To delineate the development of melatonin (MLT) production during childhood, we measured the excretion of MLT and 6-hydroxymelatonin sulfate (MLTS) in the urine of children (n = 134) from the 26th week of gestation until the age of 20 years. MLTS excretion showed a diphasic pattern with declining values in preterm babies with lowest values around term. After birth, the values remained low for the first 6 months of life. The highest values were reached between 4 and 7 years of age with a smooth but steady decline thereafter. A night-day difference was not detectable before the age of 6 months; the greatest night-day variations occurred at the time of the highest MLTS excretions. The MLT values showed an identical pattern but with amounts 1,000 times smaller; the ratio of MLTS to MLT increased from 40:1 in preterm babies to 900:1 in prepubertal children. In summary, the MLT/MLTS excretion exhibits the highest activity with respect to total secretory capacities as well as night-day differences at the time of gonadal quiescence during childhood. The strong inverse correlation of MLT and MLTS excretion with the hypothalamic-pituitary-gonadal activity points to a causal relationship between pineal gland activity and pubertal development.

The patterns of deposition and immunoreactivity of interstitial amyloid were studied in 11 pituitary glands obtained at autopsy and 9 surgically resected pituitary adenomas using Congo red staining and a panel of antisera directed against 5 major amyloid fibril proteins and all pituitary hormones. The deposition pattern of amyloid in pituitary glands differed from that in adenomas but all amyloid deposits showed an immunostaining with anti-amyloid X-light chain. The remaining antisera were immunonegative. In situ hybridization using an oligodeoxyribonucleotide-probe complementary to the mRNA coding for the constant region of human X-light chain yielded no hybridization signals in the pituitaries or pituitary adenomas, excluding local synthesis and secretion of immunoglobulins. Since no case studied suffered from generalized AX-amyloidosis and adsorption of immunoglobulins to the unknown amyloid fribril protein of the pituitary seems to be unlikely, crossreaction of the polyclonal antisera with an undefined antigen is probable. The similar immunostaining properties of amyloid deposits in "normal" pituitaries and pituitary adenomas suggest they both originate from the same precursor protein.

In a series of 39 adenomas from patients with the clinical hyperfunction syndrome of acromegaly and in one from a case of prolactinoma we studied the mRNA expression of growth hormone (GH), prolactin (PRL) and beta-human chorionic gonadotropin (HCG) by using this technique of in situ hybridization (ISH). This technique allows the direct identification and localization of cells expressing mRNA and thus synthesizing the respective hormone. The aim of our study was to demonstrate the frequent co-expression of PRL mRNA and HCG mRNA in pituitary adenomas of acromegalic patients. Probes for ISH of the above-mentioned hormones were obtained by subcloning cDNA fragments into pGEM plasmids. Subsequent Sp6-polymerase catalysed in vitro transcription with 35S-CTP revealed radiolabelled single-stranded antisense RNA probes [the probe for beta HCG detects beta-luteinizing hormone (beta LH) simultaneously because of a sequence homology of 90%]. To localize the labelled hybrids, autoradiography was carried out. Light microscopical evaluation of the tissue sections demonstrated positive signals in all cases for GH, in 80% of cases for PRL and in 25% of cases for HCG [LH] mRNA. The comparison of mRNA content shown by ISH with immunocytochemical (ICC) hormone detection revealed that in all cases the detection of GH corresponded to GH mRNA content of the cells. For PRL and HCG [LH] positive mRNA detection (ISH) and negative hormone detection (ICC) occurred in some cases (PRL 17.5%; HCG [LH] 15%). In contrast, negative mRNA detection (ISH) and positive hormone content (ICC) was also demonstrated (PRL 5%; HCG [LH] 37.5%). The remaining adenomas showed both mRNA and the respective hormone, as well as negative ISH and ICC.

DK. Detection of growth hormone, prolactin and human /J-chorionic gonadotropin mRNA in growth hormone-secreting pituitary adenomas and in prolactinsecreting pituitary adenomas by in situ hybridization using a non-isotopic detection method. Acta A non-isotopic in situ hybridization method with digoxigenin-labelled probes was used to examine growth hormone (GH), prolactin (PRL) and human \g=b\-chorionicgonadotropin (\g=b\-hCG(LH))gene expression in 63 pituitary tumours in acromegaly and 20 adenomas in hyperprolactinaemia. hCG and LH were detected simultaneously because of the extensive homology (more than 90%) of their mRNA sequences (1). A comparison with former results obtained with 35S-labelled probes shows the value of the easier and faster non-isotopic method. Additionally, immunohistochemical data are included to give even more evidence for the synthesis of the respective hormones by the tumour cells. In all 63 adenomas in acromegaly, GH mRNA was revealed in 59 PRL mRNA and in 36 \g=b\-hCG(LH)mRNA. A positive immunostaining for GH was found in all, for PRL in 40, and for \g=b\-hCG(LH) in 34 adenomas. The comparison of the two in situ hybridization methods revealed no differences concerning GH mRNA detection, but not all tumours positive after non-isotopic PRL and \g=b\-hCG(LH) mRNA detection showed signals with the radioactive method. Referring to the 20 PRL-secreting adenomas, PRL gene expression was demonstrable in all, GH mRNA in 12, and \g=b\-hCG(LH)mRNA in 2 cases. Comparing the positive results of immunohistochemistry with those of in situ hybridization, correspondence was found in 19 cases for PRL, in 5 cases for GH and in no case for \g=b\-hCG(LH). W Saeger, Department of Pathologg, Marienkrankenhaus Hamburg, Alfredstr. 9, W-2000 Hamburg 76, Germany In situ hybridization (ISH) can be used for the detection of hormone mRNA in a variety of tumour cells, as shown previously by zur Hausen et al. (2), Höfler et al. (3), Hamid et al. (4), Uhlig et al. (1) and Bäz et al. (5).Furthermore, ISH is a successful means of enriching the characterization of pituitary adenomas by immunohistochemistry (IH). The simultaneous detection of a hormone-coding mRNA by ISH and the respective protein by IH can be considered as definite evidence that the hormone has been synthesized by the cell itself and not taken up from the circulation and concentrated. For many years, ISH has been performed using radioactively labelled probes guaranteeing high specific activity of probes and low detection limits, but requiring long autoradiographic exposure times and adequate facilities to handle radioactive materials. To evade these disad¬ vantages, especially in clinical or diagnostic medicine, the use of non-isotopic alternatives has been improved. As shown by Brigati et al. (6), Jirikowski et al. (7) and Kessler (8), for example, probes can be non-isotopically labelled with biotin, 5-BrdUTP or digoxigenin and detected by an immunohistochemical method. The nonradioactive ISH procedure makes it further possible to show the localization of...

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