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Ovary |
a Department of Veterinary Science, University of Melbourne, Werribee, Victoria 3030, Australia
b Prince Henry's Institute of Medical Research, Clayton, Victoria 3168, Australia
c Oxford Brookes University, Oxford OX3 0P3, United Kingdom
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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follicle-stimulating hormone, follicular development, inhibin, ovary, ovulatory cycle
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Inhibin consists of two disulfide-linked chains (
and either the ßA or the ßB subunit) to form inhibin A or inhibin B, which is secreted as variously processed forms, with the 30-kDa dimer the most mature (and bioactive) form [12, 13]. In addition to the ß dimer, the inhibin subunit is produced, often in apparent excess [14, 15], as is the ßß dimer (activins A and B). The inhibins are primarily produced by ovarian granulosa cells, although their production is differentially regulated. In humans [9], inhibin B is thought to be produced primarily by the gonadotropin-sensitive developing antral follicle, whereas inhibin A is thought to be produced primarily by the dominant follicle. In cattle, the predominant inhibin form found in follicular fluid is inhibin A, and inhibin B levels, as measured by ELISA, are much lower [16]. Inhibin B is not detectable in bovine serum using current ELISAs [17]. However, small antral follicles that express the ßB subunit mRNA [18] may also produce significant amounts of inhibin B, which has not yet been identified in serum or plasma.
The understanding of the physiology of inhibin has been advanced by the development of serum assays. Earlier assays detected every form of inhibin containing the subunit (free subunit and inhibin dimers) [16, 19, 20]. Dimer-specific inhibin A and inhibin B assays have been developed more recently [21–23]. The need for specific dimeric inhibin assays became apparent when researchers discovered that the serum levels of free subunit were often elevated compared with dimer levels and that it was important to differentiate between the forms of inhibins A and B. Specific ELISAs for inhibin A and inhibin B assays were initially developed for humans [21, 22] and have been applied to other species with variable success, which has been attributed to the limited cross-reactivity of the assays across species. These ELISAs are sandwich assays employing two monoclonal antibodies directed to either the or ß (A or B) subunits. Knight and colleagues applied these ELISAs to ovine [10] and bovine [11] serum but utilized different subunit antibodies in the ELISA to increase the sensitivity of the ELISA to circulating inhibin A. A specific time-resolved immunofluorometric assay has also been developed for measuring inhibin A in bovine plasma [23].
Bleach and colleagues [11] applied the modified inhibin A ELISA to serum from cows throughout an estrous cycle and showed that serum inhibin A increased with follicular development (as seen in other species), decreasing to low levels following ovulation and at the initiation of luteinization. Inhibin A and FSH were negatively correlated during the follicular phase [11]. However, multiple cycles of folliculogenesis occur between ovulations in the cow, with two and three follicular wave cycles being the most common [6, 24]. The hormonal basis for these multiple cycles has been closely studied to determine the differences in the interplay between gonadal factors and both pituitary FSH and LH [6, 25].
In the present study, we utilized the inhibin A ELISA of Bleach et al. [11] to investigate the interrelationship between serum FSH and inhibin A during the estrous cycle of cows with two or three waves of ovarian follicle development. We hypothesized that the relationship between FSH as a primary ovarian stimulator and inhibin A as part of its feedback regulation differs in three- versus two-wave cycles, providing a hormonal basis for the formation of multiple follicular waves in this species.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Estrus was synchronized in a group of 20 nonlactating, multiparous Holstein-Friesian cows from the University of Melbourne's experimental herd. The cows had free access to pasture and were given supplemental cereal hay for the duration of the trial. The Victorian Institute Animal Science Ethics Committee approved this trial under Protocol 1970. Synchronization involved inserting an intravaginal progesterone device (controlled internal drug release device [CIDR]; Genetics Australia, Bacchus Marsh, Victoria) for 8 or 9 days. Estradiol benzoate (2 mg, CIDIROL; Genetics Australia) was injected concurrently with device insertion, and cloprostenol (500 µg, CIDR-PG; Genetics Australia) was injected concurrently with device removal. A second injection of estradiol benzoate (1 mg) was administered 24 h after device removal. The synchronized cows were monitored for estrus using Heat Watch sensors (DDx, Denver, CO). Each of these sensors recorded the number and duration of mounts that occurred during estrus. Onset was defined as the occurrence of the first mount of at least 3 sec duration.
Twelve of these 20 cows were in estrus in a single 24-h period and had ovulation confirmed subsequently by ovarian ultrasonography. The ovaries of each of these animals were scanned at 0700–1000 h every day from removal of the CIDR device until a second ovulation associated with behavioral estrus was confirmed at the commencement of the subsequent estrous cycle. A 7.5-MHz linear array rectal probe was used with a real time ALOKA 500 ultrasonograph. The dimensions and relative position of every follicle 
4 mm and the location and size of any corpus luteum (CL) were recorded graphically each day for each ovary.
The day of ovulation in the monitored cycle was specified as the first day the dominant follicle (>8-mm diameter) of the preceding wave was not identifiable. Ovulation was retrospectively confirmed with the visible recognition of a CL in the same position on the ovary within 96 h, concurrent with a rise in plasma concentrations of progesterone. A retrospective analysis of the follicular development within each wave identified the dominant follicle as the one that achieved the largest diameter in that wave after having been first detected at 4 mm [25].
Blood Sampling Procedures and Hormone Assays
A 10-ml blood sample was collected daily from a coccygeal tail vein or a jugular vein from each cow into a heparinized Vacutainer tube (Becton-Dickinson, Meylan, France) immediately preceding every ovarian examination. Each sample was stored on ice and then centrifuged within 2 h at 3000 rpm (1500 x g) for 15 min. The plasma was transferred into duplicate 5-ml vials and stored at -20°C until assayed.
Daily plasma samples were each assayed for inhibin A, FSH, and estradiol-17ß (E2), with every sample from an individual cow for each hormone being included in a single assay. Samples from Days 1, 2, 10, and 11 of the cycle and the days of estrus (Day 0) and ovulation were assayed for progesterone.
Commercially available RIA kits were used to quantify plasma concentrations of progesterone (Spectria; Orion Diagnostica, Espoo, Finland) and E2 (MAIA; Biochem Immunosystems, Bologne, Italy; as modified by Evans et al. [26]). The highest cross-reactivity of the progesterone assay was with pregnenolone at 3.9%; sensitivity was 0.2 ng/ml progesterone. The highest cross-reactivity recorded for the E2 assay was with estrone at 1.8%; sensitivity was 0.2 pg/ml E2. There was only one progesterone assay; its coefficient of variation (CV) was 18%. The mean interassay CV for the E2 assays was 17%, and the intraassay CV was 13.1%.
Concentrations of FSH in plasma were quantified with a cow-specific FSH RIA (kit provided by Dr. A Parlow, National Hormone and Pituitary Program, Torrence, CA) using an anti-ovine FSH rabbit IgG (AFP-C5288113) and bovine FSH standard preparation (AFP-532B). The bovine FSH (AFP-532B) was iodinated using chloramine T. Cross-reactivity was 0.04% with bovine LH and 0.06% with bovine thyroid-stimulating hormone. Sensitivity was 0.1 ng/ml for FSH. The interassay CV was 17.5%, and the intraassay CV was 6.0%.
Concentrations of inhibin A in plasma were quantified with a ELISA for bovine dimeric inhibin A [9]. The biotinylated monoclonal antibody (PPG1 14/6) specific to the bovine inhibin subunit and the E4 plates that were coated with antibody specific to the ßA subunit of inhibin A were provided by N.P. Groome (Oxford, U.K.). Sensitivity was 20 pg/ml inhibin A using a bovine inhibin standard purified at Prince Henry's Institute of Medical Research [27]. Cross-reactivity of the inhibin A bovine ELISA was <0.1% with other monomeric forms of inhibin. The interassay CV for the inhibin A ELISA was 16.5%, and the intraassay CV was 8.1%.
Statistical Analyses
Hormonal and ovarian follicle data were analyzed with respect to the day of estrus (Day 0) during the first follicular wave in the monitored cycle and with respect to the day of ovulation (Day 0) during the ovulatory wave at the end of that cycle. The day of emergence of a follicular wave was determined retrospectively as the first day that a dominant follicle was identified as being at least 4 mm in diameter.
The first follicular wave was divided into three phases based on the patterns of change in the plasma concentrations of inhibin A. The first phase was associated with the increasing concentrations of inhibin A, which occurred between the day of ovulation and the day of peak concentration. The second phase was characterized by decreasing concentrations, and the third phase was characterized by increasing concentrations associated with the emergence of a new follicular wave.
Regression analyses were used to describe the associations between FSH and inhibin A and between E2 and inhibin A in each of the cows for each of the three phases of inhibin A that occurred in the nonovulatory and ovulatory waves (SPSS 1989–1999 for Windows; SPSS, Chicago, IL). ANOVAs with repeated measures were used to quantify the significance of changes in hormone concentrations throughout the estrous cycle and changes in the average number of follicles and their size categories on Days 2, 3, and 4. Paired t-tests were used to compare selected means during nonovulatory and ovulatory waves and within each cow between these two waves (SPSS). The Levene test for an ANOVA was used to determine whether there were significant differences in the compared values. Univariate ANOVAs were used to test the significance of variation in inhibin A and FSH concentrations between animals. Planned orthogonal contrasts were undertaken between the two nonovulatory and ovulatory waves in the cows with three-wave cycles.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Analyses of the physical characteristics of follicular waves and of cycle lengths considered the number of waves during a cycle. The four cows that ovulated the second dominant follicle had a mean (±SEM) cycle length of 21.8 ± 0.3 days compared with 25.3 ± 0.9 days for the four cows that ovulated the third dominant follicle (P = 0.02). One parameter associated with the growth or size of a dominant follicle that varied between cows with two or three waves of follicular development was the interval from wave emergence to the point in the ovulatory wave when maximum diameter of the dominant follicle was attained. This interval was shorter in the third wave than in the first wave in cows that had three-wave cycles (9.0 vs. 6.8 days; P = 0.02; Table 1). The average number of follicles >3 mm in diameter on each pair of ovaries on Days 2, 3, and 4 was 9.1 ± 0.6. This number ranged from 7.0 to 12.3 for individual animals, from 8.6 to 9.9 on individual days (P > 0.1), and from 8.6 to 9.6 for cows with two- or three-wave cycles, respectively (P > 0.1; Fig. 1). The follicle population in the ovaries of cows with two waves grew more rapidly over this period of 3 days; they had 3.25 and 4.25 follicles >6 mm on Days 3 and 4 compared with only 0.75 and 3.25 large follicles for cows with three waves on the same days (P = 0.04; Fig. 1).
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The highest plasma concentration of inhibin A measured in a single plasma sample taken during the first follicular wave was 570 pg/ml, whereas the minimum nadir concentration was 150 pg/ml. This value can be compared to maximum and minimum plasma concentrations of FSH, which were 2.4 and 0.3 ng/ml, respectively. Mean plasma concentrations for individual animals over a single cycle were 245–401 pg/ml for inhibin A and 0.79–1.51 ng/ml for FSH (P < 0.001). Concentrations of both hormones varied on a daily basis within individual animals (Figs. 2–4). The differences in peak and nadir concentrations of FSH and inhibin A were usually greater among the individual animals with two-wave cycles, but there was less variation in the respective averages between the nonovulatory and ovulatory waves in the two-wave cows than in those with three-wave cycles (Table 2). Peak plasma concentrations of inhibin A were similar in both waves in cows with two-wave cycles (474 vs. 432 pg/ml; P > 0.10; Table 2), but the concentrations differed among waves in cows with three-wave cycles (332 vs. 464 pg/ml; P < 0.02; Table 2).
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The average interval from the observed emergence of the first follicular wave until peak concentration of inhibin A was shorter in cows with two-wave cycles (2.0 vs. 3.8 days; P = 0.03), but the intervals to nadir concentrations were similar (8.3 vs. 8.8 days; P = 0.63; Table 2). Once the peak plasma concentration of inhibin A had been reached during the first postovulatory wave, it began to decline and continued to do so while the new dominant follicle was in its growth phase (Figs. 2 and 3). During this phase, concentrations of FSH increased by an average of 3.3 pg per 1 pg decline in inhibin A concentration (average slope = -3.3 ± 0.76 pg; P < 0.001); seven of the eight cows showed this significant negative relationship.
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Average plasma concentrations of E2, inhibin A, and FSH declined from 1 to 2 days before ovulation (Fig. 4). The decline was most apparent in cows that ovulated the dominant follicle from the third follicular wave (225 vs. 360 pg/ml; P < 0.05). Ovulatory concentrations of inhibin A and FSH were similar following either the introductory synchronization or the spontaneous ovulation after estrus from 18 to 26 days later (Fig. 5).
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The mean and peak concentrations of inhibin A were similar during the first two nonovulatory waves in cows with three-wave cycles (262 vs. 240 pg/ml, P > 0.4; 332 vs. 292 pg/ml, P > 0.2) but were lower than the equivalent averages during the ovulatory wave (301 and 464 pg/ml, P < 0.03; Fig. 6). The comparisons for FSH were similar, with mean concentrations during the first two waves of 0.96 and 1.06 ng/ml (P > 0.50), which were lower than the mean concentration during the ovulatory wave (1.37 ng/ml; P < 0.001; Fig. 6). The peak FSH concentrations in the first two waves were both 1.31 ng/ml and were significantly different from peak concentrations in the ovulatory wave (2.1 ng/ml; P < 0.001; Fig. 6). The means and peak concentrations of inhibin A and FSH did not differ significantly between the nonovulatory and ovulatory waves in cows with two-wave cycles (Table 2).
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Peak plasma concentrations of progesterone and E2 following ovulation did not differ between the cows with two-wave and those with three-wave cycles (progesterone: 6.9 ± 0.76 ng/ml vs. 7.0 ± 1.2 ng/ml, P > 0.1; E2: 1.9 ± 0.25 pg/ml vs. 1.4 ± 0.22 pg/ml, P > 0.1). Likewise, plasma concentrations of progesterone and E2 preceding ovulation did not differ between cows with two-wave and those with three-wave cycles (progesterone: 0.14 ± 0.03 ng/ml; E2: 3.26 ± 0.41 pg/ml vs. 3.28 ± 0.61 pg/ml, P = 0.94). Increases in E2 in plasma commenced 5 days before ovulation and reached peak concentrations of 3.3 ± 0.32 pg/ml at 1.5 ± 0.27 days before ovulation. These peak levels coincided with behavioral estrus the day before ovulation. The increases in E2 commenced with the functional degeneration of the CL.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The relationship between inhibin A and FSH during the period of early follicle growth was difficult to analyze because it was so short. The mean concentrations of inhibin A and FSH both increased during the early growth phase in both the two- and three-wave cycles. Because the early growth of the follicle cohort would be stimulated by FSH, increases in the concentration of inhibin A may have been caused primarily by the increased number of newly emerged and growing follicles. FSH has also demonstrated a direct action on inhibin A production by antral follicle granulosa cells in culture [28]. The dominant follicle was approximately 8–9 mm in diameter on the day of peak plasma concentrations of inhibin A. Concentrations declined thereafter, even though average follicle diameter continued to increase to around 14 mm at Day 9 after wave emergence (Figs. 2 and 3; Table 1). Factors contributing to the decline in the plasma concentrations of inhibin A could include 1) declining production from the diminishing numbers of subordinate follicles in the cohort, 2) decreasing concentrations of FSH following a postovulatory surge associated with wave emergence and with the peak concentrations in inhibin A, and 3) a decline in the functional integrity of the nonovulatory dominant follicle as it reached its plateau phase of development.
This gradual decline in the concentrations of inhibin A was associated with increasing concentrations of FSH, as reflected by the negative slope describing the relationship between these two hormones in seven of the eight cows. A similar negative association was previously reported in heifers [11]. The emergence of the second waves was associated with increasing concentrations in FSH and inhibin A (Figs. 2 and 3).
Those cows with three-wave cycles had lower concentrations of inhibin A during the first follicle wave (Table 2) and on the days of ovulation (Fig. 5). These lower concentrations persisted into the second nonovulatory wave and also applied to FSH. In spite of these lower concentrations, the day of emergence of the second wave was similar for cows that had two- or three-wave cycles (8.8 vs. 8.5 days).
The plasma concentrations of inhibin A and FSH during the ovulatory wave did not differ significantly between cows with two-wave and those with three-wave cycles (Table 2) because cows with three-wave cycles had significantly higher concentrations of these two hormones during the ovulatory wave than during the two nonovulatory waves (Fig. 6). Similar relationships were reported in Japanese black cattle [23]. Regardless of whether the second or third dominant follicle ovulated, the period of decreasing inhibin A recognized during the first wave (phase II; Figs. 2 and 3) was not recognized following emergence of the ovulatory wave. Instead, the ovulatory wave had an extended period of increasing concentrations of inhibin A, commencing 3 days after emergence of the ovulatory follicle and in association with increasing plasma E2 (Fig. 4). This period of increasing inhibin A reached peak concentrations 2.3 days before ovulation and lasted for 5.8 days. The ovulatory follicle maintained inhibin A production during its final maturation in spite of a need to sustain rapid follicle growth preceding ovulation. The positive action of E2 on the anterior pituitary in the absence of progesterone during the ovulatory wave would have stimulated the release of FSH and LH. Concentrations of FSH increased 3 days before ovulation (Fig. 4) concomitant with E2 and inhibin A. However, the concurrently high concentrations of inhibin A may have been involved with the prevention of new wave emergence until after ovulation. Similar patterns of hormonal interactions were reported in heifers and suckled cows preceding estrus and ovulation [11, 23].
In this study, we identified periods during the development of a nonovulatory dominant follicle in the bovine estrous cycle when concentrations of inhibin A and FSH were both increasing (phases I and III) and periods when there was an inverse relationship (phase II; Figs. 2 and 3). These patterns differed from those observed during the ovulatory wave when concentrations of both hormones were elevated (Fig. 4). Those cows that had three waves of follicle development in their estrous cycles had significantly lower plasma concentrations of inhibin A on the day of ovulation (Fig. 5) and during their two nonovulatory waves (Table 2). They also had lower concentrations of FSH, except on the days of ovulation (Fig. 6). The lower concentrations of FSH may have contributed to the lower concentrations of inhibin A, which in turn may have been less effective in suppressing FSH concentrations and consequently new wave emergence. The two nonovulatory dominant follicles in these animals may not have been as functionally developed as the single nonovulatory dominant follicle in cows with two waves of follicle development during a single cycle. The inhibin A and FSH concentrations in the nonovulatory waves of a series of cycles would need to be measured to establish the reliability of the observed concentration differences associated with cows that had one or more of these waves of follicular development.
Other researchers [23, 29] have also reported that cows or heifers with three rather than two waves of follicular development have longer estrous cycles. The maximum diameter of the ovulatory follicle in lactating Holstein cows with two-wave cycles was 17.2 mm compared with 16.0 mm in cows with three-wave cycles [29]. Although only 31% of these cows had three-wave cycles, these cows had higher conception rates at 30–35 days following ovulation and artificial insemination than did those cows with two waves (81% vs. 63%) [29]. Results from our study suggest that this difference in fertility would not have been associated with differences in the plasma concentrations of inhibin A and FSH during the development of the ovulatory wave. However, both studies showed that the ovulatory follicle in a three-wave cycle is younger than that in a two-wave cycle (Table 1).
The results from this study indicate that the number of waves of ovarian follicle development that occur during the bovine estrous cycle is influenced by plasma concentrations of inhibin A and FSH measured during the first nonovulatory wave. When these concentrations were similar to those measured in the ovulatory wave, there were only two waves of follicle development. When concentrations were lower during the nonovulatory wave, there were three waves of follicle development. Regardless of differences in the concentrations of inhibin A and FSH during the nonovulatory waves, their respective concentrations were similar during the ovulatory wave and especially following luteolysis when progesterone concentrations were declining or minimal.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: K.L. Macmillan, Department of Veterinary Science, University of Melbourne, 250 Princes Hwy., Werribee, VIC 3030, Australia. FAX: 61 3 9731 2388; e-mail: k.macmillan{at}unimelb.edu.au
Received: 7 July 2002.
First decision: 12 August 2002.
Accepted: 16 September 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-subunit precursor proteins from bovine follicular fluid. Endocrinology 1989 125:2141-2149[Abstract]This article has been cited by other articles:
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  R. Webb, P. C. Garnsworthy, J.-G. Gong, and D. G. Armstrong Control of follicular growth: Local interactions and nutritional influences J Anim Sci, January 1, 2004; 82(13_suppl): E63 - 74. [Abstract] [Full Text] [PDF]
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