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Yexia Li, Yujie Jin, Yuxia Liu, Chunyan Shen, Jingxia Dong, and Jian Xu

The function of Smad3, a downstream signaling protein of the transforming growth factor β (TGFβ) pathway, in ovarian follicle development remains to be elucidated. The effects of Smad3 on ovarian granulosa cells (GCs) in rat were studied. Female rats (21 days of age Sprague–Dawley) received i.p. injections of pregnant mare serum gonadotropin, and GCs were harvested for primary culture 48 h later. These cells were engineered to overexpress or knockdown Smad3, which were validated by immunohistochemistry and western blot. The expression of proliferating cell nuclear antigen (PCNA), cyclin D2, TGFβ receptor II (TGFβRII), protein kinase A (PKA), and FSH receptor (FSHR) was also detected by western blotting. Cell cycle and apoptosis of GCs were assayed by flow cytometry. The level of estrogen secreted by GCs was detected by ELISA. Smad3 overexpression promoted estrogen production and proliferation while inhibiting apoptosis of GCs. Reduction in Smad3 by RNAi resulted in reduced estrogen production and proliferation and increased apoptosis of GCs. Manipulation of Smad3 expression also resulted in changes in FSHR and PKA expression, suggesting that the effects of Smad3 on follicle development are related to FSHR-mediated cAMP signaling.

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Jian-Jun Chang, Jing-Pian Peng, Ying Yang, Jing-Ling Wang, and Li Xu

Partial cDNA sequence coding for Microtus brandti radde (Brandt’s vole) testes-specific lactate dehydrogenase (brLDH-C4) was amplified by reverse transcription-polymerase chain reaction (RT-PCR). By inserting the product into the eukaryotic expression vector pCR3.1, pCR3.1-brLDH-C4′ was obtained as the prototype of contraceptive DNA vaccine. Immunization with pCR3.1-brLDH-C4′ in BALB/c mice generated antibodies specific to purified brLDH-C4′ and native mouse LDH-C4 protein. The birth rate of the pCR3.1-brLDH-C4′ immunized mice was found to be decreased significantly (80% lower than that of those immunized with pCR3.1). Functions of the elicited antibodies in sera from pCR3.1-brLDH-C4′ inoculated mice were further explored. The results indicated that the antibodies from the mice injected with pCR3.1-brLDH-C4′ could cause the agglutination of normal sperm suspension, while the ovarian structure and the development of ovarian follicles of these mice were not impaired, which gives a possible explanation for the immunocontraceptive effects of the pCR3.1-brLDH-C4′ DNA vaccine.

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Zi-gang Shen, Wei He, Ji Zhang, Hai-yang He, Xia Yang, Zheng-qiong Chen, Ping Yang, Jian Li, Zhi-qing Liang, Yu-zhang Wu, and Jin-tao Li

SPINLW1 (previously known as eppin (epididymal protease inhibitor)) is a target under intense scrutiny in the study of male contraceptive vaccines. B-cell-dominant epitopes are now recognized as key parts of the induction of humoral immune responses against target antigens. The generation of robust humoral responses in vivo has become a crucial problem in the development of modern vaccines. In this study, we developed a completely novel B-cell-dominant-epitope-based mimovirus vaccine, which is a kind of virus-size particulate antigen delivery system. The mimovirus successfully self-assembled from a cationic peptide containing a cell-penetrating peptide of TAT49–57 and a plasmid DNA encoding both three SPINLW1 (103–115) copies and adjuvant C3d3. The male mice were immunized with the epitope-based mimovirus vaccine, which resulted in a gradual elevation of specific serum IgG antibody levels. These reached a peak at week 4. Mating for the fertility assay showed that the mimovirus vaccine had accomplished a moderate fertility inhibition effect and investigation into the mechanism of action showed that it did so by interfering with the reproductive function of the sperm but that it did not damage the structures of the testes or cause serum testosterone to decline. Our results suggest an ideal protocol for suppressing fertility in mice by an engineered mimovirus vaccine.

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Cheng Jin, Yan Zhang, Zhi-Peng Wang, Xiu-Xia Wang, Tie-Cheng Sun, Xiao-Yu Li, Ji-Xin Tang, Jin-Mei Cheng, Jian Li, Su-Ren Chen, Shou-Long Deng, and Yi-Xun Liu

Spermatogenesis is crucial for male fertility and is therefore tightly controlled by a variety of epigenetic regulators. However, the function of enhancer of zeste homolog 2 (EZH2) in spermatogenesis and the molecular mechanisms underlying its activity remain poorly defined. Here, we demonstrate that deleting EZH2 promoted spermatogonial differentiation and apoptosis. EZH2 is expressed in spermatogonia, spermatocytes and round and elongated spermatids from stage 9 to 11 but not in leptotene and zygotene spermatocytes. Knocking down Ezh2 in vitro using a lentivirus impaired self-renewal in spermatogonial stem cells (SSCs), and the conditional knockout of Ezh2 in spermatogonial progenitors promoted precocious spermatogonial differentiation. EZH2 functions to balance self-renewal and differentiation in spermatogonia by suppressing NEUROG3 and KIT via a direct interaction that is independent of its histone methyltransferase activity. Moreover, deleting Ezh2 enhanced the activation of CASP3 in spermatids, resulting in reduced spermatozoa production. Collectively, these data demonstrate that EZH2 plays a nonclassical role in the regulation of spermatogonial differentiation and apoptosis in murine spermatogenesis.

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Rui Chen, Jian Du, Lin Ma, Li-qing Wang, Sheng-song Xie, Chang-ming Yang, Xian-yong Lan, Chuan-ying Pan, and Wu-zi Dong

MicroRNAs (miRNAs) are 18–24 nucleotides non-coding RNAs that regulate gene expression by post-transcriptional suppression of mRNA. The Chinese giant salamander (CGS, Andrias davidianus), which is an endangered species, has become one of the important models of animal evolution; however, no miRNA studies on this species have been conducted. In this study, two small RNA libraries of CGS ovary and testis were constructed using deep sequencing technology. A bioinformatics pipeline was developed to distinguish miRNA sequences from other classes of small RNAs represented in the sequencing data. We found that many miRNAs and other small RNAs such as piRNA and tsRNA were abundant in CGS tissue. A total of 757 and 756 unique miRNAs were annotated as miRNA candidates in the ovary and testis respectively. We identified 145 miRNAs in CGS ovary and 155 miRNAs in CGS testis that were homologous to those in Xenopus laevis ovary and testis respectively. Forty-five miRNAs were more highly expressed in ovary than in testis and 21 miRNAs were more highly expressed in testis than in ovary. The expression profiles of the selected miRNAs (miR-451, miR-10c, miR-101, miR-202, miR-7a and miR-499) had their own different roles in other eight tissues and different development stages of testis and ovary, suggesting that these miRNAs play vital regulatory roles in sexual differentiation, gametogenesis and development in CGS. To our knowledge, this is the first study to reveal miRNA profiles that are related to male and female CGS gonads and provide insights into sex differences in miRNA expression in CGS.

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Jian Zhang, Linlin Hao, Qian Wei, Sheng Zhang, Hui Cheng, Yanhui Zhai, Yu Jiang, Xinglan An, Ziyi Li, Xueming Zhang, and Bo Tang

Somatic cell nuclear transfer (SCNT) has been successfully used for cloning in a variety of mammalian species. However, SCNT reprogramming efficiency is relatively low, in part, due to incomplete DNA methylation reprogramming of donor cell nuclei. We previously showed that ten-eleven translocation 3 (TET3) is responsible for active DNA demethylation during preimplantation embryonic development in bovines. In this study, we constructed TET3-overexpressing cell lines in vitro and observed that the use of these fibroblasts as donor cells increased the blastocyst rate by approximately 18 percentage points compared to SCNT. The overexpression of TET3 in bovine SCNT embryos caused a decrease in the global DNA methylation level of the pluripotency genes Nanog and Oct-4, ultimately resulting in an increase in the transcriptional activity of these pluripotency genes. Moreover, the quality of bovine TET3-NT embryos at the blastocyst stage was significantly improved, and bovine TET3-NT blastocysts possessed more total number of cells and fewer apoptotic cells than the SCNT blastocysts, similar to in vitro fertilization (IVF) embryos. Nevertheless, DNA methylation of the imprinting control region (ICR) for the imprinted genes H19-IGF2 in SCNT embryos remained unaffected by TET3 overexpression, maintaining parent-specific activity for further development. Thus, the results of our study provide a promising approach to rectify incomplete epigenetic reprogramming and achieve higher cloning efficiency.

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Wen-Min Cheng, Lei An, Zhong-Hong Wu, Yu-Bo Zhu, Jing-Hao Liu, Hong-Mei Gao, Xi-He Li, Shi-Jun Zheng, Dong-Bao Chen, and Jian-Hui Tian

We recently reported that electrical activation followed by secondary chemical activation greatly enhanced the developmental competence of in vitro matured porcine oocytes fertilized by intracytoplasmic sperm injection (ICSI). We hypothesized that sperm treatment with disulfide bond reducing agents will enhance the development competence of porcine embryos produced by this ICSI procedure. We examined the effects of glutathione (GSH), dithiothreitol (DTT), GSH or DTT in combination with heparin on sperm DNA structure, paternal chromosomal integrity, pronuclear formation, and developmental competence of in vitro matured porcine oocytes after ICSI. Acridine orange staining and flow cytometry based sperm chromatin structure assay were used to determine sperm DNA integrity by calculating the cells outside the main population (COMP αT). No differences were observed in COMP αT values among GSH-treated and control groups. COMP αT values in GSH-treated groups were significantly lower than that in DTT-treated groups. Following ICSI, GSH treatments did not significantly alter paternal chromosomal integrity. Paternal chromosomal integrity in sperm treated with DTT plus or minus heparin was also the lowest among all groups. GSH-treated sperm yielded the highest rates of normal fertilization and blastocyst formation, which were significantly higher than that of control and DTT-treated groups. The majority of blastocysts derived from control and GSH-treated spermatozoa were diploid, whereas blastocysts derived from DTT-treated spermatozoa were haploid. In conclusion, sperm treatment with GSH enhanced the developmental capacity of porcine embryos produced by our optimized ICSI procedure.

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Xue-Ying Zhang, Yi-Meng Xiong, Ya-Jing Tan, Li Wang, Rong Li, Yong Zhang, Xin-Mei Liu, Xian-Hua Lin, Li Jin, Yu-Ting Hu, Zhen-Hua Tang, Zheng-Mu Wu, Feng-Hua Yin, Zheng-Quan Wang, Ye Xiao, Jian-Zhong Sheng, and He-Feng Huang

Fertilization failure often occurs during in vitro fertilization (IVF) cycles despite apparently normal sperm and oocytes. Accumulating evidence suggests that mitochondria play crucial roles in the regulation of sperm function and male fertility. 3-Nitrophthalic acid (3-NPA) can induce oxidative stress in mitochondria, and melatonin, as an antioxidant, can improve mitochondrial function by reducing mitochondrial oxidative stress. The role of sperm mitochondrial dysfunction in fertilization failure during IVF is unclear. The present study revealed that spermatozoa with low, or poor, fertilization rates had swollen mitochondria, increased mitochondria-derived ROS, and attenuated mitochondrial respiratory capacity. 3-NPA treatment enhanced mitochondrial dysfunction in sperm. Spermatozoa with poor fertilization rates, and spermatozoa treated with 3-NPA, had reduced penetration ability. The concentration of melatonin was decreased in semen samples with low and poor fertilization rates. Melatonin, not only decreased excessive mitochondria-derived ROS, but also ‘rescued’ the reduced penetration capacity of spermatozoa treated with 3-NPA. Taken together, the study suggested that mitochondria-derived ROS and mitochondrial respiratory capacity are independent bio-markers for sperm dysfunction, and melatonin may be useful in improving sperm quality and overall male fertility.