If the knowledge accumulated in the past 4 years on the reproductive role of interferon-α (IFN-α) had been available 20 years ago, then there is no doubt that this molecule would have been designated as a reproductive hormone. Eventually some workers would have expressed surprise that the molecule also exhibited properties of a lymphokine, though it might well have been argued that the findings were the result of contamination in 'pure' preparations of the hormone. But the surprise has been in the opposite direction: a material which is considered as an archetypal lymphokine has been very belatedly identified as a major embryonic signal in several mammalian species. The extent of this 'surprise' may be judged from a recent review in this Journal which described (with admirable clarity and detail) the possible role of lymphokines in endocrinology but made no mention of the fact that endocrinology has just hijacked the most
The first radioimmunoassay was described by Yalow & Berson in 1960, since when the technique has been widely used in physiological and patho-physiological studies of the protein hormones. The success in this field has led to a number of attempts to develop similar assays for the small peptide hormones (molecular weight less than 3000). However, these compounds present certain difficulties which have considerably retarded progress. The purpose of the present review is to discuss the problems of the radioimmunoassay of the neurohypophysial peptides, with particular reference to oxytocin and vasopressin.
The problems of the radioimmunoassay of small peptide hormones
There are two major problems. (1) Being of low molecular weight (about 1000) they are poor immunogens. As a result, the preparation of high-affinity antisera is considerably more exacting than in the case of larger molecules. (2) Their levels in the circulation are, in molar terms, considerably lower than those of
The size of the human fetus is primarily under genetic control. However, although there is extensive knowledge of other factors which may affect the weight of the fetus (nutrition, socio-economic class etc.) there is little information on the mechanism by which these factors affect the growth process. The matter is of considerable practical significance because much of the pathology of pregnancy is related to failure of fetal growth, or interruption of the growth process before it is complete.
The size of the human fetus, at any stage of pregnancy up to the time of delivery, is primarily determined by the size of the parents and especially the mother. Nutritional factors are also of obvious importance, especially in the later stages of pregnancy (Milner & Hill, 1984). In a typical population in a developed country, however, nutrition probably does not play a major role in determining individual variability
The human placenta secretes large quantities of specific proteins, protein hormones and steroids (Chard & Grudzinskas 1992a). Yet most of these products have no obvious function, either in the mother or the child, and it has even been suggested that placental hormones are the waste products of some underlying but ill-understood phenomenon (Gordon & Chard, 1979). Here a new hypothesis is put forward which might provide a solution to this biological enigma. It is proposed that the function of many of the specific placental products is not, as in classical endocrinology, to act as messengers and exert their efforts at distant sites. Instead, they serve to provide the placental syncytiotrophoblast with information about the maternal environment. In turn, this enables the trophoblast to function as a single, coherent tissue, both for secretion of specific products and, of far greater importance, as an organ for exchange of nutrients and waste
H. S. Wang and T. Chard
The size of the human infant at birth depends on the duration of gestation and the rate of fetal growth. Growth, and thus delivered weight, are determined by several factors: genetic constitution, nutritional status and pathological conditions (e.g. maternal diabetes, pre-eclampsia or eclampsia, smoking, infections etc.). However, despite extensive investigation, it has been difficult to identify any specific endocrine mechanism which plays an essential role in fetal growth (Chard, 1989). Recent attention has therefore focused on the autocrine and paracrine actions of growth factors, especially the insulin-like growth factors (IGFs) and their binding proteins (IGFBPs). Here, we review evidence for the role of the IGFs and IGFBPs in the control of fetal growth, and present a hypothesis that secretion of these compounds by the maternal decidua may play an important part in the control of the growth process.
Structure and function of IGFs and IGFBPs
H. S. Wang and T. Chard
Insulin-like growth factor-binding proteins (IGFBPs) in maternal and umbilical cord sera have been analysed by combinations of gel filtration chromatography, affinity cross-linking and electrophoresis. On gel filtration chromatography, the majority of circulating IGF-I in non-pregnant and pregnant women was present in the large molecular mass (150 kDa) binding proteins (IGFBP-3). In umbilical cord serum, by contrast, most IGF-I was present in the 40 kDa binding proteins (consisting of IGFBP-1 and IGFBP-2). Western blots demonstrated an apparent progressive attenuation of IGFBP-3 and IGFBP-2 in serum from pregnant women with an increase in IGFBP-1. After prior cross-linking with disuccinimidyl suberate, the 150 kDa fractions (IGFBP-3) from non-pregnant and pregnant serum showed a similar pattern on SDS-PAGE (several bands at different molecular masses). However, IGFBP-2 (one of the components of the 40 kDa fractions) was undetectable, even after cross-linking, in serum from pregnant women later than 8 weeks of gestational age and in a mixture of maternal serum at term delivery and serum from non-pregnant women. This suggests that serum IGFBP-2 was degraded by specific proteases present in pregnancy serum. Following acid treatment, the 150 kDa fractions from pregnancy serum were split into smaller subunits or fragments while the 40 kDa fractions remained unchanged, suggesting that the 40 kDa binding proteins are acid-stable. The present data demonstrate that IGFBP-3 is the principal IGFBP in pregnancy serum even though there is an apparent reduction in serum IGFBP-3 activity as revealed on Western blots. The absence of IGFBP-2 in serum from pregnant women may be due to degradation by proteases. In the fetal circulation IGFBP-1 and IGFBP-2 appear to be the major binding proteins for IGF-1.
Journal of Endocrinology (1992) 133, 149–159
R. K. Iles and T. Chard
Treatment of three β-human chorionic gonadotrophin (β-hCG)-expressing bladder tumour cell lines with interferon-α (IFN-α) (5000 U/per 106 cells) enhanced the rate of β-hCG secretion from 34·2 ±0·9 to 102·5 ± 0·1 mIU/106 cells per 72 h in cell line 5637; 111·15 ± 11·75 to 261·8± 51·75 mIU/106 cells per 72 h in cell line RT112 and 503·25 ± 28·55 to 1361·65± 110·3 mIU/106 cells per 72 h in cell line SCaBER. IFN-γ had no effect on the rate of β-hCG secretion. Both interferons reduced the growth rate of the cells: incorporation of radiolabelled thymidine was reduced by 15–45% in the presence of IFN-α and by 20–53% with IFN-γ. Enhancement of β-hCG secretion by IFN-α was dose-dependent over the range 5–50 000 U/106 cells. Analysis of cell cycle profiles by flow cytometry showed no increase in the proportion of cells in the G0G1 phase in cultures treated with IFN-α.
The conceptus of some species produces substances which are either luteotrophic or anti-luteolytic. In sheep, the corpus luteum is maintained by ovine trophoblast protein-I, which has been shown to have structural homology with human IFN-α. In primates and a few other higher mammals, early pregnancy is maintained by chorionic gonadotrophin. IFN-α is also an early product of the human conceptus. We have now shown that IFN-α enhances the ectopic production of the β-subunit of hCG by bladder tumour cells. This study suggests a direct transcription/translational effect of this cytokine on the expression of a reproductive endocrine gene.
Journal of Endocrinology (1989) 123, 501–507
T. CHARD, M. J. KITAU, and J. LANDON
A simple and rapid method is described for labelling oxytocin with 131I at a high specific activity. This method is compared with those of previous workers. A satisfactory antiserum has been raised by direct intra-lymph node injection of oxytocin adsorbed to carbon microparticles. A number of methods for separating antibody-bound from free oxytocin are described, and reasons given for preferring a procedure using ammonium sulphate precipitation. These data form the basis for developing a radioimmunoassay intended for the determination of oxytocin in human plasma.
MARION J. MARTIN, T. CHARD, and J. LANDON
A radioimmunoassay for bovine neurophysin is described. The assay has a lower limit of detection of approximately 0·5 ng/ml. Bovine neurophysin extracted and purified from acetone-dried posterior pituitary lobe powder was used for standardization, radio-iodination and immunization of rabbits. Some anti-neurophysin sera were also obtained from human subjects treated with Pitressin. Most of the antisera tested did not readily distinguish between the different neurophysin components isolated from bovine neurohypophysial tissue. The antisera cross-reacted with material extracted from whole pituitaries of a number of mammalian species, including man, pig, sheep, guinea-pig and rat, but not with the pituitary peptides from the cod or dogfish, or the egret. Other pituitary peptides, including oxytocin, vasopressin, corticotrophin, growth hormone and luteinizing hormone, did not interfere with the assay.