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Catecholamine synthesizing enzymes in the hypothalamus during the estrous cycle
Brain Research, 196 (1980)437-445
437
© Elsevier/North-Holland Biomedical Press
CATECHOLAMINE SYNTHESIZING ENZYMES IN THE HYPOTHALAMUS D U R I N G T H E ESTROUS CYCLE
LAURENCE A. CARR and JAMES L. VOOGT Department of Pharmacology and Toxicology, University of Louisville Health Sciences Center, Louisville, Ky. 40292 and Department of Physiology, University of Kansas Medical Center, Kansas City, Kans. 66103 (U.S.A.)
(Accepted March 6th, 1980) Key words: estrous cycle - - LH - - prolactin - - tyrosine hydroxylase - - dopamine-fl-hydroxylase--
catecholamine
SUMMARY The activities of tyrosine hydroxylase and dopamine-fl-hydroxylase were measured in the medial basal hypothalamus and remaining hypothalamic tissue of female rats at various times during diestrus 2, proestrus and estrus. Tyrosine hydroxylase activity in the medial basal hypothalamus was significantly lower at 12.00 h compared with other times on proestrus. This decrease preceded the elevation of serum prolactin and L H during the afternoon of proestrus. Tyrosine hydroxylase activity did not change significantly during diestrus 2 or estrus nor was it altered at any time in the remainder of the hypothalamus. Dopamine-fl-hydroxylase activity in the basal medial hypothalamus was significantly elevated at 12.00 h on proestrus and at 14.00 h on diestrus. The results provide further evidence for a decrease in dopaminergic neuron activity in the medial basal hypothalamus which may precipitate the series of events leading to the L H surge during proestrus. The increase in dopamine-fl-hydroxylase activity suggests that an increase in noradrenergic neuron activity may also be involved in triggering the release of LH.
INTRODUCTION There is extensive evidence that catecholamine-containing neuronal systems terminating in the hypothalamus may regulate or modulate the release of gonadotropins during the estrous cycle. In the rat, noradrenergic pathways appear to stimulate the release of LH. This is supported by the findings that turnover of norepinephrine is increased during proestrus in the hypothalamus 9 and subependymal
438 layer of the median eminence17. In addition, normetanephrine concentrations in the hypothalamus increased during proestrus, suggesting enhanced release of norepinephrine 1°. Administration of FLA-63 or diethyldithiocarbamate, inhibitors of dopamine-fl-hydroxylase, have been shown to block ovulation and/or LH release in cycling rats13,8~. The tuberoinfundibular dopaminergic system, on the other hand, appears to exert an inhibitory influence on LH release as suggested by decreased turnover of dopamine in these neurons during proestrus 1,17. Other studies, however, suggest that a dopaminergic stimulus is involved in the release of LH during proestrus 6,3°. The surge of LH release which occurs on the afternoon of proestrus is also accompanied by increased release of prolactin which may be mediated by a noradrenergic stimulus16 and inhibited by a dopaminergic mechanism36. The purpose of the present study was to monitor changes in the activity of two enzymes involved in the biosynthesis of catecholamines and to attempt to correlate these with the pattern of LH and prolactin release at selected times during the estrous cycle. MATERIALSAND METHODS
Animals The animals used in this study were Sprague-Dawley female rats purchased from the Sasco (Omaha, Nebr.), weighing 180-200 g. They were housed 5/cage in a temperature controlled room (21-24 °C) with the lights on at 04.00 h and off at 16.00 h. Food and water was available ad libitum. Vaginal smears were obtained daily and at least 2 consecutive 4 or 5 day cycles were recorded before the animal was used in the experiment. Animals were killed by rapid decapitation at various times during diestrus 2, proestrus and estrus.
Tissues Immediately after decapitation, the brain was removed and dissected on a cold aluminum block under a dissecting microscope. The severed end of the pituitary stalk was held with a fine forceps and 2 parallel cuts were made in an anterior direction lateral to the portal vessels, using the lateral limits of the infundibular recess as a reference. The rostral border of the medial basal hypothalamus was taken as the area where the portal vessels were no longer visible and a cut was made there. This tissue includes all of the median eminence and part of the pituitary stalk and arcuate nucleus. This method is similar to the one used by Chiocchio et al. ~. The remainder of the hypothalamus was then dissected (anterior limit: optic chiasm; posterior limit:just anterior to mammillary bodies; dorsal limit: anterior commissure). Following dissection, the medial basal hypothalamus (av. protein ----58.8/zg; approximate av. wt. -----590/~g) and remaining hypothalamic tissue (av. wt. -- 33.3 mg) were homogenized in 75/~1 and 0.5 ml, respectively, of Tris.Triton buffer (pH 6). Ten ~1 of the homogenate was removed for protein determinationis. The samples were then centrifuged at 10,000 g for 10 min.
Assay of tyrosine hydroxylase and dopamine-fl-hydroxylase Tyrosine hydroxylase activity was determined by slight alteration of the method
439 of Coyle 7 as modified by Saavedra et al. 2s. To 25 pl of tissue supernatant were added 85 /~1 of incubation mixture containing 10 F1 bovine catalase (1100 U, Sigma Chemicals) 10 pl 10 mM ferrous ammonium sulfate, 10 pl TPNH (10 mM, Sigma Chemicals), 10 pl sheep liver dihydropteridine reductase, 10 /~1 1 M potassium phosphate buffer (pH 5.5), 25/~1 Tris.Triton buffer (pH 6) and 10 #1 1.8 mM tyrosine containing 1 FCi 2,6 [3H]tyrosine (Amersham, 37 Ci/mmol). The reaction was initiated by the addition of 10 pl DL-6-methyl-5,6,7,8-tetrahydropterine (Calbiochem, 6.4 mM). After 45 min of incubation, [aH]DOPA was isolated on alumina and counted by liquid scintillation spectrometry. Dopamine-fl-hydroxylase activity was determined in 25 #1 of tissue supernatant by the method of Molinoff et al. 20 as modified by Saavedra et al. 2s.
Assay of LH and prolactin Trunk blood was obtained following decapitation and the serum was separated from the cells and stored at --20 °C for subsequent assay. Each serum sample was assayed in duplicate or triplicate for prolactin and LH by the radioimmunoassay methods of Niswender et al. 24,25. The antisera for the L H assay (GDN-15)was kindly provided by Dr. Gordon Niswender and the ovine LH for iodination (LER-1056 C2) was kindly provided by Dr. Leo E. Reichert. The remainder of the assay materials were provided by the N I A M D D Hormone Distribution Program. The reference preparations used were N I A M D D rat prolactin-RP-1 and N I A M D D rat LH-RP-1.
Data analysis Enzyme activity data and hormone data were analyzed by one-way analysis of variance and by Student's t-test.
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Fig. 1. Serum LIt and prolaetin levels at various times during the estrous cycle. Each point represents the group mean -t- 1 S.E. (n = 4-12). Thet symbolindicatesmean plasma prolactin or LH values which are significantlydifferent (P < 0.05) from values at other times on proestrus.
440 RESULTS
Serum prolactin and L H during the estrous cycle Rats were killed at 10.00, 12.00, 14.00 and 16.00 h during diestrus 2, proestrus and estrus. Proestrus was confirmed by uterine ballooning and only data from those rats that showed uterine ballooning were used for the proestrus group. Serum prolactin concentration increased significantly by 14.00 h proestrus and remained elevated at 16.00 h (Fig. 1). During diestrus 2 and estrus, prolactin levels were consistently low compared to the afternoon of proestrus. Serum LH concentration showed a similar pattern of elevation at 14.00 and 16.00h on proestrus and lower levels at all other times (Fig. 1).
Tyrosine hydroxylase and dopamine-fl-hydroxylase activity during the estrous cycle In the medial basal hypothatamus, tyrosine hydroxylase activity changed significantly during the time period of 10.00-16.00 h on proestrus (Fig. 2). Activity was significantly lower at 12.00 h than at any other time. The enzyme activity was not altered significantly during the same time period on estrus or diestrus 2. In the remainder of the hypothalamus, tyrosine hydroxylase activity did not change significantly on any of the 3 days. Significant changes in the activity of dopamine-/3-hydroxylase in the medial basal hypothalamus were found on both diestrus 2 where it reached a maximum level at 14.00 h (significantly different from 16.00 h, P < 0.05, Fig. 3) and on proestrus where the maximum level was seen at 12.00 h (significantly different from 16.00 h, P <
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Fig. 2. Tyrosine hydroxylase activity in the basal medial hypothalamus (solid line) and remainder of hypothalamic tissue (dashed line) at various times during the estrous cycle. Each point represents the group mean -4- 1 S.E. (n = 4-12). The t symbol indicates mean D O P A levels which are significantly different ( P < 0.05) from all other means on the same day.
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Fig. 3. Dopamine-fl-hydroxylase activity in the basal medial hypothalamus (solid line) and remainder of the hypothalamic tissue (dashed line) at various times during the estrous cycle. Each point represents the group mean d: S.E. (n = 5-11). The t symbol indicates mean methyloctopamine levels which are significantly different (P < 0.05) from the mean at 16.00 h on the same day.
0.05). No significant diffei-ences were found during estrus or at any time in the remainder of the hypothalamus. DISCUSSION
Several studies have attempted to detect changes in noradrenergic or dopaminergic neuron activity at various times during the estrous cycle to determine the roles of these neurons in gonadotropin release. Changes in steady-state levels of catecholaminesS, 82 are difficult to interpret since a number of dynamic biochemical mechanisms (i.e. synthesis, release, metabolism, reuptake) can bring about such changes. The use of tyrosine hydroxylase inhibitors, such as a-methyl-p-tyrosine, to measure catecholamine turnover rates may lead to some distortion of actual turnover and synthesis rates because of alterations in plasma hormone levels and subsequent feedback effects on catecholamine neurons 12. It has recently been shown that tyrosine hydroxylase activity is closely linked to the rates of catecholamine utilization and release 2 and thus may serve as an index of neuronal activity4. Since tyrosine hydroxylase is contained in both noradrenergic and dopaminergic neurons, it may be difficult to detect selective changes in a particular type of neuron 2. However, it has been demonstrated that bilateral lesions of the ventral noradrenergic bundles had no effect on either basal levels or the castration-induced increase of tyrosine hydroxylase activity in various hypothalamic nuclei, whereas dopamine-fl-hydroxylase activity was significantly reduced by the lesion 15. This suggests that most of the tyrosine hydroxylase in hypothalamic nuclei is associated with dopaminergic neurons. Although
442 alterations in dopamine-fl-hydroxylase activity were not found alter castration or hormone replacement in male rats 14 and is generally not believed to be rate-limiting in norepinephrine synthesis in the brain, recent reports suggest that dopamine-flhydroxylase may regulate norepinephrine synthesis and that its activity may change during the estrous cycle33,37. The evidence described above suggests that measurement of activity of these two enzymes under varying hormonal states may be a useful means of studying the respective roles of noradrenergic and dopaminergic neurons in the release of anterior pituitary hormones such as LH and prolactin. In agreement with the findings of many other investigators 21,3~, plasma prolactin and L H concentrations showed a sharp increase during the afternoon of proestrus. There were no significant changes in LH and prolactin during the times blood samples were obtained on diestrus 2 and estrus. However, neuronal events that influence this pattern of prolactin and LH secretion may occur many hours before the hormonal change itself. In this experiment changes in tyrosine hydroxylase and dopamine-fl-hydroxylase activity in the medial basal hypothalamus did occur during the estrous cycle, but these changes were not always correlated with changes in either LH or prolactin levels. For example, there was a significant increase in dopamine-fl-hydroxylase at 14.00 h on diestrus 2, whereas LH and prolactin levels remained unchanged throughout the time period studied. It has been shown that on the day before proestrus (diestrus 2) a rise in circulating estrogen is associated with the LH surge on proestrus3L Blockade of norepinephrine synthesis at 10.00 h on diestrus 2 decreased LH levels and blocked ovulation ~5. Thus it is possible that the increase in dopamine-flhydroxylase activity at 12.00 h on diestrus 2 reflects increased noradrenergic activity in the medial basal hypothalamus, which is necessary for the hormonal changes which occur on the following day. On the day of major hormonal changes, proestrus, tyrosine hydroxylase activity in the medial basal hypothalamus showed a 50 To decrease at 12.00 h followed by an increase during the afternoon. To our knowledge, no one has reported measurement of tyrosine hydroxylase activity during the estrous cycle. However, there are reports of enzyme changes in the hypothalamus during other endocrine manipulations. Estradiol la and testosterone 1~ have been shown to decrease tyrosine hydroxylase activity in castrated rats. The elevated estradiol levels seen during early proestrus 22 may be responsible for this decrease in tyrosine hydroxylase activity. The significance of this decrease in tyrosine hydroxylase activity followed by an increase in the afternoon with regard to LH secretion is not known. Dopamine turnover in the median eminence was found to be lower (measured in rats killed at 03.00-05.00 h) during proestrus 17 than during any other day of the cycle. Similarly, dopamine levels in the portal blood was reported to be decreased during proestrus a. These results were interpreted as support for the hypothesis that during proestrus there is a decrease in the inhibitory effect of dopamine on LH secretion. There is a great deal of pharmacological evidence that dopamine is inhibitory to LH secretion 11. There are also many pharmacological studies indicating that dopamine is stimulatory to LH via an action on L H R H 26. In the present study, the decrease in tyrosine hydroxylase activity at 12.00 h may reflect a decrease in dopaminergic activity, precipitating the series of events leading to the LH
443 surge. At the same time (12.00 h) that tyrosine hydroxylase activity is lowest during proestrus, dopamine-fl-hydroxylase activity is highest. Since this increase in dopamine-fl-hydroxylase activity is most probably a reflection of increased noradrenergic activity, this study agrees with studies showing increased norepinephrine turnover during proestrus 11,17. Pharmacological studies also suggest that norepinephrine is an essential neurotransmitter for the release of L H during proestrus 11,z9. However, it should be pointed out that an increase in noradrenergic activity during proestrus does not prove that norepinephrine is involved in the L H surge. In fact, substantial depletion of norepinephrine in the preoptic area and hypothalamus following 6hydroxydopamine treatment did not alter the estrous cycle or L H levels 2a. It is well accepted that dopamine exerts an inhibitory influence on prolactin secretion. In the present study, the only time tyrosine hydroxylase activity decreased significantly was at 12.00 h on proestrus, which was followed by an increase in prolactin. As prolactin levels increased at 14.00 and 16.00 h, tyrosine hydroxylase activity showed a parallel increase. In view of the evidence that prolactin can increase dopaminergic activity zT, it is possible that the increased tyrosine hydroxylase activity was due to the feedback of prolactin on dopamine-containing neurons in the median eminence. In conclusion, the surge of prolactin and L H on the afternoon of proestrus is preceded by a decrease in tyrosine hydroxylase activity and an increase in dopamine-flhydroxylase activity in the medial basal hypothalamus. The lack of any significant changes in enzyme activity in the remainder of the hypothalamus emphasizes the importance of catecholamines in the median eminence in the regulation of pituitary function. ACKNOWLEDGEMENTS The authors are grateful to Ms. Yie-Jane Wu for her expert technical assistance in performing these experiments. This study was supported by Public Health Service Grant HD11922.
REFERENCES 1 Ahren, K., Fuxe, K., Hamburger, L. and HiSkfelt,T., Turnover changes in the tubero-infundibular dopamine neurons during the ovarian cycle of the rat, Endocrinology, 88 (1971) 1415-1424. 2 Baeopoulos, N. G. and Bhatnagar, R. K., Correlation between tyrosine hydroxylase activity and catecholamine concentration or turnover in brain regions, J. Neurochem., 29 (1977)639-643. 3 Ben-Jonathon, N., Oliver, C., Weiner, H. J., Mical, R. S. and Porter, J. C., Dopamine in hypophysial portal plasma of the rat during the estrus cycleand throughout pregnancy, Endocrinology, 100(1977) 452-458. 4 Bustos, G., Roth, R. H., Morgenruth, V. H. and Hancke, J. L., Tyrosine hydroxylase activation and transmitter release from central noradrenergie neurons by electrical field stimulation, NaunynSchmiedeberg's Arch. exp. Path. Pharmak., 301 (1978) 149-156. 5 Chiocchio, S. R., Cannata, M. A. and Tramezzani, J. H., The sizeweight and catecholamine content of the median eminence of the rat, Brain Research, 110 (1976) 612-618. 6 Clemens, J. A., Tinsley, F. C. and Fuller, R. W., Evidence for a dopaminergic component in the seriesof neural events that lead to the pro-oestrus surge of LH, Acta endocr., Copnh., 85 (1977) 18-24.
444 7 Coyle, J. R., Tyrosine hydroxylase in rat brain - - cofactor requirements, regional and subcellular distribution, Biochem. Pharmacol., 21 (1972) 1935-1944. 8 Crowley, W. R., O'Donohue, T. L. and Jacobowitz, D. M., Changes in catecholamine content in discrete brain nuclei during the estrous cycle of the rat, Brain Research, 147 (1978) 315-326. 9 Donoso, A. O. and DeGuterriez Moyano, N. B., Adrenergic activity in hypothalamus and ovulation, Proc. Soc. exp. Biol. Med., 135 (1970) 633-635. 10 Fludder, J. M. and Tonge, S. R., Variations in the concentrations of monoamines and their metabolites in eight regions of rat brain during the estrous cycle: a basis for interactions between hormones and psychotropic drugs, J. Pharm. Pharmacol., 27 (1975) 39P. 11 Euxe, K., Influence of central catecholamines on LH-RH-containing pathways. In J. E. Tyson (Ed.), Clinics in Obstetrics and Gynaecology, Vol. 5, W. B. Saunders, London, 1978, pp. 251-269. 12 Gudelsky, G. A., Simpkins, J., Mueller, G. P., Meites, J. and Moore, K. E., Selective actions of prolactin on catecholamine turnover in the hypothalamus and on serum LH and FSH, Neuroendocrinology, 22 (1976) 206-215. 13 Kalra, S. P. and McCann, S. M., Effects of drugs modifying catecholamine synthesis on plasma LH and ovulation in the rat, Neuroendocrinology, 15 (1974) 79-91. 14 Kizer, J. S., Palkovits, M., Zivin, J., Brownstein, M., Saavedra, J. M. and Kopin, I. J., The effect of various endocrinological manipulations on tyrosine hydroxylase and dopamine-fl-hydroxylase activities in individual hypothalamic nuclei of the adult male rat, Endocrinology, 85 (1974) 799-812. 15 Kizer, J. S., Muth, E. and Jacobowitz, D. M., The effect of bilateral lesions of the ventral noradrenergic bundle on endocrine-induced changes of tyrosine hydroxylase in the rat median eminence, Endocrinology, 98 (1976) 886-893. 16 Langelier, P. and McCann, S. M., The effects of interruption of the ventral noradrenergic pathway on the proestrus discharge of prolactin in the rat, Proc. Soc. exp. Biol. Med., 154 (1977) 553-557. 17 Lofstrom, A., Catecholamine turnover alterations in discrete areas of the median eminence of the 4- and 5-day cyclic rat, Brain Research, 120 (1977) 113-131. 18 Lowry, O. H., Roseborough, N. J., Farr, A. L. and Randall, R. J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 19 Luine, V. N., McEwen, B. S. and Black, I. B., Effect of 17/4-estradiol on hypothalamic tyrosine hydroxylase activity, Brain Research, 120 (1977) 188-192. 20 Molinoff, P. B., Weinshilboum, R. and Axelrod, J., A sensitive enzymatic assay for dopamine-/;hydroxylase, J. Pharmacol. exp. Ther., 178 (1971) 425-431. 21 Neill, J. D., Effect of 'stress' on serum prolactin and luteinizing hormone levels during the estrous cycle of the rat, Endocrinology, 87 (1970) 1192-1197. 22 Nequin, L. G., Alvarez, J. and Schwartz, N. B., Measurement of serum steroid and gonadotropin levels and uterine and ovarian variables throughout 4 day and 5 day estrous cycles in the rat, Biol. Reprod., 20 (1979) 659-670. 23 Nicholson, G., Greeley, G., Humm, J., Youngblood, W. and Kizer, J. S., Lack of effect of noradrenergic denervation of the hypothalamus and medial preoptic area on the feedback regulation of gonadotropin secretion and the estrus cycle of the rat, Endocrinology, 103 (1978) 55%566. 24 Niswender, G. D., Chen, C. L., Midgley, A. R., Meites, J. and Ellis, S., Radioimmunoassay for rat prolactin, Proc. Soc. exp. Biol. Med., 130 (1969) 793-797. 25 Niswender, G. D., Midgley, A. R., Monroe, S. E. and Reichert, L., Radioimmunoassay for rat luteinizing hormone with antiovine LH serum and ovine LH 1311, Proc. Soc. exp. Biol. Med., 128 (1968) 807-811. 26 Ojeda, S. R. and McCann, S. M., Control of LH and FSH release by LH-RH: influence of putative neurotransmitters. In J. E. Tyson (Ed.), Clinics in Obstetrics and Gynaeeology, Vol. 5, W. B. Saundders, London, 1978, pp. 283-303. 27 Porter, J. C., Barnea, A., Cramer, O. M. and Parker, C. R., Hypothalamic peptide and catecholamine secretion: roles for portal and retrograde blood flow in the pituitary stalk in the release of hypothalamic dopamine and pituitary prolactin and LH. In J. E. Tyson (Ed.), Clinics in Obstetrics and Gynaeeology, Vol. 5, W. B. Saunders, London, 1978, pp. 271-282. 28 Saavedra, J. M., Brownstein, M., Palkovits, M., Kizer, J. S. and Axelrod, J., Tyrosine hydroxylase and dopamine-fl-hydroxylase: distribution in the individual rat hypothalamic nuclei, J. Neuroehem., 23 (1974) 869-871. 29 Sawyer, C. H., Some recent developments in brain-pituitary-ovarian physiology, Neuroendocrinology, 17 (1975) 97-124. 30 Schneider, H. P. G. and McCann, S. M., Mono- and indoleamines and control of LH secretion, Endocrinology, 86 (1970) 1127-1133.
445 31 Schwartz, N. B., A model for the regulation of ovulation in the rat, Recent Progress in Hormone Research, 25 (1969) 1-55. 32 Selmanoff, M. K., Pramik-Holdaway, M. J. and Weiner, R. I., Concentrations of dopamine and norepinephrinein discrete hypothalamic nuclei during the rat estrous cycle, Endocrinology, 99 (1976) 326-329. 33 Simpson, C. W., Cummins, C. and Dicara, L. V., Dopamine-/~-hydroxylase activity in rat hypothalamus during the rat estrus cycle, Pharmacol. Biochem. Behav., 3 (1975) 693-696. 34 Smith, E. R., Bowers, C. Y. and Davidson, J. M., Circulating levels of plasma gonadotropins in 4and 5-day cycling rats, Endocrinology, 93 (1973) 756-758. 35 Terasawa, E., Bridson, W. E., Davenport, J. W. and Goy, R. W., Role of brain monoamines in release of gonadotropin before proestrus in the cyclic rat, Neuroendocrinology, 18 (1975) 345-358. 36 Wiggins, J. F. and Fernstrom, J. D., L-DOPA inhibits prolactin secretion in proestrus rats, Endocrinology, 101 (1977) 469-474. 37 Wise, C. D., Belluzzi, J. D. and Stein, L., Possible role of dopamine-fl-hydroxylase in the regulation of norepinephrine biosynthesis in rat brain, Pharmacol. Biochem. Behav., 7 (1977) 549-553.
437
© Elsevier/North-Holland Biomedical Press
CATECHOLAMINE SYNTHESIZING ENZYMES IN THE HYPOTHALAMUS D U R I N G T H E ESTROUS CYCLE
LAURENCE A. CARR and JAMES L. VOOGT Department of Pharmacology and Toxicology, University of Louisville Health Sciences Center, Louisville, Ky. 40292 and Department of Physiology, University of Kansas Medical Center, Kansas City, Kans. 66103 (U.S.A.)
(Accepted March 6th, 1980) Key words: estrous cycle - - LH - - prolactin - - tyrosine hydroxylase - - dopamine-fl-hydroxylase--
catecholamine
SUMMARY The activities of tyrosine hydroxylase and dopamine-fl-hydroxylase were measured in the medial basal hypothalamus and remaining hypothalamic tissue of female rats at various times during diestrus 2, proestrus and estrus. Tyrosine hydroxylase activity in the medial basal hypothalamus was significantly lower at 12.00 h compared with other times on proestrus. This decrease preceded the elevation of serum prolactin and L H during the afternoon of proestrus. Tyrosine hydroxylase activity did not change significantly during diestrus 2 or estrus nor was it altered at any time in the remainder of the hypothalamus. Dopamine-fl-hydroxylase activity in the basal medial hypothalamus was significantly elevated at 12.00 h on proestrus and at 14.00 h on diestrus. The results provide further evidence for a decrease in dopaminergic neuron activity in the medial basal hypothalamus which may precipitate the series of events leading to the L H surge during proestrus. The increase in dopamine-fl-hydroxylase activity suggests that an increase in noradrenergic neuron activity may also be involved in triggering the release of LH.
INTRODUCTION There is extensive evidence that catecholamine-containing neuronal systems terminating in the hypothalamus may regulate or modulate the release of gonadotropins during the estrous cycle. In the rat, noradrenergic pathways appear to stimulate the release of LH. This is supported by the findings that turnover of norepinephrine is increased during proestrus in the hypothalamus 9 and subependymal
438 layer of the median eminence17. In addition, normetanephrine concentrations in the hypothalamus increased during proestrus, suggesting enhanced release of norepinephrine 1°. Administration of FLA-63 or diethyldithiocarbamate, inhibitors of dopamine-fl-hydroxylase, have been shown to block ovulation and/or LH release in cycling rats13,8~. The tuberoinfundibular dopaminergic system, on the other hand, appears to exert an inhibitory influence on LH release as suggested by decreased turnover of dopamine in these neurons during proestrus 1,17. Other studies, however, suggest that a dopaminergic stimulus is involved in the release of LH during proestrus 6,3°. The surge of LH release which occurs on the afternoon of proestrus is also accompanied by increased release of prolactin which may be mediated by a noradrenergic stimulus16 and inhibited by a dopaminergic mechanism36. The purpose of the present study was to monitor changes in the activity of two enzymes involved in the biosynthesis of catecholamines and to attempt to correlate these with the pattern of LH and prolactin release at selected times during the estrous cycle. MATERIALSAND METHODS
Animals The animals used in this study were Sprague-Dawley female rats purchased from the Sasco (Omaha, Nebr.), weighing 180-200 g. They were housed 5/cage in a temperature controlled room (21-24 °C) with the lights on at 04.00 h and off at 16.00 h. Food and water was available ad libitum. Vaginal smears were obtained daily and at least 2 consecutive 4 or 5 day cycles were recorded before the animal was used in the experiment. Animals were killed by rapid decapitation at various times during diestrus 2, proestrus and estrus.
Tissues Immediately after decapitation, the brain was removed and dissected on a cold aluminum block under a dissecting microscope. The severed end of the pituitary stalk was held with a fine forceps and 2 parallel cuts were made in an anterior direction lateral to the portal vessels, using the lateral limits of the infundibular recess as a reference. The rostral border of the medial basal hypothalamus was taken as the area where the portal vessels were no longer visible and a cut was made there. This tissue includes all of the median eminence and part of the pituitary stalk and arcuate nucleus. This method is similar to the one used by Chiocchio et al. ~. The remainder of the hypothalamus was then dissected (anterior limit: optic chiasm; posterior limit:just anterior to mammillary bodies; dorsal limit: anterior commissure). Following dissection, the medial basal hypothalamus (av. protein ----58.8/zg; approximate av. wt. -----590/~g) and remaining hypothalamic tissue (av. wt. -- 33.3 mg) were homogenized in 75/~1 and 0.5 ml, respectively, of Tris.Triton buffer (pH 6). Ten ~1 of the homogenate was removed for protein determinationis. The samples were then centrifuged at 10,000 g for 10 min.
Assay of tyrosine hydroxylase and dopamine-fl-hydroxylase Tyrosine hydroxylase activity was determined by slight alteration of the method
439 of Coyle 7 as modified by Saavedra et al. 2s. To 25 pl of tissue supernatant were added 85 /~1 of incubation mixture containing 10 F1 bovine catalase (1100 U, Sigma Chemicals) 10 pl 10 mM ferrous ammonium sulfate, 10 pl TPNH (10 mM, Sigma Chemicals), 10 pl sheep liver dihydropteridine reductase, 10 /~1 1 M potassium phosphate buffer (pH 5.5), 25/~1 Tris.Triton buffer (pH 6) and 10 #1 1.8 mM tyrosine containing 1 FCi 2,6 [3H]tyrosine (Amersham, 37 Ci/mmol). The reaction was initiated by the addition of 10 pl DL-6-methyl-5,6,7,8-tetrahydropterine (Calbiochem, 6.4 mM). After 45 min of incubation, [aH]DOPA was isolated on alumina and counted by liquid scintillation spectrometry. Dopamine-fl-hydroxylase activity was determined in 25 #1 of tissue supernatant by the method of Molinoff et al. 20 as modified by Saavedra et al. 2s.
Assay of LH and prolactin Trunk blood was obtained following decapitation and the serum was separated from the cells and stored at --20 °C for subsequent assay. Each serum sample was assayed in duplicate or triplicate for prolactin and LH by the radioimmunoassay methods of Niswender et al. 24,25. The antisera for the L H assay (GDN-15)was kindly provided by Dr. Gordon Niswender and the ovine LH for iodination (LER-1056 C2) was kindly provided by Dr. Leo E. Reichert. The remainder of the assay materials were provided by the N I A M D D Hormone Distribution Program. The reference preparations used were N I A M D D rat prolactin-RP-1 and N I A M D D rat LH-RP-1.
Data analysis Enzyme activity data and hormone data were analyzed by one-way analysis of variance and by Student's t-test.
i 600 _ 500 _1
400 z_ 300 I--O _J
o
200
~° IO0 20 m"' 0
ll|i DIESTRUS 2
PROESTRUS
ill| ESTRUS
Fig. 1. Serum LIt and prolaetin levels at various times during the estrous cycle. Each point represents the group mean -t- 1 S.E. (n = 4-12). Thet symbolindicatesmean plasma prolactin or LH values which are significantlydifferent (P < 0.05) from values at other times on proestrus.
440 RESULTS
Serum prolactin and L H during the estrous cycle Rats were killed at 10.00, 12.00, 14.00 and 16.00 h during diestrus 2, proestrus and estrus. Proestrus was confirmed by uterine ballooning and only data from those rats that showed uterine ballooning were used for the proestrus group. Serum prolactin concentration increased significantly by 14.00 h proestrus and remained elevated at 16.00 h (Fig. 1). During diestrus 2 and estrus, prolactin levels were consistently low compared to the afternoon of proestrus. Serum LH concentration showed a similar pattern of elevation at 14.00 and 16.00h on proestrus and lower levels at all other times (Fig. 1).
Tyrosine hydroxylase and dopamine-fl-hydroxylase activity during the estrous cycle In the medial basal hypothatamus, tyrosine hydroxylase activity changed significantly during the time period of 10.00-16.00 h on proestrus (Fig. 2). Activity was significantly lower at 12.00 h than at any other time. The enzyme activity was not altered significantly during the same time period on estrus or diestrus 2. In the remainder of the hypothalamus, tyrosine hydroxylase activity did not change significantly on any of the 3 days. Significant changes in the activity of dopamine-/3-hydroxylase in the medial basal hypothalamus were found on both diestrus 2 where it reached a maximum level at 14.00 h (significantly different from 16.00 h, P < 0.05, Fig. 3) and on proestrus where the maximum level was seen at 12.00 h (significantly different from 16.00 h, P <
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Fig. 3. Dopamine-fl-hydroxylase activity in the basal medial hypothalamus (solid line) and remainder of the hypothalamic tissue (dashed line) at various times during the estrous cycle. Each point represents the group mean d: S.E. (n = 5-11). The t symbol indicates mean methyloctopamine levels which are significantly different (P < 0.05) from the mean at 16.00 h on the same day.
0.05). No significant diffei-ences were found during estrus or at any time in the remainder of the hypothalamus. DISCUSSION
Several studies have attempted to detect changes in noradrenergic or dopaminergic neuron activity at various times during the estrous cycle to determine the roles of these neurons in gonadotropin release. Changes in steady-state levels of catecholaminesS, 82 are difficult to interpret since a number of dynamic biochemical mechanisms (i.e. synthesis, release, metabolism, reuptake) can bring about such changes. The use of tyrosine hydroxylase inhibitors, such as a-methyl-p-tyrosine, to measure catecholamine turnover rates may lead to some distortion of actual turnover and synthesis rates because of alterations in plasma hormone levels and subsequent feedback effects on catecholamine neurons 12. It has recently been shown that tyrosine hydroxylase activity is closely linked to the rates of catecholamine utilization and release 2 and thus may serve as an index of neuronal activity4. Since tyrosine hydroxylase is contained in both noradrenergic and dopaminergic neurons, it may be difficult to detect selective changes in a particular type of neuron 2. However, it has been demonstrated that bilateral lesions of the ventral noradrenergic bundles had no effect on either basal levels or the castration-induced increase of tyrosine hydroxylase activity in various hypothalamic nuclei, whereas dopamine-fl-hydroxylase activity was significantly reduced by the lesion 15. This suggests that most of the tyrosine hydroxylase in hypothalamic nuclei is associated with dopaminergic neurons. Although
442 alterations in dopamine-fl-hydroxylase activity were not found alter castration or hormone replacement in male rats 14 and is generally not believed to be rate-limiting in norepinephrine synthesis in the brain, recent reports suggest that dopamine-flhydroxylase may regulate norepinephrine synthesis and that its activity may change during the estrous cycle33,37. The evidence described above suggests that measurement of activity of these two enzymes under varying hormonal states may be a useful means of studying the respective roles of noradrenergic and dopaminergic neurons in the release of anterior pituitary hormones such as LH and prolactin. In agreement with the findings of many other investigators 21,3~, plasma prolactin and L H concentrations showed a sharp increase during the afternoon of proestrus. There were no significant changes in LH and prolactin during the times blood samples were obtained on diestrus 2 and estrus. However, neuronal events that influence this pattern of prolactin and LH secretion may occur many hours before the hormonal change itself. In this experiment changes in tyrosine hydroxylase and dopamine-fl-hydroxylase activity in the medial basal hypothalamus did occur during the estrous cycle, but these changes were not always correlated with changes in either LH or prolactin levels. For example, there was a significant increase in dopamine-fl-hydroxylase at 14.00 h on diestrus 2, whereas LH and prolactin levels remained unchanged throughout the time period studied. It has been shown that on the day before proestrus (diestrus 2) a rise in circulating estrogen is associated with the LH surge on proestrus3L Blockade of norepinephrine synthesis at 10.00 h on diestrus 2 decreased LH levels and blocked ovulation ~5. Thus it is possible that the increase in dopamine-flhydroxylase activity at 12.00 h on diestrus 2 reflects increased noradrenergic activity in the medial basal hypothalamus, which is necessary for the hormonal changes which occur on the following day. On the day of major hormonal changes, proestrus, tyrosine hydroxylase activity in the medial basal hypothalamus showed a 50 To decrease at 12.00 h followed by an increase during the afternoon. To our knowledge, no one has reported measurement of tyrosine hydroxylase activity during the estrous cycle. However, there are reports of enzyme changes in the hypothalamus during other endocrine manipulations. Estradiol la and testosterone 1~ have been shown to decrease tyrosine hydroxylase activity in castrated rats. The elevated estradiol levels seen during early proestrus 22 may be responsible for this decrease in tyrosine hydroxylase activity. The significance of this decrease in tyrosine hydroxylase activity followed by an increase in the afternoon with regard to LH secretion is not known. Dopamine turnover in the median eminence was found to be lower (measured in rats killed at 03.00-05.00 h) during proestrus 17 than during any other day of the cycle. Similarly, dopamine levels in the portal blood was reported to be decreased during proestrus a. These results were interpreted as support for the hypothesis that during proestrus there is a decrease in the inhibitory effect of dopamine on LH secretion. There is a great deal of pharmacological evidence that dopamine is inhibitory to LH secretion 11. There are also many pharmacological studies indicating that dopamine is stimulatory to LH via an action on L H R H 26. In the present study, the decrease in tyrosine hydroxylase activity at 12.00 h may reflect a decrease in dopaminergic activity, precipitating the series of events leading to the LH
443 surge. At the same time (12.00 h) that tyrosine hydroxylase activity is lowest during proestrus, dopamine-fl-hydroxylase activity is highest. Since this increase in dopamine-fl-hydroxylase activity is most probably a reflection of increased noradrenergic activity, this study agrees with studies showing increased norepinephrine turnover during proestrus 11,17. Pharmacological studies also suggest that norepinephrine is an essential neurotransmitter for the release of L H during proestrus 11,z9. However, it should be pointed out that an increase in noradrenergic activity during proestrus does not prove that norepinephrine is involved in the L H surge. In fact, substantial depletion of norepinephrine in the preoptic area and hypothalamus following 6hydroxydopamine treatment did not alter the estrous cycle or L H levels 2a. It is well accepted that dopamine exerts an inhibitory influence on prolactin secretion. In the present study, the only time tyrosine hydroxylase activity decreased significantly was at 12.00 h on proestrus, which was followed by an increase in prolactin. As prolactin levels increased at 14.00 and 16.00 h, tyrosine hydroxylase activity showed a parallel increase. In view of the evidence that prolactin can increase dopaminergic activity zT, it is possible that the increased tyrosine hydroxylase activity was due to the feedback of prolactin on dopamine-containing neurons in the median eminence. In conclusion, the surge of prolactin and L H on the afternoon of proestrus is preceded by a decrease in tyrosine hydroxylase activity and an increase in dopamine-flhydroxylase activity in the medial basal hypothalamus. The lack of any significant changes in enzyme activity in the remainder of the hypothalamus emphasizes the importance of catecholamines in the median eminence in the regulation of pituitary function. ACKNOWLEDGEMENTS The authors are grateful to Ms. Yie-Jane Wu for her expert technical assistance in performing these experiments. This study was supported by Public Health Service Grant HD11922.
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