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ABSTRACT

Ethane 1,2-dimethane sulphonate (EDS) selectively destroys Leydig cells in the interstitium of the testis of adult rats. The toxic activity of this compound is much less obvious in the immature rat testis. We examined the effects of EDS, its monomethyl derivative and busulphan on cultured interstitial cells, percoll-purified Leydig cells, Sertoli cells and peritubular cells derived from immature rats. The studies with interstitial cells and Leydig cells showed that EDS (40–160 μg/ml) blocked the conversion of C₂₁ and androgen precursors into testosterone and androstenedione. Higher concentrations of this compound also inhibited the production of C₂₁ steroids and the LH-induced production of cyclic AMP (cAMP). The observed effects required a latent period of at least 8 h and were slowly reversible. Isolated cells were more sensitive to EDS than monolayer cultures. Reaggregation cultures were even less sensitive. EDS was markedly more effective on immature Leydig cells than its monomethyl derivative and busulphan. In cultured Sertoli cells FSH-inducible aromatase activity, cAMP production, androgen-binding protein (ABP) production and the secretion of a paracrine factor with Leydig cell-stimulatory activity were markedly reduced by busulphan. In these cells, busulphan was clearly more active than EDS and its monomethyl derivative. The production of paracrine factors which increase ABP production and decrease FSH-inducible aromatase activity in Sertoli cells was studied as a parameter of the effects of alkane sulphonates on peritubular cells. Only busulphan markedly decreased the production of these paracrine factors. It is concluded that EDS displays a selective toxicity to Leydig cells derived from immature animals and that, apart from its effects on germ cells, busulphan may also directly impair the function of Sertoli cells and peritubular cells.

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

IGF-I mRNA has been demonstrated in testicular tissue and, more recently, localized specifically to Leydig cells. This study investigated the expression of IGF-I and side-chain cleavage enzyme (SCC) mRNA in two preparations of rat interstitial testicular cells which were separated by buoyant density into Leydig cell-enriched and -depleted fractions. RNA was prepared from interstitial cells obtained from the testes of untreated adult and immature rats and adult rats treated with human chorionic gonadotrophin (hCG) or ethane dimethanesulphonate (EDS; to destroy Leydig cells). IGF-I mRNA was detected in all samples, with five major transcripts ranging from 7·5 to 0·6 kb. Leydig cells (3β-hydroxysteroid dehydrogenase-positive and sensitive to EDS) expressed abundant IGF-I and SCC mRNAs, and levels of both were increased following hCG treatment. However, in addition, IGF-I mRNA which was derived from non-Leydig interstitial cells was detected, in the complete absence of SCC message, either in the more buoyant interstitial cells or in both interstitial cell fractions following the destruction of Leydig cells by EDS treatment. IGF-I expression in the Leydig cell-depleted cell fraction was also increased by hCG treatment, and it is therefore suggested that at least part of this non-Leydig interstitial cell IGF-I mRNA originates in Leydig cell precursors. In conclusion, Leydig cells are not the sole origin of IGF-I mRNA in the testis, and the non-Leydig cell expression may be an important component of testicular IGF-I production.