Luminescence Luteinizing Hormone/Choriogonadotropin (LH/CG) Bioassay: Measurement of Serum Bioactive LH/CG during Early Pregnancy in Human and Macaque1

Abstract

Because of the microheterogeneities of gonadotropins, measurement of immunoreactivity of these glycoproteins does not necessarily reflect changes in their bioactivity. In addition, LH bioactivities in human samples analyzed by a rodent LH bioassay have been discordant with findings based on human granulosa-luteal cells. We have isolated a human LH/choriogonadotropin (CG) receptor cDNA and expressed the recombinant protein. Using 293 cells permanently transfected with the human LH receptor cDNA and a luciferase reporter gene driven by a cAMP-dependent promoter, we have developed a luminescence LH/CG bioassay. After cells were treated with human LH or CG for 20 h, luciferase activity was measured through use of a luminometer. Luciferase activity in the cells was increased in a dose-dependent manner. In contrast, treatment with FSH, thyroid-stimulating hormone, prolactin, growth hormone, adrenocorticotropin, insulin, prostaglandins, and several neurotransmitters had no effect. Because treatment with basic fibroblast growth factor (bFGF) caused significant increases in basal luciferase activity, a fixed amount of bFGF was included in all reactions. Incubation with 0.1 to 30 microliters serum from women during different physiological states stimulated the luciferase activity in parallel with the hCG standard curve. In 4 conception cycles, bioactive LH/hCG levels began to increase 2 wk after the midcycle LH surge, followed by a logarithmic increase from 22 days on. Due to the lack of a homologous RIA for measuring CG levels in monkeys, we analyzed serum bioactive monkey CG (mCG) in macaque during early pregnancy. Bioactive mCG was detected about 12 days after the midcycle LH surge and fertile mating and persisted until Days 21-23, followed by a decline.(ABSTRACT TRUNCATED AT 250 WORDS)

Anti-androgenic endocrine disrupting activities of chlorpyrifos and piperophos

“…Using this recombinant cell line, chemicals were tested for their (anti)androgenic activities. These chemicals were then studied for their effects on steroid biosynthesis in rat Leydig cell cultures and selected chemicals were further analyzed for their cross-talk with LH receptor mediated functions using a cell-based indirect cAMP assay as was reported earlier [20,21]. The data presented here demonstrates that some widely used pesticides and pharmaceutics have (anti)androgenic properties which are not only restricted through their interactions with AR but also affects the LH receptor and cAMP cascades.…”

“…The cAMP was estimated by an indirect method using the luciferase driven cAMP response element according the method described earlier [20]. Briefly, CHO-K1 cells were transiently transfected with 1 g each of hLH receptor and CRE-luc constructs (as described earlier) using transfection reagent as per the manufacturer's instruction (Qiagen, Valencia, CA, USA).…”

Daily Immunoactive and Bioactive Human Chorionic Gonadotropin Profiles in Periimplantation Urine Samples1

“…The LH/CG cellular bioassay used was based on the cloned human fetal kidney cell line 293 that had been permanently transfected with hLH/hCG receptor and luciferase reporter gene cDNA [16]. Biologically active hCG in a sample incubated with the cells in culture is detected, following hCG binding to the LH/hCG receptor, by an increase in cAMP-mediated protein kinase A activity, the activation and expression of luciferase genes, and the subsequent luciferase substrate conversion, with a luminometer.…”

“…An LH/CG radioreceptor assay and a luminescence LH/CG cellular bioassay were developed to measure hCG molecules in pregnancy serum and to investigate the interaction of hCG with its receptor and downstream signal complex [16]. Because of the LH/CG receptor's strict binding requirement for intact molecules, hCG molecules capable of triggering the downstream signal cascade in LH/CG cellular bioassay were subsequently found to be a subset of the intact molecules measured by an immunoenzymometric assay (hCG IEMA) specific for intact hCG molecules (IhCG) and an LH/CG radioreceptor assay [17].…”

A Novel Four-Amino Acid Determinant Defines Conformational Freedom within Chorionic Gonadotropin β-Subunits

“…Recombinant hCG (Sigma) and high performance liquid chromatography-purified CG heterodimer consisting of hCG-α and mCG-β (here termed h-α/m-β CG) were used. Using HEK LH/CG-R cells expressing the human receptor and a cAMP-regulated luciferase reporter gene (12), the bioactivities of hCG and h-α/m-β CG were compared as described in Methods, generating the dose response curves shown in Fig. 5.…”