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BOR - Papers in Press, published online ahead of print October 4, 2002.
Biol Reprod 2002, 10.1095/biolreprod.102.007591
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Biology of Reproduction 67, 1840-1845 (2002)
DOI: 10.1095/biolreprod.102.007591 © 2002 Society for the Study of Reproduction, Inc.

Mechanisms of Hormone Action

Perturbation of Estradiol-Feedback Control of Luteinizing Hormone Secretion by Immunoneutralization Induces Development of Follicular Cysts in Cattle1

H. Kaneko2,a, J. Todorokib, J. Noguchia, K. Kikuchia, K. Mizoshitab, C. Kubotab, and H. Yamakuchib

a Genetic Diversity Division, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan b Cattle Breeding Development Institute Kagoshima Prefecture, Kagoshima 899-8212, Japan


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We used immunoneutralization of endogenous estradiol to investigate deficiencies in the estradiol-feedback regulation of LH secretion as a primary cause of follicular cysts in cattle. Twenty-one cows in the prostaglandin (PG) F2{alpha}-induced follicular phase were assigned to receive either 100 ml of estradiol antiserum produced in a castrated male goat (n = 11, immunized group) or the same amount of castrated male goat serum (n = 10, control group). The time of injection of the sera was designated as 0 h and Day 0. Five cows in each group were assigned to subgroups in which we determined the effects of estradiol immunization on LH secretion and follicular growth during the periovulatory period. The remaining six estradiol-immunized cows were subjected to long-term analyses of follicular growth and hormonal profiles, including evaluation of pulsatile secretion of LH. The remaining five control cows were used to determine pulsatile secretion of LH on Day 0 (follicular phase) and Day 14 (midluteal phase). The control cows exhibited a preovulatory LH surge within 48 h after injection of the control serum, followed by ovulation of the dominant follicle that had developed during the PGF2{alpha}-induced follicular phase. In contrast, the LH surge was not detected after treatment with estradiol antiserum. None of the 11 estradiol-immunized cows had ovulation of the dominant follicle, which had emerged before estradiol immunization and enlarged to more than 20 mm in diameter by Day 10. Long-term observation of the six immunized cows revealed that five had multiple follicular waves, with maximum follicular sizes of 20–45 mm at 10- to 30-day intervals for more than 50 days. The sixth cow experienced twin ovulations of the initial persistent follicles on Day 18. The LH pulse frequency in the five immunized cows that showed the long-term turnover of cystic follicles ranged from 0.81 ± 0.13 to 0.97 ± 0.09 pulses/h during the experiment, significantly (P < 0.05) higher than that in the midluteal phase of the control cows (0.23 ± 0.07). The mean LH concentration in the immunized cows was also generally higher than that in the luteal phase of the control cows. However, the LH pulse and mean concentration of LH after immunization were similar to those in the follicular phase of the control cows. Plasma concentrations of total inhibin increased (P < 0.01) concomitant with the emergence of cystic follicles and remained high during the growth of cystic follicles, whereas FSH concentrations were inversely correlated with total inhibin concentrations. In conclusion, neutralization of endogenous estradiol resulted in suppression of the preovulatory LH surge but a normal range of basal LH secretion, and this circumstance led to an anovulatory situation similar to that observed with naturally occurring follicular cysts. These findings provide evidence that lack of LH surge because of dysfunction in the positive-feedback regulation of LH secretion by estradiol can be the initial factor inducing formation of follicular cysts.

estradiol, follicle, luteinizing hormone, ovary, ovulation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cows with follicular cysts have waves of follicular growth associated with an increase in estradiol secretion [1–3] but lack a preovulatory LH surge during the growth of anovulatory follicles [3]. Exogenous injection of estradiol often fails to induce an LH surge in cows with naturally occurring or induced follicular cysts [4–8], whereas injection of GnRH can induce the release of LH [9–12]. It is widely accepted that an increase in estradiol secretion is essential for occurrence of the LH surge [13]. Taken together, these findings strongly suggest that an important physiological change in cows with cysts is the lack of an LH surge because of a functional abnormality in feedback regulation of LH secretion by estradiol. In support of this hypothesis, immunization of cattle [14, 15] or primates [16] against estradiol can induce the development of follicular cysts. However, whether alteration in LH secretion caused by a deficiency in estradiol-feedback regulation, especially the lack of an LH surge, is a cause of follicular cyst development in cattle has not been defined.

The other characteristic of LH secretion in cows with follicular cysts is high levels of basal LH secretion. Pulse frequency and mean concentration of LH in cows with cysts are greater compared with those in normal cows [17] or similar to those in the normal follicular phase [18]. The life span of the dominant follicle can be extended by increased LH pulse frequency comparable to that seen after luteolysis [19]. Thus, relatively high LH pulse frequency seems to be important for the continued excessive growth of dominant follicles. Whether this results from activation of GnRH secretion from the hypothalamus or simply from a lack of the negative-feedback effects of progesterone in anovulatory situations in cystic cows remains uncertain.

Therefore, the aim of the present study was to clarify whether a deficiency in the estradiol-feedback control of LH secretion was the initiating factor in the development of follicular cysts in cattle. We passively immunoneutralized endogenous estradiol to create a condition in which estradiol-feedback regulation was impaired. We then monitored alterations in LH secretion and follicular development.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Preparation of Estradiol Antiserum

Estradiol antiserum was raised against 1,3,5(10)-estratriene-3,17ß-diol-6-one6-(O-carboxymethyloxime) conjugated to BSA (Steraloids, Inc., Wilton, NH) in a castrated male goat [20]. Cross-reactivity was 100% for estradiol, 42% for estrone, 1.4% for estriol, and less than 0.1% for progesterone and testosterone.

Experimental Design

Animals Protocols for the use of animals in the present study were approved by the Animal Care Committee of the National Institute of Agrobiological Sciences. Twenty-one Japanese black cows with regular estrous cycles (mean body weight ± SEM, 505 ± 15.2 kg) were given two i.m. injections of prostaglandin (PG) F2{alpha} analogue (Estrumate; Sumitomo Pharm., Osaka, Japan) at 8-h intervals 10 days after estrus. Two days before the PGF2{alpha} treatment, the animals were tranquilized with 0.04 mg of xylazine (Celactal; Bayer Japan Co., Tokyo, Japan), and cannulae were inserted into their jugular veins. Forty-eight hours after the first injection of PGF2{alpha}, the animals received an i.v. bolus injection of either 100 ml of estradiol antiserum (immunized group, n = 11) or 100 ml of castrated male goat serum (control group, n = 10) through the indwelling cannula (Day 0 and 0 h = injection of sera).

Blood sampling and determination of ovarian response To clarify the effects of estradiol immunization on LH secretion and follicular growth during the periovulatory period, blood samples were taken via the indwelling jugular cannulae from five control and five immunized cows every 8 h between -72 h (Day -3) and 0 h, then every 4 h between 0 and 48 h (Day 2). The sampling interval was prolonged to every 8 h from 48 to 96 h (Day 4). The ovarian follicles of the 10 cows were examined daily between Days -3 and 10 using an ultrasound scanner (Aloka, Tokyo, Japan) with a 7.5-MHz, linear-array transducer as reported previously [21].

To assess follicular growth and hormonal profiles during a long period following estradiol immunization, the remaining six cows of the immunized group were subjected to daily blood sampling and ultrasound analysis from Days -3 to 60. For analysis of pulsatile secretion of LH, serial blood samples were collected at 15-min intervals for 8 h via the indwelling cannulae on Days 0, 5, 8, 14, 21, 31, 41, and 51. Similar serial blood samples were collected from the remaining five control cows on Day 0 (follicular phase) and on Day 14 (midluteal phase). Plasma was recovered after centrifugation of blood and stored at -30°C.

Time-Resolved Fluoroimmunoassay of Total Inhibin

Hormone preparation and antibody Concentrations of total inhibin in the plasma of the cows were determined by a competitive immunoassay using europium (Eu)-labeled inhibin A as a probe. Anti-bovine inhibin serum (TNDH-1 [22]) was used as a primary antibody. Bovine 32-kDa inhibin A was used for Eu-labeling and as a reference standard (anti-inhibin serum was provided by Dr. Taya, Tokyo University of Agriculture and Technology, Fuchu, Japan; bovine 32-kDa inhibin A was provided by Dr. Hasegawa, Kitasato University, Towada, Japan).

Assay procedures Five micrograms of bovine inhibin A were labeled with Eu-chelate of N1-(p-isothiocyanatobenzyl)-diethylenetriamine-N1,N2,N3,N3-tetraacetic acid (Eu-labeling reagent; Wallac Oy, Turku, Finland) overnight at 37°C according to the manufacturer's instructions. The Eu-labeled inhibin A was separated from the free Eu by gel filtration as reported previously [23]. Anti-bovine inhibin antiserum diluted at 1:30 000 with assay buffer (Tris-buffered saline [TBS; 0.05 M Tris/HCl, pH 7.5, and 0.15 M NaCl] containing 0.05% [w/v] BSA, 0.1% [w/v] bovine {gamma}-globulin, 0.05% [w/v] NaN3, 0.01% [v/v] Tween 40, 0.0015% [w/v] Phenol Red, and 0.02 M diethylenetriaminepentaacetic acid) was pipetted into wells of a 96-microwell plate (FluoroNunc Modules; Nalge Nunc International, Rochester, NY) coated with anti-rabbit immunoglobulin (Ig) G (Chemicon International, Inc., Temecula, CA). The wells were incubated overnight at 25°C and rinsed 10 times with wash buffer (TBS containing 0.1% [w/v] Tween 20 and 0.05% [w/v] NaN3), and then aliquots (100 µl) of the standards (0.156–10 ng/ml) and unknown samples were added. The final volume for each well was 200 µl with assay buffer. For measuring total inhibin in plasma samples, 100 µl of serum from a castrated bull, instead of 100 µl of assay buffer, were added to each well of the standards to correct for the matrix effects of bovine plasma. The wells were incubated overnight at 25°C. After incubation, the wells were washed 12 times, and Eu-labeled inhibin A (1 x 106 counts per second per 100 µl) was added to the wells. The wells were incubated for 2 h at 25°C. After the wells had been washed again 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; Wallac Oy, Turku, Finland).

Validation To determine whether different molecular-weight forms of inhibin cross-react in the fluoroimmunoassay (FIA), 20 µg of inhibin, purified from bovine follicular fluid using immunoaffinity chromatography, were fractionated by SDS-PAGE as described previously [23]. The gel was cut into 1.0-mm slices. Inhibin was extracted from each gel slice with TBS containing 5 mM EDTA under gentle shaking overnight. The gel eluates were assayed for total inhibin. The total inhibin FIA detected immunoreactivity with peak molecular-weight values of 26, 31, 54, or 108 kDa, which indicates that the FIA recognizes several molecular-weight forms of dimeric inhibin in addition to 26-kDa pro-{alpha}C [24, 25]. The detection limit of the time-resolved FIA (Tr-FIA) was 0.078 ng/ml. The intra- and interassay coefficients of variations (CVs) were 7.8% and 11.0%, respectively.

Tr-FIA of Bovine FSH and LH

The concentrations of FSH or LH in the plasma of cows were determined by competitive immunoassays using Eu-labeled FSH or LH as probes [23]. In the Tr-FIA of bovine FSH, anti-bovine FSH ß subunit serum (U.S. Department of Agriculture [USDA]-5-pool [26]) was used as a primary antibody, USDA-bFSH-I2 for Eu-labeling, and USDA-bFSH-I2 as a reference standard. In the LH Tr-FIA, anti-ovine LH serum (USDA-309-684P [27]) was used as a primary antibody, USDA-bLH-I-1 for Eu-labeling, and USDA-bLH-B5 as a reference standard. (Assay materials were provided by the USDA Animal Hormone Program, Germplasm and Gamete Physiology Laboratory, Beltsville Agricultural Research Center, Beltsville, MD.) The intra- and interassay CVs were, respectively, 4.8% and 8.9% for LH and 8.7% and 12.5% for FSH.

Estradiol Antibody Titer Determination

Changes in the concentrations of estradiol antibodies in the circulation were determined by using estradiol antiserum as a standard. Aliquots (100 µl) of the standards (0.078–10 nl/ml) and plasma samples diluted at 1:10 000 with assay buffer were pipetted into wells coated with anti-goat IgG (Chemicon). The wells were incubated overnight at 25°C. After incubation, the wells were washed 12 times, and Eu-labeled 1,3,5(10)-estratriene-3,17ß-diol-6-one6-(O-carboxymethyloxime) conjugated to BSA (2 x 106 counts per second per 100 µl) was added to the wells. Wells were incubated for 2 h at 25°C. After the wells had been washed again 12 times, 100 µl of enhancement solution were added to each well, and fluorescence was measured. Values were expressed as microliters of estradiol antiserum per milliliter of plasma.

Statistical Analyses

The occurrence of a preovulatory LH surge was defined as at least two consecutive increases in LH levels, with the highest value greater than 5 ng/ml and followed by ovulation. Pulsatile secretion of LH was analyzed with a pulsar algorithm [28]. The standard deviation criteria (G) were G(1) 5.0, G(2) 3.0, G(3) 2.0, G(4) 1.5, and G(5) 1.0. Data pertaining to hormonal profiles after estradiol immunization were subjected to ANOVA for repeated measures [29]. To compare pulse frequency and mean concentration of LH between immunized and control cows, LH levels in the two groups were subjected to one-way ANOVA. When a significant effect was obtained with the ANOVAs, the significance of the difference between means was determined by the Tukey test. All data were analyzed using the general linear models procedure of the Statistical Analysis Systems [30]. A value of P < 0.05 was considered to be significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Changes in Concentration of Estradiol Antiserum

The concentration of estradiol antiserum in the circulation was 5.4 ± 0.3 µl/ml (mean ± SEM, n = 6) 1 day after injection of estradiol antiserum (Fig. 1). It then decreased gradually thereafter. At the end of the present study, the concentration of free estradiol antiserum, which bound the Eu-labeled estradiol, was 0.5 ± 0.19 µl/ml. The half-life of the antiserum in the circulation, estimated after logarithmic transformation of the values of circulating estradiol serum, was 30.2 days.



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  		FIG. 1. Changes in plasma concentrations of free estradiol antiserum in cows that received a single i.v. injection of estradiol antiserum (Day 0 = injection of estradiol antiserum). Values are mean ± SEM (n = 6) in terms of microliters of estradiol antiserum per milliliter of plasma

Effects of Estradiol Immunization on LH Secretion and Follicular Development During the Periovulatory Phase

A preovulatory LH surge was detected in the control cows between 24 and 48 h after injection of control serum (72–96 h after the first PGF2{alpha} injection; Fig. 2a). The peak value of the LH surge was 15.5 ± 2.5 ng/ml (n = 5; Fig. 2b). In contrast, estradiol-immunized cows had no LH surge until 96 h after immunization (Fig. 2c). The dominant follicles that emerged before estradiol immunization did not ovulate but persisted and had enlarged to 21.5 ± 1.5 mm (n = 11) in diameter by Day 10. However, ovulation of dominant follicles was detected in all 10 control cows; the maximum diameter of the ovulatory follicles was 12.5 ± 1.2 mm (n = 10).



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  		FIG. 2. Changes in plasma concentrations of LH during the periovulatory period in cows that received a single i.v. injection of (a and b) control serum (CONT) or c) estradiol antiserum (A/E) 48 h after the first injection of PGF2{alpha}. The LH profile is aligned relative to injection of sera (a and c) or relative to the peak of the preovulatory LH surge (b). Values are mean ± SEM (n = 5)

Follicular Development and Hormonal Profiles During> a Long Period after Estradiol Immunization

Five of the six immunized cows subjected to long-term ovarian scanning had multiple follicular waves, with maximum follicular sizes of 20–45 mm at 10- to 30-day intervals for more than 50 days (Fig. 3a). No corpus luteum was observed in the ovary during this period. The remaining cow experienced twin ovulations from the initial persistent dominant follicles on Day 18 (Fig. 3b) and then resumed a normal interwave interval. In association with the emergence of follicular waves with cystic follicles, the circulating levels of total inhibin increased significantly (P < 0.01) and remained high during the growth of the cystic follicles (Fig. 4, a and b). Plasma concentrations of FSH were high before follicle emergence but remained low during the growth of the cystic follicles (Fig. 4, a and c). In the five immunized cows that experienced turnover of cystic follicles for more than 50 days, the LH pulse frequency ranged from 0.81 ± 0.13 pulses/h (Day 21) to 0.97 ± 0.10 pulses/h (Day 41) during the observation (Fig. 5a), comparable to the frequency observed in the follicular phase of control cows (Day 0: 0.73 ± 0.11 pulses/h). The mean concentration of LH was significantly higher in the immunized cows on Day 5 (1.23 ± 0.08 ng/ml) and between Days 21 and 51 (1.28 ± 0.08 to 1.36 ± 0.14 ng/ml) than in the midluteal phase of the controls (Day 14: 0.90 ± 0.17 ng/ml) (Fig. 5b).



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  		FIG. 3. Two patterns of follicular development following injection of estradiol antiserum (A/E; Day 0 = injection of estradiol antiserum). a) An example of a cow that had multiple follicular waves with cystic follicles. b) The cow that experienced twin ovulations of the initial persistent follicles. Open squares signify corpora lutea, and the other symbols signify follicles. Asterisks indicate ovulations



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  		FIG. 4. Development of a) cystic follicles and changes in plasma concentrations of b) total inhibin and c) FSH in estradiol-immunized cows. Values are mean ± SEM (n = 14 waves from five cows that had multiple waves with cysts). Data are aligned relative to the emergence of follicular waves with cystic follicles (Day 0 = follicular emergence)



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  		FIG. 5. The a) pulse frequency and b) mean concentrations of LH in cows after injection of estradiol antiserum. Values are mean ± SEM (n = 5 cows that had multiple waves with cysts). Day 0 indicates the day of injection of control serum or estradiol antiserum (A/E). Control Day 0 corresponds to the normal follicular phase, and Day 14 corresponds to the normal midluteal phase. Values without common superscripts are significantly (P < 0.05) different (Tukey test)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We clearly demonstrated that alteration of LH secretion by perturbation of the estradiol-feedback regulation mechanism induces formation of follicular cysts at a high rate. The lack of LH surge [3] and the failure of positive response of LH to estradiol injection [4–8] in cows with follicular cysts lead consistently to the hypothesis that follicular cysts result from a dysfunction in feedback control of LH secretion by estradiol. Several models of experimentally induced follicular cysts, including treatment with a combination of estradiol and progesterone [1, 17, 31, 32] and with estradiol [5, 6, 33, 34], have been developed. However, many of these models have provided information about the consequences of follicular cysts, but not about the mechanisms leading to their development. Moreover, the key mechanism of the previous studies seems to be the triggering of an LH surge by estradiol injection in the absence of a large follicle [5, 6], followed by alteration in hypothalamic action of estradiol, which involves a different pathway from the above hypothesis. Instead, we directly created a functional abnormality in the estradiol-feedback regulation of LH by giving a single injection of estradiol antiserum and then defined a close relationship between the development of follicular cysts and the LH profile after estradiol immunization. The resultant characteristics of the hormonal profiles and follicular development after estradiol immunization resemble those seen with spontaneously occurring follicular cysts [1–3, 18].

Immunoneutralization of estradiol attenuated the ovulation of the dominant follicle, and the follicle became persistent. Inhibition of the preovulatory LH surge occurred in estradiol-immunized cows, whereas the pulse frequency of plasma LH was comparable to that seen in the follicular phase of control cows. The above results indicate that lack of an LH surge results in the induction of follicular cysts in estradiol-immunized cows despite normal pulsatile secretion of LH. Administration of estradiol antiserum probably severely reduced the amounts of effective estradiol in the hypothalamus, which is involved in the positive-feedback regulation of LH, by eliminating free estradiol in the circulation. These circumstances inhibited the onset of an LH surge, although the ovary produced a significant quantity of estradiol. Multiple neural pathways may be responsible for the deficiency in feedback regulation of LH by estradiol, but our results suggest that a severe reduction in the utility of estradiol in the hypothalamus can cause ovarian cysts. Estrogen-receptor knockout mice have large anovulatory follicles [35, 36], which supports the above idea. Estradiol immunization induced the occurrence of multiple waves of cystic follicles for more than 50 days. Based on the observation that a substantial amount of active estradiol antiserum was still detectable in the circulation at the end of the present study, the immunized cows appeared to continue to have the capacity to neutralize endogenous estradiol, which was involved in inhibition of an LH surge for the long period.

Recent studies in cattle [37, 38] have shown that the profile of GnRH release into the cerebrospinal fluid of the third ventricle corresponds directly to that of peripheral LH. The locations of two different functional hypothalamic areas, a GnRH surge generator and a GnRH pulse generator, have been clarified [39, 40]. In the present study, the surge of LH release, but not the pulsatile secretion of LH, was altered after estradiol immunization. If the above information is considered together, then it may be possible that an alteration in the GnRH surge generator activity is involved in the occurrence of follicular cysts in cattle. Administration of progesterone to cattle with cysts results in resumption of normal cycles [18, 31] accompanied by occurrence of an LH surge [31]. Progesterone treatment also eliminates insensitivity to the positive-feedback effects of estradiol [5]. Progesterone seems to reset the responsiveness of the hypothalamus to estradiol. However, further neuroendocrine studies are required to clarify the mechanisms of control of the activity of the surge generator.

Although pulsatile secretion of LH in estradiol-immunized cattle seems to result from a lack of inhibition of progesterone in an anovulatory situation, the pulsatile secretion at the normal follicular-phase level likely is important for continued growth of follicles. Extension of growth of the dominant follicle is promoted by an LH pulse frequency comparable to that occurring after luteolysis [19, 41]. Conversely, progesterone treatment of cows with follicular cysts reduces the LH pulse frequency and induces atresia of cystic follicles [18, 31, 42, 43]. Similarly, polycystic ovary syndrome is not associated with relatively high FSH secretion in humans [44, 45]. An increase in mRNAs for the LH receptor in granulosa cells also seems to be associated with prolonged growth of cysts and increased estradiol production of cysts [46]. During the prolonged growth of follicles, high total inhibin levels are sustained—a profile similar to that of plasma inhibin A in cows with naturally occurring cysts [23]. These results suggest that production of inhibin A in granulosa cells, as well as production of estradiol [1–3, 23], is sustained by an LH pulse frequency comparable to that in the normal follicular phase and that a combination of inhibin A and estradiol establishes long-term dominance of cystic follicles by suppressing new follicular emergence.

The etiological basis of follicular cysts has not been fully clarified. Injection of ACTH inhibits an LH surge by maintaining plasma progesterone at a subluteal level for several days after luteolysis and induces the formation of persistent follicles [47, 48]. The ACTH-induced progesterone secretion originates from the adrenal gland [49], suggesting that stress can cause follicular cysts. The present study clearly demonstrated that perturbation in estradiol-feedback control of an LH surge induced the formation of follicular cysts. Whether several neurotransmitters, such as opioid peptides, mediate between stress and deficiency in estradiol-feedback regulation of LH is not yet clear.

In summary, immunoneutralization of estradiol resulted in inhibition of the LH surge, whereas pulsatile secretion of LH was within the normal range. The result was a high rate of induction of ovarian cysts. We conclude that lack of an LH surge because of a dysfunction in the positive-feedback regulation of LH is a key to the induction of follicular cysts.


   ACKNOWLEDGMENTS
 
We thank Ms. T. Aoki and Ms. E. Yamauchi for technical assistance. We are grateful to the USDA Animal Hormone Program, Germplasm and Gamete Physiology Laboratory, Beltsville Agricultural Research Center, Beltsville, MD, for providing RIA materials for bovine FSH and LH. We thank Dr. Taya, Tokyo University of Agriculture and Technology, Fuchu, Japan, for providing anti-inhibin serum and also Dr. Hasegawa, Kitasato University, Towada, Japan, for providing bovine 32-kDa inhibin.


   FOOTNOTES
 
1 Supported by a grant from the Ministry of Agriculture, Forestry and Fisheries. Back

2 Correspondence: Hiroyuki Kaneko, Genetic Diversity Division, 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 Back

Received: 29 May 2002.

First decision: 13 June 2002.

Accepted: 26 June 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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