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Ovulation in women may be predicted or detected by a simple, solid-phase, chemiluminescence immunoassay for the measurement of urinary LH. An IgG fraction of a donkey antiserum to rabbit immunoglobulins is passively adsorbed to the walls of polystyrene tubes. Human chorionic gonadotrophin-succinyl-butyl-ethyl-isoluminol and a primary antibody (rabbit anti-human LH) is incubated with samples of early morning urine. After the binding reaction, the solution is removed by aspiration. The antibody-bound fraction is washed twice with buffer. Sodium hydroxide is added and the mixture incubated. After cooling, luminescence is initiated by oxidation of the label with microperoxidase/hydrogen peroxide and the signal integrated.The method has been evaluated in terms of sensitivity, within- and between-batch precision, bias and parallelism. The time interval between the peak day of LH in EMU and maximum follicular diameter during 38 menstrual cycles was −24 h (11%), 0 h (50%), + 24 h (28%) and + 48 h (11%).

Breast milk macrophages cultured in vitro synthesized and secreted increasing amounts of protein, lysozyme, and prostaglandin E2(PGE2) into the extracellular medium. These cells were also shown to actively phagocytose labeled zymosan particles in culture. Morphologic characteristics, phagocytosis, and secretory responses of the macrophages were altered depending on the presence of various stimuli in the culture. Concanavalin A, endotoxin and zymosan particles, but not latex particles, all resulted in an increased PGE2secretion into the medium. Although total protein synthesis was not altered by any of these stimuli, Concanavalin A and endotoxin resulted in a decreased lysozyme concentration in the extracellular medium. Concanavalin A enhanced, whereas endotoxin and prior phagocytosis of latex particles inhibited phagocytosis of labeled zymosan particles. These findings indicate that phagocytosis and secretions of milk macrophages may be altered depending on the nature of the stimulating agent.

Previous studies indicated that the anti-idiotypic antibody (clone 1D5) caused an increase in uterine creatine kinase (CK) activity when administered in vivoto immature female rats, indicating that the antibody has oestrogenic-like activity. It was, therefore, of interest to investigate the structural requirements of clone 1D5to act as an oestrogen mimetic in an in vitromodel system. In the present study, the effect of clone 1D5and its proteolytic fragments, F(ab′)2, Fab′ and Fcon CK activity was examined in cultured skeletal cells having functional oestrogen receptor (ER). Incubation of female-derived calvaria cells or epiphyseal cartilage cells with clone 1D5(8·33 nm) or oestradiol (E2) (30 nm) for 24 h caused a significant increase in CK activity, indicating that clone 1D5acted as an agonist. On the other hand, incubation of male-derived calvaria cells devoid of a functional ER with clone 1D5or E2did not have any effect on CK activity. Incubation of female-derived calvaria cells with clone 1D5and E2did not result in any further increase in CK activity, whereas dihydrotestosterone (DHT) did not alter the response to clone 1D5. The CK response to clone 1D5, in female-derived calvaria cells was time- and dose-dependent and could be inhibited in a dose-dependent manner by the oestrogen antagonist tamoxifen. In contrast, the proteolytic fragments of clone 1D5, the F(ab′)2dimer (12 nm) and the Fab′ monomer (24 nm), and the Fcfragment (28 nm) did not have E2-like activity in these cells. However, while the Fab′ monomer or the Fcfragment, as well as clone 1D5, did not affect the response of the female-derived calvaria cells to E2, the F(ab′)2dimer acted like an antagonist and completely inhibited the stimulatory effect of E2or 1D5, but was unable to block the stimulatory effect of DHT on CK in male-derived calvaria cells. Collectively, these results imply that a bivalent antibody is necessary for the observed physiological responses, and that the anti-idiotypic antibody can be converted from an agonist to an antagonist by removal of the Fcportion of the antibody molecule. Furthermore, the anti-idiotypic antibody has an oestrogenic-like effect inhibited by tamoxifen only in skeletal cells capable of responding to E2.Journal of Endocrinology(1995) 145,409–416

Ovarian function in women may be monitored by a simple, solid-phase, chemiluminescence immunoassay for the measurement of oestrone-3-glucuronide in diluted urine. An IgG fraction of antiserum to oestrone-3-glucuronyl-6-bovine serum albumin is passively adsorbed to the walls of polystyrene tubes. Oestrone-3-glucuronyl-6-aminoethyl-ethyl-isoluminol is the labelled antigen. Daily samples of early morning urine are diluted in buffer (1:100; v/v), and 100 μl removed, in duplicate, for assay. After the binding reaction (1 h at 4°C), the solution is removed by aspiration. The antibody-bound fraction is washed once with buffer (400 μl). Sodium hydroxide (2n, 200 μl) is added and the mixture incubated for 60 min at 22°C. Luminescence is initiated by oxidation of the label with microperoxidase/hydrogen peroxide and the signal integrated for 10s.An evaluation of the method gave the following values: sensitivity of calibration curve 3.12 ± 0.75 pg/tube (mean ± sd). The intra-assay precision (CV%) was 9.52 (20 replicates; 38.4 ± 3.66 nmol/l) and 8.61 (15 replicates; 102.4 ± 8.82 nmol/l). The inter-assay precision (CV%) was 11.9 (mean of 4 pools−7.03, 23.16, 52.11 and 117.53 nmol/l over 2 months). The mean bias was −0.78% over the range 28 to 448 nmol/l and different aliquots (equivalent to 0.25 to 4 μl) of daily early morning urine gave results that were in good agreement. The concentration (nmol/l; mean ± sd) of oestrone-3-glucuronide in samples of early morning urine collected from healthy women during the early follicular, periovulatory and luteal phases of the menstrual cycle were 40.2 ± 9.9, 102.3 ± 39.4 and 84.3 ± 13.3 nmol/l, respectively. In addition, the results obtained from the analysis of EMU throughout 6 complete menstrual cycles were in good agreement with the values derived by radioimmunoassay (r = 0.94).

The temporal relationship between redistribution of receptors to lutropin (luteinizing hormone)/human chorionic gonadotropin in cultured rat ovarian granulosa cells and the cellular response to hormonal challenge were studied. Visualization of receptor-bound human chorionic gonadotropin by indirect immunofluorescence, with hormone-specific antibodies after fixation with 2% formaldehyde, revealed the existence of small clusters around the entire cell circumference 5--20 min after exposure to the hormone at 37 degrees C. Such small receptor aggregates were also evident if hormone incubation was at 4 degrees C or if cells were fixed with 2% formaldehyde before incubation. Larger clusters were evident after prolonged incubation with the hormone (2--4 hr) at 37 degrees C. The later change coincided with diminished cyclic AMP accumulation in respose to challenge with fresh hormone. When the fixation step was omitted and antibodies to human chorionic gonadotropin were applied after hormonal binding, acceleration of both receptor clustering and the desensitization process was observed. This maneuver also induced capping of the hormone receptors. In contrast, monovalent Fab' fragments of the antibodies were without effect. Internalization of the bound hormone in lysosomes, and subsequent degradation, was evident 8 hr after hormonal application and was not accelerated by the antibodies. It is suggested that clustering of the luteinizing hormone receptors may play a role in cellular responsiveness to the hormone. Massive aggregation of the receptors may desensitize the cell by interferring with coupling to adenylate cyclase.

OBJECTIVE`Paradoxical' responses of LH, FSH, alpha-subunits and betaLH to TRH have previously been reported in individuals with clinically non-functioning pituitary tumours (NFT). The present study was designed to assess the in vivoin vitro responses of betaFSH to TRH in NFT. We further examined the possibility that a TRH challenge with combined measurement of betaFSH and betaLH will identify a common anomalous secretory pattern in patients with NFT.
DESIGN, PATIENTS AND MEASUREMENTSForty patients with NFT underwent a standard TRH test (400 mug intravenously). Blood samples for the determination of betaFSH, betaLH, FSH and LH were collected prior to TRH as well as 15, 30, 45, 60 and 90 minutes following injection. Additionally, cultured adenomatous cells from eight of these patients were exposed to TRH in the absence and presence of octreotide and gonadotropin subunits were determined.
RESULTSTRH elicited a marked rise in circulating betaFSH in 29 of 40 individuals and in betaLH in 28 of 36 patients with NFT. In a subgroup of eight individuals whose tumours were harvested during surgery and cultured for 7-21 days, TRH increased betaFSH or betaLH and alpha-subunit release in cultured adenomatous cells in all cases, including tumours from subjects not responding to TRH in vivo. In this subgroup of patients octreotide inhibited basal betaFSH secretion but not basal betaLH secretion both in vivo and in primary cultures of NFT cells. Both the in vivoin vitro betaFSH, betaLH and alpha-subunit responses to TRH were entirely inhibited by octreotide. In all, 38 of the 40 subjects could be identified by either elevated basal betaFSH or betaLH levels and/or an abnormal rise in either betaFSH or betaLH in response to TRH.
CONCLUSIONThe measurement of basal and TRH-stimulated beta-FSH and beta-LH levels identifies an abnormal hormonal secretory pattern in the vast majority (>90%) of patients with clinically nonfunctioning pituitary tumours.