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Involvement of blood mononuclear cells in the infertility, age-associated diseases and cancer treatment

World Journal of Stem Cells 2016年第12期8卷 399-427页

作者： Antonin Bukovsky Laboratory of reproductive Biology BIOCEV Institute of Biotechnology Czech Academy of Sciences

Blood mononuclear cells consist of T cells and monocyte derived cells. Beside immunity, the blood mononuclear cells belong to the complex tissue control system(TCS), where they exhibit morphostatic function by stimulatingproliferation of tissue stem cells followed by cellular differentiation, that is stopped after attaining the proper functional stage, which differs among various tissue types. Therefore, the term immune and morphostatic system(IMS) should be implied. The TCS-mediated morphostasis also consists of vascular pericytes controlled by autonomic innervation, which is regulating the quantity of distinct tissues in vivo. Lack of proper differentiation of tissue cells by TCS causes either tissue underdevelopment, e.g., muscular dystrophy, or degenerative functional failures, e.g., type 1 diabetes and age-associated diseases. With the gradual IMS regression after 35 years of age the gonadal infertility develops, followed by a growing incidence of age-associated diseases and cancers. Without restoring an altered TCS function in a degenerative disease, the implantation of tissue-specific stem cells alone by regenerative medicine can not be successful. Transfused young blood could temporarily restore fertility to enable parenthood. The young blood could also temporarily alleviate aging diseases, and this can be extended by substances inducing IMS regeneration, like the honey bee propolis. The local and/or systemic use of honey bee propolis stopped hair and teeth loss, regressed varicose veins, improved altered hearing, and lowered high blood pressure and sugar levels. Complete regression of stage Ⅳ ovarian cancer with liver metastases after a simple elaborated immunotherapy is also reported.

关键词： Blood mononuclear cells Age-associated diseases Infertility treatment Regenerative medicine Transfusion morphostatic treatment Stem cells Tissue control system Immune system Tissue morphostasis Cancer immunotherapy

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Effect of PMSG/hCG Superovulation on Mouse Embryonic Development

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Journal of Integrative Agriculture 2013年第6期12卷 1066-1072页

作者： WU Bao-jiang XUE Hong-yan CHEN Li-ping DAI Yan-feng GUO Ji-tong LI Xi-he Inner Mongolia Saikexing reproductive biotechnology Co. Ltd. Research center for Animal Genetic Resources of Mongolia Plateau Inner Mongolia University

Kunming mouse strain is widely used in China, and the superovulation was administrated with 10 IU PMSG combined with 10 IU hCG. In this study, the effects of the exogenous gonadotropins on superovulation of Kunming mice and embryo quality derived from the superovulated mice were assessed. Female mice at 6-8-wk old were superovulated with 0, 5, 7.5 and 10 IU PMSG/hCG and mated with male mice. The embryos were retrieved at 2.5 d post coitum. No statistic difference was observed for the number of 2-cell embryos collected per mouse between control and 5 IU PMSG/hCG treatment group, but the number significantly increased for 7.5 and 10 IU PMSG/hCG treatment group （P〈0.05）. The average number of 4- cell and 8-cell embryos collected from each mouse significantly differed between control and 5, 7.5, 10 IU PMSG/hCG treatment groups （P〈0.05）. When 8-cell embryos derived from mice administrated with 0, 5, 7.5 and 10 IU PMSG/hCG were cultured in KSOM, the blastocyst development rates were 88.1, 94.7, 96.1 and 94.3%, respectively, which were similar to control （P〉0.05）. This indicated that exogenous gonadotropins have no effects on development of Kunming mouse embryos. The quality of blastocyst was assessed by labelling with Hoechst and propidium iodide for inner cell mass and trophectoderm cells, the result showed that ICM/TE ratio significantly decreased for 10 IU PMSG/hCG treatment group compared with control, 5 and 7.5 IU PMSG/hCG treatment group （P〈0.05）. This suggested that the embryo quality of Kunming mouse has been affected by high dose of gonadotropins.

关键词： PMSG mouse 8-cell embryos in vitro culture blastocysts

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Production of transgenic cashmere goat embryos expressing red fluorescent protein and containing IGF1 hair-follicle-cell specific expression cassette by somatic cell nuclear transfer

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Science China(Life Sciences) 2009年第4期52卷 390-397页

作者： BOU ShorGan Key Laboratory for Mammalian reproductive Biology and biotechnology Ministry of Education Inner Mongolia University

In the present study, cashmere goat fetal fibroblasts were transfected with pCDsR-KI, a hair-follicle-cell specific expression vector for insulin-like growth factor 1 (IGF1) that contains two markers for selection (red fluorescent protein gene and neomycin resistant gene). The transgenic fibroblasts cell lines were obtained after G418 selection. Prior to the somatic cell nuclear transfer (SCNT), the maturation rate of caprine cumulus oocytes complexes (COCs) was optimized to an in vitro maturation time of 18 h. Parthenogenetic ooctyes were used as a model to investigate the effect of two activation methods, one with calcium ionophore IA23187 plus 6-DMAP and the other with ethanol plus 6-DMAP. The cleavage rates after 48 h were respectively 88.7% and 86.4%, with no significant difference (P>0.05). There was no significant difference between the cleavage rate and the blastocyst rate in two different media (SO- Faa and CR1aa; 86.3% vs 83.9%, P>0.05 and 23.1% vs 17.2%,P>0.05). The fusion rate of a 190 V/mm group (62.4%) was significantly higher than 130 V/mm (32.8%) and 200 V/mm (42.9%), groups (P<0.05). After transgenic somatic cell nuclear transfer (TSCNT) manipulation, 203 reconstructed embryos were obtained in which the cleavage rate after in vitro development (IVD) for 48 h was 79.3% (161/203). The blastocyst rate after IVD for 7 to 9 d was 15.3% (31/203). There were 17 embryos out of 31 strongly ex- pressing red fluorescence. Two of the red fluorescent blastocysts were randomly selected to identify transgene by polymerase chain reaction. Both were positive. These results showed that: (i) RFP and Neor genes were correctly expressed indicating that transgenic somatic cell lines and positive trans- genic embryos were obtained; (ii) one more selection at the blastocyst stage was necessary although the donor cells were transgenic positive, because only partially transgenic embryos expressing red fluorescence were obtained; and (iii) through TSCNT manipulation and optim

关键词： cashmere goat insulin like growth factor 1 hair follicle fetal fibroblasts cells transgene somatic cell nuclear transfer

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Matrix attachment regions included in a bicistronic vector enhances and stabilizes follistatin gene expressions in both transgenic cells and transgenic mice

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Frontiers of Agricultural Science and Engineering 2016年第1期3卷 87-96页

作者： Xiaoming HU Jing GUO Chunling BAI Zhuying WEI Li GAO Tingmao HU Shorgan BOU Guangpeng LI Key Laboratory of National Education Ministry for Mammalian reproductive Biology and biotechnology/Key Laboratory of Herbivore reproductive biotechnology and Breeding of Ministry of Agriculture Inner Mongolia University

In the present study, follistatin(FST) gene expression vectors with either a bicistronic gene transfer cassette alone, or a bicistron gene cassette carrying a matrix attachment region(MAR) were constructed and transfected to bovine fetal fibroblasts. Evaluations of both the integration and expression of exogenous FST indicated that the p MAR-CAG-FST-IRES-Ac GFP1-poly A-MAR(pMAR-FST) vector had higher capacity to form monoclonal transgenic cells than the vector without MAR,though transient transfection and integration efficiency were similar with either construct. Remarkably, protein expression in transgenic cells with the p MAR-FST vector was significantly higher than that from the bicistronic vector. Exogenous FST was expressed in all of the p MARFST transgenic mice at F_0, F_1 and F_2. Total muscle growth in F_0 mice was significantly greater than in wild-type mice,with larger muscles in fore and hind limbs of transgenic mice. p MAR-FST transgenic mice were also found with more evenly distributed muscle bundles and thinner spaces between sarcolemma, which suggests a correlation between transgene expression-associated muscle development and the trend of muscle growth. In conclusion, a p MAR-FST vector, which excluded the resistant genes and frame structure, enhances and stabilizes FST gene expressions in both transfected cells and transgenic mice.

关键词： safety of transgenic bicistron gene transfer body transgenic mice muscle development

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Inductively coupled plasma emission spectroscopic and flame photometric analysis of goat epididymal fluid

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Asian Journal of Andrology 2000年第4期2卷 288-292页

作者： Meenakshi Gaur Vikas Pruthi Ramasare Prasad Ben M.J.Pereira reproductive Biology Laboratory Dept. of Bioscience and Biotechnology University of Roorkee Roorkee-247 667 U. P. IndiaReproductive Biology Laboratory Dept. of Bioscience and Biotechnology University of Roorkee Roorkee-247 667 U. P. IndiaReproductive Biology Laboratory Dept. of Bioscience and Biotechnology University of Roorkee Roorkee-247 667 U. P. IndiaReproductive Biology Laboratory Dept. of Bioscience and Biotechnology University of Roorkee Roorkee-247 667 U. P. India

Aim: The elemental composition of the epididymal luminal fluid (ELF) in adult goat ( Capra indica) was investigat-ed. Methods: ELF was collected by micropuncture from twelve sites along the epididymal duct. The elementalcontents was analyzed with inductively coupled plasma (ICP) emission spectroscopy, a microanalytical technique thatcan simultaneously measure many elements in minute volumes of sample. The Na and K concentrations were determinedby flame photometry. Results: ICP spectroscopy showed the presence of copper, calcium, nickel, iron, magne-sium, chromium, titanium and zinc in ELF, with fluctuating levels at different sites along the length of the ***, cobalt, lead and manganese were not fotmd. The Na^+ / K^+ ratio was seen to be higher at the initial seg-ments of the epididymis and lower at the distal. Conclusion: It is proposed that the observed characteristic distribu-tion of elements in ELF may have far reaching implications in sperm maturation and storage known to occur in the epi-didymis. (Asian J Androl 2000 Dec; 2: 288-292)

关键词： goat epididymis epididymal luminal fluid elements spectroscopy flame photometry

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Conceptus elongation in ruminants: roles of progesterone, prostaglandin, interferon tau and cortisol

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Journal of Animal Science and biotechnology 2015年第2期6卷 151-162页

作者： Kelsey Brooks Greg Burns Thomas E Spencer Department of Animal Science and center for reproductive Biology Washington State University

The majority of pregnancy loss in ruminants occurs during the first three weeks after conception, particularly during the period of conceptus elongation that occurs prior to pregnancy recognition and implantation. This review integrates established and new information on the biological role of ovarian progesterone （P4）, prostaglandins （PGs）, interferon tau （IFNT） and cortisol in endometrial function and conceptus elongation. Progesterone is secreted by the ovarian corpus luteum （CL） and is the unequivocal hormone of pregnancy. Prostaglandins （PGs） and cortisol are produced by both the epithelial cells of the endometrium and the trophectoderm of the elongating conceptus. In contrast, IFNT is produced solely by the conceptus trophectoderm and is the maternal recognition of pregnancy signa that inhibits production of luteolytic pulses of PGF2α by the endometrium to maintain the CL and thus production of P4. Available results in sheep support the idea that the individual, interactive, and coordinated actions of P4, PGs, IFNT and cortisol regulate conceptus elongation and implantation by controlling expression of genes in the endometrium and/or trophectoderm. An increased knowledge of conceptus-endometrial interactions during early pregnancy in ruminants is necessary to understand and elucidate the causes of infertility and recurrent early pregnancy loss and provide new strategies to improve fertility and thus reproductive efficiency.

关键词： Conceptus Cortisol Endometrium Interferon Prostaqlandin Ruminant

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Epigenetics and transgenerational inheritance in domesticated farm animals

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Journal of Animal Science and biotechnology 2015年第2期6卷 123-129页

作者： Amanda Feeney Eric Nilsson Michael K Skinner center for reproductive Biology School of Biological Sciences Washington State University

Epigenetics provides a molecular mechanism of inheritance that is not solely dependent on DNA sequence and that can account for non-Mendelian inheritance patterns. Epigenetic changes underlie many normal developmental processes, and can lead to disease development as well. While epigenetic effects have been studied in well-characterized rodent models, less research has been done using agriculturally important domestic animal species. This review will present the results of current epigenetic research using farm animal models （cattle, pigs, sheep and chickens）. Much of the work has focused on the epigenetic effects that environmental exposures to toxicants, nutrients and infectious agents has on either the exposed animals themselves or on their direct offspring. Only one porcine study examined epigenetic transgenerational effects; namely the effect diet micronutrients fed to male pigs has on liver DNA methylation and muscle mass in grand-offspring （F2 generation）. Healthy viable offspring are very important in the farm and husbandry industry and epigenetic differences can be associated with production traits. Therefore further epigenetic research into domestic animal health and how exposure to toxicants or nutritional changes affects future generations is imperative.

关键词： Environment EDiclenetics Pig. Review Transgenerational

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Evaluation of the expression of sperm proteins in normozoospermic and asthenozoospermic men using monoclonal antibodies

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Asian Journal of Andrology 2016年第1期18卷 108-113页

作者： Jana Capkova Alena Kubatova Lukas Ded Olina Tepla Jana Peknicova Laboratory of reproductive Biology Institute of Biotechnology Academy of Sciences of the Czech Republic v. v. i. Prague Czech Republic Clinic center IVF ISCARE Prague Czech Republic

Recent studies have shown that infertility affects estimated 15% of all couples. Male infertility is the primary or contributory cause in 60% of these cases. Consequently, the application of assisted reproduction is increasing. These methods could benefit from an extended evaluation of sperm quality. For this reason, we analyzed sperm proteins from 30 men with normal spermiograms and 30 men with asthenozoospermia. Ejaculates of both groups were tested by flow cytometry （FCM） and fluorescence with a set of well-characterized anti-human sperm Hs-monoclonal antibodies （MoAbs）, which were generated in our laboratory. No statistically significant differences were found between normospermics and asthenospermics in the expression of the sperm surface protein clusterin, evaluated with Hs-3 MoAb, and semenogelin, evaluated with Hs-9 MoAb. However, FCM revealed quantitative differences in the acrosomal proteins between normozoospermic and asthenozoospermic men, namely, in glyceraldehyde-3-phosphate dehydrogenase, evaluated with Hs-8 MoAb, valosin-containing protein, evaluated with Hs-14 MoAb, and ATP synthase （cAMP-dependent protein kinase II, PRKAR2A）, evaluated with MoAb Hs-36. Asthenozoospermic men displayed a highly reduced expression of intra-acrosomal proteins, with a likely decrease in sperm quality, and thus a negative impact on successful reproduction. Asthenozoospermia seems to be a complex disorder involving intra-acrosomal proteins.

关键词： asthenozoospermia flow cytometry fluorescence microscopy monoclonal antibodies sperm proteins

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In vivo and in vitro development of Tibetan antelope(Pantholops hodgsonii)interspecific cloned embryos

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Frontiers of Agricultural Science and Engineering 2014年第1期1卷 21-29页

作者： Guanghua SU Lei CHENG Yu GAO Kun LIU Zhuying WEI Chunling BAI Fengxia YIN Li GAO Guangpeng LI Shorgan BOU The Key Laboratory of National Education Ministry for Mammalian reproductive Biology and biotechnology Key Laboratory of Herbivore Reproductive Biotechnology and Breeding Ministry of AgricultureInner Mongolia UniversityHohhot 010021China

The Tibetan antelope is endemic to the Tibetan Plateau,China,and is now considered an endangered *** a possible rescue strategy,the development of embryos constructed by interspecies somatic cell nuclear transfer(iSCNT)was *** antelope fibroblast cells were transferred into enucleated bovine,ovine and caprine *** cloned embryos were then cultured in vitro or in the oviducts of intermediate *** than 0.5%of the reconstructed antelope-bovine embryos cultured in vitro developed to the blastocyst ***,when the cloned antelope-bovine embryos were transferred to caprine oviducts,about 1.6%of the embryos developed to the blastocyst *** contrast,only 0.7%of the antelope-ovine embryos developed to the morula stage and none developed to blastocysts in ovine *** treatment of donor cells and bovine oocytes with trichostatin A did not improve the embryo development even when cultured in the oviducts of ovine and *** the antelope-bovine embryos,constructed from oocytes treated with roscovitine or trichostatin A,were cultured in rabbit oviducts 2.3%and 14.3%developed to blastocysts,*** is concluded that although some success was achieved with the protocols used,interspecies cloning of Tibetan antelope presents difficulties still to be *** mechanisms resulting in the low embryo development need investigation and progress might require a deeper understanding of cellular reprogramming.

关键词： interspecific nuclear transfer bovine ovine caprine oviduct apoptosis

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Evaluation of deoxyribonuclease activity in seminal plasmaof ejaculated chicken semen

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Asian Journal of Andrology 2003年第3期5卷 213-216页

作者： Fuminori Sato Tomoki Soh Masa-aki Hattori Noboru Fujihara Laboratory of reproductive Physiology and biotechnology Department of Animal and Marine Bioresource SciencesFaculty of Agriculture Graduate School Kyushu University Hakozaki Fukuoka 8128581 JapanLaboratory of Reproductive Physiology and Biotechnology Department of Animal and Marine Bioresource SciencesFaculty of Agriculture Graduate School Kyushu University Hakozaki Fukuoka 8128581 JapanLaboratory of Reproductive Physiology and Biotechnology Department of Animal and Marine Bioresource SciencesFaculty of Agriculture Graduate School Kyushu University Hakozaki Fukuoka 8128581 JapanLaboratory of Reproductive Physiology and Biotechnology Department of Animal and Marine Bioresource SciencesFaculty of Agriculture Graduate School Kyushu University Hakozaki Fukuoka 8128581 Japan

Aim: To confirm the stability of exogenous genes in the generation of transgenic chickens using ejaculated chicken sperm, the deoxyribonuclease (DNase) activity was evaluated in the seminal plasma of ejaculated semen and the stability of DNA was examined by adding lipofection reagents. Methods: A PCR fragment (249 bp) of pEGFPN-1 vector was used as the DNA substrate and was incubated with the seminal plasma at 40 ℃ for 30 min. Then, the whole reaction solution was subjected to agarose gel electrophoresis and the DNA size was evaluated under UV light. Results: The DNA substrate was completely diminished after incubation with seminal plasma. However, the substrate was intact after incubation with heat-treated seminal plasma or incubation with seminal plasma in the presence of 0.5 mmol/L -5 mmol/L EDTA. The substrate was stabilized in the seminal plasma by the addition of commercially available lipofection reagents. Conclusion: The DNase activity is present in the seminal plasma of ejaculated chicken semen. However, DNA is stable in the liposomal-DNA complex.

关键词： deoxyribonucleases chickens seminal plasma sperm vectors lipofection

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