Detection of growth hormone, prolactin and human β-chorionic gonadotropin messenger RNA in growth-hormone-secreting pituitary adenomas by in situ hybridization

Abstract

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.

Detection of growth hormone, prolactin and human β-chorionic gonadotropin mRNA in growth hormone-secreting pituitary adenomas and in prolactin-secreting pituitary adenomas by in situ hybridization using a non-isotopic detection method

“…An explanation for cases of negative ISH and positive IH, which we-as well as Uhlig (1) and Bäz (5)-observed especially during studying /MiCG(LH) gene expression and hormone content, may be that the mRNA synthesis is suppressed by intracellular hormone storage or has been destroyed by endogenous ribonucleases during surgery.…”

“…As probes for in situ hybridization, fragments of cDNAs encoding GH, PRL and ¿S-hCG(LH) subcloned in pGEM (Promega) vectors by Uhlig et al (1) were used. Digoxigenin-labelled, single-stranded antisense RNA probes were obtained by in vitro transcription using Sp6 polymerase and digoxigenin-11-UTP (Boehringer, Mannheim).…”

“…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 (1) and Bäz et al. (5).…”

“…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.…”

Serum levels of β-subunit of chorionic gonadotropin in patients with pituitary tumors

“…may be present in secreting and non-secreting pituitary tumors (11)(12)(13)(14). In particular, we have demonstrated recently by immunohistochemical analysis that the majority of non-functioning pituitary adenomas (NFPA) possess a significant number of cells able to synthesize the CG molecule (14).…”