To date, the development of anticancer drugs has been conducted using two-dimensional (2D) cell culture systems. However, since cancer cells in the body are generated and developed in three-dimensional (3D) microenvironments, the use of 2D anticancer drug screening can make it difficult to accurately evaluate the anticancer effects of drug candidates. Therefore, as a step towards developing a cancer cellfriendly 3D microenvironment based on a combination of vinylsulfone-functionalized polyethylene glycol (PEG-VS) with dicysteine-containing crosslinker peptides with an intervening matrix metalloproteinase (MMP)-specific cleavage site, the types of MMPs secreted from human hepatocarcinoma HepG2 cells, a representative cancer cell, were analyzed transcriptionally and translationally. MMP3 was confirmed to be the most highly expressed protease secreted by HepG2 cells. This knowledge will be important in the design of a crosslinker necessary for the construction of PEG-based hydrogels customized for the 3D culture of HepG2 cells.

We observed MMPs expression in all sperm groups, with pro-MMP showing lower expression than active MMPs. According to the results from each freezing extender, the sperm membrane integrity (HOST: Hypoosmotic Swelling Test) analysis in TCGGD (Tris 250 mM, Citric acid 88 mM, Glucose 47 mM, Glycerol 3%, Dimethylsulpoxide 3.5 M) is 59.8 ± 0.7, TCGSD (Tris 250 mM, Citric acid 88 mM, Glucose 47 mM, Sucrose 0.1 M, Dimethylsulpoxide 3.5 M) is 59.3 ± 0.5 were significantly higher (p < 0.05) among the experimental groups. And MMPs analysis result, we observed MMPs expression in all sperm groups, with pro-MMP showing lower expression than active MMPs. The expression of active MMP-2 was the highest in sperms frozen in TCGSD and TCGD (Tris 250 mM, Citric acid 88 mM, Glucose 47 mM, Dimethylsulpoxide 3.5 M), Meanwhile, sperms from the TCGGD and TCGED (Tris 250 mM, Citric acid 88 mM, Glucose 47 mM, Ethylene glycol 3%, Dimethylsulpoxide 3.5 M) group showed lower level of active MMP-2 expression. Together, these results indicate that adding glycerol or sucrose to the sperm freezing buffer would not only suppress MMPs expression but also minimize DNA fragmentation, providing a mean to improve the success rate in the in vitro manipulation of rabbit sperms. Therefore, these results suggest that TCGGD or TCGSD extender method for freezing-thawing of rabbit sperm increased the viability after thawing.

Our study has analyzed whether inappropriate gonadotropin secretion affects the morphological changes due to the activation of intrauterine MMP. Methods A total of each 6 mice were injected with PMSG, Progesterone, and Androgen in 5 IU of intraperitoneal injection every 2 days after estrus synchronization, and morphological and MMPs expression patterns were compared after inducing hormone secretion. Also, cell survival and death related genes were compared and analyzed. The endometrium was highly developed in the PMSG, and the androgen was not developed at all. In particular, the diameter of the uterus of the Androgen group was also very narrow. MMPs activity assay in the case of PMSG was confirmed that showed low activity, whereas, progesterone and androgen In showed high activity and, in particular, very high activity of MMPs in the case of androgen in glandular cell. The expression of VEGF in the tissues of each group was different from that of MMPs. In the PMSG group, the activity of VEGF was increased in both the Myo-metrium and the endo-metrium, whereas the progesterone group showed low overall expression in the endo-metrium. Therefore, the present study showed that the activities of the endo-metrial cells and the restructuring of the endometrial cells differed according to the type of the abnormal secretory hormone. In particular, the secretion of androgen increased the activity of MMPs throughout the uterus, The endo-metrial epithelial cells are affected by the progesterone group. In conclusion, this study suggests that inappropriate gonadotropin secretion increases the functional changes of the uterus and this reconstruction may be caused by increased activity of MMPs

ompared the expression of MMPs in these oocytes and cumulus cell throughout oocytes maturated. In an attempt to investigate the effect of MMP activation and inhibitors in total protein of cumulus cell and, oocytes during oocytes maturation, we examined and monitored the localization and expression of MMPs (MMP-2 and MMP-9), TIMPs (TIMP-2 and TIMP-3), as well as their expression profiles (Real-time PCR, Gelatin Zymography and ELISA). Our results that the bovine oocytes MMP-2 and MMP-9 level was significantly associated with the rate of maturity of oocytes (P<0.05). In cumulus cell, MMP-2 was highly expressed in all stages of the oocyte’s maturation. The final oocytes maturation exhibited strong gelatinase activity. There was no significant correlation between cumulus cell MMP-9 and the maturation rate of oocytes. However, for the oocyte cytoplasm MMP-9 expression was significant correlation to the maturation oocytes. There was no significant correlation between cumulonimbus cells MMP-9 and oocyte maturation rates; however, for oocyte cytoplasm, MMP-9 expression was significantly correlated with mature oocyte. However, the TIMP-1 and TIMP-2 protein expression patterns are not correlated with the maturation rate of the oocyte. Our results suggest that MMP different expression pattern may regulate the morphological remodeling of oocyte's in the cumulus cell. Further, the MMP-2 expression has a strong relation with a higher maturation rate of the oocyte.

The major focus of this study is to analyze the expression of bovine MMPs and to monitor their activity during the estrus cycle and pregnancy. During pregnancy, MMP-2 expression was detectable around 30 days but became insignificant by 60 days, then started to increase again around 90 days and reached the maximum at 250 days. The activity of MMP-2 protein changed in accordance with its expression level. As expected, the level of TIMP-2 exhibited a reverse pattern. About MMP-9, high level expression was observed as early as 30 days and gradually increase until 90 days. Then started to decrease after 250 days. Again, the sites of MMP-9 expression were similar to those of MMP-2. On the other hand, expression of TIMP-3 remained low until 90 days but showed a small and temporal increase around 250 days. In summary, expression of different MMPs were differentially regulated during estrus cycle and pregnancy. While the expression of MMP-2 was high in estrus cycle, MMP-9 slowly takes over with the progression of pregnancy. These results indicated that the luteal tissue perform distinct functions during pregnancy and estrus. Perhaps the activity of MMP-2 is required for the structural remodeling of luteum, resulting the suppression of P4 inflow from blood. On the other hand, steady maintenance of MMP-9 throughout luteal development is important for the activation of cell proliferation, maturation and angiogenesis.

Matrix Metalloproteinases (MMP)-2 and -9 are participated in embryo development, implantation, remodeling of epithelial cell and ovulation. The objective of this study is to evaluate an impact of MMP2 and MMP9 on embryonic developmental competence as well as gene expression profiles of in vitro-produced bovine embryos. After in vitro fertilization, embryos of all groups were transferred into IVC-2 medium treated with MMP2 and MMP9 to check the optimum concentration on the basis of embryo development competence and cell numbers. The optimum concentrations for MMP2 and 9 were 1,200 ng/ml and 300 ng/ml. The blastocyst development competence was not different among 1,200 ng/ml of MMP2 vs. 300 ng/ml of MMP9 vs. combined MMP2 + 9 vs. control groups (41.46 ± 10.66 vs. 37.73 ± 8.92 vs. 45.11 ± 11.41% vs. 41.59 ± 11.88, respectively). Furthermore, the developmental competences to hatching and hatched blastocysts were not also different among the same groups (79.84 ± 12.63 vs. 83.3 ± 17.46 vs. 78.55 ± 14.48% vs. 72.02 ± 14.09). In addition, total cell number was significantly (p<0.05) greater in blastocyst treated with MMP9 300 ng/ml among all treatment groups. On the other hand, there was no significant difference of ICM vs. TE ratio in all groups. The expression of five out of six genes (i.e., MMP2, MMP9, IFNt, SSLP1 and HNRNPA2B1) was different among the groups. The expression of IFNt and HNRNPA2B1 genes was significantly greater in MMP9 (p<0.05), but there was no difference of MMP9 expression between MMP2 and MMP9 group (p>0.05). The normalized expression of MMP2 and SSLP1 was greater in MMP2 than other groups (p<0.05). In conclusion, MMPs treatment during IVC-2 medium was remarkably effected on blastocyst developmental competence and gene expression profiles that are related to embryo quality and implantation.

The main purpose of this study is to estimate the effect of adding Tea-N-Tris (TES) to the freezing buffer for miniature pig sperm. In particular, we attempted to identify the association between the MMPs expression and the fertility and viability of frozen sperm from each extender (LEY (Lactose Egg-Yolk), TLE (TES + LEY), TFGE (TES + Fructose + Glucose Egg-Yolk)). In accordance with this, Hypoosmotic Swelling Test (HOST) respond test was the lowest among sperms frozen in LEY while the highest HOST respond was observed among sperms frozen in TLE. Furthermore, we observed MMPs expression in all sperm groups, with pro-MMP showing lower expression than active MMPs. The expression of MMP-9 and MMP-2 was the highest in sperms frozen in LEY, Meanwhile, sperms from the TFGE and TLE group showed lower level of MMP-9 and MMP-2 expression in the order of TLE being the lowest. LEY group showed lower rate of blastocyst development than the TES supplement group, although the difference was not statistically significant. Meanwhile the rate of blastocyst development appeared similar when sperms from TLE and TFGE group were used for IVF. Together, these results indicate that adding Tea-N-Tris to the sperm freezing buffer only suppresses MMPs protein activation but also maximize in-vitro fertility, providing a means to improve the success rate in the in vitro manipulation of miniature pig sperm.

The main purpose of this study is to estimate the effect of adding Tea-N-Tris to the freezing buffer for miniaturepig sperm. In particular, we attempted to identify the association between the MMPs expression and the survival and viability of sperms. Prior to freezing, sperms in LEY without Tea-N-Tris showed 40.3±2.8% viability and 60.3±1.3% acrosome intact rate at 4℃. After freezing, sperms stored in LEY (lactose+Egg yolk) with Tea-N-Tris (=TLE) showed the highest viability (57.4±1.8%) and acrosome intact rate (65.6±4.6%). In accordance with this, DNA fragmentation was the highest among sperms frozen in LEY while the lowest fragmentation was observed among sperms frozen in TLE. When these sperms were used for in vitro fertilization (IVF), the LEY group showed lower rate of blastocyst development, although the difference was not statistically significant. Meanwhile the rate of blastocyst development appeared similar when sperms from TLE and TFGE (Tea-N-Tris+Fructose+ Glucose+Egg yolk) group were used for IVF. We observed MMPs expression in all sperm groups, with pro-MMP showing lower expression than active MMPs. The expression of MMP-9 and MMP-2 was the highest in sperms frozen in LEY, Meanwhile, sperms from the TFGE and TLE group showed lower level of MMP-9 and MMP-2 expression in the order of TLE being the lowest. Together, these results indicate that adding Tea-N-Tris to the sperm freezing buffer would not only suppress MMPs expression but also minimize DNA fragmentation, providing a mean to improve the success rate in the in vitro manipulation of miniaturepig sperms. * This work was supported by BioGreen 21 Program (No. PJ008029). Rural Development Administation, Republic of Korea.

Matrix metalloproteinases (MMP) play important roles in extracellular matrix (ECM) remodeling during ovarian follicular development, oocytes development and ovulation. In an attempt to investigate the effect of MMP activation in development cumulus-oocytes complexes, we examined the localization and expression of MMP, and monitored MMP expression profile. Cumulus-oocytes complexes were collected and matured in vitro for 24 hr, 36 hr and 48 hr. A mRNA expression of MMP-2, MMP-9, TIMP-2 and TIMP-3 was detected in all culture medium regardless of CC, OC and COCs. Activity of MMP-2 in the OC progressively was increased from 24 hr to 48 hr. But MMP-9 was not detected in all culture medium. The localization of MMP-2 was also measured by immunohistochemistry analysis. The MMP-2 and TIMP-2 was detected in cumulus cell and oocyte zone pellucida. Expression of MMP-2 protein in the COCs was progressively increased from 24 hr to 48 hr. However, MMP-9 protein was progressively decreased from 24 hr to 48 hr. And TIMP-2 protein was most highly expressed in the COCs 36 hr. Expression of TIMP-3 protein in the COCs was progressively increased from 24 hr to 48 hr. In conclusion, these results suggest that MMP-2 plays a role in maintaining normal maturation and development by controlling the ECM inhibitor concentration on cumulus cell and oocytes.