes were detected in MPV or PH of the follicular antrum. The loss of granulosa cells from the follicular wall can explain, at least in part, the decrease in mean pixel value and pixel heterogeneity of the follicular wall detected in ovulatory follicles. Moreover, morphologic changes indicative of atresia are typically represented by high values of number of pixel values. In conclusion, although no differences in antral characteristics of dominant v. subordinate follicles were detected, mean pixel value and pixel heterogeneity of the follicular wall were higher in dominant follicles, and pixel heterogeneity of follicular wall of ovulatory follicles decreased over days. Quantitative pixel analysis from follicular wall images using UBM could be applied to evaluate follicles in live animals. However, more studies using this technique are necessary. Research was supported by NSERC and CIHR. Luiz Pfeifer was supported by CAPES from Ministry of Education, Brazil.

Angiotensin II Signaling Promotes Follicle Growth and Dominance in Cattle

It is generally understood that angiotensin II (AngII) promotes follicle atresia in rats, although recent data suggested that this may not be true in cattle. In this study, we aimed to determine in vivo whether AngII alters follicle development in cattle, using intrafollicular injection of AngII or antagonist into the growing dominant follicle or the second largest subordinate follicle. Injection of saralasin, an AngII antagonist, into the growing dominant follicle inhibited follicular growth, and this inhibitory effect was overcome by systemic FSH supplementation. Injection of AngII into the dominant follicle did not affect follicular growth, whereas injection of AngII into the second largest follicle prevented the expected atresia of this subordinate follicle, and the treated follicle grew at the same rate as the dominant follicle for the next 24 h. Inhibition of AngII action in the dominant follicle decreased estradiol concentrations in follicular fluid and the abundance of mRNA encoding aromatase, 3β-hydroxysteroid dehydrogenase, LH receptor, and cyclinD2 in granulosa cells, with minimal effects on theca cells. The effect of AngII on aromatase mRNA levels was confirmed using an in vitro granulosa cell culture system. In conclusion, these data suggest that AngII signaling promotes follicle growth in cattle and does so by regulating genes involved in estradiol secretion and granulosa cell proliferation and differentiation.

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.

Exercise affects both ovarian follicular dynamics and hormone concentrations in mares

The objectives were to evaluate the effects of exercise on ovarian folliculogenesis and related hormones in mares. Mares (n = 11) were randomly assigned into a control (non-exercised) or treatment (exercised) group. Treatment mares (n = 5) were moderately exercised for 30 min, 6 d/wk. All mares underwent daily transrectal ultrasonographic examinations and ovarian follicles > 6 mm were measured. Blood samples were collected during the first (Cycle 1) and last (Cycle 4) cycle, and serum concentrations of cortisol, LH, and FSH were determined. Mean cortisol concentrations were elevated (P < 0.05) in exercised mares, 6.29 ± 0.22 compared with 5.62 ± 0.16 ng/dL (mean ± SEM), 30 min post exercise. There were no significant differences between groups in mean FSH concentrations; however, exercised mares had lower (17.3 ± 6.4 vs 41.1 ± 5.5 ng/mL; P < 0.05) peak LH concentrations. Furthermore, exercised mares experienced a longer (24.7 ± 0.8 vs 22.2 ± 0.8 d; P < 0.05) mean interovulatory interval for all cycles combined, fewer (P < 0.05) follicles 6 to 20 mm in diameter, and an increased (P < 0.05) number of follicles >20 mm following deviation. The dominant and largest subordinate follicle in exercised mares had a greater (P < 0.05) mean diameter on the day of deviation, suggesting delayed deviation. Exercised mares also tended (P = 0.06) to have an increased number of cycles with at least two dominant follicles compared to control (62 vs 36%, respectively), indicating a decreased ability of the largest follicle to assert dominance. Under the conditions of this study, moderately exercising mares induced higher cortisol concentrations, lowered peak LH concentrations, and altered ovarian follicular dynamics.

Supplementation of equine early spring transitional follicles with luteinizing hormone stimulates follicle growth but does not restore steroidogenic activity

This study was conducted to test the hypothesis that supplementation of growing follicles with LH during the early spring transitional period would promote the development of steroidogenically active, dominant follicles with the ability to respond to an ovulatory dose of hCG. Mares during early transition were randomly assigned to receive a subovulatory dose of equine LH (in the form of a purified equine pituitary fraction) or saline (transitional control; n = 7 mares per group) following ablation of all follicles >15 mm. Treatments were administered intravenously every 12 h from the day the largest follicle of the post-ablation wave reached 20 mm until a follicle reached >32 mm, when an ovulatory dose of hCG (3000 IU) was given. Saline-treated mares during June and July were used as ovulatory controls. In a preliminary study, injection of this pituitary fraction (eLH) to anestrus mares was followed by an increase in circulating levels of LH (P < 0.01) but not FSH (P > 0.6). Administration of eLH during early transition stimulated the growth of the dominant follicle (Group x Day, P < 0.00001), which attained diameters similar to the dominant follicle in ovulatory controls (P > 0.1). In contrast, eLH had no effect on the diameter of the largest subordinate follicle or the number of follicles >10 mm during treatment (P > 0.3). The numbers of mares that ovulated in response to hCG in transitional control, transitional eLH and ovulatory control groups (2 of 2, 3 of 5 and 7 of 7, respectively) were not significantly different (P > 0.1). However, after hCG-induced ovulation, all transitional mares returned to an anovulatory state. Circulating estradiol levels increased during the experimental period in ovulatory controls but not in transitional eLH or transitional control groups (Group x Day, P = 0.013). In addition, although progesterone levels increased after ovulation in transitional control and transitional eLH groups, levels in these two groups were lower than in the ovulatory control group after ovulation (Group, P = 0.045). In conclusion, although LH supplementation of early transitional waves beginning after the largest follicle reached 20 mm promoted growth of ovulatory-size follicles, these follicles were developmentally deficient as indicated by their reduced steroidogenic activity.

Changes in follicular blood flow and nitric oxide levels in follicular fluid during follicular deviation in cows

Two experiments were conducted to test the hypothesis that there are dynamic changes in follicular blood flow during follicular deviation and that nitric oxide (NO) in follicular fluid (FF) plays a role in regulation of follicular blood flow. In Experiment I, follicular blood flow of the two largest follicles was monitored by using Power Doppler ultrasonography during follicular deviation in sixteen follicular waves during eight estrous cycles in eight cows. Blood flow did not differ (P>0.05) between the dominant follicle (DF) and the largest subordinate follicle (SF) until the beginning of the deviation of the follicular size, but was higher (P<0.05) in DF than in the largest SF one and two days after the beginning of diameter deviation in ovulatory (n=5) and atretic (n=11) waves; respectively. In Experiment II, FF was aspirated from DF and the largest SF on the day of diameter deviation (DF, n=6; SF, n=6) and two days later (DF, n=12; SF, n=9). Nitric oxide did not differ (P>0.05) between DF and the largest SF on the day of diameter deviation but, one or two days after observed diameter deviation NO concentrations were lower (P<0.01) in DF compared to the largest SF. On the day of diameter deviation and two days later E2 levels in FF were higher (P<0.01) in DF than in the largest SF. P4 concentrations in FF were higher (P<0.05) in DF than in the largest SF on the day of diameter deviation, but did not (P>0.05) differ two days later. E2/P4 ratio in FF was the same (P>0.05) in DF and the largest SF on the day of diameter deviation, but was higher (P<0.01) in DF than in the largest SF one or two days later. In conclusion, area of follicular blood flow of DF and the largest SF increased in parallel with follicular size during follicular deviation. Furthermore, there were relationships between changes in follicular blood flow, NO concentrations and E2/P4 ratio in FF following the beginning of diameter deviation in cattle.

173 THE EFFECT OF EXERCISE ON FOLLICULOGENESIS IN MARES

The effects of exercise on follicular development in mares have not been well established; however, a combination of exercise and heat stress can negatively affect embryo recovery rate in mares. The purpose of this research was to examine the effects of exercise, independent of heat stress, on follicular development in mares. One thoroughbred and 12 quarter horse mares, aged 3 to 15 years, were used in this study. Mares were assigned to the control group or exercised at 0700 h, 6 days a week, as follows: warmed up 5 min at a walk for 3 m s–1 and then exercised 30 min, 9 m s–1 for 10 min, 13 m s–1 for 5 min, 9 m s–1 for 5 min, 13 m s–1 for 5 min, and 9 m s–1 for 5 min. Exercised mares were given a 1-month conditioning period. Rectal temperatures were recorded immediately before and after exercise. The mean rectal temperature of mares increased by 0.85°C during exercise, whereas control mares under the same environmental conditions did not experience temperature increases. Individual mares underwent a daily transrectal ultrasound examination with a 7.5-MHz linear rectal probe to monitor follicular development. Estrous cycles were normalized to 17 days for linear follicular growth analysis. Exercise had a significant impact on follicular growth at deviation, with a greater diameter of the largest (P &lt; 0.05) and second largest (P &lt; 0.01) follicles in exercised v. control mares (Table 1). Additionally, growth rate of the second largest follicle was greater in the exercised group (P &lt; 0.01). Deviation was observed in 13 of 14 cycles in control mares, whereas in exercised mares, it was clear in only 11 of 16 cycles (χ2, P = 0.086). Time of deviation was defined as the day when a difference in the growth rate between the dominant and largest subordinate follicle of a wave was first observed. The largest subordinate follicle either declined in growth rate or regressed. At Day –5 (relative to ovulation), exercised mares had significantly greater follicle diameters in both the largest and second largest follicle (P &lt; 0.05). Exercised mares had double ovulations in 4 of 16 cycles when compared with control mares, which had double ovulations in 1 of 14 cycles. No differences were found between groups in the length of the estrous cycle or ovulatory follicular diameter. Exercised mares exhibited larger diameter follicles and an increased growth rate of the largest subordinate follicle, suggesting that exercise delayed deviation. Further research is needed to assess the impact of exercise, independent of heat stress, on fertility. Table 1.Follicle dynamics in exercised v. non-exercised mares

Follicle deviation and ovulatory capacity in Bos indicus heifers

The objectives of Experiment 1 were to determine the interval from ovulation to deviation, and diameter of the dominant follicle (DF) and largest subordinate follicle (SF) at deviation in Nelore (Bos indicus) heifers by two methods (observed and calculated). Heifers (n = 12) were examined ultrasonographically every 12 h from ovulation (Day 0) to Day 5. The time of deviation and diameter of the DF and largest SF at deviation did not differ (P>0.05) between observed and calculated methods. Overall, deviation occurred 2.5+/-0.2 d (mean +/- S.E.M.) after ovulation, and diameters for DF and largest SF at deviation were 6.2+/-0.2 and 5.9 +/- 0.2 mm, respectively. Experiment 2 was designed to determine the size at which the DF acquires ovulatory capacity in B. indicus heifers. Twenty-nine heifers were monitored every 24 h by ultrasonography, from ovulation until the DF reached diameters of 7.0-8.4 mm (n=9), 8.5-10.0 mm (n=10), or >10.0 mm (n=10). At that time, heifers were treated with 25 mg of pLH and monitored by ultrasonography every 12 h for 48 h. Ovulation occurred in 3 of 9, 8 of 10, and 9 of 10 heifers, respectively (P<0.05). In summary, there was no significant difference between observed and calculated methods of determining the beginning of follicle deviation. Deviation occurred 2.5 d after ovulation when the DF reached 6.2 mm, and ovulatory capacity was acquired by DF as small as 7.0 mm.

Differentiation of the bovine dominant follicle from the cohort upregulates mRNA expression for new tissue development genes

This study was designed to identify genes that regulate the transition from FSH- to LH-dependent development in the bovine dominant follicle (DF). Serial analysis of gene expression (SAGE) was used to compare the transcriptome of granulosa cells isolated from the most oestrogenic growing cohort follicle (COH), the newly selected DF and its largest subordinate follicle (SF) which is destined for atresia. Follicle diameter, follicular fluid oestradiol (E) and E:progesterone ratio confirmed follicle identity. Results show that there are 93 transcript species differentially expressed in DF granulosa cells, but only 8 of these encode proteins known to be involved in DF development. Most characterised transcripts upregulated in the DF are from tissue development genes that regulate cell differentiation, proliferation, apoptosis, signalling and tissue remodelling. Semiquantitative real-time PCR analysis confirmed seven genes with upregulated (P< or =0.05) mRNA expression in DF compared with both COH and SF granulosa cells. Thus, the new genes identified by SAGE and real-time PCR, which show enhanced mRNA expression in the DF, may regulate proliferation (cyclin D2; CCND2), prevention of apoptosis or DNA damage (growth arrest and DNA damage-inducible, beta; GADD45B), RNA synthesis (splicing factor, arginine/serine rich 9; SFRS9) and unknown processes associated with enhanced steroidogenesis (ovary-specific acidic protein; DQ004742) in granulosa cells of DF at the onset of LH-dependent development. Further studies are required to show whether the expression of identified genes is dysregulated when abnormalities occur during DF selection or subsequent development.

Differential expression of genes for transcription factors in theca and granulosa cells following selection of a dominant follicle in cattle

Transcription factors inhibit or assist RNA polymerases in the initiation and maintenance of transcription; however, the cell specific expression and roles of transcription factors within bovine ovarian follicles during development are unknown. The aim of present study was to determine if the expression of transcription factors in theca and granulosa cells differ between the dominant and the largest subordinate follicles at different stages of the follicle wave. We used a bovine cDNA microarray to screen granulosa and theca cells from dominant and subordinate follicles for differential expression of genes coding for transcription factors. Expression was confirmed using reverse transcription polymerase chain reaction and differences in mRNA abundance further examined at Emergence, Selection and Dominance stages of the follicle wave. We have identified five genes encoding for transcription factors that have not been previously described in developing follicles with greater mRNA abundance in subordinate compared to dominant follicles. The genes (and their putative roles) are CEBP-beta (responsible for luteinization), SRF (cell survival), FKHRL1 (stimulates apoptosis), NCOR1 (modulation of the actions of the oestradiol receptor) and Midnolin (control of development via regulation of mRNA transport in cells).

Ovarian follicular and luteal dynamics in wapiti during the estrous cycle

The reproductive tracts of 13 mature hinds were examined daily by transrectal ultrasonography and blood samples were taken daily from October to January to characterize follicular, luteal, and endocrine dynamics in wapiti during the estrous season. Follicle development occurred in waves characterized by regular, synchronous development of a group of follicles in temporal succession to a surge in serum FSH concentration. The mean interovulatory interval was 21.3 ± 0.1 d, but was shorter in hinds exhibiting two follicular waves than in hinds exhibiting three and four waves ( P < 0.05). The interwave interval was similar among waves in two-wave cycles and the first wave of three-wave cycles. All other interwave intervals in three- and four-wave cycles were shorter ( P < 0.05). The maximum diameter of the dominant follicle of the first wave was similar among two-, three-, and four-wave cycles. For all other waves in three- and four-wave cycles, the maximum diameter was smaller ( P < 0.05). Corpus luteum diameter and plasma progesterone concentrations were similar between two- and three-wave cycles, but the luteal phase was longer ( P < 0.05) in four-wave cycles. The dominant follicle emerged at a diameter of 4 mm at 0.4 ± 0.1 and 0.8 ± 0.1 d before the largest and second largest subordinate follicles, respectively. The follicle destined to become dominant was larger ( P < 0.05) than the largest subordinate follicle one day after emergence, which coincided with the first significant decrease in serum FSH concentration. We concluded that the estrous cycle in wapiti is characterized by two, three, or four waves of follicular development (each preceded by a surge in circulating FSH), that there is a positive relationship between the number of waves and the duration of the cycle, and an inverse relationship between the number of waves and the magnitude of follicular dominance (diameter and duration of the dominant follicle).

Identification of Genes Involved in Apoptosis and Dominant Follicle Development During Follicular Waves in Cattle1

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.

223DEVELOPMENTAL PATTERN OF SMALL (1-3mm) ANTRAL FOLLICLES IN THE BOVINE OVARY

Knowledge about the developmental pattern of small follicles (&amp;lt;4mm) will be crucial to understanding the endogenous control of folliculogenesis and to developing methods to control it for clinical purposes. This study was designed to characterize the developmental pattern of 1–3mm follicles and to determine, retrospectively, the stage at which the future dominant follicle first attains a size advantage among follicles in the cohort. In Experiment 1, the ovaries of Hereford-cross heifers (n=18) were examined daily by high resolution transrectal ultrasonography for one natural interovulatory interval to evaluate changes in the number of 1–3-mm follicles in relation to the wave pattern of follicles ≥4mm. Interovulatory interval was divided into 2- and 3-wave based on number of waves exhibited by heifers. In Experiment 2, the ovaries of Hereford-cross cows (n=9) were examined every 6h from Day 5 to Day 13 (Day 0=ovulation) to monitor precisely the diameter changes of individual follicles ≥1mm during emergence of the second follicular wave. Data were analyzed by Proc. Mixed procedure for repeated measures (Littell RC et al., 2000 Stat in med 19, 1793–1819) in the Statistical Analysis System software package (SAS version 8.2 for MS Windows;; 2002 SAS Institute Inc. Cary, NC, USA). Results of Experiment 1 revealed a day effect (P&amp;lt;0.05) on the number of small (1–3mm) follicles, with a peak (P&amp;lt;0.05) 1 or 2 days before wave emergence (defined as the day the dominant follicle was first detected at 4mm), followed 3 to 4 days later by a peak (P&amp;lt;0.05) in the number of large follicles (≥4mm). There was an inverse relationship between the number of small and large follicles during Wave 1 (r=−0.66; P=0.05) and Wave 2 (r=−0.62; P=0.04) in 2-wave interovulatory intervals. Similarly, an inverse relation was detected between the number of small and large follicles for Wave 1 (r=−0.79; P=0.01) and Wave 3 (r=−0.90; P&amp;lt;0.01) but not for Wave 2 (r=−0.57; P=0.14) in 3-wave interovulatory intervals. The number of 1–3-mm follicles detected in anovulatory waves did not differ (P=0.53) between 2- versus 3-wave interovulatory intervals;; however, a difference (P&amp;lt;0.05) was observed between anovulatory and ovulatory waves in 3-wave interovulatory intervals but not (P=0.63) in 2-wave interovulatory intervals. Experiment 2 permitted the identification of the future dominant follicle at a diameter of 1mm and its emergence at 6–12h earlier than the largest subordinate follicle (P&amp;lt;0.01). Emergence of the future dominant (r=0.71; P=0.05) and 1st subordinate (r=0.78; P=0.02) follicles was temporally associated with a wave-eliciting rise in circulating concentrations of FSH. Growth rate of the dominant and the 1st subordinate follicle differed (P&amp;lt;0.01) from 2nd subordinate follicle at 84h after their detection at 1mm. It was concluded that small antral follicles (1–3mm) develop in a wave-like manner, and apparent selection of the dominant follicle was manifest much earlier than previously documented.

Dynamics and histological observation of first follicular wave in Japanese black cows.

To determine the turnover of the first follicular wave in Japanese black cows and quantitative immunohistological characteristics of the previously in vivo identified dominant follicle (DF) and largest subordinate follicle (SF) derived from ovariectomy on Day 7 (3 cows) and Day 10 (3 cows) (Day 0=estrus). Six cases of first follicular wave in cows were observed twice daily by ultrasound scanning. The number of follicles, diameter of DF and SF, and prevalence of apoptotic granulosa cells (GC) and theca cells (TC) were studied by TUNEL methods. At follicular wave emergence, 13.5 +/- 9.5 Class I (2-5 mm in diameter follicles) were found 12 hr after ovulation, and increased its number until Day 1 pm. Future DF and SF observed retrospectively were 4.9 +/- 0.8 mm and 4.9 +/- 0.9 mm at wave emergence. Deviation of DF and SF occurred on Day 3 pm with mean diameters of 8.9 +/- 1.3 mm and 6.8 +/- 0.9 mm, respectively. DF developed until Day 8 am with a maximum diameter of 14.4 +/- 1.8 mm (n=3) and then regressed. The follicular wall of the DF had a characteristic image of a healthy follicle on Day 7 and slightly atretic DF on Day 10, whereas SF showed heavy atresia on both Day 7 and Day 10 under HE staining. In the prevalence of apoptotic cells, DF were 4.4 +/- 1.0% and 17.9 +/- 4.9% on Day 7 and on Day 10 in GC, respectively, and 2.4 +/- 0.7% and 8.0 +/- 1.4% on Day 7 and on Day 10 in TC, respectively. These results showed that, 1) the first follicular wave in cows is characterized by 24 hr recruitment of small follicles and a gradual divergence of growth rates in future DF and SF, and 2) early regression of DF on Day 10 was preceded by severe apoptosis.

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 ≥4 mm 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 ≥3 mm 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.