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Thyrotropin-releasing hormone concentrations in different regions of the chicken brain and pituitary: an ontogenetic study
Brain Research 818 Ž1999. 260–266
Research report
Thyrotropin-releasing hormone concentrations in different regions of the chicken brain and pituitary: an ontogenetic study Kris L. Geris
a, )
, Els D’Hondt b , Eduard R. Kuhn ¨ a , Veerle M. Darras
a
a
b
Laboratory of ComparatiÕe Endocrinology, Zoological Institute, K.U. LeuÕen, Naamsestraat 61, B-3000 LouÕain, Belgium Laboratory for Neuroendocrinology and Immunological Biotechnology, K.U. LeuÕen, Naamsestraat 59, B-3000 LouÕain, Belgium Accepted 17 November 1998
Abstract The regional distribution of thyrotropin-releasing hormone ŽTRH. was studied in the chicken brain. The hypothalamus and the brain stem contained the highest concentration of TRH. Lower amounts were present in the telencephalon, the optic lobes and the cerebellum. Within the hypothalamus, TRH was most abundant in the median eminence. Other important TRH sites were the nucleus paraventricularis magnocellularis, nucleus periventricularis hypothalami, nucleus ventromedialis hypothalami, nucleus dorsomedialis hypothalami and nucleus preopticus periventricularis. On the 14th day of embryonic development ŽE14., TRH was mostly found in the brain stem. Towards hatching, TRH concentrations increased gradually in both the hypothalamic area and the brain stem. TRH concentrations in the telencephalon, optic lobes and cerebellum remained low. Pituitaries from E14 to E16 chickens were characterized by a high TRH concentration, whereas hypophyseal TRH concentrations dropped towards hatching. Our results support the hypothesis that TRH exerts both endocrine and neurocrine actions in the chicken. On the other hand, high pituitary TRH concentrations were present when hypothalamic concentrations were low and vice versa. Therefore, the chicken pituitary may function as an important source of TRH during early in ovo development at least until the moment hypothalamic control develops. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Chicken; Thyrotropin-releasing hormone; Brain; Pituitary; Nucleus; Ontogeny
1. Introduction Thyrotropin-releasing hormone ŽTRH. was the first hypophysiotropic peptide to be isolated from the hypothalamus w7x. It was called TRH because it stimulates thyrotropin ŽTSH. release from the mammalian pituitary gland. Immunocytochemical studies in rats showed that specific neurons extending exclusively from the paraventricular nucleus transport TRH to the median eminence where it is secreted into the portal blood system allowing TRH to act at the level of the pituitary w31x. As a local neurotrans-
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mitter, TRH is however, present throughout the mammalian central nervous system w19,36x. Despite its thyroid-related name, TRH has a dual site of action in the chicken. It stimulates the secretory activity of both thyrotrophs and somatotrophs w2,16x. While several research groups have investigated the potency and the mode of action of TRH at the level of the chicken pituitary w15,24x, only few data suggest a neurocrine function of TRH within the chicken brain w29x. Also, little is known about the actual presence and distribution of TRH in the chicken brain w26x. Accordingly, we screened the chicken brain Žsections and nuclei. for TRH using a sensitive and specific radioimmunoassay ŽRIA. w14x. Since several researchers showed that the mammalian pituitary is capable of synthesizing TRH w4–6,8,9,34x, the pituitary was included in this study. Finally, tissues from embryonic up to newly hatched chicks were collected and ontogenetic changes in TRH concentrations compared.
0006-8993r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 8 . 0 1 2 8 1 - 5
K.L. Geris et al.r Brain Research 818 (1999) 260–266
2. Material and methods 2.1. Animals All studies were performed on chickens of a layer strain ŽHisex White., purchased as fertilized eggs or 1-day-old male chicks ŽC1. from a local commercial hatchery ŽEuribrid, Aarschot, Belgium.. Eggs were incubated in a forced-draft incubator, at 37.88C, with increasing humidity and ventilation from day 14 onwards, under continuous lighting and a 458 rotation every hour Žstart of incubation s day 1 ŽE1... Posthatch chickens were kept in an acclimatized room with a 14Lr10D photoperiod. Water and food were available ad libitum.
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were determined using the stereotaxic atlas of Kuenzel and Masson w23x and stock brain sections stained with cresyl violet. In order to obtain the whole nucleus, punches were performed on consecutive sections. The following nuclei were collected: nucleus septalis lateralis ŽSL. and medialis ŽSM., nucleus preopticus medialis ŽPOM. and periventricularis ŽPOP., nucleus anterior Žrostralis. medialis hypothalami ŽAM., nucleus commissurae pallii ŽnCPa., nucleus paraventricularis magnocellularis ŽPVN., nucleus periventricularis hypothalami ŽPHN., nucleus rotundus ŽROT., nucleus ventromedialis hypothalami ŽVMN., nucleus dorsomedialis hypothalami ŽDMN. and the median eminence ŽME. w15x. 2.5. Measurement of TRH concentrations
2.2. Distribution in the brain To study the concentration of TRH in different brain regions, animals ŽC1; n s 10. were killed by rapid decapitation. The cranium was opened and the following brain regions were collected: telencephalon left ŽTLL. and right ŽTLR., tectum opticum Žoptic lobe. left ŽTOL. and right ŽTOR., the hypothalamic area Žhypothalamus and thalamus; HT., the cerebellum ŽCB. and the brain stem ŽBS.. Tissues were stored at y808C. Due to the small size and the fragile character of embryonic and newly hatched chicken brain, 1-week-old chickens were used to isolate specific nuclei in the brain. The total brain was carefully dissected and then immediately placed on dry ice Ž n s 8.. The pituitary was taken out of the sella tursica. Tissues were stored at y808C. 2.3. Ontogeny of TRH concentrations in the brain and the pituitary Brain regions and pituitaries were collected from E14, E16, E18, E20Žnon-pippingŽNP.., E20Žinternal pipping ŽIP.. Žper embryonic stage n s 12. and C1 Ž n s 10. chicks. In the first distribution study, no differences were found between the TRH concentration in the left or the right part of the telencephalon or of the optic lobes, so both parts were pooled, resulting in one telencephalon and one optic lobe sample per animal. Tissues were stored at y808C. 2.4. Microdissection of the nuclei The micropunch technique of Palkovits w30x was used to isolate specific brain nuclei. Serial sections of 300 mm were made by means of a cryostat, mounted on slides cleaned with chromic acid and stored at y808C. The needles ŽTP-100, Activational Systems, MI, USA. used in the punch technique were constructed of steel and the inner diameters of the needles varied from 0.2 to 2.0 mm, depending on the size of the brain nuclei to be dissected. At the beginning of the micropunch procedure, the exact coordinates of the brain nucleus that would be punched
Extraction of TRH was performed as described before w14x. In the ontogenetic study, brain regions were pooled up to stage E20 ŽIP., resulting, respectively in 6 ŽTL, TO, HT and BS. and 4 ŽCB. samples for the different brain parts. Tissues of C1 animals were used individually Ž n s 10.. Pituitaries were pooled yielding 6 ŽE14 and E16. or 4 ŽE18, E20 ŽNPrIP. and C1. samples for the different stages. TRH was labeled with the chloramine T method w14x. TRH RIA was carried out according to van Haasteren et al. w40x. Briefly, radioiodinated TRH Ž15.000 cpmrsample., antibody Žlaboratory raised, 1r10,000. Žkindly donated by Dr. T.J. Visser, Erasmus University Rotterdam., assay buffer Ž0.14 M KCl, 0.1 M KH 2 PO4 , 0.02% bovine serum albumin ŽSigma, St. Louis, USA., pH 7.5. and sample or standard Ž1.56 to 800 pg. were incubated in a final volume of 400 ml assay buffer in polystyrene tubes Ž72 h, 48C.. Separation of free and bound radioactivity was achieved by immunoprecipitation using Sac-Cel anti-rabbit globulines ŽInnogenetics, Gent, Belgium.. After 1 h of incubation at 48C and centrifugation, precipitates were counted in a g-counter ŽGammamaster, LKB.. The ED 80 and ED 20 of the RIA were respectively, 10 and 240 pg. The intra and interassay coefficients were 6.6 " 0.4% and 15.6 " 2.1%, respectively, recovery of standards in homogenates was 102.1 " 1.7%. Displacement curves resulting from serial dilutions of hypothalamic or telencephalon homogenates were parallel to the standard curve. The hypothalamic hormones somatostatin ŽSRIH. ŽSigma–Aldrich., human growth hormone ŽGH .-releasing hormone w1 – 29 x ŽhGHRH 1 – 29 ; kindly donated by Dr. T.R. Hall, Ciba-Geigy, Switzerland., synthetic rat preproTRH 160 – 169 ŽPs4; Peninsula, CA, USA., ovine corticotropin-releasing hormone ŽoCRH; UCB, Brussels. and the non-peptidyl GHsecretagogue L-692,429 w14x Žkindly donated by Dr. G. Hickey, Merck, Rahway, USA. did not cross-react in the assay. The RIA employing antiserum, a8880, shows, compared to antisera previously used w34,40,41x, much less cross-reactivity with TRH analogues that have histidine replaced by other amino acids. TRH was expressed as a
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Values represent mean " S.E.M. Statistical analysis was assessed by the general linear model of SAS Ž1985., followed by a Scheffe´ test.
detection limit of the assay. No differences were found between the right or the left part of the telencephalon or the optic lobe. In order to compare TRH proportions in the different brain regions, data from Fig. 1 were recalculated as a content Žpgrtissue; data not shown.. The actual amount of TRH in the hypothalamic region Ž269 " 32 pgrHT. was higher compared to the brain stem Ž120 " 17 pgrBS. and the telencephalon Ž126 " 15 pgrTL. content. TRH content in the other brain regions was very low Ž- 30 pgrtissue.. From the total amount of TRH in the brain, 61.8% was found in extrahypothalamic areas. Since the hypothalamic area used also contained the thalamus, this percentage will even be higher. TRH content in different brain nuclei and pituitary is shown in Fig. 2. TRH was predominantly found in the ME. Other important sources of TRH, containing approximately 30% of the TRH content in the ME, were the PVN, PHN, POP, AM, VMN and DMN. In a few animals, a low amount of TRH was recorded in the SL Ž3 out of 8: 3r8., SM Ž3r8., POM Ž2r8. and nCPa Ž4r8.. No TRH was located in the ROT. Expressing TRH content in the pituitary in pgrnucleus Ži.e., pituitary. showed that the pituitary is an important source of TRH Ž22.9 " 5.4 pgrpituitary.. The TRH content was only higher in the median eminence ŽFig. 2..
3. Results
3.2. Ontogeny of TRH concentrations in the brain and the pituitary
Fig. 1. Distribution of thyrotropin-releasing hormone ŽTRH. in the brain of 1-day-old male chickens. The collected brain sections were: telencephalon left ŽTLL. and right ŽTLR., tectum opticum Žoptic lobe. left ŽTOL. and right ŽTOR., the hypothalamic area Žhypothalamus and thalamus, HT., the cerebellum ŽCB. and the brain stem ŽBS.. Data shown are mean"S.E.M. Ž ns10.. TRH was expressed in pgrg wet weight.
concentration Žpgrg wet weight Žbrain regions. or pgrmg wet weight Žpituitary.. or as a content Žpgrnucleus.. 2.6. Statistics
3.1. Distribution in the brain Fig. 1 shows the TRH concentration in the different brain regions of 1-day-old chickens Žpgrg wet weight.. TRH was predominantly found in the hypothalamic area and the brain stem. It was also present at a considerable concentration in both parts of the telencephalon. The optic lobes and the cerebellum had a concentration close to the
In both the hypothalamic region and the brain stem, TRH concentrations rose towards the end of incubation ŽFig. 3.. During early development, TRH was mostly located in the brain stem, whereas TRH levels in the other regions were low. Hypothalamic TRH levels increased progressively during the last week of embryonic development to reach a maximum in 1-day-old chickens. A similar profile was noted for the brain stem, although no increase
Fig. 2. Distribution of TRH in several brain nuclei and the pituitary of 1-week-old chickens ŽC8.. The data shown are expressed in pg TRHrnucleus or pituitary and represent mean " S.E.M. of eight individual observations.
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Fig. 3. Ontogeny of TRH concentrations in several regions of the chicken brain during the last week of embryonic development up to 1-day-old chickens. Data shown are mean " S.E.M. Ž n s 4–10.. Since no differences were found in the TRH concentration of the several brain regions between NP and IP chicks, data of NP and IP chicks were combined ŽE20 bars.. Within one tissue, data with a common letter are not significantly different ŽScheffe, ´ P - 0.05. ŽTL: telencephalon; TO: tectum opticum; HT: hypothalamus; CB: cerebellum; BS: brain stem.. Within one tissue, a similar TRH profile is observed when the TRH data are expressed as pgrtissue.
in TRH was observed between E18 and E20, as was found in the hypothalamic region. An overall 15-fold increase was observed from E14 to C1 in hypothalamic TRH concentration, whereas brain stem TRH levels increased 6-fold. Although some changes were recorded in TRH levels of the optic lobes or the cerebellum, these were much less pronounced compared to those observed in the hypothalamus or brain stem. TRH concentrations in these brain regions remained at a low level from E14 to C1. Hypothalamic TRH content Žpgrtissue; data not shown. increased profoundly towards hatching; an opposite decrease was noticed in relative extrahypothalamic TRH
content in the brain ŽE14: 88.2%; E16: 83.3%; E18: 76.6%; E20: 59.8% and C1: 66.2%.. Unlike hypothalamic concentrations, hypophyseal TRH levels dropped towards hatching ŽFig. 4.. Embryonic stages E14–E16 were characterized by a high TRH level in the pituitary, whereas late-embryonic and newly hatched chicks had a 5- to 10-fold lower TRH concentration in the pituitary. No differences were found on day 20 between the non-pipping and internal pipping embryos. Hypophyseal TRH content dropped from 244 " 79 pgrpituitary ŽE14. to 19 " 6 pgrpituitary ŽC1..
4. Discussion
Fig. 4. Ontogenetic changes in pituitary TRH concentration Žpg TRHrmg wet weight. during the second part of embryonic development up to 1-day-old chickens Ž ns 4–6.. Data shown are mean"S.E.M. Data with a common letter are not significantly different ŽScheffe, ´ P - 0.05.. A similar TRH profile is recorded when TRH data are expressed in pgrtissue.
Using specific radioimmunoassay techniques, we have measured the amount of TRH in various regions of the chicken brain. As in the rat, large quantities of TRH were evident in the hypothalamic area. This is consistent with the role of TRH as a regulator of the release of anterior pituitary hormones. Hypothalamic TRH levels in the rat were approximately 100-fold higher than in the avian hypothalamus Ž19 and present results.. Extrahypothalamic TRH accounted for approximately 70% of the total TRH content in the chicken brain. The same proportional distribution has been recorded in mammals w19,35x. In the chicken, especially the brain stem seems characterized by a high TRH concentration. Our distribution study is well in agreement with data from Jackson and Reichlin w19x, who reported high TRH levels in the chicken hypothalamic region and brain stem, whereas low concentrations were found in the other brain regions. Immunocytochemical
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studies have also shown a widespread distributional pattern of TRH in the avian brain w21,26x. There is good evidence that only TRH synthesized in the PVN and transported to the ME regulates pituitary secretion: Ž1. lesion of the PVN extremely decreased TRH and TSH secretion in the rat w10,28,31,33x; Ž2. electrical stimulation of this specific nucleus increased the release of these peptides w10x; Ž3. thyroid hormones control TRH content exclusively in the PVN w11,37x. TRH immunoreactive perikarya and neurons have been observed respectively, in the PVN and the ME of avian hypothalamus w21,22,26x, suggesting that the same functional system exists in birds. Our data show that TRH is not only found in these areas but also in nuclei not involved in the control of pituitary secretion like the PHN, DMN and VMN. Also in rats, these nuclei were characterized by high TRH concentrations w20,25,31x. These findings, and the widespread distribution of TRH in the different brain regions, indicate that TRH does not merely act as an endocrine regulator at the level of the pituitary. In mammals, TRH has both inhibitory w32x and excitatory w3x effects on neurons in the brain, so TRH may also act as a neurotransmitter or neuromodulator in the chicken w29x. Data on the ontogenetic appearance of several hypothalamic factors in the avian brain have long been restricted to immunocytochemical studies w1,22,39x. In view of our interest in the effects TRH exerts on the pituitary, we recently studied the presence of hypothalamic TRH during chicken embryo development w13x. The present report confirms these hypothalamic data that a progressive rise in hypothalamic TRH occurs towards hatching, and further expands these findings to the other brain regions and the pituitary. At the extrahypothalamic level, major fluctuations were noticed in brain stem TRH concentrations. Due to a profound increase towards hatching, along with small changes in the other areas, the chicken brain stem contained more than 85% of the extrahypothalamic TRH content at hatching. Since no clear fluctuations in the TRH level in the other brain regions were observed, the rise in TRH in the hypothalamic area and the brain stem was not due to an overall maturation of the chicken brain. Some differences between the rise in TRH levels in both regions also suggest that two independent evolutions take place in these areas: Ž1. the increase was much more pronounced in the hypothalamic region; Ž2. the increase occurred progressively in this section, whereas in the brain stem, TRH concentrations rose during two distinct periods ŽE16–E18 and E20–C1.. The exact physiological function of the increasing TRH levels in the brain stem, as reported in the rat w35x, is still under investigation. Since hypophysectomy does not affect TRH levels in extrahypothalamic brain regions of the rat w27x, the abrupt rise in brain stem TRH does not induce changes at the level of the pituitary. Further research still has to indicate if the changes in chicken and mammalian brain stem w35x may be linked to the function of TRH as a neurotransmitter w3,29,32x.
Besides being of hypothalamic origin, several studies in mammals have shown that TRH, as other neuropeptides Žfor review, see Ref. w18x., also originates from the anterior pituitary itself. We therefore compared chicken pituitary TRH concentrations with those recorded in several nuclei. TRH pituitary content Žpgrnucleus. was higher than that within the several collected brain nuclei. This observation suggests that intrahypophyseal TRH may function as an important paracrine or autocrine factor within the pituitary in the regulation of both the thyroidal and the GH axis of the chicken. This physiological relevance of intrahypophyseal TRH is shown by the increase in pituitary TRH content in hypothyroid rats w8x. It is not likely that the accumulation of hypophyseal TRH results, in part, from the uptake of hypothalamic TRH or from a decreased intrahypophyseal degradation of TRH. In vitro rat experiments did indeed indicate that there is no appreciable uptake of exogenous TRH or w3 HxTRH into the rat pituitary beyond the binding expected for TRH on its receptors w18x. Moreover, TRH gene expression has been described in the rat pituitary w5,9x, so it is now generally accepted that TRH can be synthesized within the rat pituitary and secreted in vitro by anterior pituitary cells w5,18x. Although the proTRH peptide or its mRNA have not yet been demonstrated within the pituitary of any non-mammalian species, our data nevertheless suggest that TRH is also synthesized within the chicken pituitary. Since several studies have presented arguments for a physiological role of anterior pituitary neuropeptides w4,6,9x and the chicken pituitary contains a high level of TRH Žw17x, present results., we assessed pituitary TRH concentrations during chicken prehatch development. Hypophyseal TRH levels were high from E14 to E16, and dropped 10-fold towards hatching. In other words, embryonic pituitaries contain high levels of TRH when hypothalamic TRH concentration is low, whereas the drop in hypohyseal TRH coincides with the start of the increase in hypothalamic TRH levels. TRH of an adenohypophyseal origin may therefore be implicated in the regulation of pituitary functions, up to the moment when hypothalamic TRH levels increase. This presumed autoregulatory action of the chicken pituitary, at the start of the last week of the incubation, may be related to the late maturation of hypothalamic control, and is not caused by the absence of hormones originating from the hypothalamus since the portal blood system is already developed in E12 chicks w38x. Similar opposite profiles were recorded recently for somatostatin: hypothalamic levels increase prior to hatching w13x, whereas hypophyseal concentrations drop after embryonic day 16 w12x. To our knowledge, researchers have not yet looked into developmental changes in the hypophyseal content of hypothalamic releasing hormones in mammals. Since we showed that major changes at the level of the chicken pituitary occur Žw12x, present results. and an important paracrine role may be existing for these intrahypophyseal fluctuations w8x, it is recommended to
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study hypophyseal TRH Žand of others. concentrations during mammalian foetal development and postnatal growth. In conclusion, TRH is scattered throughout the chicken brain and is located in hypothalamic nuclei not involved in pituitary control. Our study shows that towards hatching, tissue-specific fluctuations occur in the concentration of TRH within different regions of the chicken brain. In both the hypothalamic area Žendocrine function. and in the brain stem Žneurocrine function., a profound increase was recorded during the last week of embryonic development. At the level of the pituitary, TRH concentrations dropped a few days prior to hatching. The presumable paracrinerautocrine actions of hypophyseal TRH may therefore be very important up to the moment that hypothalamic TRH control matures during in ovo development.
Acknowledgements The skillful technical assistance of Mrs. Voets, Ms. Noterdaeme, and Mr. Van Ham is gratefully acknowledged. The authors wish to thank Dr. T.J. Visser ŽTRH antiserum., Dr. G. Hickey ŽL-692,429. and Dr. T.R. Hall ŽhGHRH 1 – 29 . for their gift and Drs. S. Kotanen for reading the manuscript. K.L. Geris was supported by the Fund for Scientific Research Flanders.
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w27x T. Mitsuma, Y. Hirooka, T. Nogimori, Effects of hypophysectomy on TRH and its related peptides concentrations in various rat organs, Endocr. Regul. 28 Ž1994. 125–131. w28x M. Mori, M. Yamada, Thyroid hormones regulate the amount of thyrotrophin-releasing hormone in the hypothalamic median eminence of the rat, J. Endocrinol. 114 Ž1987. 443–448. w29x G. Nistico, D. Rotiroti, A. De Sarro, J.D. Stephenson, Behavioural electrocortical and body temperature effects after intercerebral infusion of TRH in fowls, Eur. J. Pharmacol. 50 Ž1978. 253–260. w30x M. Palkovits, Punch sampling biopsy technique, in: P.M. Conn ŽEd.., Neuroendocrine peptide methodology, Academic Press, 1989, pp. 871–879. w31x M. Palkovits, R.L. Eskay, M.J. Brownstein, The course of thyrotropin-releasing hormone fibers to the median eminence in rats, Endocrinology 110 Ž1982. 1526–1528. w32x L.P. Renaud, J.B. Martin, P. Brazeau, Depressant action of TRH, LH–RH and somatostatin on activity of central neurones, Nature 255 Ž1975. 233–235. w33x J.M.M. Rondeel, W.J. de Greef, P. Van der Schoot, P. Karels, W. Klootwijk, T.J. Visser, Effects of thyroid status and paraventricular lesions on the release of thyrotropin-releasing hormone and catecholamines into hypophysial portal blood, Endocrinology 123 Ž1988. 523–527. w34x J.M.M. Rondeel, W. Klootwijk, E. Linkels, G.A.C. van Haasteren, W.J. de Greef, T.J. Visser, Regulation of TRH-like peptide pyroglutamyl-prolineamide in the rat anterior pituitary gland, J. Endocrinol. 145 Ž1995. 43–49.
w35x J.M. Schaeffer, M.J. Brownstein, Ontogeny of TRH-like material in several regions of the rat brain, Brain Res. 182 Ž1980. 207–210. w36x T.P. Segerson, H. Hoefler, H. Childers, H.J. Wolfe, P. Wu, I.M.D. Jackson, R.M. Lechan, Localization of thyrotropin-releasing hormone prohormone messenger ribonucleic acid in rat brain: in situ hybridisation, Endocrinology 121 Ž1987. 98–107. w37x T.P. Segerson, J. Kauer, H.C. Wolfe, H. Mobtaker, P. Wu, I.M.D. Jackson, R.M. Lechan, Thyroid hormone regulates TRH biosynthesis in the paraventricular nucleus of the rat hypothalamus, Science 238 Ž1987. 78–80. w38x R.C. Thommes, R.P. Russo, Vasculogenesis in the adenohypophysis of the developing chick embryo, Growth 23 Ž1959. 205–219. w39x R.C. Thommes, J. Caliendo, J.E. Woods, Hypothalamo-adenohypophyseal-thyroid interrelationships in the chick embryo: VII. Immunocytochemical demonstration of thyrotrophin-releasing hormone, Gen. Comp. Endocrinol. 57 Ž1985. 1–9. w40x G.A.C. van Haasteren, E. Linkels, W. Klootwijk, H. Van Toor, J.M.M. Rondeel, A.P.N. Themmen, F.H. De Jong, K. Valentijn, H. Vaudry, K. Bauer, T.J. Visser, W.J. de Greef, Starvation-induced changes in hypothalamic content of pro-thyrotropin-releasing hormone ŽproTRH. mRNA and the hypothalamic release of proTRHderived peptides: role of the adrenal gland, J. Endocrinol. 145 Ž1995. 143–153. w41x T.J. Visser, W. Klootwijk, R. Doctor, G. Henneman, A new radioimmunoassay of thyrotropin-releasing hormone, FEBS Letters 83 Ž1977. 37–40.
Research report
Thyrotropin-releasing hormone concentrations in different regions of the chicken brain and pituitary: an ontogenetic study Kris L. Geris
a, )
, Els D’Hondt b , Eduard R. Kuhn ¨ a , Veerle M. Darras
a
a
b
Laboratory of ComparatiÕe Endocrinology, Zoological Institute, K.U. LeuÕen, Naamsestraat 61, B-3000 LouÕain, Belgium Laboratory for Neuroendocrinology and Immunological Biotechnology, K.U. LeuÕen, Naamsestraat 59, B-3000 LouÕain, Belgium Accepted 17 November 1998
Abstract The regional distribution of thyrotropin-releasing hormone ŽTRH. was studied in the chicken brain. The hypothalamus and the brain stem contained the highest concentration of TRH. Lower amounts were present in the telencephalon, the optic lobes and the cerebellum. Within the hypothalamus, TRH was most abundant in the median eminence. Other important TRH sites were the nucleus paraventricularis magnocellularis, nucleus periventricularis hypothalami, nucleus ventromedialis hypothalami, nucleus dorsomedialis hypothalami and nucleus preopticus periventricularis. On the 14th day of embryonic development ŽE14., TRH was mostly found in the brain stem. Towards hatching, TRH concentrations increased gradually in both the hypothalamic area and the brain stem. TRH concentrations in the telencephalon, optic lobes and cerebellum remained low. Pituitaries from E14 to E16 chickens were characterized by a high TRH concentration, whereas hypophyseal TRH concentrations dropped towards hatching. Our results support the hypothesis that TRH exerts both endocrine and neurocrine actions in the chicken. On the other hand, high pituitary TRH concentrations were present when hypothalamic concentrations were low and vice versa. Therefore, the chicken pituitary may function as an important source of TRH during early in ovo development at least until the moment hypothalamic control develops. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Chicken; Thyrotropin-releasing hormone; Brain; Pituitary; Nucleus; Ontogeny
1. Introduction Thyrotropin-releasing hormone ŽTRH. was the first hypophysiotropic peptide to be isolated from the hypothalamus w7x. It was called TRH because it stimulates thyrotropin ŽTSH. release from the mammalian pituitary gland. Immunocytochemical studies in rats showed that specific neurons extending exclusively from the paraventricular nucleus transport TRH to the median eminence where it is secreted into the portal blood system allowing TRH to act at the level of the pituitary w31x. As a local neurotrans-
) C orresponding author. [email protected]
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mitter, TRH is however, present throughout the mammalian central nervous system w19,36x. Despite its thyroid-related name, TRH has a dual site of action in the chicken. It stimulates the secretory activity of both thyrotrophs and somatotrophs w2,16x. While several research groups have investigated the potency and the mode of action of TRH at the level of the chicken pituitary w15,24x, only few data suggest a neurocrine function of TRH within the chicken brain w29x. Also, little is known about the actual presence and distribution of TRH in the chicken brain w26x. Accordingly, we screened the chicken brain Žsections and nuclei. for TRH using a sensitive and specific radioimmunoassay ŽRIA. w14x. Since several researchers showed that the mammalian pituitary is capable of synthesizing TRH w4–6,8,9,34x, the pituitary was included in this study. Finally, tissues from embryonic up to newly hatched chicks were collected and ontogenetic changes in TRH concentrations compared.
0006-8993r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 8 . 0 1 2 8 1 - 5
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2. Material and methods 2.1. Animals All studies were performed on chickens of a layer strain ŽHisex White., purchased as fertilized eggs or 1-day-old male chicks ŽC1. from a local commercial hatchery ŽEuribrid, Aarschot, Belgium.. Eggs were incubated in a forced-draft incubator, at 37.88C, with increasing humidity and ventilation from day 14 onwards, under continuous lighting and a 458 rotation every hour Žstart of incubation s day 1 ŽE1... Posthatch chickens were kept in an acclimatized room with a 14Lr10D photoperiod. Water and food were available ad libitum.
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were determined using the stereotaxic atlas of Kuenzel and Masson w23x and stock brain sections stained with cresyl violet. In order to obtain the whole nucleus, punches were performed on consecutive sections. The following nuclei were collected: nucleus septalis lateralis ŽSL. and medialis ŽSM., nucleus preopticus medialis ŽPOM. and periventricularis ŽPOP., nucleus anterior Žrostralis. medialis hypothalami ŽAM., nucleus commissurae pallii ŽnCPa., nucleus paraventricularis magnocellularis ŽPVN., nucleus periventricularis hypothalami ŽPHN., nucleus rotundus ŽROT., nucleus ventromedialis hypothalami ŽVMN., nucleus dorsomedialis hypothalami ŽDMN. and the median eminence ŽME. w15x. 2.5. Measurement of TRH concentrations
2.2. Distribution in the brain To study the concentration of TRH in different brain regions, animals ŽC1; n s 10. were killed by rapid decapitation. The cranium was opened and the following brain regions were collected: telencephalon left ŽTLL. and right ŽTLR., tectum opticum Žoptic lobe. left ŽTOL. and right ŽTOR., the hypothalamic area Žhypothalamus and thalamus; HT., the cerebellum ŽCB. and the brain stem ŽBS.. Tissues were stored at y808C. Due to the small size and the fragile character of embryonic and newly hatched chicken brain, 1-week-old chickens were used to isolate specific nuclei in the brain. The total brain was carefully dissected and then immediately placed on dry ice Ž n s 8.. The pituitary was taken out of the sella tursica. Tissues were stored at y808C. 2.3. Ontogeny of TRH concentrations in the brain and the pituitary Brain regions and pituitaries were collected from E14, E16, E18, E20Žnon-pippingŽNP.., E20Žinternal pipping ŽIP.. Žper embryonic stage n s 12. and C1 Ž n s 10. chicks. In the first distribution study, no differences were found between the TRH concentration in the left or the right part of the telencephalon or of the optic lobes, so both parts were pooled, resulting in one telencephalon and one optic lobe sample per animal. Tissues were stored at y808C. 2.4. Microdissection of the nuclei The micropunch technique of Palkovits w30x was used to isolate specific brain nuclei. Serial sections of 300 mm were made by means of a cryostat, mounted on slides cleaned with chromic acid and stored at y808C. The needles ŽTP-100, Activational Systems, MI, USA. used in the punch technique were constructed of steel and the inner diameters of the needles varied from 0.2 to 2.0 mm, depending on the size of the brain nuclei to be dissected. At the beginning of the micropunch procedure, the exact coordinates of the brain nucleus that would be punched
Extraction of TRH was performed as described before w14x. In the ontogenetic study, brain regions were pooled up to stage E20 ŽIP., resulting, respectively in 6 ŽTL, TO, HT and BS. and 4 ŽCB. samples for the different brain parts. Tissues of C1 animals were used individually Ž n s 10.. Pituitaries were pooled yielding 6 ŽE14 and E16. or 4 ŽE18, E20 ŽNPrIP. and C1. samples for the different stages. TRH was labeled with the chloramine T method w14x. TRH RIA was carried out according to van Haasteren et al. w40x. Briefly, radioiodinated TRH Ž15.000 cpmrsample., antibody Žlaboratory raised, 1r10,000. Žkindly donated by Dr. T.J. Visser, Erasmus University Rotterdam., assay buffer Ž0.14 M KCl, 0.1 M KH 2 PO4 , 0.02% bovine serum albumin ŽSigma, St. Louis, USA., pH 7.5. and sample or standard Ž1.56 to 800 pg. were incubated in a final volume of 400 ml assay buffer in polystyrene tubes Ž72 h, 48C.. Separation of free and bound radioactivity was achieved by immunoprecipitation using Sac-Cel anti-rabbit globulines ŽInnogenetics, Gent, Belgium.. After 1 h of incubation at 48C and centrifugation, precipitates were counted in a g-counter ŽGammamaster, LKB.. The ED 80 and ED 20 of the RIA were respectively, 10 and 240 pg. The intra and interassay coefficients were 6.6 " 0.4% and 15.6 " 2.1%, respectively, recovery of standards in homogenates was 102.1 " 1.7%. Displacement curves resulting from serial dilutions of hypothalamic or telencephalon homogenates were parallel to the standard curve. The hypothalamic hormones somatostatin ŽSRIH. ŽSigma–Aldrich., human growth hormone ŽGH .-releasing hormone w1 – 29 x ŽhGHRH 1 – 29 ; kindly donated by Dr. T.R. Hall, Ciba-Geigy, Switzerland., synthetic rat preproTRH 160 – 169 ŽPs4; Peninsula, CA, USA., ovine corticotropin-releasing hormone ŽoCRH; UCB, Brussels. and the non-peptidyl GHsecretagogue L-692,429 w14x Žkindly donated by Dr. G. Hickey, Merck, Rahway, USA. did not cross-react in the assay. The RIA employing antiserum, a8880, shows, compared to antisera previously used w34,40,41x, much less cross-reactivity with TRH analogues that have histidine replaced by other amino acids. TRH was expressed as a
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Values represent mean " S.E.M. Statistical analysis was assessed by the general linear model of SAS Ž1985., followed by a Scheffe´ test.
detection limit of the assay. No differences were found between the right or the left part of the telencephalon or the optic lobe. In order to compare TRH proportions in the different brain regions, data from Fig. 1 were recalculated as a content Žpgrtissue; data not shown.. The actual amount of TRH in the hypothalamic region Ž269 " 32 pgrHT. was higher compared to the brain stem Ž120 " 17 pgrBS. and the telencephalon Ž126 " 15 pgrTL. content. TRH content in the other brain regions was very low Ž- 30 pgrtissue.. From the total amount of TRH in the brain, 61.8% was found in extrahypothalamic areas. Since the hypothalamic area used also contained the thalamus, this percentage will even be higher. TRH content in different brain nuclei and pituitary is shown in Fig. 2. TRH was predominantly found in the ME. Other important sources of TRH, containing approximately 30% of the TRH content in the ME, were the PVN, PHN, POP, AM, VMN and DMN. In a few animals, a low amount of TRH was recorded in the SL Ž3 out of 8: 3r8., SM Ž3r8., POM Ž2r8. and nCPa Ž4r8.. No TRH was located in the ROT. Expressing TRH content in the pituitary in pgrnucleus Ži.e., pituitary. showed that the pituitary is an important source of TRH Ž22.9 " 5.4 pgrpituitary.. The TRH content was only higher in the median eminence ŽFig. 2..
3. Results
3.2. Ontogeny of TRH concentrations in the brain and the pituitary
Fig. 1. Distribution of thyrotropin-releasing hormone ŽTRH. in the brain of 1-day-old male chickens. The collected brain sections were: telencephalon left ŽTLL. and right ŽTLR., tectum opticum Žoptic lobe. left ŽTOL. and right ŽTOR., the hypothalamic area Žhypothalamus and thalamus, HT., the cerebellum ŽCB. and the brain stem ŽBS.. Data shown are mean"S.E.M. Ž ns10.. TRH was expressed in pgrg wet weight.
concentration Žpgrg wet weight Žbrain regions. or pgrmg wet weight Žpituitary.. or as a content Žpgrnucleus.. 2.6. Statistics
3.1. Distribution in the brain Fig. 1 shows the TRH concentration in the different brain regions of 1-day-old chickens Žpgrg wet weight.. TRH was predominantly found in the hypothalamic area and the brain stem. It was also present at a considerable concentration in both parts of the telencephalon. The optic lobes and the cerebellum had a concentration close to the
In both the hypothalamic region and the brain stem, TRH concentrations rose towards the end of incubation ŽFig. 3.. During early development, TRH was mostly located in the brain stem, whereas TRH levels in the other regions were low. Hypothalamic TRH levels increased progressively during the last week of embryonic development to reach a maximum in 1-day-old chickens. A similar profile was noted for the brain stem, although no increase
Fig. 2. Distribution of TRH in several brain nuclei and the pituitary of 1-week-old chickens ŽC8.. The data shown are expressed in pg TRHrnucleus or pituitary and represent mean " S.E.M. of eight individual observations.
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Fig. 3. Ontogeny of TRH concentrations in several regions of the chicken brain during the last week of embryonic development up to 1-day-old chickens. Data shown are mean " S.E.M. Ž n s 4–10.. Since no differences were found in the TRH concentration of the several brain regions between NP and IP chicks, data of NP and IP chicks were combined ŽE20 bars.. Within one tissue, data with a common letter are not significantly different ŽScheffe, ´ P - 0.05. ŽTL: telencephalon; TO: tectum opticum; HT: hypothalamus; CB: cerebellum; BS: brain stem.. Within one tissue, a similar TRH profile is observed when the TRH data are expressed as pgrtissue.
in TRH was observed between E18 and E20, as was found in the hypothalamic region. An overall 15-fold increase was observed from E14 to C1 in hypothalamic TRH concentration, whereas brain stem TRH levels increased 6-fold. Although some changes were recorded in TRH levels of the optic lobes or the cerebellum, these were much less pronounced compared to those observed in the hypothalamus or brain stem. TRH concentrations in these brain regions remained at a low level from E14 to C1. Hypothalamic TRH content Žpgrtissue; data not shown. increased profoundly towards hatching; an opposite decrease was noticed in relative extrahypothalamic TRH
content in the brain ŽE14: 88.2%; E16: 83.3%; E18: 76.6%; E20: 59.8% and C1: 66.2%.. Unlike hypothalamic concentrations, hypophyseal TRH levels dropped towards hatching ŽFig. 4.. Embryonic stages E14–E16 were characterized by a high TRH level in the pituitary, whereas late-embryonic and newly hatched chicks had a 5- to 10-fold lower TRH concentration in the pituitary. No differences were found on day 20 between the non-pipping and internal pipping embryos. Hypophyseal TRH content dropped from 244 " 79 pgrpituitary ŽE14. to 19 " 6 pgrpituitary ŽC1..
4. Discussion
Fig. 4. Ontogenetic changes in pituitary TRH concentration Žpg TRHrmg wet weight. during the second part of embryonic development up to 1-day-old chickens Ž ns 4–6.. Data shown are mean"S.E.M. Data with a common letter are not significantly different ŽScheffe, ´ P - 0.05.. A similar TRH profile is recorded when TRH data are expressed in pgrtissue.
Using specific radioimmunoassay techniques, we have measured the amount of TRH in various regions of the chicken brain. As in the rat, large quantities of TRH were evident in the hypothalamic area. This is consistent with the role of TRH as a regulator of the release of anterior pituitary hormones. Hypothalamic TRH levels in the rat were approximately 100-fold higher than in the avian hypothalamus Ž19 and present results.. Extrahypothalamic TRH accounted for approximately 70% of the total TRH content in the chicken brain. The same proportional distribution has been recorded in mammals w19,35x. In the chicken, especially the brain stem seems characterized by a high TRH concentration. Our distribution study is well in agreement with data from Jackson and Reichlin w19x, who reported high TRH levels in the chicken hypothalamic region and brain stem, whereas low concentrations were found in the other brain regions. Immunocytochemical
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studies have also shown a widespread distributional pattern of TRH in the avian brain w21,26x. There is good evidence that only TRH synthesized in the PVN and transported to the ME regulates pituitary secretion: Ž1. lesion of the PVN extremely decreased TRH and TSH secretion in the rat w10,28,31,33x; Ž2. electrical stimulation of this specific nucleus increased the release of these peptides w10x; Ž3. thyroid hormones control TRH content exclusively in the PVN w11,37x. TRH immunoreactive perikarya and neurons have been observed respectively, in the PVN and the ME of avian hypothalamus w21,22,26x, suggesting that the same functional system exists in birds. Our data show that TRH is not only found in these areas but also in nuclei not involved in the control of pituitary secretion like the PHN, DMN and VMN. Also in rats, these nuclei were characterized by high TRH concentrations w20,25,31x. These findings, and the widespread distribution of TRH in the different brain regions, indicate that TRH does not merely act as an endocrine regulator at the level of the pituitary. In mammals, TRH has both inhibitory w32x and excitatory w3x effects on neurons in the brain, so TRH may also act as a neurotransmitter or neuromodulator in the chicken w29x. Data on the ontogenetic appearance of several hypothalamic factors in the avian brain have long been restricted to immunocytochemical studies w1,22,39x. In view of our interest in the effects TRH exerts on the pituitary, we recently studied the presence of hypothalamic TRH during chicken embryo development w13x. The present report confirms these hypothalamic data that a progressive rise in hypothalamic TRH occurs towards hatching, and further expands these findings to the other brain regions and the pituitary. At the extrahypothalamic level, major fluctuations were noticed in brain stem TRH concentrations. Due to a profound increase towards hatching, along with small changes in the other areas, the chicken brain stem contained more than 85% of the extrahypothalamic TRH content at hatching. Since no clear fluctuations in the TRH level in the other brain regions were observed, the rise in TRH in the hypothalamic area and the brain stem was not due to an overall maturation of the chicken brain. Some differences between the rise in TRH levels in both regions also suggest that two independent evolutions take place in these areas: Ž1. the increase was much more pronounced in the hypothalamic region; Ž2. the increase occurred progressively in this section, whereas in the brain stem, TRH concentrations rose during two distinct periods ŽE16–E18 and E20–C1.. The exact physiological function of the increasing TRH levels in the brain stem, as reported in the rat w35x, is still under investigation. Since hypophysectomy does not affect TRH levels in extrahypothalamic brain regions of the rat w27x, the abrupt rise in brain stem TRH does not induce changes at the level of the pituitary. Further research still has to indicate if the changes in chicken and mammalian brain stem w35x may be linked to the function of TRH as a neurotransmitter w3,29,32x.
Besides being of hypothalamic origin, several studies in mammals have shown that TRH, as other neuropeptides Žfor review, see Ref. w18x., also originates from the anterior pituitary itself. We therefore compared chicken pituitary TRH concentrations with those recorded in several nuclei. TRH pituitary content Žpgrnucleus. was higher than that within the several collected brain nuclei. This observation suggests that intrahypophyseal TRH may function as an important paracrine or autocrine factor within the pituitary in the regulation of both the thyroidal and the GH axis of the chicken. This physiological relevance of intrahypophyseal TRH is shown by the increase in pituitary TRH content in hypothyroid rats w8x. It is not likely that the accumulation of hypophyseal TRH results, in part, from the uptake of hypothalamic TRH or from a decreased intrahypophyseal degradation of TRH. In vitro rat experiments did indeed indicate that there is no appreciable uptake of exogenous TRH or w3 HxTRH into the rat pituitary beyond the binding expected for TRH on its receptors w18x. Moreover, TRH gene expression has been described in the rat pituitary w5,9x, so it is now generally accepted that TRH can be synthesized within the rat pituitary and secreted in vitro by anterior pituitary cells w5,18x. Although the proTRH peptide or its mRNA have not yet been demonstrated within the pituitary of any non-mammalian species, our data nevertheless suggest that TRH is also synthesized within the chicken pituitary. Since several studies have presented arguments for a physiological role of anterior pituitary neuropeptides w4,6,9x and the chicken pituitary contains a high level of TRH Žw17x, present results., we assessed pituitary TRH concentrations during chicken prehatch development. Hypophyseal TRH levels were high from E14 to E16, and dropped 10-fold towards hatching. In other words, embryonic pituitaries contain high levels of TRH when hypothalamic TRH concentration is low, whereas the drop in hypohyseal TRH coincides with the start of the increase in hypothalamic TRH levels. TRH of an adenohypophyseal origin may therefore be implicated in the regulation of pituitary functions, up to the moment when hypothalamic TRH levels increase. This presumed autoregulatory action of the chicken pituitary, at the start of the last week of the incubation, may be related to the late maturation of hypothalamic control, and is not caused by the absence of hormones originating from the hypothalamus since the portal blood system is already developed in E12 chicks w38x. Similar opposite profiles were recorded recently for somatostatin: hypothalamic levels increase prior to hatching w13x, whereas hypophyseal concentrations drop after embryonic day 16 w12x. To our knowledge, researchers have not yet looked into developmental changes in the hypophyseal content of hypothalamic releasing hormones in mammals. Since we showed that major changes at the level of the chicken pituitary occur Žw12x, present results. and an important paracrine role may be existing for these intrahypophyseal fluctuations w8x, it is recommended to
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study hypophyseal TRH Žand of others. concentrations during mammalian foetal development and postnatal growth. In conclusion, TRH is scattered throughout the chicken brain and is located in hypothalamic nuclei not involved in pituitary control. Our study shows that towards hatching, tissue-specific fluctuations occur in the concentration of TRH within different regions of the chicken brain. In both the hypothalamic area Žendocrine function. and in the brain stem Žneurocrine function., a profound increase was recorded during the last week of embryonic development. At the level of the pituitary, TRH concentrations dropped a few days prior to hatching. The presumable paracrinerautocrine actions of hypophyseal TRH may therefore be very important up to the moment that hypothalamic TRH control matures during in ovo development.
Acknowledgements The skillful technical assistance of Mrs. Voets, Ms. Noterdaeme, and Mr. Van Ham is gratefully acknowledged. The authors wish to thank Dr. T.J. Visser ŽTRH antiserum., Dr. G. Hickey ŽL-692,429. and Dr. T.R. Hall ŽhGHRH 1 – 29 . for their gift and Drs. S. Kotanen for reading the manuscript. K.L. Geris was supported by the Fund for Scientific Research Flanders.
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