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Effect of ovariectomy and estrogen replacement on hypothalamic, pituitary and peripheral blood β-endorphin levels in the rat
Neuropeprides (1991) 20, 175-180 0 Longman Group UK Ltd 1991
Effect of Ovariectomy and Estrogen Replacement on Hypothalamic, Pituitary and Peripheral Blood p-Endorphin Levels in the Rat S. LE*, C.J. CHUONG*S
and T.A. PARKENING*t
*Division of Reproductive Endocrinology/Infertility, Department of Obstetrics and Gynecology; toepartment of Anatomy and Neurosciences, University of Texas Medical Branch, Galveston, TX, 77051 USA; *Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA (Reprint requests to CJC#)
Abstract-This study was initiated to detect possible changes in P-endorphin (P-EP) levels of the hypothalamus, anterior pituitary gland, and peripheral blood of rats after ovariectomy and estrogen administration. Attempts were also made to determine the correlation between peripheral and central levels of P-EP. Twenty-six Sprague-Dawley rats were decapitated. Nine had intact ovaries (Gr. INT), and 17 were ovariectomized 3 weeks before they were killed. Nine of the ovariectomized rats received estradiol benzoate (EB) (Gr. EB) and the other 8 received peanut oil (Gr. OVX) prior to the decapitation. A P-EP radioimmunoassay was used to analyze homogenates of the hypothalamus and anterior pituitary, and peripheral blood. In the hypothalamus, P-EP levels were significantly lower in Gr. INT and Gr. EB than in Gr. OVX. In the pituitary gland and peripheral blood, P-EP levels were significantly higher in Gr. INT than in Gr. OVX. Pituitary P-EP levels did not vary between Gr. OVX and Gr. EB, although P-EP levels in peripheral blood were significantly higher in Gr. EB than in Gr. OVX. No significant correlations were noted in P-EP levels between the hypothalamus, pituitary gland, and peripheral blood in either Gr. INT, Gr. OVX, or Gr. EB. It appears that EB exerts different effects on P-EP levels in the hypothalamus, anterior pituitary gland, and peripheral blood, and that P-EP levels in these regions may be independent of one another.
Introduction P-endorphin (P-EP) is synthesized primarily in the anterior and intermediate lobes of the pituitary gland and in the arcuate nucleus of the hypothalaDate received 13 June 1991 Date accepted 25 July 1991
mus (l-3). It is of interest because it may act as a modulator through which gonadal hormones exert negative feedback on gonadotropin release (4-6). Since information on the relationship between estrogen and hypothalamic, pituitary, and plasma P-EP is still lacking, our study was conducted to investigate the effects of estrogen on hypothalamic, pituitary. and plasma P-EP by examining 175
NEUROPEPTIDES
176 P-EP in these three regions at a given time after ovariectomy and estrogen replacement therapy. Currently available techniques do not afford access to the central site of synthesis and secretion of P-EP to allow direct measurement of this neuropeptide in humans. Several studies have been conducted to measure /3-EP in peripheral blood (7-16) however, the correlation between peripheral levels of P-EP and central nervous system (CNS) opioid activity remains controversial. Therefore, another purpose of our study was to determine if any correlation existed between peripheral and central levels of P-EP.
Materials and Methods Animals
Adult female Sprague-Dawley rats weighing 180200g were housed at room temperature (22-26”(Z) with the free access to rat chow and water. All rats were acclimatized in a 12-h light/dark cycle (lights on from 0700-1900h) and accustomized to daily handling for 2 weeks before the study. Vaginal smears were obtained to determine the day of the estrous cycle. Twenty-six rats were decapitated between lOOO-1200h. Nine had intact ovaries (Gr. INT), and 17 underwent ovariectomy procedures 3 weeks before they were killed. The ovaries were removed through flank incisions under ether anesthesia. The 17 ovariectomized rats were randomly divided into two groups. Nine (Gr. EB) received EB at 20&kg dissolved in 0.2~~ of peanut oil daily for 2 days intraperitoneally, and 8 (Gr. OVX) received only peanut oil. Sample collection
All animals were decapitated 10s after removal from their cages. Rats decapitated were in an area separated from the cage area so as to avoid stressing rats yet to be samples. Animals in Gr. EB and Gr. OVX were killed 24h after the second injection of EB or peanut oil. Trunk blood was collected into glass tubes containing EDTA (7.2mg/5ml blood) and immediately centrifuged at 760g for 1Omin at 4°C to separate the plasma, which was stored at -80°C for later assay. The skulls were immediately opened via an oropharyngeal approach and the brain was
removed to expose the pituitary. After the removal of the diaphragm sella, the pituitary was lifted out and placed on ice under a dissecting microscope, and the anterior lobe separated from the intermediate and posterior lobes. The anterior lobe was then weighed, and homogenized in lml of 0.2 M HCl containing 0.05 M EDTA and 0.1% sodium azide. The homogenate was centrifuged at 4000g for 30min at 4°C and the supernatant separated and stored at -80°C for later assay (17). The hypothalamus was dissected out on a plastic sheet placed on ice as a region 3mm in depth limited rostrally by the anterior margin of the optic chiasm, caudally by the mammillary bodies and laterally by the hypothalamic sulci. After weighing, the tissues were homogenized, centrifuged, and the supernatant stored the same way as for the pituitary. It is known that plasma, pituitary and brain tissues contain proteolytic enzymes which may degradate P-EP during processing of these samples. However, the addition of the enzyme inhibitor during the process of sample collection was not considered to be necessary in most studies (7,8, 14, 15, 18, 19, 20, 21). Laboratory tests
Each sample of plasma and supernatants of the pituitary and hypothalamus were assayed in duplicate for P-EP with in 1 month after sample collection. The assay was performed according to the affinity gel extraction method (Immunonuclear Corp., Stillwater, Minn, USA). A major problem known to affect the direct assay of P-EP is the non-specific binding of iodinated P-EP to high molecular weight plasma or tissue components at an alkaline pH (22). This binding interferes with the radioimmunoassay and can be circumvented by either extraction or column chromatography. Such interference does not appear to be present in acid tissue extracts and is lessened at neutral pH. Of several methods studies for the extraction of P-EP the affinity gel extraction method has been found to adequately separate P-EP from non-specific binding material. The method consisted of extracting P-EP from plasma or homogenates using specific adsorption particles (Sepharose). Rabbit anti+EP was
EFFECT OF OVARIECTOMY
Table 1
AND ESTROGEN REPLACEMENT
P-EP levels in the hypothalamus,
f3-EP (Mean f SE)
pituitary
gland and peripheral
Group fN)
Hypothalamus (PmoVmg) Pituitary (PmoVmg) Peripheral blood (PmoUL)
177
IN THE RAT
blood in the rat
INT (9)
ovx (8)
EB (9)
2.8 + 0.3” 17.7 + 2.9b 37.5 + 4.9”
6.1 f 1.6 8.5 + 1.2 18.9 f 3.3
2.5 If: 0.3” 10.8 Z!I1.7 41.3 I!Z6.0”
“P < 0.05 V.S. ovx. bP < 0.01 V.S. ovx. (N): Number of rats in each group.
coupled to Sepharose and diluted in bovine serum albumin-borate buffer containing merthiolate (lyophilized). P-EP was then eluted from the Sepharose particles with 0.025N HCl, and measured in a radioimmunoassay. The eluants were incubated in rabbit anti-p-EP serum with 1% BSA-borate buffer for 20 h at 4°C. Iodinated-P-EP was added and the mixture incubated for 20h at 4°C before adding Goat Anti-Rabbit Precipitating Complex (GAR-PPT). After 30min the mixture was centrifuged (20min at 760g) and the supernatant discarded. The precipitate was counted with a gamma scintillation counter. Synthetic human P-EP was used at prediluted concentrations to construct a standard curve correlating percent bound I-P-EP to the P-EP concentration. Counts of the standard and unknown were corrected to non-specific binding. The assay could detect
Results
In Gr. INT, 4 rats were in diestrus, 3 in estrus, and 2 in proestrus. In Gr. and Gr. OVX, the rats were also cycling before ovariectomy. Hypothalamic P-EP levels were significantly lower in Gr. INT than in Gr. OVX. In contrast, P-EP levels in the anterior pituitary gland and peripheral blood were significantly higher. When comparing P-EP levels in Gr. EB and Gr. OVX, hypothalamic levels were significantly lower, pituitary levels were higher, although not statistically significant, and peripheral blood levels were significantly higher (Table 1). No significant correlations were noted in P-EP levels between the hypothalamus and the pituitary, the hypothalamus and the peripheral blood, or the pituitary and the peripheral blood in either Gr. INT, Gr. OVX, Gr. EB or combination of the three groups (Table 2).
Table 2
Correlation gland, and peripheral
Group (N)
r*
Statistical analysis
Data was analyzed using the Student’s two tailedtest, and the computation of Pearson correlation coefficient. Only rats with complete P-EP data on the hypothalamus, pituitary gland, and peripheral blood were included in the analysis of correlations on P-EP levels of these three regions. The level of significance was P < 0.05.
between hypothalamic, pituitary blood P-EP levels in the rat
Pituitary gland V.S. hypothalamus Peripheral blood vs. hypothalamus Peripheral blood
INT (9) 0.36
OVX (5) 0.76
EB (6) 0.25
0.22
-0.13
-0.80
0.11
-0.32
-0.16
Total (20) 0.005 -0.38
0.15
V.S. pituitary gland *Pearson Correlation Coefficient. (N): Number of rats with complete p-EP data on hypothalamus, pituitary gland and peripheral All P values were >O.OS.
blood.
178 Discussion Studies (18,19,20,23-26) have been conducted on B-EP levels in the hypothalamus, pituitary gland and peripheral blood in the presence or absence of estrogen. However, most studies were conducted to examine only one or two of the above. Wilcox and Roberts (27) have shown that proopiomelanocortin (POMC) messenger ribonucleic acid levels decrease in the hypothalamus of ovariectomized rats after 3 or 14 days of estradiol treatment. Estradiol has also been shown to inhibit POMC gene transcription in ovariectomized rats within 60min after hormone administration (28). Lower l3-EP levels in hypophyseal portal blood in ovariectomized rats (20) and the stimulatory effect of estradiol on hypothalamic l3-EP release into the hypophyseal portal blood have also been reported (29,30). The simultaneous measurement of P-EP in the hypothalamus and hypophyseal portal blood could determine the location of the estrogenic effect. The presence of lower pituitary and peripheral blood l3-EP levels in Gr. OVX than in Gr. INT suggests that the synthesis of pituitary B-EP may be decreased, and that less is secreted into the peripheral circulation in the absence of estrogen. The dosages of estrogen replacement in previous studies included EB 20 l&rat/day subcutaneously for 1 or 2 days (26), 5 @at/day subcutaneously for 7 days (26), silastic capsules containing 262l.~g 17 B-estradioYm1 in sesame oil (10 mm/100 gm SW) subcutaneous implantation for 1 or 3 days (27) or for 1 or 3 weeks (18). Different dosages and durations of EB replacement resulted in different B-EP levels in various tissues (18, 25). We were interested in the effect of lower dosages and shorter duration of EB replacement, and only used about 4 CL/rat/day intraperitoneally for 2 days. Yet, we were able to restore P-EP levels in the peripheral blood of Gr. OVX to those found in Gr. INT. No statistically significant changes occurred in the l3-EP levels of the pituitary gland in these two groups. These data suggest that the synthesis of pituitary l3-EP may have been increased with no net changes in the amount stored since there may have been a concurrent increase in the secretion of l3-EP into the peripheral circulation. Although the rats in Gr. INT were in different
NEUROPEPTIDES
stages of estrous cycle, Sarkar and Yen (20) showed that there were no appreciable differences between the l3-EP-like immunoreactivity concentration in the hypophyseal portal blood either in the afternoon of estrus, or on diestrous days 1 or 2, or in the early afternoon of proestrus. The B-EPlike immunoreactivity was noted to reach a significantly lower level than at any other time of the cycle in the late afternoon of proestrus. All our rats were decapitated in the morning between lOOO1200h, and none showed a late proestrus. Therefore we assumed it was acceptable to combine the l3-EP results acquired from various tissues in Gr. INT, which were obtained at different stages of the estrous cycle. In rats, Rossier et al. (21) have shown that B-EP levels in the brain are independent of pituitary levels. Several studies (18, 19, 26, 31) have measured l3-EP concentrations in the pituitary and hypothalamus under various conditions. Their results revealed different responses in these two regions suggesting that pituitary and hypothalamic B-EP possess separate control mechanisms. In addition, hypophysectomy or adrenalectomy produced no change in the brain levels of P-EP (32, 33). Our data support this contention since no significant correlations were found in B-EP levels of these two organs. Additional information is essential, however, to determine what effect l3-EP released from the hypothalamus and transported via the hypophyseal portal blood circulation, has on the synthesis and release of P-EP in the pituitary gland. Higher levels of P-EP are usually reported in the pituitary compared to the hypothalamus. The source of higher l3-EP is mainly from the intermediate lobe, although the P-EP levels of the anterior lobe alone have also been shown to be higher than the hypothalamus (8). We only checked the B-EP levels in the anterior, but not the intermediate or the neurointermediate lobe of the pituitary gland. This might explain at least in part, why we did not find much higher levels of B-EP in the pituitary compared to the hypothalamus. The levels of l3-EP in peripheral blood supposedly reflect the amount secreted by the pituitary gland (1, 34). In patients with hypopituitarism, plasma P-EP was undetectable (35). In rats, the plasma B-EP also became undetectable
EFFECT OF OVARIECTOMY
AND ESTROGEN REPLACEMENT
after hypophysectomy (36). A significant positive correlation has been observed between the amount of P-EP found in the peripheral blood and that of the pituitary gland (34). In our study, we were unable to show any significant correlations in P-EP levels when comparing any of the three groups of rats. The presence of changing increases and decreases in the P-EP levels of the peripheral blood and pituitary gland support the proposal that the peripheral blood levels of P-EP are a reflection of its release from the pituitary gland. However, the measurement of P-EP from one sampling at a single time may not represent dynamic changes such as the synthetic rate of P-EP in the pituitary or its rate of release into the general circulation. It is also not known to what degree P-EP from other sources, such as the pancreas (37), gastrointestinal tract (38), thyroid gland (38), and mast cells (39) may be contributing to the amount of peripheral P-EP. These contributions may dilute the significant correlation between the circulating l3-EP and l3-EP content in the pituitary gland. Less data exists when attempting to correlate P-EP levels in the CNS and in the peripheral blood. Different responses to estrogen exist in these two regions (24,31). Considering that P-EP in the peripheral blood is a reflection of its release from the pituitary gland (1,34), it is doubtful that a significant correlation would exist in P-EP levels of the hypothalamus and the peripheral blood. There is a trend toward concurrent decrements and increments in P-EP levels of the peripheral blood and the anterior pituitary gland, although these are not statistically significant. In contrast, there appears to be no association in the levels of P-EP in peripheral blood and the hypothalamus. l3-EP levels in the hypothalamus, anterior pituitary gland, and peripheral blood appear to be independent of one another in the presence or absence of estrogen.
Acknowledgements We would like to express our thanks to Dr Edward R. Smith for his helpful advice on the B-endorphin radioimmunoassay. We also wish to thank MS Jo A. Rabb for the excellent editorial assistance in the preparation of the manuscript.
IN THE RAT
179
References 1. Eipper, B. A. and Mains, R. E. (1980). Structure and biosynthesis of proadrenocorticotropimendorphin and related peptides. Endocrine Reviews 1: 1-27. 2. Roberts, J. L. and Herbert, E. (1977). Characterization of a common precursor to corticotropin and B-lipotropin: of B-lipotropin peptides and their Identification arrangement relative to corticotropin in the precursor synthesized in cell-free system. Proceedings of the National Academy of Science 74: 5300-5304. 3. Liotta, D.. Gildersleeve, D., Brownstein, M. J. and Krieger. D. T. (1979). Biosynthesis in vitro of immunoreactive 31 ,OOO-dalton corticotropin/B-endorphin-like material by bovine hypothalamus. Proceedings of the National Academy of Science 76: 1448-1452. 4. Cicero. T. J.. Schainker. B. A. and Meyer, E. R. (1979). Endogenous opioids participate in the regulation of the hypothalamic-pituitary-luteinizing hormone axis and testosterone’s negative feedback control of luteinizing hormone. Endocrinology 104: 1286-1291. 5. Sylvester, P. W., Van Vugt, D. A.. Aylsworth. C. A.. Hanson, E. A. and Meites, J. (1982). Effects of morphine and naloxone on inhibition by ovarian hormones of pulsatile release of LH in ovariectomized rats. Neuroendocrinology 34: 269-273. 6. Van Vugt, D. A., Sylvester, P. W., Aylsworth, C. A. and Meites, J. (1982). Counteraction of gonadal steroid inhibition of luteinizing hormone release by naloxone. Neuroendocrinology 34: 274-278. 7. Colt, E. W. D., Wardlaw, S. and Frantz, A. G. (1981). The effect of running on plasma B-endorphin. Life Science 28: 1637-1640. 8. Carr, D. B.. Bullen. B. A., Skrinar, G. S., Arnold. M. A.. Rosenblatt, M., Beitins. I. A., Martin, J. B. and McArthur, J. W. (1981). Physical conditioning facilitates the exercise-induced secretion of B-endorphin and B-lipotropin in women. New England Journal of Medicine 305: 560-562. 9. Genazzani, A. R., Facchinetti, F., Ricci-Danero, M. G.. Parrini. D.. Petraglia, F., Larosa, R. and D’Antona. N. (1981). B-lipotropin and B-endorphin in physiological and surgical menopause. Journal of Endocrinological Investigation 4: 375-378. 10. Vrbicky. K. W., Baumstark, J. S., Wells, I. C.. Hilgers, T. W., Kable. W. T. and Elias, C. J. (1982). Evidence for the involvement of B-endorphin in the human menstrual cycle. Fertility and Sterility 38: 701-704. 11. Russell. J. B., Mitchell, D. E.. Musey. P. I. and Collins, D. C. (1984). The role of B-endorphins and catechol estrogens on the hypothalamic-pituitary axis in female athletes. Fertility and Sterility 42: 690-695. 12. Aleem, F. S. and McIntosh. T. (1984). Elevated plasma levels of B-endorphin in a group of women with polycystic ovarian disease. Fertility and Sterility 42: 686-689. 13. Laatikainen. T., Raisanen. A., Tulenheimo. A. and Salminen. K. (1985). Plasma S-endorphin and the menstrual cycle. Fertility and Sterility 44: 206-209.
180 14. Chuong, C. J., Coulam, C. B., Kao, P. C., Bergstralh, E. J. and Go, V. L. W. (1985). Neuropeptide levels in premenstrual syndrome. Fertility and Sterility 44: 760-765. 15. Tepper, R., Neri, A., Kaufman, H. Schoenfeld, A. and Ovadia, J. (1987). Obstetrics and Gynecology 70: 150-152. 16. Facchinetti, F., Martignoni, E., Petraglia, F., Sances, M. G., Nappi, G. and Ganazzani, A. R. (1987). Premenstrual fall of plasma B-endorphin in patients with premenstrual syndrome. Fertility and Sterility 47: 570-573. 17. Parkening, T. A., Collins, T. J. and Smith, E. R. (1980). Plasma and pituitary concentrations of LH, FSH and prolactin in aged female C57BU6 mice. Journal of Reproductive Fertility 58: 377-386. 18. Wardlaw, S. L., Wang, P. J. and Frantz, A. G. (1985). Regulation of B-endorphin and ACTH in the brain by estradiol. Life Science 37: 1941-1947. 19. Forman, L. J. and Estilow, S. (1988). Estrogen influences the effect of immobilization stress on immunoreactive B-endorphin levels in the female rat pituitary. Proceedings of the Society for Experimental Biological Medicine 187: 190-196. 20. Sarkar, D. K. and Yen, S. S. C. (1985). Changes in 8-endorphin-like immunoreactivity in pituitary portal blood during the estrous cycle and after ovariectomy in rats. Endocrinology 116: 2075-2079. 21. Rossier, J., Vargo, T. M., Minick, S., Long, N., Bloom, F. E. and Guillemin, R. (1977). Regional dissociation of B-endorphin and enkephalin contents in rat brain and pituitary. Proceedings of the National Academy of Science 74: 5162-5. 22. Orf, J. W., Johanson, E. N., Vanderloise, F. and Lindall, A. W. (1979). Extraction of B-endorphin from serum binding proteins. Clinical Research 27: 680A. 23. Wardlaw, S. L., Thoron, L. and Frantz, A. G. (1982). Effects of sex steroids on brain B-endorphin. Brain Research 245: 327-31. 24. Lee, S., Panerai, A. E., Bellabarba, D. and Friesen, H. G. (1980). Effect of endocrine modifications and pharmacological treatments on brain and pituitary concentrations of S-endorphin. Endocrinology 107: 245-248. 25. Forman, L. J., Sonntag, W. E., Kylka, V. W. and Meites, J. (1985). Mediation of gonadal steroids of plasma P-endorphin and LH in castrated female and male rats. Peptides 6: 835-839. 26. Forman, J. and Estilow, S. (1986). Neurotransmitters and estrogen interact to affect B-endorphin levels in castrated female rats. Peptides 7: 775-781. 27. Wilcox, J. N. and Roberts, J. L. (1985). Estrogen decreases rat hypothalamic proopiomelanocortin mess-
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28.
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enger ribonucleic acid levels. Endocrinology 117: 23922396. Roberts, J. L., Wilcox, J. N. and Blum, M. (1986). The regulation of proopiomelanocortin gene expression by estrogen in the rat hypothalamus. Neuroendocrine Molecular Biology 261. Wardlaw, S. L., Wehrenberg, W. B., Ferin, M., Antunes, J. L. and Frantz, A. F. (1982). Effect of sex steroids on b-endorphin in hypophyseal portal blood. Journal of Clinical Endocrinology and Metabolism 55: 877-881. Wehrenberg, W. B., Wardlaw, S. L., Frantz, A. G. and Ferin, M. (1982). S-endorphin in hypophyseal portal blood: variations throughout the menstrual cycle. Endocrinology 111: 879-881. Hulse, G. K. andcoleman, G. J. (1984). Effectofstresson tissue and plasma levels of immunoreactive S-endorphin in ovariectomized rats primed with oestrogen and progesterone. Journal of Endocrinology 100: 277-280. Krieger, D. T., Liotta, A. and Brownstein, M. J. (1977). Presence of corticotropin in brain of normal and hypophysectomized rats. Proceedings of the National Acadmy of Science 74: 648-652. Rossier, J., French, E., Gros. C., Minick, S., Guillemin, R. and Bloom, F. E. (1979). Adrenalectomy, dexamethasone or stress alters opioid peptide levels in rat interior pituitary but not intermediate lobe or brain. Life Science 25: 2105-2112. Rossier, J., French, E. D., Rivier, C., Ling, N., Guillemin, R. and Bloom, F. E. (1977). Footshock-induced stress increases B-endorphin levels in blood but not brain. Nature 270: 618-620. Jeffcoate, W. J., McLaughlin, L., Hope, J., Rees, L. H., Ratter, S. J. and Lowry. P. (1978). B-endorphin in human cerebrospinal fluid. Lancet 119-121. Guillemin, R., Vargo, T., Rossier, J., Minick, S., Ling, N., Rivier. C., Vale, W. and Bloom, F. (1977). B-endorphin and adrenocorticotropin are secreted concomitantly by the pituitary gland. Science 197: 1367-1369. Bruni, J. F., Watkins, W. B. and Yen, S. S. C. (1979). B-endorphin in the human pancreas. Journal of Clinical Endocrinology and Metabolism 49: 649-651. Kendall, J. W., Orwoll, E. and Gaudette, D. (1980). Extrapituitary ACTH B-endorphin in tissues of APUD series. Program 6th International Congress on Endocrinology 528. DiAugustine, R. P., Lazarus, L. H., Jahnke, G., Kahn, M. N., Erisman, M. D. and Linnoila, R. I. (1980). Corticotropin/B-endorphin immunoreactivity in rat mast cells: peptide or protease. Life Science 27: 2663-2668.
Effect of Ovariectomy and Estrogen Replacement on Hypothalamic, Pituitary and Peripheral Blood p-Endorphin Levels in the Rat S. LE*, C.J. CHUONG*S
and T.A. PARKENING*t
*Division of Reproductive Endocrinology/Infertility, Department of Obstetrics and Gynecology; toepartment of Anatomy and Neurosciences, University of Texas Medical Branch, Galveston, TX, 77051 USA; *Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA (Reprint requests to CJC#)
Abstract-This study was initiated to detect possible changes in P-endorphin (P-EP) levels of the hypothalamus, anterior pituitary gland, and peripheral blood of rats after ovariectomy and estrogen administration. Attempts were also made to determine the correlation between peripheral and central levels of P-EP. Twenty-six Sprague-Dawley rats were decapitated. Nine had intact ovaries (Gr. INT), and 17 were ovariectomized 3 weeks before they were killed. Nine of the ovariectomized rats received estradiol benzoate (EB) (Gr. EB) and the other 8 received peanut oil (Gr. OVX) prior to the decapitation. A P-EP radioimmunoassay was used to analyze homogenates of the hypothalamus and anterior pituitary, and peripheral blood. In the hypothalamus, P-EP levels were significantly lower in Gr. INT and Gr. EB than in Gr. OVX. In the pituitary gland and peripheral blood, P-EP levels were significantly higher in Gr. INT than in Gr. OVX. Pituitary P-EP levels did not vary between Gr. OVX and Gr. EB, although P-EP levels in peripheral blood were significantly higher in Gr. EB than in Gr. OVX. No significant correlations were noted in P-EP levels between the hypothalamus, pituitary gland, and peripheral blood in either Gr. INT, Gr. OVX, or Gr. EB. It appears that EB exerts different effects on P-EP levels in the hypothalamus, anterior pituitary gland, and peripheral blood, and that P-EP levels in these regions may be independent of one another.
Introduction P-endorphin (P-EP) is synthesized primarily in the anterior and intermediate lobes of the pituitary gland and in the arcuate nucleus of the hypothalaDate received 13 June 1991 Date accepted 25 July 1991
mus (l-3). It is of interest because it may act as a modulator through which gonadal hormones exert negative feedback on gonadotropin release (4-6). Since information on the relationship between estrogen and hypothalamic, pituitary, and plasma P-EP is still lacking, our study was conducted to investigate the effects of estrogen on hypothalamic, pituitary. and plasma P-EP by examining 175
NEUROPEPTIDES
176 P-EP in these three regions at a given time after ovariectomy and estrogen replacement therapy. Currently available techniques do not afford access to the central site of synthesis and secretion of P-EP to allow direct measurement of this neuropeptide in humans. Several studies have been conducted to measure /3-EP in peripheral blood (7-16) however, the correlation between peripheral levels of P-EP and central nervous system (CNS) opioid activity remains controversial. Therefore, another purpose of our study was to determine if any correlation existed between peripheral and central levels of P-EP.
Materials and Methods Animals
Adult female Sprague-Dawley rats weighing 180200g were housed at room temperature (22-26”(Z) with the free access to rat chow and water. All rats were acclimatized in a 12-h light/dark cycle (lights on from 0700-1900h) and accustomized to daily handling for 2 weeks before the study. Vaginal smears were obtained to determine the day of the estrous cycle. Twenty-six rats were decapitated between lOOO-1200h. Nine had intact ovaries (Gr. INT), and 17 underwent ovariectomy procedures 3 weeks before they were killed. The ovaries were removed through flank incisions under ether anesthesia. The 17 ovariectomized rats were randomly divided into two groups. Nine (Gr. EB) received EB at 20&kg dissolved in 0.2~~ of peanut oil daily for 2 days intraperitoneally, and 8 (Gr. OVX) received only peanut oil. Sample collection
All animals were decapitated 10s after removal from their cages. Rats decapitated were in an area separated from the cage area so as to avoid stressing rats yet to be samples. Animals in Gr. EB and Gr. OVX were killed 24h after the second injection of EB or peanut oil. Trunk blood was collected into glass tubes containing EDTA (7.2mg/5ml blood) and immediately centrifuged at 760g for 1Omin at 4°C to separate the plasma, which was stored at -80°C for later assay. The skulls were immediately opened via an oropharyngeal approach and the brain was
removed to expose the pituitary. After the removal of the diaphragm sella, the pituitary was lifted out and placed on ice under a dissecting microscope, and the anterior lobe separated from the intermediate and posterior lobes. The anterior lobe was then weighed, and homogenized in lml of 0.2 M HCl containing 0.05 M EDTA and 0.1% sodium azide. The homogenate was centrifuged at 4000g for 30min at 4°C and the supernatant separated and stored at -80°C for later assay (17). The hypothalamus was dissected out on a plastic sheet placed on ice as a region 3mm in depth limited rostrally by the anterior margin of the optic chiasm, caudally by the mammillary bodies and laterally by the hypothalamic sulci. After weighing, the tissues were homogenized, centrifuged, and the supernatant stored the same way as for the pituitary. It is known that plasma, pituitary and brain tissues contain proteolytic enzymes which may degradate P-EP during processing of these samples. However, the addition of the enzyme inhibitor during the process of sample collection was not considered to be necessary in most studies (7,8, 14, 15, 18, 19, 20, 21). Laboratory tests
Each sample of plasma and supernatants of the pituitary and hypothalamus were assayed in duplicate for P-EP with in 1 month after sample collection. The assay was performed according to the affinity gel extraction method (Immunonuclear Corp., Stillwater, Minn, USA). A major problem known to affect the direct assay of P-EP is the non-specific binding of iodinated P-EP to high molecular weight plasma or tissue components at an alkaline pH (22). This binding interferes with the radioimmunoassay and can be circumvented by either extraction or column chromatography. Such interference does not appear to be present in acid tissue extracts and is lessened at neutral pH. Of several methods studies for the extraction of P-EP the affinity gel extraction method has been found to adequately separate P-EP from non-specific binding material. The method consisted of extracting P-EP from plasma or homogenates using specific adsorption particles (Sepharose). Rabbit anti+EP was
EFFECT OF OVARIECTOMY
Table 1
AND ESTROGEN REPLACEMENT
P-EP levels in the hypothalamus,
f3-EP (Mean f SE)
pituitary
gland and peripheral
Group fN)
Hypothalamus (PmoVmg) Pituitary (PmoVmg) Peripheral blood (PmoUL)
177
IN THE RAT
blood in the rat
INT (9)
ovx (8)
EB (9)
2.8 + 0.3” 17.7 + 2.9b 37.5 + 4.9”
6.1 f 1.6 8.5 + 1.2 18.9 f 3.3
2.5 If: 0.3” 10.8 Z!I1.7 41.3 I!Z6.0”
“P < 0.05 V.S. ovx. bP < 0.01 V.S. ovx. (N): Number of rats in each group.
coupled to Sepharose and diluted in bovine serum albumin-borate buffer containing merthiolate (lyophilized). P-EP was then eluted from the Sepharose particles with 0.025N HCl, and measured in a radioimmunoassay. The eluants were incubated in rabbit anti-p-EP serum with 1% BSA-borate buffer for 20 h at 4°C. Iodinated-P-EP was added and the mixture incubated for 20h at 4°C before adding Goat Anti-Rabbit Precipitating Complex (GAR-PPT). After 30min the mixture was centrifuged (20min at 760g) and the supernatant discarded. The precipitate was counted with a gamma scintillation counter. Synthetic human P-EP was used at prediluted concentrations to construct a standard curve correlating percent bound I-P-EP to the P-EP concentration. Counts of the standard and unknown were corrected to non-specific binding. The assay could detect
Results
In Gr. INT, 4 rats were in diestrus, 3 in estrus, and 2 in proestrus. In Gr. and Gr. OVX, the rats were also cycling before ovariectomy. Hypothalamic P-EP levels were significantly lower in Gr. INT than in Gr. OVX. In contrast, P-EP levels in the anterior pituitary gland and peripheral blood were significantly higher. When comparing P-EP levels in Gr. EB and Gr. OVX, hypothalamic levels were significantly lower, pituitary levels were higher, although not statistically significant, and peripheral blood levels were significantly higher (Table 1). No significant correlations were noted in P-EP levels between the hypothalamus and the pituitary, the hypothalamus and the peripheral blood, or the pituitary and the peripheral blood in either Gr. INT, Gr. OVX, Gr. EB or combination of the three groups (Table 2).
Table 2
Correlation gland, and peripheral
Group (N)
r*
Statistical analysis
Data was analyzed using the Student’s two tailedtest, and the computation of Pearson correlation coefficient. Only rats with complete P-EP data on the hypothalamus, pituitary gland, and peripheral blood were included in the analysis of correlations on P-EP levels of these three regions. The level of significance was P < 0.05.
between hypothalamic, pituitary blood P-EP levels in the rat
Pituitary gland V.S. hypothalamus Peripheral blood vs. hypothalamus Peripheral blood
INT (9) 0.36
OVX (5) 0.76
EB (6) 0.25
0.22
-0.13
-0.80
0.11
-0.32
-0.16
Total (20) 0.005 -0.38
0.15
V.S. pituitary gland *Pearson Correlation Coefficient. (N): Number of rats with complete p-EP data on hypothalamus, pituitary gland and peripheral All P values were >O.OS.
blood.
178 Discussion Studies (18,19,20,23-26) have been conducted on B-EP levels in the hypothalamus, pituitary gland and peripheral blood in the presence or absence of estrogen. However, most studies were conducted to examine only one or two of the above. Wilcox and Roberts (27) have shown that proopiomelanocortin (POMC) messenger ribonucleic acid levels decrease in the hypothalamus of ovariectomized rats after 3 or 14 days of estradiol treatment. Estradiol has also been shown to inhibit POMC gene transcription in ovariectomized rats within 60min after hormone administration (28). Lower l3-EP levels in hypophyseal portal blood in ovariectomized rats (20) and the stimulatory effect of estradiol on hypothalamic l3-EP release into the hypophyseal portal blood have also been reported (29,30). The simultaneous measurement of P-EP in the hypothalamus and hypophyseal portal blood could determine the location of the estrogenic effect. The presence of lower pituitary and peripheral blood l3-EP levels in Gr. OVX than in Gr. INT suggests that the synthesis of pituitary B-EP may be decreased, and that less is secreted into the peripheral circulation in the absence of estrogen. The dosages of estrogen replacement in previous studies included EB 20 l&rat/day subcutaneously for 1 or 2 days (26), 5 @at/day subcutaneously for 7 days (26), silastic capsules containing 262l.~g 17 B-estradioYm1 in sesame oil (10 mm/100 gm SW) subcutaneous implantation for 1 or 3 days (27) or for 1 or 3 weeks (18). Different dosages and durations of EB replacement resulted in different B-EP levels in various tissues (18, 25). We were interested in the effect of lower dosages and shorter duration of EB replacement, and only used about 4 CL/rat/day intraperitoneally for 2 days. Yet, we were able to restore P-EP levels in the peripheral blood of Gr. OVX to those found in Gr. INT. No statistically significant changes occurred in the l3-EP levels of the pituitary gland in these two groups. These data suggest that the synthesis of pituitary l3-EP may have been increased with no net changes in the amount stored since there may have been a concurrent increase in the secretion of l3-EP into the peripheral circulation. Although the rats in Gr. INT were in different
NEUROPEPTIDES
stages of estrous cycle, Sarkar and Yen (20) showed that there were no appreciable differences between the l3-EP-like immunoreactivity concentration in the hypophyseal portal blood either in the afternoon of estrus, or on diestrous days 1 or 2, or in the early afternoon of proestrus. The B-EPlike immunoreactivity was noted to reach a significantly lower level than at any other time of the cycle in the late afternoon of proestrus. All our rats were decapitated in the morning between lOOO1200h, and none showed a late proestrus. Therefore we assumed it was acceptable to combine the l3-EP results acquired from various tissues in Gr. INT, which were obtained at different stages of the estrous cycle. In rats, Rossier et al. (21) have shown that B-EP levels in the brain are independent of pituitary levels. Several studies (18, 19, 26, 31) have measured l3-EP concentrations in the pituitary and hypothalamus under various conditions. Their results revealed different responses in these two regions suggesting that pituitary and hypothalamic B-EP possess separate control mechanisms. In addition, hypophysectomy or adrenalectomy produced no change in the brain levels of P-EP (32, 33). Our data support this contention since no significant correlations were found in B-EP levels of these two organs. Additional information is essential, however, to determine what effect l3-EP released from the hypothalamus and transported via the hypophyseal portal blood circulation, has on the synthesis and release of P-EP in the pituitary gland. Higher levels of P-EP are usually reported in the pituitary compared to the hypothalamus. The source of higher l3-EP is mainly from the intermediate lobe, although the P-EP levels of the anterior lobe alone have also been shown to be higher than the hypothalamus (8). We only checked the B-EP levels in the anterior, but not the intermediate or the neurointermediate lobe of the pituitary gland. This might explain at least in part, why we did not find much higher levels of B-EP in the pituitary compared to the hypothalamus. The levels of l3-EP in peripheral blood supposedly reflect the amount secreted by the pituitary gland (1, 34). In patients with hypopituitarism, plasma P-EP was undetectable (35). In rats, the plasma B-EP also became undetectable
EFFECT OF OVARIECTOMY
AND ESTROGEN REPLACEMENT
after hypophysectomy (36). A significant positive correlation has been observed between the amount of P-EP found in the peripheral blood and that of the pituitary gland (34). In our study, we were unable to show any significant correlations in P-EP levels when comparing any of the three groups of rats. The presence of changing increases and decreases in the P-EP levels of the peripheral blood and pituitary gland support the proposal that the peripheral blood levels of P-EP are a reflection of its release from the pituitary gland. However, the measurement of P-EP from one sampling at a single time may not represent dynamic changes such as the synthetic rate of P-EP in the pituitary or its rate of release into the general circulation. It is also not known to what degree P-EP from other sources, such as the pancreas (37), gastrointestinal tract (38), thyroid gland (38), and mast cells (39) may be contributing to the amount of peripheral P-EP. These contributions may dilute the significant correlation between the circulating l3-EP and l3-EP content in the pituitary gland. Less data exists when attempting to correlate P-EP levels in the CNS and in the peripheral blood. Different responses to estrogen exist in these two regions (24,31). Considering that P-EP in the peripheral blood is a reflection of its release from the pituitary gland (1,34), it is doubtful that a significant correlation would exist in P-EP levels of the hypothalamus and the peripheral blood. There is a trend toward concurrent decrements and increments in P-EP levels of the peripheral blood and the anterior pituitary gland, although these are not statistically significant. In contrast, there appears to be no association in the levels of P-EP in peripheral blood and the hypothalamus. l3-EP levels in the hypothalamus, anterior pituitary gland, and peripheral blood appear to be independent of one another in the presence or absence of estrogen.
Acknowledgements We would like to express our thanks to Dr Edward R. Smith for his helpful advice on the B-endorphin radioimmunoassay. We also wish to thank MS Jo A. Rabb for the excellent editorial assistance in the preparation of the manuscript.
IN THE RAT
179
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