Expression, characterisation and immunoassay of recombinant marmoset chorionic gonadotrophin dimer and beta-subunit.

A specific and sensitive ELISA for measuring marmoset chorionic gonadotrophin (mCG) in culture medium, urine and plasma was developed using a polyclonal antibody raised against recombinant mCG, tagged with six histidine molecules (rmCG-6His), as the capture antibody. A well-characterised monoclonal antibody (518B7), which was generated against bovine luteinising hormone (bLH) and has been shown to detect CG and LH in Callithrichid monkeys, was biotinylated and used as the secondary antibody. Purified rmCG, calibrated against human CG (hCG; CR127) by bioassay, or the beta-subunit (rmCGbeta), quantified from amino acid analysis and carbohydrate analysis, was used as the standard. The assay was able to detect CG activity in medium collected from cultured marmoset embryos before attachment and through to the trophoblastic vesicle stage, plasma and urine collected from pregnant marmosets, marmoset placenta and pituitary homogenates. The assay was validated and its performance compared with a bioassay based on MA10 cell response to CG, with hCG as the standard. The sensitivity was 103 pg/ml (5 pg/well) of rmCGbeta and 476 pg/ml (24 pg/well) of the heterodimer rmCG. The mean recovery of standard added to embryo culture medium, marmoset urine and plasma was 104, 112 and 92% respectively. The intra- and interassay variation was less than 10 and 16% respectively. The low cross-reactivity with cynomolgus monkey and baboon LH, their beta-subunits, cynomolgus monkey and baboon follicle-stimulating hormone and hCG suggests that the assay is specific for mCG.

Effect of cryopreservation on development and ultrastructure of preimplantation embryos from the dasyurid marsupial Sminthopsis crassicaudata.

The effect of different cryopreservation methods on the development and ultrastructure of preimplantation embryos of Sminthopsis crassicaudata, a small carnivorous marsupial and member of the family Dasyuridae, was investigated. Females were primed with 1 iu pregnant mares' serum gonadotrophin to induce oestrus and ovulation. Mating generally ensued and, approximately 6 days after priming, embryos were collected and cultured in 5% CO2 in air at 35 degrees C for 18-22 h in either Dulbecco's modified Eagles medium (DMEM) with high glucose or human tubal fluid medium (HTF), both supplemented with 10% fetal calf serum. Cleavage rates were higher in DMEM than in HTF. One slow and two ultrarapid freezing methods were used. Two out of 12 (17%) embryos cleaved in culture after freezing and thawing using the slow regimen, compared with six of 16 (38%) non-frozen controls. In addition, two of 11 (18%) embryos cleaved in culture following ultrarapid freezing and thawing by one of the two methods, compared to 31 of 41 (76%) non-frozen controls. Most of the embryos appeared morphologically normal under the light microscope after freezing and thawing by the slow regimen, but considerable variation in the degree of ultrastructural damage to the cellular organelles was evident with the transmission electron microscope. The rather low rate of cleavage after freezing and thawing was probably due, at least in part, to ultrastructural damage of the cells.