Inhibition of progesterone-mediated maturation of oocytes of Xenopus laevis by oocyte maturation inhibitor from pig follicular fluid: development of a routine assay for the inhibitor with Xenopus oocytes.

We describe an assay for oocyte maturation inhibitor (OMI) using the progesterone-mediated maturation of Xenopus oocytes. The test fraction used was a partially purified fraction from pig follicular fluid, which gave consistent inhibition of maturation in the pig oocyte assay. In the toad assay, oocytes (30-50) from each toad were pretested to determine whether satisfactory maturation was achieved because widespread animal variation was observed. Toads whose oocytes showed greater than 60% maturation in the pretest could be used directly. Toads whose oocytes showed less than 60% maturation were injected with pregnant mares serum gonadotropin (PMSG) in order to increase progesterone-mediated maturation. The dose and time after injection of PMSG before harvesting oocytes, dose and duration of progesterone exposure, and order of exposure of oocytes to OMI and progesterone were important variables. Opposing effects of OMI and progesterone were seen in oocytes from toads receiving 60 IU PMSG. In the routine assay we use animals whose oocytes show greater than 60% maturation in the pretest or animals treated with 12 IU of PMSG 4 to 7 days before use. Oocytes are exposed to the OMI fraction for 1 hr, progesterone is added, and incubations continued until controls reach maximum maturation (5 to 8 hr). The inhibition of toad oocyte maturation by OMI is reversible. The toad and mammalian oocyte assays were compared using more highly purified fractions of OMI and gave identical results.