Production in vitro of mouse early pregnancy factor and purification to homogeneity

Abstract

Early pregnancy factor (EPF) has been produced in vitro by culture of oestrous mouse oviducts and ovaries in RPMI, with the addition of prolactin and mouse embryo culture medium. The pooled harvested medium was then subjected to immunoabsorption, electrofocusing and gel filtration. A fraction was isolated with pI 6.83 and molecular weight 21 000 which was responsible for 90% of the recovered biological activity. It appeared to be homogeneous when analysed by high-performance gel-permeation chromatography. SDS treatment showed that the molecule could be split into 3 peptides of molecular weights 10 500, 7200 and 3400, the first having activity equivalent to the EPF component EPF-A from the oviduct, while the last two combined to give activity corresponding with the ovarian component EPF-B.

Early Pregnancy Factor (EPF): A Link between Fertilization and Immunomodulation

“…EPF can be detected in the rosette inhibition test in concentrations at least as low as 100 pg ml~l, i.e. 20 pg in 0·2 ml sample (Cavanagh 1984), while significant binding of labelled EPF to spleen cells is not observed below 2 ng in 0 . 2 ml sample (10 ng ml-I concentration).…”

“…With EPF isolated from sheep serum (fetal EPF) ' Wilson et al (1983) could detect 1 ng ml-I in the assay with sheep lymphocytes and anti-sheep lymphocyte serum. Cavanagh (1984) found that, with mouse lymphocytes and anti-mouse lymphocyte serum, 100 pg ml-1 mouse maternal EPF would give a positive result. With the same assay system, A. C. Cavanagh (unpublished data) compared material purified from mouse and human tissue and found that human EPF was 10 2 -10 3 times more active in the assay than was mouse EPF.…”

“…For the isolation of maternal ('early') EPF, Cavanagh (1984) used, as starting material, ovaries and oviducts from oestrous mice. These organs were cultured for 5 days in the presence of prolactin and embryo culture medium (Cavanagh et al 1982), then EPF harvested from the culture medium by immunoabsorption, electrofocusing and gel filtration.…”

“…Maternal (Cavanagh 1984), fetal (Wilson et al 1983) and tumor-derived EPF (Rolfe et al 1983a) appear to have a number of similar properties. The molecules have a monomeric size of 20000-21 000 and exhibit immunological cross-reactivity, i.e.…”

“…EPF has also been found in serum of patients with tumors of germ cell origin (Rolfe et al 1983a). Maternal, fetal and tumor EPF are of similar molecular size, exhibit immunological crossreactivity and give the same responses in the assay for EPF, but preliminary studies have suggested that they may differ in some biochemical characteristics (Wilson et al 1983;Cavanagh 1984; B. E. Rolfe, A. C. Cavanagh and H. Morton, unpublished data). 0004-9417/84/050393$02.00 EPF binds to lymphocytes and in so doing indirectly suppresses immune reactivity both in vivo and in vitro (Noonan et al 1979;Morton et al 1982a;Rolfe et al 1983b).…”