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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.

Open access

Jing Liu, Yang Wang, Peng Chen, Yue Ma, Shuo Wang, Ye Tian, Anna Wang and Danbo Wang

Previous lncRNA microarray screening found that the AC002454.1 gene was highly expressed in endometriosis (EMS), and these expression levels were highly correlated with cyclin-dependent kinase-6 (CDK6). This study investigated the expression level and correlation between AC002454.1 and CDK6 in endometrium tissues and the influence of these changes in expression upon the biological behavior of eutopic endometrial cells. We confirmed AC002454.1 and CDK6 mRNA and protein were highly expressed in ectopic and eutopic endometrial tissue from patients with EMS and were clearly correlated. In vitro, both AC002454.1 and CDK6 positively regulated the proliferation, migration and invasion ability of eutopic endometrial cells and could promote the transformation of cells from G0/G1 phase to S phase. AC002454.1 and CDK6 may have synergistic effects, thereby affecting the biological behavior of endometrial cells, and thus promote the progression of EMS.

Free access

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.

Free access

Jing Wang, Fan Wu, Qingzhen Xie, Xiaorui Liu, Fuju Tian, Wangming Xu and Jing Yang

Bacteria and viruses activate the host innate immune response via Toll-like receptor (TLR)-involved signaling and potentially cause pregnancy failure. TLR7 and TLR9 respond to single-stranded RNA (a viral intermediate) and hypomethylated CpG DNA motifs (specific molecular constituents of bacteria) respectively. In this study, we treated murine RAW264.7 cells with R837, CpG1826, or a combination of the two. RT-PCR was performed to detect cytokines, Tlr7, and Tlr9. WT and nonobese diabetic murine embryo resorption models were established by i.p. injections of TLR7 and TLR9 ligands. Neutralizing antibodies and the IL1β and TNFα inhibitors were used. The specific inhibitors anakinra and etanercept effectively prevented TLR7 and TLR9 ligand-induced embryo loss. Notably, this effect was not observed in decidual NK cell-depleted mice. Our findings suggest that anakinra and etanercept may have potential for preventing TLR7 or TLR9 ligand-induced abortion in the presence of decidual NK cells.

Free access

Huizhen Wang, Haikun Wang, Weipeng Xiong, Yongmei Chen, Quanhong Ma, Jing Ma, Yehua Ge and Daishu Han

During spermatogenesis, more than half of the differentiating spermatogenic cells undergo apoptosis before they mature into spermatozoa. Ultrastructure studies showed that the formation of lipid droplets in Sertoli cells was associated with phagocytosis of residual bodies and apoptotic germ cells by Sertoli cells. Here, a relationship between the phagocytosis of apoptotic spermatogenic cells and lipid droplet formation in Sertoli cells was studied in vitro by Oil Red O (ORO) staining. The results confirmed that the formation of lipid droplets was a result of phagocytosis of apoptotic spermatogenic cells in Sertoli cells. By comparing phagocytosis of apoptotic spermatogenic cells and thymocytes by Sertoli cells to that by macrophages, we demonstrated that the lipid droplets accumulation in phagocytes depended on phagocytosed apoptotic cell type, but not phagocyte type. However, the size of lipid droplets was related to the type of phagocytes. By this approach, we found that Sertoli cells at different postnatal stages of development had a similar phagocytic ability. These results suggested that the detection of lipid droplets by ORO staining was a practical method to evaluate the phagocytic functions of Sertoli cells in vitro. This approach could also be considered as an in vitro model to study the lipid formation, metabolism, and function in Sertoli cells.

Free access

Jiaojiao Huang, Hongyong Zhang, Jing Yao, Guosong Qin, Feng Wang, Xianlong Wang, Ailing Luo, Qiantao Zheng, Chunwei Cao and Jianguo Zhao

Accumulating evidence suggests that faulty epigenetic reprogramming leads to the abnormal development of cloned embryos and results in the low success rates observed in all mammals produced through somatic cell nuclear transfer (SCNT). The aberrant methylation status of H3K9me and H3K9me2 has been reported in cloned mouse embryos. To explore the role of H3K9me2 and H3K9me in the porcine somatic cell nuclear reprogramming, BIX-01294, known as a specific inhibitor of G9A (histone-lysine methyltransferase of H3K9), was used to treat the nuclear-transferred (NT) oocytes for 14–16 h after activation. The results showed that the developmental competence of porcine SCNT embryos was significantly enhanced both in vitro (blastocyst rate 16.4% vs 23.2%, P<0.05) and in vivo (cloning rate 1.59% vs 2.96%) after 50 nm BIX-01294 treatment. BIX-01294 treatment significantly decreased the levels of H3K9me2 and H3K9me at the 2- and 4-cell stages, which are associated with embryo genetic activation, and increased the transcriptional expression of the pluripotency genes SOX2, NANOG and OCT4 in cloned blastocysts. Furthermore, the histone acetylation levels of H3K9, H4K8 and H4K12 in cloned embryos were decreased after BIX-01294 treatment. However, co-treatment of activated NT oocytes with BIX-01294 and Scriptaid rescued donor nuclear chromatin from decreased histone acetylation of H4K8 that resulted from exposure to BIX-01294 only and consequently improved the preimplantation development of SCNT embryos (blastocyst formation rates of 23.7% vs 21.5%). These results indicated that treatment with BIX-01294 enhanced the developmental competence of porcine SCNT embryos through improvements in epigenetic reprogramming and gene expression.

Free access

BiJun Wang, Jing Li, QingLing Yang, FuLi Zhang, MengMeng Hao and YiHong Guo

This study aimed to explore the association between soluble receptor for advanced glycation end products (sRAGE) levels in follicular fluid and the number of oocytes retrieved and to evaluate the effect of sRAGE on vascular endothelial growth factor (VEGF) in granulosa cells in patients with polycystic ovarian syndrome (PCOS). Two sets of experiments were performed in this study. In part one, sRAGE and VEGF protein levels in follicular fluid samples from 39 patients with PCOS and 35 non-PCOS patients were measured by ELISA. In part two, ovarian granulosa cells were isolated from an additional 10 patients with PCOS and cultured. VEGF and SP1 mRNA and protein levels, as well as pAKT levels, were detected by real-time PCR and Western blotting after cultured cells were treated with different concentrations of sRAGE. Compared with the non-PCOS patients, patients with PCOS had lower sRAGE levels in follicular fluid. Multi-adjusted regression analysis showed that high sRAGE levels in follicular fluid predicted a lower Gn dose, more oocytes retrieved, and a better IVF outcome in the non-PCOS group. Logistic regression analysis showed that higher sRAGE levels predicted favorably IVF outcomes in the non-PCOS group. Multi-adjusted regression analysis also showed that high sRAGE levels in follicular fluid predicted a lower Gn dose in the PCOS group. Treating granulosa cells isolated from patients with PCOS with recombinant sRAGE decreased VEGF and SP1 mRNA and protein expression and pAKT levels in a dose-dependent manner.

Free access

Pengpeng Ma, Yehua Ge, Shali Wang, Jing Ma, Shepu Xue and Daishu Han

Transplantation of spermatogonial stem cells in cross-species has been widely used to study the function of Sertoli cells and the effect of phylogenetic distance between donor and recipient animals on the outcome of spermatogonial transplantation, whereas there have been only a few reports on the transplantation of testis tissue. The objective of the present study was to examine the development of grafted testes and the kinetics of spermatogenesis following syngeneic testicular transplantation in both male and female recipient Balb/c mice in an effort to establish an in vivo culture system and to compare the effects of host sex on spermatogenesis. The testes from 5-day-old Balb/c mice were transplanted under the dorsal skin of four-week-old mice. Twenty male and twenty female Balb/c mice were used as the hosts and each host received 4 grafts. The recipient mice were killed at 1, 2, 3, 5, 7, 9, 12 and 15 weeks after transplantation. The graft survival rate and graft size were measured. The status of spermatogenesis was assessed by histological analyses. The expression of the spermatid-specific Protamine-2 gene was examined by RT-PCR. Overall, 70.3% of the testicular grafts in male hosts and 67.2% in female hosts survived. All recovered grafts had increased in volume, some of them had increased by more than 30-fold. The architecture of the seminiferous tubules in female hosts appeared to be better than that in male hosts. The round spermatids were the most advanced germ cells until 15 weeks after transplantation, and no complete spermatozoon was observed in any of the grafts. The expression of protamine-2 was detected in grafts from 5 weeks posttransplantation in both male and female hosts, confirming that the spermatogenic cells differentiated into spermatids. In contrast to grafts, the testes of male hosts had a normal histological appearance. The results showed the schedule of spermatogenesis following syngeneic testicular transplantation in both male and female hosts. This model could be useful for further studies involving the endocrinology of the testis and the mechanisms of spermatogenesis.

Free access

Yongmei Chen, Huizhen Wang, Nan Qi, Hui Wu, Weipeng Xiong, Jing Ma, Qingxian Lu and Daishu Han

Mice lacking TYRO3, AXL and MER (TAM) receptor tyrosine kinases (RTKs) are male sterile. The mechanism of TAM RTKs in regulating male fertility remains unknown. In this study, we analyzed in more detail the testicular phenotype of TAM triple mutant (TAM−/−) mice with an effort to understand the mechanism. We demonstrate that the three TAM RTKs cooperatively regulate male fertility, and MER appears to be more important than AXL and TYRO3. TAM−/− testes showed a progressive loss of germ cells from elongated spermatids to spermatogonia. Young adult TAM−/− mice exhibited oligo-astheno-teratozoospermia and various morphological malformations of sperm cells. As the mice aged, the germ cells were eventually depleted from the seminiferous tubules. Furthermore, we found that TAM−/− Sertoli cells have an impaired phagocytic activity and a large number of differentially expressed genes compared to wild-type controls. By contrast, the function of Leydig cells was not apparently affected by the mutation of TAM RTKs. Therefore, we conclude that the suboptimal function of Sertoli cells leads to the impaired spermatogenesis in TAM−/− mice. The results provide novel insight into the mechanism of TAM RTKs in regulating male fertility.

Free access

Irene Ruiz-González, Jing Xu, Xiaoqiu Wang, Robert C Burghardt, Kathrin A Dunlap and Fuller W Bazer

Conceptus–endometrial communication during the peri-implantation period of pregnancy ensures establishment of pregnancy. We hypothesized that this dialog involves exosomes, ovine endogenous jaagsiekte retroviruses (enJSRV) and toll-like receptors (TLR) which regulate the secretion of interferon tau (IFNT), the pregnancy recognition signal in ruminants. First, exosomes isolated from uterine flushings from cyclic and pregnant ewes were analyzed for exosomal content and uterine expression of heat shock protein 70 (HSC70). Then, conceptus trophectoderm cells (oTr1) treated with different doses of exosomes were analyzed for the expression of genes involved in TLR-mediated cell signaling. The results revealed that exosomes contain mRNAs for enJSRV-ENV, HSC70, interleukins, and interferon (IFN)-regulatory factors. Exosomal content of enJSRV-ENV mRNA and protein decreased from days 10 and 12 to day 16 of gestation, and uterine expression of HSC70 increased in pregnant ewes compared with cyclic ewes. The oTr1 cells proliferated and secreted IFNT in a dose-dependent manner in response to exosomes from cyclic ewes. The expression of CD14, CD68, IRAK1, TRAF6, IRF6, and IRF7 mRNAs that are key to TLR-mediated expression of type 1 IFNs was significantly influenced by day of pregnancy. This study demonstrated that exosomes are liberated into the uterine lumen during the estrous cycle and early pregnancy; however, in pregnant ewes, exosomes stimulate trophectoderm cells to proliferate and secrete IFNT coordinately with regulation of TLR-mediated cell signaling. These results support our hypothesis that free and/or exosomal enJSRV act on the trophectoderm via TLR to induce the secretion of IFNT in a manner similar to that for innate immune responses of macrophages and plasmacytoid dendritic cells to viral pathogens.