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The choice of a model for studying the hypothalamus-pituitary interactions in vitro
Moleculur and Cellular Endocrmology, 45 (1986) 21-26
21
Elsevier Scientific Publishers Ireland, Ltd. MCE 01443
The choice of a model for studying the hypothalamus-pituitary interactions in vitro A. Dutt, M. Gill, S. Karanth, Division of Neuroendocrinology,
N. Lehri and H.S. Juneja
*
Institutefor Research in Reproduction (Indian Council of Medical Reseurch), Parel, Bombay 400 012 (India)
(Received 29 August 198.5;accepted 9 December 1985)
Key words; hypothalamus; pituitary; dopamine; in vitro LH; FSH; Prl.
Summary Comparative studies on the release of luteinizing hormone (LH), follicle-stimulating hormone (FSH) and prolactin (Prl) by the whole pituitary, pituitary plus hypothalamus and pituitary-hypothalamus complex (PHC) were undertaken to choose an appropriate model for studying the hypothalamus-pituitary interactions in vitro and to relate the importance of the intact neural connections between pituitary and hypothalamus on hypothalamus-pituitary interactions. Also the effect of including dopamine (DA) at 1 x lo-’ mol/l in these different in vitro systems on the release of LH, FSH and Prl was investigated. The pituitary released increasing amounts of LH and FSH at 2, 4 and 6 h but the amount of Prl released remained unchanged. The rates of release of LH, FSH and Prl by the pituitary were different and were characteristic of each hormone. Co-incubation of pituitary with hypothalamus stimulated the release of LH and FSH but inhibited the release of Prl. Pituitary-hypothalamus complex behaved almost identical to pituitary plus hypothalamus system. Inclusion of 1 X lo-’ M DA in the incubation medium stimulated the release of LH (80%) but inhibited the release of Prl (71%) by PHC. FSH was unaffected. DA had no significant effect on the release of LH, FSH and Prl by pituitary and pituitary plus hypothalamus systems. It is suggested that PHC is the system of choice for studying hypothalamus-pituitary interactions in vitro.
Introduction Several in vitro systems are available for studying metabolic functions of the hypothalamus (Rotsztejn et al., 1976; Gallardo and Ramirez, 1977; Negro-Vilar et al., 1979; Richardson et al., 1983), the pituitary (Guillemin and Vale, 1970; Arimura and Schally, 1975; Vale and Grant, 1975; Yeo et al., 1979) or the hypothalamus-pituitary interactions (Schally and Bowers, 1964; Schneider and McCann, 1969; Quijada et al., 1973; Powers * To whom correspondence should be addressed. 0303-7207/86/$03.50
and Johnson, 1984). In vitro studies on hypothalamus-pituitary interactions have utilised fragments of hypothalamus viz. basomedial hypothalamus, median eminence, ventral hypothalamus or any other hypothalamic area of defined topoghypothalamus-preoptic raphy (e.g. mediobasal area) in co-incubation with the adenohypophysis. In these studies adenohypophysis and hypothalamus are dissociated of their neural and vascular associations. In one study median eminencepituitary unit with intact portal plexus has been used (Miyachi et al., 1973). Gill et al. (1985) have successfully adopted the
0 1986 Elsevier Scientific Publishers Ireland, Ltd.
22
pituita~-h~othalamus complex (PI-K) system for in vitro studies. In this system neuroanatom~c juxtapositioning or closeness of pituitary to hypothalamus remains intact. In the present communication comparative studies on the release of LH, FSH and Prl by PHC, whole pituitary, and pituitary plus hypothalamus systems are reported. In addition the effect of dopamine added in vitro on the release of LH, FSH and Prl by these different systems is described. Materiah
and methods
A ~imals Holtzman strain adult male rats weighing 200-250 g were used. These rats were maintained in an air-conditioned facility with temperature varying from 22 to 24°C and the humidity ranging from 50 to 56%. The light schedule was 14 h light and 10 h darkness. The animals were fed a pelleted diet with water ad libitum. Dissection
of tissue
Pituitary-h~othalamus complex was dissected as described earlier (Gill et al., 1985). The rat was killed by decapitation. Its brain was exposed by a dorsal incision. The hypothal~c island was demarcated by cutting along the lateral hypothalamic sulci, posterior edge of the optic chiasma and the anterior edge of the mammillary bodies. The hypothalamic island attached to the pituitary via hypophysial stalk was lifted by an undercut 2 mm deep. Where only h~othalamus was desired, it was obtained by cutting the stalk joining the pituitary to the hypothalamus. The pituitary was carefully scooped from sella turcica after freeing it of adhering ligaments.
Dulbecco’s modified Eagle’s medium (DMEM) fortified with non-essential amino acids, 3 mM NaHCO, and 0.01 M Hepes (4-(2”hydroxyethyl)l-piperazine-ethanesulfonic acid) was used. Incubations were done at 37’C in Laxbroplastic (Laxbro, Pune, India) trays with 24 welfs of 5 ml capacity each. Each well contained 1 ml DMEM. Freshly dissected tissues were transferred into individual wells. The contents were pre-incubated for 1 h after which the spent incubation medium
was repiaced with 1 ml of fresh medium and the contexts incubated further. The incubation was stopped at desired times by removing the tissue from the incubation well. The tissue was blotted and weighed on a torsion balance. The spent incubation medium was transferred into individual tubes and stored at -30” until assayed for LH, FSH and Prl by RIA. hormone assays LH and FSH in culture medium were measured by RIA as described earlier (Nagen~~ath et al., 1982). The standard curve for LH (NIAMDDRat-LH-RP-1) ranged from 2 to 500 ng per assay tube, and for FSH (NIAM~D-Rat-FSH”RP-1) from 5 ng to 1 pg per assay tube. The inter- and intra-assay variations were 10 and 6% for FSH and 9 and 6% for LH assays, respectively. Prl was assayed as per the procedure described in the brochure supplied by the National Pituitary Agency, NIH, Bethesda, MD. The standard curve for Prl (NIA~DD-Rat-Prl-R~-2) ranged from IO pg to 12.5 ng per assay tube. Inter-assay and intra-assay variations for Prl were 14 and 596, respectively.
Levels of significance between 2 groups were computed using Student’s t-test. Results
Release of LH, FSH and Prf by different systems The amount of LH released by the whole pituitary at 2, 4 and 6 h of incubation increased progressively with incubation duration (Table 1). The rate of release was comparable at 2 and 4 h (0.27 f 0.03 and 0.25 + 0.03 pg,/h (mean i SEM), respectively) but increased significantly to 0.39 f 0.05 pg/h at 6 h. Co-incubation of pituitary with hypothalamus increased the amount and the rate of release of LH at 2, 4 and 6 h significantly as compared to the pituitary. Pituitary-hypothalamus complex secreted LH at rates comparable to pituitary plus hyp~th~amus but si~ificantly different from the pituitary. The amount of FSH released into the incubation medium increased proportionately with increased incubation duration (Table 2). The rate of
23 TABLE
1
RELEASE
OF LH BY DIFFERENT
SYSTEMS
IN VITRO
Tissues were dissected and incubated as described under time interval. Values are expressed as mean + SEM. System
Whole pituitary
Incubation
(h)
2 4 6
Materials
and methods.
Whole pituitary plus hypothalamus
Total a
Perh”
Total
Per h
Total
Per h
0.54iO.06 1.00*0.12 *** 2.34kO.3 *
0.27 k 0.03 0.25 f 0.03 0.39 f0.05 *
1.00+0.06 b 1.84+0.24 G** 4.56 f 0.48 G*
0.50 + 0.03 0.46 + 0.06 0.76+0.08 ***
0.72 f 0.08 1.84i0.28 d,* 3.9OkO.48 d.*
0.36 + 0.04 0.46 f 0.07 0.65 f 0.08 **
OF FSH BY DIFFERENT
SYSTEMS
Materials
Whole pituitary
System
Incubation
(h)
2 4 6
b P i 0.001, ’ P i 0.01,
and methods.
Incubations
Whole pituitary plus hypothalamus
were done in replicates
of 10 at each
Pituitary-hypothalamus complex
Total a
Perha
Total
Per h
Total
Per h
1.46 f 0.2 3.04kO.32 ** 5.88 f 0.6 *
0.73 f 0.1 0.76 f 0.08 0.98 f0.10
1.3 kO.06 3.04kO.32 * 8.88kO.9 b.*
0.65 f 0.03 0.76 + 0.08 1.48+0.15 *
1.2 +0.12 3.04*0.4 * 8.52 + 1.2 =,*
0.6 50.06 0.76f0.10 1.42kO.20
* P < 0.001,
a pg FSH (NIAMDD-RP-1); pituitary levels.
**P < 0.01, with respect
to FSH
=.*
levels at 2 h; b P < 0.02, ‘P i 0.05, with respect
to
3
RELEASE
OF Prl BY DIFFERENT
SYSTEMS
IN VITRO
Tissues were dissected and incubated as described under time interval. Values are expressed as mean f SEM. System
2 4 6
to LH levels at 2 h interval;
IN VITRO
Tissues were dissected and incubated as described under time interval. Values are expressed as mean f SEM.
Incubation
of 10 at each
2
RELEASE
TABLE
were done in replicates
Pituitary-hypothalamus complex
a pg LH (NIAMDD-RP-1); * P < 0.001, **P < 0.01, ***P i 0.02, with respect dP < 0.02, with respect to corresponding pituitary levels.
TABLE
Incubations
Whole pituitary
(h)
Materials
and methods.
Incubations
Whole pituitary plus hypothalamus
were done in replicates
of 10 at each
Pituitary-hypothalamus complex
Total a
Perh”
Total
Per h
Total
Per h
371.3 f 42.9 360.2 f 34.6 437.8 f 56.6
185.6 f 21.5 90.0f 8.7 ** 72.5rt 9.4*
281.4 f 22.9 294.9+31.1 401.9 f 24.3 **
140.7 f 11.5 73.7+ 7.8 * 67 f 4.1 *
227.9 f 28.9 b 345.4* 21.9 ** 270.4+ 30.7 ’
113.9* 14.4 86.4+ 5.5 45.0* 5.1 *
a ng Prl (NIAMDD-RP-2); sponding pituitary levels.
* P < 0.001,
l
* P-C 0.01, with respect
to levels at 2 h; b P -C0.02, ‘P < 0.05, with respect
to corre-
24
insignificant, probably due to large SEM. Addition of dopamine to pituitary plus hypothalamus system did not affect the release of LH and FSH. Here also dopamine effected a 15% non-significant drop in the amount of Prl released into the incubation medium. Inclusion of dopamine in the PHC system stimulated the release of LH by 80%, with no concomitant change in FSH levels. Dopamine inhibited Prl secretion by 71% and this was highly significant.
release of FSH was comparable at all the 3 time intervals. Co-incubation of pituitary with hypothalamus did not change the rate of release of FSH at 2 (0.65 + 0.03 pg/h) and 4 h (0.76 f 0.08 pg/h) but at 6 h the rate was significantly elevated (1.48 _t 0.15 pg/h). Pituitary-hypothalamus complex gave results similar to pituitary plus hypothalamus. Pituitary released comparable amounts of Prl at 2, 4 and 6 h, i.e. Prl levels did not increase with increase in incubation time (Table 3). Division of these comparable amounts by 2, 4 and 6 gave the rates of release which apparently decreased from 185.6 rig/h at 2 h, to 90 rig/h at 4 h, and to 72.5 rig/h at 6 h of incubation. Co-incubation with the hypothalamus did not suppress the amount or change the pattern of release of Prl. The amounts released by PHC at 2 and 6 h were comparable and were significantly lower than those released by the pituitary. The rate of release of Prl showed an apparent drop at 4 and 6 h, as compared to 2 h.
Discussion A study of the comparative rates of release of LH, FSH and Prl by the pituitary, pituitary plus hypothalamus and PHC systems has revealed subtle differences in mechanisms controlling the secretion of pituitary hormones. While the pituitary released increasing amounts of LH and FSH at fairly characteristic but different rates, the rate of release of Prl decreased with incubation time, thus revealing an inherent difference in the kinetics of release of LH and’ FSH from that of Prl. Such a divergence in kinetics became pronounced when the pituitary was co-incubated with hypothalamus. As compared to the pituitary, for LH, the rate of release increased at 2,4 and 6 h; for FSH, the rate increased only at 6 h while for Prl, the rate was
Effect of dopamine on the release of LH, FSH and Prl by the whole pituitary, pituitary plus hypothalamus and PHC systems Dopamine did not affect the release of LH and FSH by the pituitary (Table 4). Dopamine inhibited the release of Prl by 25% as compared to controls without dopamine, but this inhibition was
TABLE EFFECT
4 (DA) (1 X lo-' M) ON THE RATE OF RELEASE
OF DOPAMINE
OF LH, FSH AND
Prl IN VITRO
Tissues were dissected and incubated as described under Materials and methods. Incubations were done in replicates of 6. Values are expressed as mean* SEM. Dopamine was added in ascorbic acid solution. The final concentration of ascorbic acid was 0.05% per incubation. The controls received the same amount of ascorbic acid. System
Whole pituitary
Hormone released
Without DA
With DA
‘% change
Without DA
With DA
% change
Without DA
With Da
4b change
LH
0.975 k 0.298
0.878 k 0.27
- 10, NS
1.281 kO.14
1.411 f 0.524
+ 10, NS
0.832 f 0.293
1.5 *0.384
+ 80, P < 0.01
1.6 f0.7
1.5 *0.57
1.26 +0.26
1.28 kO.23
+2, NS
1.15 k 0.23
1.30 + 0.4
+13, NS
61.6 +25.1
46.4 + 26.6
50.4 f 17.2
42.7 + 17.4
65.8 + 28.2
18.8 f 8.9
- 71, P i 0.01
(ng/h) FSH (pg/h) Prl (ng/h) +. stimulation;
-,
inhibition;
Whole pituitary plus hypothalamus
-6,
NS
- 25, NS
NS, non-significant.
Pituitary-hypothalamus complex
-15,
NS
I
25
inhibited at all time intervals studied. The data suggest that factors modulating the secretion of LH and FSH by the pituitary are different from those modulating the secretion of Prl. It confirms the notion that Prl is under a tonic inhibitory control of the hypothalamus (Ben-Jonathan, 1980) and supports the contention that the release of LH and FSH may be differently modulated (Guillemin, 1978; Schally, 1978). Bergland and Page (1979) have demonstrated profused vascularization of the posterior pituitary and a crossed circulation of blood flow from posterior pituitary to adenohypophysis and vice versa. Earlier interconnections between the posterior pituitary and the adenohypophysis via short hypophysial vessels had been demonstrated by Landsmeer (1951) and Daniel and Prichard (1975). A pivotal role for posterior pituitary in the functioning of pituitary has been suggested by Peters et al. (1981). If posterior pituitary is crucial for the normal functioning of adenohypophysis then it follows that anatomic juxtapositioning or closeness between posterior pituitary and hypothalamus or between posterior pituitary and adenohypophysis must be maintained during in vitro studies. Pituitary-hypothalamus complex meets these requirements. During the present investigations the kinetics of release of LH, FSH and Prl by the pituitary and pituitary plus hypothalamus were compared to PHC. Pituitary-hypothalamus complex behaved almost identical to pituitary plus hypothalamus system and this raised a pertinent query: does PHC merit any preference over pituitary plus hypothalamus system for studying hypothalamuspituitary interactions in vitro and, if so, why? The data presented in Table 4 answer this question. Dopamine stimulated the release of LH by PHC without affecting the release of FSH. Dopamine inhibited the release of Prl by PHC. It was without any effect on the release of LH, FSH and Prl by the pituitary or pituitary plus hypothalamus systems. The data demonstrate that close anatomic juxtaposition between pituitary and hypothalamus is essential for compounds like dopamine to exert their effects. It is to be noted that dopamine failed to reduce significantly the levels of Prl secreted by pituitary. The release of Prl by pituitary is under inhibi-
tory control of posterior pituitary and it is this suppressed or basal level of Prl that is further suppressed when dopamine is added to the PHC system. Thus, the effects produced by dopamine on the pituitary and adenohypophysis are basically different. To our knowledge this is the first demonstration of inhibition of basal levels of Prl by dopamine. Dopamine has been shown to inhibit release of Prl in a wide variety of in vitro systems (Quijada et al., 1973; Rotsztejn et al., 1977; Yeo et al., 1979; Denef et al., 1980; Kramer and Hopkins, 1982). In most of these studies adenohypophysis has been used instead of whole pituitary. In some studies single cells derived from the whole pituitary instead of adenohypophysis were used. These cells were exposed to dopamine after 24 h (Denef et al., 1980) or 72 h (Kramer and Hopkins, 1982) of maintenance as primary culture in vitro. We feel that these cells responded to dopamine because preparation of single cells disrupted the architectural integrity of the pituitary resulting in the loss of control of posterior pituitary over adenohypophysis. Since posterior pituitary is the site of storage of dopamine and not of its synthesis (Ben-Jonathan, 1980) posterior pituitary cells are soon depleted of dopamine. Thus, adenohypophysial cells are not exposed to endogenous dopamine and therefore they respond to exogenous dopamine. Whether this response is physiological remains a moot question. Another question that arises is: how does dopamine exert its effect on PHC? Since dopamine does not affect the release of Prl by the pituitary it would appear that exogenous dopamine acts by stimulating the release of an endogenous prolactin inhibiting factor (endogenous dopamine?) by the hypothalamus at an appropriate site in the pituitary. Schneider and McCann (1969) have reported stimulation of LH by dopamine in co-incubations of anterior pituitary halves with stalk median eminence tissues. Dopamine was without any effect on anterior pituitary tissue alone. We did observe an inhibition of 8% in the release of LH by the pituitary on incubation with 1 x lo-’ M dopamine. The inhibition was, however, insignificant. Knigge and Joseph (1979) have observed that the whole brain with attached pituitary can be used for studying the dynamics of hypothalamic hormone synthesis and secretion in vitro. Miyachi
26
et al. (1973) have studied the release of LH and
FSH by pituitary gland (presumably whole pituitary) and pituitary-median eminence unit with intact portal plexus in flasks and in a superfusion system. Pituitary-median eminence unit released more LH and FSH than pituitary gland. Addition of dopamine to the incubation medium did not alter the release of gonadotropins by the whole pituitary but reduced the release of LH from pituitary-median eminence units. Results with whole pituitary are similar to ours in that dopamine did not affect the release of LH and FSH by the pituitary. We have observed a stimulatory effect with PHC on the release of LH while Miyachi et al. (1973) have observed inhibition of LH by dopamine in pituitary-median eminence system. This difference may be due to the nature of hypothalamic fragment that was associated with pituitary. In an isolated study we have tested the effect of dopamine on the release of LH, FSH and Prl by pituitary-median eminence dissected according to the procedure of Miyachi et al. (1973) but incubated under our standardized experimental conditions. Dopamine at 1 X lo-’ M concentration significantly inhibited the release of LH (from 5.2 + 0.77, meanf SD, to 3.8 f 0.5 pg, 27% inhibition, P < 0.01) and Prl (from 403 5 94 to 159 k 83 ng, 61% inhibition, P c 0.02) without affecting the release of FSH. Our results with PHC and pituitary-median eminence unit corroborate the findings of Miyachi et al. (1973) for pituitarymedian eminence unit). We agree with the contentions of Miyachi et al. (1973) that ‘the pituitarymedian eminence unit, even though not completely physiological, may be a more advantageous system than pituitary and median eminence incubated together’. Recently Barraclough and Wise (1982) observed that ‘only future studies using physiological models can resolve the validity of in vitro data obtained using brain tissue’. We feel that PHC is a step forward in evolving an ideal physiological model for studying hypothalamus-pituitary interactions in vitro. Acknowledgements Special reagents for radioimmunoassays were provided by the WHO and the National Institutes of Health, U.S.A. under Indo-U.S. Agreement (No. 01-051) on Science and Technology.
References Arimura, A. and Schally, A.V. (1975) In: Methods in Enzymology, Vol. 37, Eds.: B.W. G’Malley and J.G. Hardman (Academic Press, New York) pp. 233-238. Barraclough, C.A. and Wise, P.M. (1982): Endocr. Rev. 3, 91-119. Ben-Jonathan, N. (1980) J. Reprod. Fertil. 58, 501-512. Ben-Jonathan, N., Neill, M.A., Arbogast, L.A., Peters, L.L. and Hoefer, M.T. (1980) Endocrinology 106, 690-696. Bergland, R.M. and Page, R.B. (1979) Science 204, 18-24. Daniel, P.M. and Prichard, M.M.L. (1975): Acta Endocrinol. 80, Suppl. 201, l-205. Denef, C., Manet, D. and Dewals, R. (1980) Nature (London) 285, 243-246. Gallardo, E. and Ramirez, V.D. (1977) Proc. Sot. Exp. Biol. Med. 155, 79-84. Gill, M.K., Karanth, S., Dutt, A. and Juneja, H.S. (1985) Horm. Metab. Res. 17, 141-146. Guillemin, R. (1978) Science 202, 390-402. Guillemin, R. and Vale, W. (1970) In: Hypophysiotropic Hormones of the Hypothalamus: Assay and Chemistry, Ed.: J. Meites (Williams and Wilkins, Baltimore, MA) pp. 21-35. Knigge, K.M. and Joseph, S.A. (1979) Anat. Rec. 193, 590. Kramer, J.M. and Hopkins, C.R. (1982) Mol. Cell. Endocrinol. 28, 191-198. Landsmeer, J.M.F. (1951) Acta Anat. 12, 82-109. Miyachi, Y., Mecklenburg, R.S. and Lipsett, M.B. (1973) Endocrinology 93, 492-496. Nagendranath, N., Karanth, S., Sheth, A.R. and Juneja, H.S. (1982) Int. J. Androl. 5, 92-102. Negro-Vilar, A., Ojeda, S.R. and McCann, S.M. (1979) Endocrinology 104.1749-1757. Peters, L.L., Hoefer, M.T. and Ben-Jonathan, N. (1981) Science 213,659-661. Powers, C.A. and Johnson, D.C. (1984) Horm. Res. 19.117-126. Quijada, M., Illner, P., Krulich, L. and McCann, S.M. (1973) Neuroendocrinology 13, 151-163. Richardson, S.B., Nguyen, T.U. and Holiander, C.S. (1983) Am. J. Physiol. 7, E560-E566. Rotsztejn, W.H., Charli, J.L., Patton, E., Epelbaum, J. and Kordon, C. (1976) Endocrinology 99, 1663-1666. Rotsztejn, W.H., Charli, J.L., Patton, E. and Kordon, C. (1977) Endocrinology 101,1475-1483. Saavedra, J.M., Palkovits, M., Kizer, J.S., Brownstein, M. and Zivin, J.A. (1975) J. Neurochem. 25, 257-261. Schally, A.V. (1978) Science 202, 18-28. Schally, A.V. and Bowers, C.Y. (1964) Endocrinology 75, 312-320. Schneider, H.P.G. and McCann, S.M. (1969) Endocrinology 85, 121-132. Vale, W. and Grant, G. (1975) In: Methods in Enzymology, Vol. 37, Eds.: B.W. G’Malley and J.G. Hardman (Academic Press, New York) pp. 82-93. Yeo, T., Thomer, M.O., Ann Jones, Lowry, P.J. and Besser, G.M. (1979) Clin. Endocrinol. 10, 123-130.
21
Elsevier Scientific Publishers Ireland, Ltd. MCE 01443
The choice of a model for studying the hypothalamus-pituitary interactions in vitro A. Dutt, M. Gill, S. Karanth, Division of Neuroendocrinology,
N. Lehri and H.S. Juneja
*
Institutefor Research in Reproduction (Indian Council of Medical Reseurch), Parel, Bombay 400 012 (India)
(Received 29 August 198.5;accepted 9 December 1985)
Key words; hypothalamus; pituitary; dopamine; in vitro LH; FSH; Prl.
Summary Comparative studies on the release of luteinizing hormone (LH), follicle-stimulating hormone (FSH) and prolactin (Prl) by the whole pituitary, pituitary plus hypothalamus and pituitary-hypothalamus complex (PHC) were undertaken to choose an appropriate model for studying the hypothalamus-pituitary interactions in vitro and to relate the importance of the intact neural connections between pituitary and hypothalamus on hypothalamus-pituitary interactions. Also the effect of including dopamine (DA) at 1 x lo-’ mol/l in these different in vitro systems on the release of LH, FSH and Prl was investigated. The pituitary released increasing amounts of LH and FSH at 2, 4 and 6 h but the amount of Prl released remained unchanged. The rates of release of LH, FSH and Prl by the pituitary were different and were characteristic of each hormone. Co-incubation of pituitary with hypothalamus stimulated the release of LH and FSH but inhibited the release of Prl. Pituitary-hypothalamus complex behaved almost identical to pituitary plus hypothalamus system. Inclusion of 1 X lo-’ M DA in the incubation medium stimulated the release of LH (80%) but inhibited the release of Prl (71%) by PHC. FSH was unaffected. DA had no significant effect on the release of LH, FSH and Prl by pituitary and pituitary plus hypothalamus systems. It is suggested that PHC is the system of choice for studying hypothalamus-pituitary interactions in vitro.
Introduction Several in vitro systems are available for studying metabolic functions of the hypothalamus (Rotsztejn et al., 1976; Gallardo and Ramirez, 1977; Negro-Vilar et al., 1979; Richardson et al., 1983), the pituitary (Guillemin and Vale, 1970; Arimura and Schally, 1975; Vale and Grant, 1975; Yeo et al., 1979) or the hypothalamus-pituitary interactions (Schally and Bowers, 1964; Schneider and McCann, 1969; Quijada et al., 1973; Powers * To whom correspondence should be addressed. 0303-7207/86/$03.50
and Johnson, 1984). In vitro studies on hypothalamus-pituitary interactions have utilised fragments of hypothalamus viz. basomedial hypothalamus, median eminence, ventral hypothalamus or any other hypothalamic area of defined topoghypothalamus-preoptic raphy (e.g. mediobasal area) in co-incubation with the adenohypophysis. In these studies adenohypophysis and hypothalamus are dissociated of their neural and vascular associations. In one study median eminencepituitary unit with intact portal plexus has been used (Miyachi et al., 1973). Gill et al. (1985) have successfully adopted the
0 1986 Elsevier Scientific Publishers Ireland, Ltd.
22
pituita~-h~othalamus complex (PI-K) system for in vitro studies. In this system neuroanatom~c juxtapositioning or closeness of pituitary to hypothalamus remains intact. In the present communication comparative studies on the release of LH, FSH and Prl by PHC, whole pituitary, and pituitary plus hypothalamus systems are reported. In addition the effect of dopamine added in vitro on the release of LH, FSH and Prl by these different systems is described. Materiah
and methods
A ~imals Holtzman strain adult male rats weighing 200-250 g were used. These rats were maintained in an air-conditioned facility with temperature varying from 22 to 24°C and the humidity ranging from 50 to 56%. The light schedule was 14 h light and 10 h darkness. The animals were fed a pelleted diet with water ad libitum. Dissection
of tissue
Pituitary-h~othalamus complex was dissected as described earlier (Gill et al., 1985). The rat was killed by decapitation. Its brain was exposed by a dorsal incision. The hypothal~c island was demarcated by cutting along the lateral hypothalamic sulci, posterior edge of the optic chiasma and the anterior edge of the mammillary bodies. The hypothalamic island attached to the pituitary via hypophysial stalk was lifted by an undercut 2 mm deep. Where only h~othalamus was desired, it was obtained by cutting the stalk joining the pituitary to the hypothalamus. The pituitary was carefully scooped from sella turcica after freeing it of adhering ligaments.
Dulbecco’s modified Eagle’s medium (DMEM) fortified with non-essential amino acids, 3 mM NaHCO, and 0.01 M Hepes (4-(2”hydroxyethyl)l-piperazine-ethanesulfonic acid) was used. Incubations were done at 37’C in Laxbroplastic (Laxbro, Pune, India) trays with 24 welfs of 5 ml capacity each. Each well contained 1 ml DMEM. Freshly dissected tissues were transferred into individual wells. The contents were pre-incubated for 1 h after which the spent incubation medium
was repiaced with 1 ml of fresh medium and the contexts incubated further. The incubation was stopped at desired times by removing the tissue from the incubation well. The tissue was blotted and weighed on a torsion balance. The spent incubation medium was transferred into individual tubes and stored at -30” until assayed for LH, FSH and Prl by RIA. hormone assays LH and FSH in culture medium were measured by RIA as described earlier (Nagen~~ath et al., 1982). The standard curve for LH (NIAMDDRat-LH-RP-1) ranged from 2 to 500 ng per assay tube, and for FSH (NIAM~D-Rat-FSH”RP-1) from 5 ng to 1 pg per assay tube. The inter- and intra-assay variations were 10 and 6% for FSH and 9 and 6% for LH assays, respectively. Prl was assayed as per the procedure described in the brochure supplied by the National Pituitary Agency, NIH, Bethesda, MD. The standard curve for Prl (NIA~DD-Rat-Prl-R~-2) ranged from IO pg to 12.5 ng per assay tube. Inter-assay and intra-assay variations for Prl were 14 and 596, respectively.
Levels of significance between 2 groups were computed using Student’s t-test. Results
Release of LH, FSH and Prf by different systems The amount of LH released by the whole pituitary at 2, 4 and 6 h of incubation increased progressively with incubation duration (Table 1). The rate of release was comparable at 2 and 4 h (0.27 f 0.03 and 0.25 + 0.03 pg,/h (mean i SEM), respectively) but increased significantly to 0.39 f 0.05 pg/h at 6 h. Co-incubation of pituitary with hypothalamus increased the amount and the rate of release of LH at 2, 4 and 6 h significantly as compared to the pituitary. Pituitary-hypothalamus complex secreted LH at rates comparable to pituitary plus hyp~th~amus but si~ificantly different from the pituitary. The amount of FSH released into the incubation medium increased proportionately with increased incubation duration (Table 2). The rate of
23 TABLE
1
RELEASE
OF LH BY DIFFERENT
SYSTEMS
IN VITRO
Tissues were dissected and incubated as described under time interval. Values are expressed as mean + SEM. System
Whole pituitary
Incubation
(h)
2 4 6
Materials
and methods.
Whole pituitary plus hypothalamus
Total a
Perh”
Total
Per h
Total
Per h
0.54iO.06 1.00*0.12 *** 2.34kO.3 *
0.27 k 0.03 0.25 f 0.03 0.39 f0.05 *
1.00+0.06 b 1.84+0.24 G** 4.56 f 0.48 G*
0.50 + 0.03 0.46 + 0.06 0.76+0.08 ***
0.72 f 0.08 1.84i0.28 d,* 3.9OkO.48 d.*
0.36 + 0.04 0.46 f 0.07 0.65 f 0.08 **
OF FSH BY DIFFERENT
SYSTEMS
Materials
Whole pituitary
System
Incubation
(h)
2 4 6
b P i 0.001, ’ P i 0.01,
and methods.
Incubations
Whole pituitary plus hypothalamus
were done in replicates
of 10 at each
Pituitary-hypothalamus complex
Total a
Perha
Total
Per h
Total
Per h
1.46 f 0.2 3.04kO.32 ** 5.88 f 0.6 *
0.73 f 0.1 0.76 f 0.08 0.98 f0.10
1.3 kO.06 3.04kO.32 * 8.88kO.9 b.*
0.65 f 0.03 0.76 + 0.08 1.48+0.15 *
1.2 +0.12 3.04*0.4 * 8.52 + 1.2 =,*
0.6 50.06 0.76f0.10 1.42kO.20
* P < 0.001,
a pg FSH (NIAMDD-RP-1); pituitary levels.
**P < 0.01, with respect
to FSH
=.*
levels at 2 h; b P < 0.02, ‘P i 0.05, with respect
to
3
RELEASE
OF Prl BY DIFFERENT
SYSTEMS
IN VITRO
Tissues were dissected and incubated as described under time interval. Values are expressed as mean f SEM. System
2 4 6
to LH levels at 2 h interval;
IN VITRO
Tissues were dissected and incubated as described under time interval. Values are expressed as mean f SEM.
Incubation
of 10 at each
2
RELEASE
TABLE
were done in replicates
Pituitary-hypothalamus complex
a pg LH (NIAMDD-RP-1); * P < 0.001, **P < 0.01, ***P i 0.02, with respect dP < 0.02, with respect to corresponding pituitary levels.
TABLE
Incubations
Whole pituitary
(h)
Materials
and methods.
Incubations
Whole pituitary plus hypothalamus
were done in replicates
of 10 at each
Pituitary-hypothalamus complex
Total a
Perh”
Total
Per h
Total
Per h
371.3 f 42.9 360.2 f 34.6 437.8 f 56.6
185.6 f 21.5 90.0f 8.7 ** 72.5rt 9.4*
281.4 f 22.9 294.9+31.1 401.9 f 24.3 **
140.7 f 11.5 73.7+ 7.8 * 67 f 4.1 *
227.9 f 28.9 b 345.4* 21.9 ** 270.4+ 30.7 ’
113.9* 14.4 86.4+ 5.5 45.0* 5.1 *
a ng Prl (NIAMDD-RP-2); sponding pituitary levels.
* P < 0.001,
l
* P-C 0.01, with respect
to levels at 2 h; b P -C0.02, ‘P < 0.05, with respect
to corre-
24
insignificant, probably due to large SEM. Addition of dopamine to pituitary plus hypothalamus system did not affect the release of LH and FSH. Here also dopamine effected a 15% non-significant drop in the amount of Prl released into the incubation medium. Inclusion of dopamine in the PHC system stimulated the release of LH by 80%, with no concomitant change in FSH levels. Dopamine inhibited Prl secretion by 71% and this was highly significant.
release of FSH was comparable at all the 3 time intervals. Co-incubation of pituitary with hypothalamus did not change the rate of release of FSH at 2 (0.65 + 0.03 pg/h) and 4 h (0.76 f 0.08 pg/h) but at 6 h the rate was significantly elevated (1.48 _t 0.15 pg/h). Pituitary-hypothalamus complex gave results similar to pituitary plus hypothalamus. Pituitary released comparable amounts of Prl at 2, 4 and 6 h, i.e. Prl levels did not increase with increase in incubation time (Table 3). Division of these comparable amounts by 2, 4 and 6 gave the rates of release which apparently decreased from 185.6 rig/h at 2 h, to 90 rig/h at 4 h, and to 72.5 rig/h at 6 h of incubation. Co-incubation with the hypothalamus did not suppress the amount or change the pattern of release of Prl. The amounts released by PHC at 2 and 6 h were comparable and were significantly lower than those released by the pituitary. The rate of release of Prl showed an apparent drop at 4 and 6 h, as compared to 2 h.
Discussion A study of the comparative rates of release of LH, FSH and Prl by the pituitary, pituitary plus hypothalamus and PHC systems has revealed subtle differences in mechanisms controlling the secretion of pituitary hormones. While the pituitary released increasing amounts of LH and FSH at fairly characteristic but different rates, the rate of release of Prl decreased with incubation time, thus revealing an inherent difference in the kinetics of release of LH and’ FSH from that of Prl. Such a divergence in kinetics became pronounced when the pituitary was co-incubated with hypothalamus. As compared to the pituitary, for LH, the rate of release increased at 2,4 and 6 h; for FSH, the rate increased only at 6 h while for Prl, the rate was
Effect of dopamine on the release of LH, FSH and Prl by the whole pituitary, pituitary plus hypothalamus and PHC systems Dopamine did not affect the release of LH and FSH by the pituitary (Table 4). Dopamine inhibited the release of Prl by 25% as compared to controls without dopamine, but this inhibition was
TABLE EFFECT
4 (DA) (1 X lo-' M) ON THE RATE OF RELEASE
OF DOPAMINE
OF LH, FSH AND
Prl IN VITRO
Tissues were dissected and incubated as described under Materials and methods. Incubations were done in replicates of 6. Values are expressed as mean* SEM. Dopamine was added in ascorbic acid solution. The final concentration of ascorbic acid was 0.05% per incubation. The controls received the same amount of ascorbic acid. System
Whole pituitary
Hormone released
Without DA
With DA
‘% change
Without DA
With DA
% change
Without DA
With Da
4b change
LH
0.975 k 0.298
0.878 k 0.27
- 10, NS
1.281 kO.14
1.411 f 0.524
+ 10, NS
0.832 f 0.293
1.5 *0.384
+ 80, P < 0.01
1.6 f0.7
1.5 *0.57
1.26 +0.26
1.28 kO.23
+2, NS
1.15 k 0.23
1.30 + 0.4
+13, NS
61.6 +25.1
46.4 + 26.6
50.4 f 17.2
42.7 + 17.4
65.8 + 28.2
18.8 f 8.9
- 71, P i 0.01
(ng/h) FSH (pg/h) Prl (ng/h) +. stimulation;
-,
inhibition;
Whole pituitary plus hypothalamus
-6,
NS
- 25, NS
NS, non-significant.
Pituitary-hypothalamus complex
-15,
NS
I
25
inhibited at all time intervals studied. The data suggest that factors modulating the secretion of LH and FSH by the pituitary are different from those modulating the secretion of Prl. It confirms the notion that Prl is under a tonic inhibitory control of the hypothalamus (Ben-Jonathan, 1980) and supports the contention that the release of LH and FSH may be differently modulated (Guillemin, 1978; Schally, 1978). Bergland and Page (1979) have demonstrated profused vascularization of the posterior pituitary and a crossed circulation of blood flow from posterior pituitary to adenohypophysis and vice versa. Earlier interconnections between the posterior pituitary and the adenohypophysis via short hypophysial vessels had been demonstrated by Landsmeer (1951) and Daniel and Prichard (1975). A pivotal role for posterior pituitary in the functioning of pituitary has been suggested by Peters et al. (1981). If posterior pituitary is crucial for the normal functioning of adenohypophysis then it follows that anatomic juxtapositioning or closeness between posterior pituitary and hypothalamus or between posterior pituitary and adenohypophysis must be maintained during in vitro studies. Pituitary-hypothalamus complex meets these requirements. During the present investigations the kinetics of release of LH, FSH and Prl by the pituitary and pituitary plus hypothalamus were compared to PHC. Pituitary-hypothalamus complex behaved almost identical to pituitary plus hypothalamus system and this raised a pertinent query: does PHC merit any preference over pituitary plus hypothalamus system for studying hypothalamuspituitary interactions in vitro and, if so, why? The data presented in Table 4 answer this question. Dopamine stimulated the release of LH by PHC without affecting the release of FSH. Dopamine inhibited the release of Prl by PHC. It was without any effect on the release of LH, FSH and Prl by the pituitary or pituitary plus hypothalamus systems. The data demonstrate that close anatomic juxtaposition between pituitary and hypothalamus is essential for compounds like dopamine to exert their effects. It is to be noted that dopamine failed to reduce significantly the levels of Prl secreted by pituitary. The release of Prl by pituitary is under inhibi-
tory control of posterior pituitary and it is this suppressed or basal level of Prl that is further suppressed when dopamine is added to the PHC system. Thus, the effects produced by dopamine on the pituitary and adenohypophysis are basically different. To our knowledge this is the first demonstration of inhibition of basal levels of Prl by dopamine. Dopamine has been shown to inhibit release of Prl in a wide variety of in vitro systems (Quijada et al., 1973; Rotsztejn et al., 1977; Yeo et al., 1979; Denef et al., 1980; Kramer and Hopkins, 1982). In most of these studies adenohypophysis has been used instead of whole pituitary. In some studies single cells derived from the whole pituitary instead of adenohypophysis were used. These cells were exposed to dopamine after 24 h (Denef et al., 1980) or 72 h (Kramer and Hopkins, 1982) of maintenance as primary culture in vitro. We feel that these cells responded to dopamine because preparation of single cells disrupted the architectural integrity of the pituitary resulting in the loss of control of posterior pituitary over adenohypophysis. Since posterior pituitary is the site of storage of dopamine and not of its synthesis (Ben-Jonathan, 1980) posterior pituitary cells are soon depleted of dopamine. Thus, adenohypophysial cells are not exposed to endogenous dopamine and therefore they respond to exogenous dopamine. Whether this response is physiological remains a moot question. Another question that arises is: how does dopamine exert its effect on PHC? Since dopamine does not affect the release of Prl by the pituitary it would appear that exogenous dopamine acts by stimulating the release of an endogenous prolactin inhibiting factor (endogenous dopamine?) by the hypothalamus at an appropriate site in the pituitary. Schneider and McCann (1969) have reported stimulation of LH by dopamine in co-incubations of anterior pituitary halves with stalk median eminence tissues. Dopamine was without any effect on anterior pituitary tissue alone. We did observe an inhibition of 8% in the release of LH by the pituitary on incubation with 1 x lo-’ M dopamine. The inhibition was, however, insignificant. Knigge and Joseph (1979) have observed that the whole brain with attached pituitary can be used for studying the dynamics of hypothalamic hormone synthesis and secretion in vitro. Miyachi
26
et al. (1973) have studied the release of LH and
FSH by pituitary gland (presumably whole pituitary) and pituitary-median eminence unit with intact portal plexus in flasks and in a superfusion system. Pituitary-median eminence unit released more LH and FSH than pituitary gland. Addition of dopamine to the incubation medium did not alter the release of gonadotropins by the whole pituitary but reduced the release of LH from pituitary-median eminence units. Results with whole pituitary are similar to ours in that dopamine did not affect the release of LH and FSH by the pituitary. We have observed a stimulatory effect with PHC on the release of LH while Miyachi et al. (1973) have observed inhibition of LH by dopamine in pituitary-median eminence system. This difference may be due to the nature of hypothalamic fragment that was associated with pituitary. In an isolated study we have tested the effect of dopamine on the release of LH, FSH and Prl by pituitary-median eminence dissected according to the procedure of Miyachi et al. (1973) but incubated under our standardized experimental conditions. Dopamine at 1 X lo-’ M concentration significantly inhibited the release of LH (from 5.2 + 0.77, meanf SD, to 3.8 f 0.5 pg, 27% inhibition, P < 0.01) and Prl (from 403 5 94 to 159 k 83 ng, 61% inhibition, P c 0.02) without affecting the release of FSH. Our results with PHC and pituitary-median eminence unit corroborate the findings of Miyachi et al. (1973) for pituitarymedian eminence unit). We agree with the contentions of Miyachi et al. (1973) that ‘the pituitarymedian eminence unit, even though not completely physiological, may be a more advantageous system than pituitary and median eminence incubated together’. Recently Barraclough and Wise (1982) observed that ‘only future studies using physiological models can resolve the validity of in vitro data obtained using brain tissue’. We feel that PHC is a step forward in evolving an ideal physiological model for studying hypothalamus-pituitary interactions in vitro. Acknowledgements Special reagents for radioimmunoassays were provided by the WHO and the National Institutes of Health, U.S.A. under Indo-U.S. Agreement (No. 01-051) on Science and Technology.
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