Abstract

Insulin-like growth factor-binding protein (IGFBP) extraction protocols were tested for their efficacy in removing IGFBPs from bovine plasma and bovine granulosa cell culture medium compared with standard acid exclusion chromatography. Traditional extraction methods, acidification, Sep–Pak, ethanol:acetone:acetic acid (EAA) and EAA-cryoprecipitation (EAA-C), failed to remove all the IGFBPs from both granulosa cell culture medium and plasma. However, EAA and EAA-C treatment of plasma samples did give values similar to those obtained by acid exclusion HPLC, when corrected for extraction efficiency. There was an inverse relationship between insulin-like growth factor-I (IGF-I) concentration in plasma samples, as measured using HPLC chromatography, and IGF-I concentration after EAA extraction. Furthermore, the interference caused by residual IGFBPs differed between samples taken from animals given various treatments that altered peripheral IGF-I concentrations.

As for plasma samples, EAA was the most effective extraction method for culture media, but residual IGFBPs caused an overestimation of IGF-I concentrations. In culture media, but not plasma, it was possible to block the interference of IGFBPs in the IGF-I assay, in both extracted and non-extracted culture samples, by the addition of excess IGF-II. Using this assay procedure, no IGF-I production by bovine granulosa cells was detected. This was confirmed by HPLC acid chromatography.

It is concluded that HPLC extraction is needed for the accurate measurement of peripheral IGF-I concentrations. For granulosa cell culture media it is possible to measure IGF-I concentrations in non-extracted samples if the IGFBPs are blocked by adding IGF-II. Using either this assay, or after HPLC acid chromatography, no IGF-I was detected in culture media, suggesting that IGF-I is not produced by non-luteinised bovine granulosa cells.

Journal of Endocrinology (1997) 153, 231–240

Among all the hormone-secreting pituitary tumours, prolactinomas are the most frequently found in the clinic. Since dopamine is the primary inhibitor of lactotroph function, dopamine agonists represent the first-line therapy. However, a subset of patients exhibits resistance to these drugs, and therefore, alternative treatments are desired. As activins inhibit prolactin gene expression through the inhibition of Pit-1 involving the p38MAPK pathway, in the present work, we studied the local activin system as an alternative inhibitory system for lactotroph hyperplasia treatment. We used two different mouse models of prolactinoma: transgenic mice with overexpression of the human chorionic gonadotropin β-subunit (hCGβ) and mice lacking dopamine receptor type 2. In both models, females, but not males, develop lactotroph hyperplasia from the fourth month of life. We found reduced expression of pituitary activin subunits and activin receptors in hyperplastic pituitaries from both models compared with wild-type counterparts. Consequently, hyperplastic pituitaries presented a reduced activin-inhibitory action on prolactin secretion. Additionally, while female wild-type lactotrophs presented high levels of phospho-p38MAPK, it was lost in prolactinomas, concomitant with decreased activin expression, increased Pit-1 expression and tumour development. In contrast, male pituitaries express higher mRNA levels of activin subunits βA and βB, which would suggest a stronger activin inhibitory function on lactotrophs, protecting this sex from tumour development, despite genotype. The present results highlight the importance of the activin inhibitory action on lactotroph function and place the local activin system as a new target for the treatment of dopamine agonist-resistant prolactinomas.