Many strategies have been established to improve the efficiency of somatic cell nuclear transfer (SCNT), but relatively few focused on improving culture conditions. The effect of different culture media on preimplantation development of mouse nuclear transfer embryos was investigated. A modified sequential media method, named D media (M16/KSOM and CZB-EG/KSOM), was successfully established that significantly improves SCNT embryo development. Our result demonstrated that while lacking any adverse effect on in vivo fertilized embryos, the D media dramatically improves the blastocyst development of SCNT embryos compared with other commonly used media, including KSOM, M16, CZB, and αMEM. Specifically, the rate of blastocyst formation was 62.3% for D1 (M16/KSOM) versus 10–30% for the other media. An analysis of media components indicated that removing EDTA and glutamine from the media can be beneficial for early SCNT embryo development. Our results suggest that in vitro culture environment plays an important role in somatic cell reprogramming, and D media represent the most efficient culture method reported to date to support mouse SCNT early embryo development in vitro.
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Xiangpeng Dai, Jie Hao, and Qi Zhou
Ming-Huei Lin, Robert Kuo-Kuang Lee, Yuh-Ming Hwu, Chung-Hao Lu, Shian-Ling Chu, Ying-Jie Chen, Wei-Chao Chang, and Sheng-Hsiang Li
We report a secreted serine protease inhibitor Kazal-type-like (SPINKL) protein. The SPINKL protein was purified from mouse seminal vesicle secretions through a series of steps, including ion-exchange chromatography on a diethylaminoethyl-Sephacel column, gel filtration on a Sephadex G-75 column, and ion-exchange HPLC on a Q strong anion exchange column. Further analysis identified several SPINKL proteins with various N-linked carbohydrates. The SPINKL protein has six conserved cysteine residues that are nearly identical to those of members of the SPINK protein family. It was noted that the SPINKL protein showed no inhibitory activities against common serine proteases such as trypsin, chymotrypsin, subtilisin, or elastase. Spinkl mRNA and SPINKL proteins were found to be primarily expressed in seminal vesicles. Immunohistochemistry revealed that the SPINKL protein occurred in the luminal fluid and mucosal epithelium of the seminal vesicles and was regulated by testosterone. The SPINKL protein was able to bind onto sperm and enhance sperm motility. Also, it was able to suppress BSA-stimulated sperm capacitation and block sperm–oocyte interactions in vitro, suggesting that SPINKL may be a decapacitation factor.
Huan Zhang, Xiaohua Jiang, Yuanwei Zhang, Bo Xu, Juan Hua, Tieliang Ma, Wei Zheng, Rui Sun, Wei Shen, Howard J Cooke, Qiaomei Hao, Jie Qiao, and Qinghua Shi
In mammals, the primordial follicle pool, providing all oocytes available to a female throughout her reproductive life, is established perinatally. Dysregulation of primordial follicle assembly results in female reproductive diseases, such as premature ovarian insufficiency and infertility. Female mice lacking Dicer1 (Dicer), a gene required for biogenesis of microRNAs, show abnormal morphology of follicles and infertility. However, the contribution of individual microRNAs to primordial follicle assembly remains largely unknown. Here, we report that microRNA 376a (miR-376a) regulates primordial follicle assembly by modulating the expression of proliferating cell nuclear antigen (Pcna), a gene we previously reported to regulate primordial follicle assembly by regulating oocyte apoptosis in mouse ovaries. miR-376a was shown to be negatively correlated with Pcna mRNA expression in fetal and neonatal mouse ovaries and to directly bind to Pcna mRNA 3′ untranslated region. Cultured 18.5 days postcoitum mouse ovaries transfected with miR-376a exhibited decreased Pcna expression both in protein and mRNA levels. Moreover, miR-376a overexpression significantly increased primordial follicles and reduced apoptosis of oocytes, which was very similar to those in ovaries co-transfected with miR-376a and siRNAs targeting Pcna. Taken together, our results demonstrate that miR-376a regulates primordial follicle assembly by modulating the expression of Pcna. To our knowledge, this is the first microRNA–target mRNA pair that has been reported to regulate mammalian primordial follicle assembly and further our understanding of the regulation of primordial follicle assembly.
Sheng-Hsiang Li, Robert Kuo-Kuang Lee, Ming-Huei Lin, Yuh-Ming Hwu, Chung-Hao Lu, Ying-Jie Chen, Hsuan-Chiang Chen, Wen-Hwei Chang, and Wei-Chao Chang
The Ly-6 protein family refers to a group of glycophosphatidyl inositol-anchored membrane proteins with ten conserved cysteines. They are thought to be involved in cellular adhesion and signaling. Recently, a subfamily of secreted Ly-6 proteins has been identified. In the present study, we report a secreted Ly-6 protein, secreted seminal vesicle Ly-6 protein 1 (SSLP-1) purified from mouse seminal vesicles using a series of steps including ion-exchange chromatography on a diethylaminoethyl (DEAE)-Sephacel column, gel filtration on a Sephadex G-75 column, and ion-exchange HPLC on a sulfopropyl column. Further analysis demonstrated it to be a novel, previously unnamed, 17 kDa glycoprotein. N-glycosidase F treatment revealed a core protein with a molecular mass of 8720 Da. By Basic Local Alignment Search Tool Protein analysis, we found that SSLP-1 had ten conserved cysteine residues identical with other secreted Ly-6 proteins. The gene Gm191, which is located on chromosome 9, encodes SSLP-1. By Northern blotting with 21 different mouse tissues, we found that Sslp-1 mRNA was predominantly expressed in the seminal vesicle. Immunohistochemistry revealed SSLP-1 protein in the luminal fluid and mucosal epithelium of the seminal vesicles. The amount of Sslp-1 mRNA and SSLP-1 protein in the seminal vesicle was regulated by testosterone and correlated with the stage of animal maturation. The tissue-specific expression pattern suggests that SSLP-1 may play a physiological role in male mouse reproduction.