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Ovary |
Eutheria Foundation, Cross Plains, Wisconsin 53528
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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female reproductive tract, follicle, follicular development, ovary
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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During follicle growth, an extensive vascular plexus develops in the thecal layer surrounding the avascular basement membrane and granulosa layer [6]. It has been suggested that the preferential delivery of gonadotropins and nutrients via a more highly developed vascular system in individual follicles plays a role in the selection and growth of the dominant follicle [7–10] and that insufficient vascular support contributes to follicle atresia [11, 12]. However, differences among follicles in the development of the vascular network have not been implicated directly in the mechanism of follicle deviation or selection in any species, and the position of vascular changes in the cascade of events leading to diameter deviation is unknown. In this regard, however, deviation in mares is indicated morphologically not only by differential growth rate between the developing dominant and subordinate follicles, but also by an apparent expansion of the anechoic ultrasonic layer, as expressed subjectively, surrounding the granulosa of the dominant follicle [13]. This echotexture change distinguished the future dominant follicle from the future largest subordinate follicle about 1 day earlier than the beginning of diameter deviation and was attributed to increased vascularization.
Vascular endothelial growth factor (VEGF) is involved in the increase in blood vessel extension in the wall of porcine follicles [14]. Recent in vivo studies in mares showed that the follicular-fluid concentrations of VEGF increased in the future subordinate follicle after intrafollicular injection of IGF-I [4]. Furthermore, the VEGF concentrations in a follicle that was expected to become dominant decreased when concentrations of IGF-I were experimentally lowered [3]. The temporal relationships among follicular-fluid factors preceding and during follicle deviation have been studied in mares [15], but VEGF was not included. Concentrations of VEGF in equine follicular fluid were higher in the largest follicle than in the second-largest follicle 1 day after the expected beginning of deviation [4], but earlier temporal VEGF relationships are not known.
Color Doppler ultrasonography has been used for hemodynamic studies in real time [16–19]. This noninvasive tool has been used in humans [16] and cows [17] to evaluate blood flow in the wall of preovulatory follicles. In humans, blood flow determinations of individual preovulatory follicles by Doppler ultrasound provide an index on the intrafollicular environment and may be used to predict the developmental competence of the oocyte [20]. In cows, transrectal color Doppler ultrasonography demonstrated a clear difference in the vascularity of the wall of preovulatory follicles compared with anovulatory follicles [17]. Results of the above reports suggest that the color Doppler approach has potential for investigation of follicle vasculature preceding and during diameter deviation. The follicles of mares are much larger than the follicles of other farm species and women. For example, the preovulatory follicle is about 40 mm [21], 16 mm [22], and 20 mm [23] in mares, heifers, and women, respectively. Mean diameter of the largest follicle at the beginning of deviation is about 22.5 mm in mares and about 8.5 mm in cattle [1]. These diameter comparisons suggest that the larger follicles in mares have potential for study of blood flow characteristic at earlier stages than in other species. Study of the changing follicle vasculature by color Doppler ultrasonography may lead to clarification of the position of vascular changes in the sequence of events associated with the selection of the dominant follicle.
The present study tested the hypothesis that reduced blood flow in the future largest subordinate follicle versus the future dominant follicle precedes the detection of diameter deviation in mares. The hypothesis was tested by quantifying the daily changes in blood flow velocities and blood flow area within the wall of the two follicles before and during diameter deviation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Animals were handled in accordance with the Guide for Care and Use of Agricultural Animals in Agricultural Research. A total of 10 nonlactating pony mares of mixed breeds, 9–17 yr of age and weighing 290– 430 kg, were used during the ovulatory season from July to August in the Northern Hemisphere. The feeding program and the equipment and techniques for transrectal and transvaginal ultrasound scanning and manipulation of equine ovaries have been described previously [24, 25]. Before the experiment, follicles >25 mm were monitored daily by ultrasound to determine the day of ovulation.
A new follicular wave was induced by ablation of all follicles 
6 mm 10 days postovulation by ultrasound-guided transvaginal aspiration of follicle contents as described previously [24, 25]. This was done to initiate the development of a new follicular wave so that day-to-day follicle identity was not obscured by follicles from a previous wave. Aspirated follicles that refilled with fluid to 10 mm were reaspirated. When the largest follicle of the new follicular wave reached a diameter of 15 mm, all but the three largest follicles were ablated to simplify follicle identification. No difference was found in deviation or follicle growth between controls and mares in which these ablations were used [24]. Transrectal ultrasound scanning to determine follicle identity and diameters was done [25] each day until ovulation. Follicle diameter was determined by taking the average of width and length from a frozen image.
The prominence of the anechoic layer was evaluated from gray-scale B-mode images recorded separately from the color Doppler images. The scoring system was one that was previously used in follicles approaching ovulation [26] and encompassing diameter deviation [13]. The anechoic layer was identified as a gray to black band, often irregular in thickness, surrounding the granulosa. Prominence was scored arbitrarily by visual inspection from 0 (not detected) to 3 (maximal), using an average of values obtained by two operators. Slight prominence (score 1) was indicated by a thin, gray line, whereas maximal prominence was indicated by a thicker and darker line. The follicles were scored from a videotape. The operators knew mare and follicle identities but did not know the day of the beginning of diameter deviation and the score from the previous day.
Doppler Ultrasonography
The follicles were examined by transrectal pulsed-wave Doppler ultrasonography, using an ultrasound scanner (Aloka SSD-2000; Aloka America, Wallingford, CT) equipped with a finger-mounted 7.5-MHz convex transducer (UST-995-7.5). The degree of turbulence in the blood vessels is indicated by a coded-color signal; the brightness of the color is directly proportional to the velocity of flow within the blood vessels [16–19, 27]. Areas of color represent regions with a flow velocity higher than 10 mm/ sec. All scans were performed at a pulse-repetition frequency of 6 Hz. Identical color gain settings were used for all scannings. The blood flow characteristics were evaluated at the maximum diameter of the follicle. The distance between the transducer face and follicle was minimized to reduce signal attenuation. The angle of insonation was not calculated because of the small diameter of vessels, but care was taken to obtain the maximum color intensity.
Only the spectral and color modes were used in this experiment [16– 19, 27]. The spectral data were generated from a focused area as directed by the operator through placement of a 1.0-mm-wide gate over the most intensely colored area. The data were displayed in a wave form for each of three cardiac cycles. The spectral analyses determined peak systolic velocity and time-averaged maximum velocity. The latter end point represents the average maximum velocity displayed in spectral form over a time span of three cardiac cycles. Doppler spectral measurements were performed daily during each ultrasound examination. The color mode was used to determine the blood flow area in the follicle wall. Real-time B-mode/color Doppler images were stored using a digital video camera (Handycam Camcorder; Sony Electronics Inc., San Diego, CA) for blood flow area analyses in the laboratory. The tapes were viewed on the monitor of a computer. Using iMovie software (Apple, Cupertino, CA), the images were captured and saved as joint photographic experts groups (JPEG) format graphic files for further analysis. The blood flow area in the follicle wall was measured as previously described for the bovine preovulatory follicle [17]. Briefly, the JPEG graphic files were analyzed for the colored areas in the follicle wall regardless of the intensity. If a portion of the wall was not clearly delineated, the wall thickness was estimated by reference to the thickness evident in other portions of the wall. If part of a colored area extended beyond the wall into the ovary, only the part involving the wall was considered. The colored areas within the wall were changed to a single black shade using Adobe Photoshop 5.5 software (Adobe Systems, San Jose, CA). Quantification of the shaded areas was done using an NIH Image program (Version 1.62; http://rsb.info.nih.gov/nih-image/Default.html). Each black representation of the colored areas was traced, and the sum of all areas was used to determine the blood flow area within the wall displayed by the section through the maximum follicle diameter.
Follicle Designations and Groups
The follicles were identified by rank as F1, F2, and F3 (largest to smallest) according to the maximum attained diameter. Follicle identities were maintained retrospectively for data analyses. Thus, the operator did not know which follicle would later be designated F1, F2, or F3. Although only F1 and F2 were used to test the hypothesis, F3 was included to serve as the subordinate follicle in mares that developed a dominant follicle (30 mm [25]) from both F1 and F2. As a consequence, seven mares were available for the deviation study; the three mares with double dominant follicles were considered separately. The day-to-day temporal relationships among F1, F2, and F3 for diameter, echotexture, and Doppler end points were studied by placing F1 into nine diameter-range groups as follows: 5.0–7.9 mm, 8.0–10.9 mm, 11.0–13.9 mm, 14.0–16.9 mm, 17.0– 19.9 mm, 20.0–22.9 mm, 23.0–25.9 mm, 26.0–28.9, and 29.0–31.9 mm. In this portion of the study, F1, F2, and F3 were from the same day and mare within each of the nine F1-diameter ranges. For ease of communication, the nine ranges were designated as 6.5-, 9.5-, 12.5-, 15.5-, 18.5-, 21.5-, 24.5-, 27.5-, and 30.5-mm-diameter groups, respectively. Increments of 3.0 mm were used in the group assignments to represent the approximate growth of F1 during 1 day [25]. When F1 did not fall into one of the sequential range groups on a given day, the observations for the three follicles were treated as missing data, and this was done in 7% of the assignments. When F1 diameter for a given day and mare fell into a diameter group on each of 2 consecutive days, the observation closest to midrange was used, and this was done in 9% of the assignments. Doppler velocity responses were not detected in more than one follicle per diameter group in the 6.5- and 9.5-mm groups for F1 and F2 and in the 6.5- to 12.5-mm groups for F3. Therefore, the statistical analyses for F1 versus F2 excluded the 6.5- and 9.5-mm groups. For illustrative purposes, however, values of zero are included in the means shown in the figures. In summary, nine and seven diameter groups were used in the statistical comparisons of F1 and F2 for diameter and Doppler end points, respectively. An increase in the prominence of the anechoic layer did not begin in F2 until the 21.5-mm group. Therefore, only the 21.5-, 24.5-, and 27.5-mm-diameter groups were statistically analyzed for this end point.
In a second portion of the study, F1, F2, and F3 were each grouped independently of one another into the same diameter ranges that were used for F1 in the first portion of the study. Thus, in the first portion of the study, F1, F2, and F3 were compared on the same day (associated relationships), whereas in this portion, F1, at a given diameter range, was compared with F2 and F3 at a similar diameter range (independent relationships). This was done to determine if Doppler changes reflected diameter of the follicle regardless of its ranking as F1, F2, or F3. Only the 6.5-, 9.5-, 12.5-, and 15.5-mm groups were used for each follicle to minimize including an F2 or F3 that had begun to show the negative effects of the deviation mechanism.
Statistical Analyses
The diameter, echotexture, and Doppler data were examined for normality with the Kolmogorov-Smirnov test. When the normality test was significant (P < 0.05), data were transformed by either natural logarithm or square root. Follicle end points were analyzed to determine the main effects of diameter-range group, follicle (F1 versus F2), and the interaction of these two main effects using an SAS mixed procedure with a repeated statement to account for the autocorrelation between sequential measurements (8.2 version; SAS Institute Inc., Cary, NC). The diameter group at the beginning of deviation was determined for diameter, score for prominence of the anechoic layer, and each Doppler end point in which a significant group-by-follicle interaction was obtained. Diameter-range group at the beginning of deviation for each end point with a significant interaction was defined by the first diameter group in which the difference between F1 and F2 was less than the difference between F1 and F2 for the next diameter group using paired t-tests as described [15, 28, 29]. Because of the importance of a significant interaction, only the probability for the interaction is stated for each end point; however, if the interaction was not significant, the probabilities for the main effects (follicle rank, diameter group) are stated. A probability of P 
0.05 indicated that a difference was significant. All data are presented as the mean ± SEM.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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0.05) than the score in the 27.5-mm group (1.0 ± 0.2).
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The interaction for follicle (F1, F2) and diameter groups was significant (P < 0.0001) for peak systolic velocity (P < 0.05) and time-averaged maximum velocity (P < 0.04; Fig. 1). Peak systolic velocity and time-averaged velocity began to deviate between F1 and F2 in the 18.5-mm-diameter group, as indicated by a smaller (P < 0.04 and P < 0.004, respectively) difference between the two follicles in the 18.5-mm-diameter group than in the 21.5-mm-diameter group. The interaction was significant (P < 0.003) for blood flow area; deviation began in the 21.5-mm group (P < 0.009), based on the criteria used for the other end points.
When F1, F2, and F3 each were assigned independently to the 6.5-, 9.5-, 12.5-, and 15.5-mm-diameter groups, the main effect of group was significant (P < 0.0001) for each of the Doppler end points, but the effect of follicle and the follicle-by-group interaction were not (not shown). Velocity values were obtained in 0% of follicles that were 5.0–7.9 mm independent of rank and in 14% that were 8.0–10.9 mm. In contrast, a flow-area value was obtained in 30% of follicles that were 5.0–7.9 mm and in all follicles that were 8.0–10.9 mm.
There were no significant main effects of follicle (F1, F2) or diameter group or an interaction for any of the end points for F1 and F2 in the three mares with codominant follicles. When F3 was included in the analyses, there was an interaction of follicle (F1, F2, F3) and diameter group for diameter (P < 0.005), peak systolic velocity (P < 0.001), and time-averaged maximum velocity (P < 0.03; Fig. 2).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The F1-diameter group for the beginning of differential changes in F1 versus F2 in the prominence of the anechoic layer surrounding the granulosa was the same as for diameter deviation. The present results did not confirm a previous report that differential changes in the follicular wall echotexture began a day earlier than diameter deviation. However, the previous study used a different experimental approach, including normalization to observed diameter deviation in individual mares and combining the effects of several echotexture end points.
Differential blood flow velocities between F1 and F2 (velocity deviation) began an average of 6 mm (equivalent to 2 days) earlier than diameter deviation. The beginning of velocity deviation was characterized by a continued increase in velocity in F1 and the beginning of a decrease in F2. Reduced blood velocity or vascularity in the developing subordinate follicle is consistent with reports that reduced proliferation of endothelial cells in follicle capillaries and reduced theca vascularity were early events in follicle atresia in cattle [6] and sheep [33]. Mean deviation in flow area began an equivalent of 1 day after velocity deviation and 1 day before diameter deviation. These results provide the first direct indication in any species that differential blood flow changes between future dominant and subordinate follicles precede diameter deviation during follicle selection. The future dominant follicle, therefore, is well positioned by a continuation of the increasing blood supply, as well as by increased gonadotropin responsiveness [2], to continue growing despite the lower concentrations of FSH, whereas the future subordinate follicles are positioned to regress. It is known that a dominant follicle has a greater thecal vascularity and blood supply than subordinate follicles [7, 34]; however, the present results demonstrated that the decreased vascularity in the future subordinate follicle began well before the growth rate of the follicle began to diminish.
The earlier decrease for the two velocity end points than for the decrease in flow area in the future subordinate follicle (F2) is attributable to the relationships between blood velocity and area in the contracting vascular bed of a follicle that is destined to regress. The spectral analyses were made from a small area focused on the most intense color and represented by the narrow (1.0 mm) sample gate. Color for flow area determinations involved all colored area regardless of intensity. Therefore, the velocity measurements represented larger arterial vessels than for flow area, which would include vessels nearer to the capillary bed. The capillaries likely were not included in the evaluations; the scanner was set to detect blood flow velocities higher than l0 mm/sec. Reported flow rates are 0.5 mm/sec for capillaries and 5.0 mm/sec for arterioles in a mouse's ear [35]. The relationships between Doppler-detected velocities versus blood flow area also accounts for the detection of flow area in smaller follicles than for detection of velocities.
Velocity measurements that were made in 11.0- to 13.9-mm follicles were well before the beginning of diameter deviation. In this regard, a recent report [36] indicated pulsed Doppler spectrum signals were not detected in humans in follicles <12 mm or apparently well after deviation begins [1]. These species comparisons indicate that the mare model would be especially useful for study of the association between vascular changes and follicle functions, such as those associated with diameter deviation.
The detection of a diameter-range group effect but not a follicle effect (F1 versus F2) or an interaction of group and follicle for velocity or flow area when follicles were assigned to a diameter group independently of rank (F1 or F2) indicated that follicle vascularity initially increased similarly between follicles according to diameter regardless of position in the diameter hierarchy. Blood flow velocity was detected when follicles were 11.0–13.9 mm regardless of their rank. Apparently, the increasing vascularity continued independently in the two follicles until F1 and F2 were averages of 18.0 and 16.1 mm, respectively. At that time, the blood flow of F2 was negatively influenced by the vascular aspects of the deviation mechanism, beginning well before the diameter aspects of deviation.
The three mares with double dominant follicles ovulated from each follicle. There were no significant differences between F1 and F2 for diameter or any color Doppler end point. The day of deviation between F3 and the two future dominant follicles (F1 and F2) was not analyzed because of the small number of mares. However, based on inspection of the data profiles (Fig. 2), the deviations between F1 and F3 in diameter and peak systolic velocity appeared to occur on a day similar to the day of deviations for the end points between F1 and F2 in the mares with a single dominant follicle.
The results of this study implicate the end points for vascular deviation as events that preceded diameter deviation, but the temporal position of blood flow change in the series of events leading to diameter deviation is not known. Follicular-fluid concentrations of VEGF, an angiogenic factor, increase in the follicles of cattle [10] and pigs [37] as follicle diameter increases and is higher in the dominant follicle than in the subordinate follicle of mares 1 day after the expected beginning of diameter deviation [4]. Thus, follicle-produced VEGF is a candidate for a role in vascular-follicle relationships during diameter deviation. Furthermore, VEGF production increases in cultures of granulosa cells of cattle [38] and monkeys [39] when exposed to IGF-I. Most relevant to the present results, VEGF increased in the follicular-fluid of F2 in mares within 1 day after an intrafollicular injection of IGF-I at the expected beginning of deviation [4]. In addition, VEGF decreased in F1 when treated with an IGF-I-binding protein [3]. The present study demonstrated a continued increase in vascularity of the future dominant follicle but not the future subordinate follicles beginning well before diameter deviation. More study is required on the apparent temporal and functional interrelationships among changes in the IGF-I system, VEGF, and other follicular-fluid factors and the follicle vascular plexus that culminate in diameter deviation or selection of the dominant follicle.
In summary, peak systolic velocity and time-averaged maximum velocity began to decrease in the future largest subordinate follicle (F2) and continued to increase in the future dominant follicle (F1) beginning an equivalent of 2 days before the beginning of diameter deviation. Blood flow area began to change differentially between F1 and F2 1 day after the velocity changes and 1 day before the beginning of diameter deviation. The results supported the hypothesis that reduced blood flow in the future subordinate follicles precedes the detection of diameter deviation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: O.J. Ginther, Department of Animal Health and Biomedical Sciences, 1656 Linden Dr., University of Wisconsin-Madison, Madison, WI 53706. FAX: 608 262 7420; ginther{at}svm.vetmed.wisc.edu
3 Current address: Laboratory of Reproductive Endocrinology, Faculty of Agriculture, Okayama University, Tsushima Naka, Okayama 700-8530, Japan
4 E.L.G. and M.O.G. are on leave from the Departments of Veterinary and Animal Science, respectively, Federal University of Viçosa, Viçosa, Brazil
Received: 28 January 2004.
First decision: 23 February 2004.
Accepted: 25 March 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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injection in the cow. Biol Reprod 2002 66:651-658This article has been cited by other articles:
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  M A Beg and O J Ginther Follicle selection in cattle and horses: role of intrafollicular factors. Reproduction, September 1, 2006; 132(3): 365 - 377. [Abstract] [Full Text] [PDF]
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 A Martelli, P Berardinelli, V Russo, A Mauro, N Bernabo, L Gioia, M Mattioli, and B Barboni Spatio-temporal analysis of vascular endothelial growth factor expression and blood vessel remodelling in pig ovarian follicles during the periovulatory period J. Mol. Endocrinol., February 1, 2006; 36(1): 107 - 119. [Abstract] [Full Text] [PDF]
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