Session: SAT 134-163-GnRH & Gonadotroph Biology & Signaling
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Poster Board SAT-162
Superovulation is a valuable technique for developmental engineering. This technique often applies equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG) to other species, since these hormones exhibit biological activity mimic follicle stimulating hormone (FSH) and/or luteinizing hormone (LH). However, administration of the heterologous hormones has a risk to induce antibodies in the recipient. In addition, it is particularly difficult to produce activity with most similarity by usage of eCG and hCG, indicating a value to verify the activity of self-hormone. The present study aimed to produce recombinant mouse follicle stimulating hormone (rec-mFSH) and examined the biological activities.
Expression vector, pCX-mFSH, was constructed by ligation of mouse FSH subunits (alpha and beta) harboring independent promoter and polyA signal. Production of rec-mFSH was performed by transfection of the vector into the CHO cells. The rec-mFSH was collected as secreted form in culture media after 48h-cultivation. The concentration was measured by time-resolved fluorescence immunoassay (TR-FIA), and the biological activity was estimated as international unit by the granulosa cell aromatase bioassay (GAB). Eventually, activity of the superovulation was examined with ICR mice using rec-mFSH, followed by measurement of ovulation number with different number of injection and/or combined usage of excipients.
TR-FIA showed that rec-mFSH was produced at about 1.933 µg/ml in the media, and GAB confirmed that the recombinant has a biological activity at 11.19 IU/µg. We have also confirmed the activity of the rec-mFSH for the induction of superovulation. Notably, single injection with 6.87 IU rec-mFSH could not induce superovulation, but injections with 1.37 IU rec-mFSH twice a day for 3 days induced superovulation. Notably, single injection with 7.5IU rec-mFSH with polyvinylpyrrolidone, but not aluminums hydroxide gel, succeeded to induce superovulation.
We have established a system to produce the bioactive rec-mFSH in GAB and superovulation. Usage of excipients with a single injection prolonged effect of the rec-mFSH in vivo, indicating that excipients act to extend the effect of rec-mFSH by preventing a half-life of the recombinant hormone.
Nothing to Disclose: AG, TJ, KK, DK, TK, KI, TK, YK, AO
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