Search Results

ABSTRACT

IGF-I is an important local regulator of ovarian function, stimulating follicular growth and steroidogenesis in human granulosa cells. However, it is not known whether ovarian IGF-I is derived from the circulating serum pool or from local production. IGF-I peptide has only been detected in human thecal cells and not in granulosa cells. This study has used the sensitive technique of reverse transcription of mRNA followed by PCR amplification (RT/PCR) to examine IGF-I gene expression in human preovulatory granulosa cells.

Granulosa-lutein cell (GLC) samples were obtained by follicular puncture of seven women enrolled in an ovulation induction programme. Treatment had included buserelin acetate, human menopausal gonadotrophin to stimulate follicular growth and human chorionic gonadotrophin to induce ovulation. Total RNA (TRNA), extracted from the GLCs, was amplified by RT/PCR, using combinations of leader and 3′ IGF-I exon-specific primers, to yield four IGF-I gene products: IGF-IA (exons 1, 3, 4, 6), IGF-IB (exons 1, 3, 4, 5), IGF-IA′ (exons 2, 3, 4, 6) and IGF-IB′ (exons 2, 3, 4, 5). As controls from other tissues, an identical procedure was undertaken on TRNA from peripheral blood monocytes and liver. All four mRNAs were expressed in GLCs, monocytes and liver. However the pattern of IGF-I mRNA expression differed between the tissues; in liver and GLCs, the IGF-IA transcript was dominant, but in monocytes the IGF-IA′ species was the most prominent. Quantitative RT/PCR using standardization to the house-keeping gene for glyceraldehyde-3′-phosphate dehydrogenase revealed that IGF-IA mRNA was 300-fold more abundant in liver than GLCs.

This study has indicated that: i) RT/PCR can be used to detect multiple IGF-I mRNA species in small amounts of TRNA from human tissue, ii) the IGF-I gene is expressed at low level in human preovulatory GLCs and iii) there is preferential use of leader exon 1 in human GLCs and leader exon 2 in monocytes. Further studies of IGF-I gene expression by this method in other ovarian cell types and granulosa cells from the developing follicle may help to clarify the local role of IGF-I in the human ovary.

ABSTRACT

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.

ABSTRACT

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 (Bu^t)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.