In vivo oocytes grow and mature in ovarian follicles whereas oocytes are matured in vitro in plastic culture dishes with a hard surface. In vivo oocytes show a superior developmental ability to in vitro counterparts, indicating suboptimal environments of in vitro culture. This study aimed to evaluate the influence of an agarose matrix as a culture substrate during in vitro maturation (IVM) on the development of pig oocytes derived from small antral follicles (SAFs). Cumulusoocyte complexes (COCs) retrieved from SAFs were grown in a plastic culture dish without an agarose matrix and then cultured for maturation in a plastic dish coated without (control) or with a 1% or 2% (w/v) agarose hydrogel. Then, the effect of the soft agarose matrix on oocyte maturation and embryonic development was assessed by analyzing intra-oocyte contents of glutathione (GSH) and reactive oxygen species (ROS), expression of VEGFA, HIF1A , and PFKP genes, and blastocyst formation after parthenogenesis. IVM of pig COCs on a 1% (w/v) agarose matrix showed a significantly higher blastocyst formation, intra-oocyte GSH contents, and transcript abundance of VEGFA. Moreover, a significantly lower intra-oocyte ROS content was detected in oocytes matured on the 1% and 2% (w/v) agarose matrices than in control. Our results demonstrated that IVM of SAFs-derived pig oocytes on a soft agarose matrix enhanced developmental ability by improving the cytoplasmic maturation of oocytes through redox balancing and regulation of gene expression.

Generally, in vivo, primary oocytes are grown and matured into secondary oocytes in the ovarian follicles. Quality of the oocytes matured in vivo is higher than that of oocytes matured in vitro, indicating the importance of materializing the microenvironment of ovarian follicles for production of high quality oocyte. Therefore, we tried to mimic the stiffness of ovarian follicles using an agarose as a biocompatible natural polymer. Unfortunately, to date, there are no many reports on whether the quality of porcine oocytes can be increased effectively under the soft matrix. Accordingly, we tried to evaluate the effects of IVM using different mechanical properties of agarose substrate on developmental competence of porcine oocytes. Agarose substrate was constructed and cumulus-oocyte-complexes (COCs) retrieved from porcine medium antral follicles were matured on non-coated (control) culture dish or dishes coated with 1% and 2% (w/v) agarose substrate. Then, cumulus expansion, embryonic development after parthenogenetic activation, and gene expression level were analyzed and compared. As the results, significant increase in blastocyst formation and cumulus expansion were detected in COCs matured on 1% (w/v) agarose substrate compared with control. Moreover, oocytes of COCs matured on 1% (w/v) agarose substrate showed significantly higher BMP15 expression level compared with control. Pro-apoptotic gene BAX expression was significantly increased in oocytes of COCs matured on 2% (w/v) agarose substrate compared with control. In the glycolytic enzyme phosphofructokinase (PFKP) gene expression, cumulus cells of COCs matured on agarose substrate showed significantly higher PFKP expression than control while they showed significantly lower BAX expression than control. These results demonstrated that quality of porcine oocytes could be increased efficiently by the IVM of immature oocytes on the soft culture matrix using agarose.

In order to develop a protective carrier scaffolder for the external usage of medical and hygienic materials, three essential protective elements existing in nature, i.e., algin, cellulose, and calcium phosphate apatite, were investigated. The algin is a main skeletal component of sea weeds, the cellulose is of vegetables, and the calcium phosphate apatite is of vertebral animals. In the present study we select the agarose which is a derivative from algin, the cellulose fiber obtained from skin of sea squirt, calcium oxide purified from shell powder, and tricalcium phosphate apatite purchased commercially. Consequently, the agarose-cellulose hybrid was made by the hydrogen bonds intermediating the calcium phosphate apatite between agarose and cellulose molecules. As the calcium phosphate apatite is formed by the addition of calcium hydroxide into tricalcium phosphate solution, we used calcium oxide to accelerate the hybridization between the agarose and calcium phosphate apatite and also between the cellulose and calcium phosphate apatite. In the phase contrast microscopic observation the agarose-cellulose hybrid showed more compact matrix structure than the mixture of agarose and cellulose. The agarose-cellulose hybrid showed increased storage modulus but decreased loss modulus in Rheometer test compared to those of the other materials tested in this study, representing that the agarose-cellulose hybrid has the highest elasticity among them and similar water capacity to agarose. The agarose-cellulose hybrid showed the strongest antimicrobial effect in bacteria killing assay than the other materials, and also it showed a potent blood clotting effect but no immunological hypersensitivity on the human skin. From the above results we presumed that the nobel material, agarose-cellulose hybrid, is a compact scaffolding matrix which has proper elasticity, high capacity to hold substrates, and antimicrobial and blood clotting property potent enough to carry the bio-medical and hygienic materials for external treatment safely.

갈파래의 포자가 부착한 Agar와 Agarose코팅필름은 agar, agarose 또는 메탄놀의 농도에 따라 다양한 표면의 성질을 나타냈다. Sigma agarose 코팅필름에서 가장 많은 수의 포자 부착이 일어났다. Bacto agar과 그외 agarose으로 코팅한 필름에 부착한 포자는 Sigma agarose으로 코팅한 필름보다 12∼36배나 적었다. 2.5%와 5％ 의 SeaKem agarose와 Bacto agar 코팅필름의 비교에서 두 개의