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S. D. Abbot, K. Docherty, and R. N. Clayton


To determine the physiological role of the ovaries in regulation of LH subunit gene expression, levels of cytoplasmic mRNA were measured in a cDNA-RNA dot-blot hybridization assay. An increase (twofold) in α mRNA was first detected 8 days after ovariectomy and then remained stable for 4 weeks. In contrast, LH-β mRNA increased by 60–79% within 12 h of removing the ovaries and then rose progressively to six times the intact values at 3 and 4 weeks. Increases in LH-β mRNA were always greater than those of α mRNA. Oestradiol, and oestradiol plus progesterone, but not progesterone alone, prevented the rise in α and LH-β mRNA 10 days after ovariectomy.

Three days after ovariectomy, α mRNA, but not LH-β mRNA, was suppressed to below intact control values by oestradiol and oestradiol plus progesterone, indicating greater sensitivity of α mRNA to oestradiol inhibition at this stage. A single injection of oestradiol (1 μg s.c.) to rats ovariectomized 14 days previously transiently suppressed α and LH-β mRNA levels and serum LH concentrations in parallel for 1–8 h, after which high preinjection values were restored. However, pituitary LH content remained suppressed after LH mRNA levels had returned to the control values of ovariectomized rats.

In most instances there was a qualitative positive correlation between changes in α and LH-β mRNA, pituitary LH content and serum LH concentrations. LH content reflected LH-β mRNA changes more closely than those of α mRNA. However, in oestradiol-treated rats ovariectomized 10 days previously, LH content remained increased despite normalization of the LH-β and α mRNA levels, suggesting differential sensitivity to oestradiol of the gene expression and translational processes. Thus divergence of pre- and post-translational regulation of LH biosynthesis was demonstrated. These results imply an important physiological role for female sex hormones in the control of LH gene expression and LH biosynthesis.

Prolactin mRNA fell by 30–50% for the first 2 weeks after ovariectomy, but by 3 and 4 weeks values were similar to those of intact controls. Serum and pituitary prolactin levels were reduced by 50% or more at all time-points, despite normalization of mRNA. Treatment of ovariectomized rats for 10 days with oestradiol and progesterone, either alone or combined, reversed the fall in prolactin mRNA and serum and pituitary prolactin levels. These changes in prolactin gene expression and synthesis were opposite to those of LH subunits in response to the same in-vivo hormone manipulations.

Growth hormone mRNA levels were unchanged by ovariectomy, oestradiol or progesterone treatment. Levels of TSH-β mRNA increased slightly (maximum up to 50%) after ovariectomy, but were unaltered by oestradiol and progesterone treatment for 10 days. These results support the view that α mRNA changes, resulting from ovariectomy, oestradiol and progesterone treatment, occur in gonadotrophs and not thyrotrophs, which also express the α subunit gene.

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S. D. Abbot, K. Docherty, and R. N. Clayton


The control of anterior pituitary hormone gene expression by testosterone in male rat pituitaries in vivo was investigated using dot-blot mRNA-cDNA hybridization assays.

Common α subunit mRNA levels doubled by 2 days after orchidectomy and rose progressively to reach plateau levels three to four times intact control values by 2 weeks. LH-β mRNA increased significantly (≃50%) within 12h, and thereafter progressively to seven times intact control values by 3 weeks after orchidectomy. The changes in α mRNA were likely to have occurred in gonadotrophs and not thyrotrophs, since TSH-β mRNA levels were unaltered by orchidectomy. LH subunit mRNA changes were accompanied by an initial (1–4 days) decrease in pituitary LH content; thereafter, pituitary LH increased in parallel with and by a similar magnitude to the LH-β mRNA. Serum LH rises occurred before significant increases in LH subunit mRNA after orchidectomy. The lack of temporal correlation between mRNA levels and serum and pituitary LH in the early stages after removal of testosterone feedback contrasts with the good correlation when a new steady state was achieved after 3–4 weeks, and indicates differing kinetics for changes in these aspects of gonadotroph function.

An inhibitory effect of testosterone on LH subunit gene expression was confirmed by prevention of the rise in α and LH-β mRNAs when treatment commenced immediately after castration. However, pituitary LH content and serum LH levels were reduced relative to control values, suggesting additional inhibitory actions of testosterone on translational and post-translational events in gonadotrophs. A stimulatory effect of testosterone on α mRNA levels was observed between 4 and 24 h after a single injection in rats castrated 2 weeks previously, no effect being seen on LH-β mRNA. The mechanism for this action remains to be elucidated. Gene specificity of testosterone action was confirmed by unaltered levels of mRNA for prolactin, GH, TSH-β subunit and actin under all experimental conditions. No changes in pituitary content of prolactin or GH were found. We conclude that regulation of LH subunit gene expression by testosterone is an important step in control of gonadotrophin synthesis and availability for release.

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G. Saade, D. R. London, M. R. A. Lalloz, and R. N. Clayton


The effect of castration and gonadal steroid replacement on the concentrations of LH-β and α subunit and prolactin mRNA was examined in mice.

Mouse LH-β, α and prolactin mRNAs were approximately 0·8, 0·7 and 1·1 kb in size respectively. After ovariectomy, LH-β mRNA levels increased 2- to 2·5-fold, while α mRNA levels increased 2·5-fold 6 and 10 days after ovariectomy. Serum LH rose after 2 days to reach six times control values at 10 days. Pituitary LH content doubled by 8 days after ovariectomy. Prolactin mRNA levels decreased to 50–60% of control at 3, 6, 8 and 10 days after ovariectomy and parallelled the fall in serum prolaction. Pituitary prolactin content fell more slowly, to 50% of intact control values by 10 days. The increase in both LH-β and α subunit mRNA, and decrease in prolactin mRNA, and serum and pituitary hormone changes, after ovariectomy were prevented by oestradiol or oestradiol plus progesterone replacement.

Levels of LH-β mRNA increased more quickly in male than in female mice, theearliest change being seen 24 h after orchidectomy. Maximum values (two- to threefold) were found on day 6 after orchidectomy. Concentrations of α mRNA increased by 12 h to between 2 and 2·5 times control from 3 to 10 days after orchidectomy. Serum LH doubled by 12 h and was three to five times greater than control values up to 10 days. Pituitary LH content fell by 48 h before gradually increasing to intact values after 10 days. Prolactin mRNA levels decreased progressively from 2 days after orchidectomy, and this decrease was preceded by a fall in serum and pituitary prolactin which remained low throughout the experiment. Testosterone treatment attenuated the rise in α mRNA, prevented the rise in LH-β mRNA and serum LH and partially restored the decrease in prolactin mRNA seen after orchidectomy.

We conclude that in mice, as in rats and ewes, both LH-β and α subunit mRNAs are negatively regulated by gonadal steroids, whereas prolactin mRNA is positively regulated, although there are temporal differences in patterns of mRNA responses between males and females. By comparison with female rats the rise in LH-β mRNA after ovariectomy was slower in mice. Moreover, the discordant changes in pituitary LH content and LH subunit mRNAs seen in mice after castration were not observed in rats. Furthermore, pituitary prolactin and prolactin mRNA do not fall after orchidectomy of rats. The modest (50%) increase of LH-β mRNA after castration of mice suggests that an increase in mRNA is not necessarily required for increased LH production.

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D. A. Rodin, S. D. Abbot, G. Saade, and R. N. Clayton


There are significant differences between rats and mice in the gonadal regulation of several aspects of gonadotroph function. To investigate whether these extend to the pretranslational regulation of FSH synthesis by gonadal steroids, we have measured FSH-β mRNA levels following gonadectomy and sex-steroid replacement and have related these to serum and pituitary FSH as a reflection of overall hormone synthesis.

In ovariectomized rats, FSH-β mRNA levels increased by 8 h, decreased, and then rose progressively over the next 28 days. A similar pattern of response was observed in orchidectomized rats. In mice, there were progressive increases in FSH-β mRNA levels in both males and females following gonadectomy, without evidence of the early peaks observed in rats. In both species, the change in FSH-β mRNA levels after gonadectomy was greater in females than in males. These changes in FSH-β mRNA following gonadectomy were paralleled by changes in the serum FSH concentration. In ovariectomized female rats and mice, pituitary FSH stores increased by 8 h and 3 days respectively, whereas in male rats, pituitary FSH content did not rise until 10 days after orchidectomy. The most striking species difference was the marked and prolonged reduction of pituitary FSH after orchidectomy of mice.

Treatment of rats and mice from the time of ovariectomy, with a dose of oestradiol that prevents increases in serum LH, only partially attenuated the rises in FSH-β mRNA and serum FSH and did not prevent the increase in pituitary FSH content. Treatment of intact or orchidectomized rats with testosterone suppressed FSH-β mRNA levels to 50% below intact control values without affecting pituitary FSH content. In mice, testosterone treatment for 10 days reduced the post-castration increase in FSH-β mRNA by only 26%, and prevented the fall in pituitary FSH content, although the increased serum concentration of FSH was unaffected.

In conclusion: (1) there is a good correlation between FSH-β mRNA levels and overall FSH biosynthesis in male and female rats and female mice, but this relationship is less obvious in male mice where pituitary FSH stores are not increased; (2) the inability of oestradiol to prevent completely the post-ovariectomy increase in FSH-β mRNA and FSH synthesis in female rats and mice indicates either that other gonadal products are necessary or that higher doses of oestradiol are required than for complete suppression of LH synthesis; (3) whilst the post-gonadectomy increases in FSH-β mRNA are larger in the female of both species, there are no major differences between rats and mice in the regulation of FSH-β gene expression by sex steroids.

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R. N. Clayton, L. Eccleston, F. Gossard, J.-C. Thalbard, and G. Morel


There is still debate as to whether natural sequence gonadotrophin-releasing hormone (GnRH) is produced in the mammalian gonads and concerning its potential role as a paracrine modulator of gonadal function. To address this question, we have used insitu hybridization histochemistry with an oligonucleotide probe complementary to the GnRH decapeptide coding sequence, to determine the cellular site(s) of expression of the GnRH gene in rodent ovaries. GnRH mRNA was detected in granulosa and thecal cells from ovarian follicles at all stages of development (primary→Graafian), with no significant change in grain density during follicular development. The granulosa cell compartment always contained more mRNA than the thecal cell compartment. Corpora lutea expressed the GnRH gene to the same extent as thecal cells. These results indicate that preproGnRH mRNA is detectable under physiological conditions in the mammalian ovary, though whether this produces authentic GnRH decapeptide or an alternative protein product is not known. The physiological significance of these findings remains to be determined.

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K J Dudley, K Revill, R N Clayton, and W E Farrell

Investigation of the epigenome of sporadic pituitary tumours is providing a more detailed understanding of aberrations that characterise this tumour type. Early studies, in this and other tumour types adopted candidate-gene approaches to characterise CpG island methylation as a mechanism responsible for or associated with gene silencing. However, more recently, investigators have adopted approaches that do not require a priori knowledge of the gene and transcript, as example differential display techniques, and also genome-wide, array-based approaches, to ‘uncover’ or ‘unmask’ silenced genes. Furthermore, through use of chromatin immunoprecipitation as a selective enrichment technique; we are now beginning to identify modifications that target the underlying histones themselves and that have roles in gene-silencing events. Collectively, these studies provided convincing evidence that change to the tumour epigenome are not simply epiphenomena but have functional consequences in the context of pituitary tumour evolution. Our ability to perform these types of studies has been and is increasingly reliant upon technological advances in the genomics and epigenomics arena. In this context, other more recent advances and developing technologies, and, in particular, next generation or flow cell re-sequencing techniques offer exciting opportunities for our future studies of this tumour type.

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P E Clayton, R N Day, C M Silva, P Hellmann, K H Day, and M O Thorner


GH induces hepatic IGF-I synthesis by increasing transcription of its gene. IGF-I is synthesized, however, in many other tissues where the effect of GH on its gene expression is less well characterized. IGF-I and GH are produced by human lymphocytes and may function as autocrine regulators of lymphoproliferation. We have therefore used the human IM9 lymphocyte cell line to (A) define the IGF-I gene transcripts expressed and (B) investigate the effect of GH on early (protein tyrosine phosphorylation) and late (changes in IGF-I mRNA levels) events in intracellular signal transduction. Multiple IGF-I mRNA species, ranging in size from 0·9 to 5·8 kb, were detected by Northern hybridization of poly(A)+ mRNA from IM9 cells. The human IGF-I gene contains at least six exons and alternative splicing produces a number of transcripts. Solution hybridization with exon-specific riboprobes and amplification by PCR using exon-specific primers revealed that multiple transcripts were expressed in IM9 cells, and that exon 2 was the dominant leader exon.

Treatment of IM9 cells with 200 ng recombinant human (rh)GH/ml led to the specific tyrosine phosphorylation of three intracellular proteins (93, 120 and 134 kDa), which are involved in the initial signalling of the GH transduction pathway. However a solution hybridization assay using the IGF-IA specific riboprobe on IM9 cell RNA from similar experiments revealed that GH treatment did not change IGF-I gene expression.

This study has demonstrated (A) that the IGF-I gene is expressed in human IM9 lymphocytes, (B) that in contrast to other human tissue, exon 2 is the major leader exon, and (C) that rhGH induces tyrosine phosphorylation of 93, 120 and 134 kDa proteins but does not alter IGF-I gene expression. The IM9 cell may form an important model to investigate a GH transduction pathway not coupled to the IGF-I gene.

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I. S. Scott, M. K. Bennett, A. E. Porter-Goff, C. J. Harrison, B. S. Cox, C. A. Grocock, P. J. O'Shaughnessy, R. N. Clayton, R. Craven, B. J. A. Furr, and H. M. Charlton


Hypogonadal (hpg) mutant mice, with a congenital deficiency of hypothalamic gonadotrophin-releasing hormone (GnRH), and testicular feminized (tfm) mice, which lack a functional androgen receptor, were used to study the effects of the potent GnRH agonist 'Zoladex' (ICI 118630; d-Ser (But)6, Azgly10-GnRH) on pituitary and gonadal function. Zoladex (0.5 mg) in a sustained-release lactide—glycolide copolymer depot was administered subcutaneously under anaesthesia and was left in place for 7 days, after which time the effects of the drug upon pituitary and serum gonadotrophin concentrations, glycoprotein hormone subunit mRNAs and testicular morphology were investigated.

At the pituitary level, Zoladex treatment resulted in a substantial reduction in LH content in normal males, and LH content was depressed in hpg mice even below the basal levels normally found in these mutants. Pituitary LH content in the Zoladex-treated animals was depressed in the tfm groups, but not to the same levels as those found in the normal and castrated normal mice. Zoladex treatment at the time of castration prevented the post-operative elevation in serum LH associated with castration alone. In the androgen-deficient tfm mouse, Zoladex did not depress the normally elevated serum LH levels. Serum LH in the hpg animals was, in all cases, below the limit of detection of the assay.

Pituitary FSH content was depressed into the hpg range in both the normal and castrated animals, but there was no further depression in the hpg mice. The pituitary content was reduced in the tfm mice, again the effects not being as dramatic as in the normal and castrated animals. Serum FSH content, as measured by radioimmunoassay, was depressed by 50% in normal mice; there was no reduction in the hpg mice, however.

With regard to pituitary gonadotrophic hormone gene expression, Zoladex administration to normal mice caused a dramatic reduction in LHβ mRNA content, to a level approximating that found in untreated hpg mice. The drug also depressed LHβ mRNA in the castrated group to the hpg range when given at the time of castration, whereas in untreated castrated mice there was a significant increase in LHβ mRNA. In the tfm mouse, which can be considered as a model for long-term failure of androgen feedback, Zoladex again induced a fall in LHβ mRNA, but not to the same extent as in the normal and normal castrated group. Zoladex had no effect on the already low levels of LHβ mRNA found in hpg mice.

Pituitary FSHβ mRNA levels were not significantly altered by Zoladex in any of the treatment groups, whereas the drug induced a substantial rise in the common α-subunit mRNA in normal and hpg mice, to a level equalling that found in castrated tfm mice. In the latter two groups, Zoladex treatment did not result in a further increase in α-subunit mRNA above that found after castration alone, or in the untreated tfm mutant.

Treatment for 7 days with Zoladex resulted in a significant increase in testis weight, with spermatogenesis advancing beyond the first meiotic division with many round spermatids found within the seminiferous tubules. However, the interstitial cells remained atrophic and there was evidence of seminal vesicle growth. Nevertheless, there was a small but significant increase in testicular androgen content. Administration of the agonist to hypophysectomized hpg mice did not stimulate testicular or seminal vesicle growth, suggesting that the drug does not stimulate steroidogenesis via a direct action upon the testis.

Overall, the pharmacological effects of the drug appear to have turned off the transcription of the LHβ gene, with a consequent reduction in LH synthesis and probably also secretion in the longer term. With FSHβ, gene transcription was apparently unchanged and, with a substantial increase in the common α-subunit message, it would appear that the pituitary gland of Zoladex-treated animals may be predominantly biased towards FSH secretion. Although the circulating FSH levels as measured by radioimmunoassay were unaltered by Zoladex, there are several reports that GnRH agonists increase serum levels of bioactive hormones, perhaps by altering glycosylation of the FSH dimer glycoprotein.