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Regular Article |
a Genetic Diversity Department, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan
b Cattle Breeding Development Institute Kagoshima Prefecture, Kagoshima 899-8212, Japan
c Laboratory Animal Science, Kitasato University, School of Veterinary Medicine and Animal Science, Towada, Aomori 034-8628, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and ßA subunits, was specific for bovine inhibin A and did not cross-react with bovine activin A, activin AB, activin B, pro-C or human recombinant inhibin B. The detection limit of the IFMA was 3.3 pg/ml expressed in terms of bovine 32-kDa inhibin A. Dose-response curves of plasma samples obtained from intact and FSH-stimulated cows and cystic cows were parallel to the standard without any preassay processing of samples. Plasma inhibin A levels increased (P < 0.01) concomitant with emergence of nonovulatory or ovulatory follicular waves during the postpartum period. In cystic cows, plasma inhibin A was sustained at high levels for a longer period, associated with growth of persistent dominant follicles. The highest levels of inhibin A were noted during the growth phase of normal and persistent dominant follicles; however, inhibin A levels declined (P < 0.01) as these dominant follicles ceased to grow or ovulated. An inverse relationship between patterns of plasma inhibin A and FSH existed during each follicular wave in the three physiologic conditions. Increases in plasma inhibin A levels were associated with increases in plasma estradiol levels during most follicular waves; however, there was no increase in plasma estradiol level and no relationship between patterns of estradiol and FSH during follicular waves observed during the early postpartum period or midluteal phase of the estrous cycle. In conclusion, the Tr-IFMA does not require pretreatment of samples and can be used for precise measurement of bovine inhibin A without interference with free inhibin subunits. Inhibin A, produced primarily during growth of the dominant follicle, functions as a negative feedback regulator for FSH secretion throughout the postpartum period and the estrous cycle, whereas estradiol appears to have a minor role in regulation of FSH compared with inhibin A, especially during the early postpartum period and midluteal phase of the estrous cycle. The results also indicate that a persistent dominant follicle sustains inhibin A production for a longer period than the dominant follicle emerging in the estrous cycle and establishes long-term dominance by suppressing emergence of a new follicular wave.
estradiol, follicle-stimulating hormone, follicular development, inhibin
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-ßA dimer) rather than inhibin B (-ßB dimer). Passive [11–13] or active [14, 15] immunization of cyclic cattle against inhibin increases FSH secretion, whereas treatment of cattle with highly purified inhibin [16] or steroid-free bFF [17–19] decreases circulating FSH levels. Taken together, these findings imply that inhibin A produced by dominant follicles has an important role in the regulation of FSH secretion and folliculogenesis in various reproductive conditions. As yet, only one study, using a two-site ELISA, has characterized the relationship between inhibin A and FSH concentrations in serum of cattle after treatment with prostaglandin F2 to induce estrous cycles [20]. However, in other reproductive conditions, for example, during the postpartum period and in cystic cows, the relationship between circulating levels of inhibin A and FSH and follicular development has not been elucidated, and the endocrine role of inhibin A is only supposed.
Inhibin RIAs [21, 22] performed in many laboratories do not discriminate inhibin A, which has FSH-suppressing activity, from free inhibin subunits, which have no FSH-suppressing activity and exist in high concentrations in bFF [23–25] and the peripheral circulation [23]. Although several laboratories have developed a sandwich assay for inhibin A using 2 different antibodies [26–30] to overcome this major limitation in inhibin RIAs, the existing ELISA remains cumbersome because it requires preassay processing of samples by heating in the presence of SDS, followed by oxidization with hydrogen peroxide [20, 27, 30, 31]. On the other hand, europium (Eu) is a nonisotopic label for peptide hormones, and can be measured with high sensitivity by time-resolved fluorometry [32, 33]. Time-resolved immunofluorometric assays (Tr-IFMAs) with Eu have several advantages over ELISAs. In particular, the labeling procedure with Eu is simpler than that for most other enzymatic labeling procedures, and measurement of Eu is less time-consuming than ELISA [32, 33].
Therefore, this study was designed to 1) develop a sensitive and specific Tr-IFMA for bovine inhibin A that does not require pretreatment of samples and 2) clarify alterations in serum concentrations of inhibin A during the postpartum period and in cystic cows in relation to follicle turnover and secretion profile of FSH and estradiol. For comparison, the relationship between serum profiles of inhibin A and FSH throughout the intact estrous cycle was also investigated.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Protocols for the use of animals in the present study were approved by the Animal Care Committee of the National Institute of Agrobiological Sciences. Ovarian follicles of 5 suckled Japanese black cattle were examined using an ultrasound scanner (Aloka, Tokyo, Japan) with a 7.5-MHz linear array transducer, as reported previously [2]. Ultrasound scanning was performed at 2-day intervals from Day 7 postpartum until the second postpartum ovulation. The day of emergence of a new follicular wave was defined as the day on which the first member (
4 mm in diameter) of a new cohort of follicles appeared. The largest follicle in the cohort was defined as the dominant follicle if it was a minimum diameter of 8.5 mm and at least 1.5 mm larger than the next largest follicle in the cohort [34]. Blood samples were collected by venipuncture every 12 h throughout the study. Plasma was recovered after centrifugation of blood and stored at -30°C.
Estrous Cycle
Ovarian follicles of 7 parous Japanese black cattle (4–6 yr old) with regular estrous cycles were subjected to daily ultrasound analysis starting 15 days after the previous estrus and ending on the day of ovulation of the subsequent cycle. Between 24 and 48 h after onset of estrus, ovaries were examined every 4 h to detect ovulation. Blood samples were collected by venipuncture every 8 h throughout the study, and every 4 h after the onset of estrus for 48 h to characterize hormonal profiles during the periovulatory period. Plasma was recovered after centrifugation of blood and stored at -30°C.
Cows with Cystic Follicles
Growth and regression of cystic and normal follicles were examined in five Japanese cows at 2-day intervals for 60 days starting 7 days after estrus without ovulation. Cows were considered to have a follicular cyst when an anovulatory follicle larger than 20 mm persisted for more than 10 days in the absence of a corpus luteum, with accompanying cycle irregularities [35]. Daily blood samples were collected by venipuncture throughout the study. Plasma was recovered after centrifugation of blood and stored at -30°C.
Tr-IFMA of Bovine Inhibin A
Preparation of antibodies
Antibody against the inhibin subunit was purified from a goat antiserum against bovine 32-kDa inhibin (GB [11]) using an affinity column packed with 1 ml of N-hydroxysuccinimide-activated Sepharose (HiTrap NHS-activated; Amersham Pharmacia Biotech, Buckinghamshire, U.K.) to which bovine 32-kDa inhibin A was attached [36]. Immunoblot analysis demonstrated that the affinity-purified antibody recognized the subunits of various molecular forms of inhibin (described in Results). Antibody against ßA subunit was obtained from another goat antiserum (GY) against bovine 32-kDa inhibin using an affinity column (HiTrap NHS-activated; Amersham Pharmacia Biotech) to which bovine activin A (ßA-ßA dimer) was attached. Specificity of the purified ßA subunit antibody to ßA and ßB subunits was tested by immunoblotting as described previously [36]. The ßA subunit antibody cross-reacted highly with activin A (ßA-ßA dimer) compared with activin B (ßB-ßB dimer) (data not shown).
Labeling of subunit antibody
Fifty micrograms of the purified subunit antibody was incubated with 10 µg Eu-chelate of N1-(p-isothiocyanatobenzyl)-diethylenetriamine-N1,N2,N3,N3-tetraacetic acid (Eu-Labelling reagent; Wallac Oy, Turku, Finland) overnight at 37°C according to the manufacturer's instructions. The Eu-labeled antibody was separated from free Eu by gel filtration on a two-tier column (1.0 cm inner diameter, Econo-Pac column; Bio-Rad Laboratories, Hercules, CA) of Sephadex G-50 (5 cm deep; Amersham Pharmacia Biotech) and Sepharose CL-6B (5 cm deep; Amersham Pharmacia Biotech). Fractions (1 ml) were collected. Aliquots (5 µl) of each fraction, diluted at 1:100 with Tris-buffered saline (TBS; 0.05 M TrisHCl, pH 7.5, 0.15 M NaCl) containing 0.1% (w/v) 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS; Sigma, St. Louis, MO), were used to monitor fluorescence with a fluorometer (1234 DELFIA FLUOROMETER; Wallac Oy) after incubation for 5 min with 100 µl of enhancement solution (Wallac Oy). Fractions containing labeled antibody were pooled and stored at 4°C.
Assay procedure Inhibin A levels were determined based on methods previously described by Hasegawa et al. [28]. The purified ßA subunit antibody was adsorbed onto wells of a microwell 96 plate (FluoroNunc Modules; Nalge Nunc International, Rochester, NY) by incubating each well with 100 µl of the antibody preparation diluted at a concentration of 1.5 µg/ml with coating buffer (0.05 M K2HPO4 containing 0.15 M NaCl and 0.05% [w/v] sodium azide [NaN3], pH 8.9) overnight at 25°C. The wells were rinsed three times with wash buffer (TBS containing 0.1% [w/v] Tween 20 and 0.05% [w/v] NaN3), and then 200 µl of blocking buffer (0.05 M Na2HPO4, pH 8.9 containing 0.1% [w/v] BSA and 0.05% [w/v] NaN3) was added to each well, and the wells were incubated overnight at 25°C.
The assay buffer was TBS containing 0.05% (w/v) BSA, 0.1% (w/v) bovine 
-globulin, 0.05% (w/v) NaN3, 0.01% (v/v) Tween 40, 0.0015% (w/v) phenol red, and 0.02 M diethylenetriaminepentaacetic acid. Bovine 32-kDa inhibin A, purified from FF [8], was used as a reference standard. Triplicate aliquots (100 µl) of standards (10–10 000 pg/ml) and unknown samples were pipetted into ßA subunit antibody-coated wells. The final volume for each well was 200 µl with assay buffer. When inhibin A was measured in plasma samples, 100 µl of serum from a castrated bull, instead of 100 µl assay buffer, was added to each well of the standards to correct for matrix effects of bovine plasma. The wells were then incubated overnight at 25°C. After the wells had been rinsed with wash buffer (12 times), the Eu-labeled subunit antibody diluted in assay buffer (2 x 106 cps per 100 µl) was added to each well. Thereafter, wells were incubated for 3 h at 25°C. After the wells were rinsed (12 times), 100 µl of enhancement solution was added to each well, and the wells were shaken for 5 min. The fluorescence was measured with a fluorometer (1234 DELFIA FLUOROMETER).
Validation
Specificity of the IFMA was assessed by comparing the fluorescence intensity obtained in the presence of various inhibin-related proteins. The materials tested were 32-kDa inhibin A, activin A, activin AB, activin B, and free subunit of inhibin (pro-C) purified from bFF [8, 37] and human recombinant (hr) inhibin B. Parallelism was also tested by comparing dose-response curves of plasma from normal cyclic, FSH-treated, and cystic cows with that of bovine 32-kDa inhibin A. In addition, recovery of bovine 32-kDa inhibin A added to plasma from an ovariectomized cow was quantified.
Finally, to determine whether different molecular weight forms of inhibin A cross-react in the IFMA, different molecular weight forms of inhibin were isolated from bFF and then assayed by the IFMA and subjected to immunoblot analysis. Inhibin was purified from pooled bFF using an affinity column packed with 5 ml of N-hydroxysuccinimide-activated Sepharose (HiTrap NHS-activated; Amersham Pharmacia Biotech) to which 10 mg of the purified subunit antibody (GB) was attached [36]. The eluted fraction containing 20 µg of the affinity-purified inhibin was subjected to 12.5% SDS-PAGE. Rainbow colored molecular weight marker (14 300–220 000; Amersham Pharmacia Biotech) was used to estimate the molecular size of proteins. The gel was cut into 1.0-mm slices. Inhibin was extracted from each gel slice with TBS containing EDTA under gentle shaking overnight. The gel eluates were assayed for inhibin A and were subjected to immunoblot analysis. Blots were probed with the purified subunit antibody (GB) or ßA subunit antibody (R105) raised against amino acids 85–105 of the ßA subunit of bovine inhibin.
Time-Resolved Fluoroimmunoassay of Bovine FSH and LH
Hormone preparations and antibodies Concentrations of FSH or LH in plasma of cows were determined by competitive immunoassays (FSH [38], LH [39]) using Eu-labeled FSH or LH as probes. In the time-resolved fluoroimmunoassay (Tr-FIA) of bovine FSH, anti-bovine FSH ß subunit serum (USDA-5-pool) was used as a primary antibody, USDA-bFSH-I2 was used for Eu-labeling, and USDA-bFSH-I2 was used as the reference standard. In the LH Tr-FIA, anti-ovine LH serum (USDA-309-684P) was used as a primary antibody, USDA-bLH-I-1 was used for Eu-labeling, and USDA-bLH-B5 was used as the reference standard. (Assay materials were provided by United States Department of Agriculture Animal Hormone Program, Germplasm and Gamete Physiology Laboratory, Beltsville Agricultural Research Center, Beltsville, MD.)
Labeling of each hormone preparation Five micrograms of each hormone preparation was incubated with 10 µg Eu-chelate of N1-(p-isothiocyanatobenzyl)-diethylenetriamine-N1,N2,N3,N3-tetraacetic acid (Eu-Labelling reagent, Wallac Oy) overnight at 37°C according to the manufacturer's instructions. The Eu-labeled protein was separated from free Eu by gel filtration with a column (1.0 cm inner diameter, 11.5 cm, Econo-Pac column; Bio-Rad Laboratories) of Sephadex G-50 (Amersham Pharmacia Biotech).
Assay procedure Each primary antibody was pipetted into wells coated with anti-rabbit immunoglobulin G (Chemicon International Inc., Temecula, CA), and the wells were incubated overnight at 25°C for the LH assay or 34°C for the FSH assay. The wells were rinsed with wash buffer (10 times), and then standards (100 µl) and unknown samples (100–200 µl) were added and incubated overnight at 25°C for the LH assay or 34°C for the FSH assay. After incubation, the wells were washed (12 times), and Eu-labeled FSH or LH (1 x 106 cps per 100 µl) was added to wells. Wells were incubated for 2 h at 25°C for LH or for 6 h at 34°C for FSH. After the wells were washed (12 times), 100 µl of enhancement solution was added to each well, and the wells were shaken for 5 min. The fluorescence was measured with a fluorometer (1234 DELFIA FLUOROMETER). The sensitivity of the FIAs, based on a 95% confidence limit of the zero standard, was 39 pg/ml for FSH and 98 pg/ml for LH. The intraassay and interassay coefficients of variations (CVs) were 10% and 11.5% for FSH and 7.5% and 12.0% for LH, respectively.
RIA of Estradiol and Progesterone
Plasma concentrations of estradiol and progesterone were determined as described previously [40] using antisera to estradiol-17ß (GDN 244 [41]) and progesterone (GDN 337 [42]) supplied by Dr. G.D. Niswender (Colorado State University, Fort Collins, CO). In the assay for estradiol, substances that interfere with the estradiol assay were removed by a mixture of 2 ml n-hexane and 0.5 ml 50% acetonitrile (v/v) [43]. The sensitivities of the assay of estradiol and progesterone were 0.32 pg/ml and 0.025 ng/ml, respectively. The intraassay and interassay CVs were 8.9% and 10.5% for estradiol and 7.5% and 11.0% for progesterone, respectively.
Data Analyses
Because there was a difference in the timing of emergence of each follicular wave in individual cows, data were aligned relative to follicle emergence. Data pertaining to hormonal profiles and follicular growth were subjected to ANOVA for repeated measures [44]. When a significant effect was detected with the ANOVA, the significance of the difference between means was determined by the Duncan multiple range test. Regression analyses among inhibin A, FSH, and estradiol concentrations were also performed during each follicular wave. All data were analyzed using the General Linear Models or REG Procedure of the Statistical Analysis Systems (SAS Inc., Cary, NC) [45]. A value of P < 0.05 was considered to be significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The IFMA recognized bovine 32-kDa inhibin A from a concentration of 0.0033 ng/ml to a concentration of more than 10 ng/ml (Fig. 1). Cross-reactivities of bovine activin A, activin AB, activin B, and hr-inhibin B were 
0.01% (Fig. 1). Bovine pro-C also showed low cross-reactivity (1%). Serial dilutions of plasma samples from normal cyclic, FSH-stimulated, and cystic cows resulted in dose-response curves that were parallel to the standard curve generated with inhibin A (Fig. 2). Recovery of bovine 32-kDa inhibin A added into bovine ovariectomized serum was 102% at 1 ng/ml and 95% at 0.1 ng/ml. After affinity-purified bFF was fractionated by SDS-PAGE, the IFMA detected 2 high immunoreactive peaks of 31 and 54 kDa and 4 smaller peaks of 25.5, 65, 78, and 108 kDa (Fig. 3). Immunoblot analysis using the subunit and ßA subunit antibodies detected primarily the 31-, 54-, 65-, 78-, or 108-kDa band (Fig. 4), whereas only the subunit antibody recognized the 25.5-kDa band. The intraassay and interassay CVs were 6.5% and 10.5% for the inhibin A Tr-IFMA, respectively.
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Inhibin A, Estradiol, and FSH, and Follicular Dynamics During Postpartum Period of Cows
The mean ± SEM interval from parturition to the second postpartum ovulation accompanying estrous behavior was 65.0 ± 9.8 days (n = 5 cows), with a range from 45 to 80 days in beef cows that were nursing calves. Emergence of follicular waves occurred at 7- to 10-day intervals throughout the observation period. The maximum diameter of the dominant follicle between 10 and 20 days postpartum was smaller (P < 0.05) than the diameter of dominant follicles at other days after parturition (Fig. 5). Concentrations of inhibin A increased (P < 0.01) from 40–50 pg/ml before emergence of each wave to 70–110 pg/ml during the growth phase of the dominant follicles. Inhibin A levels declined (P < 0.01) as the dominant follicles ceased to grow or ovulated. Overall inhibin A concentrations in plasma increased (P < 0.05) as cows approached their first ovulation. Plasma inhibin A level was inversely correlated with FSH level (r = -0.32 to -0.4, P < 0.01) during the follicular wave at each stage after parturition. There was no significant correlation between plasma estradiol and FSH concentrations (r = -0.04, P > 0.1) during days 10–20 postpartum, although an inverse relationship was found between levels of estradiol and FSH (r = -0.35, P < 0.01) as cows approached their first ovulation.
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Inhibin A, Estradiol, and FSH, and Follicular Dynamics Throughout the Intact Estrous Cycle
There were 3 waves of follicular development throughout the estrous cycle (Fig. 6). The first wave occurred during the early luteal phase, the second wave during the midluteal phase, and the third (ovulatory) wave during the follicular phase. The mean maximum diameter of the dominant follicle in the second wave was 8.7 ± 0.3 mm (n = 7 cows), which was smaller than the dominant follicle in the first wave (9.8 ± 0.2 mm) and third wave (11.2 ± 0.3 mm). Plasma inhibin A concentration increased significantly (P < 0.01) concomitant with emergence of each wave, with a maximum value of 180–220 pg/ml and was inversely correlated with FSH concentration (r = -0.46 to -0.51, P < 0.01) during each follicular wave. An inverse relationship was found between levels of estradiol and FSH during the first wave (r = -0.37, P < 0.01) and third wave (r = -0.42, P < 0.01); however, there was no significant correlation between plasma estradiol and FSH levels during the second wave (r = 0.05, P > 0.1).
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Inhibin A, Estradiol, and FSH, and Follicular Dynamics in Cows with Cystic Follicles
The mean interval from initial detection of a follicular wave to detection of a new follicular wave was 15.5 ± 2.5 days (n = 15 waves from five cows with cystic follicles), which is a longer (P < 0.05) interwave interval than that in cows without cystic follicles (7.5 ± 0.9 days, n = 21 waves from seven cows without cysts). Concentrations of inhibin A and estradiol increased coincident with the appearance of follicular cysts (Fig. 7), and concentrations of both hormones were sustained at high levels for more than 10 days. The highest levels of inhibin A and estradiol were 310 ± 35 and 12.9 ± 1.5 pg/ml, respectively. These values were not significantly different from those associated with growth of the normal ovulatory dominant follicle in the estrous cycle (inhibin A: 220 ± 38 pg/ml; estradiol: 12.5 ± 1.8 pg/ml). There was an inverse relationship between plasma FSH level and both inhibin A (r = -0.35, P < 0.01) and estradiol (r = -0.32, P < 0.01) levels.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Several molecular weight forms of dimeric inhibin exist in bFF [8, 46–48]. The IFMA in the present study recognized the higher molecular weight precursor forms of inhibin A corresponding to 55, 65, 75, and 110 kDa, as well as fully processed 32-kDa inhibin A. Together with previous findings [46, 48] that these precursors have FSH-suppressing activity, the IFMA recognizes biologically active inhibin A. The IFMA also detected a small peak of 25.5 kDa in bFF, probably due to the presence of large amounts of 26 kDa pro-C [23–25]; however, this is negligible since the ratio of immunoreactivity of pro-C is estimated to be less than 2.5% of total inhibin A immunoreactivity. Plasma samples subjected to the ELISAs for human and bovine inhibin A need to be heated in the presence of SDS and oxidized [20, 27, 30, 31] to increase sensitivity of the immunoassays. In our IFMA, pretreatment of plasma samples is not required. The reasons for these differences between the different assay formats are unknown. However, it is possible that polyclonal antibodies raised against the whole 32-kDa inhibin in the IFMA possess a higher affinity to inhibin A compared with the monoclonal antibodies used by others [20, 27, 30, 31].
Although an endocrine role of inhibin A during the postpartum period of cows and in cystic cows has been inferred from the relationship between follicle emergence and plasma FSH profile [5–7], this is the first report that describes alterations in peripheral concentrations of inhibin A in these reproductive conditions in relation to follicle growth and profiles of plasma FSH. Concentrations of plasma inhibin A increased coincident with emergence of follicular waves, but decreased as the normal or persistent dominant follicle ceased to grow or ovulated. The present study also clearly demonstrated that an inverse relationship between plasma inhibin A and FSH levels exists during follicular waves that occur during the postpartum period and in cystic cows. These findings strongly suggest that inhibin A, primarily produced by the normal or persistent dominant follicle, functions as a negative regulator for FSH secretion throughout the postpartum period and in cystic cows. The present results, together with the previous results of Bleach at al. [20] that demonstrated that an inverse pattern of FSH and inhibin A secretion exists during the prostaglandin-synchronized estrous cycle, indicate that inhibin A has an endocrine role in various reproductive conditions of cattle.
A recent study [20] indicates that combined effects of elevated inhibin A and estradiol account for the decline in FSH during the estrous cycle; however, the present results indicate that there was no increase in plasma estradiol concentration and no relationship between estradiol and FSH concentrations during the second wave that emerged during midluteal phase. Furthermore, no relationship was found between plasma estradiol and FSH levels during the early postpartum period. These results, together with the present results showing that plasma FSH concentration is inversely related with inhibin A concentration during every follicular wave, suggest that estradiol has a minor role in the regulation of FSH secretion compared with inhibin A, especially during the midluteal phase and early postpartum period. Inhibin immunization of cows increases FSH secretion even in the presence of high levels of plasma estradiol [12, 13], which supports the suggested relative roles of inhibin A versus estradiol in negative feedback regulation of FSH. The dominant follicle that emerges during the second wave or early postpartum period seems to have low activity to produce estradiol, owing to inadequate pulsatile secretion of LH [49, 50].
The size of the largest follicle and the interval between follicular waves was much greater during follicular waves with a cystic follicle compared with follicular waves without cystic follicles. Concentrations of plasma inhibin A, as well as estradiol, were sustained at high levels associated with growth of persistent cystic follicles. Cows with follicular cysts are generally characterized by high LH secretion [6, 7, 51]. Increases in LH pulse frequency promote prolonged follicular growth and dominance associated with increased estradiol secretion [52]. Taken together, production of inhibin A is likely to be sustained during growth of the dominant follicle, which is prolonged by high LH pulse frequency. A cystic follicle (persistent dominant follicle) probably continues to produce inhibin A and estradiol and suppresses plasma FSH, which results in an extension in dominance of cystic follicles.
Based on the relationship between the plasma inhibin A profile and growth and regression of dominant follicles during follicular waves in the present and in a previous study [20], inhibin A production and release into the peripheral circulation are likely to be enhanced as dominant follicles grow, but decrease during atresia of dominant follicles. The findings are consistent with results demonstrating that amounts of follicular and ßA subunit mRNAs and size of dominant follicles are correlated positively, and that inhibin mRNAs are markedly reduced in atretic dominant follicles [53]. However, a two-site immunoradiometric assay, highly specific for 32- and 55-kDa inhibin [29], shows that inhibin A levels are high in FF of regressing dominant follicles [54]. The high intrafollicular levels of 32-kDa inhibin in atretic dominant follicles are suggested to be due to alterations in posttranslational processing of inhibin precursors that results in a selective increase in levels of 32-kDa inhibin [34, 55, 56]. At present, reasons for the discrepancy between pattern of the circulating inhibin A and intrafollicular levels of inhibin A are not readily explained.
To summarize, a simple, sensitive, and specific Tr-IFMA for bovine inhibin A in plasma was developed and validated. An inverse relationship exists between patterns of secretion of inhibin A and FSH during nonovulatory and ovulatory follicular waves that occur during the postpartum period and estrous cycle and in cows with follicular cysts, but there is no relationship between estradiol and FSH levels during the early postpartum period and midluteal phase of the estrous cycle. From these results, we conclude that nonradiometric Tr-IFMA can be used to measure alterations in secretion of inhibin A in cows reliably, and that inhibin A is a primary negative feedback hormone in various reproductive conditions of cattle.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by the Ministry of Agriculture, Forestry and Fisheries. 
2 Correspondence: Hiroyuki Kaneko, Genetic Diversity Department, National Institute of Agrobiological Sciences, Kannondai 2-1-2, Tsukuba, Ibaraki 305-8602, Japan. FAX: 81 298 38 7408; kaneko{at}nias.affrc.go.jp
Accepted: January 29, 2002.
Received: December 28, 2001.
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REFERENCES |
|---|
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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C-subunit on plasma anti-inhibin titers, plasma FSH concentrations and the incidence of multiple ovulation in heifers. Anim Reprod Sci 1996; 41:1-12 subunit and its isolation from bovine follicular fluid. J Mol Endocrinol 1989; 2:189-200-subunit precursor proteins from bovine follicular fluid. Endocrinology 1989; 125:2141-2149[Abstract] and ß subunit. J Endocrinol 1991; 129:R9-R12, ßA and ßB subunit messenger ribonucleic acids following increases in size and during different stages of differentiation or atresia of non-ovulatory follicles in cows. Biol Reprod 1994; 50:492-501[Abstract] subunit and -ßA dimer) in morphologically dominant follicles during their growing and regressing phases of development in cattle. Biol Reprod 1993; 48:268-276[Abstract]This article has been cited by other articles:
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  H Kadokawa, M Matsui, K Hayashi, N Matsunaga, C Kawashima, T Shimizu, K Kida, and A Miyamoto Peripheral administration of kisspeptin-10 increases plasma concentrations of GH as well as LH in prepubertal Holstein heifers J. Endocrinol., February 1, 2008; 196(2): 331 - 334. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Kaneko, K. Kikuchi, J. Noguchi, M. Ozawa, K. Ohnuma, N. Maedomari, and N. Kashiwazaki Effects of gonadotrophin treatments on meiotic and developmental competence of oocytes in porcine primordial follicles following xenografting to nude mice Reproduction, February 1, 2006; 131(2): 279 - 288. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. S. Burns, F. Jimenez-Krassel, J. L.H. Ireland, P. G. Knight, and J. J. Ireland Numbers of Antral Follicles During Follicular Waves in Cattle: Evidence for High Variation Among Animals, Very High Repeatability in Individuals, and an Inverse Association with Serum Follicle-Stimulating Hormone Concentrations Biol Reprod, July 1, 2005; 73(1): 54 - 62. [Abstract] [Full Text] [PDF]
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K. Y Arai, H. Kishi, S. Onodera, W. Jin, G. Watanabe, A. K Suzuki, S. Takahashi, T. Kamada, T. Nishiyama, and K. Taya Cyclic changes in messenger RNAs encoding inhibin/activin subunits in the ovary of the golden hamster (Mesocricetus auratus) J. Endocrinol., June 1, 2005; 185(3): 561 - 575. [Abstract] [Full Text] [PDF]
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C. R Burke, H. Cardenas, M. L Mussard, and M. L Day Histological and steroidogenic changes in dominant ovarian follicles during oestradiol-induced atresia in heifers Reproduction, May 1, 2005; 129(5): 611 - 620. [Abstract] [Full Text] [PDF]
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J M Haughian, O J Ginther, K Kot, and M C Wiltbank Relationships between FSH patterns and follicular dynamics and the temporal associations among hormones in natural and GnRH-induced gonadotropin surges in heifers Reproduction, January 1, 2004; 127(1): 23 - 33. [Abstract] [Full Text] [PDF]
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M. S. Medan, G. Watanabe, K. Sasaki, S. Sharawy, N. P. Groome, and K. Taya Ovarian Dynamics and Their Associations with Peripheral Concentrations of Gonadotropins, Ovarian Steroids, and Inhibin During the Estrous Cycle in Goats Biol Reprod, July 1, 2003; 69(1): 57 - 63. [Abstract] [Full Text] [PDF]
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 F. Jimenez-Krassel, M. E. Winn, D. Burns, J. L. H. Ireland, and J. J. Ireland Evidence for a Negative Intrafollicular Role for Inhibin in Regulation of Estradiol Production by Granulosa Cells Endocrinology, May 1, 2003; 144(5): 1876 - 1886. [Abstract] [Full Text] [PDF]
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H. Kaneko, J. Noguchi, K. Kikuchi, and Y. Hasegawa Molecular Weight Forms of Inhibin A and Inhibin B in the Bovine Testis Change with Age Biol Reprod, May 1, 2003; 68(5): 1918 - 1925. [Abstract] [Full Text] [PDF]
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K.I. Parker, D.M. Robertson, N.P. Groome, and K.L. Macmillan Plasma Concentrations of Inhibin A and Follicle-Stimulating Hormone Differ Between Cows with Two or Three Waves of Ovarian Follicular Development in a Single Estrous Cycle Biol Reprod, March 1, 2003; 68(3): 822 - 828. [Abstract] [Full Text] [PDF]
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H. Kaneko, J. Todoroki, J. Noguchi, K. Kikuchi, K. Mizoshita, C. Kubota, and H. Yamakuchi Perturbation of Estradiol-Feedback Control of Luteinizing Hormone Secretion by Immunoneutralization Induces Development of Follicular Cysts in Cattle Biol Reprod, December 1, 2002; 67(6): 1840 - 1845. [Abstract] [Full Text] [PDF]
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), activin A (
), activin AB (
), activin B (
), pro-
) purified from bFF, and hr-inhibin B (
)
