SUMMARY

Thyroidal uptake and release of 131I were used to study the response of pituitary autografts to locally infused synthetic thyrotrophin releasing factor (TRF) and porcine median eminence extract (MEE). Thyroidal release rates of 14% ¹³¹I/24h were maintained during continuous infusion of grafts with MEE (equivalent to 4 hypothalami/day) while 12·6% ¹³¹I/24 h was lost from thyroids of rats whose pituitary autografts were infused with 1000 ng synthetic TRF/day. Infusion of autografts with 5, 50 and 250 ng synthetic TRF/day resulted in lower, but dose-dependent, thyroidal responses suggesting that synthetic TRF could induce thyrotrophin (TSH) synthesis in the grafts.

At all doses of TRF, thyroidal ¹³¹I release was significantly increased by daily injection of cortisone (2 mg s.c.).

Local TRF stimulation of TSH release from pituitary grafts significantly increased the dose of thyroxine (T₄) needed to decrease thyroid activity, and while 1·0 μg T₄/100 g depressed activity in rats receiving saline infusions, 1·7–2·7 μg T₄/100 g was needed to inhibit ¹³¹I release in rats receiving 250 ng TRF/day, and 3 μg T₄/100 g was needed by rats receiving 1000 ng TRF/day.

Continuous TSH infusion (25–28 mu./day) into hypophysectomized rats induced thyroidal ¹³¹I release at rates similar to those in rats with TRF-stimulated pituitary autografts.

It is suggested that while synthetic TRF can enhance TSH synthesis in the pituitary its effects on TSH synthesis may normally be potentiated by other humoral substances.

SUMMARY

Thyroidal uptake and release of ¹³¹I were used to study the effects of prolonged infusion of porcine hypothalamic extract on the ability of rat pituitary autografts to secrete thyrotrophin (TSH). The extract used contained insufficient TSH to induce release of thyroidal ¹³¹I in hypophysectomized rats.

After infusion for 10 days, the time of maximal uptake of ¹³¹I and onset of ¹³¹I release was significantly shortened by infusion of hypothalamic, but not cerebral cortical extract, when compared with non-infused autografted controls. The rate of release of thyroidal ¹³¹I was significantly increased by the infusion of hypothalamic extract so that by 96–120 h after the administration of ¹³¹I the rate of release was not significantly different from that in intact controls.

Accelerated thyroidal release of ¹³¹I began 42–48 h after the application of hypothalamic extracts to pituitary autografts and fell rapidly after withdrawal of the extract. At the end of 14–17 days of infusion sections of the autografts contained aldehyde-fuchsin positive staining basophils.

Abstract

Monoclonal antibodies, specific for the βa and βb subunits of activin, were used to develop a new two-site ELISA for activin-AB. The assay had a detection limit of 0·19 ng/ml. High concentrations of activin-AB were found in bovine, ovine and porcine follicular fluids (FF), with less in human FF (1310, 1730, 688 and 7 ng/ml respectively). Recovery of spiked activin-AB standard from human, bovine and ovine FFs and from homogenized human placental extracts averaged 91%, 115%, 115% and 94% respectively. Within-plate coefficients of variation for different concentrations of activin-AB were between 1·3% and 2·67%. The between-plate coefficient of variation was 5·5%. Crossreactivity experiments showed the high specificity of the assay for activin-AB, with inhibin-A, inhibin-B, follistatin, activin-A and activin-B all cross-reacting <0·2%. Incubation with high concentrations of follistatin (500 ng/ml) prior to assay did not affect the recovery of activin-AB. Samples of bovine, porcine, ovine and human FF gave dose responses parallel to that of the standard, as did bovine granulosa cell-conditioned media. In human and porcine FF, levels of activin-A and activin-AB were similar whereas, in bovine and ovine FF, activin-A levels were approximately threefold higher than activin-AB levels. As we have reported previously for activin-A, nearly all of the endogenous activin-AB in bovine FF was detected in the eluate from gel permeation chromatography with an M _r of >700 000 indicating its association with higher molecular weight binding protein(s). By contrast, after denaturation, immunoreactive activin-AB was detected with an M _r of ∼25 000 consistent with the complete dissociation from binding proteins.

Activin-A was detected in relatively high concentrations in human FF (∼5 ng/ml), homogenized placental extracts (4·35–95·5 ng/g), sera from pregnant women (>4 ng/ml) and amniotic fluid (3–13 ng/ml), and in much lower concentrations in postmenopausal serum (500 pg/ml), normal cycle serum (100–200 pg/ml), serum from gonadotrophin-treated women (200 pg/ml) and normal adult male serum (225 pg/ml). Activin-A was also found in the culture media from explants of human amnion, chorion, maternal decidua and placenta. In marked contrast, activin-AB was undetectable (<0·19 ng/ml) in all of these samples with the exception of human FF (∼7 ng/ml).

In conclusion, we have developed a sensitive and specific ELISA to measure total (bound+free) activin-AB. Preliminary results show a more restricted distribution of this isoform compared with activin-A. The presence of high levels of both activin-A and activin-AB in FF suggests a function for both isoforms in the developing ovarian follicle.

Journal of Endocrinology (1997) 153, 221–230