Comparative analysis of primate and pig cells reveals primate-specific PINK1 expression and phosphorylation

PTEN-induced putative kinase 1(PINK1),a mitochondrial kinase that phosphorylates Parkin and other proteins,plays a crucial role in mitophagy and protection against neurodegeneration.Mutations in PINK1 and Parkin can lead to loss of function and early onset Parkinson's disease.However,there is a lack of strong in vivo evidence in rodent models to support the theory that loss of PINK1 affects mitophagy and induces neurodegeneration.Additionally,PINK1 knockout pigs(Sus scrofa)do not appear to exhibit neurodegeneration.In our recent work involving non-human primates,we found that PINK1 is selectively expressed in primate brains,while absent in rodent brains.To extend this to other species,we used multiple antibodies to examine the expression of PINK1 in pig tissues.In contrast to tissues from cynomolgus monkeys(Macaca fascicularis),our data did not convincingly demonstrate detectable PINK1expression in pig tissues.Knockdown of PINK1 in cultured pig cells did not result in altered Parkin and BAD phosphorylation,as observed in cultured monkey cells.A comparison of monkey and pig striatum revealed more PINK1-phosphorylated substrates in the monkey brain.Consistently,PINK1 knockout in pigs did not lead to obvious changes in the phosphorylation of Parkin and BAD.These findings provide new evidence that PINK1expression is specific to primates,underscoring the importance of non-human primates in investigating PINK1function and pathology related to PINK1 deficiency.

Genetically modified pigs:Emerging animal models for hereditary hearing loss

Hereditary hearing loss(HHL),a genetic disorder that impairs auditory function,significantly affects quality of life and incurs substantial economic losses for society.To investigate the underlying causes of HHL and evaluate therapeutic outcomes,appropriate animal models are necessary.Pigs have been extensively used as valuable large animal models in biomedical research.In this review,we highlight the advantages of pig models in terms of ear anatomy,inner ear morphology,and electrophysiological characteristics,as well as recent advancements in the development of distinct genetically modified porcine models of hearing loss.Additionally,we discuss the prospects,challenges,and recommendations regarding the use pig models in HHL research.Overall,this review provides insights and perspectives for future studies on HHL using porcine models.

One-step cell biomanufacturing platform:porous gelatin microcarrier beads promote human embryonic stem cell-derived midbrain dopaminergic progenitor cell differentiation in vitro and survival after transplantation in vivo

Numerous studies have shown that cell replacement therapy can replenish lost cells and rebuild neural circuitry in animal models of Parkinson’s disease.Transplantation of midbrain dopaminergic progenitor cells is a promising treatment for Parkinson’s disease.However,transplanted cells can be injured by mechanical damage during handling and by changes in the transplantation niche.Here,we developed a one-step biomanufacturing platform that uses small-aperture gelatin microcarriers to produce beads carrying midbrain dopaminergic progenitor cells.These beads allow midbrain dopaminergic progenitor cell differentiation and cryopreservation without digestion,effectively maintaining axonal integrity in vitro.Importantly,midbrain dopaminergic progenitor cell bead grafts showed increased survival and only mild immunoreactivity in vivo compared with suspended midbrain dopaminergic progenitor cell grafts.Overall,our findings show that these midbrain dopaminergic progenitor cell beads enhance the effectiveness of neuronal cell transplantation.

Effect of 43℃ treatment on expression of heat shock proteins 105, 70 and 60 in cultured monkey Sertoli cells

Aim： To examine the possible effect of heat treatment on expression of heat shock proteins （Hsps） 105, 70, and 60 in primary monkey Sertoli cells and to evaluate the possible signal pathways. Methods： Western blot analysis, realtime polymerase chain reaction （PCR）, and confocal immunohistochemistry were used to analyze mRNA and protein levels of the Hsps in response to 43~C treatment of Sertoli cells isolated from pubertal monkey testes. Results： Staining with Hoechst 33342 indicated Sertoli cells did not undergo apoptosis after heat treatment. Hspl05 was expressed in cytoplasm of untreated Sertoli cells. Both Hspl05 mRNA and protein levels were increased approximately 20-fold compared to those of the untreated controls at 12 h after heat treatment. Untreated Sertoli cells did not express Hsp70, but heat stress induced its expression in the cell cytoplasm. The time-course of changes in Hsp70 was similar to that of Hsp105. In contrast to Hsp105 and Hsp70, the change in Hsp60 expression was much less obvious. The protein level between 12 h and 48 h after heat treatment was only approximately 1.5-fold that of the untreated control. Extracellular regulated kinase （ERK） 1/2 inhibitor （U0126） or phosphoinositide kinase-3 （PI3K） inhibitor （LY294002） could partially block the response of Hspl05 and Hsp70 induced by heat treatment. Conclusion： These results indicate that the heat-induced expression of the three types of Hsp in monkey Sertoli cells might be regulated by ERK and/or PI3K signal pathways, but the profile of their expression is different, suggesting that they might have different regulatory functions in Sertoli cells.

Aberrant DNA methylation in 5′ regions of DNA methyltransferase genes in aborted bovine clones

High rate of abortion and developmental abnormalities is thought to be closely associated with inefficient epigenetic reprogramming of the transplanted nuclei during bovine cloning. It is known that one of the important mechanisms for epigenetic reprogramming is DNA methylation. DNA methylation is established and maintained by DNA methyltransferases （DNMTs）, therefore, it is postulated that the inefficient epigenetic reprogramming of transplanted nuclei may be due to abnormal expression of DNMTs. Since DNA methylation can strongly inhibit gene expression, aberrant DNA methylation of DNMT genes may disturb gene expression. But presently, it is not clear whether the methylation abnormality of DNMT genes is related to developmental failure of somatic cell nuclear transfer embryos. In our study, we analyzed methylation patterns of the 5＇ regions of four DNMT genes including Dnmt3a, Dnmt3b, Dnmtl and Dnmt2 in four aborted bovine clones. Using bisulfite sequencing method, we found that 3 out of 4 aborted bovine clones （AF1, AF2 and AF3） showed either hypermethylation or hypomethylation in the 5＇ regions of Dnmt3a and Dnmt3b, indicating that Dnmt3a and Dnmt3b genes are not properly reprogrammed. However, the individual AF4 exhibited similar methylation level and pattern to age-matched in vitro fertilized （IVF） fetuses. Besides, we found that the 5＇ regions of Dnmtl and Dnmt2 were nearly completely unmethylated in all normal adults, IVF fetuses, sperm and aborted clones. Together, our results suggest that the aberrant methylation of Dnmt3a and Dnmt3b 5＇ regions is probably associated with the high abortion of bovine clones.

Activation of extracellular signal-related kinases 1 and 2 in Sertoli cells in experimentally cryptorchid rhesus monkeys

Aim： To assess the spatiotemporal changes in the expression of extracellular signal-regulated kinases 1 and 2 （ERK1/ 2）, c-Jun N-terminal kinases （JNK） and p38 mitogen-activated protein kinases （MAPK） in response to heat stress in the cryptorchid testis, and to investigate a possible relation to Sertoli cell dedifferentiation. Methods： Immunohistochemistry and western blot were used to examine the expression and activation of ERK1/2, p38 and JNK in the cryptorchid testis at various stages after experimental cryptorchidism. Results： The abdominal temperature did not obviously change the total ERK1/2 expression but significantly activated phospho-ERK1/2 in the Sertoli cells of the cryptorchid testis. Heat stress increased total JNK expression in the Sertoli cells of the cryptorchid testis but did not activate phospho-JNK. Neither total p38 nor phospho-p38 was induced by heat stress in the Sertoli cells of the cryptorchid testis. Changes in the spatiotemporal expression of cytokeratin 18 （CK18）, a marker of immature or undifferentiated Sertoli cells, were induced in the cryptorchid testis in a pattern similar to the activation of ERK1/2. Condusion： The activation of ERK1/2 in the testis may be related to dedifferentiation of Sertoli cells under heat stress induced by experimental cryptorchidism.

Derivation of human embryonic stem cell lines from parthenogenetic blastocysts

单性生殖是之一主要、很有用，方法到导出胚胎的干细胞(转换字符) ，它可以为房间治疗是 histocompatible 房间和纸巾的重要来源。这里，我们描述从在 vitro 的线(hPES-1 和 hPES-2 ) 开发了胚囊列在后面的二个转换字符的推导和描述人的卵母细胞的单性生殖的激活。典型转换字符形态学被看见，并且转换字符标记的表示是是为碱的磷酸酶期望， octamer 有约束力的抄写因素 4，阶段特定的胚胎的抗原 3，阶段特定的胚胎的抗原 4， TRA-1-60，和 TRA-1-81，并且有否定标记例如的表示的缺席阶段特定的胚胎的抗原 1。为不同胚胎的细菌层特定的基因的表示从两根 hESC 线的胚胎植物或动物身体(EB ) 被检测，建议他们在 vitro 的区别潜力。在 vivo，然而，仅仅 hPES-1 形成了由所有三胚胎的细菌层组成的 teratoma (hPES-2 ) 。在为超过 100 个段落的连续增长以后，有趣地， hPES-1 房间仍然维持了正常 46 XX karyotype；hPES-2 在长期的段落以后显示了象染色体 translocation 那样的畸形。短双人脚踏车重复(STR ) 结果证明 hPES 线是有鸡蛋施主的基因火柴，并且基因印数据证实了这些 ES 房间的单性生殖的起源。染色体宽的 SNP 分析显示出典型地代表单性生殖的一个模式。所有这些结果表明了可行性孤立并且建立人的单性生殖的转换字符线，它提供一个重要工具因为学习 epigenetic 在一个临床的背景在转换字符以及为未来完成治疗学的干预。

Enrichment of putative human epidermal stem cells based on cell size and collagen type IV adhesiveness

人的表皮的干细胞(EpSCs ) 的丰富和鉴定具有为基本研究和临床的申请的首要的重要性。尽管为 EpSCs 的丰富的几条途径被建立了，充实可行 EpSCs 的一张纯人口仍然是一项挑战性的任务。一条改进途径值得发展提高 EpSCs 的纯净和生存能力。这里，我们报导那种房间尺寸与骨胶原类型 IV 粘着性结合了能在一条改进途径被使用充实纯、可行的人的 EpSCs。我们分开了很快支持者 keratinocytes 进从 5-7 microm (人口 A )在尺寸 的三张人口到 7-9 microm (人口 B )，到>或在直径的 =9 microm (人口 C )，并且发现人的通常认为的 EpSCs 能进一步与最小的尺寸在人口 A 被充实。在三张人口之中，人口 A 显示了 beta1-integrin 受体的最高的密度，在 G0/G1 阶段包含了房间的最高的百分比，显示出最高的原子核到细胞质比率，并且拥有了最高的殖民地形成效率(CFE ) 。当注入了鼠科的胚囊时，这些房间参予了多织物形成。与根据 beta1-integrin 抗体染色排序通常认为的 EpSCs 的一条以前的途径相比，更显著地， EpSCs 的生存能力由改进途径充实显著地被提高。我们的结果为人的 EpSCs 的丰富提供通常认为的策略，并且在干细胞生物学鼓励进一步的学习进房间尺寸的角色。

Non-genetic contributions of the sperm nucleus to embryonic development

Recent data from several laboratories have provided evidence that the newly fertilized oocyte inherits epigenetic signals from the sperm chromatin that are required for proper embryonic development. For the purposes of this review, the term epigenetic is used to describe all types of molecular information that are transmitted from the sperm cell to the embryo. There are at least six different forms of epigenetic information that have already been established as being required for proper embryogenesis in mammals or for which there is evidence that it may do so. These are （i） DNA methylation; （ii） sperm-specific histones, （iii） other chromatin-associated proteins; （iv） the perinuclear theca proteins; （v） sperm-born RNAs and, the focus of this review; and （vi） the DNA loop domain organization by the sperm nuclear matrix. These epigenetic signals should he considered when designing protocols for the manipulation and cryopreservation of spermatozoa for assisted reproductive technology as necessary components for effective fertilization and subsequent embryo development.

CENP-A Regulates Chromosome Segregation during the First Meiosis of Mouse Oocytes

Proper chromosome separation in both mitosis and meiosis depends on the correct connection between kinetochores of chromosomes and spindle microtubules. Kinetochore dysfunction can lead to unequal distribution of chromosomes during cell division and result in aneuploidy, thus kinetochores are critical for faithful segregation of chromosomes. Centromere protein A(CENP-A) is an important component of the inner kinetochore plate. Multiple studies in mitosis have found that deficiencies in CENP-A could result in structural and functional changes of kinetochores, leading to abnormal chromosome segregation, aneuploidy and apoptosis in cells. Here we report the expression and function of CENP-A during mouse oocyte meiosis. Our study found that microinjection of CENP-A blocking antibody resulted in errors of homologous chromosome segregation and caused aneuploidy in eggs. Thus, our findings provide evidence that CENP-A is critical for the faithful chromosome segregation during mammalian oocyte meiosis.

UniPron is A Fully Effective Non-hormonal Reversible Contraceptive in Baboon Model (Papio Anubis)

Objective To determine the safety and efficacy of UniPron as a reversible contraceptive.Methods Vaginal swabs were obtained before and after UniPron administration, cultured onto appropriate culture media and bacteria identification was done based on type of media used, Gram stain reactions, colony morphology and biochemical tests. Vaginal biopsy tissues were processed using paraffin wax method, stained with hematoxylin and eosin and examined under light microscopy to determine the effect of the product on vaginal tissues. The effect of UniPron on sperm was examined by mixing the product with electroejaculated spermatozoa in vitro at different concentrations. For efficacy studies, male baboons of proven fertility were mated with UniPron treated or untreated females of proven fertility during the fertile stages. Results All the five females （100%） that were treated with UniPron did not conceive and they regained total fertility when the treatment was stopped while all the controls conceived. At a concentration of 40%, UniPron completely immobilized spermatozoa in an in-vitro system. UniPron mechanism of action was by lowering the vaginal pH and on application in baboon, the pH was lowered for at least 3 h after which it went back to normal.Conclusions As we plan for a study to test UniPron as a microbicide to prevent STIs including HIV, our current study has established that this novel product is effective in contraception and harmless to vaginal tissues and vaginal microbial flora in a baboon model （Papio anubis）.

Effects of chronic heat stress on granulosa cell apoptosis and follicular atresia in mouse ovary

Background： Heat stress is known to alter follicular dynamics and granulosa cell function and may contribute to the diminished reproductive efficiency commonly observed in mammals during the summer. Although several investigators have studied heat-induced ovarian injury, few reports have focused on the effects of chronic heat stress on ovarian function and the molecular mechanisms through which it induces ovarian injury.Methods： In Exp. 1, 48 female mice were assigned to a control or heat-stressed treatment. After exposure to a constant temperature of 25 ℃ for 7, 14, 21 or 28 d（n = 6） or to 42 ℃ for 3 h per d for 7, 14, 21 or 28 d（n = 6）, the mice were euthanized and their ovaries were analyzed for follicular atresia, granulosa cell apoptosis, changes in the abundance of HSP70 protein and serum concentrations of estradiol. In Exp. 2, the expression of HSP70 and aromatase was quantified in antral follicles cultured in vitro at 37 or 42 ℃ for 24 h. In Exp. 3, granulosa cells from ovaries maintained at 37 or 41 ℃ for 2 h were analyzed for their expression of HSP70, Bim, caspase-3 and cleaved caspase-3.Results： In Exp. 1, body weight and food intake of heat-stressed mice decreased（P 〈 0.05） compared with control mice while the concentration of estradiol in serum was lower（P 〈 0.05） in heat-stressed mice than in control mice. Compared with control mice, the percentage of atretic follicles and the number of antral follicles with severe apoptotic signals were increased（P 〈 0.05） after 21 d of heat-stressed treatment. HSP70 protein was more abundant（P 〈 0.05） in heat-stressed mice than control mice. In Exp. 2, heat stress increased HSP70 and decreased aromatase proteins（P 〈 0.05） in antral follicles. In Exp. 3, TUNEL-positive granulosa cells from heat-stressed ovaries were observed concomitant with a significant increase in HSP70, Bim and cleaved caspase-3 protein.Conclusion： Heat-stress in mice decrease estradiol in serum and aromatase in antral follicles but increased number of atretic follicles and granulosa cell undergoing apoptosis which may explain the decreased fertility commonly observed in heat-stressed animals.

Induction of differentiation of human stem cells ex vivo: Toward large-scale platelet production

Platelet transfusion is one of the most reliable strategies to cure patients suffering from thrombocytopenia or platelet dysfunction.With the increasing demand for transfusion,however,there is an undersupply of donors to provide the platelet source.Thus,scientists have sought to design methods for deriving clinical-scale platelets ex vivo.Although there has been considerable success ex vivo in the generation of transformative platelets produced by human stem cells(SCs),the platelet yields achieved using these strategies have not been adequate for clinical application.In this review,we provide an overview of the developmental process of megakaryocytes and the production of platelets in vivo and ex vivo,recapitulate the key advances in the production of SC-derived platelets using several SC sources,and discuss some strategies that apply three-dimensional bioreactor devices and biochemical factors synergistically to improve the generation of large-scale platelets for use in future biomedical and clinical settings.

Progress in Understanding the Functional Roles of Extracellular Vesicles in Reproduction

Exosomes,a type of extracellular vesicles(EVs),were first discovered in sheep reticulocytes in 1983[1],and after four years,the name‘exosome’was officially given to them.A variety of cells secrete exosomes under both normal and pathological conditions.Exosomes are mainly products of themultivesicular body formed by intracellular lysosomal invagi nation,and released into the extracellular space by fusion of the outer membrane of the multivesicular body with the cell membrane.

Efficient production of pronuclear embryos in breeding and nonbreeding season for generating transgenic sheep overexpressing TLR4

Background： Brucella is a zoonotic Gram-negative pathogen that causes abortion and infertility in ruminants and humans. TLR4 is the receptor for LPS which can recognize Brucella and initiate antigen-presenting cell activities that affect both innate and adaptive immunity. Consequently, transgenic sheep over-expressing TLR4 are an suitable model to investigate the effects of TLR4 on preventing Brucellosis. In this study, we generated transgenic sheep overexpressing TLR4 and aimed to evaluate the effects of different seasons（breeding and non-breeding season） on superovulation and the imported exogenous gene on growth.Results： In total of 43 donor ewes and 166 recipient ewes in breeding season, 37 donor ewes and 144 recipient ewes in non-breeding season were selected for super-ovulation and injected embryo transfer to generate transgenic sheep.Our results indicated the no. of embryos recovered of donors and the rate of pronuclear embryos did not show any significant difference between breeding and non-breeding seasons（P 〉 0.05）. The positive rate of exogenous TLR4 tested were 21.21 % and 22.58 % in breeding and non-breeding season by Southern blot. The expression level of TLR4 in the transgenic sheep was 1.5 times higher than in the non-transgenic group（P 〈 0.05）. The lambs overexpressing TLR4 had similar growth performance with non-transgenic lambs, and the blood physiological parameters of transgenic and non-transgenic were both in the normal range and did not show any difference.Conclusions： Here we establish an efficient platform for the production of transgenic sheep by the microinjection of pronuclear embryos during the whole year. The over-expression of TLR4 had no adverse effect on the growth of the sheep.

Generation of patient-specific pluripotent stem cells and directed differentiation of embryonic stem cells for regenerative medicine

Embryonic stem（ES） cells are pluripotent cells that can give rise to derivatives of all three embryonic germ layers. Due to its characteristics, the patient-specific ES cells are of great potential for transplantation therapies. Several strategies can reprogramme somatic cells back to pluripotent stem cells： nuclear transfer, fusion with ES cells, treatment with cell extract and induction by specific factors. Considering the future clinical use, the differentiation from ES to neurons, cardiomyocytes and many other types of cells currently provide basic cognition and experience to regenerative medicine. This article will review two courses, the reprogramming of differentiated cells and the differentiation of ES cells to specific cell types.

Plasminogen Activator and Plasminogen Activator Inhibitor in Mouse Ovaries during Periovulatory Periods

Changes of ovarian tPA,uPA and PA inhibitor activities were examined in PMSG-and hCG-treatedimmature mice during periovulatory periods.The results show that 15% of the gonadotropin-treatedanimals ovulated 8 hrs after hCG administration,about 6-8 hrs earlier than in rat.It is also shownthat not only tPA activity,but also uPA activity,was regulated by gonadotropins in ovarianhomogenates and granulosa cells,and they reached maximum prior to ovulation.No measurableamount of PAI-1 activity could be detected in mouse granulosa cell conditioned medium andfollicular fluid,but considerable amount of α_2-antiplasmin,a specific inhibitor for plasmin,wasfound in follicular fluid.Cumulus-oocyte complexes contain mainly tPA.Since the ovulated eggsstill have high tPA activity,it is thought that the enzyme in the oocyte may play an important rolein implantation.