The authors thank Terry Oyama, Margaret Stobie, Greg Katayama and Perry .... McNeilly AS, Freisen HG 1978 Heterologous radioimmunoassay for rabbit ...
0013-7227/90/1273-1176$02.00/0 Endocrinology Copyright © 1990 by The Endocrine Society
Vol. 127, No. 3 Printed in U.S.A.
Gonadotropin-Releasing Hormone and Norepinephrine Release from the Rabbit Mediobasal and Anterior Hypothalamus During the Mating-Induced Luteinizing Hormone Surge* ALAN H. KAYNARD, K.-Y. FRANCIS PAU, DAVID L. HESS, AND HAROLD G. SPIES Division of Reproductive Biology and Behavior, Oregon Regional Primate Research Center, Beaverton, Oregon 97006
ABSTRACT. The release of hypothalamic GnRH in association with the mating-induced LH surge was studied in the rabbit. Push-pull perfusion (PPP) of the mediobasal (MBH) or anterior (AH) regions of the hypothalamus was performed on conscious, unrestrained does for 3 h before and 5 h after exposure to a vasectomized buck. In experiment 1, GnRH concentrations were measured by RIA in 20-min fractions of MBH-PPP. An -100fold increase in GnRH release was observed within 1 h of coitus (pre, 1.15 ± 0.29 pg/ml; peak, 106.67 ± 37.42 pg/ml; n = 6; P < 0.05). Concomitant surges of LH and PRL in the peripheral circulation were observed. In experiment 2, GnRH and norepinephrine (NE) were measured (the latter by radioenzymatic assay) in 10-min fractions of MBH-PPP. A 218% postcoital rise in NE levels (n = 5; P < 0.05) in MBH-PPP accompanied an ~50-fold peak rise in GnRH in the same samples (pre, 1.57 ± 0.23 pg/ml; peak, 76.52 ± 50.14 pg/ml; P < 0.05). MBH-NE, MBH-GnRH, LH, and PRL release began rising within 10 min
T
HE HYPOTHESIS that episodic release of GnRH into the portal vasculature is responsible for basal, pulsatile, nonsurge secretion of LH has been supported by measurement of hypothalamic-GnRH release in rats (1), sheep (2, 3), rabbits (4), and monkeys (5). In recent years, the control of preovulatory-surge secretion of LH has also been intensely investigated. Studies in the rat (6), sheep (7), monkey (8), and woman (9) have observed increases in GnRH secretion at the time of the LH surge. The rabbit represents an especially useful model for the study of GnRH/LH-surge induction since gonadotropin surges are triggered reflexively after coitus allowing the exact time of the surge to be accurately predicted. Fur-
Received March 26, 1990. Address correspondence to: Dr. Alan H. Kaynard, Divisions of Reproductive Biology, and Behavior and Neuroscience, Oregon Regional Primate Research Center, 505 N.W. 185th Avenue, Beaverton, Oregon 97006. * Publication no. 1714 of Oregon Regional Primate Research Center, supported by the National Institutes of Health Grants HD-16631, HD18185, and RR-00163, and NIH Postdoctoral Fellowship HD-07044.
of coitus. In experiment 3, GnRH was measured in 20 min fractions of AH-PPP. Coitus induced a marked rise in AHGnRH release (precoitus, 0.31 ± 0.03 pg/ml; peak, 2.25 ± 0.80 pg/ml; n = 4; P < 0.05) which differed from coitus-induced MBH-GnRH release both quantitatively (t.e. ~7-fold increase for AH us. ~50-100-fold increase for MBH; 50-min lag time for AH us. 2 ng/ml. Mean P4 peaked at 8.14 ± 1.15 ng/ml on day 7-8 day after mating. Plasma P4 in sham-mated does remained unchanged and averaged 0.76 ± 0.04 ng/ml throughout the pre- and postmating observation period. Experiment 1—increased MBH-GnRH mating
release after
Figure 2A shows the mean MBH-GnRH, LH, FSH, and PRL levels in rabbits which were mated; Fig. 2B shows comparable data for the sham-mated group. A clear and pronounced surge in GnRH release occurred in
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FIG. 2. Mean - SE levels of MBHGnRH in PPP and LH, FSH, and PRL in plasma samples from mated (panel A; n = 6) and sham-mated (panel B; n = 7) does in Exp. 1. Time points that are significantly elevated (P < 0.05) over premated levels are indicated by asterisked horizontal bracket. Vertical dashed line indicates beginning of exposure to buck {i.e. mating or sham-mating).
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GnRH AND NE RELEASE DURING PREOVULATORY SURGE
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of basal 80-90 min after mating. Except for ~2.5-3.5 h after mating, MBH-NE remained significantly elevated throughout the post-mating observation period. Individual patterns of MBH-NE release shown in Figure 4 reveal the sustained nature of the rise in mated rabbits. Shammated does expressed smaller variations in NE levels (Fig. 4, no. 288) which were shorter in duration. The pattern of MBH-GnRH release in mated does was similar to that seen in Exp. 1. The mean premating level of MBH-GnRH was 1.57 ± 0.23 pg/ml; within one sample after mating (10 min) MBH-GnRH release had risen significantly (P < 0.05) and mean MBH-GnRH peaked at 76.52 ± 50.14 pg/ml 30 min after mating (~50-fold increase, latency-to-peak 38 ± 2 min). LH secretion also mimicked that seen in Exp. 1 (basal, 0.28 ± 0.02 ng/ml; peak, 26.11 ± 9.22 ng/ml 30 min after mating; ~100-fold increase, latency-to-peak 54 ± 19 min). Due to the higher frequency of MBH-GnRH measurements in this experiment (10 min), the temporal relationship between the rises of MBH-GnRH and LH could be examined more closely. In all mated does but one, MBH-GnRH and LH rose simultaneously {i.e. during the same 10-min interval). All mated does showed the sustained, elevated pattern of MBH-GnRH release seen in Exp. 1 except no. 235 (shown in Fig. 4) that displayed a discontinuous mode of GnRH release. This animal's data account for the somewhat greater variation in mean
slowly (peak, 3.70 ± 1.19 ng/ml 90 min after mating; latency, 113 ± 19 min) and achieved a lesser relative increase (-3.5 fold). Examples of MBH-GnRH patterns from individual mated does are shown in Figure 6. Sham-mating produced no significant alteration in the release of MBH-GnRH, LH, FSH, or PRL. Mean levels of MBH-GnRH before and after sham-mating were 1.49 ± 0.30 pg/ml and 0.71 ± 0.05 pg/ml. LH levels before and after sham-mating were 0.27 ± 0.02 ng/ml and 0.24 ± 0.02 ng/ml, respectively, FSH levels were 1.68 ± 0.11 ng/ml and 1.60 ± 0.05 ng/ml, respectively, and PRL levels were 5.47 ± 0.43 ng/ml and 6.70 ± 0.46 ng/ml, respectively. Experiment 2—increased MBH-NE release after mating Figure 3 presents the mean patterns of MBH-NE, MBH-GnRH, and LH release that were observed in mated (panel A) and sham-mated (panel B) rabbits. Individual patterns of MBH-NE, MBH-GnRH and LH release for three mated and one sham-mated doe are shown in Figure 4. Mating produced an immediate rise in MBH-NE, which reached significance (P < 0.05) 1020 min after coitus (164 ± 40% of basal). Since NE was measured in perfusate pooled from two adjacent PPP samples, timing of the rise could not be determined more accurately. MBH-NE levels reached a peak of 218 ± 31%
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FIG. 4. Individual patterns of MBH-NE and MBH-GnRH in PPP and plasma LH from three mated (nos. 247, 236 and 235) and one sham-mated (no. 288) doe from Exp. 2. Note that, due to high levels, a compressed scale was used to plot MBH-GnRH values for no. 247. See Fig. 3 legend for further details.
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GnRH pattern seen in Exp. 2 compared to Exp. 1. No changes in MBH-NE, MBH-GnRH, or hormone release were observed in sham-mated animals (Figs. 3B and 4). FSH and PRL patterns in Exp. 2 were similar to those observed in Exp. 1 (data not shown). Experiment 3—Increased AH-GnRH release after mating Figure 5 depicts the patterns of mean GnRH and LH in mated does bearing PPCs with tips positioned in the AH. An increase in AH-GnRH release from a premating level of 0.31 ± 0.03 pg/ml occurred 30 min after mating and reached a peak level of 2.25 ± 0.80 pg/ml (~7-fold increase; P < 0.05) by 110 min. Mean latency-to-peak was 70 ± 14 min. Individual patterns of GnRH release for three mated does from Exp. 1 (MBH-PPP) and three
mated does from Exp. 3 (AH-PPP) are shown in Figure 6. Rabbit no. 172 illustrates the variability of the observed magnitude of MBH-GnRH surges. The surge in this animal was smaller than average (peak, ~40 pg/ml) but, as in all animals of this group, the release was sustained throughout the observation period. In all cases MBH-GnRH surges were at least an order of magnitude larger than the coitus-induced rises in AH-GnRH release; furthermore, AH-GnRH rises were intermittent rather than continuous. Hence, AH-GnRH patterns differ from MBH-GnRH patterns both quantitatively (i.e. ~7-fold increase for AH vs. ~50-100-fold increase for MBH; 50 min lag time for AH us. 10 min for MBH) and qualitatively (i.e. surge AH-GnRH release was discontinuous almost returning to baseline between peaks, while surge MBH-GnRH was far less variable). As in Exps. 1
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GnRH AND NE RELEASE DURING PREOVULATORY SURGE
1182 2.5 - AH-GnRH 2.0
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and 2, gonadotropins and PRL surged after mating (data not shown). LH and PRL rose after a 10 min delay, FSH rose more slowly after a 50 min delay. No significant changes in AH-GnRH, LH, FSH, or PRL secretion occurred in sham-mated rabbits. Discussion In the rabbit, coitus triggers a surge release of LH and FSH via a neuroendocrine reflex which is believed to
E n d o • 1990 Vol 127 • No 3
require a neurohumoral signal transmitted by the hypothalamus and received by the pituitary (25). This coitally induced LH surge triggers ovulation and thus ensures the simultaneous presence of both male and female gametes in the female reproductive tract. The technique of PPP has been used previously in our laboratory to measure GnRH release from the hypothalami of conscious rabbits (4, 18, 19). The present study has extended this technique in two ways: 1) by applying it to freely moving rabbits through the use of a tether/fluid-swivel device; and 2) by measuring catecholamine neurotransmitter release within specific brain areas. The results of this study show that a marked increase in the release of GnRH occurs in the mediobasal and anterior regions of the rabbit hypothalamus in response to coitus. This result is consistent with the hypothesis that the preovulatory surges of LH and FSH are brought about by a surge in GnRH release from hypothalamic neurons which is transmitted to the pituitary by the hypothalamohypophyseal portal circulation. The results of the present study are also consistent with PPP studies in the rabbit which have established a close linkage between hypothalamic GnRH release and basal, non-surge secretion of LH (4). Furthermore, increases in GnRH concentration in pituitary stalk plasma (26) and in PPP from the MBH (19) have been observed in conjunction with surge release of LH and ovulation induced by systemic injection of copper ions. In the past several years, the involvement of GnRH in the generation of the preovulatory surge of LH has been studied in a variety of spontaneously ovulating animals.
MBH-GnRH
AH-GnRH
FIG. 6. Individual patterns of GnRH are shown for three does from both Exp. 1 (leftpanel; MBH-PPP) and Exp. 3 (right panel; AH-PPP). Note the difference in scales used for plotting GnRH values from MBH us. AH. Vertical dashed line indicates beginning of exposure to buck (i.e. mating).
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GnRH AND NE RELEASE DURING PREOVULATORY SURGE
Increases in hypothalamic release of GnRH at the time of the LH surge have been observed in the rat (6, 27), monkey (28), sheep (7), and woman (9). Until now, no such studies have been performed in a reflexively ovulating species such as the rabbit. The relative increase in GnRH release observed in association with the preovulatory surges of other species have ranged from 2- to 6fold (6, 7, 27, 29-31). Indeed, the small magnitude of these increases in comparison to the far greater change in LH release has, in part, led investigators to postulate the involvement of additional humoral factors in the production of the LH surge [both hypothalamic (6) and ovarian (7)] and other processes such as GnRH selfpriming (29). Our results indicate that a far greater increase in MBH-GnRH release occurs in the rabbit (~50-100-fold) than has been reported for other species. This rise is proportional to that of LH during the surge (~90-fold) and is similar to that predicted by studies in sheep using exogenous GnRH and estradiol treatments to determine the pituitary requirement for production of a surge (10). As can be seen in Figure 6, some variation in the magnitude of the MBH-GnRH rise is apparent. Individual animal variation may account for these differences; alternatively, differences in the position of the PPC or condition of the PPP site may influence the recovery of MBH-GnRH from brain tissue. However, no discernable correlation was noted between MBH-GnRH levels and PPC position or histological appearance of the perfusion site. The timing of the termination of the coitally-induced MBH-GnRH and LH surges also varied between animals. LH levels began dropping before MBH-GnRH in 2 of 6 does (e.g. Figure 6 no. 172). However, in all mated rabbits, LH levels began dropping in the face of continuing high rates of MBH-GnRH release (i.e. severalfold higher than that seen during the basal, premated period) which suggests that termination of the LH surge is not the result of exhaustion of the hypothalamic GnRH-releasing apparatus. This result is predicted by studies in other animals employing exogenous GnRH treatments to induce LH surges (10). In the present study, a marked increase in the release of GnRH was observed in both the MBH and AH. Cell bodies of GnRH immunopositive nerve cells are found in both areas (32); the majority are in the preopticsuprachiasmatic areas. Large axonal bundles project from these areas to the infundibular-median eminence region. In addition to these major tracts, smaller groups of axons and single fibers are observed to project throughout the hypothalamus and the limbic areas. Immunohistological methods have shown that axon bundles within the AH become invisible shortly after copulation indicating a release of GnRH which substantially reduces internal vesicular stores (32). This conclusion has now been confirmed by our direct measurement of GnRH
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release. The release of GnRH from AH terminals was smaller and more episodic than that observed in the MBH. This is consistent with the smaller number of GnRH-neuronal terminals in this area (32). The observed episodic fluctuations of AH-GnRH release may reflect a true qualitative difference in the activity of GnRH neurons being sampled in the two areas; alternatively, the smaller magnitude of release may allow expression of underlying oscillations which are masked by the massive rate of release in the MBH. The function of GnRH being released within the AH in response to mating cannot be determined from our results, however, its linkage to copulation suggests a reproductive role. Based on studies in the rat, two functions for AH-GnRH release can be postulated. First, AH-GnRH neurons have been hypothesized to comprise a preovulatory surgegenerator in the rat (17) and thus our observation may represent a delivery of GnRH via ultra-short feedback collateral fibers of GnRH neurons projecting to the median eminence. A receptor mediated, ultra-short loop feedback mechanism controlling GnRH secretion has been demonstrated by in vivo and in vitro studies in the rat (33) and in vivo studies in the sheep (34). Second, GnRH administered to the AH of the rat can facilitate sexual behavior (35) suggesting a similar role for endogenously released GnRH in this area. Although no behavioral effects of GnRH have been demonstrated in the rabbit, we have previously shown that AH-GnRH and pituitary LH release are not necessary linked (36) indicating the function of AH-GnRH may not be neurohumoral. The increase in GnRH release seen in the MBH was accompanied by a 2-fold increase in the release of NE in this same brain area. NE is found in the rabbit hypothalamus (37) and the ability of intracerebroventricularly administered NE to evoke an LH surge (38) and adrenergic blocking agent (39) to disrupt the surge has been demonstrated. These reports, along with the observed increase in hypothalamic NE turnover rate after mating (40), establish a physiological role of NE in this process. Furthermore, we have previously measured an estrogendependent effect of locally perfused NE on MBH-GnRH release in the rabbit (18). The relatively small increase in NE release we observed is not surprising considering its extremely fast rate of degradation and re-uptake and assuming it is released within synaptic clefts and not into the less compartmented interstitial spaces around the capillary plexus of the median eminence. In addition, neuroreflexive release of LH and ovulation can still occur in rabbits whose hypothalamic-catecholaminergic system has been impaired, but not eliminated, by 6-hydroxydopamine (41). This suggests that a complete activation (i.e. massive release) of the hypothalamic NE system is not required for mating-induced GnRH release to occur.
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GnRH AND NE RELEASE DURING PREOVULATORY SURGE
Finally, coitus induced a profound and immediate surge in PRL release. This result is in contrast to a previous study by Fuchs et al. (42) which reported an immediate drop in plasma PRL after mating followed by a restoration to premating levels within an hour. Our precoital PRL values were substantially lower than those of Fuchs et al. {i.e. ~1 vs. ~15 ng/ml) indicating that our tethering system may have reduced experimental, stress induced hyperprolactinemia (43), thereby allowing detection of a normal postcoital rise in PRL. The elevated level of PRL we observed for 5 h after coitus is similar to the PRL levels Fuchs et al. reported as occurring before and 1 h after mating. We are unaware of any direct involvement of PRL in controlling gonadotropin release from the rabbit pituitary. However, in the vole, development of a functional corpus luteum is dependent
on a post-mating surge of PRL (44). Since PRL is important in the maintenance of steroidogenesis in the rabbit ovary especially with regard to the corpus luteum (45), the postcoital surge of this hormone may be important for proper ovarian response to the preovulatory gonadotropin surge and subsequent maintenance of pregnancy. In summary, we have observed a mating-induced increase in MBH-GnRH, MBH-NE and AH-GnRH release. Our data support the hypotheses that: 1) hypothalamic GnRH release into the portal circulation is the first hormonal component of the efferent limb of the ovulatory reflex in the rabbit; 2) mating-induced MBHGnRH release is related to an increase in noradrenergic tone in the hypothalamus; and 3) AH-GnRH release is enhanced following mating.
Acknowledgments The authors thank Terry Oyama, Margaret Stobie, Greg Katayama and Perry Gliessman for their technical assistance in performing RIAs, maintaining animals, and the construction and implantation of PPCs. We are grateful to Drs. William Ellinwood, Gordon Niswender, Harold Papkoff and Albert Parlow, and to the National Hormone and Pituitary Program of the NIH for providing reagent for the RIAs. We also thank Dr. Cynthia Bethea for providing reagents and expertise in setting up the REA for NE.
References 1. Levine JE, Duffy MT 1988 Simultaneous measurement of luteinizing hormone (LH)-releasing hormone, LH, and follicle-stimulating hormone release in intact and short-term castrate rats. Endocrinology 122:2211-2221 2. Levine JE, Pau K-Y F, Ramirez VD, Jackson GL 1982 Simultaneous measurement of luteinizing hormone releasing hormone and luteinizing hormone release in unanesthetized, ovariectomized sheep. Endocrinology 111:1449-1455 3. Clarke IJ, Cummins JT 1982 The temporal relationship between gonadotropin releasing hormone (GnRH) and luteinizing hormone (LH) secretion in ovariectomized ewes. Endocrinology 111:17371739
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4. Pau K-YF, Ostead KM, Hess DL, Spies HG 1986 Feedback effects of ovarian steroids on the hypothalamic-hypophyseal axis in the rabbit. Biol Reprod 35:1009-1023 5. Pau K-YF, Hess DL, Kaynard AH, Ji WZ, Gliessman PM, Spies HG 1989 Suppression of mediobasal hypothalamic GnRH and plasma LH pulsatile patterns by phentolamine in ovariectomized rhesus macaques. Endocrinology 124:891-898 6. Sutton SW, Toyama TT, Otto S, Plotsky PM 1988 Evidence that neuropeptide Y (NPY) released into the hypophyseal-portal circulation participates in priming gonadotropes to the effects of gonadotropin-releasing hormone (GnRH). Endocrinology 123: 1208-1211 7. Clark IJ, Thomas GB, Yao B, Cummins JT 1987 GnRH secretion throughout the ovine estrous cycle. Neuroendocrinology 46:82-88 8. Levine JE, Norman RL, Gleissman PM, Oyama TT, Bangsberg DR, Spies HG 1985 In vivo gonadotropin-releasing hormone and serum luteinizing hormone measurements in ovariectomized, estrogen-treated rhesus monkeys. Endocrinology 117:711-721 9. Miyake A, Kawamure J, Aono T, Kurachi K 1980 Changes in plasma LRH during the normal menstrual cycle in women. Acta Endocrinol 93:257-263
10. Kaynard AH, Malpaux B, Robinson JE, Wayne NL, Karsch FJ 1988 Importance of pituitary and neural actions of estradiol in induction of the luteinizing hormone surge in the ewe. Neuroendocrinology 48:296-303 11. Waring DW, Turgeon JL 1983 LHRH self priming of gonadotropin secretion: time course of development. Am J Physiol 244:C410C418 12. Volochin LM, Gallardo AA 1976 Effect of surgical disconnection of the medial basal hypothalamus on post-coital reflex ovulation in the rabbit. Endocrinology 99:959-962 13. Barry J 1977 Characterization and topography of LHRH neurons in the rabbit. Neurosci Lett 2:201-205 14. Bensch C, Lescure H, Dugy B, Gross C 1978 Histofuoremotrie des catecholamines de la couche externe de l'eminence mediane hypothalmique de la lapine. Ann Endocrinol 39:281-302 15. Barraclough CA, Wise PM, Selmanoff MK 1984 A role for hypothalamic catecholamines in the regulation of gonadotropin secretion. Recent Prog Horm Res 40:487-521 16. Kalra PS, Kalra SP 1986 Steroidal modulation of the regulatory neuropeptides: Luteinizing hormone, neuropeptide Y and endogenous opioid peptides. J Steroid Biochem 25:733-740 17. Ramirez VD, Feder HH, Sawyer CH 1984 The role of brain catecholamines in the regulation of LH secretion: a critical inquiry. In: Martini L, Ganong WR (eds) Frontiers in Neuroendocrinology, Raven Press, New York, vol 8:27-84 18. Pau K-YF, Spies HG 1986 Estrogen-dependent effects of norepinephrine on hypothalamic gonadotropin-releasing hormone release in the rabbit. Brain Res 399:15-23 19. Pau K-YF, Spies HG 1986 Effects of cupric acetate on hypothalamic gonadotropin-releasing hormone release in intact and ovariectomized rabbits. Neuroendocrinology 43:197-204 20. Ellinwood WE, Ronnekliev OK, Kelly MJ, Resko JA 1985 A new antiserum with conformational specificity for LHRH: usefulness for radioimmunoassay and immunocytochemistry. Peptides 6:4552 21. Surve AH, Bacso I, Brinckerhoff JH, Kirsch SJ 1976 Plasma levels of progesterone in pseudopregnant rabbits actively immunized with a progesterone-protein conjugate. Biol Reprod 15:343-349 22. Coyle JT, Henry D 1973 Catecholamines in fetal and newborn rat brain. J Neurochem 21:61-67 23. DaPrada M, Zvircher 1976 Simultaneous radioenzymatic determination of plasma and tissue adrenaline, noradrenaline and dopamine within femtomole range. Life Sci 19:1161-1174 24. Axelrod J, Tomchick R 1958 Enzymatic O-methylation of epinephrine and other catechols. J Biol Chem 233:702-705 25. Ramirez VD, Beyer C 1988 The ovarian cycle of the rabbit: Its neuroendocrine control. In: Knobil E, Neill J (eds) Physiology of Reproduction. Raven Press, New York, pp 1873-1892 26. Tsou RC, Dailey RA, McLanahan cS, Parent AD, Tindall GT, Neill JD 1977 Luteinizing hormone-releasing hormone (LHRH) levels in pituitary stalk plasma during the preovulatory gonadotro-
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GnRH AND NE RELEASE DURING PREOVULATORY SURGE pin surge of rabbits. Endocrinology 101:534-539 27. Sarkar DK, Chiappa SA, Fink G, Sherwood NM 1976 Gonadotrophin-releasing hormone surge in pro-oestrous rats. Nature 264:461463 28. Neill JD, Patton JM, Dailey RA, Tsou RC, Tindall GT 1977 Luteinizing hormone-releasing hormone (LH-RH) in pituitary stalk blood of rhesus monkeys: relationship to levels of LH release. Endocrinology 101:430-434 29. Levine JE, Ramirez VD 1982 Luteinizing hormone-releasing hormone release during the rat estrous cycle and after ovariectomy, as estimated with push-pull cannulae. Endocrinology 111:14391448 30. Miyake S, Tasaka K, Sakumoto T, Kawamura Y, Aono T 1983 Estrogen induces the release of luteinizing hormone-releasing hormone in normal cyclic women. J Clin Endocrinol Metab 56:11001102 31. Park O-K, Ramirez VD 1989 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 50:66-72 32. Flerko B, Setalo G, Vigh S, Arimura A, Schally AV 1978 The luteinizing hormone-releasing hormone (LH-RH) neuron system in the rat and rabbit. In: Scott DE, Kozlowski GP, Weindl A (eds) Brain Endocrine Interaction, III. Neural Hormones and Reproduction. Third International Symposium, Wurzburg, 1977, Karger, Basel, pp 108-116 33. DePaolo LV, King RV, Carrillo AJ 1987 In vivo and in vitro examination of an autoregulatory mechanism for luteinizing hormone releasing hormone. Endocrinology 120:272-279 34. Naylor AM, Porter DWF, Lincoln DW 1989 Inhibitory effect of central LHRH on LH secretion in the ovariectomized ewe. Neuroendocrinology 49:531-536 35. Moss RL, McCann SM, Dudley CA 1975 Releasing hormones and sexual behavior. Prog Brain Res 42:26-46 36. Kaynard AH, Pau K-YF, Spies HG Suppression of medial basal hypothalamic GnRH and pituitary LH release in pseudopregnant
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