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The purpose of this study was to evaluate the effect of chronic immobilization on the hypophysial-adrenal and hypophysial-gonadal axes of adult male rhesus monkeys and the effect such manipulation has on the ability of these axes to respond to exogenous corticotropin, gonadotropin, and GnRH administration. A comparison was also made of the effects of immobilization on testosterone secretion at periods of low (April) and high (November) gonadal activity in this animal. Adult male rhesus monkeys were immobilized in a horizontal position for periods of up to 20 days during March/April. The function of the hypophysial-adrenal and hypophysial-gonadal axes was studied by monitoring plasma levels of cortisol, 17-hydroxylated precursors, 11 deoxycortisol, and testosterone during the period of restraint. Groups of immobilized and control animals also received iv injections of ACTH, FSH, and LH or LHRH on day 18 of the experiment. An additional group of animals was immobilized for 20 days, but did not receive exogenous hormone treatment. This group was used for comparison of seasonal differences in testosterone secretion with another group of animals subjected to the same treatment in November. During the first 3 h of immobilization, levels of cortisol, 17-hydroxylated precursors, and 11-deoxycortisol increased markedly from initial levels. Cortisol levels remained elevated for 3 days, whereas levels of the other three adrenal hormones declined to near-initial levels within 24 h. Testosterone levels declined steadily during the first 6 h of immobilization in males studied at a time of high testicular activity (November), while an increase during the first hour of restraint followed by a decline during the next 3 days were observed in males studied during a period of low testicular activity (April). Animals injected with ACTH on day 18 of immobilization had cortisol levels similar to those of control animals, but other groups of animals restrained for a similar period exhibited a lower level of plasma testosterone than controls after the injection of FSH and LH or LHRH. These data suggest that adaptation to stress results in a reduced demand for corticosteroid production and that the adrenals of chronically stressed animals are capable of responding to exogenous corticotropin, or alternatively, the immobilization imposed was stressful for only a limited time, and after a few days, animals no longer reacted as in response to stress. Also, secretion of testosterone in male monkeys is markedly influenced by the functional state of the gonads at the time of stress initiation.

Noncross-reactive monoclonal antibodies specific for human chorionic gonadotropin (hCG) were obtained after pre-selection for submolecular specificity with a synthetic peptide immunogen. Mice were immunized with a synthetic peptide representing a segment unique to the beta-subunit of hCG (amino acid residues 109-145), conjugated to diphtheria toxoid. We then derived nine different hybridomas that secreted monoclonal antibodies reactive with both native hCG and isolated C-terminal peptide, after somatic cell hybridization of immune spleen cells with a nonsecretory myeloma cell line. None of the nine monoclonal antibodies, termed beta-hCG-CTPa1----a9, reacted with hLH, hFSH, or hTSH, although these pituitary hormones display extensive amino acid sequence homology with hCG. The noncross-reactive anti-beta-hCG monoclonal antibodies show apparent association constants on the order of 10(9) to 10(10) M-1. A sandwich-type enzyme-linked immunosorbent assay was set up with cut-off values of around 5 mIU/ml. These antibodies might have important implications for: a) improving the diagnosis and clinical management of pregnancy; b) monitoring the course of development of carcinomas which secrete the hormone, through in vitro assays or in vivo radioimmunodetection; c) evaluating the antibodies' therapeutic potential against such carcinomas; d) studying the biologic functions of the C-terminal segment of beta-hCG; and e) addressing the anti-fertility effect of antibodies raised against that segment.

As a part of a programme to develop a fertility regulating vaccine, antibodies specific for human chorionic gonadotrophin were raised by immunizing rabbits with a synthetic 37 amino acid C-terminal peptide of beta hCG conjugated to tetanus toxoid as carrier, and using Bordetella pertussis or Freund's complete adjuvant as adjuvants. Lack of cross-reactivity of the antibodies with human luteinizing hormone was determined by direct binding in a radioimmunoassay and by immunofluorescence on adult human pituitary sections. Not only did the antibodies bind to native hormone in a radioimmunoassay but they also neutralized the biological activity of hCG as measured by an in vivo bioassay. Rabbits which had been injected with the conjugate with B. pertussis as adjuvant made antibodies of comparable affinity to those animals which had been immunized with the antigen in Freund's complete adjuvant, though the latter group did produce more antibodies.

Human placental villous trophoblast is known to be unreactive with W6/32 and other monoclonal antibodies recognizing monomorphic determinants of human class I MHC heavy chains, whereas extravillous cytotrophoblast in the placental bed is W6/32-reactive by immunohistology. We have now demonstrated, in contrast, that syncytiotrophoblast is the only cellular component of baboon early placental villous tissue which is reactive with any of these antibodies. Radioimmunoprecipitation of detergent-solubilized baboon placental membrane preparations, and subsequent SDS-PAGE, has shown the W6/32-reactive component to have an m.w. of 41,000 and to be associated with beta 2-microglobulin, whereas baboon peripheral lymphocytes express 45,000 m.w. W6/32-reactive antigens comparable with the HLA-A,B,C heavy chains of human lymphocytes.

Implantation of pelleted agonistic analogs of luteinizing hormone-releasing hormone (LH-RH) into six pregnant baboons, ranging between 14 and 21 days after mating in individual animals, resulted in two abortions: one at 39 and the other at 67 days after mating. Plasma progesterone levels were low in treated animals through the 23rd day after mating, in spite of rising levels of plasma chorionic gonadotropin. Plasma levels of progesterone then rose rapidly to normal in five of six animals. Plasma estradiol levels were low from treatment on in the late-aborting animal but were normal until 5 days before delivery in the early abortant. The profile of circulating chorionic gonadotropin was truncated in treated animals, which may be the first indication of a paradoxical effect of LH-RH agonist on the trophoblast.

Sensitive immunoassays have been applied to WHO reference bank sera from fertile and infertile women in order to assess any naturally occurring antibody reactive with isolated human placental trophoblast membranes or two separate trophoblast protein products (hCG and SP1). A very low incidence of antibody reactive with solubilised trophoblast membrane was detected, and no significant antibody to either hCG or SP1 could be detected. Infertile states represented within this serum bank appear unlikely to involve adverse immune reactions to trophoblast.

Four baboons were primed with diazotized beta human chorionic gonadotrophin and boosted with diazotized C-terminal beta human chorionic gonadotrophin peptide, and the changes in antibody amount and affinity determined using a double isotope modified Farr assay, using labelled human chorionic gonadotrophin as the antigen. The degree of cross-reaction with human luteinizing hormone was also determined. Although appreciable reactivity with luteinizing hormone was observed soon after immunization, this declined rapidly during the response. At the time intervals studied, there was a progressive increase in affinity of antibodies to human chorionic gonadotrophin until day 248 after priming. At day 313, in two of the animals, there was a decrease in affinity from 1.04 X 10(11) to 6.80 X 10(10) and 1.05 X 10(11) to 4.93 X 10(10) l/mol, whereas in the other two baboons there was a further increase in antibody affinity. At corresponding time intervals, there was a steady decrease in values of total antibody binding sites. To determine the overall effect of the maturation of affinity with a decrease in antibody amount on biological efficacy, the theoretical amount of chorionic gonadotrophin that would be neutralized was calculated. We computed that in all instances, over 99% of a peak concentration of chorionic gonadotrophin that could be in circulation in a pregnant baboon would be neutralized. This was in excellent agreement with results of mating experiments in these baboons. In over forty cycles studied, none of the matings resulted in a sustained pregnancy.

Ultrastructural, autoradiographic, immunofluorescent and biochemical techniques were used to characterize primary cultures of term placental cytotrophoblast in order to gain insight into the differentiation and secretory capacities of the cellular component of human trophoblast. Trypsin treatment of placental villi allowed isolation of a predominantly cytotrophoblast cell population that maintained viability up to 13 weeks in monolayer culture. Autoradiographic studies of tritiated thymidine incorporation identified a smaller diameter mononucleated cell population that was mitotically active and developed into larger diameter mononucleated cells and into multinucleated cells during culture. Ultrastructurally, cultured cells formed desmosomes, had an extensive network of cytoplasmic microfilaments and contained the organelles for hormone synthesis and secretion. These cells secreted steroid hormones, secreted Schwangerschafts protein I, actively incorporated tritiated glycoprotein precursors and expressed surface immunoreactivity for the beta-subunit of human chorionic gonadotrophin (hCG). However, medium concentrations of hCG and human placental lactogen dropped rapidly to undetectable levels after 14 days in primary culture. Cells grown beyond confluence differentiated into 1 to 2 mm structures with a villus-like histology. Our studies indicate that cytotrophoblast can secrete steroids, cytotrophoblast differentiation occurs in vitro in the absence of maternal tissues, hCG synthesis occurs in cultured cytotrophoblast and medium concentrations of placental protein hormones are not the best indicators of cell viability for cultures of cytotrophoblast.