Evidence for a Luteinizing Hormone Surge Center in the Hypothalamus of the Pig'. Robert R. Kraeling, 2,3 Bennett Johnson,3 C. Richard Barb,3 and George B.
BIOLOGY OF REPRODUCTION 58, 1199-1205 (1998)
Evidence for a Luteinizing Hormone Surge Center in the Hypothalamus of the Pig' Robert R. Kraeling, 2,3 Bennett Johnson,3 C. Richard Barb, 3 and George B. Rampacek 4 United States Department of Agriculture, 3 R.B. Russell Agricultural Research Center, Athens, Georgia 30605 Animal and Dairy Science Department, 4 University of Georgia, Athens, Georgia 30602 fore the LH surge is required for accumulation of adequate stores of LH in the anterior pituitary gland. However, it is possible that estradiol-induced surge secretion of LH simply results from loss of the negative feedback inhibition of estradiol on the pulse generator, either by dissipation of estradiol from the system or by development of refractoriness to estradiol, rather than from subsequent positive feedback stimulation of a specific surge generator. Britt et al. [2] reported that pulsatile administration of GnRH from 0 through 96 h after administration of estradiol benzoate (EB) failed to induce an LH surge in gilts passively immunized against GnRH. However, if pulsatile GnRH was administered from 54 through 96 h in similarly treated gilts, an LH surge was induced. In addition, Kesner et al. [3] demonstrated that an LH surge failed to occur in OVX gilts receiving pulsatile GnRH injections from 0 through 48 h after EB injection. Therefore, there may not be a distinct preovulatory surge secretion of GnRH in the pig as has been documented for the rat [4-7], sheep [8], monkey [9, 10], and mare [11]. In that case, an LH surge would occur only after termination of low-dose administration of estradiol, which would inhibit pulsatile LH secretion and therefore mimic the negative phase of the LH surge. Thus, the objective of this experiment was to determine whether the estradiol-induced LH surge is a result of both a negative and positive feedback action on the hypothalamic-pituitary unit. To test this idea, a low dose of EB was injected every 24 h from 0 through 24, 48, 72, or 96 h in OVX gilts. In addition, data were utilized from two dose titration trials conducted to select the minimal effective dose of EB, administered chronically by Alzet osmotic pumps, that would suppress pulsatile LH secretion without inducing a preovulatory-like LH surge in OVX gilts for future experimentation.
ABSTRACT Studies were conducted to determine whether there is an LH surge generator in the hypothalamus of the pig. In experiment 1, 157-day-old ovariectomized (OVX) gilts received 1.5 Fig estradiol benzoate (EB)/kg BW i.m. every 12 h from 0 through 24, 48, 72, or 96 h. Blood was sampled every 6 h from 3 to 36 h and every 3 h from 36 through 144 h. One of 3, 4 of 4, 4 of 4, and 2 of 3 gilts displayed an LH surge after treatment for 24, 48, 72, and 96 h, respectively. With the exception that time to maximum LH concentration was greater in gilts treated for 96 h than in those treated for 72 h (p < 0.05), parameters of the surge were similar among all gilts. In experiment 2a, an Alzet osmotic pump containing EB or vehicle was inserted s.c. behind an ear of 124-day-old OVX gilts, resulting in the following daily doses of EB: 0, 0.75, 1.50, or 3.00 tFg/kg BW. Blood was sampled at 0, 2, 4, 6, and 8 h and every 8 h thereafter through 168 h to evaluate surge secretion of LH, and every 15 min for 8 h starting at 168 h to evaluate pulsatile LH secretion. Zero of 3, 0 of 2, 3 of 3, and 3 of 3 gilts displayed an LH surge after 0, 0.75, 1.50, and 3.00 Ig EB/kg BW, respectively. Parameters of the surge were similar among gilts. Pulsatile LH secretion, evaluated 7 days after pump insertion, was significantly suppressed for estradiol-treated gilts compared to controls. In experiment 2b, at 182 days of age, 10 gilts used in experiment 2a plus 2 additional gilts in the original group prepared but not used for experiment 2a, were randomly assigned in groups (n = 3) to the following daily doses of EB: 0, 0.19, 0.38, or 0.75 ,ug/kg BW, administered again by osmotic pump. Treatment and blood-sampling schedules were the same as in experiment 2a. Zero, 0, 1, and 2 gilts displayed an LH surge after treatment with 0, 0.19, 0.38, and 0.75 ig EB/kg BW, respectively. Parameters of the surge were similar among gilts that displayed an LH surge. Pulsatile LH secretion was significantly suppressed for estradioltreated gilts compared to controls. Thus, the LH surge resulted from positive feedback stimulation of a specific surge generator rather than attenuation or dissipation of negative feedback inhibition of estradiol on a pulse generator.
MATERIALS AND METHODS All procedures performed on live animals were approved by the Animal Care and Use Committee of both the Richard B. Russell Research Center, Agricultural Research Service, USDA, and the University of Georgia. The crossbred (Chester x Yorkshire x Hampshire x Duroc) gilts used in this study were maintained at an ambient temperature of 22°C and an artificial photoperiod of 12L: 12D in individual pens in an environmentally controlled building and were fed 2 kg of a corn-soybean meal diet (14% crude protein) once per day.
INTRODUCTION Surge secretion of LH results after an apparent stimulation of an LH surge generator within the central nervous system (CNS) by elevated estrogen secretion from preovulatory follicles. A single injection of estradiol in ovariectomized (OVX) gilts caused immediate suppression of serum LH concentrations for 48-72 h followed by a sustained, preovulatory-like, surge of LH [1]. Kraeling and Barb [1] concluded that an LH pulse generator and an LH surge generator exist within the CNS of the pig and indicated that suppression of LH secretion beAccepted December 18, 1997. Received October 27, 1997. 'Mention of a trade name, proprietary product, or specific equipment does not constitute a guarantee or warranty by the USDA and does not imply its approval to the exclusion of other products that may be suitable. 'Correspondence: R.R. Kraeling, Animal Physiology Research Unit, USDA/ARS, R.B. Russell Ag Res Ctr, PO Box 5677, Athens, GA 306045677. FAX: (706) 546-3586.
Experiment 1
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Fourteen prepubertal gilts, which had previously displayed an estradiol-induced LH surge, were OVX at 143 days of age. A cannula was placed non-surgically into a jugular vein at 157 days of age and 101.4 ± 2.7 kg BW. Starting at 0800 h (0 h) the next day, gilts received 1.5 jig EB (Sigma Chemical Co., St. Louis, MO)/kg BW i.m. every 12 h from 0 through 24 h (n = 3), 48 h (n = 4), 72 h (n = 4), or 96 h (n = 3).
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TIME (HOURS) FIG. 1. Mean serum estradiol concentrations for prepubertal OVX gilts after receiving 1.5 I.g EB/kg BW i.m. every 12 h from a) 0 through 24 h (n = 3), b) 0 through 48 h (n = 4), c) 0 through 72 h (n = 4), and d) 0 through 96 h (n = 3). The vehicle for EB injection was corn oil. Blood samples were collected every 6 h from Hour 0 to Hour 36 and every 3 h from Hour 36 through Hour 144. All blood serum samples were assayed for LH, and samples collected at 0, 24, 48, 72, 96, and 120 h were assayed for estradiol. Experiment 2a Fourteen prepubertal gilts were OVX at 111 days of age. Eleven of these gilts were randomly assigned to one of the
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FIG. 2. Mean serum LH concentrations for prepubertal OVX gilts after receiving 1.5 jIg EB/kg BW i.m. every 12 h from a) 0 through 48 h (n = 3), b) 0 through 72 h (n = 4), and c) 0 through 96 h (n = 2). following daily doses of EB: control (n = 3), 0.75 [xg/kg BW (n = 2), 1.50 g/kg BW (n = 3), or 3.00 g/kg BW (n = 3). At 124 days of age, a cannula was placed nonsurgically into a jugular vein, and an Alzet osmotic pump, model 2ML2 (Alza Corporation, Palo Alto, CA), containing the appropriate dose of EB or vehicle was inserted s.c. behind the right ear (Time 0). Blood samples were collected via jugular cannula at Hours 0, 2, 4, 6, and 8 and every 8 h thereafter through Hour 168 to evaluate surge secretion of LH. To evaluate pulsatile LH secretion, blood samples were collected every 15 min for 8 h starting at Hour 168. All blood samples were
TABLE 1. Time to emergence of the LH surge, time to maximum serum LH concentration, magnitude of the LH surge, and total LH secreted in OVX gilts injected daily with 1.5 ILg EB/kg BW for 24, 48, 72, or 96 h.
Treatment (h) 24 48 72 96 b
No. of gilts displaying LH surge 0 3 4 2
of 3 of 4 of 4 of 3
Mean + SE. Less than 72 h (p < 0.05).
Time to emergence of surge (h),
Time to maximum LH concentration (h)a
Magnitude of surge (ng/ml)a
Total LH secreted (ng/ml120 h)a
70 + 6 74 + 6 66 + 0
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3.9 + 1.4 3.0 + 0.6 2.4 _+0.4
35.5 + 8.6 31.4 + 6.1 27.6 + 0.3
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Blood samples were allowed to clot overnight at 4C. Samples were then centrifuged at 1800 x g for 15 min, and serum was collected and frozen at -20 0 C until time of assay. LH hormone RIAs were performed as previously described [12, 13]. Intraassay and interassay coefficients of variation, obtained by replicating a single serum pool containing 0.93 ng LH/ml eight times in 6 consecutive assays were 8.7% and 5.4%, respectively. Assay sensitivity was 0.15 ng/ml. Preparations used for iodination and standard curves were USDA-p-LH-B-1. Serum estradiol was also quantified by RIA [14]. Assay sensitivity was 0.3 pg/tube. Intraassay and interassay coefficients of variation, obtained by replicating a single serum pool containing 42.5 pg estradiol/ml six times in 8 consecutive assays, were 16.4% and 20.2%, respectively.
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assayed for LH, and samples collected at Hours 0, 2, 4, 6, 8, and every 8 h thereafter were assayed for estradiol. Animals were 57.2 2.3 kg BW at the start of treatment. Alzet osmotic pumps, model 2ML2, which delivered 2 ml per day, were filled with the appropriate dose of EB in polypropylene glycol vehicle or of vehicle alone and submerged in physiological saline for 48 h before use. Experiment 2b On the basis of results of experiment 2a, it was necessary to perform another dose-response study at lower doses.
The following characterization of an LH surge has been reported previously [15]. Mean baseline serum LH concentration was calculated for each individual animal from samples collected from 24 to 60 h after EB injection. Magnitude of the LH surge was the maximum serum LH concentration after EB injection. Time of emergence of the LH surge was the time when serum LH concentration was equal to or greater than three standard deviations above mean baseline concentration and was followed by increasing serum LH concentrations in subsequent samples. Termination of the LH surge was the time when serum LH concentration was again equal to that at Time 0. The pattern of serum LH concentrations after EB injection was classified as a surge if magnitude was at least 50% greater than the serum LH concentration at Time 0 and the time from emergence to termination of the surge was at least 20 h. Total LH secreted for each individual gilt was expressed as ng LH/(ml-120 h) for experiment 1 and ng LH(ml 152 h) for experiments 2a and 2b, which was a summation of the values determined from 24 h through 144 h after EB injection in experiment 1 and from 24 h through 176 h after EB injection in experiments 2a and 2b, respectively. Total LH secreted was assumed to reflect the amount of LH released from the pituitary gland during the EB-induced surge secretion of LH. In the sequential blood sampling period in experiments 2a and 2b, samples were considered to be part
TABLE 2. Time to emergence of the LH surge, time to maximum serum LH concentration, magnitude of the LH surge, and total LH secreted in OVX gilts administered 0, 0.75, 1.50, or 3.00 vIg EB/kg BW daily for 7 days. Treatment (pIg EB/kg BW per day) 0 0.75 1.50 3.00 a Mean
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of 3 of 2 of 3 of 3
Time to emergence of surge (h)a
Time to maximum LH concentration (h)a
Magnitude of surge (ng/ml) a
67 + 3 64 + 0
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of a pulse if the hormone concentration was 50% greater than a previous nadir [16]. Basal serum LH concentrations were then calculated after removal of all values that were associated with serum LH pulses. Data for time to emergence of the LH surge, time to maximum LH, and total LH secretion during the LH surge in experiments 1, 2a, and 2b and mean serum LH concentration, basal serum LH concentration, number of LH pulses, and LH pulse magnitude for the sequential blood sampling period in experiments 2a and 2b were subjected to analysis of variance, and differences between treatment means were determined using single degree of freedom preplanned comparisons using least-square means [17]. RESULTS Experiment 1 Mean serum estradiol concentrations were above 20 pg/ml at Hour 48 for all gilts and at Hour 72 for gilts treated
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TIME (HOURS) FIG. 5. Mean serum estradiol concentrations for prepubertal OVX gilts during administration of a) 0 Lg EB/kg BW (n = 3), b) 0.19 RpgEB/kg BW (n = 3), c) 0.38 p.g EB/kg BW (n = 3), and d) 0.75 ILg EB/kg BW (n = 3) daily for 7 days administered by Alzet osmotic pump.
for 48, 72, and 96 h (Fig. 1). None of 3, 3 of 4, 4 of 4, and 2 of 3 gilts displayed an LH surge after treatment for 24, 48, 72, and 96 h, respectively (Table 1). In those gilts that displayed an LH surge, with the exception that time to maximum LH concentration was greater in gilts treated for 96 h than in those treated for 72 h (p < 0.05), parameters
TABLE 3. Mean and basal serum LH concentrations and number and magnitude of LH pulses in OVX gilts during infusion of 0, 0.75, 1.50, or 3.00 I.g EB/kg BW per day for 7 days. Treatment (.Lg EB/kg BW per day)
No. of gilts
Mean (ng/ml)
Basal (ng/ml)a
Number of pulses/8 ha
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1203
LUTEINIZING HORMONE SURGE CENTER IN THE PIG
TABLE 4. Time to emergence of the LH surge, time to maximum serum LH concentration, magnitude of the LH surge, and total LH secreted in OVX gilts administered 0, 0.19, 0.38, or 0.75 /Rg EB/kg BW daily for 7 days. Treatment (I.g EB/kg BW per day)
No. of gilts displaying LH surge
Time to emergence of surge (h)a
Time to maximum LH concentration (h)a
0 of3 0of3 1 of 3 2 of 3
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112 104 8
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Magnitude of surge (ng/ml)a
Total LH secreted (ng/ml 14 h)a
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of the surge were similar among all gilts regardless of the length of EB treatment (Fig. 2 and Table 1). Experiment 2a Mean serum estradiol concentrations were above 20 pg/ml at Hour 48 for gilts receiving 1.5 or 3.0 pxg EB/kg BW and at Hour 72 for gilts receiving 3.0 ,pg EB/kg BW (Fig. 3). None of 3, 0 of 2, 3 of 3, and 3 of 3 gilts displayed an LH surge after treatment with 0, 0.75, 1.50, and 3.00 ptg EB/kg, respectively (Table 2). Time to emergence of the LH surge, time to maximum LH concentration, magnitude of the LH surge, and total LH secreted were similar among gilts regardless of the dose of EB administered (Fig. 4 and Table 2). Parameters of pulsatile LH secretion are presented in Table 3. Mean serum LH concentration (p < 0.0005), basal serum LH concentration (p < 0.0005), number of LH pulses (p < 0.005), and pulse magnitude (p < 0.05) were lower for all estradiol-treated gilts compared to controls.
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Experiment 2b Mean serum estradiol concentrations were above 20 pg/ml at Hour 48 and 72 for gilts receiving 0.38 or 0.75 Ixg EB/kg BW (Fig. 5). None of 3, 0 of 3, 1 of 3, and 2 of 3 gilts displayed an LH surge after treatment with 0, 0.19, 0.38, and 0.75 jig EB/kg BW, respectively (Table 4). In those gilts that displayed an LH surge, time to emergence of the LH surge, time to maximum LH concentration, magnitude of the LH surge, and total LH secreted were similar among gilts regardless of dose of EB administered (Fig. 6 and Table 4). Parameters of pulsatile LH secretion are presented in Table 5. Mean serum LH concentration (p < 0.0005), basal serum LH concentration (p < 0.005), number of LH pulses (p < 0.005), and pulse magnitude (p < 0.05) were lower for all estradiol-treated gilts compared to controls. DISCUSSION It is generally accepted that the increasing serum concentration of estradiol secreted by developing preovulatory follicles stimulates the preovulatory surge of LH during the estrous cycle. It is known that signals from the CNS are required to elicit the preovulatory LH surge in the pig, since various methods used to block signals from the CNS, such as anesthesia with pentobarbitone sodium [18], hypophysial stalk transection [19], oral administration of the dithiocarbamoylhydrazine derivative, AIMAX [13], or immunization with GnRH antiserum [2], also blocked surge secretion of LH. However, the preovulatory LH surge may be only a manifestation of simply an attenuation or total loss of the negative feedback inhibition of estradiol on a pulse generator, either by dissipation of estradiol from the system or by development of refractoriness to estradiol, rather than of subsequent positive feedback stimulation of a specific surge generator. Suppression of LH secretion before the LH surge may permit the pituitary gland to accumulate adequate stores of LH for the surge and/or to develop greater sensitivity to resumed pulsatile GnRH secretion. We assume that inhibition of nilkatile T.l-I
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FIG. 6. Mean serum LH concentrations for prepubertal OVX gilts during administration of a) 0.38 Ig EB/kg BW (n = 1) and b) 0.75 jLg EB/kg BW (n = 2) daily for 7 days administered by Alzet osmotic pump.
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1204
KRAELING ET AL.
TABLE 5. Mean and basal serum LH concentrations and number and magnitude of LH pulses in OVX gilts during infusion of 0, 0.19, 0.38, or 0.75 jIg EB/kg BW per day for 7 days. Treatment (lg EB/kg BW per day) 0 0.19 0.38 0.75
No. of gilts
Mean (ng/ml)a
Basal (ng/ml)a
Number of pulses/8 ha
Pulse magnitude (ng/ml)a
3 3 3 3
0.88 ± 0.13 0.19 0.03 d d 0.22 ± 0.02 d 0.17 ± 0.01
0.73 0.17 0.19 0.16
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± SE. Less than 0 jig/kg per day (p < 0.05). c Less than 0 Lg/kg per day (p < 0.005). d Less than 0 ig/kg per day (p < 0.0005). b
Mean serum LH concentrations decreased to approximately 0.4 ng/ml or less by Hour 24 after the first EB injection rather than by Hour 6. Time to emergence of the surge was approximately 71 h rather than approximately 54 h, and time to maximum serum LH concentration was approximately 86 h rather than approximately 66 h. Therefore, since the pulse generator was under prolonged suppression by continuous administration of EB, the subsequent LH surge in all the gilts must have resulted from the activation of a surge generator. The LH surge was not blocked and did not occur at different times in each treatment group relative to the first estradiol injection in experiment 1, 2a, or 2b, as supported by data presented in Tables 1, 2, and 4; and inhibition of pulsatile LH secretion was still evident on the seventh day of EB treatment in experiments 2a and 2b, as supported by data presented in Tables 3 and 5. Therefore, the LH surge was the result of a positive feedback stimulation of a specific surge generator rather than attenuation or dissipation of the negative feedback inhibition of estradiol on the pulse generator. Because the characteristics of the LH surge were similar among all gilts, we conclude that there is a distinct surge generator within the CNS of the pig that must be stimulated by estradiol in order for the LH surge to occur. However, in addition, suppression of pulsatile LH secretion before the LH surge is necessary to permit the pituitary gland to accumulate adequate stores of LH for the surge and/or to develop greater sensitivity to resumed surge secretion of GnRH. These concepts are supported by Kesner et al. [3], who demonstrated that estradiol failed to induce surge secretion of LH in OVX gilts given pulsatile GnRH from 0 through 48 h after estradiol, and also by Britt and coworkers [2], who demonstrated that in gilts passively immunized against GnRH and given estradiol, pulsatile administration of a GnRH analogue from 0 through 96 h failed to stimulate an LH surge but that an LH surge occurred after pulsatile administration of GnRH analogue from 54 through 96 h after estradiol injection. Results of Kesner et al. [3] and Britt et al. [2] indicate that LH secretion must be suppressed for more than 48 h in order for an LH surge to occur. Results from the present study demonstrate that estradiol exhibits both a negative and positive feedback action on LH secretion. Although comparison of serum estradiol concentration between experiments 2a and 2b is confounded with time, generally, serum estradiol concentrations for gilts receiving 0.75 pig/kg BW per day were greater in experiment 2b than in experiment 2a. In addition, serum estradiol concentrations were greater for gilts that received 0.38 pxg/kg BW per day in experiment 2b than for gilts receiving 0.75 g/kg BW per day in experiment 2a. These results support those of Elsaesser et al. [29] and Christenson et al.
[30], who reported that the metabolic clearance rate of estradiol was greater in immature gilts than in peripubertal gilts. Moreover, serum estradiol concentrations must reach the putative threshold of 20 pg/ml and be maintained at that level for approximately 24-48 h in order for an LH surge to occur. This indirect approach to determining whether surge secretion of LH is generated by surge secretion of GnRH is necessary because a direct method of measuring GnRH output from the hypothalamus during the LH surge has not been successfully developed in the pig as it has in the rat, sheep, mare, and monkey. Leshin et al. [31] described temporal profiles of endogenous GnRH and LH secretion in conscious, OVX gilts by simultaneously sampling push-pull perfusates of the anterior pituitary gland and blood from the jugular vein. However, this technique was not successful during the estradiol-induced LH surge because the presence of the cannula appeared to inhibit surge secretion of LH (unpublished results). REFERENCES 1. Kraeling RR, Barb CR. Hypothalamic control of gonadotropin and prolactin secretion in pigs. J Reprod Fertil Suppl 1990; 40:3-17. 2. Britt JH, Esbenshade KL, Ziecik AJ. Roles of estradiol and gonadotropin-releasing hormone in controlling negative and positive feedback associated with the luteinizing hormone surge in ovariectomized pigs. Biol Reprod 1991; 45:478-485. 3. Kesner JS, Price-Taras EA, Kraeling RR, Rampacek GB, Barb CR. Negative feedback as an obligatory antecedent to the estradiol-induced luteinizing hormone surge in ovariectomized pigs. Biol Reprod 1989; 41:409-413. 4. Levine JE, Ramirez VD. In vivo release of luteinizing hormone-releasing hormone estimated with push-pull cannulae from the mediobasal hypothalami of ovariectomized, steroid-primed rats. Endocrinology 1980; 107:1782-1790. 5. Park O-K, Ramirez VD. Spontaneous changes in LHRH release during the rat estrous cycle, as measured with repetitive push-pull perfusions of the pituitary gland in the same female rats. Neuroendocrinology 1989; 50:66-72. 6. Levine JE, Ramirez VD. Luteinizing hormone-releasing hormone release during the rat estrous cycle and after ovariectomy, as estimated with push-pull cannulae. Endocrinology 1982; 111:1439-1448. 7. Sarkar DK, Chiappa SA, Fink G. GnRH surge in pro-oestrous rats. Nature 1976; 264:461-463. 8. Caraty A, Evans NP, Fabre-Nys CJ, Karsch FJ. The preovulatory gonadotrophin-releasing hormone surge: a neuroendocrine signal for ovulation. J Reprod Fertil Suppl 1995; 49:245-255. 9. Xia L, Van Vugt D, Alston EJ, Luckhaus J, Ferin M. A surge of gonadotropin-releasing hormone accompanies the estradiol-induced gonadotropin surge in the rhesus monkey. Endocrinology 1992; 131: 2812-2820. 10. Pau K-YF, Berria M, Hess DL, Spies HG. Preovulatory gonadotropinreleasing hormone surge in ovarian-intact rhesus macaques. Endocrinology 1993; 133:1650-1656. 11. Irvine CHG, Alexander SL. The dynamics of gonadotrophin-releasing hormone, LH and FSH secretion during the spontaneous ovulatory
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