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Dong Han, Xin-Yan Cao, Hui-Li Wang, Jing-Jing Li, Yan-Bo Wang and Jing-He Tan

Although studies suggest that the low competence of oocytes from prepubertal animals is due to their insufficient cytoplasmic maturation and that FSH improves oocyte maturation possibly by retarding meiotic progression and allowing more time for cytoplasmic maturation, the mechanisms by which puberty and gonadotropins regulate meiotic progression require additional detailed studies. For the first time, we observed that while meiotic progression was significantly slower, the maturation-promoting factor (MPF) activity of oocytes was significantly higher in prepubertal than in adult mice. To resolve this contradiction, we specified the molecules regulating the MPF activity and their localization during oocyte maturation in prepubertal and adult mice primed with or without gonadotropins. Our tests using corresponding enzyme regulators suggested that while activities of protein kinase A were unaffected, the activity of adenylate cyclase (ADCY) and phosphodiesterase increased while cell division cycle 2 homolog A (CDC2A) decreased significantly after puberty. While most of the adult oocytes had CDC2A protein concentrated in the germinal vesicle (GV) region, the majority of prepubertal oocytes showed no nuclear concentration of CDC2A. Maximally priming mice with equine chorionic gonadotropin brought the above parameters of prepubertal oocytes close to those in adult oocytes. Together, the results suggest that puberty and gonadotropin control oocyte meiotic progression mainly by regulating the ADCY activity and the concentration of the activated MPF toward the GV region.

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Tian-Hong Zhu, Shao-Jie Ding, Tian-Tian Li, Li-Bo Zhu, Xiu-Feng Huang and Xin-Mei Zhang

Endometriosis is an estrogen-dependent disease. Previous research has shown that abnormal enzymes associated with estrogen (E2) metabolism and an increased number of mast cells (MCs) in endometriomas are implicated in the pathogenesis of endometriosis. However, it remains unclear how MCs mediate the role of E2 in endometriosis. Accordingly, we investigated whether E2 was associated with the number of MCs, and the rate of degranulation, in local ovarian endometriomas, as well as the role of E2 on MCs during the pathogenesis of endometriosis. Using enzyme-linked immunosorbent assay and immunohistochemistry, we found that concentrations of E2, and the number and activity of MCs, were significantly higher in ovarian endometriomas than in controls, and that these parameters were correlated with the severity of endometriosis-associated dysmenorrhea. By measuring the release of hexosaminidase, we found that the rate of RBL2H3 cell degranulation increased after E2 treatment. Furthermore, activation of RBL2H3 cells by E2 was found to trigger the release of biologically active nerve growth factor, which promotes neurite outgrowth in PC12 cells and also sensitizes dorsal root ganglion cells via upregulation of Nav1.8 and transient receptor potential cation channel (subfamily V member 1) expression levels. When treated with E2, endometriotic cells could promote RBL2H3 cell recruitment by upregulating expression levels of stem cell factor, transforming growth factor-β and monocyte chemoattractant protein-1; these observations were not evident with control endometrial cells. Thus, elevated E2 concentrations may be a key factor for degranulation and recruitment of MCs in ovarian endometriomas, which play a key role in endometriosis-associated dysmenorrhea.

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Jiang Wen, Juan Liu, Guangqi Song, Limei Liu, Bo Tang and Ziyi Li

6-Bromoindirubin-3′-oxime (BIO), which is one of the glycogen synthase kinase 3 inhibitors and a key regulator of numerous signaling pathways, was reported to be capable of maintaining the pluripotency of human and mouse embryonic stem cells. Presently, it is unknown whether BIO can influence the derivation of porcine embryonic germ (EG) cells. In this study, porcine primordial germ cells (PGCs) were isolated from gonads of 24- and 28-day embryos, and were then treated with BIO either individually or in combination with other cytokines (stem cell factor (SCF), leukemia inhibitory factor (LIF), and fibroblast growth factor (FGF); abbreviated as ‘3F’), and the effects of the treatment on the proliferation ability of porcine PGCs at early stage were examined using 5-bromo-2-deoxyuridine (Brdu) immunostaining assay. After continuous culture, the effects on the efficiency of porcine undifferentiated EG cells in the third passage and differentiated EG cells from embryoid bodies were examined as well. The results obtained through the observation of the Brdu-labeled PGCs indicated that BIO in combination with 3F resulted in a significant increase in the mitosis index, and also indicated that the BIO in combination with 3F had a higher efficiency in promoting the formation of porcine EG colony derived from porcine day 24 PGCs than BIO used either individually or in combination with LIF. In addition, BIO in combination with 3F exhibited the apparent anti-differentiation activity by reversing the differentiated EG cells to the undifferentiated status. Our results demonstrate that BIO in combination with SCF, LIF, and FGF could significantly contribute to the establishment of a porcine EG cell colony and maintain the undifferentiated status.

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Bo Zheng, Jun Yu, Yueshuai Guo, Tingting Gao, Cong Shen, Xi Zhang, Hong Li and Xiaoyan Huang

The cellular nucleic acid-binding protein (CNBP), also known as zinc finger protein 9, is a highly conserved zinc finger protein that is strikingly conserved among vertebrates. Data collected from lower vertebrates showed that CNBP is expressed at high levels and distributed in the testes during spermatogenesis. However, the location and function of CNBP in mammalian testes are not well known. Here, by neonatal mouse testis culture and spermatogonial stem cells (SSC) culture methods, we studied the effect of CNBP knockdown on neonatal testicular development. Our results revealed that CNBP was mainly located in the early germ cells and Sertoli cells. Knockdown of CNBP using morpholino in neonatal testis culture caused disruption of seminiferous tubules, mislocation of Sertoli cells and loss of germ cells, which were associated with the aberrant Wnt/β-catenin pathway activation. However, knockdown of CNBP in SSC culture did not affect the survival of germ cells. In conclusion, our study suggests that CNBP could maintain testicular development by inhibiting the Wnt/β-catenin pathway, particularly by influencing Sertoli cells.

Open access

Cai Chen, Han Wu, Dan Shen, Saisai Wang, Li Zhang, Xiaoyan Wang, Bo Gao, Tianwen Wu, Bichun Li, Kui Li and Chengyi Song

The similarities and differences of small RNAs in seminal plasma, epididymal sperm and ejaculated sperm remain largely undefined. We conducted a systematic comparative analysis of small RNA profiles in pig ejaculated sperm, epididymal sperm and seminal plasma and found that the diversity distribution of small RNA species was generally similar, whereas the abundance of small RNAs is dramatically different across the three libraries; miRNAs and small RNAs derived from rRNA, tRNA, small nuclear RNA, 7SK RNA, NRON RNA and cis-regulatory RNA were enriched in the three libraries, but piRNA was absent. A large population of small RNAs from ejaculated sperm are ejaculated sperm specific, and only 8–30% of small RNAs overlapped with those of epididymal sperm or seminal plasma and a small proportion (5–18%) of small RNAs were shared in the three libraries, suggesting that, in addition to the testes, sperm RNAs may also originate from seminal plasma, epididymis as well as other resources. Most miRNAs were co-distributed but differentially expressed across the three libraries, with epididymal sperm exhibiting the highest abundance, followed by ejaculated sperm and seminal plasma. The prediction of target genes of the top 10 highly expressed miRNAs across the three libraries revealed that these miRNAs may be involved in spermatogenesis, zygote development and the interaction between the environment and animals. Our study provides the first description of the similarities and differences of small RNA profiles in ejaculated sperm, epididymal sperm and seminal plasma and indicates that sperm RNA may have origins other than the testes.

Free access

Tong Sun, Shi-Jie Li, Hong-Lu Diao, Chun-Bo Teng, Hong-Bin Wang and Zeng-Ming Yang

Cyclooxygenase (COX), a rate-limiting enzyme that produces prostaglandins (PGs) from arachidonic acid, exists in two isoforms, COX-1 and COX-2. PGE2 synthase (PGES) is a terminal prostanoid synthase and can enzymatically convert the cyclooxygenase product PGH2 to PGE2, including two isoforms: microsomal PGES (mPGES) and cytosolic PGES (cPGES). cPGES is predominantly linked with COX-1 to promote the immediate response. mPGES is preferentially coupled with the inducible COX-2 to promote delayed PGE2 generation. COX-2-deficient female mice are infertile with abnormalities in ovulation, fertilization, implantation and decidualization. The aim of this study was to examine immunohistochemically the expression pattern of COX-1, COX-2, mPGES and cPGES proteins in the endometrium of the rhesus monkey during the menstrual cycle. COX-1 immunostaining was mainly localized in the luminal epithelium and glandular epithelium near the lumen, and detected in all the stages during the menstrual cycle. COX-2 immunostaining was mainly localized in the luminal and glandular epithelium, and strongly shown during the mid-luteal phase (days 16 and 20) of the menstrual cycle. There was a strong cPGES immunostaining in the luminal and glandular epithelium on days 12, 16, 20 and 25 of the menstrual cycle. mPGES immunostaining was strongly detected in the glandular epithelium on days 20 and 25 of the menstrual cycle. These data suggest that the coupling of cPGES and COX-1 in the luminal epithelium may be responsible for the synthesis of PGE2 in monkey endometrium, and the coupling of mPGES and COX-2 in the glandular epithelium may be of importance for preparing the receptive endometrium.

Free access

Xiaoxiao Hou, Jun Liu, Zhiren Zhang, Yanhui Zhai, Yutian Wang, Zhengzhu Wang, Bo Tang, Xueming Zhang, Liguang Sun and Ziyi Li

DNA methylation and histone modification play important roles in the development of mammalian embryos. Cytochalasin B (CB) is an actin polymerization inhibitor that can significantly affect cell activity and is often used in studies concerning cytology. In recent years, CB is also commonly being used in in vitro experiments on mammalian embryos, but few studies have addressed the effect of CB on the epigenetic modification of embryonic development, and the mechanism underlying this process is also unknown. This study was conducted to investigate the effects of CB on DNA methylation and histone modification in the development of parthenogenetically activated porcine embryos. Treatment with 5 μg/mL CB for 4 h significantly increased the cleavage rate, blastocyst rate and total cell number of blastocysts. However, the percentage of apoptotic cells and the expression levels of the apoptosis-related genes BCL-XL, BAX and CASP3 were significantly decreased. Treatment with CB significantly decreased the expression levels of DNMT1, DNMT3a, DNMT3b, HAT1 and HDAC1 at the pronuclear stage and promoted the conversion of 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC). After CB treatment, the level of AcH3K9 was upregulated and the level of H3K9me3 was downregulated. When combined with Scriptaid and 5-Aza-Cdr, CB further improved the embryonic development competence and decreased the expression of BCL-XL, BAX and CASP3. In conclusion, these results suggest that CB could improve embryonic development and the quality of the blastocyst by improving the epigenetic modification during the development of parthenogenetically activated embryos.

Free access

Rui-Song Ye, Meng Li, Chao-Yun Li, Qi-En Qi, Ting Chen, Xiao Cheng, Song-Bo Wang, Gang Shu, Li-Na Wang, Xiao-Tong Zhu, Qing-Yan Jiang, Qian-Yun Xi and Yong-Liang Zhang

FSH plays an essential role in processes involved in human reproduction, including spermatogenesis and the ovarian cycle. While the transcriptional regulatory mechanisms underlying its synthesis and secretion have been extensively studied, little is known about its posttranscriptional regulation. A bioinformatics analysis from our group indicated that a microRNA (miRNA; miR-361-3p) could regulate FSH secretion by potentially targeting the FSHB subunit. Herein, we sought to confirm these findings by investigating the miR-361-3p-mediated regulation of FSH production in primary pig anterior pituitary cells. Gonadotropin-releasing hormone (GnRH) treatment resulted in an increase in FSHB synthesis at both the mRNA, protein/hormone level, along with a significant decrease in miR-361-3p and its precursor (pre-miR-361) levels in time- and dose-dependent manner. Using the Dual-Luciferase Assay, we confirmed that miR-361-3p directly targets FSHB. Additionally, overexpression of miR-361-3p using mimics significantly decreased the FSHB production at both the mRNA and protein levels, with a reduction in both protein synthesis and secretion. Conversely, both synthesis and secretion were significantly increased following miR-361-3p blockade. To confirm that miR-361-3p targets FSHB, we designed FSH-targeted siRNAs, and co-transfected anterior pituitary cells with both the siRNA and miR-361-3p inhibitors. Our results indicated that the siRNA blocked the miR-361-3p inhibitor-mediated upregulation of FSH, while no significant effect on non-target expression. Taken together, our results demonstrate that miR-361-3p negatively regulates FSH synthesis and secretion by targeting FSHB, which provides more functional evidence that a miRNA is involved in the direct regulation of FSH.

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Chun-Bo Teng, Hong-Lu Diao, Hong Ma, Jing Cong, Hao Yu, Xing-Hong Ma, Li-Bin Xu and Zeng-Ming Yang

Signal transducer and activator of transcription 3 (Stat3), a member of the Stat family, is specifically activated during mouse embryo implantation. The aim of this study was to investigate the expression, activation and regulation of Stat3 in rat uterus during early pregnancy, pseudopregnancy, delayed implantation and artificial decidualization. Stat3 mRNA was highly expressed in the luminal epithelium on day 5 and in the luminal epithelium and underlying stromal cells at implantation sites on day 6 of pregnancy. There was a strong level of Stat3 protein expression and phosphorylation in the stromal cells near the lumen and in the luminal epithelium on day 5 of pregnancy, which was similar to day 5 of pseudopregnancy. In the afternoon of day 6, the strong level of Stat3 phosphorylation was detected only in the luminal epithelium. Stat3 was highly expressed and activated in the decidual cells from days 7 to 9 of pregnancy and under artificial decidualization in the present study. Our results suggest that the strong level of Stat3 activation in the luminal epithelium and underlying stromal cells during the pre-implantation period may be important for establishing uterine receptivity as in mice, and the high level of Stat3 expression and activation in decidual cells may play a role during decidualization.

Free access

Fei Gao, Jiyu Guan, Limei Liu, Sheng Zhang, Peipei An, Anran Fan, Guangqi Song, Peng Zhang, Tianchuang Zhao, Bo Tang, Xueming Zhang and Ziyi Li

The Wilms' tumour 1 (WT1) gene originally identified as a tumour suppressor associated with WTs encodes a zinc finger-containing transcription factor that is expressed in multiple tissues and is an important regulator of cellular and organ growth, proliferation, development, migration and survival. However, there is a deficiency of data regarding the expression and function of WT1 during oocyte maturation and preimplantation embryonic development. Herein, we sought to define the expression characteristics and functions of WT1 during oocyte maturation and preimplantation embryonic development in pigs. We show that WT1 is expressed in porcine oocytes and at all preimplantation stages in embryos generated by ICSI. We then evaluated the effects of down-regulating WT1 expression at germinal vesicle and early ICSI stages using a recombinant plasmid (pGLV3-WT1-shRNA). Down-regulation of WT1 did not affect oocyte maturation but significantly decreased preimplantation embryonic development and increased apoptosis in blastocysts. These results indicate that WT1 plays important roles in the development of porcine preimplantation embryos.