Progress in characterization of pre-implantation factor in embryo cultures and in vivo.

PROBLEM: Pre-implantation factor (PIF), a small, embryo-derived peptide is detected in the maternal serum prior to implantation and is associated with successful pregnancy outcome. However, its identity is not known. METHOD OF STUDY: PIF was isolated from mouse embryo conditioned media and from pregnant porcine sera, using high-performance liquid chromatography (HPLC) followed by mass spectrometry. RESULTS: Conditioned culture media was separated by gel filtration chromatography followed by reversed phase chromatography. At each step, PIF activity was determined by the lymphocyte/platelet binding autorosette assay (LPBA). Mass spectrometry yielded a single peak with a mass of 1300 Da. The peptide is, however, present in very low concentrations (fM), which has so far precluded complete identification. Pregnant porcine sera that exhibit potent PIF activity were deproteinated by acetone and further fractionated by reversed phase HPLC. Active fractions contain peptides of molecular masses 523 and 551 Da. CONCLUSION: PIF, likely to be peptides, represents a novel substance related to pregnancy initiation and maintenance.

Mitochondrial binding of triiodothyronine (T3). Demonstration by electron-microscopic radioautography of dispersed liver cells.

To assess the distribution of the thyroid hormone triiodothyronine (T3) within intact living cells, freshly prepared dispersed rat hepatocytes were incubated with [125I]-T3 for periods of 5 min and 30 min. Light- and electron-microscopic (EM) radioautography was carried out to determine the distribution of grains over the isolated cells. Both procedures showed the grains distributed almost entirely over the cytoplasmic matrix rather than the nucleus. Grain counts under the EM were compared with expectation based on established quantitative methods. Only the mitochondria showed obvious and statistically significant grain counts, whereas the nucleus failed to accumulate grains in excess of expectations by chance alone based on area. The findings support the existence of mitochondrial binding of T3, presumably a prerequisite for its action in direct stimulation of the mitochondria.

Thyroid function in gestational trophoblastic neoplasia: evidence that the thyrotropic activity of chorionic gonadotropin mediates the thyrotoxicosis of choriocarcinoma.

An investigation was made of thyroid function in 20 patients with gestational trophoblastic neoplasia. Two patients were judged to be overtly thyrotoxic on the basis of the symptoms and physical findings; both patients had widely metastatic choriocarcinoma, markedly increased serum T4 levels (21.4 and 27.7 micrograms/100 ml), and extremely high levels of serum human chorionic gonadotropin (hCG) (3,220 and 6,720 IU/ml) relative to those of normal gestation(< 100 IU/ml). Three other patients had moderately increased serum T4 levels (13 to 17.1 micrograms/100 ml), moderately increased serum hCG levels (110 to 310 IU/ml), and findings on clinical examination which suggested euthyroidism. Using the mouse thyroid bioassay, we found that the biologic characteristics of the thyroid-stimulating factor were those of purified hCG, and that the levels of thyroid-stimulating activity in both serum and urine correlated closely with the levels of hCG. These results provide evidence that the thyroid-stimulating activity intrinsic to the hCG molecule plays the central pathophysiologic role in choriocarcinoma-associated thyrotoxicosis.

Thyroid-stimulating activity of chorionic gonadotropin and luteinizing hormone.

While recent evidence indicates that the hCG molecule has intrinsic thyroid-stimulating activity (TSA), it is not clear whether a thyrotropic molecule other than hCG accounts for some of the TSA apparent in the crude or highly purified hCG. To determine if a thyrotropic factor is excluded from crude urinary hCG during purification of hCG, the ratio of interstitial cell-stimulating activity (ICSA) to TSA was determined in the starting material used for hCG preparation as well as in the highly purified hCG preparation. The ratio of the two biological activities did not change significantly during purification, suggesting that no factor present in crude hCG other than hCG itself accounts for the TSA. The highly purified hCG preparation was gel-filtered on Sephadex G-100 and the main protein peak was divided into three fractions. The ratio of TSA to ICSA was the same in each fraction, further indicating that these activities are intrinsic to the same molecule. If hCG has intrinsic thyrotropic activity, as these data indicate, then thyrotropic activity would be expected to be a secondary biological activity of LH, since there are strong structural and functional similarities between LH and hCG. In order to assess the LH molecule for intrinsic TSA, an LH preparation with minimal TSH contamination was prepared by recombining subunits exhibiting minimal TSH immunoreactivity. The LH molecule formed from the recombination of highly purified hCG alpha and ovine LH beta subunits exhibited TSA in the bioassay that was 25 times greater than that expected based on the immunoreactive TSH contamination. There was no evidence to support the existance of a thyrotropic factor other than hCG in either crude or highly purified hCG preparations. Our finding that a hybrid LH molecule structurally similar to hCG with potent ICSA also exhibits intrinsic TSA further extends and supports the hypothesis that TSA is an intrinsic property of the hCG molecule.

The TRH stimulation test in endemic goitre in Greece.

The serum T4=T4(D), T3 in vitro uptake=RT3U and TSH were measured before and 30 min after the injection of 200 mug of TRH in 30 subjects from an iodine deficient area of Central Greece. These persons were divided in two groups of 16 goitrous and 14 nongoitrous patients and the results from each group compared to each other and also with the same parameters obtained from 14 controls subjects, all nongoitrous and from a nonendemic area. It has been found that in this area with a mild iodine deficiency serum T4 is the only hormone decreased whereas RT3U and TSH both before and 30 min after TRH injection are normal. There existed however a negative correlation between serum T4 and TSH in the goitrous group. Why the nongoitrous iodine deficient persons achieve the same compensation without thyroid gland enlargement remains unsolved but it may be stated from the present results that goitre development should be regarded as a maladaptation rather than a compensatory process.