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Neutral aminopeptidase activity levels during the estrous cycle and the pregnancy in the hypothalamus and the pituitary of the rat
Life Sciences, Vol. Printed in the U S A
52, pp.
1629-1632
Pergamon
Press
NEUTRAL AMINOPEPTIDASE ACTIVITY LEVELS DURING THE ESTROUS CYCLE AND THE PREGNANCY IN THE HYPOTHALAMUS AND THE PITUITARY OF THE RAT J.M. de Gandarias, J. Irazusta, E. Echevarrfa and L. Casis* Department of Physiology. Medical School.University of the Basque Country. P.O. Box 699. Bilbao. Spain.
(Received
in final
form March 9, 1993)
Summary Neutral aminopeptidase activity levels during the estrous cycle and pregnancy, in the hypothalamus and the pituitary of experimental animals, was studied in this research. The cyclic stages were the estrous, diestrous and proestrous (at morning and afternoon). The pregnancy phases were the 2nd, 7th, 14th and 20th day postinsemination. The parturition day was also studied. The higher cyclic enzyme levels were found in the proestrous stage (afternoon). No differences between estrous and diestrous were observed. During pregnancy, there was a rise at 7th and 14th day, with a decrease in the 20th day. In the hypothalamus, significant increases after parturition were shown, the latter not being observed in the pituitary. In general, the changes seem to be parallel to those of the luteinizing hormone. The cyclic alterations in the secretion of gonadotropins (Gn) during the estrous cycle in rats are influenced by changes in the plasma concentrations of sex steroids. These feedback actions of steroid hormones are mediated by the hypothalamus and by the changing responsiveness of the pituitary to LH-RH. It has been described that L-Cystine-arylamidase, which is capable of inactivating LH-RH in the hypothalamus and the pituitary, could play a role in the regulation of gonadotropin release (1). This aminopeptidase activity was found to behave, during the estrous cycle, inversely with respect to the release of gonadotropins from the anterior pituitary, showing its lower levels during the proestrous stage (2). Most recently, it has been shown that neutral and basic aminopeptidase activities do not behave inversely but in a parallel way with respect to the release of Gn during the estrous cycle of the rat, the enzyme levels being higher during the proestrous than in the estrous stage (3,4). The changes in neutral and basic enzymatic activities seem to be specific to the brain areas more directly related to reproduction (5). These changes also run parallel to the LH levels after orchydectomy and ovariectomy (6). In this paper, we present the levels of a soluble neutral aminopeptidase activity (nAAP) during the estrous cycle and during different stages of pregnancy in order to provide more information on the possible feedback mechanisms of the hormonal surge from the hypothalamicpituitary axis.
* To whom correspondence should be sent.
Copyright
0024-3205/93 $6.00 + .00 © 1993 Pergamon Press Ltd All rights reserved.
1630
n A A P C h a n g e s in L H L e v e l s
Vol.
52, No. 20,
1993
Material and Methods Female Sprague-Dawley rats, bred in our colony and maintained under conditions of controlled illumination (lights on from 07.00 to 19.00) and temperature (24 -°C), with unlimited access to water and standard rat chow, were used in this investigation. All the rats involved were 3 months old: this is important in view of age-related changes of aminopeptidase activities from two weeks to three months (7), without further changes in older animals (8). Timing of the estrous cycle was determined by examining dally vaginal smears and only rats showing two or more regular cycles were selected for the experiments. The cyclic animals were grouped into estrous (at 10.00 h), diestrous (at 10.00 h) and proestrous (at 10.00 and 15.00 h) groups (n=10 each). The pregnancy stages were as follows: 2, 7, 14 and 20 days postinsemination (n=10 each) (day of insemination was considered day zero of gestation). Also the parturition day (n=10) was included in the research. The animals were set under Equithensin anesthesia (2 ml/kg body wt) and perfused with saline plus 50 mmol/l phosphate buffer, pH 7.4, from the left cardiac ventricle. After perfusion, the hypothalamus (taken by dissection) and the pituitary gland were homogenized (in 10 mmol/l Tris HC1, pH 7.4) and ultracentrifuged (100.000 g, 35 min). The resulting supernatant was used for the analysis of nAAP and proteins. Aminopeptidase activity was fluorimetrically measured in triplicate, using Leu-2naphthylamide as substrate, by the method of Greenberg (9) but with the recent modifications described by Alba et al. (10). The results were recorded as units of nAAP per milligram of protein (mean + SEM). One unit of nAAP is the amount of enzyme that hydrolyzes one picomol of aminoacyl-2-naphthylamide per minute. Protein concentration was measured (in triplicate) by the method of Bradford (11). Differences between means were calculated by the Scheff6's test, and comparison between groups by ANOVA test. Results and Discussion Figure 1 shows the nAAP during the estrous cycle and pregnancy in the hypothalamus. ANOVA test detected significant differences throughout the cycle (p<0.001) and pregnancy (p<0.01). During the estrous cycle, there were no differences between estrous and diestrous, but the levels of the proestrous (morning p<0.05, afternoon p<0.005) were significantly higher than the stages mentioned above. The enzyme levels in the middle phase of the pregnancy (7 and 14 days) were higher (not significant) than those of the 2nd and 20th. There was a significant rise after parturition (p<0.05) when compared with the estrous stage. Figure 2 shows the nAAP in the pituitary gland during the cycle and pregnancy. ANOVA test shows significant differences throughout the estrous cycle (p<0.001) and the pregnancy (p<0.005). As with the hypothalamus, no significant differences between estrous and diestrous were observed, but the levels of the proestrous phase (afternoon p<0.005) were higher than those of the estrous. During pregnancy, the levels in the 7th and 14th days were clear but not significantly higher than those of the 2nd. The enzyme levels on the 20th day were significantly lower than those of the 7th day (p<0.05). No changes after parturition were appreciated. These results show how in the estrous cycle the higher levels of nAAP are found in the proestrous. In this cyclic stage, the enzyme levels were higher in the afternoon than in the morning. It is well established that in the rat LH response, as evoked by a standard stimulus of LH-RH, increases steadily during the estrous cycle, reaching maximal values during the early afternoon of proestrous (12,13).
Vol.
52, NO.
20,
1993
nAAP C h a n g e s
in LH L e v e l s
1631
160 140 120 1O0 80 60 4-0 20 0 E
D
Pm
Po
2
7
14
20
Part
Fig. 1 Neutral aminopeptidase activity during the estrous cycle (E= estrous; D= diestrous; Pm= proestrous morning; Pa= proestrous afternoon) and pregnancy (2, 7, 14, 20=- days; Part= parturition) in the hypothalamus. 120
1 ©0
80
60
40
20
0
,
E
.
.
.
D
.
.
Pm
.
.
.
Pc
.
.
.
2
7
14
20
Part
Fig. 2 Neutral aminopeptidase activity during the estrous cycle (E= estrous; D= diestrous; Pro= proestrous morning; Pa= proestrous afternoon) and pregnancy (2, 7, 14, 20=- days; Part= parturition) in the pituitary gland. Also changes in aminopeptidase activity throughout the pregnancy were observed. The maintenance in the levels of enzyme activity produced on the second day after insemination (compared with estrus stage) and the significant rise in the postpartum day (compared also with estrous stage), likewise are parallel to the LH secretion (14). The increase in the activity after parturition was not observed in the pituitary. However, it is necessary to note that the LH surge from the pituitary during the estrous cycle and in the parturition are differentially regulated (14). With respect to the clear, but in some cases not significant, increases in nAAP observed during the middle stages of pregnancy, the general increase in the levels of pituitary hormones during these phases must be noted. In summary, the results obtained in this research shows how soluble neutral aminopeptidase activity changes (in general) its activity parallel with the levels of LH. With these results, to date, it is not possible to assign a specific role for this enzyme activity in the regulation of gonadotropin, but with the observed changes, it could be that the enzyme action is not altogether directed at the metabolic conlrol of LH. Acknowledgments We would like to thank Prof. David Hallett for his revision of the manuscript.
1632
nAAP C h a n g e s
in LH L e v e l s
Vol.
52, No.
20,
1993
References 1. H. KUHL, H.-D. TAUBERT, Acta Endocrinol. 78 634-648 (1975). 2. H. KUHL, C. ROSNIATOWSKI, H.-D.TAUBERT, Acta Endocrinol. 87 476-484 (1978). 3. J.M. de GANDARIAS, L. CASIS, J. IRAZUSTA, E. ECHEVARRIA, G. ARECHAGA, M. RAMIREZ, Acta Endocrinol. 121 671-673 (1989). 4. J.M. de GANDARIAS, M. RAMIREZ, E. ECHEVARRIA, J. IRAZUSTA, L. CASIS, Gen. Physiol. Biophys. 9 385-389 (1990). 5. J.M. de GANDARIAS, E. ECHEVARRIA, J. IRAZUSTA, L. CASIS, Exp. Clin. Endocrinol. 99 64-67 (1992). 6. J.M. de GANDARIAS, M. RAMIREZ, J. ZULAICA, L. CASIS, Horm. metabol. Res. 21 285-286 (1989). 7. J.M. de GANDARIAS, M. RAMIREZ, J. ZULAICA, C. IRIBAR, L. CASIS, J. Biochem. 105 44-46 (1989). 8. J.M. de GANDARIAS, L. CASIS, R. MUGICA, G. ARECHAGA, M. RAMIREZ, Rev. esp. Fisiol. 45 89-94 (1989). 9. L.J. GREENBERG, Biochem. Biophys. Res. Commun. 9 430-435 (1962). 10. F. ALBA, C. IRIBAR, M. RAMIREZ, C. ARENAS, Arch. Neurobiol. 52 169-173 (1989). 11. M.M.BRADFORD, Analyt.Biochem. 72 248-254 (1976). 12. F. NAKAMURA, K. TAYA, S. SASAMOTO, F. YOSHIMURA, Acta Anat. 124 104-110 (1985). 13. G. WATANABE, K. TAYA, S. SASAMOTO, J. Endocrinol. 26 151-157 (1990). 14. T.R. KOITER, G.C.J. van der SCHAAF-VERDONK, G.A. SCHUILING, J. Endocrinol. 115 247-254 (1987).
52, pp.
1629-1632
Pergamon
Press
NEUTRAL AMINOPEPTIDASE ACTIVITY LEVELS DURING THE ESTROUS CYCLE AND THE PREGNANCY IN THE HYPOTHALAMUS AND THE PITUITARY OF THE RAT J.M. de Gandarias, J. Irazusta, E. Echevarrfa and L. Casis* Department of Physiology. Medical School.University of the Basque Country. P.O. Box 699. Bilbao. Spain.
(Received
in final
form March 9, 1993)
Summary Neutral aminopeptidase activity levels during the estrous cycle and pregnancy, in the hypothalamus and the pituitary of experimental animals, was studied in this research. The cyclic stages were the estrous, diestrous and proestrous (at morning and afternoon). The pregnancy phases were the 2nd, 7th, 14th and 20th day postinsemination. The parturition day was also studied. The higher cyclic enzyme levels were found in the proestrous stage (afternoon). No differences between estrous and diestrous were observed. During pregnancy, there was a rise at 7th and 14th day, with a decrease in the 20th day. In the hypothalamus, significant increases after parturition were shown, the latter not being observed in the pituitary. In general, the changes seem to be parallel to those of the luteinizing hormone. The cyclic alterations in the secretion of gonadotropins (Gn) during the estrous cycle in rats are influenced by changes in the plasma concentrations of sex steroids. These feedback actions of steroid hormones are mediated by the hypothalamus and by the changing responsiveness of the pituitary to LH-RH. It has been described that L-Cystine-arylamidase, which is capable of inactivating LH-RH in the hypothalamus and the pituitary, could play a role in the regulation of gonadotropin release (1). This aminopeptidase activity was found to behave, during the estrous cycle, inversely with respect to the release of gonadotropins from the anterior pituitary, showing its lower levels during the proestrous stage (2). Most recently, it has been shown that neutral and basic aminopeptidase activities do not behave inversely but in a parallel way with respect to the release of Gn during the estrous cycle of the rat, the enzyme levels being higher during the proestrous than in the estrous stage (3,4). The changes in neutral and basic enzymatic activities seem to be specific to the brain areas more directly related to reproduction (5). These changes also run parallel to the LH levels after orchydectomy and ovariectomy (6). In this paper, we present the levels of a soluble neutral aminopeptidase activity (nAAP) during the estrous cycle and during different stages of pregnancy in order to provide more information on the possible feedback mechanisms of the hormonal surge from the hypothalamicpituitary axis.
* To whom correspondence should be sent.
Copyright
0024-3205/93 $6.00 + .00 © 1993 Pergamon Press Ltd All rights reserved.
1630
n A A P C h a n g e s in L H L e v e l s
Vol.
52, No. 20,
1993
Material and Methods Female Sprague-Dawley rats, bred in our colony and maintained under conditions of controlled illumination (lights on from 07.00 to 19.00) and temperature (24 -°C), with unlimited access to water and standard rat chow, were used in this investigation. All the rats involved were 3 months old: this is important in view of age-related changes of aminopeptidase activities from two weeks to three months (7), without further changes in older animals (8). Timing of the estrous cycle was determined by examining dally vaginal smears and only rats showing two or more regular cycles were selected for the experiments. The cyclic animals were grouped into estrous (at 10.00 h), diestrous (at 10.00 h) and proestrous (at 10.00 and 15.00 h) groups (n=10 each). The pregnancy stages were as follows: 2, 7, 14 and 20 days postinsemination (n=10 each) (day of insemination was considered day zero of gestation). Also the parturition day (n=10) was included in the research. The animals were set under Equithensin anesthesia (2 ml/kg body wt) and perfused with saline plus 50 mmol/l phosphate buffer, pH 7.4, from the left cardiac ventricle. After perfusion, the hypothalamus (taken by dissection) and the pituitary gland were homogenized (in 10 mmol/l Tris HC1, pH 7.4) and ultracentrifuged (100.000 g, 35 min). The resulting supernatant was used for the analysis of nAAP and proteins. Aminopeptidase activity was fluorimetrically measured in triplicate, using Leu-2naphthylamide as substrate, by the method of Greenberg (9) but with the recent modifications described by Alba et al. (10). The results were recorded as units of nAAP per milligram of protein (mean + SEM). One unit of nAAP is the amount of enzyme that hydrolyzes one picomol of aminoacyl-2-naphthylamide per minute. Protein concentration was measured (in triplicate) by the method of Bradford (11). Differences between means were calculated by the Scheff6's test, and comparison between groups by ANOVA test. Results and Discussion Figure 1 shows the nAAP during the estrous cycle and pregnancy in the hypothalamus. ANOVA test detected significant differences throughout the cycle (p<0.001) and pregnancy (p<0.01). During the estrous cycle, there were no differences between estrous and diestrous, but the levels of the proestrous (morning p<0.05, afternoon p<0.005) were significantly higher than the stages mentioned above. The enzyme levels in the middle phase of the pregnancy (7 and 14 days) were higher (not significant) than those of the 2nd and 20th. There was a significant rise after parturition (p<0.05) when compared with the estrous stage. Figure 2 shows the nAAP in the pituitary gland during the cycle and pregnancy. ANOVA test shows significant differences throughout the estrous cycle (p<0.001) and the pregnancy (p<0.005). As with the hypothalamus, no significant differences between estrous and diestrous were observed, but the levels of the proestrous phase (afternoon p<0.005) were higher than those of the estrous. During pregnancy, the levels in the 7th and 14th days were clear but not significantly higher than those of the 2nd. The enzyme levels on the 20th day were significantly lower than those of the 7th day (p<0.05). No changes after parturition were appreciated. These results show how in the estrous cycle the higher levels of nAAP are found in the proestrous. In this cyclic stage, the enzyme levels were higher in the afternoon than in the morning. It is well established that in the rat LH response, as evoked by a standard stimulus of LH-RH, increases steadily during the estrous cycle, reaching maximal values during the early afternoon of proestrous (12,13).
Vol.
52, NO.
20,
1993
nAAP C h a n g e s
in LH L e v e l s
1631
160 140 120 1O0 80 60 4-0 20 0 E
D
Pm
Po
2
7
14
20
Part
Fig. 1 Neutral aminopeptidase activity during the estrous cycle (E= estrous; D= diestrous; Pm= proestrous morning; Pa= proestrous afternoon) and pregnancy (2, 7, 14, 20=- days; Part= parturition) in the hypothalamus. 120
1 ©0
80
60
40
20
0
,
E
.
.
.
D
.
.
Pm
.
.
.
Pc
.
.
.
2
7
14
20
Part
Fig. 2 Neutral aminopeptidase activity during the estrous cycle (E= estrous; D= diestrous; Pro= proestrous morning; Pa= proestrous afternoon) and pregnancy (2, 7, 14, 20=- days; Part= parturition) in the pituitary gland. Also changes in aminopeptidase activity throughout the pregnancy were observed. The maintenance in the levels of enzyme activity produced on the second day after insemination (compared with estrus stage) and the significant rise in the postpartum day (compared also with estrous stage), likewise are parallel to the LH secretion (14). The increase in the activity after parturition was not observed in the pituitary. However, it is necessary to note that the LH surge from the pituitary during the estrous cycle and in the parturition are differentially regulated (14). With respect to the clear, but in some cases not significant, increases in nAAP observed during the middle stages of pregnancy, the general increase in the levels of pituitary hormones during these phases must be noted. In summary, the results obtained in this research shows how soluble neutral aminopeptidase activity changes (in general) its activity parallel with the levels of LH. With these results, to date, it is not possible to assign a specific role for this enzyme activity in the regulation of gonadotropin, but with the observed changes, it could be that the enzyme action is not altogether directed at the metabolic conlrol of LH. Acknowledgments We would like to thank Prof. David Hallett for his revision of the manuscript.
1632
nAAP C h a n g e s
in LH L e v e l s
Vol.
52, No.
20,
1993
References 1. H. KUHL, H.-D. TAUBERT, Acta Endocrinol. 78 634-648 (1975). 2. H. KUHL, C. ROSNIATOWSKI, H.-D.TAUBERT, Acta Endocrinol. 87 476-484 (1978). 3. J.M. de GANDARIAS, L. CASIS, J. IRAZUSTA, E. ECHEVARRIA, G. ARECHAGA, M. RAMIREZ, Acta Endocrinol. 121 671-673 (1989). 4. J.M. de GANDARIAS, M. RAMIREZ, E. ECHEVARRIA, J. IRAZUSTA, L. CASIS, Gen. Physiol. Biophys. 9 385-389 (1990). 5. J.M. de GANDARIAS, E. ECHEVARRIA, J. IRAZUSTA, L. CASIS, Exp. Clin. Endocrinol. 99 64-67 (1992). 6. J.M. de GANDARIAS, M. RAMIREZ, J. ZULAICA, L. CASIS, Horm. metabol. Res. 21 285-286 (1989). 7. J.M. de GANDARIAS, M. RAMIREZ, J. ZULAICA, C. IRIBAR, L. CASIS, J. Biochem. 105 44-46 (1989). 8. J.M. de GANDARIAS, L. CASIS, R. MUGICA, G. ARECHAGA, M. RAMIREZ, Rev. esp. Fisiol. 45 89-94 (1989). 9. L.J. GREENBERG, Biochem. Biophys. Res. Commun. 9 430-435 (1962). 10. F. ALBA, C. IRIBAR, M. RAMIREZ, C. ARENAS, Arch. Neurobiol. 52 169-173 (1989). 11. M.M.BRADFORD, Analyt.Biochem. 72 248-254 (1976). 12. F. NAKAMURA, K. TAYA, S. SASAMOTO, F. YOSHIMURA, Acta Anat. 124 104-110 (1985). 13. G. WATANABE, K. TAYA, S. SASAMOTO, J. Endocrinol. 26 151-157 (1990). 14. T.R. KOITER, G.C.J. van der SCHAAF-VERDONK, G.A. SCHUILING, J. Endocrinol. 115 247-254 (1987).