Changes in Responses to GnRH on Luteinizing Hormone and Follicle Stimulating Hormone Secretion in Prepubertal Heifers

Transcription

1 Journal of Reproduction and Development, Vol. 48, No. 6, 2002 Original Changes in Responses to GnRH on Luteinizing Hormone and Follicle Stimulating Hormone Secretion in Prepubertal Heifers Ken NAKADA 1), Yukikazu ISHIKAWA 1), Toshihiko NAKAO 1)# and Yutaka SAWAMUKAI 1) 1) Department of Veterinary Obstetrics and Gynecology, School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu, Hokkaido , Japan # Present: Graduate School for International Development and Cooperation, Hiroshima University, Higashi-Hiroshima, Hiroshima , Japan Abstract. This study was carried out to investigate changes in pituitary response to GnRH on gonadotrophin secretion in prepubertal heifers. A total of 50 prepubertal Holstein-Friesian heifers were treated with 1 µg/kg GnRH intravenously at 1, 2, 4, 6 and 8 months of age, and plasma samples were collected from the jugular vein at 0 to 360 min after GnRH treatment. Plasma concentrations of LH and FSH were measured by RIA. Increase in concentrations of LH and FSH, induced by GnRH, were observed in all heifers from 1 month of age, and the concentrations of LH and FSH at 30 min after GnRH treatment were significantly greater than those before GnRH treatment in heifers of all ages. The time from the GnRH administration to the appearance of the peak of LH and FSH rise was similar at each age and, was prolonged with age. The peak concentration and the amount of release from the pituitary of LH rise increased with age, while those of FSH had a tendency to decrease with age. These results indicate that the pituitary gland already has reactivity to GnRH in heifers by 1 month of age, that the capacity of LH release to GnRH in the pituitary gland develops with age, and that the regulatory mechanism for the secretion of LH and FSH develops differently in prepubertal heifers. This study suggests that the development of the capacity to secrete LH secretion in response to GnRH in the pituitary gland before puberty is one of the factors for deciding the time of the onset of puberty in heifers. Key words: GnRH, LH, Pituitary gland, Puberty (J. Reprod. Dev. 48: , 2002) t the puberty, LH surge release in response to GnRH surge release occurs in the pituitary gland, and then the rapid burst increase in peripheral LH concentration induces the first ovulation from fully-grown follicles in the ovary. Before the first ovulation at puberty, the hypothalamus-pituitary-gonad axis as main regulating system of reproduction has to govern. It is generally considered that the final component of the reproductive endocrine system to develop, that Accepted for publication: May 24, 2002 Correspondence: K. Nakada regulates the time of the onset of puberty in heifers and ewes, is the hypothalamus [1 3]. The hypothalamic content of GnRH and the number of pituitary receptors for GnRH do not change during sexual maturation in heifers [4]. The final component for the onset of puberty is thought to be the development of a GnRH releasing pattern develop like in sexually mature cattle. The pattern of GnRH release decides secretion of gonadotrophin in the pituitary gland [5, 6]. Peripheral circulating hormones, estradiol-17β and inhibin, directly or indirectly regulate GnRH and

2 546 NAKADA et al. gonadotrophin secretion in prepubertal heifers [7 9]. Nakada et al. [10] clarified the changes in the peripheral plasma concentrations of reproductive hormones of the heifer from birth to puberty. The plasma estradiol-17β concentration was low in value until 1 month before puberty, and then gradually increased until puberty. Nevertheless, peripheral estradiol in peripubertal heifers was lower than that in sexually mature cattle and was thought to be a potent ovarian factor inhibiting tonic LH secretion [7, 8]. The strong negative feedback action of estradiol on LH secretion decreased around puberty [11]. As a result, the increase in pulse frequency and basal concentration of LH stimulates follicular growth and the activity of steroid hormone synthesis in the ovary of prepubertal heifers from 50 days before puberty [4]. Peripheral concentrations of estradiol-17β from follicles increase following follicular growth around puberty. Nakada et al. [12] showed that responsiveness of estradiol on LH surge secretion already had in 3 months of age and increased with age in prepubertal heifers. The time from increase in peripheral estradiol-17β concentration to the appearance of LH rise shortened and the peak concentration of LH surge increased with age in prepubertal heifers [12]. The time and the peak concentration of LH rise after estradiol treatment in heifers of 9 months of age became the same as those in sexually mature cattle [12]. These changes with sexual maturation are thought to be one of the most important factors for the appearing time of sequential reproductive events, such as estrous behavior, mating or insemination, ovulation and fertilization. Nevertheless, what it relates to the changes in estradiol positive effect on LH surge release has not yet been clarified. Recently, for the effective utilization of bioresources, cattle in a nonreproductive cycle or immature stage have been tried to use as donors of embryos or unfertilized oocytes for in vitro fertilization or nuclear transfer [13 17]. In the case of immature or prepubertal heifers, follicular growth in the ovary and ovulation are stimulated by treatment with exogenous gonadotrophin and GnRH reagent. However, it is not possible to obtain an expecting good result from the ovarian and/or pituitary responsiveness to these reagents without stabilizing in prepubertal heifers. One problem is thought that the responsiveness to these hormone reagents in prepubertal heifers is quite different from that in sexually mature cattle. The changes in the responsiveness in reproductive organs to reproductive hormones during sexual maturation give us good information, when the superovulation technique is applied in sexually immature heifers. The present study was designed to clarify the changes in the responsiveness of gonadotrophin secretion in the pituitary gland in response to GnRH stimulation in prepubertal heifers, and to discuss the relevance to the changes in the responsiveness of LH in the pituitary gland of the changes in other peripheral hormone concentrations. Materials and Methods Animals and treatments Fifty Holstein-Friesian heifers were used for this study and divided into five groups, 10 head in each of 1, 2, 4, 6 and 8 months of age. Animals were maintained in paddocks and were fed to allow body weight gains of 0.65 kg/d. During blood sample collection, animals were restrained in stanchions to which they had been previously acclimated. Except during the sampling periods, feed and water were provided ad libitum. All heifers were healthy and in good condition during this study. One micrograms per 1 kg body weight of GnRH (Biogenesis, UK) in 5 ml saline were intravenously injected into heifers at 1, 2, 4, 6 and 8 months of age. Blood sampling Blood samples were obtained from the jugular vein at 30 min before GnRH treatment, and from GnRH treatment (0) to 360 min after GnRH treatment. Plasma was immediately removed by centrifugation (1700 g, 20 min) and stored at 30 C until required for hormone assay. Assay procedures Assay of plasma concentrations of LH involved anti-ovine LH serum [18], USDA-bLH-B-6 for radioiodination and USDA-bLH-B-6 as a reference standard (modified Echternkamp et al. [19]). Assay of plasma concentrations of FSH involved antibovine FSH β-subunit antiserum (USDA-5-pool), USDA-bFSH-I-2 for radioiodination and USDAbFSH-I-2 as a reference standard (modified Bolt

3 PITUITARY RESPONSE TO GnRH IN PREPUBERTAL HEIFERS 547 and Rollins [20]). The sensitivity of the assay for LH and FSH was 7.2 pg and 6.3 pg per tube, respectively, and the intra- and inter-assay coefficients of variation, calculated according to the methods employed by Rodbard [21], were 8.6% and 11.3% for LH and 4.9% and 12.2% for FSH, respectively. Statistical analysis Results were subjected to analysis of variance for repeated measures [22]. When a significant effect was obtained using analysis of variance, the significance of the difference between two means was tested using Student s t-test. When more than two means were compared, the significance of the difference between means was determined by Duncan s multiple range test. All data were analyzed using the Excel Statistical Analysis Systems. A value of P<0.05 was considered to be significant. Results Changes in LH responses after GnRH treatment Changes in plasma concentration of LH after GnRH treatment in prepubertal heifers are shown in Fig. 1, and the characteristics of LH responses to GnRH are listed in Table 1. The basal concentrations of LH at just before GnRH treatment gradually increased with age. The responsiveness of LH release to GnRH was observed in all heifers of 1 month of age. The peripheral concentration of LH at 30 min after intravenous injection of GnRH was significantly higher than that before injection in heifers of all ages, and gradually increased with age from 2.68 ± 0.55 ng/ml at 1 month to ± 1.46 ng/ml at 8 months of age. The persistence of Fig. 1. Changes in plasma LH concentrations in prepubertal heifers after GnRH treatment. (A) 1, (B) 2, (C) 4, (D) 6 and (E) 8 months of age are shown. Arrows show the time of GnRH injection. Asterisks indicate that values are significantly higher than the value at the time of GnRH treatment (time=0) in each group. Values are mean ± SEM for 10 heifers in a group. Table 1. Characteristics of changes in plasma LH concentrations in response to GnRH treatment in prepubertal heifers Months of age n Basal [LH] d Time to LH Peak [LH] Ratio peak/basal Peak increment (ng/ml) peak (min) e (ng/ml) [LH] (ng/ml) f AUC g ± 0.11* a 36 ± 4 a 2.89 ± 0.72 a 6.68 ± ± 0.71 a ± 92.9 a ± 0.11 a 36 ± 4 a 4.07 ± 0.76 a 9.36 ± ± 0.79 a ± 57.2 a ± 0.16 ab 48 ± 11 ab 5.98 ± 0.72 ab ± ± 0.59ab ± ab ± 0.12 bc 45 ± 11 a 9.63 ± 2.89 bc 9.12 ± ± 2.80 bc ± b ± 0.21 c 72 ± 14 b ± 2.48 c ± ± 2.35 c ± c *: mean ± S.E.M. a, b, c: Values in same columns with different letter (P<0.05). d: LH concentrations. e: Time from GnRH treatment to appearance of LH peak. f: Concentration of increment from basal LH (time 0) to peak of LH rise after GnRH treatment. g: Areas under the curve, The values are accumulated LH concentrations from 0 to 360 min after GnRH treatment.

4 548 NAKADA et al. significant increase in LH concentration to the basal concentration was within 90 min at 1 and 2 months of age, and 120 min at more than 4 months of age. The time to the appearance of the peak of LH rise after GnRH treatment became longer with age and the time in 8 months heifers was significantly longer than that in other ages. The concentrations of the peak of LH rise and the amount of LH release for 360 min after GnRH treatment significantly increased with age and these values at 8 months of age were more than 4 fold greater than those at 1 month of age. Furthermore, the ratio of peak LH concentration to basal LH concentration was not significantly different among ages, although the increment from the basal concentration to the peak concentration of LH rise gradually increased with age. Changes in FSH responses after GnRH treatment Changes in plasma concentration of FSH after GnRH treatment in prepubertal heifers are shown in Fig. 2, and the characteristics of FSH responses to GnRH are listed in Table 2. The basal concentrations of FSH at just before GnRH treatment gradually decreased with age. The responsiveness of FSH release to GnRH was observed in all heifers of 1 month of age. The peripheral concentration of FSH at 30 min after intravenous injection of GnRH was significantly higher than that before injection in heifers of all ages, and gradually decreased with age from 1.71 ± 0.23 ng/ml at 1 month to 0.74 ± 0.11 ng/ml at 8 months of age. The FSH concentrations at 30 min after GnRH were significantly different between 1 or 2, and 4 to 8 months of age. The persistence of significant increase in FSH concentration to the basal concentration was within 120 min in heifers, Fig. 2. Changes in plasma FSH concentrations in prepubertal heifers after GnRH treatment. (A) 1, (B) 2, (C) 4, (D) 6 and (E) 8 months of age are shown. Arrows show the time of GnRH injection. Asterisks indicate that values are significantly higher than the value at the time of GnRH treatment (time=0) in each group. Values are mean ± SEM for 10 heifers in a group. Table 2. Characteristics of changes in plasma FSH concentrations in response to GnRH treatment in prepubertal heifers Basal [FSH] d Time to FSH peak Peak [FSH] Ratio peak/basal Peak increment Months of age n (ng/ml) (min) e (ng/ml) [FSH] (ng/ml) f AUC g ± 0.08* a 36 ± 4 ab 1.81 ± ± ± ± ± ± 3 a 1.59 ± ± ± ± ± ± ± ± ± ± ± 0.03 b 54 ± 11 b 1.24 ± ± ± ± ± ± 13 c 1.10 ± ± ± ± 24.7 *: means ± S.E.M. a, b, c: Values in same columns with different letter (P<0.05). d: FSH concentrations. e: Time from GnRH treatment to appearance of FSH peak. f: Concentration of increment from basal FSH (time 0) to peak of FSH rise after GnRH treatment. g: Areas under the curve, The values are accumulated FSH concentrations from 0 to 360 min after GnRH treatment.

5 PITUITARY RESPONSE TO GnRH IN PREPUBERTAL HEIFERS 549 except at 2 months of age. The time to the appearance of the peak of FSH rise after GnRH treatment became longer with age and the time in 8 months heifers was significantly longer than that in other ages. The concentrations of the peak of FSH rise and the amount of FSH release for 360 min after GnRH treatment gradually decreased with age. The peak concentration of FSH at 8 months was 60% of that at 1 months of age. The ratio of peak FSH concentration to basal FSH concentration and increment from the basal concentration to the peak concentration of FSH rise did not change with age. Discussion The results obtained from this study indicate that prepubertal heifers from 1 month of age can respond to GnRH with LH and FSH surge release. In both LH and FSH responses to GnRH, the time of the appearance of the peak concentration after GnRH injection was similar in the same age groups in prepubertal heifers, and the concentrations of LH and FSH 30 min after intravenous injection of GnRH were significantly higher than those at the time of injection in all age groups. These findings indicate that GnRH can stimulate gonadotrophin release via GnRH receptors on gonadotropes, that were the cells of origin for gonadotrophin synthesis, and that the response to GnRH in the anterior pituitary gland exists in relative young sexually immature female cattle. Foster [23] showed that sheep fetus in the latter half of gastation could release LH and FSH after exogenous GnRH treatment. Mueller et al. [24] demonstrated that the responsiveness in the pituitary to GnRH increased in ewes until 3 weeks of age. In cattle, GnRH administration induced LH and FSH release from 1 month of age [3], and the numbers of pituitary receptors and binding affinity for GnRH did not change during sexual maturation [4]. These previous data show that GnRH receptors already exist in the anterior pituitary gland at birth in ruminants. Our data support these previous results, and taken together they suggest that cattle, which are the ruminants like sheep, have responsiveness to GnRH during the fetus stage, and at least by 1 month of age. In our study, the peak concentration of LH rise induced by injection of GnRH gradually increased with age in prepubertal heifers. Kinder et al. [11] showed that the strong negative feedback action of estradiol on LH secretion was observed in prepubertal heifers and then decreased around puberty. Day et al. [4] indicated that the increase in pulse frequency and basal concentration of LH in prepubertal heifers from 50 days before puberty stimulates follicular growth and activity of steroid hormone synthesis in the ovaries. GnRH, which is released in the median eminence, stimulates gonadotrophin synthesis in gonadotropes in the pituitary gland. LH, synthesized by GnRH, is stored in small vesicles in gonadotropes and is then released from gonadotropes into peripheral blood in a one-to-one mode, by GnRH stimulation [5]. The pattern of GnRH release has 2 types: that one is pulse, and the other is surge [25 28]. In this study, after a sufficient amount of GnRH was injected singly to prepubertal heifers, surge LH release from the pituitary gland was observed after a transient increase in GnRH concentration. Therefore, it is possible to regard the increase in the LH concentration 30 min after GnRH administration with the age as corresponding with an increase in LH accumulation in the pituitary gland, indicating that the LH accumulation in the pituitary gland increases with age in prepubertal heifers. The peak concentration of LH rise and the areas under the curve (AUC) for 360 min after GnRH administration, as total amount of LH release, increased with the age, too. Furthermore, the time from GnRH administration to the LH peak appearance and the duration of the rise to basal LH concentration prolonged with age. These results also indicate that LH is accumulated in the pituitary gland and that the responsiveness for LH release to GnRH increases with age. The results concerning the changes in FSH concentrations after GnRH administration showed that there was a tendency for the peak FSH concentration and AUC of FSH rise to decrease with age. In sexually mature cattle, the main regulating factors of FSH secretion are GnRH as stimulating factor, and inhibin and estradiol-17β as inhibiting factors [5, 29]. Kaneko et al. [9] showed that inhibin had inhibitory effects on FSH secretion from 60 days of age. In our result, the basal FSH concentration in heifers until 4 months of age was higher than that in cyclic cows, thus it is thought that the circulating FSH concentration in immature heifers is sufficient to induce new follicular growth. Evans et al. [30] indicated that follicular wave with

6 550 NAKADA et al. preceding FSH rise was observed in heifers from 2 weeks of age, and that the diameter of the largest follicle in ovaries gradually increased with age in prepubertal heifers until 8 months of age. Nakada et al. [10] showed that regular FSH rises with 7- to 8- day intervals were observed from 10 days to 8 months of age, and that the peak concentration of the FSH rise gradually decreased with age in prepubertal heifers until 8 months of age. These results indicate that there are some factors inhibiting FSH secretion in the pituitary gland from ovarian follicles with follicular growth in prepubertal heifers, and that the inhibiting effects on FSH secretion by the factors grow stronger with age in prepubertal heifers from 10 days to at least 8 months of age. In an in vitro study on male rats, administration of GnRH pulses at a relatively rapid frequency (e.g., every 30 minutes) elicited maximal stimulation of gonadotropin α and LH β subunit mrna, whereas less frequent administration (e.g., every 120 minutes or more) failed to affect the expression of those mrnas [31, 32]. In contrast, lower frequencies of GnRH administration clearly signalled expression of the FSH β subunit mrna, whereas more rapid frequencies had no effect on FSH β mrna [31, 32]. In prepubertal heifers, frequencies of LH pulses increased with age [4]. These results suggest that GnRH pulse frequency increases with age, and that FSH synthesis in the pituitary grand decreases with age in prepubertal heifers. Therefore, the results of our study are in agreement with these findings since the FSH peak concentrations and AUC after GnRH stimulation gradually decreased with age in prepubertal heifers from 1 month to 8 months of age. In summary, these data indicate that gonadotrophin secretion in response to GnRH on is available in prepubertal heifers at a relatively young age, and that LH secretion in response to GnRH stimulation increases and FSH secretion decreases with age in prepubertal heifers. These are perhaps the mechanisms responsible for prepubertal decline in negative effects of ovarian factors on secretion of LH, and for prepubertal incline in negative ones on secretion of FSH in the pituitary gland of prepubertal heifers. Future studies to evaluate the importance of these changes for regulation of the timing of onset of puberty as well as the role of other hormones in the pubertal process of heifers are required. Acknowledgements We are grateful to Dr. Y. Mori (Laboratory of Veterinary Ethology, University of Tokyo, Japan) for providing antiserum to ovine LH (YM#18); and to Dr. D. J. Bolt (USDA Animal Hormone Program, USA) for supplying generous amounts of FSH and LH assay reagents, and of antiserum to bovine FSH β-subunit (USDA-5-pool). We also thank Mr. Kenji Yagi for his technical assistance, and the staff of the dairy farm of Rakuno Gakuen University for caring for the animals. This work was supported in part by a Grant-in-Aid to Cooperative Research from Rakuno Gakuen University, by a grant from the Morinaga Hoshi-Kai, and by a Sasakawa Scientific Research Grant from The Japan Science Society. References 1. Foster DL, Ryan KD, Goodman RL, Legan SJ, Karsch FJ, Yellon SM. Delayed puberty in lambs chronically treated with oestradiol. J Reprod Fertil 1986; 78: Kinder JE, Day ML, Kittok RJ. Endocrine regulation of puberty in cows and ewes. J Reprod Fertil Suppl 1987; 34: Schams D, Schallenberger E, Gombe S, Karg H. Endocrine patterns associated with puberty in male and female cattle. J Reprod Fertil Suppl 1981; 30: Day ML, Imakawa K, Wolfe PL, Kittok RJ, Kinder JE. Endocrine mechanisms of puberty in heifers. Role of hypothalamo-pituitary estradiol receptors in the negative feedback of estradiol on luteinizing hormone secretion. Biol Reprod 1987; 37: Roche JF. Control and regulation of folliculogenesis a symposium in perspective. Rev Reprod 1996; 1: McNeilly JR, Brown P, Clark AJ, McNeilly AS. Gonadotrophin-releasing hormone modulation of gonadotrophins in the ewe: evidence for differential effects on gene expression and hormone secretion. J Mol Endocrinol 1991; 7: Day ML, Imakawa K, Garcia Winder M, Zalesky DD, Schanbacher BD, Kittok RJ, Kinder JE.

7 PITUITARY RESPONSE TO GnRH IN PREPUBERTAL HEIFERS 551 Endocrine mechanisms of puberty in heifers: estradiol negative feedback regulation of luteinizing hormone secretion. Biol Reprod 1984; 31: Dodson SE, McLeod BJ, Haresign W, Peters AR, Lamming GE. Endocrine changes from birth to puberty in the heifer. J Reprod Fertil 1988; 82: Kaneko H, Nakada K, Akagi S, Watanabe G, Taya K, Hasegawa Y. Effects of immunoneutralization of inhibin on FSH secretion in prepubertal calves. Biol Reprod Suppl 1996; 54: Nakada K, Moriyoshi M, Nakao T, Watanabe G, Taya K. Changes in concentrations of plasma immunoreactive follicle-stimulating hormone, luteinizing hormone, estradiol-17beta, testosterone, progesterone, and inhibin in heifers from birth to puberty. Domest Anim Endocrinol 2000; 18: Kinder JE, Bergfeld EG, Wehrman ME, Peters KE, Kojima FN. Endocrine basis for puberty in heifers and ewes. J Reprod Fertil Suppl 1995; 49: Nakada K, Moriyoshi M, Nakao T. Changes in peripheral levels of luteinizing hormone and follicle stimulating hormone in prepubertal heifers after estradiol treatment. J Reprod Dev 2001; 47: Fry RC, Simpson TL, Squires TJ. Ultrasonically guided transvaginal oocyte recovery from calves treated with or without GnRH. Theriogenology 1998; 49: Seneda MM, Esper CR, Garcia JM, Oliveira JA, Vantini R. Relationship between follicle size and ultrasound-guided transvaginal oocyte recovery. Anim Reprod Sci 2001; 67: Bousquet D, Twagiramungu H, Morin N, Brisson C, Carboneau G, Durocher J. In vitro embryo production in the cow: an effective alternative to the conventional embryo production approach. Theriogenology 1999; 51: Ooe M, Rajamahendran R, Boediono A, Suzuki T. Ultrasound-guided follicle aspiration and IVF in dairy cows treated with FSH after removal of the dominant follicle at different stages of the estrous cycle [corrected]. J Vet Med Sci 1997; 59: Meintjes M, Bellow MS, Broussard JR, Paul JB, Godke RA. Transvaginal aspiration of oocytes from hormone-treated pregnant beef cattle for in vitro fertilization. J Anim Sci 1995; 73: Mori Y, Kano Y. Changes in plasma concentrations of LH, progesterone and oestradiol in relation to the occurrence of luteolysis, oestrus and time of ovulation in the Shiba goat. J Reprod Fertil 1984; 72: Echternkamp SE, Bolt DJ, Hawk HW. Ovarian and pituitary hormones in blood of progestogen-treated ewes. J Anim Sci 1976; 42: Bolt DJ, Rollins R. Development and application of a radioimmunoassay for bovine follicle-stimulating hormone. J Anim Sci 1983; 56: Rodbard D. Statistical quality control and routine data processing for radioimmunoassay and immunoradiometric assays. Clin Chem 1974; 20: Glantz SA, Slinker BK. Primer of applicated Regression and Analysis of Variance. N. Y.: McGraw-Hill, Foster DL. Puberty in the sheep. In: Knobil E, Neill JD (eds.), The Physiology of Reproduction. N. Y.: Raven Press Ltd., Mueller PL, Sklar CA, Gluckman PD, Kaplan SL, Grumbach MM. Hormone ontogeny in the ovine fetus. IX. Luteinizing hormone and folliclestimulating hormone response to luteinizing hormone-releasing factor in mid- and late gestation and in the neonate. Endocrinology 1981; 108: Domanski E, Chomicka LK, Ostrowska A, Gajewska A, Mateusiak K. Release of luteinizing hormone-releasing hormone, β-endorphin and noradrenaline by the nucleus infundibularis/ median eminence during preovulatory GnRH surge. Neuroendocrinology 1991; 54: Clarke IJ, Thomas GB, Yao B, Cummings JT. GnRH secretion throughout the ovine estrous cycle. Neuroendocrinology 1987; 46: Goodman RL. Neuroendocrine control of the ovine estrous cycle. In: Knobil E, Neill JD (eds.), The Physiology of Reproduction. N. Y.: Raven Press Ltd., Moenter SM, Caraty A, Lacatelli A, Karsch FJ. Pattern of gonadotropin-releasing hormone (GnRH) secretion leading up to ovulation in the ewe: existance of a preovulatory GnRH surge. Endocrinology 1991; 129: Kaneko H, Kishi H, Watanabe G, Taya K, Sasamoto S, Hasegawa Y. Changes in plasma concentrations of immunoreactive inhibin, estradiol and FSH associated with follicular waves during the estrous cycle of cow. J Reprod Dev 1995; 41: Evans AC, Adams GP, Rawlings NC. Follicular and hormonal development in prepubertal heifers from 2 to 36 weeks of age. J Reprod Fertil 1994; 102: Haisenleder DJ, Katt JA, Ortolano GA, Elgewely MR, Duncan JA, Dee C, Marshall JC. Influence of ponadotropin-releasing hormone pulse amplitude, frequency, and treatment duration on the regulation of luteinizing hormone (LH) subunit messenger ribonucleic acids and LH secretion. Mol Endocrinol 1988; 2: Marshall JC, Dalkin AC, Haisenleder DJ, Paul SJ, Ortolanl GA, Kelch RP. Gonadotropin-releasing hormone pulses: regulators of gonadotropin synthesis and ovulatory cycles. Recent Prog Horm Res 1991; 47: