Sericin accelerates the production of hyaluronan and decreases the incidence of polyspermy fertilization in bovine oocytes during in vitro maturation

Abstract

Fetal bovine serum (FBS) has been widely used as a supplement in the maturation medium of bovine oocytes in vitro. However, serum contains many undefined factors and is potentially infectious to humans and animals. As a serum replacement, we evaluated the feasibility of using the silk protein, sericin, derived from the cocoons of silkworm. To examine the rates of oocyte maturation and fertilization, cumulus-oocyte complexes were cultured in TCM-199 supplemented with 0.01%, 0.05%, 0.1% or 0.15% sericin or 5% FBS. The sizes of the perivitelline space that might relate to polyspermy, the expressions of Has2 and CD44 mRNA, the amount of hyaluronan (hyaluronic acid: HA) contained in the oocytes and the rates of blastocyst formation following insemination were then compared between the oocytes cultured with 0.05% sericin and 5% FBS, because the polyspermy rates in oocytes cultured with 0.05% sericin were significantly lower than in those cultured with 5% FBS. After in vitro maturation (IVM), the mean size of the perivitelline space was significantly greater in oocytes cultured with sericin than in those cultured with FBS, although the rates of nuclear maturation, fertilization and blastocyst formation of oocytes under both IVM conditions were not significantly different. The expression of HAS2 and CD44 mRNA and the amount of HA in the denuded oocytes cultured with 0.05% sericin were significantly greater than in those cultured with FBS. These results indicate the feasibility of sericin as an alternative protein supplement for IVM in bovine oocytes.

Fig. 1.

Bovine oocytes cultured for 0 (a) or 22 h in TCM-199 medium with 0.05% sericin (b) or 5% FBS (c). The perivitelline space is not present in the oocyte immediately after collection from the antral follicle (a). The perivitelline space (arrow) is larger in the oocytes cultured with sericin (b) than in those cultured with FBS (c). Scale bar = 50 µm.

Fig. 2.

Quantitative real-time RT-PCR analysis of bovine HAS2 and CD44 mRNAs in the oocytes and cumulus cells cultured for 22 h in TCM199 with 0.05% sericin or 5% FBS. The expression of these mRNAs was normalized to the expression of GAPDH measured in the same RNA preparation. Results of three independent experiments are summarized and expressed as the mean ± SEM. Different letters above the bars indicate significant differences (P<0.05).