A regimen of twice daily metyrapone injections (100 mg/kg), resulted in pharmacological adrenalectomy of pregnant rats and fetuses in utero, i.e. depression of plasma corticosterone and elevation of plasma adrenocorticotropic hormone (ACTH). Toxicity was minimal on days 14-17 of pregnancy, and increased with higher maternal weight and pregnancy progression. Corticotropin releasing hormone (CRH) messenger RNA abundance in the pregnant adults increased significantly within 48 h of metyrapone initiation. No change in CRH gene expression in the paraventricular nucleus of fetuses (days 17-18) was seen, even after 72 h of the regimen. This is compatible with the independence of CRH gene expression of glucocorticoid feedback in the fetal rat

Baram, TZ

Schultz, L

Baram, Tallie Z

Schultz, Linda

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UC IrvineUC Irvine Previously Published WorksTitleCRH gene expression in the fetal rat is not increased after pharmacological adrenalectomyPermalinkhttps://escholarship.org/uc/item/6bx7x4kkJournalNeuroscience Letters, 142(2)ISSN0304-3940AuthorsBaram, Tallie ZSchultz, LindaPublication Date1992-08-01DOI10.1016/0304-3940(92)90376-iCopyright InformationThis work is made available under the terms of a Creative Commons Attribution License, availalbe at https://creativecommons.org/licenses/by/4.0/ Peer reviewedeScholarship.org Powered by the California Digital LibraryUniversity of California,Veuroscience Letter.s'. 142 (1992) 215 218 215 : 1992 Elsevier Scientific Publishers lreland Ltd. All rights reserved 0304-3940/92/$ 05.00 NSL. 08830 CRH gene expression in the fetal rat is not increased after pharmacological adrenalectomy Tallie Z. B a r a m  and  L inda  Schul tz  Department ~/ Neurology, University qf Southern Cal(/ornia. Division ~/' Neurok~gy. Childrens Ho,wital Los Angeles. Lo~ ,4n~eh'.~. CA ( USA ) (Received 15 November 1991; Revised version received 5 May 1992: Accepted 12 May 1992) Key words': Corticotropin releasing factor: Messenger RNA: Development; Paraventricular nucleus: Glucocorticoid feedback A regimen of twice daily metyrapone injections (100 mg/kg), resulted in pharmacological adrenalectomy of pregnant rats and fetuses in utero, i.e. depression of plasma corticosterone and elevation of plasma adrenocorlicotropic hormone (ACTH). Toxicity was minimal on days 14 17 of pregnancy, and increased with higher maternal weight and pregnancy progression. Corticotropin releasing hormone (CRH) messenger RNA abun- dance in the pregnant adults increased significantly within 48 h of metyrapone initiation. No change in CRH gene expression in the paraventricular nucleus of fetuses (days 17 18) was seen, even alter 72 h of the regimen. This is compatible with the independence o1" CRH gene expression of glucocorticoid feedback in the fetal rat. The mechanisms controlling corticotropin releasing hormone (CRH) gene expression in the developing rat brain have not been defined. The onset of  messenger RNA ( C R H - m R N A )  synthesis occurs on the 17th fetal day, and after increasing in the late fetal period, de- creases markedly around the time of birth [2, 7]. Whether the onset and the hiatus in C R H  gene expression are intrinsic, constitutively regulated phenomena,  or are modulated via negative feedback by circulating adreno- corticotropic hormone (ACTH) or corticosterone, is un- known. This study was designed to examine the effects of eliminating the negative corticosterone (CORT) feed- back on the brain-adrenal  axis in the fetal rat. Our goal was to study the effects of  functional fetal adrenalectomy on C R H  gene expression in the fetal rat, in comparison with the pregnant adult. Forty t imed-pregnancy Sprague Dawley derived rats, weighing less than 350 g, were kept on a 12 h light/dark cycle and allowed unlimited access to lab chow and water. Pregnancy was dated by the presence of a vaginal plug (day 0). Gestation lasted for 21 days. Pharmacologi- cal adrenalectomy schedule [14] was started on the 14th- 18th day of pregnancy, and continued for 48 or 72 h, as previously described [4]. Briefly, pregnant rats were in- jected subcutaneously with dimethylsulfoxide (DMSO) ('orre~v~ondence: T.Z. Baram, Division of Neurology 82. Childrens Hospital Los Angeles, RO. Box 54700, Los Angeles, CA 90054-0700. USA. vehicle or 100mg/kg metyrapone (Sigma, St. Louis, MO), every 12 h. On the morning of sacrifice, metyrap- one was followed, at 09.15 h, by 100 mg/kg aminoglu- tethimide. Rats were decapitated at 10.00 h and uteri quickly har- vested. Maternal and pooled 07 = 5) fetal trunk blood was obtained as described previously, and assayed for CORT and A C T H  [4]. Adult brains and fetal heads were frozen immediately on powdered dry ice and kept frozen at -80°C.  Frozen brains were cut into 20/,tin coronal sections and mounted on gelatin-coated slides. Brain regions were identified by established landmarks [1]. Prior to in situ hybridization (ISH), sections were brought to room temperature, air-dried and fixed for 20 rain in fresh 4% buffered PBS-paraformaldehyde. Sections were dehy- drated through increasing ethanol concentrations, rehy- drated, exposed to 0.5% acetic anhydride 0.1 M trietha- nolamine (pH=8), then dehydrated through 100% etha- nol [2, 3]. Details of  oligonucleotide probe generation and of ISH, modified from Young [19], have been described [2, 3]. Briefly, prehybridization for I h in hybridization buffer lacking dextran sulfate (0.2 ml/slice), was followed by a 20 h ISH at 40°C [2, 3]. Reaction volume was 0.03 ml/slice, under a coverslip in a humidity chamber. Following serial washes, hybridized sections were proc- essed for autoradiography [2, 3]. A 60 nucleotide syn- thetic probe corresponding to the codons for the 20 21h  3 0 0  V .... ! L I ~ 300~ I I--- C) 1 0 0  .4 0 16 17 ! 8  D a y  o f  p r e g n a n c : ,  T r e a t e d  KEEl] C o n t  co l  n :3 n : 3  -7 Fig. 1. Plasma ACTH in pregnant rats treated with metyrapone for 48 h. Pharmacological adrenalectomy was achieved by mctyrapone in- jections (see text). ACTH levels of  treated animals were significantly different from those of  controls: P<0.05. F_~ L i',/e ~ w ' e l  ,4~  I~Sl!] S ic  k 1 0 0 [  I ~e'~]d N D~I~'~UDS SICk clead k eo~ 2 GO I Z a L g~ 2c o ~e i l 01 S ] 7 (9<I ~8 ' 5 ] '  D a y  o t  p r e g n a n t )  1 Fig. 2. Toxicity of  metyrapone, given ['or 48 72 h. as a function of gestational day in pregnant rat and fetuses. Day of  sacrifice is indicated. Number of rats are in brackets, +, an additional rat in this group was not pregnant. COOH-amino acids of CRH [17] was labelled on the 3' end with dATp-35S. Specific activity of labelled probes was 5-8 x 108 dpm//tg. The complementary 'sense' strand was similarly generated and labelled. Serial sections of the hypothalamus at the level of the paraventricular nucleus (PVN) were examined. For quantitative studies, 5 sections with the maximal CRH mRNA, derived from at least 3 rats each from 2 separate litters (for fetuses), were compared [2, 3]. Images were obtained and optical density (O.D.) determined over the PVN and over the parietal cortex as described previously [2, 3]. To allow for variability in background (cortex) O.D., the O.D. ratio of PVN to parietal cortex was util- ized as a measure of C R H - m R N A  abundance, and means and standard deviations for 10 ratios were deter- mined. For adult rats, sections derived from comparable levels in the PVN were also dipped in emulsion (NTB-2, Kodak), and exposed for 1 week. They were subjected to darkfield microscopy and analysis. Brainpaste standards were used to ascertain that measurements were in the linear range [2]. Analysis of variance (Minitab, Univer- sity Park, PA), was used to assess the significance of dif- ferences between experimental groups. Effect of  pharmacological adrenalectomy on maternal and fetal levels of CORT and A CTH. Plasma ACTH in- creased significantly (P<0.05) in metyrapone-treated rats (Fig. 1). This effect persisted for 72 h. The metyrapone regimen decreased plasma CORT levels 85--95%, e.g. plasma CORT in treated adults was 5.9 + 1.6/tg/dl, com- pared with 30.2 _+ 3.7/tg/dl in controls 48 h after initia- tion of the regimen [4]. The reduction in CORT levels was maximal by 24 h, and persisted for at least 72 h [4]. Toxici O, of  the metyrapone regimen as a Jimction o,1' pregnancy day. Metyrapone injections were well toler- ated by rats on the 14th 17th pregnancy days. Rats weighing less than 350 g by day 17 of pregnancy (11/14), as well as the two animals found not to be pregnant, appeared well (Fig. 2). Abnormal fetal appearance or activity, fetal demise or poor maternal activity were seen in heavier animals (3/10, day 17) and worsened with pro- gression of pregnancy (Fig. 2). Therefore, following these preliminary results, no experiments were con- ducted beyond the 18th gestational day, to avoid animal stress and malaise. Furthermore, CRH-mRNA was ana- lyzed only in adult and fetal rats which appeared healthy and indistinguishable from DMSO-injected controls in terms of weight, activity etc. Only treated adult and fetal rats with (pooled, for fetuses) CORT levels lower than 5 ¢tg/dl were analyzed [18]. Effects of metyrapone treatment h7 CRF-mRNA in fetal and pregnant rats. At the end of 48 h of metyrapone treatment, CRH-mRNA in the PVN of pregnant rats in- creased significantly (Fig. 3). In fetal rats aged 17-18 days, subjected to 48 or 72 h of the regimen, no change in CRH-mRNA abundance was evident. Fig. 3 demon- strates the quantitative analysis of CRH-mRNA abun- dance in pregnant and fetal rats. Metyrapone "pharma- × 2 8 u Z > 13. E~ © Fig. 3. Effect of  metyrapone-injection regimen oil CRH-mRNA in PVN of  pregnant and fetal rats. Metyrapone was initiated 48 h prior to sacrifice. Bars indicate means and standard errors. Control (C) and treated (M) fetal values are not statistically significant. Adult treated group (M) differs significantly (P<0.05) from control (C). "sense' de- notes otigonucleotide complementary to CRH probe, 217 Fig. 4. Photomicrographs of the PVN of 18 day fetal rats following ISH with an oligonucleotide complementary to CRH-messenger RNA. A: treated: 48 h of metyrapone administration. B: control. PVN, paraventricular nucleus. Bar - I ram. See text for details of 1SH. cological adrenalectomy' was initiated on the 16th gesta- tional day, and animals were sacrificed 48 h later. The significant (P<0.05) increase in C R H - m R N A  abundance in the pregnant adults rats is contrasted with the lack of change in CRH-gene expression in the 18 day fetuses. Fig. 4 shows photomicrographs of coronal brain sections at the level of the PVN, of fetal rats subjected to the same regimen. In Fig. 5, control and treated adult PVN are shown. Darkfield micrographs of comparable PVN lev- els mainly show an increase in intensity of C R H - m R N A  signal, and not an increase in number of cells expressing CRH. These experiments provide evidence that significant reduction of plasma CORT levels in the fetal rat does not result in the expected enhancement of CRH gene expres- sion. This is in contradistinction to the reported effect in the adult [9, I1, 18, 19], as well as to our findings in pregnant rats. CORT levels decreased to 5/ag/dl, levels reported in surgically adrenalectomized rats, and result- ing in enhanced CRH gene expression in adult animals [18]. Specifically, Swanson and Simmons [18] have shown that in the adult rat, adrenalectomy results in in- creased C R H - m R N A  in the PVN within 48 h, while other investigators, using either ISH or Northern blot analysis, have documented elevation of CRH gent ex- pression in the PVN a week t\~llowing adrenalectomy [9, 11, 18]. Swanson and Simmons [18] found lhat CORT levels greater than 5/ag/dl were necessary for inhibition of adrenalectomy-induced increase in CRH-mRNA.  Beyer Fig. 5. Darkficld photomicrographs of comparable levels of the paraventricular nucleus or" treated (A) and control (B) pregnant r a t s  subjected to ISH forCRH-mRNA, lncreased number ofgrains per cell is seen in thc trcatcd animal (star). Bar : 1001m3. 21~ et al. [5] reported restoration of control PVN-CRH- mRNA with lower CORT levels (2.9/.zg/dl), but utilized Northern blot analysis of whole PVN. The levels of plasma CORT achieved with the metyrapone regimen should result in minimal occupa- tion of GC receptors [15]. The enhanced CRH gene ex- pression in the pregnant rats suggests that metyrapone- induced GC depletion was adequate to eliminate its neg- ative feedback on CRH synthesis. Furthermore, non- specific stress secondary to the regimen would be ex- pected to increase fetal CORT and CRH mRNA [12] neither of which was observed. Metyrapone administration is associated with the stress of injection and handling [8] which, present also in vehicle-injected controls, is unlikely to account for in- creased CRH-mRNA in the pregnant rats. Metyrapone has been shown to accelerate the response of the adreno- cortical system to adrenalectomy [8], but this effect, sig- nilicant within hours of adrenalectomy, was nol ob- served 2 3 days later [8]. The etfective metyrapone dose in pregnant rats ( 100 mg/kg) is significantly lower than in adult males (200-300 mg/kg) [14]. This fact, as well as the correlation of morbidity and body weight, is probably secondary to the altered pharmacokinetics of the drug during late gestation in the pregnant rat. We found no temporal alteration of CRH gene expres- sion by pharmacological adrenalectomy in the fetal rat. Metyrapone administration starting on the 14th or 15th fetal day did not result in an onset of CRH-gene expres- sion prior to the reported [2, 7] 17th fetal day (not shown). Our findings are compatible with the lack of a GC- negative feedback effect on CRH-gene expression in the fetal rat. GC receptors have been demonstrated by Northern blot analysis of total fetal brain RNA [10]. The lack of negative feedback may thus be a consequence of decreased receptor function [13], or the immaturity of transduction mechanisms beyond the steroid-receptor complex interaction. 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CRH gene expression in the fetal rat is not increased after pharmacological adrenalectomy

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CRH gene expression in the fetal rat is not increased after pharmacological adrenalectomy.

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