Normal and neoplastic growth of the prostate gland are dependent on androgen receptor （AR） expression and function. Androgenic activation of the AR, in association with its coregulatory factors, is the classical pat...Normal and neoplastic growth of the prostate gland are dependent on androgen receptor （AR） expression and function. Androgenic activation of the AR, in association with its coregulatory factors, is the classical pathway that leads to transcriptional activity of AR target genes. Alternatively, cytoplasmic signaling crosstalk of AR by growth factors, neurotrophic peptides, cytokines or nonandrogenic hormones may have important roles in prostate carcinogenesis and in metastatic or androgen-independent （AI） progression of the disease. In addition, cross-modulation by various nuclear transcription factors acting through basal transcriptional machinery could positively or negatively affect the AR or AR target genes expression and activity. Androgen ablation leads to an initial favorable response in a significant number of patients; however, almost invariably patients relapse with an aggressive form of the disease known as castration-resistant or hormone-refractory prostate cancer （PCa）. Understanding critical molecular events that lead PCa cells to resist androgen-deprivation therapy is essential in developing successful treatments for hormone-refractory disease. In a significant number of hormone-refractory patients, the AR is overexpressed, mutated or genomically amplified. These genetic alterations maintain an active presence for a highly sensitive AR, which is responsive to androgens, antiandrogens or nonandrogenic hormones and collectively confer a selective growth advantage to PCa cells. This review provides a brief synopsis of the AR structure, AR coregulators, posttranslational modifications of AR, duality of AR function in prostate epithelial and stromal cells, AR-dependent signaling, genetic changes in the form of somatic and germline mutations and their known functional significance in PCa cells and tissues.展开更多
The rapid developments of science and technology in China over recent decades, particularly in biomedical research, have brought forward serious challenges regarding ethical governance. Recently, Jian-kui HE, a Chines...The rapid developments of science and technology in China over recent decades, particularly in biomedical research, have brought forward serious challenges regarding ethical governance. Recently, Jian-kui HE, a Chinese scientist, claimed to have "created" the first gene-edited babies, designed to be naturally immune to the human immunodeficiency virus(HIV). The news immediately triggered widespread criticism, denouncement, and debate over the scientific and ethical legitimacy of HE’s genetic experiments. China’s guidelines and regulations have banned germline genome editing on human embryos for clinical use because of scientific and ethical concerns, in accordance with the international consensus. HE’s human experimentation has not only violated these Chinese regulations, but also breached other ethical and regulatory norms. These include questionable scientific value, unreasonable risk-benefit ratio, illegitimate ethics review, invalid informed consent, and regulatory misconduct. This series of ethical failings of HE and his team reveal the institutional failure of the current ethics governance system which largely depends on scientist’s self-regulation. The incident highlights the need for urgent improvement of ethics governance at all levels, the enforcement of technical and ethical guidelines, and the establishment of laws relating to such bioethical issues.展开更多
The field of reproductive biology has undergone significant developments in the last decade. The notion that there is a fixed reserve pool of oocytes before birth was established by Zuckerman in 1951. However, in 2004...The field of reproductive biology has undergone significant developments in the last decade. The notion that there is a fixed reserve pool of oocytes before birth was established by Zuckerman in 1951. However, in 2004, an article published in nature challenged this central dogma of mammalian reproductive biology. Tilly's group reported the existence of ovarian germline stem cells(GSCs) in postnatal ovaries of mice and suggested that the bone marrow could be an extragonadal source of ovarian GSCs. These findings were strongly criticized; however, several independent groups have sincesuccessfully isolated and characterized ovarian GSCs in postnatal mice. The ovarian GSCs are located in the ovarian surface epithelium and express markers of undifferentiated GSCs. When transplanted into mouse ovaries, mouse ovarian GSCs could differentiate and produce embryos and offspring. Similarly, in a recent study, ovarian GSCs were found to be present in the ovaries of women of reproductive age. Conversely, there is increasing evidence that stem cells responsible for maintaining a healthy state in normal tissue may be a source of some cancers, including ovarian cancer. Cancer stem cells(CSCs) have been found in many tissues, including ovaries. Some researchers have suggested that ovarian cancer may be a result of the transformation and dysfunction of ovarian GSCs with self-renewal properties. Drug resistant and metastasisgenerating CSCs are responsible for many important problems affecting ovarian cancer patients. Therefore, the identification of CSCs will provide opportunities for the development of new therapeutic strategies for treatments for infertility and ovarian cancer. In this article, we summarize the current understanding of ovarian GSCs in adult mammals, and we also discuss whether there is a relationship between GSCs and CSCs.展开更多
AIM: To analyze the prevalence of germline MLH1 and MSH2 gene mutations and evaluate the clinical characteristics of Hungarian hereditary non-polyposis colorectal cancer (HNPCC) families. METHODS: Thirty-six kindreds ...AIM: To analyze the prevalence of germline MLH1 and MSH2 gene mutations and evaluate the clinical characteristics of Hungarian hereditary non-polyposis colorectal cancer (HNPCC) families. METHODS: Thirty-six kindreds were tested for mutations using conformation sensitive gel electrophoreses, direct sequencing and also screening for genomic rearrangements applying multiplex ligation-dependent probe amplifi cation (MLPA). RESULTS: Eighteen germline mutations (50%) were identifi ed, 9 in MLH1 and 9 in MSH2. Sixteen of these sequence alterations were considered pathogenic, the remaining two were non-conservative missense alterations occurring at highly conserved functional motifs. The majority of the defi nite pathogenic mutations (81%, 13/16) were found in families fulfilling the stringent Amsterdam Ⅰ/Ⅱ criteria, including three rearrangements revealed by MLPA (two in MSH2 and one in MLH1). However, in three out of sixteen HNPCC-suspected families (19%), a disease-causing alteration could be revealed. Furthermore, nine mutations described here are novel, and none of the sequence changes were found in more than one family.CONCLUSION: Our study describes for the f irst time the prevalence and spectrum of germline mismatch repair gene mutations in Hungarian HNPCC and suspected-HNPCC families. The results presented here suggest that clinical selection criteria should be relaxed and detection of genomic rearrangements should be included in genetic screening in this population.展开更多
We are entering an exciting epoch in livestock biotechnology during which the fundamental approaches(such as transgenesis, spermatozoa cryopreservation and artificial insemination) will be enhanced based on the modern...We are entering an exciting epoch in livestock biotechnology during which the fundamental approaches(such as transgenesis, spermatozoa cryopreservation and artificial insemination) will be enhanced based on the modern understanding of the biology of spermatogonial stem cells(SSCs) combined with the outstanding recent advances in genomic editing technologies and in vitro cell culture systems. The general aim of this review is to outline comprehensively the promising applications of SSC manipulation that could in the nearest future find practical application in livestock breeding. Here, we will focus on 1) the basics of mammalian SSC biology;2) the approaches for SSC isolation and purification;3) the available in vitro systems for the stable expansion of isolated SSCs;4) a discussion of how the manipulation of SSCs can accelerate livestock transgenesis;5) a thorough overview of the techniques of SSC transplantation in livestock species(including the preparation of recipients for SSC transplantation,the ultrasonographic-guided SSC transplantation technique in large farm animals, and the perspectives to improve further the SSC transplantation efficiency), and finally, 6) why SSC transplantation is valuable to extend the techniques of spermatozoa cryopreservation and/or artificial insemination. For situations where no reliable data have yet been obtained for a particular livestock species, we will rely on the data obtained from studies conducted in rodents because the knowledge gained from rodent research is translatable to livestock species to a great extent. On the other hand, we will draw special attention to situations where such translation is not possible.展开更多
Only a small number of cells in adult tissues （the stem cells） possess the ability to self-renew at every cell division, while producing differentiating daughter cells to maintain tissue homeostasis for an organism...Only a small number of cells in adult tissues （the stem cells） possess the ability to self-renew at every cell division, while producing differentiating daughter cells to maintain tissue homeostasis for an organism＇s lifetime. The Drosophila ovary harbors three different types of stem cell populations （germline stem cell （GSC）, somatic stem cell （SSC） and escort stem cell （ESC）） located in a simple anatomical structure known as germarium, rendering it one of the best model systems for studying stem cell biology due to reliable stem cell identification and available sophisticated genetic tools for manipulating gene functions. Particularly, the niche for the GSC is among the first and best studied ones, and studies on the GSC and its niche have made many unique contributions to a better understanding of relationships between stem cells and their niche. So far, both the GSC and the SSC have been shown to be regulated by extrinsic factors originating from their niche and intrinsic factors functioning within. Multiple signaling pathways are required for controlling GSC and SSC self-renewal and differentiation, which provide unique opportunities to investigate how multiple signals from the niche are interpreted in the stem cell. Since the Drosophila ovary contains three types of stem cells, it also provides outstanding opportunities to study how multiple stem cells in a given tissue work collaboratively to contribute to tissue function and maintenance. This review highlights recent major advances in studying Drosophila ovarian stem cells and also discusses future directions and challenges.展开更多
Background: The prevalence of Lynch syndrome and screening strategies for this disorder in Chinese patients with endometrial cancer have seldom been investigated. Such data would be essential for the screening, preven...Background: The prevalence of Lynch syndrome and screening strategies for this disorder in Chinese patients with endometrial cancer have seldom been investigated. Such data would be essential for the screening, prevention, genetic counseling, and treatment of Lynch syndrome. The purpose of this prospective study was to determine the accuracy of the mismatch repair (MMR) protein immunohistochemistry (IHC), microsatellite instability (MSI) test, and clinical diagnostic criteria in screening for Lynch syndrome-associated endometrial cancer (LS-EC) in a prospective Chinese cohort. Methods: All patients with newly diagnosed endometrial cancer (EC) were evaluated using clinical diagnostic criteria (Amsterdam II criteria and the revised Bethesda guidelines), MSI test, and IHC of MMR proteins in tumor tissues. For all patients, the screening results were compared with results of germline sequencing for pathogenic variants of MMR genes. Results: Between December 2017 and August 2018, a total of 111 unselected patients with newly diagnosed EC were enrolled. Six patients (5.4%) harbored a pathogenic germline mutation of MMR genes: 1 had a mutation in MutL homolog 1 (MLH1), 2 in MutS homolog 2 (MSH2), and 3 in MutS homolog 6 (MSH6). The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for identifying LS-EC were 33.3%, 88.6%, 14.3%, and 95.9%, for the clinical criteria, 66.7%, 75.0%, 14.3%, and 97.3% for IHC of MMR proteins, 100%, 89.9%, 33.3%, and 100% for MSI test, and 100%, 72.4%, 20.0% and 100% for combined IHC and MSI test, respectively. The combination of IHC and MSI test had higher sensitivity and PPV than the clinical criteria (p = 0.030). MSI test and IHC were highly concordant for LS-EC screening (73/77, 94.8%). Conclusion: The accuracy of the combination of IHC of MMR proteins and MSI test for screening LS among Chinese patients with EC was superior to that of the clinical criteria.展开更多
Objective: Germline alterations in the breast cancer susceptibility genes type 1 and 2, BRCA1 and BRCA2, predispose individuals to hereditary cancers, including breast, ovarian, prostate, pancreatic, and stomach cance...Objective: Germline alterations in the breast cancer susceptibility genes type 1 and 2, BRCA1 and BRCA2, predispose individuals to hereditary cancers, including breast, ovarian, prostate, pancreatic, and stomach cancers.Accumulating evidence suggests inherited genetic susceptibility to lung cancer.The present study aimed to survey the prevalence of pathogenic germline BRCA mutations(gBRCAm) and explore the potential association between gBRCAm and disease onset in Chinese advanced non-small cell lung cancer(NSCLC) patients.Methods: A total of 6,220 NSCLC patients were screened using capture-based ultra-deep targeted sequencing to identify patients harboring germline BRCA1/2 mutations.Results: Out of the 6,220 patients screened, 1.03%(64/6,220) of the patients harbored the pathogenic gB RCAm, with BRCA2 mutations being the most predominant mutations(49/64, 76.5%).Patients who developed NSCLC before 50 years of age were more likely to carry gBRCAm(P = 0.036).Among the patients harboring classic lung cancer driver mutations, those with concurrent gBRCAm were significantly younger than those harboring the wild-type gBRCA(P = 0.029).By contrast, the age of patients with or without concurrent gBRCAm was comparable to those of patients without the driver mutations(P = 0.972).In addition, we identified EGFR-mutant patients with concurrent gBRCAm who showed comparable progression-free survival but significantly longer overall survival(P = 0.002) compared to EGFR-mutant patients with wild-type germline BRCA.Conclusions: Overall, our study is the largest survey of the prevalence of pathogenic gBRCAm in advanced Chinese NSCLC patients.Results suggested a lack of association between germline BRCA status and treatment outcome of EGFR-TKI.In addition,results showed a positive correlation between pathogenic gB RCAm and an early onset of NSCLC.展开更多
The mutation rate is a pivotal biological characteristic,intricately governed by natural selection and historically garnering considerable attention.Recent advances in high-throughput sequencing and analytical methodo...The mutation rate is a pivotal biological characteristic,intricately governed by natural selection and historically garnering considerable attention.Recent advances in high-throughput sequencing and analytical methodologies have profoundly transformed our understanding in this domain,ushering in an unprecedented era of mutation rate research.This paper aims to provide a comprehensive overview of the key concepts and methodologies frequently employed in the study of mutation rates.It examines various types of mutations,explores the evolutionary dynamics and associated theories,and synthesizes both classical and contemporary hypotheses.Furthermore,this review comprehensively explores recent advances in understanding germline and somatic mutations in animals and offers an overview of experimental methodologies,mutational patterns,molecular mechanisms,and driving forces influencing variations in mutation rates across species and tissues.Finally,it proposes several potential research directions and pressing questions for future investigations.展开更多
Gastric cancer is one of the leading causes of cancer-related deaths worldwide. Among which, about 1%–3% of gastric cancer patients were characterized by inherited gastric cancer predisposition syndromes, knowing as ...Gastric cancer is one of the leading causes of cancer-related deaths worldwide. Among which, about 1%–3% of gastric cancer patients were characterized by inherited gastric cancer predisposition syndromes, knowing as hereditary diffuse gastric cancer（HDGC）. Studies reported that CDH1 germline mutations are the main cause of HDGC. With the help of rapid development of genetic testing technologies and data analysis tools, more and more researchers focus on seeking candidate susceptibility genes for hereditary cancer syndromes. In addition, National Comprehensive Cancer Network（NCCN） guidelines recommend that the patients of HDGC carrying CDH1 mutations should undergo prophylactic gastrectomy or routine endoscopic surveillances. Therefore, genetic counseling plays a key role in helping individuals with pathogenic mutations make appropriate risk management plans. Moreover, experienced and professional genetic counselors as well as a systematic multidisciplinary team（MDT） are also required to facilitate the development of genetic counseling and benefit pathogenic mutation carriers who are in need of regular and standardized risk management solutions. In this review, we provided an overview about the germline mutations of several genes identified in HDGC, suggesting that these genes may potentially act as susceptibility genes for this malignant cancer syndrome. Furthermore, we introduced information for prevention, diagnosis and risk management of HDGC. Investigations on key factors that may have effect on risk management decision-making and genetic data collection of more cancer syndrome family pedigrees are required for the development of HDGC therapeutic strategies.展开更多
AIM: To explore germline hypermethylation of the tumor suppressor genes MLH1, CDH1 and P16INK4a in suspected cases of hereditary gastric cancer (GC). METHODS: A group of 140 Chinese GC patients in whom the primary can...AIM: To explore germline hypermethylation of the tumor suppressor genes MLH1, CDH1 and P16INK4a in suspected cases of hereditary gastric cancer (GC). METHODS: A group of 140 Chinese GC patients in whom the primary cancer had developed before the age of 60 or who had a familial history of cancer were screened for germline hypermethylation of the MLH1, CDH1 and P16INK4a tumor suppressor genes. GenomicDNA was extracted from peripheral blood leukocytes and modified by sodium bisulfite. The treated DNA was then subjected to bisulfi te DNA sequencing for a specif ic region of the MLH1 promoter. The methylation status of CDH1 or P16INK4a was assayed using methylation-specif ic PCR. Clonal bisulf ite allelic sequencing in positive samples was performed to obtain a comprehensive analysis of the CpG island methylation status of these promoter regions. RESULTS: Methylation of the MLH1 gene promoter was detected in the peripheral blood DNA of only 1/140 (0.7%) of the GC patient group. However, this methylation pattern was mosaic rather than the allelic pattern which has previously been reported for MLH1 in hereditary non-polyposis colorectal cancer (HNPCC) patients. We found that 10% of the MLH1 alleles in the peripheral blood DNA of this patient were methylated, consistent with 20% of cells having one methylated allele. No germline promoter methylation of the CDH1 or P16INK4a genes was detected. CONCLUSION: Mosaic germline epimutation of the MLH1 gene is present in suspected hereditary GC patients in China but at a very low level. Germline epimutation of the CDH1 or P16INK4a gene is not a frequent event.展开更多
Maize is the most important agricultural crop used for food, feed, and biofuel as well as a raw material for industrial products such as packaging material. To increase yield and to overcome hybridization barriers, st...Maize is the most important agricultural crop used for food, feed, and biofuel as well as a raw material for industrial products such as packaging material. To increase yield and to overcome hybridization barriers, studies of maize gamete development, the pollen tube journey, and fertilization mechanisms were initiated more than a century ago. In this review, we summarize and discuss our current understanding of the regulatory components for germline development including sporogenesis and gametogenesis, the pro- gamic phase of pollen germination and pollen tube growth and guidance, as well as fertilization mecha- nisms consisting of pollen tube arrival and reception, sperm cell release, fusion with the female gametes, and egg cell activation. Mechanisms of asexual seed development are not considered here. While only a few molecular players involved in these processes have been described to date and the underlying mech- anisms are far from being understood, maize now represents a spearhead of reproductive research for all grass species. Recent development of essentially improved transformation and gene-editing systems may boost research in this area in the near future.展开更多
Genomes are incredibly dynamic within diverse eukaryotes and programmed genome rearrangements(PGR)play important roles in generating genomic diversity.However,genomes and chromosomes in metazoans are usually large in ...Genomes are incredibly dynamic within diverse eukaryotes and programmed genome rearrangements(PGR)play important roles in generating genomic diversity.However,genomes and chromosomes in metazoans are usually large in size which prevents our understanding of the origin and evolution of PGR.To expand our knowledge of genomic diversity and the evolutionary origin of complex genome rearrangements,we focus on ciliated protists(ciliates).Ciliates are single-celled eukaryotes with highly fragmented somatic chromosomes and massively scrambled germline genomes.PGR in ciliates occurs extensively by removing massive amounts of repetitive and selfish DNA elements found in the silent germline genome dur-ing development of the somatic genome.We report the partial germline genomes of two spirotrich ciliate species,namely Strombidium cf.sulcatum and Halteria grandinella,along with the most compact and highly fragmented somatic genome for S.cf.sulcatum.We provide the first insights into the genome rearrangements of these two species and compare these features with those of other ciliates.Our analyses reveal:(1)DNA sequence loss through evolution and during PGR in S.cf.sulcatum has combined to produce the most compact and efficient nanochromosomes observed to date;(2)the compact,transcriptome-like somatic genome in both species results from extensive removal of a relatively large number of shorter germline-specific DNA sequences;(3)long chromosome breakage site motifs are duplicated and retained in the somatic genome,revealing a complex model of chromosome fragmentation in spirotrichs;(4)gene scrambling and alternative pro-cessing are found throughout the core spirotrichs,offering unique opportunities to increase genetic diversity and regulation in this group.展开更多
Double sex and mab-3-related transcription factor 1(Dmrt1),which is expressed in goat male germline stem cells(mGSCs)and Sertoli cells,is one of the most conserved transcription factors involved in sex determination.I...Double sex and mab-3-related transcription factor 1(Dmrt1),which is expressed in goat male germline stem cells(mGSCs)and Sertoli cells,is one of the most conserved transcription factors involved in sex determination.In this study,we highlighted the role of Dmrt1 in balancing the innate immune response in goat mGSCs.Dmrt1 recruited promyelocytic leukemia zinc finger(Plzf),also known as zinc finger and BTB domain-containing protein 16(Zbtb16),to repress the Toll-like receptor 4(TLR4)-dependent inflammatory signaling pathway and nuclear factor(NF)-κB.Knockdown of Dmrt1 in seminiferous tubules resulted in widespread degeneration of germ and somatic cells,while the expression of proinflammatory factors were significantly enhanced.We also demonstrated that Dmrt1 stimulated proliferation of mGSCs,but repressed apoptosis caused by the immune response.Thus,Dmrt1 is sufficient to reduce inflammation in the testes,thereby establishing the stability of spermatogenesis and the testicular microenvironment.展开更多
Germlines in plants are formed de novo during post-embryonic development, while little is known about the mechanism that controls this process. In Arabidopsis, the earliest gene controlling this process is SPOROCYTELE...Germlines in plants are formed de novo during post-embryonic development, while little is known about the mechanism that controls this process. In Arabidopsis, the earliest gene controlling this process is SPOROCYTELESS （SPL）. A decade ago, we showed that loss of SPL function abolished sporogenesis in both male and female organs of Arabidopsis. However, its function is unclear up to now. In this study, we showed that SPL belongs to a novel transcription repressor family specific in embryophyte, which consists of 173 members in the land plants so far. All of them contain a conserved SPL-motif in their N-terminal and an ethylene-responsive element binding factor-associated amphiphilic repression （EAR） motif in the C-terminal, therefore designated as SPL-like, EAR-containing proteins （SPEARs）. Consis- tently, SPL acts as a transcriptional repressor in yeast and tobacco cells, and SPEAR proteins are able to form homodimer and/or het- erodimer with each other in vitro. Furthermore, SPEARs interact with the TOPLESS （TPL） co-repressors via the EAR motif and TCP family transcription factors in yeast cells. Together, we propose that SPL and SPEARs most likely belong to a novel transcription repressor family in land plants which may play a variety of developmental roles in plants.展开更多
MET tyrosine kinase and its ligand,hepatocyte growth factor（HGF）,play a pivotal role in the activties of tumor cells.A germline missense variant in exon 2 of the MET gene,N375S（rs33917957 A〉G）,may alter the bindi...MET tyrosine kinase and its ligand,hepatocyte growth factor（HGF）,play a pivotal role in the activties of tumor cells.A germline missense variant in exon 2 of the MET gene,N375S（rs33917957 A〉G）,may alter the binding affinity of MET for HGF and thus modify the risk of tumorigenesis.In this study,we performed a case-control study to assess the association between N375S and gastric cancer risk in 1,681 gastric cancer cases and 1,858 cancer-free controls.Logistic regression analysis was applied to estimate crude and adjusted odds ratios（ORs） and 95% confidence intervals（CIs） for the associations between genotypes and gastric cancer risk.We found that MET N375S variant genotypes（NS/SS） were associated with a significantly decreased risk of gastric cancer（OR = 0.78,95% CI = 0.63-0.96,P = 0.021） compared with the wildtype homozygote（NN）.The finding indicates that this germline variant in MET may decrease gastric cancer susceptibility in Han Chinese.展开更多
Pluripotent stem cells are unspecialized cells withunlimited self-renewal, and they can be triggered to differentiate into desired specialized cell types. These features provide the basis for an unlimited cell source ...Pluripotent stem cells are unspecialized cells withunlimited self-renewal, and they can be triggered to differentiate into desired specialized cell types. These features provide the basis for an unlimited cell source for innovative cell therapies. Pluripotent cells also allow to study developmental pathways, and to employ them or their differentiated cell derivatives in pharmaceutical testing and biotechnological applications. Via blastocyst complementation, pluripotent cells are a favoured tool for the generation of genetically modified mice. The recently established technology to generate an induced pluripotency status by ectopic co-expression of the transcription factors Oct4, Sox2, Klf4 and c-Myc allows to extending these applications to farm animal species, for which the derivation of genuine embryonic stem cells was not successful so far. Most induced pluripotent stem(i PS) cells are generated by retroviral or lentiviral transduction of reprogramming factors. Multiple viral integrations into the genome may cause insertional mutagenesis and may increase the risk of tumour formation. Non-integration methods have been reported to overcome the safety concerns associated with retro and lentiviral-derived i PS cells, such as transient expression of the reprogramming factors using episomal plasmids, and direct delivery of reprogramming m RNAs or proteins. In this review, we focus on the mechanisms of cellular reprogramming and current methods used to induce pluripotency. We also highlight problems associated with the generation of i PS cells. An increased understanding of the fundamental mechanisms underlying pluripotency and refining the methodology of i PS cell generation will have a profound impact on future development and application in regenerative medicine and reproductive biotechnology of farm animals.展开更多
Background: Gonocytes give rise to spermatogonial stem cells, and thereby play an essential role in establishing spermatogenesis. Optimized culture conditions for gonocytes provide an opportunity for their study and i...Background: Gonocytes give rise to spermatogonial stem cells, and thereby play an essential role in establishing spermatogenesis. Optimized culture conditions for gonocytes provide an opportunity for their study and in vitro manipulation for potential application in reproductive technologies. Using six experiments in a step-wise design, we examined the effects of several culture conditions on the maintenance, proliferation, and colony formation of porcine gonocytes. Testis cells from neonatal piglets were cultured for 7 d in DMEM supplemented with 10% fetal bovine serum. The examined culture conditions included using different cell seeding densities, gonocyte proportions, incubation temperatures, sampling strategies, and medium changing regimens.Results: Confluency of cel s was optimal(>90% by ~6 d) when 3.0 × 104 testis cel s/cm2 containing ~40% gonocytes were used. Incubating the cel s at 35 °C or 37 °C resulted in similar cel number and viability at confluency, but incubation at 35 °C resulted in a delayed confluency. In the first 2 d of culture, gonocytes remained mostly floating in the medium and gradual y settled over the next 5 d. Consequently, not changing the medium for 7 d(as opposed to changing it every 2 d) led to a significant increase in the number of gonocyte colonies by reducing the loss of "floating gonocytes".Conclusion: We found that gonocytes require the presence of a critical minimum number of somatic cel s for settlement, and can proliferate and form growing colonies even in a basic medium. Large numbers of viable gonocytes remain floating in the medium for several days. The optimized culture conditions in the present study included seeding with 3.0 × 104 testis cel s/cm2 containing ~40% gonocytes, incubating at 37 °C, and without changing the medium in the first week, which can result in improved colony formation of porcine gonocytes.展开更多
文摘Normal and neoplastic growth of the prostate gland are dependent on androgen receptor （AR） expression and function. Androgenic activation of the AR, in association with its coregulatory factors, is the classical pathway that leads to transcriptional activity of AR target genes. Alternatively, cytoplasmic signaling crosstalk of AR by growth factors, neurotrophic peptides, cytokines or nonandrogenic hormones may have important roles in prostate carcinogenesis and in metastatic or androgen-independent （AI） progression of the disease. In addition, cross-modulation by various nuclear transcription factors acting through basal transcriptional machinery could positively or negatively affect the AR or AR target genes expression and activity. Androgen ablation leads to an initial favorable response in a significant number of patients; however, almost invariably patients relapse with an aggressive form of the disease known as castration-resistant or hormone-refractory prostate cancer （PCa）. Understanding critical molecular events that lead PCa cells to resist androgen-deprivation therapy is essential in developing successful treatments for hormone-refractory disease. In a significant number of hormone-refractory patients, the AR is overexpressed, mutated or genomically amplified. These genetic alterations maintain an active presence for a highly sensitive AR, which is responsive to androgens, antiandrogens or nonandrogenic hormones and collectively confer a selective growth advantage to PCa cells. This review provides a brief synopsis of the AR structure, AR coregulators, posttranslational modifications of AR, duality of AR function in prostate epithelial and stromal cells, AR-dependent signaling, genetic changes in the form of somatic and germline mutations and their known functional significance in PCa cells and tissues.
基金Project supported by the National Natural Science Foundation of China(No.L1824000)
文摘The rapid developments of science and technology in China over recent decades, particularly in biomedical research, have brought forward serious challenges regarding ethical governance. Recently, Jian-kui HE, a Chinese scientist, claimed to have "created" the first gene-edited babies, designed to be naturally immune to the human immunodeficiency virus(HIV). The news immediately triggered widespread criticism, denouncement, and debate over the scientific and ethical legitimacy of HE’s genetic experiments. China’s guidelines and regulations have banned germline genome editing on human embryos for clinical use because of scientific and ethical concerns, in accordance with the international consensus. HE’s human experimentation has not only violated these Chinese regulations, but also breached other ethical and regulatory norms. These include questionable scientific value, unreasonable risk-benefit ratio, illegitimate ethics review, invalid informed consent, and regulatory misconduct. This series of ethical failings of HE and his team reveal the institutional failure of the current ethics governance system which largely depends on scientist’s self-regulation. The incident highlights the need for urgent improvement of ethics governance at all levels, the enforcement of technical and ethical guidelines, and the establishment of laws relating to such bioethical issues.
文摘The field of reproductive biology has undergone significant developments in the last decade. The notion that there is a fixed reserve pool of oocytes before birth was established by Zuckerman in 1951. However, in 2004, an article published in nature challenged this central dogma of mammalian reproductive biology. Tilly's group reported the existence of ovarian germline stem cells(GSCs) in postnatal ovaries of mice and suggested that the bone marrow could be an extragonadal source of ovarian GSCs. These findings were strongly criticized; however, several independent groups have sincesuccessfully isolated and characterized ovarian GSCs in postnatal mice. The ovarian GSCs are located in the ovarian surface epithelium and express markers of undifferentiated GSCs. When transplanted into mouse ovaries, mouse ovarian GSCs could differentiate and produce embryos and offspring. Similarly, in a recent study, ovarian GSCs were found to be present in the ovaries of women of reproductive age. Conversely, there is increasing evidence that stem cells responsible for maintaining a healthy state in normal tissue may be a source of some cancers, including ovarian cancer. Cancer stem cells(CSCs) have been found in many tissues, including ovaries. Some researchers have suggested that ovarian cancer may be a result of the transformation and dysfunction of ovarian GSCs with self-renewal properties. Drug resistant and metastasisgenerating CSCs are responsible for many important problems affecting ovarian cancer patients. Therefore, the identification of CSCs will provide opportunities for the development of new therapeutic strategies for treatments for infertility and ovarian cancer. In this article, we summarize the current understanding of ovarian GSCs in adult mammals, and we also discuss whether there is a relationship between GSCs and CSCs.
基金Supported by the Hungarian Research Grants OTKA T-046570, NKFPI-00024/2005 and ETT 397/2006
文摘AIM: To analyze the prevalence of germline MLH1 and MSH2 gene mutations and evaluate the clinical characteristics of Hungarian hereditary non-polyposis colorectal cancer (HNPCC) families. METHODS: Thirty-six kindreds were tested for mutations using conformation sensitive gel electrophoreses, direct sequencing and also screening for genomic rearrangements applying multiplex ligation-dependent probe amplifi cation (MLPA). RESULTS: Eighteen germline mutations (50%) were identifi ed, 9 in MLH1 and 9 in MSH2. Sixteen of these sequence alterations were considered pathogenic, the remaining two were non-conservative missense alterations occurring at highly conserved functional motifs. The majority of the defi nite pathogenic mutations (81%, 13/16) were found in families fulfilling the stringent Amsterdam Ⅰ/Ⅱ criteria, including three rearrangements revealed by MLPA (two in MSH2 and one in MLH1). However, in three out of sixteen HNPCC-suspected families (19%), a disease-causing alteration could be revealed. Furthermore, nine mutations described here are novel, and none of the sequence changes were found in more than one family.CONCLUSION: Our study describes for the f irst time the prevalence and spectrum of germline mismatch repair gene mutations in Hungarian HNPCC and suspected-HNPCC families. The results presented here suggest that clinical selection criteria should be relaxed and detection of genomic rearrangements should be included in genetic screening in this population.
基金supported by the S grant of the Ministry of Education,Youth and Sport(MEYS)of Czech Republicsupported by the Primus Research Programme PRIMUS/17/MED/16 of the Charles University
文摘We are entering an exciting epoch in livestock biotechnology during which the fundamental approaches(such as transgenesis, spermatozoa cryopreservation and artificial insemination) will be enhanced based on the modern understanding of the biology of spermatogonial stem cells(SSCs) combined with the outstanding recent advances in genomic editing technologies and in vitro cell culture systems. The general aim of this review is to outline comprehensively the promising applications of SSC manipulation that could in the nearest future find practical application in livestock breeding. Here, we will focus on 1) the basics of mammalian SSC biology;2) the approaches for SSC isolation and purification;3) the available in vitro systems for the stable expansion of isolated SSCs;4) a discussion of how the manipulation of SSCs can accelerate livestock transgenesis;5) a thorough overview of the techniques of SSC transplantation in livestock species(including the preparation of recipients for SSC transplantation,the ultrasonographic-guided SSC transplantation technique in large farm animals, and the perspectives to improve further the SSC transplantation efficiency), and finally, 6) why SSC transplantation is valuable to extend the techniques of spermatozoa cryopreservation and/or artificial insemination. For situations where no reliable data have yet been obtained for a particular livestock species, we will rely on the data obtained from studies conducted in rodents because the knowledge gained from rodent research is translatable to livestock species to a great extent. On the other hand, we will draw special attention to situations where such translation is not possible.
文摘Only a small number of cells in adult tissues （the stem cells） possess the ability to self-renew at every cell division, while producing differentiating daughter cells to maintain tissue homeostasis for an organism＇s lifetime. The Drosophila ovary harbors three different types of stem cell populations （germline stem cell （GSC）, somatic stem cell （SSC） and escort stem cell （ESC）） located in a simple anatomical structure known as germarium, rendering it one of the best model systems for studying stem cell biology due to reliable stem cell identification and available sophisticated genetic tools for manipulating gene functions. Particularly, the niche for the GSC is among the first and best studied ones, and studies on the GSC and its niche have made many unique contributions to a better understanding of relationships between stem cells and their niche. So far, both the GSC and the SSC have been shown to be regulated by extrinsic factors originating from their niche and intrinsic factors functioning within. Multiple signaling pathways are required for controlling GSC and SSC self-renewal and differentiation, which provide unique opportunities to investigate how multiple signals from the niche are interpreted in the stem cell. Since the Drosophila ovary contains three types of stem cells, it also provides outstanding opportunities to study how multiple stem cells in a given tissue work collaboratively to contribute to tissue function and maintenance. This review highlights recent major advances in studying Drosophila ovarian stem cells and also discusses future directions and challenges.
基金This study was supported by the Chinese Academy of Medical Sciences Initiative for Innovative Medicine(CAMS-2017-I2M-1-002)by the National Science-technology Support Plan Projects(2015BAI13B04).
文摘Background: The prevalence of Lynch syndrome and screening strategies for this disorder in Chinese patients with endometrial cancer have seldom been investigated. Such data would be essential for the screening, prevention, genetic counseling, and treatment of Lynch syndrome. The purpose of this prospective study was to determine the accuracy of the mismatch repair (MMR) protein immunohistochemistry (IHC), microsatellite instability (MSI) test, and clinical diagnostic criteria in screening for Lynch syndrome-associated endometrial cancer (LS-EC) in a prospective Chinese cohort. Methods: All patients with newly diagnosed endometrial cancer (EC) were evaluated using clinical diagnostic criteria (Amsterdam II criteria and the revised Bethesda guidelines), MSI test, and IHC of MMR proteins in tumor tissues. For all patients, the screening results were compared with results of germline sequencing for pathogenic variants of MMR genes. Results: Between December 2017 and August 2018, a total of 111 unselected patients with newly diagnosed EC were enrolled. Six patients (5.4%) harbored a pathogenic germline mutation of MMR genes: 1 had a mutation in MutL homolog 1 (MLH1), 2 in MutS homolog 2 (MSH2), and 3 in MutS homolog 6 (MSH6). The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for identifying LS-EC were 33.3%, 88.6%, 14.3%, and 95.9%, for the clinical criteria, 66.7%, 75.0%, 14.3%, and 97.3% for IHC of MMR proteins, 100%, 89.9%, 33.3%, and 100% for MSI test, and 100%, 72.4%, 20.0% and 100% for combined IHC and MSI test, respectively. The combination of IHC and MSI test had higher sensitivity and PPV than the clinical criteria (p = 0.030). MSI test and IHC were highly concordant for LS-EC screening (73/77, 94.8%). Conclusion: The accuracy of the combination of IHC of MMR proteins and MSI test for screening LS among Chinese patients with EC was superior to that of the clinical criteria.
基金supported by grant from the National Natural Science Foundation of China (Grant No.81502699)
文摘Objective: Germline alterations in the breast cancer susceptibility genes type 1 and 2, BRCA1 and BRCA2, predispose individuals to hereditary cancers, including breast, ovarian, prostate, pancreatic, and stomach cancers.Accumulating evidence suggests inherited genetic susceptibility to lung cancer.The present study aimed to survey the prevalence of pathogenic germline BRCA mutations(gBRCAm) and explore the potential association between gBRCAm and disease onset in Chinese advanced non-small cell lung cancer(NSCLC) patients.Methods: A total of 6,220 NSCLC patients were screened using capture-based ultra-deep targeted sequencing to identify patients harboring germline BRCA1/2 mutations.Results: Out of the 6,220 patients screened, 1.03%(64/6,220) of the patients harbored the pathogenic gB RCAm, with BRCA2 mutations being the most predominant mutations(49/64, 76.5%).Patients who developed NSCLC before 50 years of age were more likely to carry gBRCAm(P = 0.036).Among the patients harboring classic lung cancer driver mutations, those with concurrent gBRCAm were significantly younger than those harboring the wild-type gBRCA(P = 0.029).By contrast, the age of patients with or without concurrent gBRCAm was comparable to those of patients without the driver mutations(P = 0.972).In addition, we identified EGFR-mutant patients with concurrent gBRCAm who showed comparable progression-free survival but significantly longer overall survival(P = 0.002) compared to EGFR-mutant patients with wild-type germline BRCA.Conclusions: Overall, our study is the largest survey of the prevalence of pathogenic gBRCAm in advanced Chinese NSCLC patients.Results suggested a lack of association between germline BRCA status and treatment outcome of EGFR-TKI.In addition,results showed a positive correlation between pathogenic gB RCAm and an early onset of NSCLC.
文摘The mutation rate is a pivotal biological characteristic,intricately governed by natural selection and historically garnering considerable attention.Recent advances in high-throughput sequencing and analytical methodologies have profoundly transformed our understanding in this domain,ushering in an unprecedented era of mutation rate research.This paper aims to provide a comprehensive overview of the key concepts and methodologies frequently employed in the study of mutation rates.It examines various types of mutations,explores the evolutionary dynamics and associated theories,and synthesizes both classical and contemporary hypotheses.Furthermore,this review comprehensively explores recent advances in understanding germline and somatic mutations in animals and offers an overview of experimental methodologies,mutational patterns,molecular mechanisms,and driving forces influencing variations in mutation rates across species and tissues.Finally,it proposes several potential research directions and pressing questions for future investigations.
基金supported by Beijing Municipal Administration of Hospitals’ Youth Program (QML20151003)Beijing Municipal Administration of Hospitals Clinical Medicine Development of Special Funding Support (ZYLX201701)Inner Mongolia Science & Technology Plan (kjt13sf04)
文摘Gastric cancer is one of the leading causes of cancer-related deaths worldwide. Among which, about 1%–3% of gastric cancer patients were characterized by inherited gastric cancer predisposition syndromes, knowing as hereditary diffuse gastric cancer（HDGC）. Studies reported that CDH1 germline mutations are the main cause of HDGC. With the help of rapid development of genetic testing technologies and data analysis tools, more and more researchers focus on seeking candidate susceptibility genes for hereditary cancer syndromes. In addition, National Comprehensive Cancer Network（NCCN） guidelines recommend that the patients of HDGC carrying CDH1 mutations should undergo prophylactic gastrectomy or routine endoscopic surveillances. Therefore, genetic counseling plays a key role in helping individuals with pathogenic mutations make appropriate risk management plans. Moreover, experienced and professional genetic counselors as well as a systematic multidisciplinary team（MDT） are also required to facilitate the development of genetic counseling and benefit pathogenic mutation carriers who are in need of regular and standardized risk management solutions. In this review, we provided an overview about the germline mutations of several genes identified in HDGC, suggesting that these genes may potentially act as susceptibility genes for this malignant cancer syndrome. Furthermore, we introduced information for prevention, diagnosis and risk management of HDGC. Investigations on key factors that may have effect on risk management decision-making and genetic data collection of more cancer syndrome family pedigrees are required for the development of HDGC therapeutic strategies.
基金Supported by The National Natural Science Foundation of China, No. 30972535the Natural Science Foundation of Jiangsu, China, No. BK2008269the Fundamental Research Funds for the Central Universities of China, No. 1112021402
文摘AIM: To explore germline hypermethylation of the tumor suppressor genes MLH1, CDH1 and P16INK4a in suspected cases of hereditary gastric cancer (GC). METHODS: A group of 140 Chinese GC patients in whom the primary cancer had developed before the age of 60 or who had a familial history of cancer were screened for germline hypermethylation of the MLH1, CDH1 and P16INK4a tumor suppressor genes. GenomicDNA was extracted from peripheral blood leukocytes and modified by sodium bisulfite. The treated DNA was then subjected to bisulfi te DNA sequencing for a specif ic region of the MLH1 promoter. The methylation status of CDH1 or P16INK4a was assayed using methylation-specif ic PCR. Clonal bisulf ite allelic sequencing in positive samples was performed to obtain a comprehensive analysis of the CpG island methylation status of these promoter regions. RESULTS: Methylation of the MLH1 gene promoter was detected in the peripheral blood DNA of only 1/140 (0.7%) of the GC patient group. However, this methylation pattern was mosaic rather than the allelic pattern which has previously been reported for MLH1 in hereditary non-polyposis colorectal cancer (HNPCC) patients. We found that 10% of the MLH1 alleles in the peripheral blood DNA of this patient were methylated, consistent with 20% of cells having one methylated allele. No germline promoter methylation of the CDH1 or P16INK4a genes was detected. CONCLUSION: Mosaic germline epimutation of the MLH1 gene is present in suspected hereditary GC patients in China but at a very low level. Germline epimutation of the CDH1 or P16INK4a gene is not a frequent event.
文摘Maize is the most important agricultural crop used for food, feed, and biofuel as well as a raw material for industrial products such as packaging material. To increase yield and to overcome hybridization barriers, studies of maize gamete development, the pollen tube journey, and fertilization mechanisms were initiated more than a century ago. In this review, we summarize and discuss our current understanding of the regulatory components for germline development including sporogenesis and gametogenesis, the pro- gamic phase of pollen germination and pollen tube growth and guidance, as well as fertilization mecha- nisms consisting of pollen tube arrival and reception, sperm cell release, fusion with the female gametes, and egg cell activation. Mechanisms of asexual seed development are not considered here. While only a few molecular players involved in these processes have been described to date and the underlying mech- anisms are far from being understood, maize now represents a spearhead of reproductive research for all grass species. Recent development of essentially improved transformation and gene-editing systems may boost research in this area in the near future.
基金funded by the Laoshan Laboratory(No.LSKJ202203202)the National Natural Science Foundation of China(32270539,31922013,31961123002)+3 种基金the Natural Science Foundation of Shandong Province(ZR2020JQ13)Royal Society/NSFC International Exchanges Cost Share Project(IEC\NSFC\201024)National Institutes of Health(P40OD010964)the Fundamental Research Funds for the Central Universities(202141004).
文摘Genomes are incredibly dynamic within diverse eukaryotes and programmed genome rearrangements(PGR)play important roles in generating genomic diversity.However,genomes and chromosomes in metazoans are usually large in size which prevents our understanding of the origin and evolution of PGR.To expand our knowledge of genomic diversity and the evolutionary origin of complex genome rearrangements,we focus on ciliated protists(ciliates).Ciliates are single-celled eukaryotes with highly fragmented somatic chromosomes and massively scrambled germline genomes.PGR in ciliates occurs extensively by removing massive amounts of repetitive and selfish DNA elements found in the silent germline genome dur-ing development of the somatic genome.We report the partial germline genomes of two spirotrich ciliate species,namely Strombidium cf.sulcatum and Halteria grandinella,along with the most compact and highly fragmented somatic genome for S.cf.sulcatum.We provide the first insights into the genome rearrangements of these two species and compare these features with those of other ciliates.Our analyses reveal:(1)DNA sequence loss through evolution and during PGR in S.cf.sulcatum has combined to produce the most compact and efficient nanochromosomes observed to date;(2)the compact,transcriptome-like somatic genome in both species results from extensive removal of a relatively large number of shorter germline-specific DNA sequences;(3)long chromosome breakage site motifs are duplicated and retained in the somatic genome,revealing a complex model of chromosome fragmentation in spirotrichs;(4)gene scrambling and alternative pro-cessing are found throughout the core spirotrichs,offering unique opportunities to increase genetic diversity and regulation in this group.
基金This work was supported by the China National Basic Research Program(2016YFA0100203)National Natural Science Foundation of China(31572399Detail,32072806,32072815,32002246)+3 种基金State Key Lab of Reproductive Regulation&Breeding of Grassland Livestock(SKL-OT-201801)Science and Technology Major Project of Inner Mongolia Autonomous Region of China(ZDZX2018065)and Shaanxi Province Science and Technology Innovation Team(2019TD-036)The authors thank Dr.John Clotaire Daguia Zambe for helpful comments about this paper,Jia Fang for the PGL3-NF-κB luciferase reporter plasmid,and Dong-Xue Che for bioinformatics analysis.
文摘Double sex and mab-3-related transcription factor 1(Dmrt1),which is expressed in goat male germline stem cells(mGSCs)and Sertoli cells,is one of the most conserved transcription factors involved in sex determination.In this study,we highlighted the role of Dmrt1 in balancing the innate immune response in goat mGSCs.Dmrt1 recruited promyelocytic leukemia zinc finger(Plzf),also known as zinc finger and BTB domain-containing protein 16(Zbtb16),to repress the Toll-like receptor 4(TLR4)-dependent inflammatory signaling pathway and nuclear factor(NF)-κB.Knockdown of Dmrt1 in seminiferous tubules resulted in widespread degeneration of germ and somatic cells,while the expression of proinflammatory factors were significantly enhanced.We also demonstrated that Dmrt1 stimulated proliferation of mGSCs,but repressed apoptosis caused by the immune response.Thus,Dmrt1 is sufficient to reduce inflammation in the testes,thereby establishing the stability of spermatogenesis and the testicular microenvironment.
基金financially supported by the National Nature Science Foundation of China(No.30921003)Major Research Plan from the Ministry of Science and Technology of China(No.2013CB945100)
文摘Germlines in plants are formed de novo during post-embryonic development, while little is known about the mechanism that controls this process. In Arabidopsis, the earliest gene controlling this process is SPOROCYTELESS （SPL）. A decade ago, we showed that loss of SPL function abolished sporogenesis in both male and female organs of Arabidopsis. However, its function is unclear up to now. In this study, we showed that SPL belongs to a novel transcription repressor family specific in embryophyte, which consists of 173 members in the land plants so far. All of them contain a conserved SPL-motif in their N-terminal and an ethylene-responsive element binding factor-associated amphiphilic repression （EAR） motif in the C-terminal, therefore designated as SPL-like, EAR-containing proteins （SPEARs）. Consis- tently, SPL acts as a transcriptional repressor in yeast and tobacco cells, and SPEAR proteins are able to form homodimer and/or het- erodimer with each other in vitro. Furthermore, SPEARs interact with the TOPLESS （TPL） co-repressors via the EAR motif and TCP family transcription factors in yeast cells. Together, we propose that SPL and SPEARs most likely belong to a novel transcription repressor family in land plants which may play a variety of developmental roles in plants.
基金supported by grants from National Natural Science Foundation of China(No.81001276 and No.81072380)a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions
文摘MET tyrosine kinase and its ligand,hepatocyte growth factor（HGF）,play a pivotal role in the activties of tumor cells.A germline missense variant in exon 2 of the MET gene,N375S（rs33917957 A〉G）,may alter the binding affinity of MET for HGF and thus modify the risk of tumorigenesis.In this study,we performed a case-control study to assess the association between N375S and gastric cancer risk in 1,681 gastric cancer cases and 1,858 cancer-free controls.Logistic regression analysis was applied to estimate crude and adjusted odds ratios（ORs） and 95% confidence intervals（CIs） for the associations between genotypes and gastric cancer risk.We found that MET N375S variant genotypes（NS/SS） were associated with a significantly decreased risk of gastric cancer（OR = 0.78,95% CI = 0.63-0.96,P = 0.021） compared with the wildtype homozygote（NN）.The finding indicates that this germline variant in MET may decrease gastric cancer susceptibility in Han Chinese.
基金Supported by CREST fellowship from Department of Biotechnology,Ministry of Science and Technology,Government of India(DK)International fellowship for Ph D from ICAR(TRT),Government of IndiaInternational training in generation of i PS cells from NAIP,ICAR,Government of India(TA)
文摘Pluripotent stem cells are unspecialized cells withunlimited self-renewal, and they can be triggered to differentiate into desired specialized cell types. These features provide the basis for an unlimited cell source for innovative cell therapies. Pluripotent cells also allow to study developmental pathways, and to employ them or their differentiated cell derivatives in pharmaceutical testing and biotechnological applications. Via blastocyst complementation, pluripotent cells are a favoured tool for the generation of genetically modified mice. The recently established technology to generate an induced pluripotency status by ectopic co-expression of the transcription factors Oct4, Sox2, Klf4 and c-Myc allows to extending these applications to farm animal species, for which the derivation of genuine embryonic stem cells was not successful so far. Most induced pluripotent stem(i PS) cells are generated by retroviral or lentiviral transduction of reprogramming factors. Multiple viral integrations into the genome may cause insertional mutagenesis and may increase the risk of tumour formation. Non-integration methods have been reported to overcome the safety concerns associated with retro and lentiviral-derived i PS cells, such as transient expression of the reprogramming factors using episomal plasmids, and direct delivery of reprogramming m RNAs or proteins. In this review, we focus on the mechanisms of cellular reprogramming and current methods used to induce pluripotency. We also highlight problems associated with the generation of i PS cells. An increased understanding of the fundamental mechanisms underlying pluripotency and refining the methodology of i PS cell generation will have a profound impact on future development and application in regenerative medicine and reproductive biotechnology of farm animals.
基金financially supported by grants from the Natural Science of Engineering Research and Council(NSERC)of Canada awarded to A.HonaramoozGraduate student scholarships to A.H.Awang-Junaidi were provided by the Ministry of Higher Education of Malaysia,the University of Saskatchewan College of Graduate and Postdoctoral Studiesthe University of Saskatchewan Western College of Veterinary Medicine
文摘Background: Gonocytes give rise to spermatogonial stem cells, and thereby play an essential role in establishing spermatogenesis. Optimized culture conditions for gonocytes provide an opportunity for their study and in vitro manipulation for potential application in reproductive technologies. Using six experiments in a step-wise design, we examined the effects of several culture conditions on the maintenance, proliferation, and colony formation of porcine gonocytes. Testis cells from neonatal piglets were cultured for 7 d in DMEM supplemented with 10% fetal bovine serum. The examined culture conditions included using different cell seeding densities, gonocyte proportions, incubation temperatures, sampling strategies, and medium changing regimens.Results: Confluency of cel s was optimal(>90% by ~6 d) when 3.0 × 104 testis cel s/cm2 containing ~40% gonocytes were used. Incubating the cel s at 35 °C or 37 °C resulted in similar cel number and viability at confluency, but incubation at 35 °C resulted in a delayed confluency. In the first 2 d of culture, gonocytes remained mostly floating in the medium and gradual y settled over the next 5 d. Consequently, not changing the medium for 7 d(as opposed to changing it every 2 d) led to a significant increase in the number of gonocyte colonies by reducing the loss of "floating gonocytes".Conclusion: We found that gonocytes require the presence of a critical minimum number of somatic cel s for settlement, and can proliferate and form growing colonies even in a basic medium. Large numbers of viable gonocytes remain floating in the medium for several days. The optimized culture conditions in the present study included seeding with 3.0 × 104 testis cel s/cm2 containing ~40% gonocytes, incubating at 37 °C, and without changing the medium in the first week, which can result in improved colony formation of porcine gonocytes.
